Science.gov

Sample records for labeled single-stranded dna

  1. Method for producing labeled single-stranded nucleic acid probes

    DOEpatents

    Dunn, John J.; Quesada, Mark A.; Randesi, Matthew

    1999-10-19

    Disclosed is a method for the introduction of unidirectional deletions in a cloned DNA segment. More specifically, the method comprises providing a recombinant DNA construct comprising a DNA segment of interest inserted in a cloning vector, the cloning vector having an f1 endonuclease recognition sequence adjacent to the insertion site of the DNA segment of interest. The recombinant DNA construct is then contacted with the protein pII encoded by gene II of phage f1 thereby generating a single-stranded nick. The nicked DNA is then contacted with E. coli Exonuclease III thereby expanding the single-stranded nick into a single-stranded gap. The single-stranded gapped DNA is then contacted with a single-strand-specific endonuclease thereby producing a linearized DNA molecule containing a double-stranded deletion corresponding in size to the single-stranded gap. The DNA treated in this manner is then incubated with DNA ligase under conditions appropriate for ligation. Also disclosed is a method for producing single-stranded DNA probes. In this embodiment, single-stranded gapped DNA, produced as described above, is contacted with a DNA polymerase in the presence of labeled nucleotides to fill in the gap. This DNA is then linearized by digestion with a restriction enzyme which cuts outside the DNA segment of interest. The product of this digestion is then denatured to produce a labeled single-stranded nucleic acid probe.

  2. Solvent-modified ultrafast decay dynamics in conjugated polymer/dye labeled single stranded DNA

    NASA Astrophysics Data System (ADS)

    Kim, Inhong; Kang, Mijeong; Woo, Han Young; Oh, Jin-Woo; Kyhm, Kwangseuk

    2015-07-01

    We have investigated that organic solvent (DMSO, dimethyl sulfoxide) modifies energy transfer efficiency between conjugated polymers (donors) and fluorescein-labeled single stranded DNAs (acceptors). In a mixture of buffer and organic solvent, fluorescence of the acceptors is significantly enhanced compared to that of pure water solution. This result can be attributed to change of the donor-acceptor environment such as decreased hydrophobicity of polymers, screening effect of organic solvent molecules, resulting in an enhanced energy transfer efficiency. Time-resolved fluorescence decay of the donors and the acceptors was modelled by considering the competition between the energy harvesting Foerster resonance energy transfer and the energy-wasting quenching. This enables to quantity that the Foerster distance (R0 = 43.3 Å) and resonance energy transfer efficiency (EFRET = 58.7 %) of pure buffer solution become R0 = 38.6 Å and EFRET = 48.0 % when 80% DMSO/buffer mixture is added.

  3. Single strand DNA functionalized single wall carbon nanotubes as sensitive electrochemical labels for arsenite detection.

    PubMed

    Wang, Yonghong; Wang, Ping; Wang, Yiqiang; He, Xiaoxiao; Wang, Kemin

    2015-08-15

    In this work, a simple and sensitive electrochemical strategy for arsenite detection based on the ability of arsenite bound to single-strand DNA (ssDNA) and the signal transduction of single wall carbon nanotubes (SWCNTs) is developed. To realize this purpose, the ssDNA/SWCNTs complexes were formed at first by making ssDNA wrapped around SWCNTs via π-stacking. In the presence of arsenite, the arsenite could strongly bind with the G/T bases of ssDNA and decrease the π-π interaction between ssDNA and SWCNTs, resulting in a certain amount of ssDNA dissociating from the complexes. The separated SWCNTs were selectively assembled on the self-assembled monolayer (SAM) modified Au electrode. Then the SWCNTs onto the SAM-modified Au electrode substantially restored heterogeneous electron transfer that was almost totally blocked by the SAM. The assembled SWCNTs could generate a considerably sensitive and specific tactic for signal transduction, which was related to the concentration of the arsenite. Through detecting the currents mediated by SWCNTs, a linear response to concentration of arsenite ranging from 0.5 to 10ppb and a detection limit of 0.5ppb was readily achieved with desirable specificity and sensitivity. Such a SWCNTs-based biosensor creates a simple, sensitive, nonradioactive route for detection of arsenite. In addition, this demonstration provides a new approach to fabrication of stable biosensors with favorable electrochemical properties believed to be appealing to electroanalytical applications.

  4. Thermophoresis of single stranded DNA.

    PubMed

    Reineck, Philipp; Wienken, Christoph J; Braun, Dieter

    2010-01-01

    The manipulation and analysis of biomolecules in native bulk solution is highly desired; however, few methods are available. In thermophoresis, the thermal analog to electrophoresis, molecules are moved along a microscopic temperature gradient. Its theoretical foundation is still under debate, but practical applications for analytics in biology show considerable potential. Here we measured the thermophoresis of highly diluted single stranded DNA using an all-optical capillary approach. Temperature gradients were created locally by an infrared laser. The thermal depletion of oligonucleotides of between 5 and 50 bases in length were investigated by fluorescence at various salt concentrations. To a good approximation, the previously tested capacitor model describes thermophoresis: the Soret coefficient linearly depends on the Debye length and is proportional to the DNA length to the power of 0.35, dictated by the conformation-based size scaling of the diffusion coefficient. The results form the basis for quantitative DNA analytics using thermophoresis.

  5. DNA sequencing by a single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

    SciTech Connect

    Goodwin, P.M.; Schecker, J.A.; Wilkerson, C.W.; Hammond, M.L.; Ambrose, W.P.; Jett, J.H.; Martin, J.C.; Marrone, B.L.; Keller, R.A. ); Haces, A.; Shih, P.J.; Harding, J.D. )

    1993-01-01

    We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.

  6. DNA sequencing by a single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

    SciTech Connect

    Goodwin, P.M.; Schecker, J.A.; Wilkerson, C.W.; Hammond, M.L.; Ambrose, W.P.; Jett, J.H.; Martin, J.C.; Marrone, B.L.; Keller, R.A.; Haces, A.; Shih, P.J.; Harding, J.D.

    1993-02-01

    We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.

  7. 18F-labeled Single-Stranded DNA Aptamer for PET Imaging of Protein Tyrosine Kinase-7 Expression

    PubMed Central

    Wang, Lu; Wang, Zhe; Yang, Xiangyu; Dewhurst, Andrew; Ma, Ying; Zhu, Guizhi; Niu, Gang; Kiesewetter, Dale O.; Vasdev, Neil; Liang, Steven H.; Chen, Xiaoyuan

    2016-01-01

    Protein tyrosine kinase-7 (PTK7), a member of receptor tyrosine kinase superfamily initially identified as colon carcinoma kinase-4 (CCK-4), is highly expressed in various human malignancies. Its expression was found to correlate with aggressive biological behaviors such as increased cell proliferation, invasiveness and migration. Despite the importance and unmet need of imaging PTK7 in vivo, there is currently no clinically-relevant method to visualize tumoral PTK7 expression noninvasively such as PET or SPECT. This study aims to develop a specific, selective and high affinity PET radioligand based on single-stranded DNA (ssDNA) aptamer to address this challenge. Methods Sgc8, a 41-oligonucleotide that targets to PTK7, was labeled with F-18 using a two-step radiochemical synthesis, which featured a direct one-step radiofluorination on the distinctive spirocyclic hypervalent iodine(III) precursor to give 18F-fluorobenzyl azide followed by copper mediated “click” conjugation with Sgc8-alkyne. 18F-Sgc8 was evaluated in vitro and in vivo in two cell lines, HCT116 and U87MG, which express high and low amounts of PTK7, respectively. Results Sgc8 was labeled efficiently with F-18 in an isolated radiochemical yield of 62 ± 2%, non-decay-corrected (ndc) based on 18F-fluorobenzyl azide. 18F-Tr-Sgc8 was found to possess high affinity binding to both cell lines, with IC50 values for HCT116 as 2.7 ± 0.6 nM and U87MG as 16.9 ± 2.1 nM. In vivo PET imaging clearly visualized PTK7 expression in HCT116 xenografted mice with tumor uptake of 0.76 ± 0.09 %ID/g at 30 min post-injection (p.i.) for the subcutaneous tumor model and greater than 1.5 %ID/g for the liver metastasis model. U87MG xenograft tumors had much lower tracer accumulation (0.13 ± 0.06 %ID/g at 30 min p.i.), which was consistent with the lower expression of PTK7 in this tumor model. The labeled aptamer was rapidly cleared from the blood through the kidneys and bladder to give high tumor-to-blood and tumor

  8. A signal-on fluorescent aptasensor based on single-stranded DNA-sensitized luminescence of terbium (III) for label-free detection of breast cancer cells.

    PubMed

    Cai, Shuxian; Li, Guangwen; Zhang, Xi; Xia, Yaokun; Chen, Mei; Wu, Dongzhi; Chen, Qiuxiang; Zhang, Jing; Chen, Jinghua

    2015-06-01

    Breast cancer is the most common type of malignant tumor in women. Recently, it has been shown that detection of breast cancer tumor cells outside the primitive tumor is an effective early diagnosis with great prognostic and clinical utility. For this purpose, we developed a signal-on fluorescence aptasensor for label-free, facile and sensitive detection of MCF-7 breast cancer cells. Due to target-aptamer specific recognition and single-stranded DNA-sensitized luminescence of terbium (III), the proposed aptasensor exhibits excellent sensitivity with detection limit as low as 70 cells mL(-1). Compared with common organic dyes and the emerging nano-technological probes, the combination of terbium (III) and single-stranded DNA signal probe (Tb(3+)-SP) serves as a more powerful bio-probe because of its stable optical property, good biocompatibility and free from complex synthesis. The feasibility investigations have illustrated the potential applicability of this aptasensor for selective and sensitive detection of MCF-7 breast cancer cells. Moreover, this proposed aptasensor can be also extended for the determination of other tumor cancers or bio-molecules by altering corresponding aptamers. Taken together, this easy-to-perform aptasensor may represent a promising way for early screening and detection of tumor cancers or other bio-molecules in clinical diagnosis.

  9. Recombination in Eukaryotic Single Stranded DNA Viruses

    PubMed Central

    Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-01-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803

  10. Recombination in eukaryotic single stranded DNA viruses.

    PubMed

    Martin, Darren P; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-09-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution.

  11. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel

    1991-01-01

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

  12. Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

    DOEpatents

    Gray, J.W.; Pinkel, D.

    1991-07-02

    A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations. No Drawings

  13. Single-Stranded DNA Curtains for Studying Homologous Recombination.

    PubMed

    Ma, C J; Steinfeld, J B; Greene, E C

    2017-01-01

    Homologous recombination is an important pathway involved in the repair of double-stranded DNA breaks. Genetic studies form the foundation of our knowledge on homologous recombination. Significant progress has also been made toward understanding the biochemical and biophysical properties of the proteins, complexes, and reaction intermediates involved in this essential DNA repair pathway. However, heterogeneous or transient recombination intermediates remain extremely difficult to assess through traditional ensemble methods, leaving an incomplete mechanistic picture of many steps that take place during homologous recombination. To help overcome some of these limitations, we have established DNA curtain methodologies as an experimental platform for studying homologous DNA recombination in real-time at the single-molecule level. Here, we present a detailed overview describing the preparation and use of single-stranded DNA curtains in applications related to the study of homologous DNA recombination with emphasis on recent work related to the study of the eukaryotic recombinase Rad51.

  14. Diffusion of human Replication Protein A along single stranded DNA

    PubMed Central

    Nguyen, Binh; Sokoloski, Joshua; Galletto, Roberto; Elson, Elliot L.; Wold, Marc S.; Lohman, Timothy M.

    2014-01-01

    Replication Protein A (RPA) is a eukaryotic single stranded (ss) DNA binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA-DNA interactions, we combined ensemble and single molecule fluorescence approaches to examine human RPA diffusion along ssDNA and find that an hRPA hetero-trimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D1 ~5000 nucleotide2s−1 at 37°C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA. PMID:25058683

  15. Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

    PubMed Central

    Simon, J R; Moore, P D

    1987-01-01

    Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA. Images PMID:3302673

  16. Dynamics of single-stranded DNA tethered to a solid

    NASA Astrophysics Data System (ADS)

    Radiom, Milad; Paul, Mark R.; Ducker, William A.

    2016-06-01

    Tethering is used to deliver specific biological and industrial functions. For example, single-stranded DNA (ssDNA) is tethered to polymerases and long sequences of double-stranded DNA (dsDNA) during replication, and to solids in DNA microarrays. However, tethering ssDNA to a large object limits not only the available ssDNA conformations, but also the range of time-scales over which the mechanical responses of ssDNA are important. In this work we examine the effect of tethering by measurement of the mechanical response of ssDNA that is tethered at each end to two separate atomic force microscope cantilevers in aqueous solution. Thermal motion of the cantilevers drives the ends of the ssDNA chain at frequencies near 2 kHz. The presence of a tethered molecule makes a large difference to the asymmetric cross-correlation of two cantilevers, which enables resolution of the mechanical properties in our experiments. By analysis of the correlated motion of the cantilevers we extract the friction and stiffness of the ssDNA. We find that the measured friction is much larger than the friction that is usually associated with the unencumbered motion of ssDNA. We also find that the measured relaxation time, ∼30 μs, is much greater than prior measurements of the free-molecule relaxation time. We attribute the difference to the loss of conformational possibilities as a result of constraining the ends of the ssDNA.

  17. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase.

    PubMed

    Gansauge, Marie-Theres; Gerber, Tobias; Glocke, Isabelle; Korlević, Petra; Lippik, Laurin; Nagel, Sarah; Riehl, Lara Maria; Schmidt, Anna; Meyer, Matthias

    2017-01-23

    DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3' biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude.

  18. π-ELECTRONS in a Single Strand of Dna:

    NASA Astrophysics Data System (ADS)

    Iguchi, Kazumoto

    We revisit the problem of the electronic properties of a single strand of DNA, formulating the Hückel approximation for π-electrons in both the sugar-phosphate backbone chain and the π-stacking of nitrogenous bases in a single strand of DNA where the nitrogenous bases are adenine (A), guanine (G), cytosine (C) and thymine (T), respectively. We calculate the electronic band structure of π-electrons: (i) in the single nitrogenous base molecules such as A, G, C and T, (ii) in the single sugar-phosphate molecule, (iii) in the single nucleotide systems such as A, G, C, T with the single sugar-phosphate group, and (iv) in the system of a single strand of DNA with an infinite repetition of a nucleotide such as A, G, C and T, respectively. We find the following: In the case of (i), there is an energy gap between the energy levels for the HOMO and LUMO in the nitrogenous base. This guarantees the semiconducting character of the bases as a mother material. In the case of (ii), there are the HOMO localized at the oxygen site with a double bond and the LUMO localized around the phosphorus atom, which have a quite large energy gap. In the case of (iii), the energy levels for the HOMO and LUMO of the nitrogenous base remain almost the same as those of the nucleotide, while those of the sugar-phosphate group remain the same as well. The HOMO of the sugar-phosphate group exists right below the HOMO of the nitrogenous base. Therefore, comparing the energy levels for the HOMOs of the nitrogenous base group with those of the sugar-phosphate group, the nitrogenous base group behaves as a donor while the sugar-phosphate group behaves as an acceptor. In the case of (iv), there are energy bands and band gaps for the extended states in the nitrogenous base group and the sugar-phosphate group as well as the discrete levels for the localized states at the phosphate site in the spectrum. There is a transition from semiconductor to semimetal as the π-electron hopping between the

  19. Two classes of single-stranded regions evident in deproteinized preparations of replicating DNA isolated from mammalian cells

    SciTech Connect

    Stewart, B.W.; Kavallaris, M.; Catchpoole, D.; Norris, M.D. )

    1991-02-01

    In DNA isolated from proliferating human lymphoblastoid CCRF-CEM cells which had been pulse-labeled by exposure to (3H)thymidine for periods from 30 s to 10 min, single-stranded regions were analyzed by caffeine-gradient elution from benzoylated DEAE-cellulose. Two classes of structural defect were evident. Some replicating DNA exhibited single-stranded regions of approximately 200 nucleotides, while most newly incorporated radioactivity was associated with DNA containing single-stranded regions from 900 to approximately 4000 nucleotides. The distribution of thymidine-derived radioactivity did not suggest sequential or preferential labeling of these DNA fractions as the incorporation time was varied. The findings may be correlated with recent proposals regarding the structural basis of eukaryotic DNA replication.

  20. Renaturation of complementary single-stranded DNA circles: complete rewinding facilitated by the DNA untwisting enzyme.

    PubMed Central

    Champoux, J J

    1977-01-01

    Renaturation of two complementary single-stranded circles should be limited by topological constraints against the rewinding of the DNA helix. If a mixture of complementary single-stranded rings is annealed and then treated with the DNA untwisting enzyme, the DNA circles completely renature as judged by (i) the presence of interlocked rings that sediment at 53 S in alkali, (ii) the buoyant density of the renatured DNA in CsCl gradients containing ethidium bromide, and (iii) the resistance of the product to the single-strand-specific S1 nuclease. Therefore, the DNA untwisting enzyme is able to provide a transient single-strand break that is sufficient to allow the two strands to completely rewind. The possibility that the untwisting enzyme might facilitate the initiation of the process of genetic recombination is discussed. PMID:202951

  1. Factors affecting SFHR gene correction efficiency with single-stranded DNA fragment

    SciTech Connect

    Tsuchiya, Hiroyuki; Harashima, Hideyoshi; Kamiya, Hiroyuki . E-mail: hirokam@pharm.hokudai.ac.jp

    2005-11-04

    A 606-nt single-stranded (ss) DNA fragment, prepared by restriction enzyme digestion of ss phagemid DNA, improves the gene correction efficiency by 12-fold as compared with a PCR fragment, which is the conventional type of fragment used in the small fragment homologous replacement method [H. Tsuchiya, H. Harashima, H. Kamiya, Increased SFHR gene correction efficiency with sense single-stranded DNA, J. Gene Med. 7 (2005) 486-493]. To reveal the characteristic features of this gene correction with the ss DNA fragment, the effects on the gene correction in CHO-K1 cells of the chain length, 5'-phosphate, adenine methylation, and transcription were studied. Moreover, the possibility that the ss DNA fragment is integrated into the target DNA was examined with a radioactively labeled ss DNA fragment. The presence of methylated adenine, but not the 5'-phosphate, enhanced the gene correction efficiency, and the optimal length of the ss DNA fragment ({approx}600 nt) was determined. Transcription of the target gene did not affect the gene correction efficiency. In addition, the target DNA recovered from the transfected CHO-K1 cells was radioactive. The results obtained in this study indicate that length and adenine methylation were important factors affecting the gene correction efficiency, and that the ss DNA fragment was integrated into the double-stranded target DNA.

  2. Super-resolution mbPAINT for optical localization of single-stranded DNA.

    PubMed

    Chen, Jixin; Bremauntz, Alberto; Kisley, Lydia; Shuang, Bo; Landes, Christy F

    2013-10-09

    We demonstrate the application of superlocalization microscopy to identify sequence-specific portions of single-stranded DNA (ssDNA) with sequence resolution of 50 nucleotides, corresponding to a spatial resolution of 30 nm. Super-resolution imaging was achieved using a variation of a single-molecule localization method, termed as "motion blur" point accumulation for imaging in nanoscale topography (mbPAINT). The target ssDNA molecules were immobilized on the substrate. Short, dye-labeled, and complementary ssDNA molecules stochastically bound to the target ssDNA, with repeated binding events allowing super-resolution. Sequence specificity was demonstrated via the use of a control, noncomplementary probe. The results support the possibility of employing relatively inexpensive short ssDNAs to identify gene sequence specificity with improved resolution in comparison to the existing methods.

  3. Characterization of a mitochondrial protein binding to single-stranded DNA.

    PubMed Central

    Mignotte, B; Barat, M; Mounolou, J C

    1985-01-01

    A DNA-binding protein from Xenopus laevis oocyte mitochondria which has been found associated with the D-loop also shows a strong preference for single-stranded DNA. The binding to polynucleotides is dependent on the base composition, but no sequence specificity was found. This protein, called mtSSB, binds tightly and cooperatively to single-stranded DNA. By its amino-acid composition and its binding properties it appears to be similar to the single-stranded DNA-binding proteins found in prokaryotes. PMID:4039816

  4. Characterization of single stranded telomeric DNA-binding proteins in cultured soybean (Glycine max) cells.

    PubMed

    Kwon, Chian; Kwon, Kisang; Chung, In Kwon; Kim, Soon Young; Cho, Myeon Haeng; Kang, Bin Goo

    2004-06-30

    We have identified and characterized a protein factor in soybean (Glycine max) nuclear extracts that binds to plant single stranded telomeric DNA repeats. A single DNA-protein complex was detected in gel retardation assays using synthetic telomeres and nuclear extracts. The protein forming this complex was designated soy-bean (Glycine max) single stranded telomeric DNA-binding protein (Gm-STBP). Gm-STBP binds to single stranded telomeric DNA containing more than two repeats. It does not bind to Tetrahymena, human or mutated plant telomere sequences, and its binding activity is not affected by RNase treatment. Gm-STBP activity gradually decreased after suspension cultures entered stationary phase. A slower migrating band was formed with extracts of earlier and later phases of soybean suspension cultures. Our findings suggest that binding of Gm-STBP to plant single stranded telomeric DNA may play a role in the proper functioning of telomeres during development.

  5. ELECTRON MICROSCOPY OF SINGLE-STRANDED DNA: CIRCULARITY OF DNA OF BACTERIOPHAGE PHI-X174.

    PubMed

    FREIFELDER, D; KLEINSCHMIDT, A K; SINSHEIMER, R L

    1964-10-09

    The single-stranded DNA of coliphage X174 has been examined with the electron microscope by a modification of the protein-monolayer-adsorption technique. The molecules were found to be circular with a total length of 1.77 +/- 0.13 microns.

  6. Efficient Production of Single-Stranded Phage DNA as Scaffolds for DNA Origami

    PubMed Central

    2015-01-01

    Scaffolded DNA origami enables the fabrication of a variety of complex nanostructures that promise utility in diverse fields of application, ranging from biosensing over advanced therapeutics to metamaterials. The broad applicability of DNA origami as a material beyond the level of proof-of-concept studies critically depends, among other factors, on the availability of large amounts of pure single-stranded scaffold DNA. Here, we present a method for the efficient production of M13 bacteriophage-derived genomic DNA using high-cell-density fermentation of Escherichia coli in stirred-tank bioreactors. We achieve phage titers of up to 1.6 × 1014 plaque-forming units per mL. Downstream processing yields up to 410 mg of high-quality single-stranded DNA per one liter reaction volume, thus upgrading DNA origami-based nanotechnology from the milligram to the gram scale. PMID:26028443

  7. DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

    NASA Astrophysics Data System (ADS)

    Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

    2016-02-01

    Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

  8. Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

    PubMed Central

    Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

    2013-01-01

    DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

  9. 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.

  10. Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

    PubMed Central

    Putterman, D G; Casadevall, A; Boyle, P D; Yang, H L; Frangione, B; Day, L A

    1984-01-01

    The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, f1) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNA-binding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses. Images PMID:6422463

  11. Quantum dynamics of a hole migration through DNA: A single strand DNA model.

    PubMed

    Shirmovsky, S Eh

    2016-10-01

    A model predicting the behavior of a hole acting on the DNA strand was investigated. The hole-DNA interaction on the basis of a quantum-classical, non-linear DNA single strand model was described. The fact that a DNA molecule is formed by a furanose ring as its sugar, phosphate group and bases was taken into consideration. Based on the model, results were obtained for the probability of a hole location on the DNA base sequences, such as GTTGGG, GATGTGGG, GTTGTTGGG as well as on the sugar-phosphate groups mated with them.

  12. Ferric and cupric ions requirement for DNA single-strand breakage by H2O2.

    PubMed

    Tachon, P

    1989-01-01

    Hydrogen peroxide (H2O2), was able to nick the replicative form of the phage fd, without the addition of a reducing agent or of a metal. This DNA single-strand breakage decreased with an increase of the ionic strength, suggesting that H2O2 reacted with traces of metal bound to DNA. When cupric of ferric ions were added, the rate of DNA single-strand breakage by H2O2 greatly increased and it was 20-30 times faster with cupric than with ferric ions. The addition of EDTA at an equimolar ratio or in excess of metal prevented partially DNA single-strand cleavage by H2O2 in the presence of ferric ions and completely when cupric ions were used. Superoxide dismutase prevented DNA single-strand breakage by H2O2 and ferric ions. On the contrary, with cupric ions and H2O2, the addition of superoxide dismutase increased the rate of DNA single-strand breakage. That superoxide dismutase was acting catalytically was shown by the loss of its effects after heat inactivation of the enzyme. The results of the present study show that besides its involvement in the Fenton reaction, H2O2 is able to reduce the metal bound to DNA, generating the superoxide anion radical or/and its protonated form, the perhydroxyl radical involved in DNA nicking. On the other hand, the ability of cuprous ions unlike ferrous ions to dismutate the superoxide radical may explain some differences observed between iron and copper in the DNA single-strand breakage by H2O2.

  13. Role of Single-Stranded DNA Binding Activity of T Antigen in Simian Virus 40 DNA Replication

    PubMed Central

    Wu, Chunxiao; Roy, Rupa; Simmons, Daniel T.

    2001-01-01

    We have previously mapped the single-stranded DNA binding domain of large T antigen to amino acid residues 259 to 627. By using internal deletion mutants, we show that this domain most likely begins after residue 301 and that the region between residues 501 and 550 is not required. To study the function of this binding activity, a series of single-point substitutions were introduced in this domain, and the mutants were tested for their ability to support simian virus 40 (SV40) replication and to bind to single-stranded DNA. Two replication-defective mutants (429DA and 460EA) were grossly impaired in single-stranded DNA binding. These two mutants were further tested for other biochemical activities needed for viral DNA replication. They bound to origin DNA and formed double hexamers in the presence of ATP. Their ability to unwind origin DNA and a helicase substrate was severely reduced, although they still had ATPase activity. These results suggest that the single-stranded DNA binding activity is involved in DNA unwinding. The two mutants were also very defective in structural distortion of origin DNA, making it likely that single-stranded DNA binding is also required for this process. These data show that single-stranded DNA binding is needed for at least two steps during SV40 DNA replication. PMID:11222709

  14. Reagentless, Electrochemical Approach for the Specific Detection of Double- and Single-Stranded DNA Binding Proteins

    PubMed Central

    Ricci, Francesco; Bonham, Andrew J.; Mason, Aaron C.; Reich, Norbert O.; Plaxco, Kevin W.

    2009-01-01

    Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins. PMID:19199570

  15. Zn2+ blocks annealing of complementary single-stranded DNA in a sequence-selective manner

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A simple low-temperature EDTA-free agarose gel electrophoresis procedure (LTEAGE) coupled with UV-Vis spectrum and fluorescence quenching analyses was developed and the Zn2+-single-stranded (ss) DNA interaction was investigated under near-physiological conditions. It was found that Zn2+ blocked the...

  16. Selective binding of single-stranded DNA-binding proteins onto DNA molecules adsorbed on single-walled carbon nanotubes.

    PubMed

    Nii, Daisuke; Hayashida, Takuya; Yamaguchi, Yuuki; Ikawa, Shukuko; Shibata, Takehiko; Umemura, Kazuo

    2014-09-01

    Single-stranded DNA-binding (SSB) proteins were treated with hybrids of DNA and single-walled carbon nanotubes (SWNTs) to examine the biological function of the DNA molecules adsorbed on the SWNT surface. When single-stranded DNA (ssDNA) was used for the hybridization, significant binding of the SSB molecules to the ssDNA-SWNT hybrids was observed by using atomic force microscopy (AFM) and agarose gel electrophoresis. When double-stranded DNA (dsDNA) was used, the SSB molecules did not bind to the dsDNA-SWNT hybrids in most of the conditions that we evaluated. A specifically modified electrophoresis procedure was used to monitor the locations of the DNA, SSB, and SWNT molecules. Our results clearly showed that ssDNA/dsDNA molecules on the SWNT surfaces retained their single-stranded/double-stranded structures.

  17. Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus

    PubMed Central

    Wadsworth, Ross I. M.; White, Malcolm F.

    2001-01-01

    Single-stranded DNA binding proteins (SSBs) play central roles in cellular and viral processes involving the generation of single-stranded DNA. These include DNA replication, homologous recombination and DNA repair pathways. SSBs bind DNA using four ‘OB-fold’ (oligonucleotide/oligosaccharide binding fold) domains that can be organised in a variety of overall quaternary structures. Thus eubacterial SSBs are homotetrameric whilst the eucaryal RPA protein is a heterotrimer and euryarchaeal proteins vary significantly in their subunit compositions. We demonstrate that the crenarchaeal SSB protein is an abundant protein with a unique structural organisation, existing as a monomer in solution and multimerising on DNA binding. The protein binds single-stranded DNA distributively with a binding site size of ~5 nt per monomer. Sulfolobus SSB lacks the zinc finger motif found in the eucaryal and euryarchaeal proteins, possessing instead a flexible C-terminal tail, sensitive to trypsin digestion, that is not required for DNA binding. In comparison with Escherichia coli SSB, the tail may play a role in protein–protein interactions during DNA replication and repair. PMID:11160923

  18. Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

    PubMed Central

    Hong, Ka Lok; Sooter, Letha J.

    2015-01-01

    Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed. PMID:26199940

  19. Monitoring Single-Stranded DNA Secondary Structure Formation by Determining the Topological State of DNA Catenanes

    PubMed Central

    Liang, Xingguo; Kuhn, Heiko; Frank-Kamenetskii, Maxim D.

    2006-01-01

    Single-stranded DNA (ssDNA) has essential biological functions during DNA replication, recombination, repair, and transcription. The structure of ssDNA must be better understood to elucidate its functions. However, the available data are too limited to give a clear picture of ssDNA due to the extremely capricious structural features of ssDNA. In this study, by forming DNA catenanes and determining their topology (the linking number, Lk) through the electrophoretic analysis, we demonstrate that the studies of catenanes formed from two ssDNA molecules can yield valuable new information about the ssDNA secondary structure. We construct catenanes out of two short (60/70 nt) ssDNA molecules by enzymatic cyclization of linear oligodeoxynucleotides. The secondary structure formed between the two DNA circles determines the topology (the Lk value) of the constructed DNA catenane. Thus, formation of the secondary structure is experimentally monitored by observing the changes of linking number with sequences and conditions. We found that the secondary structure of ssDNA is much easier to form than expected: the two strands in an internal loop in the folded ssDNA structure prefer to braid around each other rather than stay separately forming a loop, and a duplex containing only mismatched basepairs can form under physiological conditions. PMID:16461397

  20. X-ray photoelectron spectroscopy of γ-ray-irradiated single-stranded DNA

    NASA Astrophysics Data System (ADS)

    Lee, Eunmo; Hong, W.; Han, J. H.; Choi, D. M.; Lee, Cheol Eui; Kim, H. D.; Kim, J.

    2015-07-01

    The effects of γ-ray irradiation on herring sperm single-stranded DNA have been studied by using X-ray photoelectron spectroscopy (XPS) in the view of the bonding configurations and the structural modifications. The significant changes in the hydrogen, carbon, nitrogen, and phosphorous bonding energies, as revealed by the XPS analysis, indicate that electron transfers result in the creation of radicals and in DNA strand breaks.

  1. DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans

    PubMed Central

    Hickman, Alison Burgess; James, Jeffrey A; Barabas, Orsolya; Pasternak, Cécile; Ton-Hoang, Bao; Chandler, Michael; Sommer, Suzanne; Dyda, Fred

    2010-01-01

    Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd2+) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity. PMID:20890269

  2. The single-stranded DNA-binding protein of Escherichia coli.

    PubMed Central

    Meyer, R R; Laine, P S

    1990-01-01

    The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake. PMID:2087220

  3. PET Imaging of Tenascin-C with a Radiolabeled Single-Stranded DNA Aptamer

    PubMed Central

    Jacobson, Orit; Yan, Xuefeng; Niu, Gang; Weiss, Ido D.; Ma, Ying; Szajek, Lawrence P.; Shen, Baozhong; Kiesewetter, Dale O.; Chen, Xiaoyuan

    2017-01-01

    Tenascin-C is an extracellular matrix glycoprotein that is expressed by injured tissues and by various cancers. Recent publications showed that tenascin-C expression by cancer lesions predicts tumor growth, metastasis, and angiogenesis, suggesting tenascin-C as a potential therapeutic target. Currently there is no noninvasive method to determine tumoral tenascin-C expression in vivo. To address the need for an agent to image and quantify tenascin-C, we report the development of a radioactive PET tracer based on a tenascin-C–specific single-stranded DNA aptamer (tenascin-C aptamer). Methods Tenascin-C aptamer was radiolabeled with 18F and 64Cu. PET imaging studies for the evaluation of tumor uptake and pharmacokinetics of tenascin-C aptamer were performed in comparison to a nonspecific scrambled aptamer (Sc aptamer). Results The labeled tenascin-C aptamer provided clear visualization of tenascin-C–positive but not tenascin-C–negative tumors. The uptake of tenascin-C aptamer was significantly higher than that of Sc aptamer in tenascin-C–positive tumors. The labeled tenascin-C aptamer had fast clearance from the blood and other nonspecific organs through the kidneys, resulting in high tumor contrast. Conclusion Our data suggest that suitably labeled tenascin-C aptamer can be used as a PET tracer to image tumor expression of tenascin-C with a high tumor-to-background ratio and might provide insightful and personalized medical data that will help determine appropriate treatment and monitoring. PMID:25698784

  4. Dissociative Electron Attachment to Phosphoric Acid Esters: The Direct Mechanism for Single Strand Breaks in DNA

    SciTech Connect

    Koenig, Constanze; Kopyra, Janina; Bald, Ilko; Illenberger, Eugen

    2006-07-07

    We use dibutyl phosphate to simulate the behavior of the phosphate group in DNA towards the attack of low energy electrons. We find that the compound undergoes effective dissociative electron attachment within a low energy resonant feature at 1 eV and a further resonance peaking at 8 eV. The dissociative electron attachment (DEA) reactions are associated with the direct cleavage of the C-O and the P-O bond but also the excision of the PO{sup -}, PO{sub 3}{sup -}, H{sub 2}PO{sub 3}{sup -} units. For the phosphate group coupled in the DNA network these reactions represent single strand breaks. We hence propose that the most direct mechanism of single strand breaks occurring in DNA at subexcitation energies (<4 eV) is due to DEA directly to the phosphate group.

  5. Equilibrium binding of single-stranded DNA to the secondary DNA binding site of the bacterial recombinase RecA.

    PubMed

    Gourves, A S; Defais, M; Johnson, N P

    2001-03-30

    The bacterial recombinase RecA forms a nucleoprotein filament in vitro with single-stranded DNA (ssDNA) at its primary DNA binding site, site I. This filament has a second site, site II, which binds ssDNA and double-stranded DNA. We have investigated the binding of ssDNA to the RecA protein in the presence of adenosine 5'-O-(thiotriphosphate) cofactor using fluorescence anisotropy. The RecA protein carried out DNA strand exchange with a 5'-fluorescein-labeled 32-mer oligonucleotide. The anisotropy signal was shown to measure oligonucleotide binding to RecA, and the relationship between signal and binding density was determined. Binding of ssDNA to site I of RecA was stable at high NaCl concentrations. Binding to site II could be described by a simple two-state equilibrium, K = 4.5 +/- 1.5 x 10(5) m(-1) (37 degrees C, 150 mm NaCl, pH 7.4). The reaction was enthalpy-driven and entropy-opposed. It depended on salt concentration and was sensitive to the type of monovalent anion, suggesting that anion-dependent protein conformations contribute to ssDNA binding at site II.

  6. A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology

    PubMed Central

    Ander, Marcel; Subramaniam, Sivaraman; Fahmy, Karim; Stewart, A. Francis; Schäffer, Erik

    2015-01-01

    Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA–protein interaction. PMID:26271032

  7. Noncovalent Sidewall Functionalization of Carbon Nanotubes by Biomolecules: Single-stranded DNA and Hydrophobin

    NASA Astrophysics Data System (ADS)

    Taeger, Sebastian; Xuang, Li Yi; Günther, Katrin; Mertig, Michael

    2005-09-01

    Single-stranded DNA (ssDNA) is known to disperse individual carbon nanotubes (CNT) into aqueous suspensions. But other biomolecules are able to do so as well. We demonstrate a protein-assisted CNT dispersion by using hydrophobin. The yields of the suspensions are monitored by optical absorption spectroscopy (OAS). We perform atomic force microscopy (AFM) studies of DNA- and hydrophobin-functionalized CNT with a resolution that allows us to identify individual molecules attached to isolated CNT. We control the density of DNA on the nanotubes by the DNA:CNT ratio, and observe stable suspensions of CNT with surprisingly low surface coverages.

  8. Method for nucleic acid hybridization using single-stranded DNA binding protein

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1996-01-01

    Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe. The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample (potentially including the nucleic acid sequence) with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution (i.e., one in which the t.sub.1/2 of renaturation is longer than 3 weeks) of single-stranded DNA greater than 500 fold (i.e., to a t.sub.1/2 less than 60 min, preferably less than 5 min, and most preferably about 1 min.) in the absence of nucleotide triphosphates.

  9. Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

    PubMed

    Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

    2013-10-01

    Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

  10. Complementary addressed modification and cleavage of a single stranded DNA fragment with alkylating oligonucleotide derivatives.

    PubMed Central

    Vlassov, V V; Zarytova, V F; Kutiavin, I V; Mamaev, S V; Podyminogin, M A

    1986-01-01

    A single stranded DNA fragment was modified with alkylating derivatives of oligonucleotides complementary to a certain nucleotide sequences in the fragment. The derivatives carried aromatic 2-chloroethylamino groups at their 3'- or 5'-terminal nucleotide residues. Some of the derivatives carried both alkylating group and intercalating phenazine group which stabilized complementary complexes. It was found that these oligonucleotide derivatives modify the DNA fragment in a specific way near the target complementary nucleotide sequences, and the DNA fragment can be cleaved at the alkylated nucleotides positions. Alkylating derivatives carrying phenazine groups were found to be the most efficient in reaction with the DNA fragment. Images PMID:3714471

  11. Single-strand DNA detection using a planar photonic-crystal-waveguide-based sensor.

    PubMed

    Toccafondo, V; García-Rupérez, J; Bañuls, M J; Griol, A; Castelló, J G; Peransi-Llopis, S; Maquieira, A

    2010-11-01

    We report an experimental demonstration of single-strand DNA (ssDNA) detection at room temperature using a photonic-crystal-waveguide-based optical sensor. The sensor surface was previously biofunctionalized with ssDNA probes to be used as specific target receptors. Our experiments showed that it is possible to detect these hybridization events using planar photonic-crystal structures, reaching an estimated detection limit as low as 19.8 nM for the detection of the complementary DNA strand.

  12. Towards quantitative viromics for both double-stranded and single-stranded DNA viruses

    PubMed Central

    Roux, Simon; Solonenko, Natalie E.; Dang, Vinh T.; Poulos, Bonnie T.; Schwenck, Sarah M.; Goldsmith, Dawn B.; Coleman, Maureen L.; Breitbart, Mya

    2016-01-01

    Background Viruses strongly influence microbial population dynamics and ecosystem functions. However, our ability to quantitatively evaluate those viral impacts is limited to the few cultivated viruses and double-stranded DNA (dsDNA) viral genomes captured in quantitative viral metagenomes (viromes). This leaves the ecology of non-dsDNA viruses nearly unknown, including single-stranded DNA (ssDNA) viruses that have been frequently observed in viromes, but not quantified due to amplification biases in sequencing library preparations (Multiple Displacement Amplification, Linker Amplification or Tagmentation). Methods Here we designed mock viral communities including both ssDNA and dsDNA viruses to evaluate the capability of a sequencing library preparation approach including an Adaptase step prior to Linker Amplification for quantitative amplification of both dsDNA and ssDNA templates. We then surveyed aquatic samples to provide first estimates of the abundance of ssDNA viruses. Results Mock community experiments confirmed the biased nature of existing library preparation methods for ssDNA templates (either largely enriched or selected against) and showed that the protocol using Adaptase plus Linker Amplification yielded viromes that were ±1.8-fold quantitative for ssDNA and dsDNA viruses. Application of this protocol to community virus DNA from three freshwater and three marine samples revealed that ssDNA viruses as a whole represent only a minor fraction (<5%) of DNA virus communities, though individual ssDNA genomes, both eukaryote-infecting Circular Rep-Encoding Single-Stranded DNA (CRESS-DNA) viruses and bacteriophages from the Microviridae family, can be among the most abundant viral genomes in a sample. Discussion Together these findings provide empirical data for a new virome library preparation protocol, and a first estimate of ssDNA virus abundance in aquatic systems. PMID:28003936

  13. Reversible accumulation of double- and single-stranded DNA breaks in DNA in growth-arrested cells

    SciTech Connect

    S'yakste, N.I.; S'yakste, T.G.; Zaleskaya, N.D.

    1987-01-01

    The authors study the possibility of the formation and repair of double-stranded breaks in DNA during a change in the proliferative status of cells. Jungarian hamster fibroblasts, transformed by SV-40 virus, were cultured in Carrel's flasks in a nutrient mixture containing Eagle's medium, lactalbumin hydrolysate, and bovine serum in the ratio of 4.5:4.5:1. DNA was labelled by the addition of 0.4 MBq/ml of tritium-thymidine to the incubation medium for 2-7 days. To arrest cell growth, the monolayer formed seven days after seeding was placed in medium with the serum concentration lowered to 1%, and incubated for 7-9 days. The cells were restimulated for division with fresh complete medium. The mitotic index was 20% in the exponentially growing cultures while in the resting cultures it was 1-2% and rose to 16% 2 days after stimulation. Double-stranded DNA breaks were determined by neutral elution of DNA; single-stranded breaks were measured by the alkaline DNA uncoiling method with fixation of hydroxyapatite. The formation of double-stranded DNA breaks and their repair, in response to a change in the proliferative status of the cell, were discovered for the first time.

  14. FEN1 participates in repair of the 5'-phosphotyrosyl terminus of DNA single-strand breaks.

    PubMed

    Kametani, Yukiko; Takahata, Chiaki; Narita, Takashi; Tanaka, Kiyoji; Iwai, Shigenori; Kuraoka, Isao

    2016-01-01

    Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells.

  15. 'A' forms of RNAs in single strands, duplexes and RNA-DNA hybrids.

    PubMed Central

    Broyde, S; Hingerty, B

    1978-01-01

    Helical parameters have been calculated for the 'A' form minimum energy conformations of ApA, CpC, GpG, UpU, GpC and UpA. The helix geometries are base sequence dependent. The single strands are narrower and more tightly wound than that duplex RNA-11 form. 9-12 kcal./mole are needed to convert these single strands to the RNA-11 conformation. However, in some sequences other 'A' type conformers capable of complementary base pairing may be formed at lower energetic cost. There is substantially more base stacking in the calculated single strands than in the RNA-11 conformation. Calculated intrastrand base stacking energies reflect these differences, and also are sequence dependent. The 'A' form RNA subunits differ from the analogous DNAs in possessing a larger rise per residue, needed to accomodate the 2'-OH. RNA-DNA hybrids are consequently more likely to be in the 'A-RNA than in the 'A'-DNA conformation, although the base sequence determines the extent of the preference. PMID:693318

  16. Relative rates of repair of single-strand breaks and postirradiation DNA degradation in normal and induced cells of Escherichia coli.

    PubMed Central

    Pollard, E C; Fugate, J K

    1978-01-01

    Labeled DNA from irradiated Excherichia coli cells has been studied on an alkaline sucrose gradient without acid precipitation of the DNA. This enables the observation of both DNA repair and DNA degradation. The use of a predose of ultraviolet light (UV) causes induction of an inhibitor of postirradiation DNA degradation in lex+ strains. The effect of this induction on both the repair of single-strand breaks and DNA degradation has been followed in strains WU3610 (uvr+) and WU3610-89 (uvr-). The repair process is more rapid than the degradation, and when degradation is inhibited more repair is apparent. Cells that are lex- (Bs-1 and AB2474) cannot be induced for inhibition of degradation. Nevertheless, by observation at short times repair can be seen clearly. This repaired DNA is degraded, suggesting that the signal for DNA degradation is not a single-strand break. PMID:365253

  17. Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein

    SciTech Connect

    Ryzhikov, Mikhail; Koroleva, Olga; Postnov, Dmitri; Tran, Andrew; Korolev, Sergey

    2011-08-25

    RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.

  18. Discovery of a novel circular single-stranded DNA virus from porcine faeces.

    PubMed

    Sikorski, Alyssa; Argüello-Astorga, Gerardo R; Dayaram, Anisha; Dobson, Renwick C J; Varsani, Arvind

    2013-01-01

    A large number of novel single-stranded DNA (ssDNA) viruses have been characterised from various environmental sources in the last 5 years. The bulk of these have been from faecal sources, and faecal sampling is an ideal non-invasive pathogen sampling method. We characterised a novel ssDNA from a porcine faecal sample from Cass Basin of the South Island of New Zealand. The novel viral genome has two large open reading frames (ORFs), which are bidirectionally transcribed and separated by intergenic regions. The largest ORF has some degree of similarity (<30 %) to the putative capsid protein of chimpanzee stool-associated circular ssDNA virus (ChiSCV) and pig stool-associated single-stranded DNA virus (PigSCV), whereas the second-largest ORF has high similarity to the putative replication-associated protein (Rep) of ChiSCV (~50 %) and bovine stool-associated circular DNA virus (BoSCV; ~30 %). Based on genome architecture, location of putative stem-loop like elements, and maximum-likelihood phylogenetic analysis of the gene encoding the Rep protein, the novel isolate belongs to the same family of ssDNA viruses as ChiSCV and BoSCV.

  19. DNA Binding of Centromere Protein C (CENPC) Is Stabilized by Single-Stranded RNA

    PubMed Central

    Du, Yaqing; Topp, Christopher N.; Dawe, R. Kelly

    2010-01-01

    Centromeres are the attachment points between the genome and the cytoskeleton: centromeres bind to kinetochores, which in turn bind to spindles and move chromosomes. Paradoxically, the DNA sequence of centromeres has little or no role in perpetuating kinetochores. As such they are striking examples of genetic information being transmitted in a manner that is independent of DNA sequence (epigenetically). It has been found that RNA transcribed from centromeres remains bound within the kinetochore region, and this local population of RNA is thought to be part of the epigenetic marking system. Here we carried out a genetic and biochemical study of maize CENPC, a key inner kinetochore protein. We show that DNA binding is conferred by a localized region 122 amino acids long, and that the DNA-binding reaction is exquisitely sensitive to single-stranded RNA. Long, single-stranded nucleic acids strongly promote the binding of CENPC to DNA, and the types of RNAs that stabilize DNA binding match in size and character the RNAs present on kinetochores in vivo. Removal or replacement of the binding module with HIV integrase binding domain causes a partial delocalization of CENPC in vivo. The data suggest that centromeric RNA helps to recruit CENPC to the inner kinetochore by altering its DNA binding characteristics. PMID:20140237

  20. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element Specific for Bromacil

    PubMed Central

    Williams, Ryan M.; Kulick, Amanda R.; Yedlapalli, Srilakshmi; Battistella, Louisa; Hajiran, Cyrus J.; Sooter, Letha J.

    2014-01-01

    Bromacil is a widely used herbicide that is known to contaminate environmental systems. Due to the hazards it presents and inefficient detection methods, it is necessary to create a rapid and efficient sensing device. Towards this end, we have utilized a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for bromacil. We have identified one MRE with high affinity (Kd = 9.6 nM) and specificity for bromacil compared to negative targets of selection and other pesticides. The selected ssDNA MRE will be useful as the sensing element in a field-deployable bromacil detection device. PMID:25400940

  1. Orientation discrimination of single-stranded DNA inside the α-hemolysin membrane channel

    NASA Astrophysics Data System (ADS)

    Mathé, Jérôme; Aksimentiev, Aleksei; Nelson, David R.; Schulten, Klaus; Meller, Amit

    2005-08-01

    We characterize the voltage-driven motion and the free motion of single-stranded DNA (ssDNA) molecules captured inside the ≈1.5-nm α-hemolysin pore, and show that the DNA-channel interactions depend strongly on the orientation of the ssDNA molecules with respect to the pore. Remarkably, the voltage-free diffusion of the 3‧-threaded DNA (in the trans to cis direction) is two times slower than the corresponding 5‧-threaded DNA having the same poly(dA) sequence. Moreover, the ion currents flowing through the blocked pore with either a 3‧-threaded DNA or 5‧ DNA differ by ≈30%. All-atom molecular dynamics simulations of our system reveal a microscopic mechanism for the asymmetric behavior. In a confining pore, the ssDNA straightens and its bases tilt toward the 5‧ end, assuming an asymmetric conformation. As a result, the bases of a 5‧-threaded DNA experience larger effective friction and forced reorientation that favors co-passing of ions. Our results imply that the translocation process through a narrow pore is more complicated than previously believed and involves base tilting and stretching of ssDNA molecules inside the confining pore. Author contributions: K.S. and A.M. designed research; J.M., A.A., D.R.N., K.S., and A.M. performed research; J.M., A.A., and A.M. analyzed data; J.M., A.A., D.R.N., K.S., and A.M. wrote the paper; A.A. and K.S. performed molecular dynamics simulations; and D.R.N. performed calculations.This paper was submitted directly (Track II) to the PNAS office.Abbreviations: α-HL, α-hemolysin MD, molecular dynamics; ssDNA, single-stranded DNA.

  2. Detection of DNA single-strand breaks induced by procarcinogens in Chinese hamster ovary cells cocultured with rat hepatocytes

    SciTech Connect

    Yang, K.H.; Shin, C.G.; Choe, S.Y.; Kim, D.H.

    1984-01-01

    DNA single-strand breaks induced by procarcinogens were detected in Chinese hamster overy (CHO) cell cocultured with adult rat hepatocytes. Freshly isolated adult rat hepatocytes were added to the CHO cell culture prelabeled with (/sup 3/H) thymidine. After allowing the hepatocytes to attach on or near the CHO cells, aflatoxin B/sub 1/ or benzo(a)pyrene was added to the culture and incubated for the desired time. DNA single-strand breaks in CHO cells were measured by the alkaline elution technique. Aflatoxin B/sub 1/ induced some DNA single-strand breaks in CHO cells cultured alone, but in coculture system with hepatocytes the number of DNA single-strand breaks increased greatly. The magnitude of the increase was related to the dose and the time of exposure to aflatoxin B/sub 1/. Addition of proteinase-K to the cell lysates increased the elution of DNA compared to that of samples without proteinase-K. Benzo(a)pyrene did not induce any DNA single-strand breaks in CHO cells in the absence of liver cells, but a significant number of single-strand breaks were detected in the coculture system.

  3. Replication Protein A Prohibits Diffusion of the PCNA Sliding Clamp along Single-Stranded DNA

    PubMed Central

    2017-01-01

    The replicative polymerases cannot accommodate distortions to the native DNA sequence such as modifications (lesions) to the native template bases from exposure to reactive metabolites and environmental mutagens. Consequently, DNA synthesis on an afflicted template abruptly stops upon encountering these lesions, but the replication fork progresses onward, exposing long stretches of the damaged template before eventually stalling. Such arrests may be overcome by translesion DNA synthesis (TLS) in which specialized TLS polymerases bind to the resident proliferating cell nuclear antigen (PCNA) and replicate the damaged DNA. Hence, a critical aspect of TLS is maintaining PCNA at or near a blocked primer/template (P/T) junction upon uncoupling of fork progression from DNA synthesis by the replicative polymerases. The single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), coats the exposed template and might prohibit diffusion of PCNA along the single-stranded DNA adjacent to a blocked P/T junction. However, this idea had yet to be directly tested. We recently developed a unique Cy3-Cy5 Forster resonance energy transfer (FRET) pair that directly reports on the occupancy of DNA by PCNA. In this study, we utilized this FRET pair to directly and continuously monitor the retention of human PCNA at a blocked P/T junction. Results from extensive steady state and pre-steady state FRET assays indicate that RPA binds tightly to the ssDNA adjacent to a blocked P/T junction and restricts PCNA to the upstream duplex region by physically blocking diffusion of PCNA along ssDNA. PMID:28177605

  4. Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

    PubMed Central

    Gallardo, Ignacio F.; Zhou, Yi; Gong, Fade; Yang, Soo-Hyun; Wold, Marc S.; Miller, Kyle M.; Paull, Tanya T.

    2016-01-01

    Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells. PMID:26884156

  5. Self-assembly of complex two-dimensional shapes from single-stranded DNA tiles.

    PubMed

    Wei, Bryan; Vhudzijena, Michelle K; Robaszewski, Joanna; Yin, Peng

    2015-05-08

    Current methods in DNA nano-architecture have successfully engineered a variety of 2D and 3D structures using principles of self-assembly. In this article, we describe detailed protocols on how to fabricate sophisticated 2D shapes through the self-assembly of uniquely addressable single-stranded DNA tiles which act as molecular pixels on a molecular canvas. Each single-stranded tile (SST) is a 42-nucleotide DNA strand composed of four concatenated modular domains which bind to four neighbors during self-assembly. The molecular canvas is a rectangle structure self-assembled from SSTs. A prescribed complex 2D shape is formed by selecting the constituent molecular pixels (SSTs) from a 310-pixel molecular canvas and then subjecting the corresponding strands to one-pot annealing. Due to the modular nature of the SST approach we demonstrate the scalability, versatility and robustness of this method. Compared with alternative methods, the SST method enables a wider selection of information polymers and sequences through the use of de novo designed and synthesized short DNA strands.

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

    PubMed

    Kim, Kyoung-Ho; Bae, Jin-Woo

    2011-11-01

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

  7. A novel single-stranded DNA detection method based on organic semiconductor heterojunction

    NASA Astrophysics Data System (ADS)

    Gu, Wen; Liu, Hongbo; Zhang, Xia; Zhang, Hao; Chen, Xiong; Wang, Jun

    2016-12-01

    We demonstrate a novel DNA detection method with low-cost and disposable advantages by utilizing F16CuPc/CuPc planar organic heterojunction device. Single-stranded DNA (ssDNA) molecules have been well immobilized on the surface of CuPc film observed by atomic force microscopy, producing an obvious electrical response of the device. The conductivity of the organic heterojunction film was significantly increased by ssDNA immobilization because ssDNA molecules brought additional positive charges at heterojunction interface. Furthermore, the thickness dependence of CuPc upper layer on the electrical response was studied to optimize the sensitivity. This study will be helpful for the development of organic heterojunction based biosensors.

  8. Generation of DNA single-strand displacement by compromised nucleotide excision repair

    PubMed Central

    Godon, Camille; Mourgues, Sophie; Nonnekens, Julie; Mourcet, Amandine; Coin, Fréderic; Vermeulen, Wim; Mari, Pierre-Olivier; Giglia-Mari, Giuseppina

    2012-01-01

    Nucleotide excision repair (NER) is a precisely coordinated process essential to avoid DNA damage-induced cellular malfunction and mutagenesis. Here, we investigate the mechanistic details and effects of the NER machinery when it is compromised by a pathologically significant mutation in a subunit of the repair/transcription factor TFIIH, namely XPD. In contrast to previous studies, we find that no single- or double-strand DNA breaks are produced at early time points after UV irradiation of cells bearing a specific XPD mutation, despite the presence of a clear histone H2AX phosphorylation (γH2AX) signal in the UV-exposed areas. We show that the observed γH2AX signal can be explained by the presence of longer single-strand gaps possibly generated by strand displacement. Our in vivo measurements also indicate a strongly reduced TFIIH-XPG binding that could promote single-strand displacement at the site of UV lesions. This finding not only highlights the crucial role of XPG's interactions with TFIIH for proper NER, but also sheds new light on how a faulty DNA repair process can induce extreme genomic instability in human patients. PMID:22863773

  9. Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA

    SciTech Connect

    Lee, B.L.; Murakami, A.; Blake, K.R.; Lin, S.B.; Miller, P.S.

    1988-05-03

    Oligodeoxyribonucleoside methylphosphonates derivatized at the 5' end with 4'-(amino-alkyl)-4,5',8-trimethylpsoralen were prepared. The interaction of these psoralen-derivatized methyl-phosphonate oligomers with synthetic single-stranded DNAs 35 nucleotides in length was studied. Irradiation of a solution containing the 35-mer and its complementary methylphosphonate oligomer at 365 nm gave a cross-linked duplex produced by cycloaddition between the psoralen pyrone ring of the derivatized methylphosphonate oligomer and a thymine base of the DNA. Photoadduct formation could be reversed by irradiation at 254 nm. The rate and extent of cross-linking were dependent upon the length of the aminoalkyl linker between the trimethylpsoralen group and the 5' end of the methylphosphonate oligomer. Methylphosphonate oligomers derivatized with 4'-((N-(2-aminoethyl)amino)methyl)-4,5',8-trimethylpsoralen gave between 70% and 85% cross-linked product when irradiated for 20 min at 4 /sup 0/C. These results suggest that the methylphosphonate oligomers undergo both cross-linking and deactivation reactions when irradiated at 365 nm. The cross-linking reaction was dependent upon the fidelity of base-pairing interactions between the methylphosphonate oligomers and the single-stranded DNA. The extent and sequence specificity of photoinduced cross-linking, combined with the known ability of methylphosphonate oligomers to be taken up by living cells, suggest that psoralen-derivatized oligonucleoside methylphosphonates may be useful probes of cellular gene expression.

  10. Electron attachment to the cytosine-centered DNA single strands: does base stacking matter?

    PubMed

    Gu, Jiande; Wang, Jing; Leszczynski, Jerzy

    2012-02-02

    Electron attachment to the trimer of nucleotide, dGpdCpdG, has been investigated by a quantum mechanical approach at a reliable level of theory. The study of the electron attached dGpdCpdG species demonstrates that cytosine contained DNA single strands have a strong tendency to capture low-energy electrons and to form electronically stable cytosine-centered radical anions. The comparative study of the model molecules pdCpdG and dGpdCp reveals that base stacking has little contribution to the adiabatic electron affinity (AEA) of cytosine in DNA single strands. Additionally, the base-base stacking does not affect the vertical detachment energy (VDE) of the cytosine-centered radicals. Intrastrand H-bonding is found to be critical in increasing the values of the AEA and VDE. However, base-base stacking is revealed to be important in enlarging the vertical electron affinity (VEA) of cytosine. The electron attachment to the cytosine moiety intensifies the intrastrand H-bonding between the neighboring G and C bases. This process disrupts the base-base stacking interaction in the radical anion of dGpdCpdG.

  11. Novel Circular Single-Stranded DNA Viruses among an Asteroid, Echinoid and Holothurian (Phylum: Echinodermata).

    PubMed

    Jackson, Elliot W; Bistolas, Kalia S I; Button, Jason B; Hewson, Ian

    2016-01-01

    Echinoderms are prone to large population fluctuations that can be mediated by pervasive disease events. For the majority of echinoderm disease events the causative pathogen is unknown. Viruses have only recently been explored as potential pathogens using culture-independent techniques though little information currently exists on echinoderm viruses. In this study, ten circular ssDNA viruses were discovered in tissues among an asteroid (Asterias forbesi), an echinoid (Strongylocentrotus droebachiensis) and a holothurian (Parastichopus californicus) using viral metagenomics. Genome architecture and sequence similarity place these viruses among the rapidly expanding circular rep-encoding single stranded (CRESS) DNA viral group. Multiple genomes from the same tissue were no more similar in sequence identity to each other than when compared to other known CRESS DNA viruses. The results from this study are the first to describe a virus from a holothurian and continue to show the ubiquity of these viruses among aquatic invertebrates.

  12. Novel Circular Single-Stranded DNA Viruses among an Asteroid, Echinoid and Holothurian (Phylum: Echinodermata)

    PubMed Central

    Jackson, Elliot W.; Bistolas, Kalia S. I.; Button, Jason B.; Hewson, Ian

    2016-01-01

    Echinoderms are prone to large population fluctuations that can be mediated by pervasive disease events. For the majority of echinoderm disease events the causative pathogen is unknown. Viruses have only recently been explored as potential pathogens using culture-independent techniques though little information currently exists on echinoderm viruses. In this study, ten circular ssDNA viruses were discovered in tissues among an asteroid (Asterias forbesi), an echinoid (Strongylocentrotus droebachiensis) and a holothurian (Parastichopus californicus) using viral metagenomics. Genome architecture and sequence similarity place these viruses among the rapidly expanding circular rep-encoding single stranded (CRESS) DNA viral group. Multiple genomes from the same tissue were no more similar in sequence identity to each other than when compared to other known CRESS DNA viruses. The results from this study are the first to describe a virus from a holothurian and continue to show the ubiquity of these viruses among aquatic invertebrates. PMID:27855181

  13. Using surface-enhanced Raman spectroscopy to probe for genetic markers on single-stranded DNA

    NASA Astrophysics Data System (ADS)

    Moody, Benjamin; Leotaud, John; McCarty, Gregory S.

    2010-03-01

    Methods capable of quickly and inexpensively collecting genetic information are of increasing importance. We report a method of using surface-enhanced Raman spectroscopy to probe single-stranded DNA for genetic markers. This unique approach is used to analyze unmodified genes of moderate length for genetic markers by hybridizing native test oligonucleotides into a surface-enhanced Raman complex, vastly increasing detection sensitivity as compared to traditional Raman spectroscopy. The Raman complex is formed by sandwiching the test DNA between 40-nm gold nanoparticles and a photolithographically defined gold surface. With this design, we are able to collect characteristic Raman spectra about the test DNA and to detect genetic markers such as single-nucleotide polymorphisms (SNPs) and polymorphic regions. Results show that strands containing one of three different types of polymorphism can be differentiated using statistically significant trends regarding Raman intensity.

  14. Spermine and spermidine protection of plasmid DNA against single-strand breaks induced by singlet oxygen.

    PubMed Central

    Khan, A U; Di Mascio, P; Medeiros, M H; Wilson, T

    1992-01-01

    Oxidative damage to DNA induced by singlet molecular oxygen (1O2*) includes single-strand breaks, which the biologically occurring 1O2* quenchers spermine and spermidine are shown to prevent. These polyamines at a physiological concentration (10 mM) reduce the percentage of the open circular form of pBR322 plasmid DNA, which is generated at the expense of the native supercoiled form when the plasmids are incubated with a chemical source of 1O2*, the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate. Spermine and spermidine can be expected to protect DNA against other damaging effects of 1O2*. Images PMID:1454831

  15. Single-stranded DNA phages: from early molecular biology tools to recent revolutions in environmental microbiology.

    PubMed

    Székely, Anna J; Breitbart, Mya

    2016-03-01

    Single-stranded DNA (ssDNA) phages are profoundly different from tailed phages in many aspects including the nature and size of their genome, virion size and morphology, mutation rate, involvement in horizontal gene transfer, infection dynamics and cell lysis mechanisms. Despite the importance of ssDNA phages as molecular biology tools and model systems, the environmental distribution and ecological roles of these phages have been largely unexplored. Viral metagenomics and other culture-independent viral diversity studies have recently challenged the perspective of tailed, double-stranded DNA (dsDNA) phages, dominance by demonstrating the prevalence of ssDNA phages in diverse habitats. However, the differences between ssDNA and dsDNA phages also substantially limit the efficacy of simultaneously assessing the abundance and diversity of these two phage groups. Here we provide an overview of the major differences between ssDNA and tailed dsDNA phages that may influence their effects on bacterial communities. Furthermore, through the analysis of 181 published metaviromes we demonstrate the environmental distribution of ssDNA phages and present an analysis of the methodological biases that distort their study through metagenomics.

  16. Surface modification of solid-state nanopores for sticky-free translocation of single-stranded DNA.

    PubMed

    Tang, Zhipeng; Lu, Bo; Zhao, Qing; Wang, Jiajun; Luo, Kaifu; Yu, Dapeng

    2014-11-12

    Nanopore technology is one of the most promising approaches for fast and low-cost DNA sequencing application. Single-stranded DNA detection is primary objective in such device, while solid-state nanopores remain less explored than their biological counterparts due to bio-molecule clogging issue caused by surface interaction between DNA and nanopore wall. By surface coating a layer of polyethylene glycol (PEG), solid-state nanopore can achieve long lifetime for single-stranded DNA sticky-free translocation at pH 11.5. Associated with elimination of non-specific binding of molecule, PEG coated nanopore presents new surface characteristic as less hydrophility, lower 1/f noise, and passivated surface charge responsiveness on pH. Meanwhile, conductance blockage of single-stranded DNA is found to be deeper than double-stranded DNA, which can be well described by a string of blobs model for a quasi-equilibrium state polymer in constraint space.

  17. recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein.

    PubMed Central

    McEntee, K; Weinstock, G M; Lehman, I R

    1980-01-01

    The single-stranded DNA-binding protein of Escherichia coli significantly alters the strand assimilation reaction catalyzed by recA protein [McEntee, K., Weinstock, G. M. & Lehman, I. R. (1979) Proc. Natl. Acad. Sci. USA 76, 2615--2619]. The binding protein (i) increases the rate and extent of strand assimilation into homologous duplex DNA, (ii) enhances the formation of a complex between recA protein and duplex DNA in the presence of homologous or heterologous single-stranded DNA, (iii) reduces the rate and extent of ATP hydrolysis catalyzed by recA protein in the presence of single-stranded DNA, (iv) reduces the high concentration of recA protein required for strand assimilation, and (v) permits detection of strand assimilation in the presence of the ATP analog, adenosine 5'-O-(O-thiotriphosphate). Single-stranded DNA-binding protein purified from a binding protein mutant (lexC) is considerably less effective than wild-type binding protein in stimulating strand assimilation, a result which suggests that single-stranded DNA-binding protein participates in general recombination in vivo. PMID:6244589

  18. Single-strand DNA library preparation improves sequencing of formalin-fixed and paraffin-embedded (FFPE) cancer DNA

    PubMed Central

    Stiller, Mathias; Sucker, Antje; Griewank, Klaus; Aust, Daniela; Baretton, Gustavo Bruno; Schadendorf, Dirk; Horn, Susanne

    2016-01-01

    DNA derived from formalin-fixed and paraffin-embedded (FFPE) tissue has been a challenge to large-scale genomic sequencing, due to its low quality and quantities. Improved techniques enabling the genome-wide analysis of FFPE material would be of great value, both from a research and clinical perspective. Comparing a single-strand DNA library preparation method originally developed for ancient DNA to conventional protocols using double-stranded DNA derived from FFPE material we obtain on average 900-fold more library molecules and improved sequence complexity from as little as 5 ng input DNA. FFPE DNA is highly fragmented, usually below 100bp, and up to 60% of reads start after or end prior to adenine residues, suggesting that crosslinks predominate at adenine residues. Similar to ancient DNA, C > T substitutions are slightly increased with maximum rates up to 3% at the ends of molecules. In whole exome sequencing of single-strand libraries from lung, breast, colorectal, prostate and skin cancers we identify known cancer mutations. In summary, we show that single-strand library preparation enables genomic sequencing, even from low amounts of degraded FFPE DNA. This method provides a clear advantage both in research and clinical settings, where FFPE material (e.g. from biopsies) often is the only source of DNA available. Improving the genetic characterization that can be performed on conventional archived FFPE tissue, the single-strand library preparation allows scarce samples to be used in personalized medicine and enables larger sample sizes in future sequencing studies. PMID:27463017

  19. Single-stranded DNA scanning and deamination with Single molecule resolution

    NASA Astrophysics Data System (ADS)

    Rueda, David

    2012-04-01

    Over the past decade, single-molecule fluorescence resonance energy transfer spectroscopy (smFRET) has become an increasingly popular tool to study the structural dynamics of biopolymers, such as DNA, RNA and proteins. The most attractive aspect of single-molecule experiments is that, unlike ensemble-averaged techniques, they directly reveal the structural dynamics of individual molecules, which would otherwise be hidden in ensemble-averaged experiments. Here, we will present a novel single molecule assay to study, for the first time, scanning of an enzyme (APOBEC3G, involved in the defense against HIV) on single stranded DNA (ssDNA). We have investigated the ssDNA scanning and activity of Apo3G with smFRET. Our data show that Apo3G scans ssDNA randomly and bidirectionally with average excursion lengths of ˜ 10 å and ˜1 s-1 scanning rates. Apo3G quasi-localization is observed on highly reactive motifs located near the one end of the ssDNA. Motif-dependent ssDNA bending is also observed, where the bending is maximal for highly reactive targets located near the DNA end. Interestingly, both the Apo3G scanning and Apo3G-induced ssDNA bending is reduced with lowered ionic strength, indicating that Apo3G motion on ssDNA is facilitated by salt by reducing `electrostatic friction'. Although scanning is random, asymmetric catalytic orientation may be the reason for Apo3G directional activity.

  20. Molecular Genetic and Biochemical Characterization of the Vaccinia Virus I3 Protein, the Replicative Single-Stranded DNA Binding Protein

    PubMed Central

    Greseth, Matthew D.; Boyle, Kathleen A.; Bluma, Matthew S.; Unger, Bethany; Wiebe, Matthew S.; Soares-Martins, Jamaria A.; Wickramasekera, Nadi T.; Wahlberg, James

    2012-01-01

    Vaccinia virus, the prototypic poxvirus, efficiently and faithfully replicates its ∼200-kb DNA genome within the cytoplasm of infected cells. This intracellular localization dictates that vaccinia virus encodes most, if not all, of its own DNA replication machinery. Included in the repertoire of viral replication proteins is the I3 protein, which binds to single-stranded DNA (ssDNA) with great specificity and stability and has been presumed to be the replicative ssDNA binding protein (SSB). We substantiate here that I3 colocalizes with bromodeoxyuridine (BrdU)-labeled nascent viral genomes and that these genomes accumulate in cytoplasmic factories that are delimited by membranes derived from the endoplasmic reticulum. Moreover, we report on a structure/function analysis of I3 involving the isolation and characterization of 10 clustered charge-to-alanine mutants. These mutants were analyzed for their biochemical properties (self-interaction and DNA binding) and biological competence. Three of the mutant proteins, encoded by the I3 alleles I3-4, -5, and -7, were deficient in self-interaction and unable to support virus viability, strongly suggesting that the multimerization of I3 is biologically significant. Mutant I3-5 was also deficient in DNA binding. Additionally, we demonstrate that small interfering RNA (siRNA)-mediated depletion of I3 causes a significant decrease in the accumulation of progeny genomes and that this reduction diminishes the yield of infectious virus. PMID:22438556

  1. DNA single strand breaks in peripheral blood lymphocytes induced by three nitroimidazole derivatives.

    PubMed

    Rodriguez Ferreiro, Gisell; Cancino Badías, Lourdes; Lopez-Nigro, Marcela; Palermo, Ana; Mudry, Marta; González Elio, Prieto; Carballo, Marta Ana

    2002-06-14

    Tinidazole (TNZ), ornidazole (ONZ) and metronidazole (MTZ) are antiparasitic drugs (nitroimidazole derivatives) that have proven to be effective against Trichomonas vaginalis, Entoamoeba histolytica, Giardia lamblia and Helicobacter pylori. The reduction of the nitro group and the generation of short-lived reactive intermediates are the basis of its parasiticidal activity. This reduction is associated with its mutagenic activity in bacteria, although in mammalian cells DNA damage seems to be related to the production of reactive oxygen species (ROS). Using alkaline single cell electrophoresis, a significant increase in single strand breaks and alkali labile sites in human peripheral blood lymphocytes (PBL) exposed to MTZ, ONZ and TNZ at 10, 100 and 500 microg/ml is observed. MTZ causes less damage, especially at higher concentrations, when compared with TNZ, the most harmful of the drugs tested. These findings suggest that primary damage is induced under aerobic conditions and confirms that these nitroimidazoles are DNA damaging agents.

  2. Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes.

    PubMed

    Ma, Enbo; Harrington, Lucas B; O'Connell, Mitchell R; Zhou, Kaihong; Doudna, Jennifer A

    2015-11-05

    Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.

  3. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

    PubMed Central

    Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

    2015-01-01

    Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

  4. Molecular-basis of single-walled carbon nanotube recognition by single-stranded DNA.

    PubMed

    Roxbury, Daniel; Mittal, Jeetain; Jagota, Anand

    2012-03-14

    Hybrids of biological molecules and single-walled carbon nanotubes (SWCNT) have proven useful for SWCNT sorting and are enabling several biomedical applications in sensing, imaging, and drug delivery. In the DNA-SWCNT system, certain short (10-20mer) sequences of single-stranded DNA recognize specific SWCNT, allowing the latter to be sorted from a chirality diverse mixture. (1) However, little is known about the DNA secondary structures that underlie their recognition of SWCNTs. Using replica exchange molecular dynamics (REMD) of multiple strands on a single SWCNT, we report that DNA forms ordered structures on SWCNTs that are strongly DNA sequence and SWCNT dependent. DNA sequence (TAT)(4) on its recognition partner, the (6,5) SWCNT, (1) forms an ordered right-handed helically wrapped barrel, stabilized by intrastrand, self-stitching hydrogen bonds and interstrand hydrogen bonding. The same sequence on the larger diameter (8,7)-SWCNT forms a different and less-stable structure, demonstrating SWCNT selectivity. In contrast, homopolymer (T)(12), with weaker tendency for intrastrand hydrogen bonding, forms a distinctly left-handed wrap on the (6,5)-SWCNT, demonstrating DNA sequence specificity. Experimental measurements show that (TAT)(4) selectively disperses smaller diameter SWCNTs more efficiently than (T)(12), establishing a relationship between recognition motifs and binding strength. The developing understanding of DNA secondary structure on nanomaterials can shed light on a number of issues involving hybrids of nanomaterials and biological molecules, including nanomedicine, health-effects of nanomaterials, and nanomaterial processing.

  5. Translocation of E. coli RecQ helicase on single-stranded DNA.

    PubMed

    Rad, Behzad; Kowalczykowski, Stephen C

    2012-04-03

    A member of the SF2 family of helicases, Escherichia coli RecQ, is involved in the recombination and repair of double-stranded DNA breaks and single-stranded DNA (ssDNA) gaps. Although the unwinding activity of this helicase has been studied biochemically, the mechanism of translocation remains unclear. To this end, using ssDNA of varying lengths, the steady-state ATP hydrolysis activity of RecQ was analyzed. We find that the rate of ATP hydrolysis increases with DNA length, reaching a maximum specific activity of 38 ± 2 ATP/RecQ/s. Analysis of the rate of ATP hydrolysis as a function of DNA length implies that the helicase has a processivity of 19 ± 6 nucleotides on ssDNA and that RecQ requires a minimal translocation site size of 10 ± 1 nucleotides. Using the T4 phage encoded gene 32 protein (G32P), which binds ssDNA cooperatively, to decrease the lengths of ssDNA gaps available for translocation, we observe a decrease in the rate of ATP hydrolysis activity that is related to lattice occupancy. Analysis of the activity in terms of the average gap sizes available to RecQ on the ssDNA coated with G32P indicates that RecQ translocates on ssDNA on average 46 ± 11 nucleotides before dissociating. Moreover, when bound to ssDNA, RecQ hydrolyzes ATP in a cooperative fashion, with a Hill coefficient of 2.1 ± 0.6, suggesting that at least a dimer is required for translocation on ssDNA. We present a kinetic model for translocation by RecQ on ssDNA based on this characterization.

  6. High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein.

    PubMed

    Morten, Michael J; Gamsjaeger, Roland; Cubeddu, Liza; Kariawasam, Ruvini; Peregrina, Jose; Penedo, J Carlos; White, Malcolm F

    2017-03-01

    Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.

  7. Single-stranded DNA-binding proteins: multiple domains for multiple functions.

    PubMed

    Dickey, Thayne H; Altschuler, Sarah E; Wuttke, Deborah S

    2013-07-02

    The recognition of single-stranded DNA (ssDNA) is integral to myriad cellular functions. In eukaryotes, ssDNA is present stably at the ends of chromosomes and at some promoter elements. Furthermore, it is formed transiently by several cellular processes including telomere synthesis, transcription, and DNA replication, recombination, and repair. To coordinate these diverse activities, a variety of proteins have evolved to bind ssDNA in a manner specific to their function. Here, we review the recognition of ssDNA through the analysis of high-resolution structures of proteins in complex with ssDNA. This functionally diverse set of proteins arises from a limited set of structural motifs that can be modified and arranged to achieve distinct activities, including a range of ligand specificities. We also investigate the ways in which these domains interact in the context of large multidomain proteins/complexes. These comparisons reveal the structural features that define the range of functions exhibited by these proteins.

  8. Genetic spell-checking: gene editing using single-stranded DNA oligonucleotides.

    PubMed

    Rivera-Torres, Natalia; Kmiec, Eric B

    2016-02-01

    Single-stranded oligonucleotides (ssODNs) can be used to direct the exchange of a single nucleotide or the repair of a single base within the coding region of a gene in a process that is known, generically, as gene editing. These molecules are composed of either all DNA residues or a mixture of RNA and DNA bases and utilize inherent metabolic functions to execute the genetic alteration within the context of a chromosome. The mechanism of action of gene editing is now being elucidated as well as an understanding of its regulatory circuitry, work that has been particularly important in establishing a foundation for designing effective gene editing strategies in plants. Double-strand DNA breakage and the activation of the DNA damage response pathway play key roles in determining the frequency with which gene editing activity takes place. Cellular regulators respond to such damage and their action impacts the success or failure of a particular nucleotide exchange reaction. A consequence of such activation is the natural slowing of replication fork progression, which naturally creates a more open chromatin configuration, thereby increasing access of the oligonucleotide to the DNA template. Herein, how critical reaction parameters influence the effectiveness of gene editing is discussed. Functional interrelationships between DNA damage, the activation of DNA response pathways and the stalling of replication forks are presented in detail as potential targets for increasing the frequency of gene editing by ssODNs in plants and plant cells.

  9. Involvement of histidine in complex formation of PriB and single-stranded DNA.

    PubMed

    Fujiyama, Saki; Abe, Yoshito; Takenawa, Taichi; Aramaki, Takahiko; Shioi, Seijiro; Katayama, Tsutomu; Ueda, Tadashi

    2014-02-01

    PriB is a basic 10-kDa protein that acts as a facilitator in PriA-dependent replication restart in Escherichia coli. PriB has an OB-fold dimer structure and exhibits single-stranded DNA (ssDNA)-binding activities similar to single-stranded binding protein (SSB). In this study, we examined PriB's interaction with ssDNA (oligo-dT35, -dT15, and -dT7) using heteronuclear NMR analysis. Interestingly, (1)H or (15)N chemical shift changes of the PriB main-chain showed two distinct modes using oligo-dT35. The chemical shift perturbation sites in the primary mode were consistent with the main contact site in PriB-ssDNA, which was previously determined by crystal structure analysis. The results also suggested that approximately 8nt in ssDNA was the main contact site to PriB. In the secondary mode, residues in the α-helix region (His57-Ser65) and in β4-loop3-β5 were mainly perturbed. On the other hand, we examined the state of ssDNA by FRET using 5'-Cy3- and 3'-Cy5-modified oligo-dT35. As the PriB concentration increased, two-step saturation curves were observed in the FRET assay, suggesting a compact structure of ssDNA. Moreover, we confirmed two-step PriB binding to oligo-dT35 using EMSA. The pH dependence of FRET suggested contribution of the His residues. Therefore, we prepared His mutants of PriB and found that His64 in the α-helix region contributed to the second interaction between PriB and ssDNA using FRET and EMSA. Thus, from a structural standpoint, we suggested the role of His64 on the compactness of the PriB-ssDNA complex and on the positive cooperativity of PriB.

  10. Controlling the translocation of single-stranded DNA through alpha-hemolysin ion channels using viscosity.

    PubMed

    Kawano, Ryuji; Schibel, Anna E P; Cauley, Christopher; White, Henry S

    2009-01-20

    Translocation of single-stranded DNA through alpha-hemolysin (alpha-HL) channels is investigated in glycerol/water mixtures containing 1 M KCl. Experiments using glass nanopore membranes as the lipid bilayer support demonstrate that the translocation velocities of poly(deoxyadenylic acid), poly(deoxycytidylic acid), and poly(deoxythymidylic acid) 50-mers are decreased by a factor of approximately 20 in a 63/37 (vol %) glycerol/water mixture, relative to aqueous solutions. The ion conductance of alpha-HL and the entry rate of the polynucleotides into the protein channel also decrease with increasing viscosity. Precise control of translocation parameters by adjusting viscosity provides a potential means to improve sequencing methods based on ion channel recordings.

  11. Plant somatic hybrid cytoplasmic DNA characterization by single-strand conformation polymorphism.

    PubMed

    Olivares-Fuster, Oscar; Hernández-Garrido, María; Guerri, José; Navarro, Luis

    2007-06-01

    Unlike maternal inheritance in sexual hybridization, plant somatic hybridization allows transfer, mixing and recombination of cytoplasmic genomes. In addition to the use of somatic hybridization in plant breeding programs, application of this unique tool should lead to a better understanding of the roles played by the chloroplastic and mitochondrial genomes in determining agronomically important traits. The nucleotide sequences of cytoplasmic genomes are much more conserved than those of nuclear genomes. Cytoplasmic DNA composition in somatic hybrids is commonly elucidated either by length polymorphism analysis of restricted genome regions amplified with universal primers (PCR-RF) or by hybridization of total DNA using universal cytoplasmic probes. In this study, we demonstrate that single-stranded conformational polymorphism (SSCP) analysis is a powerful, quick and easy alternative method for cytoplasmic DNA characterization of somatic hybrids, especially for mitochondrial DNA. The technique allows detection of polymorphisms based on both size and sequence of amplified targets. Twenty-two species of the subfamily Aurantioideae were analyzed with eight universal primers (four from chloroplastic and four from mitochondrial regions). Differences in chloroplastic DNA composition were scored in 98% of all possible two-parent combinations, and different mitochondrial DNA profiles were found in 87% of them. Analysis by SSCP was also successfully used to characterize somatic hybrids and cybrids obtained by fusion of Citrus sinensis (L.) Osb. and C. excelsa Wester protoplasts.

  12. Viral Single-Strand DNA Induces p53-Dependent Apoptosis in Human Embryonic Stem Cells

    PubMed Central

    Hirsch, Matthew L.; Fagan, B. Matthew; Dumitru, Raluca; Bower, Jacquelyn J.; Yadav, Swati; Porteus, Matthew H.; Pevny, Larysa H.; Samulski, R. Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication. PMID:22114676

  13. Human replication protein A binds single-stranded DNA in two distinct complexes.

    PubMed Central

    Blackwell, L J; Borowiec, J A

    1994-01-01

    Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes. Images PMID:8196638

  14. RecA-ssDNA filaments supercoil in the presence of single-stranded DNA-binding protein

    SciTech Connect

    Shi Weixian; Larson, Ronald G. . E-mail: rlarson@umich.edu

    2007-06-08

    Using atomic force microscopy (AFM), we find that RecA-single-stranded DNA (RecA-ssDNA) filaments, in the presence of single-stranded DNA-binding (SSB) protein, organize into left-handed bundles, which differ from the previously reported disordered aggregates formed when SSB is excluded from the reaction. In addition, we see both left- and right-handedness on bundles of two filaments. These two-filament supercoils, individual filaments, and other smaller bundles further organize into more complicated bundles, showing overall left-handedness which cannot be explained by earlier arguments that presumed supercoiling is absent in RecA-ssDNA filaments. This novel finding and our previous results regarding supercoiling of RecA-double-stranded DNA (RecA-dsDNA) filaments are, however, consistent with each other and can possibly be explained by the intrinsic tendency of RecA-DNA filaments, in their fully coated form, to order themselves into helical bundles, independent of the DNA inside the filaments (ssDNA or dsDNA). RecA-RecA interactions may dominate the bundling process, while the original conformation of DNA inside filaments and other factors (mechanical properties of filaments, concentration of filaments, and Mg{sup 2+} concentration) could contribute to the variation in the appearance and pitch of supercoils. The tendency of RecA-DNA filaments to form ordered supercoils and their presence during strand exchange suggest a possible biological importance of supercoiled filaments.

  15. Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers

    SciTech Connect

    Freeman, S.E.; Blackett, A.D.; Monteleone, D.C.; Setlow, R.B.; Sutherland, B.M.; Sutherland, J.C.

    1986-10-01

    The authors have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controller scanner. The medium lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single stand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single stand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determined pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single stand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amendable to radioactive labeling.

  16. Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

    PubMed Central

    Tucker, Kimberly P; Parsons, Rachel; Symonds, Erin M; Breitbart, Mya

    2011-01-01

    Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem. PMID:21124487

  17. Novel Single-Stranded DNA Virus Genomes Recovered from Chimpanzee Feces Sampled from the Mambilla Plateau in Nigeria

    PubMed Central

    Walters, Matthew; Bawuro, Musa; Christopher, Alfred; Knight, Alexander; Kraberger, Simona; Stainton, Daisy; Chapman, Hazel

    2017-01-01

    ABSTRACT Metagenomic approaches are rapidly expanding our knowledge of the diversity of viruses. In the fecal matter of Nigerian chimpanzees we recovered three gokushovirus genomes, one circular replication-associated protein encoding single-stranded DNA virus (CRESS), and a CRESS DNA molecule. PMID:28254982

  18. Translocation of single stranded DNA through the α-hemolysin protein nanopore in acidic solutions

    PubMed Central

    de Zoysa, Ranulu Samanthi S.; Krishantha, D.M. Milan; Zhao, Qitao; Gupta, Jyoti; Guan, Xiyun

    2012-01-01

    The effect of acidic pH on the translocation of single-stranded DNA through the α-hemolysin pore is investigated. Two significantly different types of events, i.e., deep blockades and shallow blockades, are observed at low pH. The residence times of the shallow blockades are not significantly different from those of the DNA translocation events obtained at or near physiological pH, while the deep blockades have much larger residence times and blockage amplitudes. With a decrease in the pH of the electrolyte solution, the percentage of the deep blockades in the total events increases. Furthermore, the mean residence time of these long-lived events is dependent on the length of DNA, and also varies with the nucleotide base, suggesting that they are appropriate for use in DNA analysis. In addition to be used as an effective approach to affect DNA translocation in the nanopore, manipulation of the pH of the electrolyte solution provides a potential means to greatly enhance the sensitivity of nanopore stochastic sensing. PMID:21997574

  19. Selection and characterization of single stranded DNA aptamers for the hormone abscisic Acid.

    PubMed

    Grozio, Alessia; Gonzalez, Victor M; Millo, Enrico; Sturla, Laura; Vigliarolo, Tiziana; Bagnasco, Luca; Guida, Lucrezia; D'Arrigo, Cristina; De Flora, Antonio; Salis, Annalisa; Martin, Elena M; Bellotti, Marta; Zocchi, Elena

    2013-10-01

    The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 μM and 0.80 ± 0.07 μM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.

  20. Multiple Layers of Chimerism in a Single-Stranded DNA Virus Discovered by Deep Sequencing

    PubMed Central

    Krupovic, Mart; Zhi, Ning; Li, Jungang; Hu, Gangqing; Koonin, Eugene V.; Wong, Susan; Shevchenko, Sofiya; Zhao, Keji; Young, Neal S.

    2015-01-01

    Viruses with single-stranded (ss) DNA genomes infect hosts in all three domains of life and include many medically, ecologically, and economically important pathogens. Recently, a new group of ssDNA viruses with chimeric genomes has been discovered through viral metagenomics. These chimeric viruses combine capsid protein genes and replicative protein genes that, respectively, appear to have been inherited from viruses with positive-strand RNA genomes, such as tombusviruses, and ssDNA genomes, such as circoviruses, nanoviruses or geminiviruses. Here, we describe the genome sequence of a new representative of this virus group and reveal an additional layer of chimerism among ssDNA viruses. We show that not only do these viruses encompass genes for capsid proteins and replicative proteins that have distinct evolutionary histories, but also the replicative genes themselves are chimeras of functional domains inherited from viruses of different families. Our results underscore the importance of horizontal gene transfer in the evolution of ssDNA viruses and the role of genetic recombination in the emergence of novel virus groups. PMID:25840414

  1. Multicopy single-stranded DNA directs intestinal colonization of enteric pathogens

    DOE PAGES

    Elfenbein, Johanna R.; Knodler, Leigh A.; Nakayasu, Ernesto S.; ...

    2015-09-14

    Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking itsmore » retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.« less

  2. Multicopy Single-Stranded DNA Directs Intestinal Colonization of Enteric Pathogens

    SciTech Connect

    Elfenbein, Johanna R.; Knodler, Leigh A.; Nakayasu, Ernesto S.; Ansong, Charles; Brewer, Heather M.; Bogomolnaya, Lydia; Adams, L. Garry; McClelland, Michael; Adkins, Joshua N.; Andrews-Polymenis, Helene L.; Fang, Ferric C.

    2015-09-14

    Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.

  3. Multicopy single-stranded DNA directs intestinal colonization of enteric pathogens

    SciTech Connect

    Elfenbein, Johanna R.; Knodler, Leigh A.; Nakayasu, Ernesto S.; Ansong, Charles; Brewer, Heather M.; Bogomolnaya, Lydia; Adams, L. Garry; McClelland, Michael; Adkins, Joshua N.; Andrews-Polymenis, Helene L.; Fang, Ferric C.

    2015-09-14

    Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.

  4. The adsorption of short single-stranded DNA oligomers to mineral surfaces.

    PubMed

    Cleaves, H James; Crapster-Pregont, Ellen; Jonsson, Caroline M; Jonsson, Christopher L; Sverjensky, Dimitri A; Hazen, Robert A

    2011-06-01

    We studied the adsorption of short single-stranded deoxyribonucleic acid (ssDNA) oligomers, of approximately 30 nucleotides (nt) in length, of varying sequence, adenine+guanine+cytosine (AGC) content, and propensity to form secondary structure, to equal surface area samples of olivine, pyrite, calcite, hematite, and rutile in 0.1M NaCl, 0.05M pH 8.1 KHCO(3) buffer. Although the mineral surfaces have widely varying points of zero charge, under these conditions they show remarkably similar adsorption of ssDNA regardless of oligomer characteristics. Mineral surfaces appear to accommodate ssDNA comparably, or ssDNA oligomers of this length are able to find binding sites of comparable strength and density due to their flexibility, despite the disparate surface properties of the different minerals. This may partially be due charge shielding by the ionic strength of the solutions tested, which are typical of many natural environments. These results may have some bearing on the adsorption and accumulation of biologically derived nucleic acids in sediments as well as the abiotic synthesis of nucleic acids before the origin of life.

  5. Single strand DNA specificity analysis of human nucleoside diphosphate kinase B.

    PubMed

    Agou, F; Raveh, S; Mesnildrey, S; Véron, M

    1999-07-09

    Nucleoside diphosphate kinases (NDP kinases) form a family of oligomeric enzymes present in all organisms. Eukaryotic NDP kinases are hexamers composed of identical subunits (approximately 17 kDa). A distinctive property of human NDPK-B encoded by the gene nm23-H2 is its ability to stimulate the gene transcription. This property is independent of its catalytic activity and is possibly related to the role of this protein in cellular events including differentiation and tumor metastasis. In this paper, we report the first characterization of human NDPK-B.DNA complex formation using a filter-binding assay and fluorescence spectroscopy. We analyzed the binding of several oligonucleotides mimicking the promoter region of the c-myc oncogene including variants in sequence, structure, and length of both strands. We show that NDPK-B binds to single-stranded oligonucleotides in a nonsequence specific manner, but that it exhibits a poor binding activity to double-stranded oligonucleotides. This indicates that the specificity of recognition to DNA is a function of the structural conformation of DNA rather than of its specific sequence. Moreover, competition experiments performed with all nucleotides provide evidence for the contribution of the six active sites in the DNA.protein complex formation. We propose a mechanism through which human NDPK-B could stimulate transcription of c-myc or possibly other genes involved in cellular differentiation.

  6. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Atrazine

    PubMed Central

    Williams, Ryan M.; Crihfield, Cassandra L.; Gattu, Srikanth; Holland, Lisa A.; Sooter, Letha J.

    2014-01-01

    Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples. PMID:25196435

  7. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

    PubMed

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

    2015-06-05

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  8. Substrate-assisted 2D DNA lattices and algorithmic lattices from single-stranded tiles.

    PubMed

    Kim, Junghoon; Ha, Tai Hwan; Park, Sung Ha

    2015-08-07

    We present a simple route to circumvent kinetic traps which affect many types of DNA nanostructures in their self-assembly process. Using this method, a new 2D DNA lattice made up of short, single-stranded tile (SST) motifs was created. Previously, the growth of SST DNA assemblies was restricted to 1D (tubes and ribbons) or finite-sized 2D (molecular canvases). By utilizing the substrate-assisted growth method, sets of SSTs were designed as unit cells to self-assemble into periodic and aperiodic 2D lattices which continuously grow both along and orthogonal to the helical axis. Notably, large-scale (∼1 μm(2)) fully periodic 2D lattices were fabricated using a minimum of just 2 strand species. Furthermore, the ability to create 2D lattices from a few motifs enables certain rules to be encoded into these SSTs to carry out algorithmic self-assembly. A set of these motifs was designed to execute simple 1-input 1-output COPY and NOT algorithms, the space-time manifestations which were aperiodic 2D algorithmic SST lattices. The methodology presented here can be straightforwardly applied to other motifs which fall into this type of kinetic trap to create novel DNA crystals.

  9. Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

    SciTech Connect

    Neto, J.L. Siqueira; Lira, C.B.B.; Giardini, M.A.; Khater, L.; Perez, A.M.; Peroni, L.A.; Reis, J.R.R. dos; Freitas-Junior, L.H.; Ramos, C.H.I.; Cano, M.I.N. . E-mail: micano@ibb.unesp.br

    2007-06-29

    Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres.

  10. Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

    PubMed

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.

  11. Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

    PubMed Central

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  12. QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

    EPA Science Inventory

    Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.

    ABSTRACT

    DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

  13. Single-molecule elasticity of single-stranded DNA, a model flexible polyelectrolyte

    NASA Astrophysics Data System (ADS)

    McIntosh, Dustin B.

    Understanding the structure of unfolded, flexible polyelectrolytes is important for our comprehension of basic processes in molecular biology (e.g., RNA and protein folding) and our ability to exploit the polymers in technology (e.g., in self-assembled nanostructures). Here, we investigate the structure of single single-stranded DNA molecules and their interactions with ions using magnetic tweezers. Our data reveal that single-stranded DNA is not well-described by ideal polymer models such as the Worm-Like Chain. At low force, we report the first experimental observation of a nonlinear elastic regime revealing the relevance of long-range excluded volume effects. At high force, the extension scales as a logarithm in monovalent salt. Molecular dynamics simulations indicate that this logarithmic regime is the result of ion-stabilized wrinkles at short-length scales along the polymer backbone. Addition of divalent salt to the buffer results in enhanced elasticity indicating increased wrinkling or polymer ''wrapping" around the divalent ions. Using a thermodynamic identity, we are able to count ions as they are released into the bulk upon polymer elongation. We find that ssDNA releases significantly more ions than dsDNA. We posit that the recently termed ''Snake-Like Chain" model (Ullner, J. Phys. Chem B (2003)) for flexible polyelectrolytes may explain these observations. As a first step towards characterizing biologically relevant nucleic acid structures, we measure the effects of base-stacking on ssDNA elasticity. We find that base-stacking in poly(dA) significantly enhances the rigidity of the polymer as evidenced by the low-force elasticity. The unstacking transition of poly(dA) at high force reveals that the intrinsic electrostatic tension on the molecule varies significantly more weakly on salt concentration than predictions from mean-field models. Further, we provide a model-independent estimate of the free energy difference between stacked and unstacked nucleic

  14. Sodium azide-induced DNA single-strand breaks and DNA-protein crosslinks in barley embryos

    SciTech Connect

    Veleminsky, J.; Kleinhofs, A.; Nilan, R.A.

    1982-01-01

    Barley (Hordeum vulgare L., cv. Himalaya) embryos germinated for two days in sterile culture were exposed to 5 mM and 10 mM NaN/sub 3/ in pH 3 buffer for 2 h. (/sup 3/H) DNA from the isolated nuclei was analyzed by alkaline elution from PVC filters. DNA single-strand breaks and DNA-protein crosslinks, detected by proteinase K digestion, were detected both immediately and 24 h after the azide treatment. Repair of these lesions during 24 h of posttreatment incubation of embryos in nutrient medium was not observed.

  15. Analysis of sequence variation in Gnathostoma spinigerum mitochondrial DNA by single-strand conformation polymorphism analysis and DNA sequence.

    PubMed

    Ngarmamonpirat, Charinthon; Waikagul, Jitra; Petmitr, Songsak; Dekumyoy, Paron; Rojekittikhun, Wichit; Anantapruti, Malinee T

    2005-03-01

    Morphological variations were observed in the advance third stage larvae of Gnathostoma spinigerum collected from swamp eel (Fluta alba), the second intermediate host. Larvae with typical and three atypical types were chosen for partial cytochrome c oxidase subunit I (COI) gene sequence analysis. A 450 bp polymerase chain reaction product of the COI gene was amplified from mitochondrial DNA. The variations were analyzed by single-strand conformation polymorphism and DNA sequencing. The nucleotide variations of the COI gene in the four types of larvae indicated the presence of an intra-specific variation of mitochondrial DNA in the G. spinigerum population.

  16. An automated microfluidic system for single-stranded DNA preparation and magnetic bead-based microarray analysis

    PubMed Central

    Wang, Shuaiqin; Sun, Yujia; Liu, Yan; Xiang, Guangxin; Wang, Lei; Cheng, Jing; Liu, Peng

    2015-01-01

    We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-μl pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, “amplicon-in-answer-out” operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis. PMID:25825617

  17. Absorption by DNA single strands of adenine isolated in vacuo: The role of multiple chromophores

    NASA Astrophysics Data System (ADS)

    Nielsen, Lisbeth Munksgaard; Pedersen, Sara Øvad; Kirketerp, Maj-Britt Suhr; Nielsen, Steen Brøndsted

    2012-02-01

    The degree of electronic coupling between DNA bases is a topic being up for much debate. Here we report on the intrinsic electronic properties of isolated DNA strands in vacuo free of solvent, which is a good starting point for high-level excited states calculations. Action spectra of DNA single strands of adenine reveal sign of exciton coupling between stacked bases from blueshifted absorption bands (˜3 nm) relative to that of the dAMP mononucleotide (one adenine base). The bands are blueshifted by about 10 nm compared to those of solvated strands, which is a shift similar to that for the adenine molecule and the dAMP mononucleotide. Desolvation has little effect on the bandwidth, which implies that inhomogenous broadening of the absorption bands in aqueous solution is of minor importance compared to, e.g., conformational disorder. Finally, at high photon energies, internal conversion competes with electron detachment since dissociation of the bare photoexcited ions on the microsecond time scale is measured.

  18. Structural Basis of Detection and Signaling of DNA Single-Strand Breaks by Human PARP-1

    PubMed Central

    Eustermann, Sebastian; Wu, Wing-Fung; Langelier, Marie-France; Yang, Ji-Chun; Easton, Laura E.; Riccio, Amanda A.; Pascal, John M.; Neuhaus, David

    2015-01-01

    Summary Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins. PMID:26626479

  19. Structural Basis of Detection and Signaling of DNA Single-Strand Breaks by Human PARP-1.

    PubMed

    Eustermann, Sebastian; Wu, Wing-Fung; Langelier, Marie-France; Yang, Ji-Chun; Easton, Laura E; Riccio, Amanda A; Pascal, John M; Neuhaus, David

    2015-12-03

    Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1's function remained obscure; inherent dynamics of SSBs and PARP-1's multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1's signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodification in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.

  20. Structural basis of detection and signaling of DNA single-strand breaks by human PARP-1

    DOE PAGES

    Eustermann, Sebastian; Wu, Wing -Fung; Langelier, Marie -France; ...

    2015-11-25

    Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformabilitymore » of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Finally, our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.« less

  1. Structural basis of detection and signaling of DNA single-strand breaks by human PARP-1

    SciTech Connect

    Eustermann, Sebastian; Wu, Wing -Fung; Langelier, Marie -France; Yang, Ji -Chun; Easton, Laura E.; Riccio, Amanda A.; Pascal, John M.; Neuhaus, David

    2015-11-25

    Poly(ADP-ribose)polymerase 1 (PARP-1) is a key eukaryotic stress sensor that responds in seconds to DNA single-strand breaks (SSBs), the most frequent genomic damage. A burst of poly(ADP-ribose) synthesis initiates DNA damage response, whereas PARP-1 inhibition kills BRCA-deficient tumor cells selectively, providing the first anti-cancer therapy based on synthetic lethality. However, the mechanism underlying PARP-1’s function remained obscure; inherent dynamics of SSBs and PARP-1’s multi-domain architecture hindered structural studies. Here we reveal the structural basis of SSB detection and how multi-domain folding underlies the allosteric switch that determines PARP-1’s signaling response. Two flexibly linked N-terminal zinc fingers recognize the extreme deformability of SSBs and drive co-operative, stepwise self-assembly of remaining PARP-1 domains to control the activity of the C-terminal catalytic domain. Automodifcation in cis explains the subsequent release of monomeric PARP-1 from DNA, allowing repair and replication to proceed. Finally, our results provide a molecular framework for understanding PARP inhibitor action and, more generally, allosteric control of dynamic, multi-domain proteins.

  2. Sequence-specific cleavage of single-stranded DNA: oligodeoxynucleotide-EDTA X Fe(II).

    PubMed Central

    Dreyer, G B; Dervan, P B

    1985-01-01

    The synthesis of a DNA hybridization probe 19 nucleotides in length, equipped with the metal chelator EDTA at C-5 of thymidine in position 10 (indicated by T*) is described. DNA-EDTA 1 has the sequence 5'-T-A-A-C-G-C-A-G-T*-C-A-G-G-C-A-C-C-G-T-3', which is complementary to a 19-nucleotide sequence in the plasmid pBR322. In the presence of Fe(II), O2, and dithiothreitol, DNA-EDTA 1 affords specific cleavage (25 degrees C, pH 7.4, 60 min) at its complementary sequence in a heat-denatured 167-base-pair restriction fragment. Cleavage occurs over a range of 16 nucleotides at the site of hybridization of 1, presumably due to a diffusible reactive species. No other cleavage sites are observed in the 167-base-pair restriction fragment. The procedure used to synthesize DNA-EDTA probes is based on the incorporation of a thymidine modified at C-5 with the triethyl ester of EDTA. By using routine phosphoramidite procedures, thymidine-EDTA can be incorporated into oligodeoxynucleotides of any desired length and sequence. Because the efficiency of the DNA cleavage reaction is dependent on the addition of both Fe(II) and reducing agent (dithiothreitol), the initiation of the cleavage reaction can be controlled. These DNA-EDTA X Fe(II) probes should be useful for the sequence-specific cleavage of single-stranded DNA (and most likely RNA) under mild conditions. Images PMID:3919391

  3. Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes.

    PubMed Central

    Vámosi, G; Gohlke, C; Clegg, R M

    1996-01-01

    Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5' ends with 5-carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the single-stranded as well as for the double-stranded DNA molecules. Two states are fluorescent, with lifetimes in the range of 0.5-1 ns and 2.5-3 ns. A third state of TMRh-DNA does not fluoresce (a dark species of TMRh-DNA). The distribution of the TMRh-DNA molecules among these three states is strongly temperature and ionic strength dependent. Estimates are made of some reaction parameters of the multistate model. The results are discussed in terms of the photophysics of TMRh, and consequences of the multiple conformers of TMRh-DNA for studies involving fluorescence studies with TMRh-labeled DNA are considered. PMID:8842236

  4. Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease.

    PubMed

    Yeeles, Joseph T P; van Aelst, Kara; Dillingham, Mark S; Moreno-Herrero, Fernando

    2011-06-24

    AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase assays and our understanding of helicase mechanisms in general.

  5. Distinct circular single-stranded DNA viruses exist in different soil types.

    PubMed

    Reavy, Brian; Swanson, Maud M; Cock, Peter J A; Dawson, Lorna; Freitag, Thomas E; Singh, Brajesh K; Torrance, Lesley; Mushegian, Arcady R; Taliansky, Michael

    2015-06-15

    The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.

  6. Suppression of FOXM1 Transcriptional Activities via a Single-Stranded DNA Aptamer Generated by SELEX.

    PubMed

    Xiang, Qin; Tan, Guixiang; Jiang, Xia; Wu, Kuangpei; Tan, Weihong; Tan, Yongjun

    2017-03-30

    The transcription factor FOXM1 binds to its consensus sequence at promoters through its DNA binding domain (DBD) and activates proliferation-associated genes. The aberrant overexpression of FOXM1 correlates with tumorigenesis and progression of many cancers. Inhibiting FOXM1 transcriptional activities is proposed as a potential therapeutic strategy for cancer treatment. In this study, we obtained a FOXM1-specific single stranded DNA aptamer (FOXM1 Apt) by SELEX with a recombinant FOXM1 DBD protein as the target of selection. The binding of FOXM1 Apt to FOXM1 proteins were confirmed with electrophoretic mobility shift assays (EMSAs) and fluorescence polarization (FP) assays. Phosphorthioate-modified FOXM1 Apt (M-FOXM1 Apt) bound to FOXM1 as wild type FOXM1 Apt, and co-localized with FOXM1 in nucleus. M-FOXM1-Apt abolished the binding of FOXM1 on its consensus binding sites and suppressed FOXM1 transcriptional activities. Compared with the RNA interference of FOXM1 in cancer cells, M-FOXM1 Apt repressed cell proliferation and the expression of FOXM1 target genes without changing FOXM1 levels. Our results suggest that the obtained FOXM1 Apt could be used as a probe for FOXM1 detection and an inhibitor of FOXM1 transcriptional functions in cancer cells at the same time, providing a potential reagent for cancer diagnosis and treatment in the future.

  7. Suppression of FOXM1 Transcriptional Activities via a Single-Stranded DNA Aptamer Generated by SELEX

    PubMed Central

    Xiang, Qin; Tan, Guixiang; Jiang, Xia; Wu, Kuangpei; Tan, Weihong; Tan, Yongjun

    2017-01-01

    The transcription factor FOXM1 binds to its consensus sequence at promoters through its DNA binding domain (DBD) and activates proliferation-associated genes. The aberrant overexpression of FOXM1 correlates with tumorigenesis and progression of many cancers. Inhibiting FOXM1 transcriptional activities is proposed as a potential therapeutic strategy for cancer treatment. In this study, we obtained a FOXM1-specific single stranded DNA aptamer (FOXM1 Apt) by SELEX with a recombinant FOXM1 DBD protein as the target of selection. The binding of FOXM1 Apt to FOXM1 proteins were confirmed with electrophoretic mobility shift assays (EMSAs) and fluorescence polarization (FP) assays. Phosphorthioate-modified FOXM1 Apt (M-FOXM1 Apt) bound to FOXM1 as wild type FOXM1 Apt, and co-localized with FOXM1 in nucleus. M-FOXM1-Apt abolished the binding of FOXM1 on its consensus binding sites and suppressed FOXM1 transcriptional activities. Compared with the RNA interference of FOXM1 in cancer cells, M-FOXM1 Apt repressed cell proliferation and the expression of FOXM1 target genes without changing FOXM1 levels. Our results suggest that the obtained FOXM1 Apt could be used as a probe for FOXM1 detection and an inhibitor of FOXM1 transcriptional functions in cancer cells at the same time, providing a potential reagent for cancer diagnosis and treatment in the future. PMID:28358012

  8. Stretching and Controlled Motion of Single-Stranded DNA in Locally-Heated Solid-State Nanopores

    PubMed Central

    Belkin, Maxim; Maffeo, Christopher; Wells, David B.

    2013-01-01

    Practical applications of solid-state nanopores for DNA detection and sequencing require the electrophoretic motion of DNA through the nanopores to be precisely controlled. Controlling the motion of single-stranded DNA presents a particular challenge, in part because of the multitude of conformations that a DNA strand can adopt in a nanopore. Through continuum, coarse-grained and atomistic modeling, we demonstrate that local heating of the nanopore volume can be used to alter the electrophoretic mobility and conformation of single-stranded DNA. In the nanopore systems considered, the temperature near the nanopore is modulated via a nanometer-size heater element that can be radiatively switched on and off. The local enhancement of temperature produces considerable stretching of the DNA fragment confined within the nanopore. Such stretching is reversible, so that the conformation of DNA can be toggled between compact (local heating is off) and extended (local heating is on) states. The effective thermophoretic force acting on single-stranded DNA in the vicinity of the nanopore is found to be sufficiently large (4–8 pN) to affect such changes in the DNA conformation. The local heating of the nanopore volume is observed to promote single-file translocation of DNA strands at transmembrane biases as low as 10 mV, which opens new avenues for using solid-state nanopores for detection and sequencing of DNA. PMID:23876013

  9. DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe

    PubMed Central

    Nandakumar, Jayakrishnan; Cech, Thomas R.

    2012-01-01

    Eukaryotic chromosome ends are protected from illicit DNA joining by protein–DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA. PMID:21911358

  10. Single strand DNA binding proteins 1 and 2 protect newly replicated telomeres

    PubMed Central

    Gu, Peili; Deng, Wei; Lei, Ming; Chang, Sandy

    2013-01-01

    Human single-strand (ss) DNA binding proteins 1 and 2 (hSSB1 and 2) are components of the hSSB1/2-INTS3-C9orf80 heterotrimeric protein complex shown to participate in DNA damage response and maintenance of genome stability. However, their roles at telomeres remain unknown. Here, we generated murine SSB1 conditional knockout mice and cells and found that mSSB1 plays a critical role in telomere end protection. Both mSSB1 and mSSB2 localize to a subset of telomeres and are required to repair TRF2-deficient telomeres. Deletion of mSSB1 resulted in increased chromatid-type fusions involving both leading- and lagging-strand telomeric DNA, suggesting that it is required for the protection of G-overhangs. mSSB1's interaction with INTS3 is required for its localization to damaged DNA. mSSB1 interacts with Pot1a, but not Pot1b, and its association with telomeric ssDNA requires Pot1a. mSSB1Δ/Δ mice die at birth with developmental abnormalities, while mice with the hypomorphic mSSB1F/F allele are born alive and display increased sensitivity to ionizing radiation (IR). Our results suggest that mSSB1 is required to maintain genome stability, and document a previously unrecognized role for mSSB1/2 in the protection of newly replicated leading- and lagging-strand telomeres. PMID:23459151

  11. Unraveling the motion of single-stranded DNA binding proteins on DNA using force and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Ha, Taekjip

    2012-02-01

    Single-stranded DNA binding (SSB) proteins bind to and control the accessibility of single stranded (ss) DNA generated as a transient intermediate during a variety of cellular processes. For subsequent DNA processing, however, such a tightly wrapped, high-affinity protein--DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. Here we show, using single-molecule two- and three-colour fluorescence resonance energy transfer, that SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes. In addition, using an optomechanical tool combining single-molecule fluorescence and force methods, we probed how proteins with such a large binding site size (˜ 65 nucleotides) can migrate rapidly on DNA and how protein-protein interactions and tension may modulate the motion. We observed force-induced progressive unravelling of ssDNA from the SSB surface between 1 and 6 pN, followed by SSB dissociation at ˜10 pN, and obtained experimental evidence of a reptation mechanism for protein movement along DNA wherein a protein slides via DNA bulge formation and propagation. SSB diffusion persists even when bound with RecO, and at forces under which the fully wrapped state is perturbed, suggesting that even in crowded cellular conditions SSB can act as a sliding platform to recruit and carry its interacting proteins for use in DNA replication, recombination and repair.

  12. Covalent Adduct Formation between the Antihypertensive Drug Hydralazine and Abasic Sites in Double- and Single-Stranded DNA

    PubMed Central

    2015-01-01

    Hydralazine (4) is an antihypertensive agent that displays both mutagenic and epigenetic properties. Here, gel electrophoretic, mass spectroscopic, and chemical kinetics methods were used to provide evidence that medicinally relevant concentrations of 4 rapidly form covalent adducts with abasic sites in double- and single-stranded DNA under physiological conditions. These findings raise the intriguing possibility that the genotoxic properties of this clinically used drug arise via reactions with an endogenous DNA lesion rather than with the canonical structure of DNA. PMID:25405892

  13. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule

    SciTech Connect

    Changela, A.; Digate, R.J.; Mondragon, A.

    2010-03-05

    A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes - DNA topoisomerases - alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 {angstrom} resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.

  14. In Vivo Conversion of the Single-Stranded DNA of the Kilham Rat Virus to a Double-Stranded Form

    PubMed Central

    Salzman, Lois Ann; White, Wesley

    1973-01-01

    Kilham rat virus (KRV) contains linear, single-stranded DNA in the virion. The fate of radioactive viral DNA was followed after infection of monolayer cells. Within 60 min after infection of cells, 28 to 42% of the parental viral DNA is converted to a new form. This new DNA form is believed to be double stranded and linear on the basis of its sedimentation in neutral and alkaline sucrose gradients, elution from hydroxyapatite columns, its buoyant density in equilibrium CsCl density gradients, and appearance in the electron microscope. The double-stranded linear KRV DNA may be analogous to the replicative form of certain bacteriophages, including φX174, which contain single-stranded circular genomes. Images PMID:4347430

  15. Application of motif-based tools on evolutionary analysis of multipartite single-stranded DNA viruses.

    PubMed

    Wang, Hsiang-Iu; Chang, Chih-Hung; Lin, Po-Heng; Fu, Hui-Chuan; Tang, Chuanyi; Yeh, Hsin-Hung

    2013-01-01

    Multipartite viruses contain more than one distinctive genome component, and the origin of multipartite viruses has been suggested to evolve from a non-segmented wild-type virus. To explore whether recombination also plays a role in the evolution of the genomes of multipartite viruses, we developed a systematic approach that employs motif-finding tools to detect conserved motifs from divergent genomic regions and applies statistical approaches to select high-confidence motifs. The information that this approach provides helps us understand the evolution of viruses. In this study, we compared our motif-based strategy with current alignment-based recombination-detecting methods and applied our methods to the analysis of multipartite single-stranded plant DNA viruses, including bipartite begomoviruses, Banana bunchy top virus (BBTV) (consisting of 6 genome components) and Faba bean necrotic yellows virus (FBNYV) (consisting of 8 genome components). Our analysis revealed that recombination occurred between genome components in some begomoviruses, BBTV and FBNYV. Our data also show that several unusual recombination events have contributed to the evolution of BBTV genome components. We believe that similar approaches can be applied to resolve the evolutionary history of other viruses.

  16. Single-stranded DNA conformation polymorphism at the Rdl locus in Hypothenemus hampei (Coleoptera: Scolytidae).

    PubMed

    Borsa, P; Coustau, C

    1996-02-01

    The homologue of the resistance to dieldrin gene (Rdl) in Drosophila melanogaster was cloned and sequenced in the scolytid beetle Hypothenemus hampei, a coffee pest resistant to cyclodiene insecticides in New Caledonia. The amino acid sequence of the Rdl exon no. 7 protein product in H. hampei was identical to that in D. melanogaster and showed the same amino acid change as that characterizing susceptible vs. resistant D. melanogaster. Samples from natural H. hampei populations (from Asia, the Pacific Islands, Africa and Central America), from reference susceptible (S) and resistant (R) laboratory strains, and from their hybrid progenies, were analysed at the Rdl locus using single-stranded DNA conformation polymorphism on polymerase chain reaction products. The susceptible allele was the only allele present in all samples from natural populations except in the only resistant population known to date (Ponerihouen, New Caledonia). Females and some males obtained at F1 from R x S crosses were heterozygous at the Rdl locus, confirming that this local mate competing species is diplo-diploid.

  17. Carboplatin enhances the production and persistence of radiation-induced DNA single-strand breaks

    SciTech Connect

    Yang, L.; Douple, E.B.; O`Hara, J.A.; Wang, H.J.

    1995-09-01

    Fluorometric analysis of DNA unwinding and alkaline elution were used to investigate the production and persistence of DNA single-strand breaks (SSBs) in Chinese hamster V79 and xrs-5 cells treated with the chemotherapeutic agent carboplatin in combination with radiation. Carboplatin was administered to cells before irradiation in hypoxic conditions, or the drug was added immediately after irradiation during the postirradiation recovery period in air. The results of DNA unwinding studies suggest that carboplatin enhances the production of radiation-induced SSBs in hypoxic V79 cells and xrs-5 cells by a factor of 1.86 and 1.83, respectively, when combined with radiation compared to the SSBs produced by irradiation alone. Carboplatin alone did not produce a measureable number of SSBs. Alkaline elution profiles also indicated that the rate of elution of SSBs was higher in cells treated with the carboplatin is present after irradiation and during the postirradiation recovery period, the rejoining of radiation-induced SSBs by a factor of 1.46 in V79 cells with 20 Gy irradiation and by a factor of 2.02 in xrs-5 cells with 20 Gy irradiation. When carboplatin is present after irradiation and during the postirradiation recovery period, the rejoining of radiation-induced SSBs is inhibited during this postirradiation incubation period (radiopotentiation) with a relative inhibition factor at 1 h postirradiation of 1.25 in V79 cells and 1.15 in xrs-5 cells. An increased production and persistence of SSBs resulting from the interaction of carboplatin with radiation may be an important step in the mechanism responsible for the potentiated cell killing previously from studies in animal tumors and in cultured cells. 31 refs., 7 figs.

  18. The structure of DdrB from Deinococcus: a new fold for single-stranded DNA binding proteins

    PubMed Central

    Sugiman-Marangos, Seiji; Junop, Murray S.

    2010-01-01

    Deinococcus spp. are renowned for their amazing ability to recover rapidly from severe genomic fragmentation as a result of exposure to extreme levels of ionizing radiation or desiccation. Despite having been originally characterized over 50 years ago, the mechanism underlying this remarkable repair process is still poorly understood. Here, we report the 2.8 Å structure of DdrB, a single-stranded DNA (ssDNA) binding protein unique to Deinococcus spp. that is crucial for recovery following DNA damage. DdrB forms a pentameric ring capable of binding single-stranded but not double-stranded DNA. Unexpectedly, the crystal structure reveals that DdrB comprises a novel fold that is structurally and topologically distinct from all other single-stranded binding (SSB) proteins characterized to date. The need for a unique ssDNA binding function in response to severe damage, suggests a distinct role for DdrB which may encompass not only standard SSB protein function in protection of ssDNA, but also more specialized roles in protein recruitment or DNA architecture maintenance. Possible mechanisms of DdrB action in damage recovery are discussed. PMID:20129942

  19. Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

    NASA Astrophysics Data System (ADS)

    Shokri, Leila; Rouzina, Ioulia; Williams, Mark C.

    2009-06-01

    Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork.

  20. A Comparison of Two Single-Stranded DNA Binding Models by Mutational Analysis of APOBEC3G

    PubMed Central

    Shindo, Keisuke; Li, Ming; Gross, Phillip J.; Brown, William L.; Harjes, Elena; Lu, Yongjian; Matsuo, Hiroshi; Harris, Reuben S.

    2012-01-01

    APOBEC3G is the best known of several DNA cytosine deaminases that function to inhibit the replication of parasitic genetic elements including the lentivirus HIV. Several high-resolution structures of the APOBEC3G catalytic domain have been generated, but none reveal how this enzyme binds to substrate single-stranded DNA. Here, we constructed a panel of APOBEC3G amino acid substitution mutants and performed a series of biochemical, genetic, and structural assays to distinguish between “Brim” and “Kink” models for single-strand DNA binding. Each model predicts distinct sets of interactions between surface arginines and negatively charged phosphates in the DNA backbone. Concordant with both models, changing the conserved arginine at position 313 to glutamate abolished both catalytic and restriction activities. In support of the Brim model, arginine to glutamate substitutions at positions 213, 215, and 320 also compromised these APOBEC3G activities. Arginine to glutamate substitutions at Kink model residues 374 and 376 had smaller effects. These observations were supported by A3G catalytic domain-ssDNA chemical shift perturbation experiments. The overall data set is most consistent with the Brim model for single-stranded DNA binding by APOBEC3G. PMID:24832226

  1. Single-stranded DNA library preparation uncovers the origin and diversity of ultrashort cell-free DNA in plasma

    PubMed Central

    Burnham, Philip; Kim, Min Seong; Agbor-Enoh, Sean; Luikart, Helen; Valantine, Hannah A.; Khush, Kiran K.; De Vlaminck, Iwijn

    2016-01-01

    Circulating cell-free DNA (cfDNA) is emerging as a powerful monitoring tool in cancer, pregnancy and organ transplantation. Nucleosomal DNA, the predominant form of plasma cfDNA, can be adapted for sequencing via ligation of double-stranded DNA (dsDNA) adapters. dsDNA library preparations, however, are insensitive to ultrashort, degraded cfDNA. Drawing inspiration from advances in paleogenomics, we have applied a single-stranded DNA (ssDNA) library preparation method to sequencing of cfDNA in the plasma of lung transplant recipients (40 samples, six patients). We found that ssDNA library preparation yields a greater portion of sub-100 bp nuclear genomic cfDNA (p 10−5, Mann-Whitney U Test), and an increased relative abundance of mitochondrial (10.7x, p 10−5) and microbial cfDNA (71.3x, p 10−5). The higher yield of microbial sequences from this method increases the sensitivity of cfDNA-based monitoring for infections following transplantation. We detail the fragmentation pattern of mitochondrial, nuclear genomic and microbial cfDNA over a broad fragment length range. We report the observation of donor-specific mitochondrial cfDNA in the circulation of lung transplant recipients. A ssDNA library preparation method provides a more informative window into understudied forms of cfDNA, including mitochondrial and microbial derived cfDNA and short nuclear genomic cfDNA, while retaining information provided by standard dsDNA library preparation methods. PMID:27297799

  2. Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB).

    PubMed

    van Loon, Barbara; Samson, Leona D

    2013-03-01

    Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair.

  3. Complex formation between p53 and replication protein A inhibits the sequence-specific DNA binding of p53 and is regulated by single-stranded DNA.

    PubMed Central

    Miller, S D; Moses, K; Jayaraman, L; Prives, C

    1997-01-01

    Human replication protein A (RP-A) (also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. Potentially important to both these functions, it is also capable of complex formation with the tumor suppressor protein p53. Here we show that although p53 is unable to prevent RP-A from associating with a range of single-stranded DNAs in solution, RP-A is able to strongly inhibit p53 from functioning as a sequence-specific DNA binding protein when the two proteins are complexed. This inhibition, in turn, can be regulated by the presence of various lengths of single-stranded DNAs, as RP-A, when bound to these single-stranded DNAs, is unable to interact with p53. Interestingly, the lengths of single-stranded DNA capable of relieving complex formation between the two proteins represent forms that might be introduced through repair and replicative events. Increasing p53 concentrations can also overcome the inhibition by steady-state levels of RP-A, potentially mimicking cellular points of balance. Finally, it has been shown previously that p53 can itself be stimulated for site-specific DNA binding when complexed through the C terminus with short single strands of DNA, and here we show that p53 stays bound to these short strands even after binding a physiologically relevant site. These results identify a potential dual role for single-stranded DNA in the regulation of DNA binding by p53 and give insights into the p53 response to DNA damage. PMID:9121469

  4. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides

    PubMed Central

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B.

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9. PMID:27609304

  5. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

    PubMed

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B

    2016-09-09

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.

  6. Non-uniform binding of single-stranded DNA binding proteins to hybrids of single-stranded DNA and single-walled carbon nanotubes observed by atomic force microscopy in air and in liquid

    NASA Astrophysics Data System (ADS)

    Umemura, Kazuo; Ishizaka, Kei; Nii, Daisuke; Izumi, Katsuki

    2016-12-01

    Using atomic force spectroscopy (AFM), we observed hybrids of single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWNTs) with or without protein molecules in air and in an aqueous solution. This is the first report of ssDNA-SWNT hybrids with proteins in solution analyzed by AFM. In the absence of protein, the height of the ssDNA-SWNT hybrids was 1.1 ± 0.3 nm and 2.4 ± 0.6 nm in air and liquid, respectively, suggesting that the ssDNA molecules adopted a flexible structure on the SWNT surface. In the presence of single-stranded DNA binding (SSB) proteins, the heights of the hybrids in air and liquid increased to 6.4 ± 3.1 nm and 10.0 ± 4.5 nm, respectively. The AFM images clearly showed binding of the SSB proteins to the ssDNA-SWNT hybrids. The morphology of the SSB-ssDNA-SWNT hybrids was non-uniform, particularly in aqueous solution. The variance of hybrid height was quantitatively estimated by cross-section analysis along the long-axis of each hybrid. The SSB-ssDNA-SWNT hybrids showed much larger variance than the ssDNA-SWNT hybrids.

  7. Direct Amplification of Single-Stranded DNA for Pyrosequencing using Linear-After-The-Exponential (LATE)-PCR

    PubMed Central

    Salk, Jesse J.; Sanchez, J Aquiles; Pierce, Kenneth E.; Rice, John E.; Soares, Kevin C.; Wangh, Lawrence J.

    2006-01-01

    Pyrosequencing is a highly effective method for quantitatively genotyping short genetic sequences, but is currently hampered by a labor intensive sample preparation process designed to isolate single-stranded DNA from double-stranded products generated by conventional PCR. Here LATE-PCR is introduced as an efficient and potentially automatable method of directly amplifying single-stranded DNA for pyrosequencing, thus eliminating the need for solid-phase sample preparation and reducing the risk of laboratory contamination. These improvements are illustrated for SNP genotyping applications including an integrated, single cell-through-sequencing assay to detect a mutation at the globin IVS-110 site that is frequently responsible for β-Thalassemia. PMID:16540077

  8. 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

  9. Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli.

    PubMed Central

    Moreau, P L

    1988-01-01

    Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA. PMID:2836358

  10. RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

    PubMed Central

    Maréchal, Alexandre; Zou, Lee

    2015-01-01

    The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications. PMID:25403473

  11. [Characterization of recombinant single-stranded DNA-binding protein from Escherichia coli and its application in accurate pyrosequencing].

    PubMed

    Wang, Jianping; Zou, Bingjie; Chen, Zhiyao; Ma, Yinjiao; Xu, Shu; Zhou, Guohua

    2011-10-01

    We expressed recombinant single-stranded DNA-binding protein (r-SSBP) from Escherichia coli with the molecular weight of 24-kDa by using genetic engineering strategy, and demonstrated the single-stranded DNA (ssDNA)-binding activity of r-SSBP by electrophoretic mobility shift assay (EMSA). To further characterize r-SSBP, we studied the effects of r-SSBP on melting temperature (T(m)) of DNA. The results showed that r-SSBP could bind to ssDNA, and lower the T(m) of DNA, especially for single-base mismatched DNA. Therefore, r-SSBP significantly increased the T(m) difference between single-base mismatched DNA and perfect matched DNA. These results are very beneficial for single-nucleotide polymorphism detection. Moreover, we applied r-SSBP in high sensitive pyrosequencing system developed by our group. The results suggest that the r-SSBP decreased non-specific signals, corrected the proportion of signal peak height and improved the performance of pyrosequencing.

  12. TTV, a new human virus with single stranded circular DNA genome.

    PubMed

    Hino, Shigeo

    2002-01-01

    TT virus (TTV) was found in 1997 from a hepatitis patient without virus markers. However, the real impact of TTV on liver diseases remains uncertain to date. Due to the lack of suitable cell systems to support the growth of TTV, the biology of TTV is still obscure. This review tries to summarise the current status of TTV on aspects other than the taxonomic diversity of TTV. TTV was the first human virus with a single stranded circular DNA genome. TTV was considered to be a member of Circoviridae, but others suggested it conformed to a new family. TTV is distinct from ambisense viruses in the genus Circovirus, since the former genome is negative stranded. The genome structure of TTV is more related to chicken anaemia virus in the genus Gyrovirus, however, the sequence similarity is minimal except for a short stretch at 3816-3851 of TA278. Currently the working group is proposing the full name for TTV as TorqueTenoVirus and the TTV-like mini virus as TorqueTenoMiniVirus (TTMV) in a new genus Anellovirus (ring). TTVs are prevalent in non-human primates and human TTV can cross-infect chimpanzees. Furthermore, TTV sequences have been detected in chickens, pigs, cows and sheep. TTV can be transmitted by mother-to-child infection. However, within a year after birth, the prevalence reaches the same level for children born to both TTV-positive and TTV-negative mothers even without breast-feeding. The non-coding region surrounding a short 113 nt GC-rich stretch and occupying approximately one-third of the genome is considered to contain the putative replication origin. Three mRNAs are expressed by TTV, 3.0 and 1.2 and 1.0 kb species. A protein translated from the 3.0 kb mRNA is considered to be the major capsid protein as well as replicase. The nature of the proteins translated by the other two mRNAs are still putative.

  13. The Adsorption of Short Single-Stranded DNA Oligomers on Mineral Surfaces

    NASA Astrophysics Data System (ADS)

    Kopstein, M.; Sverjensky, D. A.; Hazen, R. M.; Cleaves, H. J.

    2009-12-01

    Previous studies have described feasible pathways for the synthesis of simple organic building blocks such as formaldehyde and hydrogen cyanide, and their reaction to form more complex biomolecules such as nucleotide bases, amino acids and sugars (Miller and Orgel 1974, Miller and Cleaves 2006). However, the polymerization of monomers into a useful genetic material remains problematic (Orgel 2004). Organic building blocks were unlikely to polymerize from very dilute aqueous solution in the primitive oceans. Mineral surface adsorption has been suggested as a possible mechanism for concentrating the necessary building blocks (Bernal 1951). This study focused on the adsorption behavior of single-stranded DNA homo-oligomers of adenine and thymine (including the monomers, dimers, tetramers, hexamers, octomers, and decamers) with five different mineral surfaces (pyrite, rutile, hematite, olivine and calcite). Adsorption was studied in 0.1 M pH 8.1 KHCO3 with0.05 M NaCl as background electrolyte. Solutions were mixed for 24 hours at room temperature, centrifuged and the supernatants analyzed by UV/visible spectrophotometry. Equilibrium solution concentrations were measured and used to determine the number of moles adsorbed per square meter. Langmuir isotherms were constructed using the experimental data. It was found that adenine-containing molecules tend to bind much more strongly than thymine-containing molecules. It was also found that the number of moles adsorbed at saturation tends to fall with increasing chain length, while adsorption affinity tends to rise. Oligomer length appears to affect adsorption more than the mineral type. These results may have implications for the primordial organization of the first nucleic acid molecules as the persistence of extra-cellular nucleic acids in the environment. References Bernal, J. D. (1951) The Physical Basis of Life (Routledge, London). Miller S.L. and Cleaves, H.J. (2006) Prebiotic chemistry on the primitive Earth. In

  14. Peptidic determinants and structural model of human NDP kinase B (Nm23-H2) bound to single-stranded DNA.

    PubMed

    Raveh, S; Vinh, J; Rossier, J; Agou, F; Véron, M

    2001-05-22

    Isoform B of human NDP kinase (NDPK-B) was previously identified as a transcription factor stimulating in vitro and ex vivo the transcription of the c-myc oncogene, which involves this enzyme in carcinogenesis. We have studied the enzymatic properties of NDPK-B in the presence of several single-stranded oligonucleotides. We show that the oligonucleotides are competitive inhibitors of the catalytic activity, indicating that the active site acts as a binding template for the anchorage of the oligonucleotide. Furthermore, the presence of a guanine at the 3'-end of several different aptamers increases its affinity 10-fold. To define the surface of the protein contacting the DNA within the nucleoprotein complex, we used single nanosecond laser pulses as the cross-linking reagent and MALDI-TOF mass spectrometry to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Using 11-mer and 30-mer single-stranded oligonucleotides, the same three different nucleopeptides were identified after irradiation of the complexes, indicating a common binding mode for these two aptamers. Taken together, these results allowed us to propose a structural model of NDPK-B bound to single-stranded DNA.

  15. The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

    PubMed

    Ramachandran, Aparna; Nandakumar, Divya; Deshpande, Aishwarya P; Lucas, Thomas P; R-Bhojappa, Ramanagouda; Tang, Guo-Qing; Raney, Kevin; Yin, Y Whitney; Patel, Smita S

    2016-08-05

    Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.

  16. The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

    SciTech Connect

    Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.; Volkert, Michael R.

    2004-02-01

    Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

  17. Analysis of guanine oxidation products in double-stranded DNA and proposed guanine oxidation pathways in single-stranded, double-stranded or quadruplex DNA.

    PubMed

    Morikawa, Masayuki; Kino, Katsuhito; Oyoshi, Takanori; Suzuki, Masayo; Kobayashi, Takanobu; Miyazawa, Hiroshi

    2014-02-10

    Guanine is the most easily oxidized among the four DNA bases, and some guanine-rich sequences can form quadruplex structures. In a previous study using 6-mer DNA d(TGGGGT), which is the shortest oligomer capable of forming quadruplex structures, we demonstrated that guanine oxidation products of quadruplex DNA differ from those of single-stranded DNA. Therefore, the hotooxidation products of double-stranded DNA (dsDNA) may also differ from that of quadruplex or single-stranded DNA, with the difference likely explaining the influence of DNA structures on guanine oxidation pathways. In this study, the guanine oxidation products of the dsDNA d(TGGGGT)/d(ACCCCA) were analyzed using HPLC and electrospray ionization-mass spectrometry (ESI-MS). As a result, the oxidation products in this dsDNA were identified as 2,5-diamino-4H-imidazol-4-one (Iz), 8-oxo-7,8-dihydroguanine (8oxoG), dehydroguanidinohydantoin (Ghox), and guanidinohydantoin (Gh). The major oxidation products in dsDNA were consistent with a combination of each major oxidation product observed in single-stranded and quadruplex DNA. We previously reported that the kinds of the oxidation products in single-stranded or quadruplex DNA depend on the ease of deprotonation of the guanine radical cation (G•+) at the N1 proton. Similarly, this mechanism was also involved in dsDNA. Deprotonation in dsDNA is easier than in quadruplex DNA and more difficult in single-stranded DNA, which can explain the formation of the four oxidation products in dsDNA.

  18. Dissociation of single-strand DNA: single-walled carbon nanotube hybrids by Watson-Crick base-pairing.

    PubMed

    Jung, Seungwon; Cha, Misun; Park, Jiyong; Jeong, Namjo; Kim, Gunn; Park, Changwon; Ihm, Jisoon; Lee, Junghoon

    2010-08-18

    It has been known that single-strand DNA wraps around a single-walled carbon nanotube (SWNT) by pi-stacking. In this paper it is demonstrated that such DNA is dissociated from the SWNT by Watson-Crick base-pairing with a complementary sequence. Measurement of field effect transistor characteristics indicates a shift of the electrical properties as a result of this "unwrapping" event. We further confirm the suggested process through Raman spectroscopy and gel electrophoresis. Experimental results are verified in view of atomistic mechanisms with molecular dynamics simulations and binding energy analyses.

  19. Conformational transitions and stop-and-go nanopore transport of single-stranded DNA on charged graphene

    NASA Astrophysics Data System (ADS)

    Shankla, Manish; Aksimentiev, Aleksei

    2014-10-01

    Control over interactions with biomolecules holds the key to applications of graphene in biotechnology. One such application is nanopore sequencing, where a DNA molecule is electrophoretically driven through a graphene nanopore. Here we investigate how interactions of single-stranded DNA and a graphene membrane can be controlled by electrically biasing the membrane. The results of our molecular dynamics simulations suggest that electric charge on graphene can force a DNA homopolymer to adopt a range of strikingly different conformations. The conformational response is sensitive to even very subtle nucleotide modifications, such as DNA methylation. The speed of DNA motion through a graphene nanopore is strongly affected by the graphene charge: a positive charge accelerates the motion, whereas a negative charge arrests it. As a possible application of the effect, we demonstrate stop-and-go transport of DNA controlled by the charge of graphene. Such on-demand transport of DNA is essential for realizing nanopore sequencing.

  20. Induction of single strand scission in bacteriophage phi X174 replicative form I DNA by mitomycin C.

    PubMed

    Ueda, K; Morita, J; Komano, T

    1981-03-01

    The action of mitomycin C on double-stranded replicative form I DNA (RF I DNA; supercoiled, covalently closed, circular duplex DNA) of bacteriophage phi X174 was investigated using the technique of agarose gel electrophoresis. Mitomycin C reduced with sodium hydrosulfite (sodium dithionite, Na2S2O4) caused single strand scission in phi X174 RF I DNA in the presence of Cu2+. Cu2+ was essential for this DNA cleave action, and other transition metal ions such as Fe2+, Fe3+, Mn2+, Co2+ and Zn2+ were of no effect. This DNA strand scission was inhibited by catalase (EC 1.11.1.6) and various radical scavengers. This DNA strand scission was caused by free oxygen radicals generated during autoxidation of reduced mitomycin C in the presence of Cu2+.

  1. Dynamic Mechanism of Single-Stranded DNA Encapsulated into Single-Wall Carbon Nanotubes: A Molecular Dynamics Simulation Study

    NASA Astrophysics Data System (ADS)

    Xing, Yan-Fei; Yang, Chuan-Lu; Mo, Yong-Fang; Wang, Mei-Shan; Ma, Xiao-Guang

    2014-02-01

    Hybrids of single-walled carbon nanotubes (SWCNTs) and biological molecules have been utilized for numerous applications in sensing, imaging, and drug delivery. By molecular dynamics simulation, we investigate the encapsulation of single-strand DNA (ssDNA) containing eight adenine bases with (17,17)-(12,12) SWCNTs. The effects of the diameter and length of SWCNTs on the encapsulation process are explored with the calculated curves of the center-of-mass distance, the van der Waals interaction between the ssDNA and SWCNT, the root-mean-square deviation of the ssDNA, and the radius of gyration of the ssDNA. The free energy of the encapsulated ssDNA for each SWCNT is also obtained via steered molecular dynamics simulation. The most suitable SWCNT for encapsulating the ssDNA is also suggested.

  2. Stretching single-stranded DNA: interplay of electrostatic, base-pairing, and base-pair stacking interactions.

    PubMed Central

    Zhang, Y; Zhou, H; Ou-Yang, Z C

    2001-01-01

    Recent single-macromolecule observations revealed that the force/extension characteristics of single-stranded DNA (ssDNA) are closely related to solution ionic concentration and DNA sequence composition. To understand this, we studied the elastic property of ssDNA through the Monte Carlo implementation of a modified freely jointed chain (FJC), with electrostatic, base-pairing, and base-pair stacking interactions all incorporated. The simulated force-extension profiles for both random and designed sequences have attained quantitative agreements with the experimental data. In low-salt solution, electrostatic interaction dominates, and at low forces, the molecule can be more easily aligned than an unmodified FJC. In high-salt solution, secondary hairpin structure appears in ssDNA by the formation of base pairs between complementary bases, and external stretching causes a hairpin-coil structural transition, which is continuous for ssDNA made of random sequences. In designed sequences such as poly(dA-dT) and poly(dG-dC), the stacking potential between base pairs encourages the aggregation of base pairs into bulk hairpins and makes the hairpin-coil transition a discontinuous (first-order) process. The sensitivity of elongation to the base-pairing rule is also investigated. The comparison of modeling calculations and the experimental data suggests that the base pairing of single-stranded polynucleotide molecules tends to form a nested and independent planar hairpin structure rather than a random intersecting pattern. PMID:11463654

  3. Base damage within single-strand DNA underlies in vivo hypermutability induced by a ubiquitous environmental agent.

    PubMed

    Chan, Kin; Sterling, Joan F; Roberts, Steven A; Bhagwat, Ashok S; Resnick, Michael A; Gordenin, Dmitry A

    2012-01-01

    Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA) is more prone to damage than double-strand DNA (dsDNA), due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA-specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5' to 3' enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3' ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2'-deoxyuracil intermediate in DNA that was resistant to excision by uracil-DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions accurately.

  4. Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative form in dna mutants of Escherichia coli.

    PubMed

    Kodaira, K I; Taketo, A

    1977-05-17

    Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.

  5. The intrinsically disordered linker of E. coli SSB is critical for the release from single-stranded DNA.

    PubMed

    Tan, Hui Yin; Wilczek, Luke A; Pottinger, Sasheen; Manosas, Maria; Yu, Cong; Nguyenduc, Trong; Bianco, Piero R

    2017-04-01

    The Escherichia coli single stranded DNA binding protein (SSB) is crucial for DNA replication, recombination and repair. Within each process, it has two seemingly disparate roles: it stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing and, forms complexes with a group of proteins known as the SSB-interactome. Key to both roles is the C-terminal, one-third of the protein, in particular the intrinsically disordered linker (IDL). Previously, they have shown using a series of linker deletion mutants that the IDL links both ssDNA and target protein binding by mediating interactions with the oligosaccharide/oligonucleotide binding fold in the target. In this study, they examine the role of the linker region in SSB function in a variety of DNA metabolic processes in vitro. Using the same linker mutants, the results show that in addition to association reactions (either DNA or protein), the IDL is critical for the release of SSB from DNA. This release can be under conditions of ssDNA competition or active displacement by a DNA helicase or recombinase. Consistent with their previous work these results indicate that SSB linker mutants are defective for SSB-SSB interactions, and when the IDL is removed a terminal SSB-DNA complex results. Formation of this complex inhibits downstream processing of DNA by helicases such as RecG or PriA as well as recombination, mediated by RecA. A model, based on the evidence herein, is presented to explain how the IDL acts in SSB function.

  6. Deceleration of single-stranded DNA passing through a nanopore using a nanometre-sized bead structure

    NASA Astrophysics Data System (ADS)

    Goto, Yusuke; Haga, Takanobu; Yanagi, Itaru; Yokoi, Takahide; Takeda, Ken-Ichi

    2015-11-01

    DNA sequencing with a solid-state nanopore requires a reduction of the translocation speeds of single-stranded DNA (ssDNA) over 10 μs/base. In this study, we report that a nanometre-sized bead structure constructed around a nanopore can reduce the moving speed of ssDNA to 270 μs/base by adjusting the diameter of the bead and its surface chemical group. This decelerating effect originates from the strong interaction between ssDNA and the chemical group on the surface of the bead. This nanostructure was simply prepared by dip coating in which a substrate with a nanopore was immersed in a silica bead solution and then dried in an oven. As compared with conventional approaches, our novel method is less laborious, simpler to perform and more effective in reducing ssDNA translocation speed.

  7. Discovery, Prevalence, and Persistence of Novel Circular Single-Stranded DNA Viruses in the Ctenophores Mnemiopsis leidyi and Beroe ovata.

    PubMed

    Breitbart, Mya; Benner, Bayleigh E; Jernigan, Parker E; Rosario, Karyna; Birsa, Laura M; Harbeitner, Rachel C; Fulford, Sidney; Graham, Carina; Walters, Anna; Goldsmith, Dawn B; Berger, Stella A; Nejstgaard, Jens C

    2015-01-01

    Gelatinous zooplankton, such as ctenophores and jellyfish, are important components of marine and brackish ecosystems and play critical roles in aquatic biogeochemistry. As voracious predators of plankton, ctenophores have key positions in aquatic food webs and are often successful invaders when introduced to new areas. Gelatinous zooplankton have strong impacts on ecosystem services, particularly in coastal environments. However, little is known about the factors responsible for regulating population dynamics of gelatinous organisms, including biological interactions that may contribute to bloom demise. Ctenophores are known to contain specific bacterial communities and a variety of invertebrate parasites and symbionts; however, no previous studies have examined the presence of viruses in these organisms. Building upon recent studies demonstrating a diversity of single-stranded DNA viruses that encode a replication initiator protein (Rep) in aquatic invertebrates, this study explored the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata collected from the Skidaway River Estuary and Savannah River in Georgia, USA. Using rolling circle amplification followed by restriction enzyme digestion, this study provides the first evidence of viruses in ctenophores. Investigation of four CRESS-DNA viruses over an 8-month period using PCR demonstrated temporal trends in viral prevalence and indicated that some of the viruses may persist in ctenophore populations throughout the year. Although future work needs to examine the ecological roles of these ctenophore-associated viruses, this study indicates that viral infection may play a role in population dynamics of gelatinous zooplankton.

  8. Quenching of Single-Walled Carbon Nanotube Fluorescence by Dissolved Oxygen Reveals Selective Single-Stranded DNA Affinities.

    PubMed

    Zheng, Yu; Bachilo, Sergei M; Weisman, R Bruce

    2017-04-13

    The selective interactions between short oligomers of single-stranded DNA (ssDNA) and specific structures of single-walled carbon nanotubes have been exploited in powerful methods for nanotube sorting. We report here that nanotubes coated with ssDNA also display selective interactions through the selective quenching of nanotube fluorescence by dissolved oxygen. In aqueous solutions equilibrated under 1 atm of O2, emission intensity from semiconducting nanotubes is reduced by between 9 and 40%, varying with the combination of ssDNA sequence and nanotube structure. This quenching reverses promptly and completely on removal of dissolved O2 and may be due to physisorption on nanotube surfaces. Fluorescence quenching offers a simple, nondestructive approach for studying the structure-selective interactions of ssDNA with single-walled carbon nanotubes and identifying recognition sequences.

  9. Single-stranded DNA detection by solvent-induced assemblies of a metallo-peptide-based complex

    NASA Astrophysics Data System (ADS)

    Das, Priyadip; Reches, Meital

    2016-05-01

    DNA detection is highly important for the sensitive sensing of different pathogenic bacteria and viruses. The major challenge is to create a sensor that can selectively detect very small concentrations of DNA without the need for amplification or complicated equipment. Different technologies such as optical, electrochemical and microgravimetric approaches can detect DNA fragments. Here we show, for the first time, the use of self-assembled nanostructures generated by a metallo-peptide as an optical sensing platform for DNA detection. The system can selectively detect single stranded DNA fragments by fluorescence measurements as it can discriminate even one base mismatch and can perform in the presence of other interfering proteins. This system may be useful in lab-on-a-chip applications.DNA detection is highly important for the sensitive sensing of different pathogenic bacteria and viruses. The major challenge is to create a sensor that can selectively detect very small concentrations of DNA without the need for amplification or complicated equipment. Different technologies such as optical, electrochemical and microgravimetric approaches can detect DNA fragments. Here we show, for the first time, the use of self-assembled nanostructures generated by a metallo-peptide as an optical sensing platform for DNA detection. The system can selectively detect single stranded DNA fragments by fluorescence measurements as it can discriminate even one base mismatch and can perform in the presence of other interfering proteins. This system may be useful in lab-on-a-chip applications. Electronic supplementary information (ESI) available: Peptide and receptor synthesis, characterization of the final and intermediate products, experimental details and additional figures including SEM, TEM, DLS, XRD, UV analysis and AFM topographic analysis. See DOI: 10.1039/c5nr07714a

  10. Construction, characterization, and selected site-specific mutagenesis of an anti-single-stranded DNA single-chain autoantibody.

    PubMed

    Rumbley, C A; Denzin, L K; Yantz, L; Tetin, S Y; Voss, E W

    1993-06-25

    Single-chain antibodies are comprised of immunoglobulin light and heavy chain variable domains joined through a polypeptide linker. A single-chain autoantibody, containing the 14-amino acid 212-polypeptide linker (GSTSGSGKSSEGKG), was constructed based on the light and heavy chain variable region gene sequences of anti-single-stranded DNA autoantibody BV04-01 (IgG2b, kappa). Following protein expression in Escherichia coli, denaturation, refolding, and affinity purification, single-chain autoantibody 04-01 binding with single-stranded DNA and poly(dT) was characterized in solid-phase and solution-phase assays. Homopolymer ligand binding results demonstrated that single-chain autoantibody 04-01 possessed anti-DNA binding properties similar to BV04-01 IgG and Fab fragments. Based on x-ray crystallographic analyses of BV04-01, site-specific mutagenesis studies were conducted on 2 residues (L32Tyr and H100aTrp) involved in aromatic stacking interactions with the middle thymidine of a (dT)3 ligand.

  11. Detection of DNA single-strand breaks during the repair of UV damage in xeroderma pigmentosum cells

    SciTech Connect

    Fornace, A.J. Jr.; Seres, D.S.

    1983-01-01

    In this investigation, xeroderma pigmentosum (XP) fibroblasts, XP12BE, were uv-irradiated and then incubated with cytosine arabinoside and hydroxyurea for 4 hr to inhibit the polymerase step of DNA excision repair. By alkaline elution, DNA single-strand breaks (SSB) were detected in XP cells with this regimen with an efficiency of 0.1-0.2 SSB per 10/sup 9/ daltons of DNA per J m/sup -2/. There was an approximately linear relation between the SSB frequency and uv dose over a range of 0.2 to 25 J m/sup -2/. This effect was approximately two orders of magnitude greater in excision-proficient normal human fibroblasts than in XP cells. These results support the conclusion that a low residual level of DNA excision repair occurs in XP group A cells and that the SSB generated during this repair can be accumulated with this polymerase inhibitor.

  12. Charge transport and photoresponses in a single-stranded DNA/single-walled carbon nanotube composite film

    NASA Astrophysics Data System (ADS)

    Hong, Wonseon; Lee, Eunmo; Kue Park, Jun; Eui Lee, Cheol

    2013-06-01

    Electrical conductivity and photoresponse measurements have been carried out on a single-stranded DNA (ssDNA)/single-walled carbon nanotube (SWNT) composite film in comparison to those of a SWNT film. While the temperature-dependent electrical conductivity of the pristine SWNT film was described well by the combined mechanism of a three-dimensional variable-range hopping and hopping conduction, that of the ssDNA/SWNT composite film followed a fluctuation-induced tunneling model. Besides, competition of photoexcited charge carrier generation and oxygen adsorption/photodesorption in the photoresponses of the films was observed and discussed in view of the role of the DNA wrapping. Thus, the biopolymer coating of the SWNTs is shown to play a significant role in modifying the charge dynamics of the composite system.

  13. Single-primer-limited amplification: a method to generate random single-stranded DNA sub-library for aptamer selection.

    PubMed

    He, Chao-Zhu; Zhang, Kun-He; Wang, Ting; Wan, Qin-Si; Hu, Piao-Ping; Hu, Mei-Di; Huang, De-Qiang; Lv, Nong-Hua

    2013-09-01

    The amplification of a random single-stranded DNA (ssDNA) library by polymerase chain reaction (PCR) is a key step in each round of aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX), but it can be impeded by the amplification of by-products due to the severely nonspecific hybridizations among various sequences in the PCR system. To amplify a random ssDNA library free from by-products, we developed a novel method termed single-primer-limited amplification (SPLA), which was initiated from the amplification of minus-stranded DNA (msDNA) of an ssDNA library with reverse primer limited to 5-fold molar quantity of the template, followed by the amplification of plus-stranded DNA (psDNA) of the msDNA with forward primer limited to 10-fold molar quantity of the template and recovery of psDNA by gel excision. We found that the amount of by-products increased with the increase of template amount and thermal cycle number. With the optimized template amount and thermal cycle, SPLA could amplify target ssDNA without detectable by-products and nonspecific products and could produce psDNA 16.1 times as much as that by asymmetric PCR. In conclusion, SPLA is a simple and feasible method to efficiently generate a random ssDNA sub-library for aptamer selection.

  14. A novel, evolutionarily conserved gene family with putative sequence-specific single-stranded DNA-binding activity.

    PubMed

    Castro, Patricia; Liang, Hong; Liang, Jan C; Nagarajan, Lalitha

    2002-07-01

    Complete and partial deletions of chromosome 5q are recurrent cytogenetic anomalies associated with aggressive myeloid malignancies. Earlier, we identified an approximately 1.5-Mb region of loss at 5q13.3 between the loci D5S672 and D5S620 in primary leukemic blasts. A leukemic cell line, ML3, is diploid for all of chromosome 5, except for an inversion-coupled translocation within the D5S672-D5S620 interval. Here, we report the development of a bacterial artificial chromosome (BAC) contig to define the breakpoint and the identification of a novel gene SSBP2, the target of disruption in ML3 cells. A preliminary evaluation of SSBP2 as a tumor suppressor gene in primary leukemic blasts and cell lines suggests that the remaining allele does not undergo intragenic mutations. SSBP2 is one of three members of a closely related, evolutionarily conserved, and ubiquitously expressed gene family. SSBP3 is the human ortholog of a chicken gene, CSDP, that encodes a sequence-specific single-stranded DNA-binding protein. SSBP3 localizes to chromosome 1p31.3, and the third member, SSBP4, maps to chromosome 19p13.1. Chromosomal localization and the putative single-stranded DNA-binding activity suggest that all three members of this family are capable of potential tumor suppressor activity by gene dosage or other epigenetic mechanisms.

  15. APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure.

    PubMed

    Holtz, Colleen M; Sadler, Holly A; Mansky, Louis M

    2013-07-01

    Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (i.e., APOBEC3G or A3G) is an evolutionarily conserved cytosine deaminase that potently restricts human immunodeficiency virus type 1 (HIV-1), retrotransposons and other viruses. A3G has a nucleotide target site specificity for cytosine dinucleotides, though only certain cytosine dinucleotides are 'hotspots' for cytosine deamination, and others experience little or no editing by A3G. The factors that define these critical A3G hotspots are not fully understood. To investigate how A3G hotspots are defined, we used an in vitro fluorescence resonance energy transfer-based oligonucleotide assay to probe the site specificity of A3G. Our findings strongly suggest that the target single-stranded DNA (ssDNA) secondary structure as well as the bases directly 3' and 5' of the cytosine dinucleotide are critically important A3G recognition. For instance, A3G cannot readily deaminate a cytosine dinucleotide in ssDNA stem structures or in nucleotide base loops composed of three bases. Single-stranded nucleotide loops up to seven bases in length were poor targets for A3G activity unless cytosine residues flanked the cytosine dinucleotide. Furthermore, we observed that A3G favors adenines, cytosines and thymines flanking the cytosine dinucleotide target in unstructured regions of ssDNA. Low cytosine deaminase activity was detected when guanines flanked the cytosine dinucleotide. Taken together, our findings provide the first demonstration that A3G cytosine deamination hotspots are defined by both the sequence context of the cytosine dinucleotide target as well as the ssDNA secondary structure. This knowledge can be used to better trace the origins of mutations to A3G activity, and illuminate its impact on processes such as HIV-1 genetic variation.

  16. Chromatin collapse during caspase-dependent apoptotic cell death requires DNA fragmentation factor, 40-kDa subunit-/caspase-activated deoxyribonuclease-mediated 3'-OH single-strand DNA breaks.

    PubMed

    Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Sánchez-Osuna, María; Casanelles, Elisenda; García-Belinchón, Mercè; Comella, Joan X; Yuste, Victor J

    2013-03-29

    Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD(-/-) cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3'-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3'-OH ends in single-strand rather than double-strand DNA nicks/breaks.

  17. Chemical shift changes provide evidence for overlapping single-stranded DNA and XPA binding sites on the 70 kDa subunit of human replication protein A

    SciTech Connect

    Daughdrill, Gary W.; Buchko, Garry W.; Botuyan, Maria V.; Arrowsmith, Cheryl H.; Wold, Marc S.; Kennedy, Michael A.; Lowry, David F.

    2003-07-15

    Replication protein A (RPA) is a heterotrimeric single-stranded DNA (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, NMR spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1-326 (hRPA701-326), and a fragment of the human XPA protein, residues 98-219 (XPA-MBD). Intensity changes were observed for amide resonances in the 1H-15N correlation spectrum of uniformly 15N-labeled hRPA701-326 after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA binding domain that is between residues 183 and 296 of the hRPA701-326 fragment. The hRPA701-326 residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA binding domain to identify the binding surface with XPA-MBD. The XPA-MBD binding surface showed significant overlap with an ssDNA binding surface that was previously identified using NMR spectroscopy and X-ray crystallography.

  18. Effects of proton irradiation on single-stranded DNA studied by using X-ray photoelectron spectroscopy

    NASA Astrophysics Data System (ADS)

    Lee, E.; Lee, Cheol Eui; Han, J. H.

    2016-08-01

    X-ray photoelectron spectroscopy (XPS) has been employed in order to study the effects of proton irradiation on herring sperm single-stranded DNA. Systematic changes of the chemical shifts in the C, N, O, and P XPS line components as functions of the irradiation dose were observed, indicative of the bonding configurations in the DNA system. While the C 1 s XPS lines showed weak blueshifts, the N 1 s, O 1 s, and P 2 p XPS lines showed blueshifts with a marked dependence on the irradiation dose in a prominent manner. Our results show that linear energy transfer by charged particles and photons may have distinct molecular-level effects as the C 1 s, N 1 s, O 1 s, and P 2 p XPS lines showed redshifts in our previous study of effects of the γ-ray irradiation on the same system.

  19. Agrobacterium rhizogenes GALLS Protein Substitutes for Agrobacterium tumefaciens Single-Stranded DNA-Binding Protein VirE2

    PubMed Central

    Hodges, Larry D.; Cuperus, Josh; Ream, Walt

    2004-01-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2. PMID:15126468

  20. Nerve growth factor inhibits the synthesis of a single-stranded DNA binding protein in pheochromocytoma cells (clone PC12).

    PubMed Central

    Biocca, S; Cattaneo, A; Calissano, P

    1984-01-01

    Arrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activity. Images PMID:6585787

  1. Discovery, Prevalence, and Persistence of Novel Circular Single-Stranded DNA Viruses in the Ctenophores Mnemiopsis leidyi and Beroe ovata

    PubMed Central

    Breitbart, Mya; Benner, Bayleigh E.; Jernigan, Parker E.; Rosario, Karyna; Birsa, Laura M.; Harbeitner, Rachel C.; Fulford, Sidney; Graham, Carina; Walters, Anna; Goldsmith, Dawn B.; Berger, Stella A.; Nejstgaard, Jens C.

    2015-01-01

    Gelatinous zooplankton, such as ctenophores and jellyfish, are important components of marine and brackish ecosystems and play critical roles in aquatic biogeochemistry. As voracious predators of plankton, ctenophores have key positions in aquatic food webs and are often successful invaders when introduced to new areas. Gelatinous zooplankton have strong impacts on ecosystem services, particularly in coastal environments. However, little is known about the factors responsible for regulating population dynamics of gelatinous organisms, including biological interactions that may contribute to bloom demise. Ctenophores are known to contain specific bacterial communities and a variety of invertebrate parasites and symbionts; however, no previous studies have examined the presence of viruses in these organisms. Building upon recent studies demonstrating a diversity of single-stranded DNA viruses that encode a replication initiator protein (Rep) in aquatic invertebrates, this study explored the presence of circular, Rep-encoding single-stranded DNA (CRESS-DNA) viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata collected from the Skidaway River Estuary and Savannah River in Georgia, USA. Using rolling circle amplification followed by restriction enzyme digestion, this study provides the first evidence of viruses in ctenophores. Investigation of four CRESS-DNA viruses over an 8-month period using PCR demonstrated temporal trends in viral prevalence and indicated that some of the viruses may persist in ctenophore populations throughout the year. Although future work needs to examine the ecological roles of these ctenophore-associated viruses, this study indicates that viral infection may play a role in population dynamics of gelatinous zooplankton. PMID:26733971

  2. Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

    PubMed Central

    Harley, Matthew J.; Schildbach, Joel F.

    2003-01-01

    Conjugative plasmid transfer is an important mechanism for diversifying prokaryotic genomes and disseminating antibiotic resistance. Relaxases are conjugative plasmid-encoded proteins essential for plasmid transfer. Relaxases bind and cleave one plasmid strand site- and sequence-specifically before transfer of the cleaved strand. TraI36, a domain of F plasmid TraI that contains relaxase activity, binds a plasmid sequence in single-stranded form with subnanomolar KD and high sequence specificity. Despite 91% amino acid sequence identity, TraI36 domains from plasmids F and R100 discriminate between binding sites. The binding sites differ by 2 of 11 bases, but both proteins bind their cognate site with three orders of magnitude higher affinity than the other site. To identify specificity determinants, we generated variants having R100 amino acids in the F TraI36 background. Although most retain F specificity, the Q193R/R201Q variant binds the R100 site with 10-fold greater affinity than the F site. The reverse switch (R193Q/Q201R) in R100 TraI36 confers a wild-type F specificity on the variant. Nonadditivity of individual amino acid and base contributions to recognition suggests that the specificity difference derives from multiple interactions. The F TraI36 crystal structure shows positions 193 and 201 form opposite sides of a pocket within the binding cleft, suggesting binding involves knob-into-hole interactions. Specificity is presumably modulated by altering the composition of the pocket. Our results demonstrate that F-like relaxases can switch between highly sequence-specific recognition of different sequences with minimal amino acid substitution. PMID:14504391

  3. Actinomycin D binds strongly to d(TGTCATTG), a single-stranded DNA devoid of GpC sites.

    PubMed

    Chen, F M; Sha, F

    2001-05-01

    Despite the absence of the GpC sequence and complete self-complementarity, d(CGTCGTCG) has recently been shown to bind strongly to actinomycin D (ACTD) with a binding density of about one drug molecule per strand. To further elucidate the nature of such a binding, studies are herein made with single-base G --> A and C --> T replacements in d(CGTCGTCG) to identify the DNA bases that play important roles in the strong ACTD binding of this oligomer. On the basis of these results, the octamer d(TGTCATTG) has been identified as a potentially strong ACTD binder. Indeed, binding titration confirms such an expectation and reveals an ACTD binding constant of about 1 x 10(7) M(-1) and a binding density of roughly 0.8 drug molecule per DNA strand for this strong binding mode. Similar binding studies with single-base substitutions on d(TGTCATTG) further reveal the relative importance of the C and G bases on its ACTD binding, with the 3'-terminus G appearing to be the most crucial base. Further base substitutions lead to the conclusion that these C and G bases act in concert rather than individually in the ACTD binding of d(TGTCATTG). Spectral comparisons with the apparently single-stranded GpC-containing d(TGCTTTG) led to the proposal of a speculated monomeric hairpin binding model to account for the experimental observations. This model makes use of the notion that ACTD prefers to have the 3'-sides of both G bases stacking on the opposite faces of its planar phenoxazone chromophore, a principle akin to its classic preference for the GpC sequence in duplex form. The finding that ACTD can bind strongly to single-stranded DNA of special sequence motifs may have important implications.

  4. High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy

    PubMed Central

    Short, Judith M.; Liu, Yang; Chen, Shaoxia; Soni, Neelesh; Madhusudhan, Mallur S.; Shivji, Mahmud K.K.; Venkitaraman, Ashok R.

    2016-01-01

    Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5–5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51–DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease. PMID:27596592

  5. Sequence-based separation of single-stranded DNA using nucleotides in capillary electrophoresis: focus on phosphate.

    PubMed

    Zhang, Xueru; McGown, Linda B

    2013-06-01

    DNA analysis has widespread applicability in biology, medicine, biotechnology, and forensics. DNA separation by length is readily achieved using sieving gels in electrophoresis. Separation by sequence is less simple, generally requiring adequate differences in native or induced conformation or differences in thermal or chemical stability of the strands that are hybridized prior to measurement. We previously demonstrated separation of four single-stranded DNA 76-mers that differ by only a few A-G substitutions based solely on sequence using guanosine-5'-monophosphate (GMP) in the running buffer. We attributed separation to the unique self-assembly of GMP to form higher order structures. Here, we examine an expanded set of 76-mers designed to probe the mechanism of the separation and effects of experimental conditions. We were surprised to find that other ribonucleotides achieved the similar separation to GMP, and that some separation was achieved using sodium phosphate instead of GMP. Potassium phosphate achieved almost as good separations as the ribonucleotides. This suggests that the separation medium provides a physicochemical environment for the DNA that effects strand migration in a sequence-selective manner. Further investigation is needed to determine whether the mechanism involves specific interactions between the phosphates and the DNA strands or is a result of other properties of the separation medium. Phosphate generally has been avoided in DNA separations by capillary gel electrophoresis because its high ionic strength exacerbates Joule heating. Our results suggest that phosphate compounds should be examined for separation of DNA based on sequence.

  6. Enhancement of RecA-mediated self-assembly in DNA nanostructures through basepair mismatches and single-strand nicks

    NASA Astrophysics Data System (ADS)

    Corbett, Sybilla Louise; Sharma, Rajan; Davies, Alexander Giles; Wälti, Christoph

    2017-01-01

    The use of DNA as a structural material for nanometre-scale construction has grown extensively over the last decades. The development of more advanced DNA-based materials would benefit from a modular approach enabling the direct assembly of additional elements onto nanostructures after fabrication. RecA-based nucleoprotein filaments encapsulating short ssDNA have been demonstrated as a tool for highly efficient and fully programmable post-hoc patterning of duplex DNA scaffold. However, the underlying assembly process is not fully understood, in particular when patterning complex DNA topologies. Here, we report the effect of basepair-mismatched regions and single-strand nicks in the double-stranded DNA scaffold on the yield of RecA-based assembly. Significant increases in assembly yield are observed upon the introduction of unpaired basepairs directly adjacent to the assembly region. However, when the unpaired regions were introduced further from the assembly site the assembly yield initially decreased as the length of the unpaired region was increased. These results suggest that an unpaired region acts as a kinetic trap for RecA-based nucleoprotein filaments, impeding the assembly mechanism. Conversely, when the unpaired region is located directly adjacent to the assembly site, it leads to an increase in efficiency of RecA patterning owing to increased breathing of the assembly site.

  7. Enhancement of RecA-mediated self-assembly in DNA nanostructures through basepair mismatches and single-strand nicks

    PubMed Central

    Corbett, Sybilla Louise; Sharma, Rajan; Davies, Alexander Giles; Wälti, Christoph

    2017-01-01

    The use of DNA as a structural material for nanometre-scale construction has grown extensively over the last decades. The development of more advanced DNA-based materials would benefit from a modular approach enabling the direct assembly of additional elements onto nanostructures after fabrication. RecA-based nucleoprotein filaments encapsulating short ssDNA have been demonstrated as a tool for highly efficient and fully programmable post-hoc patterning of duplex DNA scaffold. However, the underlying assembly process is not fully understood, in particular when patterning complex DNA topologies. Here, we report the effect of basepair-mismatched regions and single-strand nicks in the double-stranded DNA scaffold on the yield of RecA-based assembly. Significant increases in assembly yield are observed upon the introduction of unpaired basepairs directly adjacent to the assembly region. However, when the unpaired regions were introduced further from the assembly site the assembly yield initially decreased as the length of the unpaired region was increased. These results suggest that an unpaired region acts as a kinetic trap for RecA-based nucleoprotein filaments, impeding the assembly mechanism. Conversely, when the unpaired region is located directly adjacent to the assembly site, it leads to an increase in efficiency of RecA patterning owing to increased breathing of the assembly site. PMID:28112216

  8. Investigation of the nanoviscosity effect of a G-quadruplex and single-strand DNA using fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Lee, Dongkeun; Kim, Minjung; Kim, Soo Yong; Shin, Hyosup; Kim, Sok Won; Park, Inho

    2015-01-01

    Guanine (G)-quadruplexes are of interest because of their presence in the telomere sequence and the oncogene promoter region. Their diffusion and change of structure, especially in high viscosity solutions, are important for understanding their dynamics. G-quadruplexes may have less effective viscosity (nanoviscosity) when they are smaller than the solvent molecules. In this paper, we report the difference in the diffusion dynamics of the G-rich DNA sequences of single-strand DNA (ssDNA) and the G-quadruplex in aqueous, sucrose, and polyethylene glycol (PEG) solutions. From experiments with aqueous and sucrose solutions, we confirm that a simple diffusion model according to the viscosity is appropriate. In the PEG experiments, the nanoviscosity effect is observed according to PEG's molecular weight. In the PEG 200 solution, both the ssDNA and the G-quadruplex possess macroviscosity. In the PEG 10 000 solution, the G-quadruplex possesses nanoviscosity and the ssDNA possesses macroviscosity, whereas, in the PEG 35 000 solution, both ssDNA and the G-quadruplex possess nanoviscosity. The experimental results are consistent with the theoretical predictions.

  9. Enhancement of RecA-mediated self-assembly in DNA nanostructures through basepair mismatches and single-strand nicks.

    PubMed

    Corbett, Sybilla Louise; Sharma, Rajan; Davies, Alexander Giles; Wälti, Christoph

    2017-01-23

    The use of DNA as a structural material for nanometre-scale construction has grown extensively over the last decades. The development of more advanced DNA-based materials would benefit from a modular approach enabling the direct assembly of additional elements onto nanostructures after fabrication. RecA-based nucleoprotein filaments encapsulating short ssDNA have been demonstrated as a tool for highly efficient and fully programmable post-hoc patterning of duplex DNA scaffold. However, the underlying assembly process is not fully understood, in particular when patterning complex DNA topologies. Here, we report the effect of basepair-mismatched regions and single-strand nicks in the double-stranded DNA scaffold on the yield of RecA-based assembly. Significant increases in assembly yield are observed upon the introduction of unpaired basepairs directly adjacent to the assembly region. However, when the unpaired regions were introduced further from the assembly site the assembly yield initially decreased as the length of the unpaired region was increased. These results suggest that an unpaired region acts as a kinetic trap for RecA-based nucleoprotein filaments, impeding the assembly mechanism. Conversely, when the unpaired region is located directly adjacent to the assembly site, it leads to an increase in efficiency of RecA patterning owing to increased breathing of the assembly site.

  10. Defective DNA Ligation during Short-Patch Single-Strand Break Repair in Ataxia Oculomotor Apraxia 1 ▿

    PubMed Central

    Reynolds, John J.; El-Khamisy, Sherif F.; Katyal, Sachin; Clements, Paula; McKinnon, Peter J.; Caldecott, Keith W.

    2009-01-01

    Ataxia oculomotor apraxia 1 (AOA1) results from mutations in aprataxin, a component of DNA strand break repair that removes AMP from 5′ termini. Despite this, global rates of chromosomal strand break repair are normal in a variety of AOA1 and other aprataxin-defective cells. Here we show that short-patch single-strand break repair (SSBR) in AOA1 cell extracts bypasses the point of aprataxin action at oxidative breaks and stalls at the final step of DNA ligation, resulting in the accumulation of adenylated DNA nicks. Strikingly, this defect results from insufficient levels of nonadenylated DNA ligase, and short-patch SSBR can be restored in AOA1 extracts, independently of aprataxin, by the addition of recombinant DNA ligase. Since adenylated nicks are substrates for long-patch SSBR, we reasoned that this pathway might in part explain the apparent absence of a chromosomal SSBR defect in aprataxin-defective cells. Indeed, whereas chemical inhibition of long-patch repair did not affect SSBR rates in wild-type mouse neural astrocytes, it uncovered a significant defect in Aptx−/− neural astrocytes. These data demonstrate that aprataxin participates in chromosomal SSBR in vivo and suggest that short-patch SSBR arrests in AOA1 because of insufficient nonadenylated DNA ligase. PMID:19103743

  11. Identification of several clades of novel single-stranded circular DNA viruses with conserved stem-loop structures in pig feces

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Metagenomic analysis of fecal samples collected from swine with diarrhea detected sequences encoding a replication initiator protein (Rep) typically found in small circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses. Complete nucleotide sequences of ten novel genomes were cloned and determ...

  12. Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing

    PubMed Central

    Lecomte, Emilie; Tournaire, Benoît; Cogné, Benjamin; Dupont, Jean-Baptiste; Lindenbaum, Pierre; Martin-Fontaine, Mélanie; Broucque, Frédéric; Robin, Cécile; Hebben, Matthias; Merten, Otto-Wilhelm; Blouin, Véronique; François, Achille; Redon, Richard; Moullier, Philippe; Léger, Adrien

    2015-01-01

    Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles. PMID:26506038

  13. Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing.

    PubMed

    Lecomte, Emilie; Tournaire, Benoît; Cogné, Benjamin; Dupont, Jean-Baptiste; Lindenbaum, Pierre; Martin-Fontaine, Mélanie; Broucque, Frédéric; Robin, Cécile; Hebben, Matthias; Merten, Otto-Wilhelm; Blouin, Véronique; François, Achille; Redon, Richard; Moullier, Philippe; Léger, Adrien

    2015-10-27

    Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles.

  14. Detection of short single-strand DNA homopolymers with ultrathin Si3N4 nanopores.

    PubMed

    Ma, Jian; Qiu, Yinghua; Yuan, Zhishan; Zhang, Yin; Sha, Jingjie; Liu, Lei; Sun, Litao; Ni, Zhonghua; Yi, Hong; Li, Deyu; Chen, Yunfei

    2015-08-01

    A series of nanopores with diameters ranging from 2.5 to 63 nm are fabricated on a reduced Si3N4 membrane by focused ion beam and high energy electron beam. Through measuring the blocked ionic currents for DNA strands threading linearly through those solid-state nanopores, it is found that the blockade ionic current is proportional to the square of the hydrodynamic diameter of the DNA strand. With the nanopore diameter reduced to be comparable with that of DNA strands, the hydrodynamic diameter of the DNA becomes smaller, which is attributed to the size confinement effects. The duration time for the linear DNA translocation events increases monotonically with the nanopore length. By comparing the spatial configurations of DNA strands through nanopores with different diameters, it is found that the nanopore with large diameter has enough space to allow the DNA strand to translocate through with complex conformation. With the decrease of the nanopore diameter, the folded part of the DNA is prone to be straightened by the nanopore, which leads to the increase in the occurrence frequency of the linear DNA translocation events. Reducing the diameter of the nanopore to 2.5 nm allows the detection and discrimination of three nucleotide "G" and three nucleotide "T" homopolymer DNA strands based on differences in their physical dimensions.

  15. Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities.

    PubMed

    Guo, Yang; Kragelund, Birthe B; White, Malcolm F; Peng, Xu

    2015-06-19

    The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b (gp17) and ORF436 (gp18) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both encoding proteins of unknown function and forming an operon with ORF207 (gp19). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5' → 3' ssDNA exonuclease activity, in addition to the previously demonstrated ssDNA endonuclease activity. Further, in vitro pull-down assay demonstrated interactions between gp17 and gp18 and between gp18 and gp19 with the former being mediated by the intrinsically disordered C-terminus of gp17. The strand-displacement replication mode proposed previously for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair.

  16. Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated▿

    PubMed Central

    Dostál, Lubomír; Schildbach, Joel F.

    2010-01-01

    Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal ∼300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer. PMID:20435720

  17. Yields of single-strand breaks in double-stranded calf thymus DNA irradiated in aqueous solution in the presence of oxygen and scavengers

    SciTech Connect

    Udovicic, Lj.; Mark, F.; Bothe, E.

    1994-11-01

    Yields of radiation-induced single-strand breaks in double-stranded calf thymus DNA have been measured as a function of OH scavenger concentration in N{sub 2}O/O{sub 2}-saturated aqueous solution. The experimental data are well represented by a theoretical model based on non-homogeneous reaction kinetics, without the need to adjust any parameter. The good agreement between experimental and theoretical data is taken as evidence that, in the presence of oxygen, the main effect of added scavengers with respect to the formation of single-strand breaks in double-stranded DNA is OH radical scavenging. 30 refs., 3 figs., 1 tab.

  18. The Telomere Binding Protein Cdc13 and the Single-Stranded DNA Binding Protein RPA Protect Telomeric DNA from Resection by Exonucleases.

    PubMed

    Greetham, Matthew; Skordalakes, Emmanuel; Lydall, David; Connolly, Bernard A

    2015-09-25

    The telomere is present at the ends of all eukaryotic chromosomes and usually consists of repetitive TG-rich DNA that terminates in a single-stranded 3' TG extension and a 5' CA-rich recessed strand. A biochemical assay that allows the in vitro observation of exonuclease-catalyzed degradation (resection) of telomeres has been developed. The approach uses an oligodeoxynucleotide that folds to a stem-loop with a TG-rich double-stranded region and a 3' single-stranded extension, typical of telomeres. Cdc13, the major component of the telomere-specific CST complex, strongly protects the recessed strand from the 5'→3' exonuclease activity of the model exonuclease from bacteriophage λ. The isolated DNA binding domain of Cdc13 is less effective at shielding telomeres. Protection is specific, not being observed in control DNA lacking the specific TG-rich telomere sequence. RPA, the eukaryotic single-stranded DNA binding protein, also inhibits telomere resection. However, this protein is non-specific, equally hindering the degradation of non-telomere controls.

  19. Salt Dependence of the Radius of Gyration and Flexibility of Single-stranded DNA in Solution probed by Small-angle X-ray Scattering

    SciTech Connect

    Sim, Adelene Y.L.; Lipfert, Jan; Herschlag, Daniel; Doniach, Sebastian

    2012-07-06

    Short single-stranded nucleic acids are ubiquitous in biological processes and understanding their physical properties provides insights to nucleic acid folding and dynamics. We used small angle x-ray scattering to study 8-100 residue homopolymeric single-stranded DNAs in solution, without external forces or labeling probes. Poly-T's structural ensemble changes with increasing ionic strength in a manner consistent with a polyelectrolyte persistence length theory that accounts for molecular flexibility. For any number of residues, poly-A is consistently more elongated than poly-T, likely due to the tendency of A residues to form stronger base-stacking interactions than T residues.

  20. Charge enhancement of single-stranded DNA in negative electrospray ionization using the supercharging reagent meta-nitrobenzyl alcohol.

    PubMed

    Brahim, Bessem; Alves, Sandra; Cole, Richard B; Tabet, Jean-Claude

    2013-12-01

    Charge enhancement of single-stranded oligonucleotide ions in negative ESI mode is investigated. The employed reagent, meta-nitrobenzyl alcohol (m-NBA), was found to improve total signal intensity (Itot), increase the highest observed charge states (zhigh), and raise the average charge states (zavg) of all tested oligonucleotides analyzed in negative ESI. To quantify these increases, signal enhancement ratios (SER1%) and charge enhancement coefficients (CEC1%) were introduced. The SER1%, (defined as the quotient of total oligonucleotide ion abundances with 1% m-NBA divided by total oligonucleotide abundance without m-NBA) was found to be greater than unity for every oligonucleotide tested. The CEC1% values (defined as the average charge state in the presence of 1% m-NBA minus the average charge state in the absence of m-NBA) were found to be uniformly positive. Upon close inspection, the degree of charge enhancement for longer oligonucleotides was found to be dependent upon thymine density (i.e., the number and the location of phospho-thymidine units). A correlation between the charge enhancement induced by the presence of m-NBA and the apparent gas-phase acidity (largely determined by the sequence of thymine units but also by the presence of protons on other nucleobases) of multiply deprotonated oligonucleotide species, was thus established. Ammonium cations appeared to be directly involved in the m-NBA supercharging mechanism, and their role seems to be consistent with previously postulated ESI mechanisms describing desorption/ionization of single-stranded DNA into the gas phase.

  1. Charge Enhancement of Single-Stranded DNA in Negative Electrospray Ionization Using the Supercharging Reagent Meta-nitrobenzyl Alcohol

    NASA Astrophysics Data System (ADS)

    Brahim, Bessem; Alves, Sandra; Cole, Richard B.; Tabet, Jean-Claude

    2013-12-01

    Charge enhancement of single-stranded oligonucleotide ions in negative ESI mode is investigated. The employed reagent, meta-nitrobenzyl alcohol (m-NBA), was found to improve total signal intensity (Itot), increase the highest observed charge states (zhigh), and raise the average charge states (zavg) of all tested oligonucleotides analyzed in negative ESI. To quantify these increases, signal enhancement ratios (SER1%) and charge enhancement coefficients (CEC1%) were introduced. The SER1%, (defined as the quotient of total oligonucleotide ion abundances with 1 % m-NBA divided by total oligonucleotide abundance without m-NBA) was found to be greater than unity for every oligonucleotide tested. The CEC1% values (defined as the average charge state in the presence of 1 % m-NBA minus the average charge state in the absence of m-NBA) were found to be uniformly positive. Upon close inspection, the degree of charge enhancement for longer oligonucleotides was found to be dependent upon thymine density (i.e., the number and the location of phospho-thymidine units). A correlation between the charge enhancement induced by the presence of m-NBA and the apparent gas-phase acidity (largely determined by the sequence of thymine units but also by the presence of protons on other nucleobases) of multiply deprotonated oligonucleotide species, was thus established. Ammonium cations appeared to be directly involved in the m-NBA supercharging mechanism, and their role seems to be consistent with previously postulated ESI mechanisms describing desorption/ionization of single-stranded DNA into the gas phase.

  2. Evidence that single-stranded DNA breaks are a normal feature of koala sperm chromatin, while double-stranded DNA breaks are indicative of DNA damage.

    PubMed

    Zee, Yeng Peng; López-Fernández, Carmen; Arroyo, F; Johnston, Stephen D; Holt, William V; Gosalvez, Jaime

    2009-08-01

    In this study, we have used single and double comet assays to differentiate between single- and double-stranded DNA damage in an effort to refine the interpretation of DNA damage in mature koala spermatozoa. We have also investigated the likelihood that single-stranded DNA breakage is part of the natural spermiogenic process in koalas, where its function would be the generation of structural bends in the DNA molecule so that appropriate packaging and compaction can occur. Koala spermatozoa were examined using the sperm chromatin dispersion test (SCDt) and comet assays to investigate non-orthodox double-stranded DNA. Comet assays were conducted under 1) neutral conditions; and 2) neutral followed by alkaline conditions (double comet assay); the latter technique enabled simultaneous visualisation of both single-stranded and double-stranded DNA breaks. Following the SCDt, there was a continuum of nuclear morphotypes, ranging from no apparent DNA fragmentation to those with highly dispersed and degraded chromatin. Dispersion morphotypes were mirrored by a similar diversity of comet morphologies that could be further differentiated using the double comet assay. The majority of koala spermatozoa had nuclei with DNA abasic-like residues that produced single-tailed comets following the double comet assay. The ubiquity of these residues suggests that constitutive alkali-labile sites are part of the structural configuration of the koala sperm nucleus. Spermatozoa with 'true' DNA fragmentation exhibited a continuum of comet morphologies, ranging from a more severe form of alkaline-susceptible DNA with a diffuse single tail to nuclei that exhibited both single- and double-stranded breaks with two comet tails.

  3. Defective DNA single-strand break repair is responsible for senescence and neoplastic escape of epithelial cells

    PubMed Central

    Nassour, Joe; Martien, Sébastien; Martin, Nathalie; Deruy, Emeric; Tomellini, Elisa; Malaquin, Nicolas; Bouali, Fatima; Sabatier, Laure; Wernert, Nicolas; Pinte, Sébastien; Gilson, Eric; Pourtier, Albin; Pluquet, Olivier; Abbadie, Corinne

    2016-01-01

    The main characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that unlike fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA damage response (DDR), but accumulate oxidative-stress-induced DNA single-strand breaks (SSBs). These breaks remain unrepaired because of a decrease in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that engage a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Importantly, the default in SSB repair also leads to the emergence of post-senescent transformed and mutated precancerous cells. In human-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a decline of PARP1, whereas DDR foci accumulate mainly in dermal fibroblasts. These findings point SSBs as a DNA damage encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis. PMID:26822533

  4. APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase

    PubMed Central

    Suspène, Rodolphe; Sommer, Peter; Henry, Michel; Ferris, Stéphane; Guétard, Denise; Pochet, Sylvie; Chester, Ann; Navaratnam, Naveenan; Wain-Hobson, Simon; Vartanian, Jean-Pierre

    2004-01-01

    In the absence of the viral vif gene, human immunodeficiency virus (HIV) may be restricted by the APOBEC3G gene on chromosome 22. The role of the HIV Vif protein is to exclude host cell APOBEC3G from the budding virion. As APOBEC3G shows sequence homology to cytidine deaminases, it is presumed that in the absence of Vif, cytidine residues in the cDNA are deaminated yielding uracil. It is not known if additional proteins mediate APOBEC3G function or if deamination occurs in concert with reverse transcription. This report describes an in vitro assay showing that Baculovirus derived APOBEC3G alone extensively deaminates cDNA independently of reverse transcriptase. It reproduces the dinucleotide context typical of G → A hypermutants derived from a Δvif virus. By using an RNaseH– form of reverse transcriptase, it was shown that the cDNA has to be free of its RNA template to allow deamination. APOBEC3G deamination of dC or dCTP was not detected. In short, APOBEC3G is a single-stranded DNA cytidine deaminase capable of restricting retroviral replication. PMID:15121899

  5. Characterization of a complete genome of a circular single-stranded DNA virus from porcine stools in Korea.

    PubMed

    Kim, A Reum; Chung, Hee Chun; Kim, Hye Kwon; Kim, Eun Ok; Nguyen, Van Giap; Choi, Min Gyung; Yang, Hye Jung; Kim, Jung Ah; Park, Bong Kyun

    2014-02-01

    Porcine circular single-stranded DNA viruses have been just identified from swine feces in Korea. This virus was mentioned as bovine stool-associated circular DNA virus (BoSCV)-like virus discovered from porcine stools. However, the thorough characteristics of the virus were not identified. Therefore, this research focuses on finding a full genome sequence and analyzing the genetic features of the virus. The virus, now called porcine stool-associated circular DNA virus in Korea (PoSCV Kor), consists of 2,589 bases forming circular structure. It has two major ORFs inversely encoding replicase and capsid protein, with each stem-loop structure between 5' ends and 3' ends of the two putative ORFs. This characteristics is the same as PoSCV in New Zealand, but different from chimpanzee stool-associated circular virus (ChiSCVs) and BoSCV, which have one stem-loop structure. Therefore, it would be sure that PoSCV Kor is very similar to PoSCV in respect to the genetic aspect; the same number of nucleotide bases and the amino acid identity of replicase and capsid protein (96 and 93 %, respectively). This fact could be certified through the finding that PoSCV Kor and PoSCV are in the same cluster by phylogenetic analysis based on the comparison with full-sequences of other circular ssDNA viruses.

  6. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

    SciTech Connect

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

    2015-04-22

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  7. Synergistic decrease of DNA single-strand break repair rates in mouse neural cells lacking both Tdp1 and aprataxin

    PubMed Central

    El-Khamisy, Sherif F.; Katyal, Sachin; Patel, Poorvi; Ju, Limei; McKinnon, Peter J.; Caldecott, Keith W.

    2009-01-01

    Ataxia oculomotor apraxia-1 (AOA1) is an autosomal recessive neurodegenerative disease that results from mutations of aprataxin (APTX). APTX associates with the DNA single- and double-strand break repair machinery and is able to remove AMP from 5′-termini at DNA strand breaks in vitro. However, attempts to establish a DNA strand break repair defect in APTX-defective cells have proved conflicting and unclear. We reasoned that this may reflect that DNA strand breaks with 5′-AMP represent only a minor subset of breaks induced in cells, and/or the availability of alternative mechanisms for removing AMP from 5′-termini. Here, we have attempted to increase the dependency of chromosomal single- and double-strand break repair on aprataxin activity by slowing the rate of repair of 3′-termini in aprataxin-defective neural cells, thereby increasing the likelihood that the 5′-termini at such breaks become adenylated and/or block alternative repair mechanisms. To do this, we generated a mouse model in which APTX is deleted together with tyrosyl DNA phosphodiesterase (TDP1), an enzyme that repairs 3′-termini at a subset of single-strand breaks (SSBs), including those with 3′-topoisomerase-1 (Top1) peptide. Notably, the global rate of repair of oxidative and alkylation-induced SSBs was significantly slower in Tdp1−/−/Aptx−/− double knockout quiescent mouse astrocytes compared with Tdp1−/− or Aptx−/− single knockouts. In contrast, camptothecin-induced Top1-SSBs accumulated to similar levels in Tdp1−/− and Tdp1−/−/Aptx−/− double knockout astrocytes. Finally, we failed to identify a measurable defect in double-strand break repair in Tdp1−/−, Aptx−/− or Tdp1−/−/Aptx−/− astrocytes. These data provide direct evidence for a requirement for aprataxin during chromosomal single-strand break repair in primary neural cells lacking Tdp1. PMID:19303373

  8. Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

    PubMed

    Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

    2014-02-01

    Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

  9. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

    Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

  10. Rolling-circle replication of UV-irradiated duplex DNA in the phi X174 replicative-form----single-strand replication system in vitro

    SciTech Connect

    Shavitt, O.; Livneh, Z.

    1989-06-01

    Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substrate DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers.

  11. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    PubMed Central

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S.

    2012-01-01

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. 20/34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access substrate DNA cytosines. PMID:22181350

  12. First-in-class small molecule inhibitors of the single-strand DNA cytosine deaminase APOBEC3G.

    PubMed

    Li, Ming; Shandilya, Shivender M D; Carpenter, Michael A; Rathore, Anurag; Brown, William L; Perkins, Angela L; Harki, Daniel A; Solberg, Jonathan; Hook, Derek J; Pandey, Krishan K; Parniak, Michael A; Johnson, Jeffrey R; Krogan, Nevan J; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A; Harris, Reuben S

    2012-03-16

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

  13. LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells.

    PubMed

    van Ravesteyn, Thomas W; Dekker, Marleen; Fish, Alexander; Sixma, Titia K; Wolters, Astrid; Dekker, Rob J; Te Riele, Hein P J

    2016-04-12

    Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.

  14. LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

    PubMed Central

    van Ravesteyn, Thomas W.; Dekker, Marleen; Fish, Alexander; Sixma, Titia K.; Wolters, Astrid; Dekker, Rob J.; te Riele, Hein P. J.

    2016-01-01

    Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype. PMID:26951689

  15. Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein

    PubMed Central

    Hopkins, Max; Kailasan, Shweta; Cohen, Allison; Roux, Simon; Tucker, Kimberly Pause; Shevenell, Amelia; Agbandje-McKenna, Mavis; Breitbart, Mya

    2014-01-01

    The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein–protein or viral–host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles. PMID:24694711

  16. Diversity of environmental single-stranded DNA phages revealed by PCR amplification of the partial major capsid protein.

    PubMed

    Hopkins, Max; Kailasan, Shweta; Cohen, Allison; Roux, Simon; Tucker, Kimberly Pause; Shevenell, Amelia; Agbandje-McKenna, Mavis; Breitbart, Mya

    2014-10-01

    The small single-stranded DNA (ssDNA) bacteriophages of the subfamily Gokushovirinae were traditionally perceived as narrowly targeted, niche-specific viruses infecting obligate parasitic bacteria, such as Chlamydia. The advent of metagenomics revealed gokushoviruses to be widespread in global environmental samples. This study expands knowledge of gokushovirus diversity in the environment by developing a degenerate PCR assay to amplify a portion of the major capsid protein (MCP) gene of gokushoviruses. Over 500 amplicons were sequenced from 10 environmental samples (sediments, sewage, seawater and freshwater), revealing the ubiquity and high diversity of this understudied phage group. Residue-level conservation data generated from multiple alignments was combined with a predicted 3D structure, revealing a tendency for structurally internal residues to be more highly conserved than surface-presenting protein-protein or viral-host interaction domains. Aggregating this data set into a phylogenetic framework, many gokushovirus MCP clades contained samples from multiple environments, although distinct clades dominated the different samples. Antarctic sediment samples contained the most diverse gokushovirus communities, whereas freshwater springs from Florida were the least diverse. Whether the observed diversity is being driven by environmental factors or host-binding interactions remains an open question. The high environmental diversity of this previously overlooked ssDNA viral group necessitates further research elucidating their natural hosts and exploring their ecological roles.

  17. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    SciTech Connect

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S.

    2012-04-04

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

  18. Discovery of two novel viruses expands the diversity of single-stranded DNA and single-stranded RNA viruses infecting a cosmopolitan marine diatom.

    PubMed

    Kimura, Kei; Tomaru, Yuji

    2015-02-01

    Recent studies have suggested that diatom viruses are an important factor affecting diatom population dynamics, which in turn are important in considering marine primary productivity. The marine planktonic diatom Chaetoceros tenuissimus Meunier is a cosmopolitan species and often causes blooms off the western coast of Japan. To date, two viruses, C. tenuissimus DNA virus (CtenDNAV) type I and CtenRNAV type I, have been identified that potentially affect C. tenuissimus population dynamics in the natural environment. In this study, we successfully isolated and characterized two additional novel viruses (CtenDNAV type II and CtenRNAV type II). This paper reports the basic characteristics of these new viruses isolated from surface water or sediment from the Hiroshima Bay, Japan. The physiological and morphological characteristics of the two new viruses were similar to those of the previously isolated viruses. However, the amino acid sequences of the structural proteins of CtenDNAV type II and CtenRNAV type II were clearly distinct from those of both type I viruses, with identity scores of 38.3% and 27.6%, respectively. Our results suggest that at least four genetically distinct viruses sharing the same diatom host are present in western Japan and affect the population dynamics of C. tenuissimus. Moreover, the result that CtenRNAV type II lysed multiple diatom species indicates that RNA viruses may affect various diatom populations in the natural environment.

  19. The binding mode of human nucleoside diphosphate kinase B to single-strand DNA.

    PubMed

    Agou, F; Raveh, S; Véron, M

    2000-06-01

    In this paper, we studied the interaction of the human isoform B of nucleoside diphosphate kinase (NDP kinase B) with the nuclease hypersensitive element (NHE) present in the promoter element of the c-myc oncogene. The DNA-binding properties of NDP kinase B and other NDP kinases are compared and the nucleotide requirement for binding are discussed. Using quantitative methods, we identified the DNA-binding sites on the protein and we proposed a structural model for a complex of one hexameric NDP kinase B with an oligonucleotide.

  20. Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

    SciTech Connect

    Kovacs, E.; Langemann, H. )

    1990-11-01

    Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of (3H)thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h.

  1. The alkaline elution technique for measuring DNA single strand breaks: increased reliability and sensitivity.

    PubMed

    Koch, C J; Giandomenico, A R

    1994-07-01

    The alkaline elution procedure is noted, on the one hand, for its sensitivity in the detection of DNA damage, but on the other hand, for its extreme variability and inconsistency. These deficiencies in the technique have been traced to incomplete exchanges of the various solutions used in the cell rinse and lysis and DNA rinse and elution portions of the procedure. Solutions to the above indicated problems involve several changes to the standard procedure, including, at the cellular stage, the complete removal of rinsing solutions followed by the addition of lysis solution at 0 degrees C. After standard cell lysis and alkaline rinse of the DNA, the alkaline rinse solution is replaced by elution solution at high flow rate to allow a uniform starting time for the elution, which then proceeds as a nearly first-order function of time (not elution volume). Using gamma-rays as the damaging agent, reproducibility within and between experiments is easily comparable to radiation survival itself, and typically, duplicate elution channels within an experiment provide nearly identical information without the need for internal standards and complex ratiometric analysis techniques. The procedures described allow the reproducible assessment of radiation damage to cellular DNA at doses much below 1 Gy and allow the study of repair processes down to similar levels of residual damage. This unexpected increase in technique sensitivity may be caused by maintenance of near-freezing temperatures during cell manipulation and addition of lysis solutions or to more complete and uniform lysis.

  2. 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

  3. Cloning DNA restriction endonuclease fragments with protruding single-stranded ends.

    PubMed

    Wartell, R M; Reznikoff, W S

    1980-05-01

    A new method of in vitro recombination was employed to construct plasmids containing lac promoter fragments 64 bp and 144 bp long. The 64 bp HpaII-HhaI fragment contains the binding site for the catabolite activator protein (CAP). The HpaII-HaeIII 144 bp fragment includes the binding sites for RNA polymerase, the lac repressor and CAP. The method utilizes the ability of T4 DNA polymerase to make flush-ended DNA either by filling in a recessed 3'-end or by exonucleolytic removal of a protruding 3'-end. The treated fragments were then blunt-end ligated to the filled-in EcoRI cloning sites of the plasmids pVH51 and pBR322 using T4 ligase. In this process, the EcoRI sites were regenerated on the fragment ends thus facilitating the subsequent isolation of the fragments from their cloning vectors.

  4. Theoretical Study of the Transpore Velocity Control of Single-Stranded DNA

    PubMed Central

    Qian, Weixin; Doi, Kentaro; Uehara, Satoshi; Morita, Kaito; Kawano, Satoyuki

    2014-01-01

    The electrokinetic transport dynamics of deoxyribonucleic acid (DNA) molecules have recently attracted significant attention in various fields of research. Our group is interested in the detailed examination of the behavior of DNA when confined in micro/nanofluidic channels. In the present study, the translocation mechanism of a DNA-like polymer chain in a nanofluidic channel was investigated using Langevin dynamics simulations. A coarse-grained bead-spring model was developed to simulate the dynamics of a long polymer chain passing through a rectangular cross-section nanopore embedded in a nanochannel, under the influence of a nonuniform electric field. Varying the cross-sectional area of the nanopore was found to allow optimization of the translocation process through modification of the electric field in the flow channel, since a drastic drop in the electric potential at the nanopore was induced by changing the cross-section. Furthermore, the configuration of the polymer chain in the nanopore was observed to determine its translocation velocity. The competition between the strength of the electric field and confinement in the small pore produces various transport mechanisms and the results of this study thus represent a means of optimizing the design of nanofluidic devices for single molecule detection. PMID:25116683

  5. Quiescent human peripheral blood lymphocytes do not contain a sizable amount of preexistent DNA single-strand breaks

    SciTech Connect

    Boerrigter, M.E.; Mullaart, E.; van der Schans, G.P.; Vijg, J.

    1989-02-01

    Sedimentation of nucleoids through neutral sucrose density gradients has shown that nucleoids isolated from phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) sediment faster than nucleoids derived from quiescent lymphocytes, which was attributed to rejoining of DNA single-strand breaks (SSB) present in the resting cells. We isolated PBL from donors and determined the amount of SSB in nonradiolabeled, untreated resting and PHA-stimulated cells by applying the alkaline filter elution technique. Calibration was based on dose-dependent induction of SSB by /sup 60/Co-gamma-radiation. Quiescent cells did not contain a sizable amount of SSB. Mitogen-stimulated cells showed equally low amounts of SSB per cell. The present study indicates that the interpretation of the results obtained with the nucleoid sedimentation technique concerning the supposed rejoining of SSB in PHA-stimulated human lymphocytes is incorrect. Other, equally sensitive, techniques such as alkaline filter elution appear to be preferable for studies on DNA damage and repair.

  6. Viral recombination blurs taxonomic lines: examination of single-stranded DNA viruses in a wastewater treatment plant

    PubMed Central

    Pearson, Victoria M.; Caudle, S. Brian

    2016-01-01

    Understanding the structure and dynamics of microbial communities, especially those of economic concern, is of paramount importance to maintaining healthy and efficient microbial communities at agricultural sites and large industrial cultures, including bioprocessors. Wastewater treatment plants are large bioprocessors which receive water from multiple sources, becoming reservoirs for the collection of many viral families that infect a broad range of hosts. To examine this complex collection of viruses, full-length genomes of circular ssDNA viruses were isolated from a wastewater treatment facility using a combination of sucrose-gradient size selection and rolling-circle amplification and sequenced on an Illumina MiSeq. Single-stranded DNA viruses are among the least understood groups of microbial pathogens due to genomic biases and culturing difficulties, particularly compared to the larger, more often studied dsDNA viruses. However, the group contains several notable well-studied examples, including agricultural pathogens which infect both livestock and crops (Circoviridae and Geminiviridae), and model organisms for genetics and evolution studies (Microviridae). Examination of the collected viral DNA provided evidence for 83 unique genotypic groupings, which were genetically dissimilar to known viral types and exhibited broad diversity within the community. Furthermore, although these genomes express similarities to known viral families, such as Circoviridae, Geminiviridae, and Microviridae, many are so divergent that they may represent new taxonomic groups. This study demonstrated the efficacy of the protocol for separating bacteria and large viruses from the sought after ssDNA viruses and the ability to use this protocol to obtain an in-depth analysis of the diversity within this group. PMID:27781171

  7. PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

    PubMed

    Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

    2013-06-14

    Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

  8. Organophosphorus pesticides detection using broad-specific single-stranded DNA based fluorescence polarization aptamer assay.

    PubMed

    Zhang, Cunzheng; Wang, Li; Tu, Zhui; Sun, Xing; He, Qinghua; Lei, Zhaojing; Xu, Chongxin; Liu, Yuan; Zhang, Xiao; Yang, Jingyi; Liu, Xianjin; Xu, Yang

    2014-05-15

    An approach is developed to detect the organophosphorus pesticides via competitive binding to a recombinant broad-specificity DNA aptamer with a molecular beacon (MB), the binding of the MB to the aptamer results in the activation of a fluorescent signal, which can be measured for pesticide quantification. Aptamers selected via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) were structurally modified and truncated to narrow down the binding region of the target, which indicated that loops of the aptamer contributed different functions for different chemical recognition. Thereafter, a variant fused by two different minimum functional structures, was clarified with broad specificity and increased affinity. Further molecular docking and molecular dynamics simulations was conducted to understand the molecular interaction between DNA structure and chemicals. 3D modeling revealed a hot spot area formed by 3 binding sites, forces including hydrogen bonds and van der Waals interactions appear to play a significant role in enabling and stabilizing the binding of chemicals. Finally, an engineered aptamer based approach for the detection of organophosphorus pesticides was successfully applied in a test using a real sample, the limit of quantification (LOQ) for phorate, profenofos, isocarbophos, and omethoate reached 19.2, 13.4, 17.2, and 23.4 nM (0.005 mg L(-1)), respectively.

  9. Dragonfly cyclovirus, a novel single-stranded DNA virus discovered in dragonflies (Odonata: Anisoptera).

    PubMed

    Rosario, Karyna; Marinov, Milen; Stainton, Daisy; Kraberger, Simona; Wiltshire, Elizabeth J; Collings, David A; Walters, Matthew; Martin, Darren P; Breitbart, Mya; Varsani, Arvind

    2011-06-01

    Dragonfly cyclovirus (DfCyV), a new species of ssDNA virus discovered using viral metagenomics in dragonflies (family Libellulidae) from the Kingdom of Tonga. Metagenomic sequences of DfCyV were similar to viruses of the recently proposed genus Cyclovirus within the family Circoviridae. Specific PCRs resulted in the recovery of 21 DfCyV genomes from three dragonfly species (Pantala flavescens, Tholymis tillarga and Diplacodes bipunctata). The 1741 nt DfCyV genomes share >95 % nucleotide identity and are classified into 11 subtypes representing a single strain. The DfCyV genomes share 48-63 % genome-wide nucleotide identity with cycloviruses identified in human faecal samples. Recombination analysis revealed three recombinant DfCyV genomes, suggesting that recombination plays an important role in cyclovirus evolution. To our knowledge, this is the first report of a circular ssDNA virus identified in insects, and the data may help elucidate evolutionary links among novel Circoviridae recently identified in animals and environmental samples.

  10. The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA

    PubMed Central

    Hayner, Jaclyn N.; Douma, Lauren G.; Bloom, Linda B.

    2014-01-01

    Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. PMID:25159615

  11. Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins

    PubMed Central

    Xu, Zhixiong; Meng, Xianzhang; Cai, Ying; Liang, Hong; Nagarajan, Lalitha; Brandt, Stephen J.

    2007-01-01

    The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and β-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins. PMID:17437998

  12. Interaction between adenovirus DNA-binding protein and single-stranded polynucleotides studied by circular dichroism and ultraviolet absorption.

    PubMed

    van Amerongen, H; van Grondelle, R; van der Vliet, P C

    1987-07-28

    The adenovirus DNA-binding protein (AdDBP) is a multifunctional protein required for viral DNA replication and control of transcription. We have studied the binding of AdDBP to single-stranded M13 DNA and to the homopolynucleotides poly(rA), poly(dA), and poly(dT) by means of circular dichroism (CD) and optical density (OD) measurements. The binding to all these polynucleotides was strong and nearly stoichiometric. Titration experiments showed that the size of the binding site is 9-11 nucleotides long for M13 DNA, poly(dA), and poly(rA). A higher value (15.0 +/- 0.8) was found for poly(dT). Pronounced changes in the circular dichroism and optical density spectra were observed upon binding of AdDBP. In general, both the positive peak around 260-270 nm and the negative peak around 240-250 nm in the CD spectra decreased in intensity, and a shift of the crossover point to longer wavelengths was found. The OD spectra observed upon binding of AdDBP are remarkably similar to those obtained with prokaryotic helix-destabilizing proteins like bacteriophage T4 gene 32 protein and fd gene 5 protein. The data can best be interpreted by assuming that the AdDBP-polynucleotide complex has a regular, rigid, and extended configuration that satifies two criteria: (1) a considerable tilt of the bases in combination with (2) a small rotation per base and/or a shift of the bases closer to the helix axis.

  13. Ionic strength-dependent persistence lengths of single-stranded RNA and DNA.

    PubMed

    Chen, Huimin; Meisburger, Steve P; Pabit, Suzette A; Sutton, Julie L; Webb, Watt W; Pollack, Lois

    2012-01-17

    Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths (l(p)) of ssRNA. We observe valence and ionic strength-dependent differences in l(p) in a direct comparison between 40-mers of deoxythymidylate (dT(40)) and uridylate (rU(40)) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo.

  14. Ionic strength-dependent persistence lengths of single-stranded RNA and DNA

    PubMed Central

    Chen, Huimin; Meisburger, Steve P.; Pabit, Suzette A.; Sutton, Julie L.; Webb, Watt W.; Pollack, Lois

    2012-01-01

    Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths (lp) of ssRNA. We observe valence and ionic strength-dependent differences in lp in a direct comparison between 40-mers of deoxythymidylate (dT40) and uridylate (rU40) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo. PMID:22203973

  15. Conserved region 3 of the adenovirus type 5 DNA-binding protein is important for interaction with single-stranded DNA.

    PubMed Central

    Neale, G A; Kitchingman, G R

    1990-01-01

    The adenovirus-encoded single-stranded DNA-binding protein (DBP) functions in viral DNA replication and several aspects of RNA metabolism. Previous studies (G. A. M. Neale and G. R. Kitchingman, J. Biol. Chem. 264:3153-3159, 1989) have defined three highly conserved regions in the carboxy-terminal domain of the protein (amino acids 178 to 186, 322 to 330, and 464 to 475) that may be involved in the binding of the protein to single-stranded DNA. We examined the role of conserved region 3 (464 to 475) by constructing nine classes of point mutants with from one to four amino acid changes. The point mutants were tested for their ability to assist adeno-associated virus DNA replication. All nine differed from wild-type DBP; seven were essentially nonfunctional, whereas two had 55 and 145%, respectively, of the wild-type DBP helper activity. Three of the mutants were found to be temperature sensitive, with significantly greater helper activity at 33 degrees C than at 37 degrees C. All nine mutants produced essentially wild-type levels of protein. One monoclonal antibody against the DBP, termed 2/4, did not immunoprecipitate the mutant DBPs as well as wild-type DBP, indicating either that the antibody recognized sequences around CR3 or that the conformation of the protein around the epitope recognized by 2/4 had changed. Two of the three temperature-sensitive DBP mutants bound to single-stranded DNA-cellulose with the same affinity as wild-type DBP at 4 degrees C; the remaining mutants all showed reduced affinity. These results demonstrated that many of the residues within conserved region 3 of the DBP are important for interaction of the protein with nucleic acid. Images PMID:2296078

  16. Single-stranded DNA oligomers stimulate error-prone alternative repair of DNA double-strand breaks through hijacking Ku protein

    PubMed Central

    Yuan, Ying; Britton, Sébastien; Delteil, Christine; Coates, Julia; Jackson, Stephen P.; Barboule, Nadia; Frit, Philippe; Calsou, Patrick

    2015-01-01

    In humans, DNA double-strand breaks (DSBs) are repaired by two mutually-exclusive mechanisms, homologous recombination or end-joining. Among end-joining mechanisms, the main process is classical non-homologous end-joining (C-NHEJ) which relies on Ku binding to DNA ends and DNA Ligase IV (Lig4)-mediated ligation. Mostly under Ku- or Lig4-defective conditions, an alternative end-joining process (A-EJ) can operate and exhibits a trend toward microhomology usage at the break junction. Homologous recombination relies on an initial MRN-dependent nucleolytic degradation of one strand at DNA ends. This process, named DNA resection generates 3′ single-stranded tails necessary for homologous pairing with the sister chromatid. While it is believed from the current literature that the balance between joining and recombination processes at DSBs ends is mainly dependent on the initiation of resection, it has also been shown that MRN activity can generate short single-stranded DNA oligonucleotides (ssO) that may also be implicated in repair regulation. Here, we evaluate the effect of ssO on end-joining at DSB sites both in vitro and in cells. We report that under both conditions, ssO inhibit C-NHEJ through binding to Ku and favor repair by the Lig4-independent microhomology-mediated A-EJ process. PMID:26350212

  17. Top3-Rmi1 DNA single-strand decatenase is integral to the formation and resolution of meiotic recombination intermediates.

    PubMed

    Kaur, Hardeep; De Muyt, Arnaud; Lichten, Michael

    2015-02-19

    The topoisomerase III (Top3)-Rmi1 heterodimer, which catalyzes DNA single-strand passage, forms a conserved complex with the Bloom's helicase (BLM, Sgs1 in budding yeast). This complex has been proposed to regulate recombination by disassembling double Holliday junctions in a process called dissolution. Top3-Rmi1 has been suggested to act at the end of this process, resolving hemicatenanes produced by earlier BLM/Sgs1 activity. We show here that, to the contrary, Top3-Rmi1 acts in all meiotic recombination functions previously associated with Sgs1, most notably as an early recombination intermediate chaperone, promoting regulated crossover and noncrossover recombination and preventing aberrant recombination intermediate accumulation. In addition, we show that Top3-Rmi1 has important Sgs1-independent functions that ensure complete recombination intermediate resolution and chromosome segregation. These findings indicate that Top3-Rmi1 activity is important throughout recombination to resolve strand crossings that would otherwise impede progression through both early steps of pathway choice and late steps of intermediate resolution.

  18. Transient suppression of MLH1 allows effective single-nucleotide substitution by single-stranded DNA oligonucleotides.

    PubMed

    Dekker, Marleen; de Vries, Sandra; Aarts, Marieke; Dekker, Robert; Brouwers, Conny; Wiebenga, Oliver; de Wind, Niels; Cantelli, Erika; Tonelli, Roberto; Te Riele, Hein

    2011-10-01

    Short synthetic single-stranded oligodeoxyribonucleotides (ssODNs) can be used to introduce subtle modifications into the genome of mouse embryonic stem cells (ESCs). We have previously shown that effective application of ssODN-mediated gene targeting in ESC requires (transient) suppression of DNA mismatch repair (MMR). However, whereas transient down-regulation of the mismatch recognition protein MSH2 allowed substitution of 3 or 4 nucleotides, 1 or 2 nucleotide substitutions were still suppressed. We now demonstrate that single- or dinucleotide substitution can effectively be achieved by transient down-regulation of the downstream MMR protein MLH1. By exploiting highly specific real-time PCR, we demonstrate the feasibility of substituting a single basepair in a non-selectable gene. However, disabling the MMR machinery may lead to inadvertent mutations. To obtain insight into the mutation rate associated with transient MMR suppression, we have compared the impact of transient and constitutive MMR deficiency on the repair of frameshift intermediates at mono- and dinucleotide repeats. Repair at these repeats relied on the substrate specificity and functional redundancy of the MSH2/MSH6 and MSH2/MSH3 MMR complexes. MLH1 knockdown increased the level of spontaneous mutagenesis, but modified ESCs remained germ line competent. Thus, transient MLH1 suppression provides a valuable extension of the MSH2 knockdown strategy, allowing rapid generation of mice carrying single basepair alterations in their genome.

  19. Development of a Single Stranded DNA Aptamer as a Molecular Probe for LNCap Cells Using Cell-SELEX

    PubMed Central

    Almasi, Faezeh; Mousavi Gargari, Seyed Latif; Bitaraf, Fatemeh; Rasoulinejad, Samaneh

    2016-01-01

    Background: Nowadays, highly specific aptamers generated by cell SELEX technology (systematic evolution of ligands by exponential enrichment) are being applied for early detection of cancer cells. Prostate Specific Membrane Antigen (PSMA), over expressed in prostate cancer, is a highly specific marker and therefore can be used for diagnosis of the prostate cancer cells. The aim of the present study was to select single-stranded DNA aptamers against LNCap cells highly expressing PSMA, using cell–SELEX method which can be used as a diagnostic tool for the detection of prostate cancer cells. Methods: After 10 rounds of cell-SELEX, DNA aptamers were isolated against PSMA using LNCaP cells as a target and PC-3 cell lines for counter SELEX. Five DNA aptamers with more than 70% affinity were selected up on flow cytometry analysis of positive clones. Results: Dissociation constants of two selected sequences (A12-B1) were estimated in the range of 33.78±3.77 and 57.49±2.214 pmol, respectively. Conserved secondary structures of A12 and B1 sequences suggest the necessity of these structures for binding with high affinity to native PSMA. Comparison of the secondary structures of our isolated aptamers and aptamer A10 obtained by protein SELEX showed similar stem-loop structures which could be responsible for the recognition of PSMA on LNCap cell surface. Conclusion: Our results indicated that selected aptamers may turn out to be ideal candidates for the development of a detection tool and also can be used in targeted drug delivery for future smart drugs. PMID:27563422

  20. Short-term splenic impact of single-strand DNA functionalized multi-walled carbon nanotubes intraperitoneally injected in rats.

    PubMed

    Clichici, Simona; Biris, Alexandru Radu; Catoi, Cornel; Filip, Adriana; Tabaran, Flaviu

    2014-04-01

    In recent years, a great deal of studies have focused on the possible toxicity of carbon nanotubes (CNT), as a result of their potential applications in the field of nanotechnologies. The investigation of spleen toxicity is part of the carbon nanotubes-induced toxicity assessment. In this study, we investigated the possible toxic effects of CNT on the rat spleen, after intraperitoneally (i.p.) administration of a single dose [1.5 ml; 2 mg multi-walled (MW) CNT per body weight (bw)] of multi-walled carbon nanotubes (exterior diameter 15-25 nm, interior diameter 10-15 nm, surface 88 m(2) g(-1) ) functionalized 1:1 with single-strand DNA (ss-DNA-MWCNT, 270 mg l(-1) ). CNT functionalization with DNA determines a stable dispersion in the body fluids. For the detection of carbon nanotubes in the spleen, Raman spectroscopy, histopathologic examination, confocal microscopy and transmission electron microscopy (TEM) were performed at different time points (1, 6, 24, 48 and 144 h) after MWCNT administration. The dynamics of oxidative stress parameters (malondialdehyde, protein carbonyls and reduced glutathione), along with nitrosative stress parameters (nitric oxide, inducible NO synthase), the pro-inflammatory cytokines [interleukin-(IL)-1β] and the number of cells expressing caspase 3 and proliferating cell nuclear antigen (PCNA) were assessed. Our results indicate that, after i.p. administration, MWCNT translocate progressively in the spleen, with a peak of concentration after 48 h, and determine lymphoid hyperplasia and an increase in the number of cells which undergo apoptosis, in parallel with the enhancement of the mitosis in the white pulp and with transient alterations of oxidative stress and inflammation that need further investigations for a longer period of monitoring.

  1. Novel function of the poly(c)-binding protein α-CP2 as a transcriptional activator that binds to single-stranded DNA sequences.

    PubMed

    Kang, Duk-Hee; Song, Kyu Young; Wei, Li-Na; Law, Ping-Yee; Loh, Horace H; Choi, Hack Sun

    2013-11-01

    α-complex protein 2 (α-CP2) is known as an RNA-binding protein that interacts in a sequence-specific manner with single-stranded polycytosine [poly(C)]. This protein is involved in various post-transcriptional regulations, such as mRNA stabilization and translational regulation. In this study, the full-length mouse α-CP2 gene was expressed in an insoluble form with an N-terminal histidine tag in Escherichia coli and purified for homogeneity using affinity column chromatography. Its identity was confirmed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Recombinant α-CP2 was expressed and refolded. The protein folding conditions for denatured α-CP2 were optimized. DNA and RNA electrophoretic mobility shift assays demonstrated that the recombinant α-CP2 is capable of binding to both single-stranded DNA and RNA poly(C) sequences. Furthermore, plasmids expressing α-CP2 activated the expression of a luciferase reporter when co-transfected with a single-stranded (pGL-SS) construct containing a poly(C) sequence. To our knowledge, this study demonstrates for the first time that α-CP2 functions as a transcriptional activator by binding to a single-stranded poly(C) sequence.

  2. Switching from single-stranded to double-stranded DNA limits the unwinding processivity of ring-shaped T7 DNA helicase.

    PubMed

    Jeong, Yong-Joo; Rajagopal, Vaishnavi; Patel, Smita S

    2013-04-01

    Phage T7 helicase unwinds double-stranded DNA (dsDNA) by encircling one strand while excluding the complementary strand from its central channel. When T7 helicase translocates on single-stranded DNA (ssDNA), it has kilobase processivity; yet, it is unable to processively unwind linear dsDNA, even 60 base-pairs long. Particularly, the GC-rich dsDNAs are unwound with lower amplitudes under single-turnover conditions. Here, we provide evidence that T7 helicase switches from ssDNA to dsDNA during DNA unwinding. The switching propensity is higher when dsDNA is GC-rich or when the 3'-overhang of forked DNA is <15 bases. Once helicase encircles dsDNA, it travels along dsDNA and dissociates from the end of linear DNA without strand separation, which explains the low unwinding amplitude of these substrates. Trapping the displaced strand with ssDNA binding protein or changing its composition to morpholino oligomer that does not interact with helicase increases the unwinding amplitude. We conclude that the displaced strand must be continuously excluded and kept away from the central channel for processive DNA unwinding. The finding that T7 helicase can switch from ssDNA to dsDNA binding mode during unwinding provides new insights into ways of limiting DNA unwinding and triggering fork regression when stalled forks need to be restarted.

  3. Replication of simian virus 40 DNA after UV irradiation: evidence of growing fork blockage and single-stranded gaps in daughter strands

    SciTech Connect

    Mezzina, M.; Menck, C.F.M.; Courtin, P.; Sarasin, A.

    1988-11-01

    The molecular mechanisms of in vivo inhibition of mammalian DNA replication by exposure to UV light (at 254 nm) was studied in monkey and human cells infected with simian virus 40. Analysis of viral DNA by electron microscopy and sucrose gradients confirmed that the presence of UV-induced lesions severely blocks DNA synthesis, and thus the conversion of replicative intermediates (RIs) into fully replicated form I DNA is inhibited by UV irradiation. These blocked RI molecules present several special features when visualized by electron microscopy. In excision repair-proficient monkey and human cells they are composed of a double-stranded circular DNA with a double-stranded tail whose size corresponds to the average interpyrimidine dimer distance, as determined by the dimer-specific T4 endonuclease V. In excision repair-deficient human cells from patients with xeroderma pigmentosum, UV-irradiated RIs present a Carins-like structure similar to that observed for replicating molecules obtained from unirradiated infected cells. Single-stranded gaps are visualized in the replicated portions of UV-irradiated RI molecules; such regions are detected and clearly distinguishable from double-stranded DNA when probed by a specific single-stranded DNA-binding protein such as the bacteriophage T4 gene 32 product. Consistent with the presence of gaps in UV-irradiated RI molecules, single-strand-specific S1 nuclease digestion causes a shift in their sedimentation properties when analyzed in neutral sucrose gradients compared with undamaged molecules.

  4. Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA*

    PubMed Central

    Ciesielski, Grzegorz L.; Bermek, Oya; Rosado-Ruiz, Fernando A.; Hovde, Stacy L.; Neitzke, Orrin J.; Griffith, Jack D.; Kaguni, Laurie S.

    2015-01-01

    The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems. PMID:26446790

  5. Water film thickness-dependent conformation and diffusion of single-strand DNA on poly(ethylene glycol)-silane surface

    PubMed Central

    Park, Jae Hyun; Aluru, N. R.

    2010-01-01

    In this paper, we investigate, using molecular dynamics simulations, the conformation and diffusion of longer and shorter single-strand DNA (ssDNA) as a function of water film thickness. While the conformation of the shorter ssDNA is significantly affected and the diffusion is suppressed with reduction in water film thickness, the conformation and diffusion of the longer DNA is not influenced. We explain our observations by considering the competition between stacking interaction of bases and solvation tendency of ssDNA. This paper suggests an approach to control the surface motion of ssDNA in nanoscale water films using film thickness. PMID:20404938

  6. Transient oxidative stress and inflammation after intraperitoneal administration of multiwalled carbon nanotubes functionalized with single strand DNA in rats

    SciTech Connect

    Clichici, Simona; Biris, Alexandru Radu; Tabaran, Flaviu; Filip, Adriana

    2012-03-15

    Multi-walled carbon nanotubes (MWCNTs) are widely used for nanotechnology. Their impact on living organisms is, however, not entirely clarified. Oxidative stress and inflammation seem to be the key mechanisms involved in MWCNTs' cytotoxicity. Until present, pulmonary and skin models were the main tested experimental designs to assess carbon nanotubes' toxicity. The systemic administration of MWCNTs is essential, with respect for future medical applications. Our research is performed on Wistar rats and is focused on the dynamics of oxidative stress parameters in blood and liver and pro-inflammatory cytokines in liver, after single dose (270 mg l{sup −1}) ip administration of MWCNTs (exterior diameter 15–25 nm, interior diameter 10–15 nm, surface 88 m{sup 2} g{sup −1}) functionalized with single strand DNA (ss-DNA). The presence of MWCNTs in blood was assessed by Raman spectroscopy, while in liver histological examination and confocal microscopy were used. It was found that ss-DNA-MWCNTs induce oxidative stress in plasma and liver, with the return of the tested parameters to normal values, 6 h after ip injection of nanotubes, with the exception of reduced glutathione in plasma. The inflammatory cytokines (TNF-α, IL-1β) had a similar pattern of evolution. We also assessed the level of ERK1/2 and the phosphorylation of p65 subunit of NF-kB in liver that had a transient increase and returned to normal at the end of the tested period. Our results demonstrate that ss-DNA-MWCNTs produce oxidative stress and inflammation, but with a transient pattern. Given the fact that antioxidants modify the profile not only for oxidative stress, but also of inflammation, the dynamics of these alterations may be of practical importance for future protective strategies. -- Highlights: ► ss-DNA-MWCNTs ip administration induce oxidative stress in plasma and liver. ► ss-DNA-MWCNTs ip administration determine liver inflammation. ► ERK1/2 and p65 phosphorylated NF-KB increase

  7. Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

    PubMed Central

    Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

    2017-01-01

    CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and

  8. Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

    PubMed

    Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

    2017-01-01

    CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and

  9. Surface area as a dose metric for carbon black nanoparticles: A study of oxidative stress, DNA single-strand breakage and inflammation in rats

    NASA Astrophysics Data System (ADS)

    Chuang, Hsiao-Chi; Chen, Li-Chen; Lei, Yu-Chen; Wu, Kuen-Yuh; Feng, Po-Hao; Cheng, Tsun-Jen

    2015-04-01

    Combustion-derived nanoparticles are characterised by a high surface area (SA) per mass. SA is proposed to regulate the bioreactivity of nanoparticles; however, the dose metric for carbon black remains controversial. To determine the relationships between bioreactivity and SA, male spontaneously hypertensive rats were exposed to carbon black (CB) nanoparticles (15, 51 and 95 nm) via intratracheal instillation for 24 h. Pulmonary exposure to CB resulted in a significant increase in systemic 8-hydroxy-2‧-deoxyguanosine (8-OHdG), DNA single-strand breakages in peripheral blood cells and pulmonary cell infiltration in rats. The oxidative potential and particularly the corresponding SA of CB were correlated with the level of 8-OHdG, DNA single-strand breakages and pulmonary cell infiltration in rats. We conclude that SA is an important dose metric for CB that can regulate oxidative stress and DNA damage in rats. Furthermore, this observation was more significant for smaller sized CB.

  10. Helix geometry of single stranded DNA 'A' and 'B' forms from minimum energy conformations of dimeric subunits.

    PubMed Central

    Hingerty, B; Broyde, S

    1978-01-01

    Low energy conformations with dihedral angles similar to those occurring in fibers of the 'A' and 'B' forms of DNAs have been calculated for the deoxydinucleoside phosphates dApdA, dCpdC, dTpdT, dGpdG and dGpdC (1-3). These conformers have been used as building blocks for generating larger single stranded polymers, whose helical parameters we have calculated. We find that single stranded 'A' and 'B' form helices tend to be narrower and more tightly wound than the duplexes obtained in fibers (4,5). This is consistent with experimental observations on single stranded fibers of poly (rC) (6). We also find that the different sequences have different helix geometries. In addition, it is observed that large variations in helix geometry for a given sequence are achievable at little energetic cost. PMID:643603

  11. Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G.C-->T.A transversions in simian kidney cells.

    PubMed Central

    Moriya, M

    1993-01-01

    A single-stranded shuttle vector has been developed for the purpose of investigating translesional events in mammalian cells. The vector is designed to permit site-specific introduction of defined DNA lesions between a gene for neomycin resistance and its promoter. Efficiencies of translesional synthesis in simian kidney cells (COS) and Escherichia coli are established by determining the number of neomycin- and ampicillin-resistant colonies recovered, respectively, after introduction of a modified vector. Fidelity of translesional synthesis is evaluated by analyzing the nucleotide sequence of progeny phagemid DNA in the region corresponding to the lesion site. This experimental system, capable of detecting mutagenic and nonmutagenic events at and adjacent to the lesion site, was used to establish the mutagenic potential of a single 8-oxoguanine residue in DNA. This modified base, produced by attack of reactive oxygen species on cellular DNA, did not cause a decrease in the number of transformants when single-stranded DNA containing the lesion replicated in COS cells or E. coli. The predominant mutations observed (> 78%) were G-->T transversions targeted to the site of the lesion. The mutation frequencies for this event were 2.5-4.8% in COS cells and 1.8% in E. coli. It is concluded that a single-stranded shuttle vector, utilized in conjunction with a site-specific approach, can be used to investigate translesional events in mammalian cells and in bacteria. Images PMID:8430083

  12. Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G.C-->T.A transversions in simian kidney cells.

    PubMed

    Moriya, M

    1993-02-01

    A single-stranded shuttle vector has been developed for the purpose of investigating translesional events in mammalian cells. The vector is designed to permit site-specific introduction of defined DNA lesions between a gene for neomycin resistance and its promoter. Efficiencies of translesional synthesis in simian kidney cells (COS) and Escherichia coli are established by determining the number of neomycin- and ampicillin-resistant colonies recovered, respectively, after introduction of a modified vector. Fidelity of translesional synthesis is evaluated by analyzing the nucleotide sequence of progeny phagemid DNA in the region corresponding to the lesion site. This experimental system, capable of detecting mutagenic and nonmutagenic events at and adjacent to the lesion site, was used to establish the mutagenic potential of a single 8-oxoguanine residue in DNA. This modified base, produced by attack of reactive oxygen species on cellular DNA, did not cause a decrease in the number of transformants when single-stranded DNA containing the lesion replicated in COS cells or E. coli. The predominant mutations observed (> 78%) were G-->T transversions targeted to the site of the lesion. The mutation frequencies for this event were 2.5-4.8% in COS cells and 1.8% in E. coli. It is concluded that a single-stranded shuttle vector, utilized in conjunction with a site-specific approach, can be used to investigate translesional events in mammalian cells and in bacteria.

  13. The DNA-Binding Domain of Human PARP-1 Interacts with DNA Single-Strand Breaks as a Monomer through Its Second Zinc Finger

    PubMed Central

    Eustermann, Sebastian; Videler, Hortense; Yang, Ji-Chun; Cole, Paul T.; Gruszka, Dominika; Veprintsev, Dmitry; Neuhaus, David

    2011-01-01

    Poly(ADP-ribose)polymerase-1 (PARP-1) is a highly abundant chromatin-associated enzyme present in all higher eukaryotic cell nuclei, where it plays key roles in the maintenance of genomic integrity, chromatin remodeling and transcriptional control. It binds to DNA single- and double-strand breaks through an N-terminal region containing two zinc fingers, F1 and F2, following which its C-terminal catalytic domain becomes activated via an unknown mechanism, causing formation and addition of polyadenosine-ribose (PAR) to acceptor proteins including PARP-1 itself. Here, we report a biophysical and structural characterization of the F1 and F2 fingers of human PARP-1, both as independent fragments and in the context of the 24-kDa DNA-binding domain (F1 + F2). We show that the fingers are structurally independent in the absence of DNA and share a highly similar structural fold and dynamics. The F1 + F2 fragment recognizes DNA single-strand breaks as a monomer and in a single orientation. Using a combination of NMR spectroscopy and other biophysical techniques, we show that recognition is primarily achieved by F2, which binds the DNA in an essentially identical manner whether present in isolation or in the two-finger fragment. F2 interacts much more strongly with nicked or gapped DNA ligands than does F1, and we present a mutational study that suggests origins of this difference. Our data suggest that different DNA lesions are recognized by the DNA-binding domain of PARP-1 in a highly similar conformation, helping to rationalize how the full-length protein participates in multiple steps of DNA single-strand breakage and base excision repair. PMID:21262234

  14. Secreted single-stranded DNA is involved in the initial phase of biofilm formation by Neisseria gonorrhoeae.

    PubMed

    Zweig, Maria; Schork, Sabine; Koerdt, Andrea; Siewering, Katja; Sternberg, Claus; Thormann, Kai; Albers, Sonja-Verena; Molin, Søren; van der Does, Chris

    2014-04-01

    Neisseria gonorrhoeae is an obligate human pathogen that colonizes the genital tract and causes gonorrhoea. Neisseria gonorrhoeae can form biofilms during natural cervical infections, on glass and in continuous flow-chamber systems. These biofilms contain large amounts of extracellular DNA, which plays an important role in biofilm formation. Many clinical isolates contain a gonococcal genetic island that encodes a type IV secretion system (T4SS). The T4SS of N. gonorrhoeae strain MS11 secretes ssDNA directly into the medium. Biofilm formation, studied in continuous flow-chamber systems by confocal laser scanning microscopy (CLSM), was strongly reduced, especially in the initial phases of biofilm formation, in the presence of Exonuclease I, which specifically degrades ssDNA or in a ΔtraB strain that does not secrete ssDNA. To specifically detect ssDNA in biofilms using CLSM, a novel method was developed in which thermostable fluorescently labelled ssDNA- and ss/dsDNA-binding proteins were used to visualize ssDNA and total DNA in biofilms and planktonic cultures. Remarkably, mainly dsDNA was detected in biofilms of the ssDNA secreting strain. We conclude that the secreted ssDNA facilitates initial biofilm formation, but that the secreted ssDNA is not retained in mature biofilms.

  15. A Model to Investigate Single-Strand DNA Responses in G1 Human Cells via a Telomere-Targeted, Nuclease-Deficient CRISPR-Cas9 System

    PubMed Central

    Crefcoeur, Remco P.; Zgheib, Omar; Halazonetis, Thanos D.

    2017-01-01

    DNA replication stress has the potential to compromise genomic stability and, therefore, cells have developed elaborate mechanisms to detect and resolve problems that may arise during DNA replication. The presence of single-stranded DNA (ssDNA) is often associated with DNA replication stress and serves as a signal for both checkpoint and repair responses. In this study, we exploited a CRISPR-Cas9 system to induce regions of ssDNA in the genome. Specifically, single-guide RNAs bearing sequence complementarity to human telomeric repeats, were used to target nuclease-deficient Cas9 (dCas9) to telomeres. Such targeting was associated with the formation of DNA-RNA hybrids, leaving one telomeric DNA strand single-stranded. This ssDNA then recruited DNA repair and checkpoint proteins, such as RPA, ATRIP, BLM and Rad51, at the telomeres. Interestingly, targeting of all these proteins to telomeric ssDNA was observed even in cells that were in the G1 phase of the cell cycle. Therefore, this system has the potential to serve as a platform for further investigation of DNA replication stress responses at specific loci in the human genome and in all phases of the cell cycle. PMID:28046023

  16. Characterization of the phage phi 29 protein p5 as a single-stranded DNA binding protein. Function in phi 29 DNA-protein p3 replication.

    PubMed Central

    Martín, G; Lázaro, J M; Méndez, E; Salas, M

    1989-01-01

    The phage phi 29 protein p5, required in vivo in the elongation step of phi 29 DNA replication, was highly purified from Escherichia coli cells harbouring a gene 5-containing plasmid and from phi 29-infected Bacillus subtilis. The protein was characterized as the gene 5 product by amino acid analysis and NH2-terminal sequence determination. The purified protein p5 was shown to bind to single-stranded DNA and to protect it against nuclease degradation. No effect of protein p5 was observed either on the formation of the p3-dAMP initiation complex or on the rate of elongation. However, protein p5 greatly stimulated phi 29 DNA-protein p3 replication at incubation times where the replication in the absence of p5 leveled off. Images PMID:2499869

  17. Isolation of cDNA encoding a binding protein specific to 5'-phosphorylated single-stranded DNA with G-rich sequences.

    PubMed Central

    Mizuta, T R; Fukita, Y; Miyoshi, T; Shimizu, A; Honjo, T

    1993-01-01

    We have isolated the cDNA encoding a binding protein to the sequence motif of the immunoglobulin S mu region by the southwestern method. The binding protein designated S mu bp-2 specifically binds to 5'-phosphorylated single-stranded DNA containing 5'-G and GGGG stretches. The amino acid sequence deduced from the cDNA sequence showed that the S mu bp-2 belongs to the putative helicase superfamily which is involved in replication, recombination and repair. Expression of S mu bp-2 mRNA is ubiquitous and augmented in spleen cells stimulated with lipopolysaccharide and interleukin 4 which also induce class switching. The S mu bp-2 gene is conserved among vertebrates. Possible involvement of S mu bp-2 in class switching is discussed. Images PMID:8493094

  18. The HRDC domain of E. coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling.

    PubMed

    Harami, Gábor M; Nagy, Nikolett T; Martina, Máté; Neuman, Keir C; Kovács, Mihály

    2015-06-11

    DNA-restructuring activities of RecQ-family helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom's syndrome (BLM) helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme.

  19. Characterization and application to the detection of single-stranded DNA binding protein of fluorescent DNA-templated copper/silver nanoclusters.

    PubMed

    Lan, Guo-Yu; Chen, Wei-Yu; Chang, Huan-Tsung

    2011-09-21

    A simple strategy for the preparation of strongly fluorescent and stable DNA-Cu/Ag NCs from reduction of AgNO(3) and Cu(NO(3))(2) by NaBH(4) in the presence of DNA having a sequence 5'-CCCTTAATCCCC-3' has been demonstrated. Fluorescence, absorption, X-ray photoelectron spectroscopy (XPS), and electrospray ionization-mass spectrometry (ESI-MS) measurements have been applied to the characterization of the DNA-Cu/Ag NCs. The ESI-MS data reveal that each DNA-Cu/Ag NC contained 2 Ag and 1 Cu atoms. The interactions among DNA with the Ag and Cu atoms are further supported by the data of low-temperature fluorescence. In the presence of Cu(2+) ions, the reaction time is 1.5 h, which is much shorter than that (120 h) for the preparation of Ag-DNA NCs that are prepared in a mixture of AgNO(3), NaBH(4) and DNA without containing Cu(2+) ions. Relative to the DNA-Ag NCs, the DNA-Cu/Ag NCs have greater fluorescence (quantum yield 51.2% vs. 11.5%). The DNA-Cu/Ag NCs are highly sensitive and selective for the detection of single-stranded DNA binding protein (SSB), with a linear range 1-50 nM and a limit of detection 0.2 nM at a signal-to-ratio of 3.

  20. Molecular cloning of MSSP-2, a c-myc gene single-strand binding protein: characterization of binding specificity and DNA replication activity.

    PubMed Central

    Takai, T; Nishita, Y; Iguchi-Ariga, S M; Ariga, H

    1994-01-01

    We have previously reported the human cDNA encoding MSSP-1, a sequence-specific double- and single-stranded DNA binding protein [Negishi, Nishita, Saëgusa, Kakizaki, Galli, Kihara, Tamai, Miyajima, Iguchi-Ariga and Ariga (1994) Oncogene, 9, 1133-1143]. MSSP-1 binds to a DNA replication origin/transcriptional enhancer of the human c-myc gene and has turned out to be identical with Scr2, a human protein which complements the defect of cdc2 kinase in S.pombe [Kataoka and Nojima (1994) Nucleic Acid Res., 22, 2687-2693]. We have cloned the cDNA for MSSP-2, another member of the MSSP family of proteins. The MSSP-2 cDNA shares highly homologous sequences with MSSP-1 cDNA, except for the insertion of 48 bp coding 16 amino acids near the C-terminus. Like MSSP-1, MSSP-2 has RNP-1 consensus sequences. The results of the experiments using bacterially expressed MSSP-2, and its deletion mutants, as histidine fusion proteins suggested that the binding specificity of MSSP-2 to double- and single-stranded DNA is the same as that of MSSP-1, and that the RNP consensus sequences are required for the DNA binding of the protein. MSSP-2 stimulated the DNA replication of an SV40-derived plasmid containing the binding sequence for MSSP-1 or -2. MSSP-2 is hence suggested to play an important role in regulation of DNA replication. Images PMID:7838710

  1. Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase.

    PubMed

    Byrd, Alicia K; Matlock, Dennis L; Bagchi, Debjani; Aarattuthodiyil, Suja; Harrison, David; Croquette, Vincent; Raney, Kevin D

    2012-07-13

    Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA (ssDNA) that forms due to thermal fraying. Here, we show that Dda translocates unidirectionally on ssDNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single-molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to the applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and that the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (V(un)) by the velocity of translocation on ssDNA in nucleotides per second (V(trans)). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the "active" range. In the case of Dda, the average DNA unwinding velocity was 257±42 bp/s, and the average translocation velocity was 267±15 nt/s. The V(un)/V(trans) value of 0.96 places Dda in a unique category of being an essentially "perfectly" active helicase.

  2. Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

    PubMed Central

    Jose, Davis; Weitzel, Steven E.; Baase, Walter A.; Michael, Miya M.; von Hippel, Peter H.

    2015-01-01

    We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex. PMID:26275774

  3. Crystal structure of a DNA aptamer bound to PvLDH elucidates novel single-stranded DNA structural elements for folding and recognition

    PubMed Central

    Choi, Sung-Jin; Ban, Changill

    2016-01-01

    Structural elements are key elements for understanding single-stranded nucleic acid folding. Although various RNA structural elements have been documented, structural elements of single-stranded DNA (ssDNA) have rarely been reported. Herein, we determined a crystal structure of PvLDH in complex with a DNA aptamer called pL1. This aptamer folds into a hairpin-bulge contact by adopting three novel structural elements, viz, DNA T-loop-like motif, base–phosphate zipper, and DNA G·G metal ion zipper. Moreover, the pL1:PvLDH complex shows unique properties compared with other protein:nucleic acid complexes. Generally, extensive intermolecular hydrogen bonds occur between unpaired nucleotides and proteins for specific recognitions. Although most protein-interacting nucleotides of pL1 are unpaired nucleotides, pL1 recognizes PvLDH by predominant shape complementarity with many bridging water molecules owing to the combination of three novel structural elements making protein-binding unpaired nucleotides stable. Moreover, the additional set of Plasmodium LDH residues which were shown to form extensive hydrogen bonds with unpaired nucleotides of 2008s does not participate in the recognition of pL1. Superimposition of the pL1:PvLDH complex with hLDH reveals steric clashes between pL1 and hLDH in contrast with no steric clashes between 2008s and hLDH. Therefore, specific protein recognition mode of pL1 is totally different from that of 2008s. PMID:27725738

  4. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    NASA Astrophysics Data System (ADS)

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-01

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  5. Structure-based analysis of the interaction between the simian virus 40 T-antigen origin binding domain and single-stranded DNA.

    PubMed

    Meinke, Gretchen; Phelan, Paul J; Fradet-Turcotte, Amélie; Bohm, Andrew; Archambault, Jacques; Bullock, Peter A

    2011-01-01

    The origin-binding domain (OBD) of simian virus 40 (SV40) large T-antigen (T-Ag) is essential for many of T-Ag's interactions with DNA. Nevertheless, many important issues related to DNA binding, for example, how single-stranded DNA (ssDNA) transits along the T-Ag OBD, have yet to be established. Therefore, X-ray crystallography was used to determine the costructure of the T-Ag OBD bound to DNA substrates such as the single-stranded region of a forked oligonucleotide. A second structure of the T-Ag OBD crystallized in the presence of poly(dT)(12) is also reported. To test the conclusions derived from these structures, residues identified as being involved in binding to ssDNA by crystallography or by an earlier nuclear magnetic resonance study were mutated, and their binding to DNA was characterized via fluorescence anisotropy. In addition, these mutations were introduced into full-length T-Ag, and these mutants were tested for their ability to support replication. When considered in terms of additional homology-based sequence alignments, our studies refine our understanding of how the T-Ag OBDs encoded by the polyomavirus family interact with ssDNA, a critical step during the initiation of DNA replication.

  6. Structure-Based Analysis of the Interaction between the Simian Virus 40 T-Antigen Origin Binding Domain and Single-Stranded DNA

    SciTech Connect

    G Meinke; P Phelan; A Fradet-Turcotte; A Bohm; J Archambault; P Bullock

    2011-12-31

    The origin-binding domain (OBD) of simian virus 40 (SV40) large T-antigen (T-Ag) is essential for many of T-Ag's interactions with DNA. Nevertheless, many important issues related to DNA binding, for example, how single-stranded DNA (ssDNA) transits along the T-Ag OBD, have yet to be established. Therefore, X-ray crystallography was used to determine the costructure of the T-Ag OBD bound to DNA substrates such as the single-stranded region of a forked oligonucleotide. A second structure of the T-Ag OBD crystallized in the presence of poly(dT){sub 12} is also reported. To test the conclusions derived from these structures, residues identified as being involved in binding to ssDNA by crystallography or by an earlier nuclear magnetic resonance study were mutated, and their binding to DNA was characterized via fluorescence anisotropy. In addition, these mutations were introduced into full-length T-Ag, and these mutants were tested for their ability to support replication. When considered in terms of additional homology-based sequence alignments, our studies refine our understanding of how the T-Ag OBDs encoded by the polyomavirus family interact with ssDNA, a critical step during the initiation of DNA replication.

  7. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules.

    PubMed

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  8. Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

    SciTech Connect

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-07-28

    Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

  9. DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

    PubMed Central

    Sugiyama, Tomohiko; New, James H.; Kowalczykowski, Stephen C.

    1998-01-01

    Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast. PMID:9600915

  10. The physical determinants of the DNA conformational landscape: an analysis of the potential energy surface of single-strand dinucleotides in the conformational space of duplex DNA

    PubMed Central

    Elsawy, Karim M.; Hodgson, Michael K.; Caves, Leo S. D.

    2005-01-01

    A multivariate analysis of the backbone and sugar torsion angles of dinucleotide fragments was used to construct a 3D principal conformational subspace (PCS) of DNA duplex crystal structures. The potential energy surface (PES) within the PCS was mapped for a single-strand dinucleotide model using an empirical energy function. The low energy regions of the surface encompass known DNA forms and also identify previously unclassified conformers. The physical determinants of the conformational landscape are found to be predominantly steric interactions within the dinucleotide backbone, with medium-dependent backbone-base electrostatic interactions serving to tune the relative stability of the different local energy minima. The fidelity of the PES to duplex DNA properties is validated through a correspondence to the conformational distribution of duplex DNA crystal structures and the reproduction of observed sequence specific propensities for the formation of A-form DNA. The utility of the PES is demonstrated through its succinct and accurate description of complex conformational processes in simulations of duplex DNA. The study suggests that stereochemical considerations of the nucleic acid backbone play a role in determining conformational preferences of DNA which is analogous to the role of local steric interactions in determining polypeptide secondary structure. PMID:16214808

  11. Use of single-strand conformation polymorphism of amplified 16S rDNA for grouping of bacteria isolated from foods.

    PubMed

    Takahashi, Hajime; Kimura, Bon; Tanaka, Yuichiro; Mori, Mayumi; Yokoi, Asami; Fujii, Tateo

    2008-04-01

    The grouping method for isolated strains from foods using single-strand conformation polymorphism (SSCP) after PCR amplification of a portion of 16S rDNA was developed. This method was able to group the strains from various food samples based on 16S rDNA sequence. As 97.8% of the isolated strains from various foods were grouped correctly, use of the PCR-SSCP method enables the prompt and labor-saving analysis of microbial population of food-derived bacterial strains. Advantages in speed and accuracy of bacterial population identification by the PCR-SSCP method have practical application for food suppliers and testing laboratories.

  12. Probing the Salt Concentration Dependent Nucelobase Distribution in a Single-Stranded DNA-Single-Walled Carbon Nanotube Hybrid with Molecular Dynamics.

    PubMed

    Ghosh, Soumadwip; Patel, Nisheet; Chakrabarti, Rajarshi

    2016-01-28

    The hybrids of single-walled carbon nanotube (SWCNT) and single stranded DNA (ssDNA) are novel nanoscale materials having remarkable applications in nanotechnology. The absorption of nucleobases on the surface of a SWCNT depends strongly on the ionic strength of the medium. In this paper, using atomistic molecular dynamics we have shown that at low salt concentration ssDNA wraps on the surface of SWCNT through hydrophobic π-π stacking between the DNA bases and the sp(2)-hybridized carbon atoms of the carbon nanotube. At high salt concentration, however, the DNA molecule adopts a partially folded structure and the ssDNA-SWCNT wrapping gets weakened significantly due to the self-stacking of the DNA bases. Our study can find relevance in CNT mediated gene delivery processes where subsequent unwrapping of the gene from its carrier is anticipated across the cell membrane regulated by an existing salt concentration gradient.

  13. Chimeras between single-stranded DNA-binding proteins from Escherichia coli and Mycobacterium tuberculosis reveal that their C-terminal domains interact with uracil DNA glycosylases.

    PubMed

    Handa, P; Acharya, N; Varshney, U

    2001-05-18

    Uracil, a promutagenic base in DNA can arise by spontaneous deamination of cytosine or incorporation of dUMP by DNA polymerase. Uracil is removed from DNA by uracil DNA glycosylase (UDG), the first enzyme in the uracil excision repair pathway. We recently reported that the Escherichia coli single-stranded DNA binding protein (SSB) facilitated uracil excision from certain structured substrates by E. coli UDG (EcoUDG) and suggested the existence of interaction between SSB and UDG. In this study, we have made use of the chimeric proteins obtained by fusion of N- and C-terminal domains of SSBs from E. coli and Mycobacterium tuberculosis to investigate interactions between SSBs and UDGs. The EcoSSB or a chimera containing its C-terminal domain interacts with EcoUDG in a binary (SSB-UDG) or a ternary (DNA-SSB-UDG) complex. However, the chimera containing the N-terminal domain from EcoSSB showed no interactions with EcoUDG. Thus, the C-terminal domain (48 amino acids) of EcoSSB is necessary and sufficient for interaction with EcoUDG. The data also suggest that the C-terminal domain (34 amino acids) of MtuSSB is a predominant determinant for mediating its interaction with MtuUDG. The mechanism of how the interactions between SSB and UDG could be important in uracil excision repair pathway has been discussed.

  14. Phototriggered formation and repair of DNA containing a site-specific single strand break of the type produced by ionizing radiation or AP lyase activity.

    PubMed

    Zhang, K; Taylor, J S

    2001-01-09

    DNA strand breaks are produced by a variety of agents and processes such as ionizing radiation, xenobiotics, oxidative metabolism, and enzymatic processing of DNA base damage. One of the major types of strand breaks produced by these processes is a single nucleotide gap terminating in 5'- and 3'-phosphates. Previously, we had developed a method for sequence-specifically producing such phosphate-terminated strand breaks in an oligodeoxynucleotide by way of two photochemically activated (caged) building blocks placed in tandem. We now report the design and synthesis of a single caged building block consisting of 1,3-(2-nitrophenyl)-1,3-propanediol, for producing phosphate-terminated strand breaks, and its use producing such a break at a specific site in a double-stranded circular DNA vector. To produce the site-specific break in a duplex vector, a primer containing the caged single strand break was extended opposite the single strand form of a circular DNA vector followed by enzymatic ligation and purification. The single strand break could then be formed in quantitative yield by irradiation of the vector with 365 nm light. In contrast to a previous study, it was found that the strand break can be repaired by Escherichia coli DNA polymerase I and E. coli DNA ligase alone, though less efficiently than in the presence of the 3'-phosphate processing enzyme E. coli endonuclease IV. Repair in the absence of endonuclease IV could be attributed to hydrolysis of the 3'-phosphate in the presence of dNTP and to a lesser extent to exonucleolytic removal of the 3'-phosphate-bearing terminal nucleotide by way of the 3' --> 5' exonuclease activity of polymerase I. This work demonstrates that specialized 3'-end processing enzymes such as endonuclease IV or exonuclease III are not absolutely required for repair of phosphate-terminated gaps. In addition to preparing single strand breaks, the caged building block described should also be useful for preparing double strand breaks and

  15. Single-strand breaks in DNA of various organs of mice induced by methyl methanesulfonate and dimethylsulfoxide determined by the alkaline unwinding technique

    SciTech Connect

    Solveig Walles, S.A.; Erixon, K.

    1984-03-01

    The method for determination of single-strand breaks (SSB) in DNA by the technique of alkaline unwinding and hydroxylapatite chromatography has been applied for cell nuclei from organs of mice. Male mice were given methyl methane-sulfonate (MMS) and dimethylsulfoxide (DMSO) by i.p. administration. Cell nuclei were prepared from various organs and then lysed in alkali. The amount of DNA was determined by fluorometry using 4',6-diamidino-2-phenylindole.2HCl. The relative level of SSB in DNA was determined in various organs (liver, kidney, lung, spleen, testis and brain) 1-24 h after administration of the agent. After MMS-treatment the number of SSB in DNA increased to about the same extent in all organs 1 h post-treatment but then decreased by time. The SSB persisted for the longest time in brain- and lung-DNA. DMSO induced SSB only in DNA of kidney.

  16. Mcm4 C-terminal domain of MCM helicase prevents excessive formation of single-stranded DNA at stalled replication forks

    PubMed Central

    Nitani, Naoki; Yadani, Chie; Yabuuchi, Hayato; Masukata, Hisao; Nakagawa, Takuro

    2008-01-01

    The minichromosome maintenance (MCM) helicase, composed of subunits Mcm2–7, is essential for the initiation and elongation phases of DNA replication. Even when DNA synthesis is blocked, MCM continues DNA unwinding to some extent for activation of the replication checkpoint and then stops. However, the mechanism of regulation of MCM-helicase activity remains unknown. Here, we show that truncation of the Mcm4 C-terminal domain (CTD) in fission yeast results in hypersensitivity to replication block caused by dNTP depletion. The truncation mcm4-c84 does not affect the activation of the replication checkpoint pathway but delays its attenuation during recovery from replication block. Two dimensional gel electrophoresis showed that mcm4-c84 delays the disappearance of replication intermediates, indicating that the Mcm4 CTD is required for efficient recovery of stalled replication forks. Remarkably, chromatin immunoprecipitation revealed that mcm4-c84 brings about an increase rather than a decrease in the association of the single-stranded DNA-binding protein RPA to stalled forks, and MCM and the accessory complex GINS are unaffected. These results suggest that the Mcm4 CTD is required to suspend MCM-helicase activity after the formation of single-stranded DNA sufficient for checkpoint activation. PMID:18753627

  17. Structural Basis for Dimer Formation of Human Condensin Structural Maintenance of Chromosome Proteins and Its Implications for Single-stranded DNA Recognition*

    PubMed Central

    Uchiyama, Susumu; Kawahara, Kazuki; Hosokawa, Yuki; Fukakusa, Shunsuke; Oki, Hiroya; Nakamura, Shota; Kojima, Yukiko; Noda, Masanori; Takino, Rie; Miyahara, Yuya; Maruno, Takahiro; Kobayashi, Yuji; Ohkubo, Tadayasu; Fukui, Kiichi

    2015-01-01

    Eukaryotic structural maintenance of chromosome proteins (SMC) are major components of cohesin and condensins that regulate chromosome structure and dynamics during cell cycle. We here determine the crystal structure of human condensin SMC hinge heterodimer with ∼30 residues of coiled coils. The structure, in conjunction with the hydrogen exchange mass spectrometry analyses, revealed the structural basis for the specific heterodimer formation of eukaryotic SMC and that the coiled coils from two different hinges protrude in the same direction, providing a unique binding surface conducive for binding to single-stranded DNA. The characteristic hydrogen exchange profiles of peptides constituted regions especially across the hinge-hinge dimerization interface, further suggesting the structural alterations upon single-stranded DNA binding and the presence of a half-opened state of hinge heterodimer. This structural change potentially relates to the DNA loading mechanism of SMC, in which the hinge domain functions as an entrance gate as previously proposed for cohesin. Our results, however, indicated that this is not the case for condensins based on the fact that the coiled coils are still interacting with each other, even when DNA binding induces structural changes in the hinge region, suggesting the functional differences of SMC hinge domain between condensins and cohesin in DNA recognition. PMID:26491021

  18. A sensitive method for the quantification of virion-sense and complementary-sense DNA strands of circular single-stranded DNA viruses

    PubMed Central

    Rodríguez-Negrete, Edgar A.; Sánchez-Campos, Sonia; Cañizares, M. Carmen; Navas-Castillo, Jesús; Moriones, Enrique; Bejarano, Eduardo R.; Grande-Pérez, Ana

    2014-01-01

    Circular single-stranded DNA (ssDNA) viruses are the smallest viruses known to infect eukaryotes. High recombination and mutation rates have conferred these viruses with an evolutionary potential that has facilitated their emergence. Their damaging effects on livestock (circoviruses) and crops (geminiviruses and nanoviruses), and the ubiquity of anelloviruses in human populations and other mammalian species, have resulted in increased interest in better understanding their epidemiology and infection mechanisms. Circular ssDNA viral replication involves the synthesis of dsDNA intermediates containing complementary-sense (CS) and virion-sense (VS) strands. Precise quantification of VS and CS accumulation during viral infections can provide insights into the molecular mechanisms underlying viral replication and the host invasion process. Although qPCR protocols for quantifying viral molecules exist, none of them discriminate VS and CS strands. Here, using a two-step qPCR protocol we have quantified VS and CS molecule accumulation during the infection process of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae). Our results show that the VS/CS strand ratio and overall dsDNA amounts vary throughout the infection process. Moreover, we show that these values depend on the virus-host combination, and that most CS strands are present as double-stranded molecules. PMID:25241765

  19. New Type of Papillomavirus and Novel Circular Single Stranded DNA Virus Discovered in Urban Rattus norvegicus Using Circular DNA Enrichment and Metagenomics.

    PubMed

    Hansen, Thomas Arn; Fridholm, Helena; Frøslev, Tobias Guldberg; Kjartansdóttir, Kristín Rós; Willerslev, Eske; Nielsen, Lars Peter; Hansen, Anders Johannes

    2015-01-01

    Rattus norvegicus (R. norvegicus) are ubiquitous and their presence has several effects on the human populations in our urban areas on a global scale. Both historically and presently, this close interaction has facilitated the dissemination of many pathogens to humans, making screening for potentially zoonotic and emerging viruses in rats highly relevant. We have investigated faecal samples from R. norvegicus collected from urban areas using a protocol based on metagenomic enrichment of circular DNA genomes and subsequent sequencing. We found a new type of papillomavirus, with a L1 region 82% identical to that of the known R. norvegicus Papillomavirus 2. Additionally, we found 20 different circular replication associated protein (Rep)-encoding single stranded DNA (CRESS-DNA) virus-like genomes, one of which has homology to the replication-associated gene of Beak and feather disease virus. Papillomaviruses are a group of viruses known for their carcinogenic potential, and although they are known to infect several different vertebrates, they are mainly studied and characterised in humans. CRESS-DNA viruses are found in many different environments and tissue types. Both papillomaviruses and CRESS-DNA viruses are known to have pathogenic potential and screening for novel and known viruses in R. norvegicus could help identify viruses with pathogenic potential.

  20. Structure–function relationships of two paralogous single-stranded DNA-binding proteins from Streptomyces coelicolor: implication of SsbB in chromosome segregation during sporulation

    PubMed Central

    Paradzik, Tina; Ivic, Nives; Filic, Zelimira; Manjasetty, Babu A.; Herron, Paul; Luic, Marija; Vujaklija, Dusica

    2013-01-01

    The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Å resolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions. PMID:23393191

  1. Comparative Study of different msDNA (multicopy single-stranded DNA) structures and phylogenetic comparison of reverse transcriptases (RTs): evidence for vertical inheritance.

    PubMed

    Das, Rasel; Shimamoto, Tadashi; Hosen, Sultan Mohammad Zahid; Arifuzzaman, Mohammad

    2011-01-01

    The multi-copy single-stranded DNA (msDNA) is yielded by the action of reverse transcriptase of retro-element in a wide range of pathogenic bacteria. Upon this phenomenon, it has been shown that msDNA is only produced by Eubacteria because many Eubacteria species contained reverse transcriptase in their special retro-element. We have screened around 111 Archaea at KEGG (Kyoto Encyclopedia of Genes and Genomes) database available at genome net server and observed three Methanosarcina species (M.acetivorans, M.barkeri and M.mazei), which also contained reverse transcriptase in their genome sequences. This observation of reverse transcriptase in Archaea raises questions regarding the origin of this enzyme. The evolutionary relationship between these two domains of life (Eubacteria and Archaea) hinges upon the phenomenon of retrons. Interestingly, the evolutionary trees based on the reverse transcriptases (RTs) and 16S ribosomal RNAs point out that all the Eubacteria RTs were descended from Archaea RTs during their evolutionary times. In addition, we also have shown some significant structural features among the newly identified msDNA-Yf79 in Yersinia frederiksenii with other of its related msDNAs (msDNA-St85, msDNA-Vc95, msDNA-Vp96, msDNA-Ec78 and msDNA-Ec83) from pathogenic bacteria. Together the degree of sequence conservation among these msDNAs, the evolutionary trees and the distribution of these ret (reverse transcriptase) genes suggest a possible evolutionary scenario. The single common ancestor of the organisms of Eubacteria and Archaea subgroups probably achieved this ret gene during their evolution through the vertical descent rather than the horizontal transformations followed by integration into this organism genome by a mechanism related to phage recognition and/or transposition.

  2. Deoxyribonuclease I generates single-stranded gaps in chromatin deoxyribonucleic acid.

    PubMed

    Riley, D E

    1980-06-24

    Production of 10-base multiple DNA ladder fragments during DNase I digestion of chromatin is explained by a model which does not involve site-specific nicking by the DNase I. This model was tested because it explains why 10-base (actually 10.4 base) multiple-related fragments are paradoxically generated by both endonucleolytic (DNase I) and exonucleolytic (exonuclease III) mechanisms. This new model also explains the phenomenon of substantial single-stranded DNA production during DNase I digestion of chromatin. The latter phenomenon has been widely observed but is not explained by previous models. The single-stranded gap model to be presented makes testable predictions. Primarily, these are that DNase I produces single-stranded gaps in chromatin DNA and that the termini of 10-base multiple ladder fragments are separated by single-stranded gaps. Single-stranded gap production by DNase I was confirmed by a number of methods. Sensitivity of ladder band components (from DNase I but not staphylococcal nuclease digests) to S1 nuclease suggested that the ladder fragments themselves may compose a significant portion of these gaps. Separation of ladder fragment termini by single-stranded gaps was verified by demonstrating both resistance to the nick-specific NAD+-dependent ligase and sensitivity to T4 ligase which can ligate across gaps. Many single-stranded gaps, occurring both individually and clusters, were observed by electron microscopy using either cytochrome c labeling (where the gaps) are thinner than duplex) or gene 32 protein labeling (gaps thicker than duplex). Gap sizes were estimated by protecting them with gene 32 protein and digesting away unprotected duplexes. By this method, gap sizes fall into a ladder distribution (from 10 or 20 bases up to 120 bases), which, at least in the region of the shorter sizes, clearly indicates the sizes of single-stranded gaps formed in chromatin by DNase I.

  3. Accumulation of polycyclic aromatic hydrocarbon-induced single strand breaks is attributed to slower rejoining processes by DNA polymerase inhibitor, cytosine arabinoside in CHO-K1 cells

    SciTech Connect

    Park, Jongkun ); Lee, Jungsup; Lee, Hyungho; Choi, Insoon; Park, Sangdai )

    1991-01-01

    The authors demonstrate a successful induction of DNA single breaks in CHO-K1 cells by cocultivation with mouse embryonic fibroblasts (MEF) during exposure to benzo(a)pyrene (BP) or 3-methylcholanthrene (MC). When compared to those induced by methyl methanesulfonate (MMS), the DNA single strand breaks induced by BP and MC were markedly accumulated by post-incubation with cytosine arabinoside (araC) and were much more delayed in their rejoining. These results suggest that the active metabolites of BP or MC produced by cocultivation with MEF or microsomal fraction (S-15) result in the formation of large DNA adducts which require an active participation of DNA polymerase {alpha}({delta}) in the polymerization step of excision repair for their removal.

  4. Ortho-stabilized 18F-azido click agents and application in PET imaging of single-stranded DNA aptamer

    PubMed Central

    Wang, Lu; Jacobson, Orit; Avdic, Din; Rotstein, Benjamin H.; Weiss, Ido D.; Collier, Lee

    2015-01-01

    Azido 18F-arenes are important and versatile building blocks for radiolabeling of biomolecules via Huisgen cycloaddition (‘click chemistry’) in positron emission tomography (PET). However, routine access of such clickable agents is challenged by inefficient multi-step and esoteric radiochemical approaches. Herein we describe a high-yielding direct radiofluorination for azido 18F-arenes by developing an oxygen ortho-stabilized iodonium derivative (OID). This OID strategy addresses an unmet need for a reliable azido 18F-arene clickable agent in bioconjugation reactions. A ssDNA aptamer is radiolabeled and visualized in a xenograft mouse model of human colon cancer by PET, which demonstrates a convenient and highly efficient way of labeling biomolecules and tracking them by OID approach. PMID:26308650

  5. DNA single strand breakage, DNA adducts, and sister chromatid exchange in lymphocytes and phenanthrene and pyrene metabolites in urine of coke oven workers.

    PubMed Central

    Popp, W; Vahrenholz, C; Schell, C; Grimmer, G; Dettbarn, G; Kraus, R; Brauksiepe, A; Schmeling, B; Gutzeit, T; von Bülow, J; Norpoth, K

    1997-01-01

    OBJECTIVES: To investigate the specificity of biological monitoring variables (excretion of phenanthrene and pyrene metabolites in urine) and the usefulness of some biomarkers of effect (alkaline filter elution, 32P postlabelling assay, measurement of sister chromatid exchange) in workers exposed to polycyclic aromatic hydrocarbons (PAHs). METHODS: 29 coke oven workers and a standardised control group were investigated for frequencies of DNA single strand breakage, DNA protein cross links (alkaline filter elution assay), sister chromatid exchange, and DNA adducts (32P postlabelling assay) in lymphocytes. Phenanthrene and pyrene metabolites were measured in 24 hour urine samples. 19 different PAHs (including benzo(a)pyrene, pyrene, and phenanthrene) were measured at the workplace by personal air monitoring. The GSTT1 activity in erythrocytes and lymphocyte subpopulations in blood was also measured. RESULTS: Concentrations of phenanthrene, pyrene, and benzo(a)pyrene in air correlated well with the concentration of total PAHs in air; they could be used for comparisons of different workplaces if the emission compositions were known. The measurement of phenanthrene metabolites in urine proved to be a better biological monitoring variable than the measurement of 1-hydroxypyrene. Significantly more DNA strand breaks in lymphocytes of coke oven workers were found (alkaline filter elution assay); the DNA adduct rate was not significantly increased in workers, but correlated with exposure to PAHs in a semiquantitative manner. The number of sister chromatid exchanges was lower in coke oven workers but this was not significant; thus counting sister chromatid exchanges was not a good variable for biomonitoring of coke oven workers. Also, indications for immunotoxic influences (changes in lymphocyte subpopulations) were found. CONCLUSIONS: The measurement of phenanthrene metabolites in urine seems to be a better biological monitoring variable for exposure to PAHs than

  6. Single-stranded DNA fragments of insect-specific nuclear polyhedrosis virus act as selective DNA insecticides for gypsy moth control.

    PubMed

    Oberemok, Volodymyr V; Skorokhod, Oleksii A

    2014-07-01

    This paper focuses on the DNA insecticides as a novel preparation against gypsy moth (Lymantria dispar) based on DNA fragments of the anti-apoptotic gene of its nuclear polyhedrosis virus. It was found that the external application of a solution with two single-stranded DNA fragments from BIR and RING domains of LdMNPV (L.dispar multicapsid nuclear polyhedrosis virus) IAP-3 (inhibitor of apoptosis) gene induces a significantly higher mortality of gypsy moth caterpillars in comparison with the application of the control solutions. This effect does not depend on the infection of caterpillars with LdMNPV. The results also show that DNA insecticides based on LdMNPV IAP-3 gene fragments can be selective in action, and at least are not harmful to tobacco hornworm (Manduca sexta) and black cutworm (Agrotis ipsilon). Part of the gypsy moth genome cloned with the fragments of BIR and RING domains of LdMNPV IAP-3 gene as primers, has an overlap with the corresponding part of the LdMNPV IAP-3 gene and L.dispar IAP-1 mRNA for an inhibitor of apoptosis protein with the high cover by query, allows assuming that we cloned a part of gypsy moth anti-apoptosis gene. This finding gives the grounding that proposed here DNA insecticides might act through the blocking of the mechanisms involved in post transcriptional expression of insect anti-apoptosis genes. The results show the insecticidal potential of the viral genome fragments that can be used to create safe and relatively fast-acting DNA insecticides to control the quantity of gypsy moth populations, important task for forestry and agriculture.

  7. Hsp70 translocates to the nuclei and nucleoli, binds to XRCC1 and PARP-1, and protects HeLa cells from single-strand DNA breaks.

    PubMed

    Kotoglou, Polychronis; Kalaitzakis, Alexandros; Vezyraki, Patra; Tzavaras, Theodore; Michalis, Lampros K; Dantzer, Francoise; Jung, Jae U; Angelidis, Charalampos

    2009-07-01

    For many years, there has been uncertainty concerning the reason for Hsp70 translocation to the nucleus and nucleolus. Herein, we propose that Hsp70 translocates to the nucleus and nucleoli in order to participate in pathways related to the protection of the nucleoplasmic DNA or ribosomal DNA from single-strand breaks. The absence of Hsp70 in HeLa cells, via Hsp70 gene silencing (knockdown), indicated the essential role of Hsp70 in DNA integrity. Therefore, HeLa Hsp70 depleted cells were very sensitive in heat treatment and their DNA breaks were multiple compared to that of control HeLa cells. The molecular mechanism with which Hsp70 performs its role at the level of nucleus and nucleolus during stress was examined. Hsp70 co-localizes with PARP1 in the nucleus/nucleoli as was observed in confocal studies and binds to the BCRT domain of PARP1 as was revealed with protein-protein interaction assays. It was also found that Hsp70 binds simultaneously to XRCC1 and PARP-1, indicating that Hsp70 function takes place at the level of DNA repair and possibly at the base excision repair system. Making a hypothetical model, we have suggested that Hsp70 is the molecule that binds and interrelates with PARP1 creating the repair proteins simultaneously, such as XRCC1, at the single-strand DNA breaks. Our data partially clarify a previously unrecognized cellular response to heat stress. Finally, we can speculate that Hsp70 plays a role in the quality and integrity of DNA.

  8. Hsp70 translocates to the nuclei and nucleoli, binds to XRCC1 and PARP-1, and protects HeLa cells from single-strand DNA breaks

    PubMed Central

    Kotoglou, Polychronis; Kalaitzakis, Alexandros; Vezyraki, Patra; Tzavaras, Theodore; Michalis, Lampros K.; Dantzer, Francoise; Jung, Jae U.

    2008-01-01

    For many years, there has been uncertainty concerning the reason for Hsp70 translocation to the nucleus and nucleolus. Herein, we propose that Hsp70 translocates to the nucleus and nucleoli in order to participate in pathways related to the protection of the nucleoplasmic DNA or ribosomal DNA from single-strand breaks. The absence of Hsp70 in HeLa cells, via Hsp70 gene silencing (knockdown), indicated the essential role of Hsp70 in DNA integrity. Therefore, HeLa Hsp70 depleted cells were very sensitive in heat treatment and their DNA breaks were multiple compared to that of control HeLa cells. The molecular mechanism with which Hsp70 performs its role at the level of nucleus and nucleolus during stress was examined. Hsp70 co-localizes with PARP1 in the nucleus/nucleoli as was observed in confocal studies and binds to the BCRT domain of PARP1 as was revealed with protein–protein interaction assays. It was also found that Hsp70 binds simultaneously to XRCC1 and PARP-1, indicating that Hsp70 function takes place at the level of DNA repair and possibly at the base excision repair system. Making a hypothetical model, we have suggested that Hsp70 is the molecule that binds and interrelates with PARP1 creating the repair proteins simultaneously, such as XRCC1, at the single-strand DNA breaks. Our data partially clarify a previously unrecognized cellular response to heat stress. Finally, we can speculate that Hsp70 plays a role in the quality and integrity of DNA. PMID:19089598

  9. Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific single-stranded DNA and improved real-time detection

    PubMed Central

    Pierce, Kenneth E.; Sanchez, J. Aquiles; Rice, John E.; Wangh, Lawrence J.

    2005-01-01

    Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX ≥ 5°C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis. PMID:15937116

  10. Effect of temperature on the low-frequency vibrational spectrum and relative structuring of hydration water around a single-stranded DNA.

    PubMed

    Chakraborty, Kaushik; Bandyopadhyay, Sanjoy

    2015-01-07

    Molecular dynamics simulations of the single-stranded DNA oligomer (5'-CGCGAAT TCGCG-3') in aqueous solution have been carried out at different temperatures between 160 K and 300 K. The effects of temperature on the low-frequency vibrational spectrum and local structural arrangements of water molecules hydrating the DNA strand have been explored in detail. The low-frequency density of states distributions reveal that increasingly trapped transverse water motions play a dominant role in controlling the band corresponding to O⋯O⋯O bending or transverse oscillations of hydration water at supercooled temperatures. In addition, presence of a broad band around 260 (±20) cm(-1) under supercooled conditions indicates transformation from high density liquid-like structuring of hydration water at higher temperatures to that of a low density liquid at lower temperatures. It is found that long-range correlations between the supercooled hydration water molecules arise due to such local structural transition around the DNA oligomer.

  11. Nonadditivity in the recognition of single-stranded DNA by the schizosaccharomyces pombe protection of telomeres 1 DNA-binding domain, Pot1-DBD.

    PubMed

    Croy, Johnny E; Altschuler, Sarah E; Grimm, Nicole E; Wuttke, Deborah S

    2009-07-28

    The Schizosaccharomyces pombe protection of telomeres 1 (SpPot1) protein recognizes the 3' single-stranded ends of telomeres and provides essential protective and regulatory functions. The ssDNA-binding activity of SpPot1 is conferred by its ssDNA-binding domain, Pot1-DBD (residues 1-389), which can be further separated into two distinct domains, Pot1pN (residues 1-187) and Pot1pC (residues 188-389). Here we show that Pot1pC, like Pot1pN, can function independently of Pot1-DBD and binds specifically to a minimal nonameric oligonucleotide, d(GGTTACGGT), with a K(D) of 400 +/- 70 nM (specifically recognized nucleotides in bold). NMR chemical shift perturbation analysis indicates that the overall structures of the isolated Pot1pN and Pot1pC domains remain intact in Pot1-DBD. Furthermore, alanine scanning reveals modest differences in the ssDNA-binding contacts provided by isolated Pot1pN and within Pot1-DBD. Although the global character of both Pot1pN and Pot1pC is maintained in Pot1-DBD, chemical shift perturbation analysis highlights localized structural differences within the G1/G2 and T3/T4 binding pockets of Pot1pN in Pot1-DBD, which correlate with its distinct ssDNA-binding activity. Furthermore, we find evidence for a putative interdomain interface on Pot1pN that mediates interactions with Pot1pC that ultimately result in the altered ssDNA-binding activity of Pot1-DBD. Together, these data provide insight into the mechanisms underlying the activity and regulation of SpPot1 at the telomere.

  12. Double strand break unwinding and resection by the mycobacterial helicase-nuclease AdnAB in the presence of single strand DNA-binding protein (SSB).

    PubMed

    Unciuleac, Mihaela-Carmen; Shuman, Stewart

    2010-11-05

    Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3' to 5' DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevented from reannealing or promoting ongoing ATP hydrolysis. We found that the AdnAB motor catalyzed processive unwinding of 2.7-11.2-kbp linear duplex DNAs at a rate of ∼250 bp s(-1), while hydrolyzing ∼5 ATPs per bp unwound. Crippling the AdnA phosphohydrolase active site did not affect the rate of unwinding but lowered energy consumption slightly, to ∼4.2 ATPs bp(-1). Mutation of the AdnB phosphohydrolase abolished duplex unwinding, consistent with a model in which the "leading" AdnB motor propagates a Y-fork by translocation along the 3' DNA strand, ahead of the "lagging" AdnA motor domain. By tracking the resection of the 5' and 3' strands at the DSB ends, we illuminated a division of labor among the AdnA and AdnB nuclease modules during dsDNA unwinding, whereby the AdnA nuclease processes the unwound 5' strand to liberate a short oligonucleotide product, and the AdnB nuclease incises the 3' strand on which the motor translocates. These results extend our understanding of presynaptic DSB processing by AdnAB and engender instructive comparisons with the RecBCD and AddAB clades of bacterial helicase-nuclease machines.

  13. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins.

    PubMed

    Rosario, Karyna; Schenck, Ryan O; Harbeitner, Rachel C; Lawler, Stephanie N; Breitbart, Mya

    2015-01-01

    Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA) viruses that encode a conserved replication initiator protein (Rep) in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA) viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap) encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs), which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses.

  14. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins

    PubMed Central

    Rosario, Karyna; Schenck, Ryan O.; Harbeitner, Rachel C.; Lawler, Stephanie N.; Breitbart, Mya

    2015-01-01

    Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA) viruses that encode a conserved replication initiator protein (Rep) in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA) viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap) encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs), which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses. PMID:26217327

  15. Localization of specific sequences and DNA single-strand breaks in individual UV-A-irradiated human lymphocytes by COMET FISH

    NASA Astrophysics Data System (ADS)

    Bock, Claudia; Rapp, Alexander; Dittmar, Heike; Monajembashi, Shamci; Greulich, Karl-Otto

    1999-01-01

    The COMET assay, a single cell electrophoresis technique which allows to separate electrophoretically fractionated DNA according to size has been combined with fluorescence in situ hybridization (FISH) which allows to localize specific genes or gene regions. This combination (COMET FISH) allows the detection of DNA single strand breaks in specific regions of the genome of human lymphocytes at the single cell level. Various types of DNA probes, e.g. centromere-, (alpha) - satellite-, telomere-, whole chromosome-, single copy- and region specific DNA probes have been used to investigate whether the UV-A induced DNA single strand breaks are distributed randomly all over the human genome or induced at specific sites ('hot spots'). In the investigated human peripheral blood lymphocytes all but one centromere reveal low sensitivity for UV-A irradiation (500 kJ/m2), while telomeres are randomly distributed over COMET heads and tails. The human chromosome 1 is fractionated by irradiation, but remains in the COMET head, indicating an only moderate degree of fractionation. Among three tested single copy probes, c- myc, p53 and p58, the p53 gene located on chromosome 17p13.1 and the p58 gene (1p36) appear to be located in UV-A stable regions of the human genome in 95% of 65 investigated lymphocytes. In contrast, the c-myc proto-oncogene (8q24) is found in the COMET tail in 90% of the 27 investigated lymphocytes and thus appears to be more sensitive to UV-A irradiation.

  16. IgA containing circulating immune complexes and IgA anti-single stranded DNA antibodies in patients with obstructive jaundice.

    PubMed Central

    Ohshio, G; Furukawa, F; Sekita, K; Manabe, T; Tobe, T; Hamashima, Y

    1985-01-01

    Elevated levels of IgA containing circulating immune complexes (IgA-CIC) and IgG containing (IgG-) CIC were detected in patients with obstructive jaundice due to biliary tract stones and/or tumour of biliary tract or pancreas. Levels of serum IgA were also elevated, and correlated with the levels of IgA-CIC. The levels of IgA and IgG anti-single stranded (ss) DNA were elevated, and there was a significant correlation between the levels of IgA-CIC and IgA anti-ssDNA antibodies. The cause of IgA-CIC increase in patients with obstructive jaundice might be due not only to simple obstruction of biliary tract but also to other factors such as a tissue destruction. PMID:3872190

  17. Polymerase chain reaction-single strand conformation polymorphism analyses of nuclear and chloroplast DNA provide evidence for recombination, multiple introductions and nascent speciation in the Caulerpa taxifolia complex.

    PubMed

    Meusnier, I; Valero, M; Destombe, C; Godé, C; Desmarais, E; Bonhomme, F; Stam, W T; Olsen, J L

    2002-11-01

    Independent lines of evidence support an Australian origin for the Mediterranean populations of the tropical alga Caulerpa taxifolia. To complement previous biogeographical studies based on nuclear rDNA internal transcribed spacer (ITS), a new chloroplast marker was developed--the cp 16S rDNA intron-2. Sequence variability for both nuclear and chloroplast markers were assessed in 110 individuals using single strand conformation polymorphism. Comparison of intrapopulation genetic diversity between invasive Mediterranean and 'native' Australian populations revealed the occurrence of two divergent and widespread clades. The first clade grouped nontropical invasive populations with inshore-mainland populations from Australia, while the second clustered all offshore-island populations studied so far. Despite our finding of nine distinct nuclear and five distinct chloroplast profiles, a single nucleocytoplasmic combination was characteristic of the invasive populations and sexual reproduction was found to be very rare. C. taxifolia is clearly a complex of genetically and ecologically differentiated sibling species or subspecies.

  18. DNA single-strand breaks, double-strand breaks, and crosslinks in rat testicular germ cells: Measurements of their formation and repair by alkaline and neutral filter elution

    SciTech Connect

    Bradley, M.O.; Dysart, G. )

    1985-06-01

    This work describes a neutral and alkaline elution method for measuring DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-DNA crosslinks in rat testicular germ cells after treatments in vivo or in vitro with both chemical mutagens and gamma-irradiation. The methods depend upon the isolation of testicular germ cells by collagenase and trypsin digestion, followed by filtration and centrifugation. {sup 137}Cs irradiation induced both DNA SSBs and DSBs in germ cells held on ice in vitro. Irradiation of the whole animal indicated that both types of DNA breaks are induced in vivo and can be repaired. A number of germ cell mutagens induced either DNA SSBs, DSBs, or cross-links after in vivo and in vitro dosing. These chemicals included methyl methanesulfonate, ethyl methanesulfonate, ethyl nitrosourea, dibromochlorpropane, ethylene dibromide, triethylene melamine, and mitomycin C. These results suggest that the blood-testes barrier is relatively ineffective for these mutagens, which may explain in part their in vivo mutagenic potency. This assay should be a useful screen for detecting chemical attack upon male germ-cell DNA and thus, it should help in the assessment of the mutagenic risk of chemicals. In addition, this approach can be used to study the processes of SSB, DSB, and crosslink repair in DNA of male germ cells, either from all stages or specific stages of development.

  19. Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex.

    PubMed

    Das, Devashish; Faridounnia, Maryam; Kovacic, Lidija; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2017-02-17

    The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble.

  20. A new system for the amplification of biological signals: RecA and complimentary single strand DNA probes on a leaky surface acoustic wave biosensor.

    PubMed

    Zhang, Liqun; Wang, Yunxia; Chen, Ming; Luo, Yang; Deng, Kun; Chen, Dong; Fu, Weiling

    2014-10-15

    This research describes a new amplification signals system of the leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor for the simple, sensitive and rapid detection of the target double-stranded DNA (dsDNA). The system consists of a RecA protein-coated complementary single-stranded DNA (cssDNA) probe complex that amplifies the biological signal to improve the sensitivity of the biosensor. The bis-PNA probe for detecting HPV was first immobilized on a gold surface membrane of the detection channel. After the probe was completely hybridized with the corresponding target DNA, different concentrations of the "RecA protein-complementary single strand DNA probe" were added to react with the bis-PNA/dsDNA complex. The phase shift of the LSAW biosensors, which was measured and found to be most significant when the RecA protein was 45 μg/mL and the ATPγS was 2.5 mmol/L. Compared with other concentrations (P<0.01) of RecA and ATPγS, the value of the phase shift was (11.74 ± 1.03) degrees and the ratio of the phase shift and hybridization time clearly outperformed that of the other concentrations. Compared to the direct hybridization of the bis-PNA probe and the target DNA sequence, the sensitivity was effectively improved and the detection time was significantly shortened. PNA binding adjacent to the area of the target sequence homologous to the probe significantly increased the yield of the hybridization reaction between the PNA/dsDNA complex and the RecA protein-coated cssDNA probe. In this condition, the phase shift was significantly obvious and the detection time was significantly shortened. In conclusion, the combination of the RecA protein-coated cssDNA probe and the LSAW bis-PNA biosensor provides sensitivity and simple and rapid detection of clinical trace pathogenic microorganisms.

  1. Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells.

    PubMed Central

    Galli, A; Schiestl, R H

    1998-01-01

    Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage. PMID:9649517

  2. Evidence for involvement of multiple iron species in DNA single-strand scission by H2O2 in HL-60 cells.

    PubMed

    Byrnes, R W

    1996-01-01

    Some of the properties of cellular iron species which react with H2O2 to cause DNA single-strand breaks in HL-60 cells were characterized in control cells and in cells made deficient of iron using 4,7-phenylsulfonyl-1,10-phenanthroline (bathophenanthroline disulfonic acid or BPS) and ascorbate. Single-strand breaks were measured using alkaline elution of DNA of cells treated at 4 degrees to minimize repair during treatment. Strand breakage in the presence of 10% serum was only 40% of that in the absence of serum. This effect was traced to reaction of H2O2 with metals, most likely iron, in serum. Dimethyl sulfoxide (Me2SO) inhibited a maximum of 65% of breaks in control cells. The diffusion distance from the site of generation of hydroxyl radicals to the site of reaction with DNA for the Me2SO-inhibitable fraction was 6.9 nm. There was no significant alteration in the fraction of Me2SO-inhibitable strand breaks or in diffusion distance in iron-deficient cells, though total strand breaks decreased by 70%. When the effect of extracellular iron in serum was taken into account, 60 microM orthophenanthroline (OP) inhibited a maximum of 85% of strand breaks. In cells pretreated with 60 microM OP, the Me2SO-inhibitable fraction of the remaining strand breaks decreased to 32%, while the diffusion distance decreased to 4.1 nm. These data indicate the existence of a number of different iron species, as characterized by overlapping but not coincidental inhibition by OP and Me2SO, and by differing diffusion distances.

  3. Plant transformation by Agrobacterium tumefaciens: modulation of single-stranded DNA-VirE2 complex assembly by VirE1.

    PubMed

    Frenkiel-Krispin, Daphna; Wolf, Sharon Grayer; Albeck, Shira; Unger, Tamar; Peleg, Yoav; Jacobovitch, Jossef; Michael, Yigal; Daube, Shirley; Sharon, Michal; Robinson, Carol V; Svergun, Dmitri I; Fass, Deborah; Tzfira, Tzvi; Elbaum, Michael

    2007-02-09

    Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.

  4. Identification of diverse circular single-stranded DNA viruses in adult dragonflies and damselflies (Insecta: Odonata) of Arizona and Oklahoma, USA.

    PubMed

    Dayaram, Anisha; Potter, Kristen A; Pailes, Roberta; Marinov, Milen; Rosenstein, Dana D; Varsani, Arvind

    2015-03-01

    Next generation sequencing and metagenomic approaches are commonly used for the identification of circular replication associated protein (Rep)-encoding single stranded (CRESS) DNA viruses circulating in various environments. These approaches have enabled the discovery of some CRESS DNA viruses associated with insects. In this study we identified and recovered 31 viral genomes which represent 24 distinct CRESS DNA viruses from seven dragonfly species (Rhionaeschna multicolor, Erythemis simplicicollis, Erythrodiplax fusca, Libellula quadrimaculata, Libellula saturata, Pachydiplax longipennis, and Pantala hymenaea) and two damselfly species (Ischnura posita, Ischnura ramburii) sampled in various locations in the states of Arizona and Oklahoma of the United States of America (USA). We also identified Sclerotinia sclerotiorum hypovirulence-associated DNA virus-1 (SsHADV-1) in P. hymenaea, E. simplicicollis and I. ramburii sampled in Oklahoma, which is the first report of SsHADV-1 in the New World. The genome architectures of the CRESS DNA viruses recovered vary, but they all have at least two major open reading frames (ORFs) that have either a bidirectional or unidirectional arrangement. Four of the viral genomes recovered, in addition to the three isolates of SsHADV-1, show similarities to viruses of the proposed gemycircularvirus group. Analysis of the Rep encoded by the remaining 24 viral genomes reveals that these are highly diverse and allude to the fact that they represent novel CRESS DNA viruses.

  5. Evolution of eukaryotic single-stranded DNA viruses of the Bidnaviridae family from genes of four other groups of widely different viruses

    NASA Astrophysics Data System (ADS)

    Krupovic, Mart; Koonin, Eugene V.

    2014-06-01

    Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. However, viruses of the family Bidnaviridae, instead of the endonuclease, encode a protein-primed type B DNA polymerase (PolB) and hence break this pattern. We investigated the provenance of all bidnavirus genes and uncover an unexpected turbulent evolutionary history of these unique viruses. Our analysis strongly suggests that bidnaviruses evolved from a parvovirus ancestor from which they inherit a jelly-roll capsid protein and a superfamily 3 helicase. The radiation of bidnaviruses from parvoviruses was probably triggered by integration of the ancestral parvovirus genome into a large virus-derived DNA transposon of the Polinton (polintovirus) family resulting in the acquisition of the polintovirus PolB gene along with terminal inverted repeats. Bidnavirus genes for a receptor-binding protein and a potential novel antiviral defense modulator are derived from dsRNA viruses (Reoviridae) and dsDNA viruses (Baculoviridae), respectively. The unusual evolutionary history of bidnaviruses emphasizes the key role of horizontal gene transfer, sometimes between viruses with completely different genomes but occupying the same niche, in the emergence of new viral types.

  6. Effective cross sections for production of single-strand breaks in plasmid DNA by 0.1 to 4.7 eV electrons.

    PubMed

    Panajotovic, Radmila; Martin, Frédéric; Cloutier, Pierre; Hunting, Darel; Sanche, Léon

    2006-04-01

    We determined effective cross sections for production of single-strand breaks (SSBs) in plasmid DNA [pGEM 3Zf(-)] by electrons of 10 eV and energies between 0.1 and 4.7 eV. After purification and lyophilization on a chemically clean tantalum foil, dry plasmid DNA samples were transferred into a high-vacuum chamber and bombarded by a monoenergetic electron beam. The amount of the circular relaxed DNA in the samples was separated from undamaged molecules and quantified using agarose gel electrophoresis. The effective cross sections were derived from the slope of the yield as a function of exposure and had values in the range of 10(-15)- 10(-14) cm2, giving an effective cross section of the order of 10(-18) cm2 per nucleotide. Their strong variation with incident electron energy and the resonant enhancement at 1 eV suggest that considerable damage is inflicted by very low-energy electrons to DNA, and it indicates the important role of pi* shape resonances in the bond-breaking process. Furthermore, the fact that the energy threshold for SSB production is practically zero implies that the sensitivity of DNA to electron impact is universal and is not limited to any particular energy range.

  7. Evolution of eukaryotic single-stranded DNA viruses of the Bidnaviridae family from genes of four other groups of widely different viruses

    PubMed Central

    Krupovic, Mart; Koonin, Eugene V.

    2014-01-01

    Single-stranded (ss)DNA viruses are extremely widespread, infect diverse hosts from all three domains of life and include important pathogens. Most ssDNA viruses possess small genomes that replicate by the rolling-circle-like mechanism initiated by a distinct virus-encoded endonuclease. However, viruses of the family Bidnaviridae, instead of the endonuclease, encode a protein-primed type B DNA polymerase (PolB) and hence break this pattern. We investigated the provenance of all bidnavirus genes and uncover an unexpected turbulent evolutionary history of these unique viruses. Our analysis strongly suggests that bidnaviruses evolved from a parvovirus ancestor from which they inherit a jelly-roll capsid protein and a superfamily 3 helicase. The radiation of bidnaviruses from parvoviruses was probably triggered by integration of the ancestral parvovirus genome into a large virus-derived DNA transposon of the Polinton (polintovirus) family resulting in the acquisition of the polintovirus PolB gene along with terminal inverted repeats. Bidnavirus genes for a receptor-binding protein and a potential novel antiviral defense modulator are derived from dsRNA viruses (Reoviridae) and dsDNA viruses (Baculoviridae), respectively. The unusual evolutionary history of bidnaviruses emphasizes the key role of horizontal gene transfer, sometimes between viruses with completely different genomes but occupying the same niche, in the emergence of new viral types. PMID:24939392

  8. Role of the external NH2 linker on the conformation of surface immobilized single strand DNA probes and their SERS detection

    NASA Astrophysics Data System (ADS)

    He, Lijie; Langlet, Michel; Stambouli, Valerie

    2017-03-01

    The conformation and topological properties of DNA single strand probe molecules attached on solid surfaces are important, notably for the performances of devices such as biosensors. Commonly, the DNA probes are tethered to the surface using external linkers such as NH2. In this study, the role and influence of this amino-linker on the immobilization way and conformation of DNA probes on Ag nanoparticle surface is emphasized using Surface Enhanced Raman Spectroscopy (SERS). We compare the SERS spectra and their reproducibility in the case of two groups of DNA polybase probes which are polyA, polyC, polyT, and polyG. In the first group, the polybases exhibit an external NH2 functional linker while in the second group the polybases are NH2-free. The results show that the reproducibility of SERS spectra is enhanced in the case of the first group. It leads us to propose two models of polybase conformation on Ag surface according to the presence or the absence of the external NH2 linker. In the presence of the NH2 external linker, the latter would act as a major anchoring point. As a result, the polybases are much ordered with a less random orientation than in the case of NH2-free polybases. Consequently, in view of further in situ hybridization for biosensing applications, it is strongly recommended to use NH2 linker functionalized DNA probes.

  9. Interferon-α and interleukin-12 are induced, respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic cells.

    PubMed

    Katashiba, Yuichi; Miyamoto, Rie; Hyo, Akira; Shimamoto, Keiko; Murakami, Naoko; Ogata, Makoto; Amakawa, Ryuichi; Inaba, Muneo; Nomura, Shosaku; Fukuhara, Shirou; Ito, Tomoki

    2011-02-01

    Dendritic cells (DCs) are initiators of innate immunity and acquired immunity as cells linking these two bio-defence systems through the production of cytokines such as interferon-α (IFN-α) and interleukin-12 (IL-12). Nucleic acids such as DNA from damaged cells or pathogens are important activators not only for anti-microbial innate immune responses but also in the pathogenesis of IFN-related autoimmune diseases. Plasmacytoid DCs are regarded as the main effectors for the DNA-mediated innate immunity by possessing DNA-sensing toll-like receptor 9 (TLR9). We here found that double-stranded DNA (dsDNA) complexed with lipotransfectants triggered activation of human monocyte-derived DCs (moDCs), leading to the preferential production of IFN-α but not IL-12. This indicates that myeloid DCs also function as supportive effectors against the invasion of pathogenic microbes through the DNA-mediated activation in innate immunity. The dsDNA with lipotransfectants can be taken up by moDCs without co-localization of endosomal LAMP1 staining, and the dsDNA-mediated IFN-α production was not impaired by chloroquine. These findings indicate that moDC activation by dsDNA does not involve the endosomal TLR pathway. In contrast, single-stranded RNA (ssRNA) stimulated moDCs to secrete IL-12 but not IFN-α. This process was inhibited by chloroquine, suggesting an involvement of the TLR pathway in ssRNA-mediated moDC activation. As might be inferred from our findings, myeloid DCs may function as a traffic control between innate immunity via IFN-α production and acquired immunity via IL-12 production, depending on the type of nucleic acids. Our results provide a new insight into the biological action of myeloid DCs underlying the DNA-mediated activation of protective or pathogenic immunity.

  10. Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR Green I.

    PubMed

    Xiang, Dong-Shan; Zhou, Guo-Hua; Luo, Ming; Ji, Xing-Hu; He, Zhi-Ke

    2012-08-21

    We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.

  11. Identification of anisakid nematodes with zoonotic potential from Europe and China by single-strand conformation polymorphism analysis of nuclear ribosomal DNA.

    PubMed

    Zhu, X Q; Podolska, M; Liu, J S; Yu, H Q; Chen, H H; Lin, Z X; Luo, C B; Song, H Q; Lin, R Q

    2007-11-01

    Using genetic markers defined previously in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA), isotopic, and non-isotopic polymerase-chain-reaction-coupled single-strand conformation polymorphism (SSCP) were utilized to identify each of three anisakid species [Anisakis simplex (s.l.), Contracaecum osculatum (s.l.), and Hysterothylacium aduncum] from different host species and geographical locations in Poland and Sweden. While subtle microheterogeneity was observed within each of Anisakis simplex (s.l.) and H. aduncum, distinct SSCP profiles were displayed for each of the three species, allowing identification and differentiation of the three taxa. Subsequent sequencing of the ITS-1 and ITS-2 rDNA revealed that A. simplex (s.l.) represented Anisakis simplex s.s. and Contracaecum osculatum (s.l.) represented C. osculatum C. Application of the non-isotopic SSCP assay of ITS-2 to larval anisakid samples from different hosts and geographical locations in China revealed three distinct SSCP profiles, one of which was consistent with that of A. simplex (s.l.), and the other two had different SSCP profiles from that of C. osculatum C and H. aduncum. Sequencing of the ITS-1 and ITS-2 rDNA for representative Chinese anisakid samples examined revealed three anisakid species in China, i.e., Anisakis typica, Anisakis pegreffii, and Hysterothylacium sp. These molecular tools will be useful for identification and investigation of the ecology of anisakid nematodes in China and elsewhere.

  12. In vitro synthesis of large peptide molecules using glucosylated single-stranded bacteriophage T4D DNA template.

    PubMed Central

    Hulen, C; Legault-Demare, J

    1975-01-01

    Denatured Bacteriophage T4D DNA is able to stimulate aminoacid incorporation into TCA-precipitable material in an in vitro protein synthesis system according to base DNA sequences. Newly synthesized polypeptides remain associated with ribosomes and have a molecular weight in range of 15,000 to 45,000 Daltons. PMID:1052527

  13. RecQ helicase translocates along single-stranded DNA with a moderate processivity and tight mechanochemical coupling.

    PubMed

    Sarlós, Kata; Gyimesi, Máté; Kovács, Mihály

    2012-06-19

    Maintenance of genome integrity is the major biological role of RecQ-family helicases via their participation in homologous recombination (HR)-mediated DNA repair processes. RecQ helicases exert their functions by using the free energy of ATP hydrolysis for mechanical movement along DNA tracks (translocation). In addition to the importance of translocation per se in recombination processes, knowledge of its mechanism is necessary for the understanding of more complex translocation-based activities, including nucleoprotein displacement, strand separation (unwinding), and branch migration. Here, we report the key properties of the ssDNA translocation mechanism of Escherichia coli RecQ helicase, the prototype of the RecQ family. We monitored the pre-steady-state kinetics of ATP hydrolysis by RecQ and the dissociation of the enzyme from ssDNA during single-round translocation. We also gained information on the translocation mechanism from the ssDNA length dependence of the steady-state ssDNA-activated ATPase activity. We show that RecQ occludes 18 ± 2 nt on ssDNA during translocation. The hydrolysis of ATP is noncooperative in the presence of ssDNA, indicating that RecQ active sites work independently during translocation. In the applied conditions, the enzyme hydrolyzes 35 ± 4 ATP molecules per second during ssDNA translocation. RecQ translocates at a moderate processivity, with a mean run length of 100-320 nt on ssDNA. The determined tight mechanochemical coupling of 1.1 ± 0.2 ATP consumed per nucleotide traveled indicates an inchworm-type mechanism.

  14. Specificity of mutagenesis by 4-aminobiphenyl: mutations at G residues in bacteriophage M13 DNA and G-->C transversions at a unique dG(8-ABP) lesion in single-stranded DNA.

    PubMed

    Verghis, S B; Essigmann, J M; Kadlubar, F F; Morningstar, M L; Lasko, D D

    1997-12-01

    Mutagenesis by the human bladder carcinogen 4-aminobiphenyl (ABP) was studied in single-stranded DNA from a bacteriophage M13 cloning vector. In comparison to ABP lesions in double-stranded DNA, lesions in single-stranded DNA were approximately 70-fold more mutagenic and 50-fold more genotoxic. Sequencing analysis of ABP-induced mutations in the lacZ gene revealed exclusively base-pair substitutions, with over 80% of the mutations occurring at G sites; the G at position 6310 accounted for 25% of the observed mutations. Among the sequence changes at G sites, G-->T transversions predominated, followed by G-->C transversions and G-->A transitions. In order to further elucidate the mutagenic mechanism of ABP, an oligonucleotide containing the major DNA adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG(8-ABP)), was situated within the PstI site of a single-stranded M13 genome. After in vivo replication of the adduct containing ABP-modified and control (unadducted) genomes, the mutational frequency and mutational specificity of the dG(8-ABP) lesion were determined. The targeted mutational efficiency was approximately 0.01%, and the primary mutation observed was the G-->C transversion. Thus dG(8-ABP), albeit weakly mutagenic at the PstI site, can contribute to the mutational spectrum of ABP lesions.

  15. The role of molecular structure of sugar-phosphate backbone and nucleic acid bases in the formation of single-stranded and double-stranded DNA structures.

    PubMed

    Poltev, Valeri; Anisimov, Victor M; Danilov, Victor I; Garcia, Dolores; Sanchez, Carolina; Deriabina, Alexandra; Gonzalez, Eduardo; Rivas, Francisco; Polteva, Nina

    2014-06-01

    Our previous DFT computations of deoxydinucleoside monophosphate complexes with Na(+)-ions (dDMPs) have demonstrated that the main characteristics of Watson-Crick (WC) right-handed duplex families are predefined in the local energy minima of dDMPs. In this work, we study the mechanisms of contribution of chemically monotonous sugar-phosphate backbone and the bases into the double helix irregularity. Geometry optimization of sugar-phosphate backbone produces energy minima matching the WC DNA conformations. Studying the conformational variability of dDMPs in response to sequence permutation, we found that simple replacement of bases in the previously fully optimized dDMPs, e.g. by constructing Pyr-Pur from Pur-Pyr, and Pur-Pyr from Pyr-Pur sequences, while retaining the backbone geometry, automatically produces the mutual base position characteristic of the target sequence. Based on that, we infer that the directionality and the preferable regions of the sugar-phosphate torsions, combined with the difference of purines from pyrimidines in ring shape, determines the sequence dependence of the structure of WC DNA. No such sequence dependence exists in dDMPs corresponding to other DNA conformations (e.g., Z-family and Hoogsteen duplexes). Unlike other duplexes, WC helix is unique by its ability to match the local energy minima of the free single strand to the preferable conformations of the duplex.

  16. 5-Aminolevulinic acid induces single-strand breaks in plasmid pBR322 DNA in the presence of Fe2+ ions.

    PubMed

    Onuki, J; Medeiros, M H; Bechara, E J; Di Mascio, P

    1994-02-22

    5-Aminolevulinic acid (ALA), a heme precursor accumulated in chemical and inborn porphyrias, has been demonstrated to produce reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes and subcellular structures. Exposure of plasmid pBR322 DNA to ALA (0.01-3 mM) in the presence of 10 microM Fe2+ ions causes DNA single-strand breaks (ssb), revealed by agarose gel electrophoresis as an increase in the proportion of the open circular form (75 +/- 7.5% at 3 mM ALA) at the expense of the supercoiled form. Addition of either anti-oxidant enzymes such as superoxide dismutase (10 micrograms/ml) and catalase (20 micrograms/ml), or a metal chelator (DTPA, 2.5 mM), or a HO. scavenger (mannitol, 100 mM) inhibited the damage (by 30, 45, 55, and 81%, respectively), evidencing the involvement of O2-., H2O2 and HO. (by the Haber-Weiss reaction) in this process. Hydrogen peroxide (100 microM) or Fe2+ (10 microM) alone were of little effect on the extent of DNA ssb. The present data may shed light on the correlation reported between primary liver-cell carcinoma and intermittent acute porphyria.

  17. Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family Geminiviridae.

    PubMed

    Loconsole, Giuliana; Saldarelli, Pasquale; Doddapaneni, Harshavardhan; Savino, Vito; Martelli, Giovanni P; Saponari, Maria

    2012-10-10

    In the attempt to identify the causal agent of Citrus chlorotic dwarf disease (CCDD), a virus-like disorder of citrus, the small RNA fraction and total DNA from symptomatic citrus plants were subjected to high-throughput sequencing. DNA fragments deriving from an apparently new geminivirus-like agent were found and assembled by NGS to re-construct the entire viral genome. The newly identified virus has a circular single-stranded DNA genome comprising five open reading frames (ORFs) with sequence homologies with those encoded by geminiviruses. PCR and qPCR assays were successfully used for determining its presence in the CCDD-affected plants obtained by graft propagation. The larger genome size (3.64 vs. 2.5-3.0 kb) and a number of differences in its structural organization, identified this virus as a highly divergent member of the family Geminiviridae, to which the provisional name of Citrus chlorotic dwarf-associated virus (CCDaV) is assigned.

  18. The 4-nitroquinoline 1-oxide mutational spectrum in single stranded DNA is characterized by guanine to pyrimidine transversions.

    PubMed

    Fronza, G; Campomenosi, P; Iannone, R; Abbondandolo, A

    1992-03-25

    4-Nitroquinoline-1-oxide is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. In ds or ss damaged DNA the ratio C8/N2 adducts is 1:2 and 8-10:1, respectively. In bacteria and yeast 4NQO has been shown to be a base substitution mutagen acting at G residues inducing mainly G to A transitions. We determined the mutational spectrum induced by the 4NQO metabolite, acetoxy-4-aminoquinoline 1-oxide, in the M13lacZ'/E. coli lacZ delta M15 alpha complementation assay using ssDNA. Among 68 Ac-4HAQO induced mutants, G to Pyr transversion was the most frequent base substitution observed. By comparison with dsDNA based systems, our data suggest that dGuo-C8-AQO induces G to Pyr transversions. A mechanism to explain how this lesion may induce transversions is proposed.

  19. Genetic Transformation and Mutagenesis Via Single-Stranded DNA in the Unicellular, Diazotrophic Cyanobacteria of the Genus Cyanothece

    SciTech Connect

    Min, Hongtao; Sherman, Louis A.

    2010-11-01

    We describe a genetic system for producing specific gene knockouts in Cyanothece sp. Strain PCC 7822 using a ssDNA technique (37). The first fully segregated mutant was in the nifK gene (DnifK) and the mutant was unable to grow on a medium lacking combined nitrogen and produced virtually no hydrogen.

  20. Association of circulating antibodies against double-stranded and single-stranded DNA with thyroid autoantibodies in Graves' disease and Hashimoto's thyroiditis patients.

    PubMed

    Pedro, A B P; Romaldini, J H; Americo, C; Takei, K

    2006-01-01

    The occurrence of antinuclear antibody (ANA), rheumatoid factor (RF), antibodies to double-stranded DNA (anti-dsDNA) and to single-stranded DNA (anti-ssDNA) was investigated in 51 patients with autoimmune thyroid diseases (AITD), and in 25 matched control subjects. In comparison with controls, the prevalence of anti-dsDNA was 74.5% in AITD patients (p=0.0001), 82.0% in 39 hyperthyroid Graves' disease (GD) (p=0.0001), and 50.0% in 12 euthyroid Hashimoto's thyroiditis (HT) patients (p=0.0001). The prevalence of anti-ssDNA was 90.1% in AITD (94.8% in GD and 75% in HT; p=0.001). The concentration of both anti-dsDNA and anti-ssDNA were higher (p=0.002) in AITD, in GD (p=0.001), and in HT (p=0.01) patients than in controls. Two patients with AITD were identified as positive for ANA. RF was detected in 4 AITD patients. Positive correlation was noted between anti-dsDNA with T4 (p=0.001), T3 (p=0.002), thyroid peroxidase antibody (anti-TPO) (p=0.0001), and TSH (p=0.001) values but not with thyroglobulin antibody (anti-Tg). Serum anti-ssDNA values were also correlated with T3 (p=0.0001), TSH (p=0.003), and anti-TPO (p=0.0001). However, by using a multiple regression analysis only anti-TPO remained associated with anti-dsDNA and both anti-Tg and anti-TPO with anti-ssDNA values. The predisposition to develop systemic autoimmune disorders is not influenced by thyroid hormones. The elevated prevalence of serum anti-dsDNA and anti-ssDNA in AITD patients points out that we must be aware of the risk for predisposition for the development of other systemic autoimmune diseases.

  1. Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability.

    PubMed

    Feng, Yuqing; Love, Robin P; Ara, Anjuman; Baig, Tayyba T; Adolph, Madison B; Chelico, Linda

    2015-11-06

    APOBEC3H is a deoxycytidine deaminase that can restrict the replication of HIV-1 in the absence of the viral protein Vif that induces APOBEC3H degradation in cells. APOBEC3H exists in humans as seven haplotypes (I-VII) with different cellular stabilities. Of the three stable APOBEC3H haplotypes (II, V, and VII), haplotypes II and V occur most frequently in the population. Despite APOBEC3H being a bona fide restriction factor, there has been no comparative biochemical characterization of APOBEC3H haplotypes. We characterized the ssDNA scanning mechanisms that haplotypes II and V use to search their ssDNA substrate for cytosine-containing deamination motifs. APOBEC3H haplotype II was able to processively deaminate multiple cytosines in a single enzyme-substrate encounter by using sliding, jumping, and intersegmental transfer movements. In contrast, APOBEC3H haplotype V exhibited diminished sliding and intersegmental transfer abilities but was able to jump along ssDNA. Due to an Asp or Glu at amino acid 178 differentiating these APOBEC3H haplotypes, the data indicated that this amino acid on helix 6 contributes to processivity. The diminished processivity of APOBEC3H haplotype V did not result in a reduced efficiency to restrict HIV-1 replication in single-cycle infectivity assays, suggesting a redundancy in the contributions of jumping and intersegmental transfer to mutagenic efficiency. Optimal processivity on ssDNA also required dimerization of APOBEC3H through the β2 strands. The findings support a model in which jumping can compensate for deficiencies in intersegmental transfer and suggest that APOBEC3H haplotypes II and V induce HIV-1 mutagenesis efficiently but by different mechanisms.

  2. Detection of Benzo[a]pyrene-Guanine Adducts in Single-Stranded DNA using the α-Hemolysin Nanopore

    PubMed Central

    Perera, Rukshan T.; Fleming, Aaron M.; Johnson, Robert P.; Burrows, Cynthia J.; White, Henry S.

    2017-01-01

    The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2′-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5′ or 3′ directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples. PMID:25629967

  3. Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore.

    PubMed

    Perera, Rukshan T; Fleming, Aaron M; Johnson, Robert P; Burrows, Cynthia J; White, Henry S

    2015-02-20

    The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2'-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5' or 3' directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

  4. Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore

    NASA Astrophysics Data System (ADS)

    Perera, Rukshan T.; Fleming, Aaron M.; Johnson, Robert P.; Burrows, Cynthia J.; White, Henry S.

    2015-02-01

    The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2‧-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5‧ or 3‧ directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

  5. Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique--isoelectric focusing of proteins and a single-stranded DNA fragment.

    PubMed

    Walowski, Britta; Hüttner, Wilhelm; Wackerbarth, Hainer

    2011-11-01

    Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (μFFE) chip with a filling capacity of 9.5 μL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm(-1) and 422 V cm(-1), depending on the application.

  6. Borrelia burgdorferi cp32 BpaB Modulates Expression of the Prophage NucP Nuclease and SsbP Single-Stranded DNA-Binding Protein

    PubMed Central

    Chenail, Alicia M.; Jutras, Brandon L.; Adams, Claire A.; Burns, Logan H.; Bowman, Amy; Verma, Ashutosh

    2012-01-01

    The Borrelia burgdorferi BpaB proteins of the spirochete's ubiquitous cp32 prophages are DNA-binding proteins, required both for maintenance of the bacteriophage episomes and for transcriptional regulation of the cp32 erp operons. Through use of DNase I footprinting, we demonstrate that BpaB binds the erp operator initially at the sequence 5′-TTATA-3′. Electrophoretic mobility shift assays indicated that BpaB also binds with high affinity to sites located in the 5′ noncoding regions of two additional cp32 genes. Characterization of the proteins encoded by those genes indicated that they are a single-stranded DNA-binding protein and a nuclease, which we named SsbP and NucP, respectively. Chromatin immunoprecipitation indicated that BpaB binds erp, ssbP, and nucP in live B. burgdorferi. A mutant bacterium that overexpressed BpaB produced significantly higher levels of ssbP and nucP transcript than did the wild-type parent. PMID:22730122

  7. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Clostridium difficile Toxin B and Sensitive Detection in Human Fecal Matter

    PubMed Central

    Maher, Eamonn; Williams, Ryan M.; Sooter, Letha J.

    2015-01-01

    Toxin B is one of the major virulence factors of Clostridium difficile, a bacterium that is responsible for a significant number of diarrhea cases in acute care settings. Due to the prevalence of C. difficile induced diarrhea, rapid and correct diagnosis is crucial in the disease management. In this study, we have employed a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for toxin B. At the end of the 12-round selection, one MRE with high affinity (Kd = 47.3 nM) for toxin B was identified. The selected MRE demonstrated low cross binding activities on negative targets: bovine serum albumin, Staphylococcus aureus alpha toxin, Pseudomonas aeruginosa exotoxin A, and cholera toxin of Vibrio cholera. A modified sandwich ELISA assay was developed utilizing the selected ssDNA MRE as the antigen capturing element and achieved a sensitive detection of 50 nM of toxin B in human fecal preparations. PMID:25734010

  8. In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

    PubMed Central

    Hong, Ka L.; Battistella, Luisa; Salva, Alysia D.; Williams, Ryan M.; Sooter, Letha J.

    2015-01-01

    Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved. PMID:25633102

  9. Anti-cardiolipin antibodies in neurological disorders: cross-reaction with anti-single stranded DNA activity.

    PubMed Central

    Colaço, C B; Scadding, G K; Lockhart, S

    1987-01-01

    Antiphospholipid (PL) antibodies have been detected in sera from patients with chronic neurological diseases associated with disorders of immunity. In an isotype specific radioimmunoassay for anti-cardiolipin (CL) antibodies, we found IgM anti-CL (greater than 2 s.d. above mean of controls) in 17/25 (68%) patients with myasthenia gravis (MG), 8/25 (32%) with the Lambert-Eaton myasthenic syndrome (LEMS), 5/17 (29%) with multiple sclerosis and 3/11 (27%) cases of migraine. IgG anti-CL was only found in low titres in sera from 10 patients with MG and three with LEMS. Significant anti-CL activity could not be detected in sera from nine patients with acute Guillain-Barré Syndrome (GBS), 12 chronic cases of epilepsy, 8/9 with oat cell carcinoma and 9/10 with acute stroke. Further tests on 39 sera with the highest anti-CL activity, from all of the above disease groups, showed a significant correlation between IgM anti-CL and IgM anti-ss DNA activities. In a series of competitive inhibition assays six sera from patients with MG were shown to have a proportion of both specific and cross-reactive IgM anti-CL and IgM anti-ss DNA antibodies. Anti-phospholipid antibodies occur in certain neurological diseases, at lower titres than seen in SLE, yet their cross-reactive binding to ss DNA suggests similar antibacterial origins as have been proposed for lupus auto-antibodies. In the absence of overt infection they might reflect a breakdown of tolerance for non-organ specific membrane antigens in diseases with predominantly organ specific membrane bound putative autoimmunogens. PMID:3498571

  10. Targeted guanine oxidation by a dinuclear copper(II) complex at single stranded/double stranded DNA junctions.

    PubMed

    Li, Lei; Murthy, Narasimha N; Telser, Joshua; Zakharov, Lev N; Yap, Glenn P A; Rheingold, Arnold L; Karlin, Kenneth D; Rokita, Steven E

    2006-09-04

    A dinuclear copper(II) complex [Cu(II)2(PD'O-)(H2O)2](ClO4)3 (5) with terminal Cu(II)-H(2)O moieties and a Cu...Cu distance of 4.13 A (X-ray structure) has been synthesized and characterized by EPR spectroscopy (ferromagnetic coupling observed) and cyclic voltammetry. Dizinc(II) and mononuclear copper(II) analogues [Zn(II)2(PD'O-)(H2O)2]3+ (7) and [Cu(II)(mPD'OH)(H2O)]2+ (6), respectively, have also been synthesized and structurally characterized. Reacting 5/MPA/O(2) (MPA = 3-mercaptopropionic acid) with DNA leads to a highly specific oxidation of guanine (G) at a junction between single- and double-stranded DNA. Mass spectrometric analysis of the major products indicates a gain of +18 and +34 amu relative to initial DNA strands. The most efficient reaction requires G at the first and second unpaired positions of each strand extending from the junction. Less reaction is observed for analogous targets in which the G cluster is farther from the junction or contains less than four Gs. Consistent with our previous systems, the multinuclear copper center is required for selective reaction; mononuclear complex 6 is not effective. Hydrogen peroxide as a substitute for MPA/O2 also does not lead to activity. Structural analysis of a [Cu(II)2(PD'O-)(G)]3+ complex (8) and dizinc analogue [Zn(II)(2)(PD'O-)(G)](ClO4)3 (9) (G = guanosine) reveals coordination of the G O6 and N7 atoms with the two copper (or zinc) centers and suggests that copper-G coordination likely plays a role in recognition of the DNA target. The Cu2-O2 intermediate responsible for guanine oxidation appears to be different from that responsible for direct-strand scission induced by other multinuclear copper complexes; the likely course of reaction is discussed.

  11. Repression of the Defense Gene PR-10a by the Single-Stranded DNA Binding Protein SEBF

    PubMed Central

    Boyle, Brian; Brisson, Normand

    2001-01-01

    The potato pathogenesis-related gene PR-10a is transcriptionally activated in response to pathogen infection or elicitor treatment. Characterization of the cis-acting elements of the PR-10a promoter revealed the presence of a silencing element between residues −52 and −27 that contributes to transcriptional regulation. In this study, we have isolated a silencing element binding factor (SEBF) from potato tuber nuclei that binds to the coding strand of the silencing element in a sequence-specific manner. The consensus binding site of SEBF, PyTGTCNC, is present in a number of PR genes and shows striking similarity to the auxin response element. Mutational analysis of the PR-10a promoter revealed an inverse correlation between the in vitro binding of SEBF and the expression of PR-10a. SEBF was purified to homogeneity from potato tubers, and sequencing of the N terminus of the protein led to the isolation of a cDNA clone. Sequence analysis revealed that SEBF is homologous with chloroplast RNA binding proteins that possess consensus sequence–type RNA binding domains characteristic of heterogenous nuclear ribonucleoproteins (hnRNPs). Overexpression of SEBF in protoplasts repressed the activity of a PR-10a reporter construct in a silencing element–dependent manner, confirming the role of SEBF as a transcriptional repressor. PMID:11701886

  12. Pleolipoviridae, a newly proposed family comprising archaeal pleomorphic viruses with single-stranded or double-stranded DNA genomes.

    PubMed

    Pietilä, Maija K; Roine, Elina; Sencilo, Ana; Bamford, Dennis H; Oksanen, Hanna M

    2016-01-01

    Viruses infecting archaea show a variety of virion morphotypes, and they are currently classified into more than ten viral families or corresponding groups. A pleomorphic virus morphotype is very common among haloarchaeal viruses, and to date, several such viruses have been isolated. Here, we propose the classification of eight such viruses and formation of a new family, Pleolipoviridae (from the Greek pleo for more or many and lipos for lipid), containing three genera, Alpha-, Beta-, and Gammapleolipovirus. The proposal is currently under review by the International Committee on Taxonomy of Viruses (ICTV). The members of the proposed family Pleolipoviridae infect halophilic archaea and are nonlytic. They share structural and genomic features and differ from any other classified virus. The virion of pleolipoviruses is composed of a pleomorphic membrane vesicle enclosing the genome. All pleolipoviruses have two major structural protein species, internal membrane and spike proteins. Although the genomes of the pleolipoviruses are single- or double-stranded, linear or circular DNA molecules, they share the same genome organization and gene synteny and show significant similarity at the amino acid level. The canonical features common to all members of the proposed family Pleolipoviridae show that they are closely related and thus form a new viral family.

  13. Efficient single-strand cleavage of DNA mediated by a MnIIIMnIV-based artificial nuclease.

    PubMed

    Qian, Jing; Yu, Shasha; Wang, Wenjun; Wang, Liping; Tian, Jinlei; Yan, Shiping

    2014-02-14

    A water-soluble Mn(IV) 1,4,7-triazacyclononane complex, [Mn(IV)2L2(μ-O)2](ClO4)2·2H2O (1), was prepared to serve as a nuclease mimic (L = 1,4,7-triazacyclononane-N-acetate). Complex 1 was readily synthesized from the highly water soluble ligand (L), with Mn(III) salt, [Mn3O(MeCO2)7]·3H2O in basic condition, and characterized by X-ray, IR, electronic spectroscopy, cyclic voltammetry and magnetic susceptibility as well as ESI-MS. The bond valence sum (BVS) analysis and magnetic data suggest that 1 is a Mn(IV)-μ-O2-Mn(IV) species. The electrospray mass spectrum and electronic spectrum of 1 in aqueous solution indicates that dinuclear Mn complex [Mn(III)Mn(IV)L2(μ-O)2](+) (2) is the active species. A predominantly hydrolytic cleavage mechanism was confirmed through experiments performed in the presence of various radical scavengers, T4 ligase and under anaerobic conditions. The kinetic aspects of DNA cleavage under pseudo- or true-Michaelis-Menten conditions were also detailed, kinetic parameters (kcat, KM, Vmax) were calculated to be 6.27 h(-1), 7.35 × 10(-5) M, 4.6 × 10(-4) M h(-1); 0.683 h(-1), 1.93 × 10(-5) M, 1.32 × 10(-5) M h(-1) for 2, respectively.

  14. Cationic phosphoramidate α-oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis C virus IRES-mediated translation

    PubMed Central

    Michel, Thibaut; Martinand-Mari, Camille; Debart, Françoise; Lebleu, Bernard; Robbins, Ian; Vasseur, Jean-Jacques

    2003-01-01

    A potential means to improve the efficacy of steric-blocking antisense oligonucleotides (ON) is to increase their affinity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electrostatic repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucleoside linkages into ON with a non-natural α-anomeric configuration. Cationic α-ON bound with high affinity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modifications, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic α-ON and their RNA targets were not substrates for RNase H. The penalty in Tm inflicted by a single mismatch, however, was high; suggesting that they are well suited as sequence-specific, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have confirmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic α-ON in cell culture. PMID:12954764

  15. Insights into electron tunneling across hydrogen-bonded base-pairs in complete molecular circuits for single-stranded DNA sequencing.

    PubMed

    Lee, Myeong H; Sankey, Otto F

    2009-01-21

    We report a first-principles study of electron ballistic transport through a molecular junction containing deoxycytidine-monophosphate (dCMP) connected to metal electrodes. A guanidinium ion and guanine nucleobase are tethered to gold electrodes on opposite sides to form hydrogen bonds with the dCMP molecule providing an electric circuit. The circuit mimics a component of a potential device for sequencing unmodified single-stranded DNA. The molecular conductance is obtained from DFT Green's function scattering methods and is compared to estimates from the electron tunneling decay constant obtained from the complex band structure. The result is that a complete molecular dCMP circuit of 'linker((CH(2))(2))-guanidinium-phosphate-deoxyribose-cytosine-guanine' has a very low conductance (of the order of fS) while the hydrogen-bonded guanine-cytosine base-pair has a moderate conductance (of the order of tens to hundreds of nS). Thus, while the transverse electron transfer through base-pairing is moderately conductive, electron transfer through a complete molecular dCMP circuit is not. The gold Fermi level is found to be aligned very close to the HOMO for both the guanine-cytosine base-pair and the complete molecular dCMP circuit. Results for two different plausible geometries of the hydrogen-bonded dCMP molecule reveal that the conductance varies from fS for an extended structure to pS for a slightly compressed structure.

  16. Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism.

    PubMed

    Carter, Sidney D; Vigasová, Dana; Chen, Jiang; Chovanec, Miroslav; Aström, Stefan U

    2009-07-21

    Double-strand breaks (DSBs) represent the most severe DNA lesion a cell can suffer, as they pose the risk of inducing loss of genomic integrity and promote oncogenesis in mammals. Two pathways repair DSBs, nonhomologous end joining (NHEJ) and homologous recombination (HR). With respect to mechanism and genetic requirements, characterization of these pathways has revealed a large degree of functional separation between the two. Nej1 is a cell-type specific regulator essential to NHEJ in Saccharomyces cerevisiae. Srs2 is a DNA helicase with multiple roles in HR. In this study, we show that Nej1 physically interacts with Srs2. Furthermore, mutational analysis of Nej1 suggests that the interaction was strengthened by Dun1-dependent phosphorylation of Nej1 serines 297/298. Srs2 was previously shown to be recruited to replication forks, where it promotes translesion DNA synthesis. We demonstrate that Srs2 was also efficiently recruited to DSBs generated by the HO endonuclease. Additionally, efficient Srs2 recruitment to this DSB was dependent on Nej1, but independent of mechanisms facilitating Srs2 recruitment to replication forks. Functionally, both Nej1 and Srs2 were required for efficient repair of DSBs with 15-bp overhangs, a repair event reminiscent of a specific type of HR called single-strand annealing (SSA). Moreover, absence of Rad51 suppressed the SSA-defect in srs2 and nej1 strains. We suggest a model in which Nej1 recruits Srs2 to DSBs to promote NHEJ/SSA-like repair by dismantling inappropriately formed Rad51 nucleoprotein filaments. This unexpected link between NHEJ and HR components may represent cross-talk between DSB repair pathways to ensure efficient repair.

  17. Nej1 recruits the Srs2 helicase to DNA double-strand breaks and supports repair by a single-strand annealing-like mechanism

    PubMed Central

    Carter, Sidney D.; Vigašová, Dana; Chen, Jiang; Chovanec, Miroslav; Åström, Stefan U.

    2009-01-01

    Double-strand breaks (DSBs) represent the most severe DNA lesion a cell can suffer, as they pose the risk of inducing loss of genomic integrity and promote oncogenesis in mammals. Two pathways repair DSBs, nonhomologous end joining (NHEJ) and homologous recombination (HR). With respect to mechanism and genetic requirements, characterization of these pathways has revealed a large degree of functional separation between the two. Nej1 is a cell-type specific regulator essential to NHEJ in Saccharomyces cerevisiae. Srs2 is a DNA helicase with multiple roles in HR. In this study, we show that Nej1 physically interacts with Srs2. Furthermore, mutational analysis of Nej1 suggests that the interaction was strengthened by Dun1-dependent phosphorylation of Nej1 serines 297/298. Srs2 was previously shown to be recruited to replication forks, where it promotes translesion DNA synthesis. We demonstrate that Srs2 was also efficiently recruited to DSBs generated by the HO endonuclease. Additionally, efficient Srs2 recruitment to this DSB was dependent on Nej1, but independent of mechanisms facilitating Srs2 recruitment to replication forks. Functionally, both Nej1 and Srs2 were required for efficient repair of DSBs with 15-bp overhangs, a repair event reminiscent of a specific type of HR called single-strand annealing (SSA). Moreover, absence of Rad51 suppressed the SSA-defect in srs2 and nej1 strains. We suggest a model in which Nej1 recruits Srs2 to DSBs to promote NHEJ/SSA-like repair by dismantling inappropriately formed Rad51 nucleoprotein filaments. This unexpected link between NHEJ and HR components may represent cross-talk between DSB repair pathways to ensure efficient repair. PMID:19571008

  18. The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides.

    PubMed

    Rivera-Torres, Natalia; Strouse, Bryan; Bialk, Pawel; Niamat, Rohina A; Kmiec, Eric B

    2014-01-01

    With recent technological advances that enable DNA cleavage at specific sites in the human genome, it may now be possible to reverse inborn errors, thereby correcting a mutation, at levels that could have an impact in a clinical setting. We have been developing gene editing, using single-stranded DNA oligonucleotides (ssODNs), as a tool to direct site specific single base changes. Successful application of this technique has been demonstrated in many systems ranging from bacteria to human (ES and somatic) cells. While the frequency of gene editing can vary widely, it is often at a level that does not enable clinical application. As such, a number of stimulatory factors such as double-stranded breaks are known to elevate the frequency significantly. The majority of these results have been discovered using a validated HCT116 mammalian cell model system where credible genetic and biochemical readouts are available. Here, we couple TAL-Effector Nucleases (TALENs) that execute specific ds DNA breaks with ssODNs, designed specifically to repair a missense mutation, in an integrated single copy eGFP gene. We find that proximal cleavage, relative to the mutant base, is key for enabling high frequencies of editing. A directionality of correction is also observed with TALEN activity upstream from the target base being more effective in promoting gene editing than activity downstream. We also find that cells progressing through S phase are more amenable to combinatorial gene editing activity. Thus, we identify novel aspects of gene editing that will help in the design of more effective protocols for genome modification and gene therapy in natural genes.

  19. High sensitivity of the single-strand conformation polymorphism method for detecting sequence variations in the low-density lipoprotein receptor gene validated by DNA sequencing.

    PubMed

    Jensen, H K; Jensen, L G; Hansen, P S; Faergeman, O; Gregersen, N

    1996-08-01

    We designed oligonucleotide primer pairs to amplify the promoter region, the translated exon sequences, and the flanking intron sequences of all 18 exons of the LDL receptor gene to compare the ability of the PCR single-strand conformation polymorphism (PCR-SSCP) method with semiautomated solid-phase genomic DNA sequencing to detect sequence variations. In 20 apparently unrelated Danish patients with a clinical diagnosis of heterozygous familial hypercholesterolemia (FH), we identified 13 different mutations in the LDL receptor gene: two silent (C331C, N494 N); five missense (W66G, E119K, T383P, W556S, T7051); one nonsense (W23X); three splice-site (313 + 1G-->A, 1061-8T-->C, 1846-1G-->A); and two frameshift (335del10, 1650delG) mutations. Four of these mutations, N494 N, T383P, 1061-8T-->C, and W556S, have not been reported earlier. The pathogenicity of the T383P, 1061-8T-->C, and W556S mutations remains to be established by in vitro mutagenesis and transfection studies. One patient had three mutations (335del10, 1061-8T-->C, and T705I) on the same allele. Further, nine well-known polymorphisms were detectable with this methodological setup. Direct DNA sequencing of the PCR products used for the SSCP analysis did not reveal any sequence variations not detected by the PCR-SSCP method. In two patients we did not detect any mutation by either method. We conclude that the PCR-SSCP analysis, performed as described here, is as sensitive and efficient as DNA sequencing in the ability to identify the sequence variations in the LDL receptor gene of the patients with heterozygous FH of this study.

  20. The induction/loss of the oxidant-resistant phenotype of Chinese hamster ovary (CHO) cell variants does not correlate with sensitivity to DNA single strand breakage by hydrogen peroxide.

    PubMed

    Sestili, P; Cattabeni, F; Cantoni, O

    1994-11-01

    Hydrogen peroxide resistant variants of Chinese hamster ovary (CHO) cells characterized by different levels of resistance to growth inhibition induced by the oxidant displayed a decreased susceptibility to the induction of DNA single strand breakage by hydrogen peroxide. Resistance to DNA damage, however, was maximal in cells resistant to killing by low concentrations of H2O, and did not increase further in cells characterized by a much higher resistance to the toxic action of the oxidant. Different sensitivities to the induction of DNA single strand breakage observed in wild type and resistant sublines were related to a decreased susceptibility/differential depletion of H2O2, rather than being dependent on different velocities in DNA repair processes. Growth of resistant cells in the absence of H2O2 resulted in a rapid loss of resistance to induction of DNA strand scission by H2O2. Cells retained resistance to the growth-inhibitory effect of the oxidant under conditions where resistance to the production of DNA single strand breaks was lost. Experiments aimed at elucidating the molecular basis for resistance to DNA damage induction by H2O2 have demonstrated that this effect is dependent upon the catalase activity of the specific sublines as well as on their different total protein content.

  1. Different Mutagenic Potential of HIV-1 Restriction Factors APOBEC3G and APOBEC3F Is Determined by Distinct Single-Stranded DNA Scanning Mechanisms

    PubMed Central

    Ara, Anjuman; Love, Robin P.; Chelico, Linda

    2014-01-01

    The APOBEC3 deoxycytidine deaminase family functions as host restriction factors that can block replication of Vif (virus infectivity factor) deficient HIV-1 virions to differing degrees by deaminating cytosines to uracils in single-stranded (−)HIV-1 DNA. Upon replication of the (−)DNA to (+)DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils, thereby inducing C/G→T/A mutations that can functionally inactivate HIV-1. Although both APOBEC3F and APOBEC3G are expressed in cell types HIV-1 infects and are suppressed by Vif, there has been no prior biochemical analysis of APOBEC3F, in contrast to APOBEC3G. Using synthetic DNA substrates, we characterized APOBEC3F and found that similar to APOBEC3G; it is a processive enzyme and can deaminate at least two cytosines in a single enzyme-substrate encounter. However, APOBEC3F scanning movement is distinct from APOBEC3G, and relies on jumping rather than both jumping and sliding. APOBEC3F jumping movements were also different from APOBEC3G. The lack of sliding movement from APOBEC3F is due to an 190NPM192 motif, since insertion of this motif into APOBEC3G decreases its sliding movements. The APOBEC3G NPM mutant induced significantly less mutations in comparison to wild-type APOBEC3G in an in vitro model HIV-1 replication assay and single-cycle infectivity assay, indicating that differences in DNA scanning were relevant to restriction of HIV-1. Conversely, mutation of the APOBEC3F 191Pro to 191Gly enables APOBEC3F sliding movements to occur. Although APOBEC3F 190NGM192 could slide, the enzyme did not induce more mutagenesis than wild-type APOBEC3F, demonstrating that the unique jumping mechanism of APOBEC3F abrogates the influence of sliding on mutagenesis. Overall, we demonstrate key differences in the impact of APOBEC3F- and APOBEC3G-induced mutagenesis on HIV-1 that supports a model in which both the processive DNA scanning mechanism and preferred deamination motif (APOBEC3F, 5

  2. Analyses of the interaction between the origin binding domain from simian virus 40 T antigen and single-stranded DNA provide insights into DNA unwinding and initiation of DNA replication.

    PubMed

    Reese, Danielle K; Meinke, Gretchen; Kumar, Anuradha; Moine, Stephanie; Chen, Kathleen; Sudmeier, James L; Bachovchin, William; Bohm, Andrew; Bullock, Peter A

    2006-12-01

    DNA helicases are essential for DNA metabolism; however, at the molecular level little is known about how they assemble or function. Therefore, as a model for a eukaryotic helicase, we are analyzing T antigen (T-ag) the helicase encoded by simian virus 40. In this study, nuclear magnetic resonance (NMR) methods were used to investigate the transit of single-stranded DNA (ssDNA) through the T-ag origin-binding domain (T-ag OBD). When the residues that interact with ssDNA are viewed in terms of the structure of a hexamer of the T-ag OBD, comprised of residues 131 to 260, they indicate that ssDNA passes over one face of the T-ag OBD and then transits through a gap in the open ring structure. The NMR-based conclusions are supported by an analysis of previously described mutations that disrupt critical steps during the initiation of DNA replication. These and related observations are discussed in terms of the threading of DNA through T-ag hexamers and the initiation of viral DNA replication.

  3. Analyses of the Interaction between the Origin Binding Domain from Simian Virus 40 T Antigen and Single-Stranded DNA Provide Insights into DNA Unwinding and Initiation of DNA Replication▿

    PubMed Central

    Reese, Danielle K.; Meinke, Gretchen; Kumar, Anuradha; Moine, Stephanie; Chen, Kathleen; Sudmeier, James L.; Bachovchin, William; Bohm, Andrew; Bullock, Peter A.

    2006-01-01

    DNA helicases are essential for DNA metabolism; however, at the molecular level little is known about how they assemble or function. Therefore, as a model for a eukaryotic helicase, we are analyzing T antigen (T-ag) the helicase encoded by simian virus 40. In this study, nuclear magnetic resonance (NMR) methods were used to investigate the transit of single-stranded DNA (ssDNA) through the T-ag origin-binding domain (T-ag OBD). When the residues that interact with ssDNA are viewed in terms of the structure of a hexamer of the T-ag OBD, comprised of residues 131 to 260, they indicate that ssDNA passes over one face of the T-ag OBD and then transits through a gap in the open ring structure. The NMR-based conclusions are supported by an analysis of previously described mutations that disrupt critical steps during the initiation of DNA replication. These and related observations are discussed in terms of the threading of DNA through T-ag hexamers and the initiation of viral DNA replication. PMID:17005644

  4. Leishmania major: genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer.

    PubMed

    Tashakori, Mahnaz; Mahnaz, Tashakori; Kuhls, Katrin; Katrin, Kuhls; Al-Jawabreh, Amer; Amer, Al-Jawabreh; Mauricio, Isabel L; Isabel, Mauricio; Schönian, Gabriele; Gabriele, Schönian; Farajnia, Safar; Safar, Farajnia; Alimohammadian, Mohammad Hossein; Hossein, Alimohammadian Mohammad

    2006-04-01

    Protozoan parasites of Leishmania major are the causative agents of cutaneous leishmaniasis in different parts of Iran. We applied PCR-based methods to analyze L. major parasites isolated from patients with active lesions from different geographic areas in Iran in order to understand DNA polymorphisms within L. major species. Twenty-four isolates were identified as L. major by RFLP analysis of the ribosomal internal transcribed spacer 1 (ITS1) amplicons. These isolates were further studied by single-strand conformation polymorphism (SSCP) analysis and sequencing of ITS1 and ITS2. Data obtained from SSCP analysis of the ITS1 and ITS2 loci revealed three and four different patterns among all studied samples, respectively. Sequencing of ITS1 and ITS2 confirmed the results of SSCP analysis and showed the potential of the PCR-SSCP method for assessing genetic heterogeneity within L. major. Different patterns in ITS1 were due to substitution of one nucleotide, whereas in ITS2 the changes were defined by variation in the number of repeats in two polymorphic microsatellites. In total five genotypic groups LmA, LmB, LmC, LmD and LmE were identified among L. major isolates. The most frequent genotype, LmA, was detected in isolates collected from different endemic areas of cutaneous leishmaniasis in Iran. Genotypes LmC, LmD and LmE were found only in the new focus of CL in Damghan (Semnan province) and LmB was identified exclusively among isolates of Kashan focus (Isfahan province). The distribution of genetic polymorphisms suggests the existence of distinct endemic regions of L. major in Iran.

  5. Aqueous extracts of selenium-fertilized broccoli increase selenoprotein activity and inhibit DNA single-strand breaks, but decrease the activity of quinone reductase in Hepa 1c1c7 cells.

    PubMed

    Keck, Anna-Sigrid; Finley, John W

    2006-05-01

    Depending on growth conditions, broccoli may be enriched in the isothiocyanate sulforaphane and/or the mineral selenium (Se); both compounds may play an important role in the reduction of intracellular oxidative stress and chronic disease prevention. Sulforaphane up-regulates transcription of Phase II detoxification proteins (e.g. quinone reductase [QR]), whereas Se is needed for the production of thioredoxin reductase (TR) and glutathione peroxidase-1 (GPx1), both of which exhibit antioxidant activity. The objective of the present study was to determine whether the fertilization of broccoli with Se increases the antioxidant ability of broccoli. Hydrogen peroxide-induced DNA single-strand breaks (measured by single cell electrophoresis, Comet assay) and activity of antioxidant enzymes (GPx, TR and QR) were measured in mouse hepatoma cells (Hepa 1c1c7 cells) treated with purified sulforaphane, sodium selenite or extracts of selenized broccoli. When supplied separately as chemically pure substances, sodium selenite was more effective than sulforaphane for reduction of single-strand breaks. Se-fertilized broccoli extracts were the most effective for reduction of DNA single-strand breaks, and extracts that contained 0.71 microM Se and 0.08 microM sulforaphane inhibited 94% of DNA single-strand breaks. A significant positive association (r = 0.81, p = 0.009) between GPx1 activity and inhibition of DNA single-strand breaks as well as a 24h lag time between addition of Se, sulforaphane or broccoli extract and inhibition of single-strand breaks suggests that some of the antioxidant protection is mediated through selenoproteins. Conversely, fertilization of broccoli with Se decreased the ability of broccoli extract to induce QR activity. These results demonstrate that Se and sulforaphane, alone or as a component of broccoli, may help decrease oxidative stress. They further suggest that Se is the most important for decreasing oxidative stress, but maximizing the Se content

  6. Effect of long-term removal of iron from asbestos by desferrioxamine B on subsequent mobilization by other chelators and induction of DNA single-strand breaks.

    PubMed

    Chao, C C; Aust, A E

    1994-01-01

    The long-term removal of iron from crocidolite or amosite by desferrioxamine B (DF) at pH 7.5 or 5.0 was studied. Crocidolite or amosite (1 mg/ml) was suspended in 50 mM NaCl at pH 7.5 or 5.0 with the addition of 1 mM DF for up to 90 days. Although the rate of iron mobilization decreased with time, iron was continuously mobilized from both forms of asbestos at pH 5.0 or 7.5. The amount of iron mobilized from crocidolite was at least twice that mobilized from amosite at either pH. Iron was mobilized more rapidly from crocidolite at pH 5.0 than at 7.5 for the first 15 days, but at later times the amount being mobilized at pH 7.5 became equal to or slightly greater than that at 5.0. For amosite, the mobilization at pH 5.0 was always greater than that at pH 7.5. Next, the effect of iron removal from asbestos by DF on subsequent iron mobilization by a second chelator (EDTA or citrate) and on induction of DNA single-strand breaks (SSBs) was studied. Asbestos, treated for up to 15 days with DF at pH 7.5, was washed to remove ferrioxamine and excess DF, then incubated with EDTA or citrate (1 mM). The rates of iron mobilization from both forms of asbestos by a second chelator decreased as more and more iron was removed by DF. Induction of DNA SSBs also decreased, reflecting the unavailability of iron to catalyze the damage. The results suggest three things. First, if long-term mobilization of iron from asbestos occurs in vivo as has been observed in vitro, it may play a role in the long-term biological effects of asbestos. Second, more rapid mobilization of iron from asbestos fibers may occur when the fibers are phagocytized by cells and maintained in phagosomes where the pH is 4.0-5.0. Third, treatment of asbestos by iron chelators, such as DF, prior to exposure to cultured cells or whole animals, may reduce the biological effects of asbestos resulting from iron, but may not completely eliminate them.

  7. Direct observation of single flexible polymers using single stranded DNA†

    PubMed Central

    Brockman, Christopher; Kim, Sun Ju

    2012-01-01

    Over the last 15 years, double stranded DNA (dsDNA) has been used as a model polymeric system for nearly all single polymer dynamics studies. However, dsDNA is a semiflexible polymer with markedly different molecular properties compared to flexible chains, including synthetic organic polymers. In this work, we report a new system for single polymer studies of flexible chains based on single stranded DNA (ssDNA). We developed a method to synthesize ssDNA for fluorescence microscopy based on rolling circle replication, which generates long strands (>65 kb) of ssDNA containing “designer” sequences, thereby preventing intramolecular base pair interactions. Polymers are synthesized to contain amine-modified bases randomly distributed along the backbone, which enables uniform labelling of polymer chains with a fluorescent dye to facilitate fluorescence microscopy and imaging. Using this approach, we synthesized ssDNA chains with long contour lengths (>30 μm) and relatively low dye loading ratios (~1 dye per 100 bases). In addition, we used epifluorescence microscopy to image single ssDNA polymer molecules stretching in flow in a microfluidic device. Overall, we anticipate that ssDNA will serve as a useful model system to probe the dynamics of polymeric materials at the molecular level. PMID:22956981

  8. A single-stranded DNA binding protein from mouse tumor cells specifically recognizes the C-rich strand of the (AGG:CCT)n repeats that can alter DNA conformation.

    PubMed Central

    Muraiso, T; Nomoto, S; Yamazaki, H; Mishima, Y; Kominami, R

    1992-01-01

    A protein that binds to a synthetic oligonucleotide of (CCT)12 has been purified from Ehrlich ascites tumor cells by a (CCT)12 affinity chromatography. The protein (p70) has an apparent molecular mass of 70 kDa, as assayed by Southwestern analysis. A competition experiment revealed that p70 binds to (CCT)12, (CCCT)8 and (CCTCCCT)6, but not to (CTT)12, (CT)16 and (CCTGCCT)6, suggesting that p70 has a sequence-specificity. The complementary (AGG)12 and the double stranded DNA did not show the binding. It is also confirmed by S1 nuclease analysis that the (AGG:CCT)12 duplex takes a single-stranded conformation in the absence of the protein. This raises a possibility that the duplex forms two single-stranded loops in chromosomes, the C-rich strand being bound to p70. Structural analysis of the resulting (AGG)12 strand by non-denaturing polyacrylamide gel electrophoresis demonstrated the presence of slower and faster migrated conformers in a neutral pH buffer containing 50 mM NaCl at 5 degrees C. The ratio was dependent on the DNA concentration. Both conformers disappeared in the absence of NaCl. This suggests that (AGG)12 can form intra- and inter-molecular complexes by non-Watson-Crick, guanine:guanine base-pairing. The possible biological function of the (AGG:CCT)n duplex and the p70 is discussed. Images PMID:1480484

  9. Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones.

    PubMed Central

    Altiok, S; Groner, B

    1993-01-01

    Transcription of the beta-casein gene in mammary epithelial cells is regulated by the lactogenic hormones insulin, glucocorticoids, and prolactin. The DNA sequence elements in the promoter which confer the action of the hormones on the transcriptional machinery and the nuclear proteins binding to this region have been investigated. We found that 221 nucleotides of promoter sequence 5' of the RNA start site are sufficient to mediate the induction of a chloramphenicol acetyltransferase reporter gene in transfected HC11 mammary epithelial cells. Deletion of 5' sequences to position -183 results in a construct with enhanced basal activity which still retains inducibility. A -170 beta-casein promoter-chloramphenicol acetyltransferase construct has very low transcriptional activity, which indicates the presence of a negative regulatory in the region between -221 and -183 and a positive regulatory element between -183 and -170. Band shift analysis showed that the promoter region between -194 and -163 specifically binds two nuclear proteins. The proteins are sequence-specific, single-stranded DNA-binding proteins which exclusively recognize the upper DNA strand and most likely play a repressing role in transcription. DNA binding activity of these nuclear proteins was observed only in nuclear extracts from mammary glands of mice in late pregnancy and postlactation, not during lactation. Hormonal control of the DNA binding activity of these proteins was also observed in the mammary epithelial cell line HC11. Mixing experiments showed that extracts from mammary tissue of lactating mice and from lactogenic hormone-treated HC11 cells contain an activity which can suppress the DNA binding of the single-stranded DNA-binding proteins.2+ identical specificity to the single-stranded DNA. Images PMID:8246951

  10. Synthesis of circular double-stranded DNA having single-stranded recognition sequence as molecular-physical probe for nucleic acid hybridization detection based on atomic force microscopy imaging.

    PubMed

    Nakano, Koji; Matsunaga, Hideshi; Murata, Masaharu; Soh, Nobuaki; Imato, Toshihiko

    2009-08-01

    A new class of DNA probes having a mechanically detectable tag is reported. The DNA probe, which consists of a single-stranded recognition sequence and a double-stranded circular DNA entity, was prepared by polymerase reaction. M13mp18 single strand and a 32mer oligodeoxynucleotide whose 5'-end is decorated with the recognition sequence were used in combination as template and primer, respectively. We have successfully demonstrated that the DNA probe is useful for bioanalytical purposes: by deliberately attaching target DNA molecules onto Au(111) substrates and by mechanically reading out the tag-entity using a high-resolution microscopy including atomic force microscopy, visualization/detection of the individual target/probe DNA conjugate was possible simply yet straightforwardly. The present DNA probe can be characterized as a 100%-nucleic acid product material. It is simply available by one-pod synthesis. A surface topology parameter, image roughness, has witnessed its importance as a quantitative analysis index with particular usability in the present visualization/detection method.

  11. OligArch: A software tool to allow artificially expanded genetic information systems (AEGIS) to guide the autonomous self-assembly of long DNA constructs from multiple DNA single strands

    PubMed Central

    Bradley, Kevin M

    2014-01-01

    Summary Synthetic biologists wishing to self-assemble large DNA (L-DNA) constructs from small DNA fragments made by automated synthesis need fragments that hybridize predictably. Such predictability is difficult to obtain with nucleotides built from just the four standard nucleotides. Natural DNA's peculiar combination of strong and weak G:C and A:T pairs, the context-dependence of the strengths of those pairs, unimolecular strand folding that competes with desired interstrand hybridization, and non-Watson–Crick interactions available to standard DNA, all contribute to this unpredictability. In principle, adding extra nucleotides to the genetic alphabet can improve the predictability and reliability of autonomous DNA self-assembly, simply by increasing the information density of oligonucleotide sequences. These extra nucleotides are now available as parts of artificially expanded genetic information systems (AEGIS), and tools are now available to generate entirely standard DNA from AEGIS DNA during PCR amplification. Here, we describe the OligArch (for "oligonucleotide architecting") software, an application that permits synthetic biologists to engineer optimally self-assembling DNA constructs from both six- and eight-letter AEGIS alphabets. This software has been used to design oligonucleotides that self-assemble to form complete genes from 20 or more single-stranded synthetic oligonucleotides. OligArch is therefore a key element of a scalable and integrated infrastructure for the rapid and designed engineering of biology. PMID:25161743

  12. [Effect of dimethyl sulfoxide on the extent of DNA single-strand breaks and alkali-labile sites induced by 365 nm UV-radiation in human blood lymphocyte nucleoids].

    PubMed

    Smetanina, N M; Pustovalova, M V; Osipov, A N

    2014-01-01

    It is shown that exposure of 365 nm UV radiation at doses of 10, 20 and 50 kJ/m2 induces a dose-dependent increase in DNA single-strand breaks and alkali-labile sites (SSB and ALS) detected by comet and halo assays in human blood lymphocyte nucleoids. Adding 10% dimethyl sulfoxide (DMSO) reduces the SSB and ALS yields--in 3 times. A strong drop in the output of UV-A-induced SSB and ALS in lymphocyte nucleoids in the presence of DMSO shows the leading role of *OH radicals in this DNA damage formation under exposure to 365-nm UV-radiation.

  13. Diepoxybutane induces the formation of DNA-DNA rather than DNA-protein cross-links, and single-strand breaks and alkali-labile sites in human hepatocyte L02 cells.

    PubMed

    Wen, Ying; Zhang, Pan-Pan; An, Jing; Yu, Ying-Xin; Wu, Ming-Hong; Sheng, Guo-Ying; Fu, Jia-Mo; Zhang, Xin-Yu

    2011-11-01

    1,3-Butadiene (BD) is an air pollutant and a known carcinogen. 1,2,3,4-Diepoxybutane (DEB), one of the major in vivo metabolites of BD, is considered the ultimate culprit of BD mutagenicity/carcinogenicity. DEB is a bifunctional alkylating agent, being capable of inducing the formation of monoalkylated DNA adducts and DNA cross-links, including DNA-DNA and DNA-protein cross-links (DPC). In the present study, we investigated DEB-caused DNA cross-links and breaks in human hepatocyte L02 cells using comet assay. With alkaline comet assay, it was observed that DNA migration increased with the increase of DEB concentration at lower concentrations (10-200μM); however, at higher concentrations (200-1000μM), DNA migration decreased with the increase of DEB concentration. This result indicated the presence of cross-links at >200μM, which was confirmed by the co-treatment experiments using the second genotoxic agents, tert-butyl hydroperoxide and methyl methanesulfonate. At 200μM, which appeared as a threshold, the DNA migration-retarding effect of cross-links was just observable by the co-treatment experiments. At <200μM, the effect of cross-links was too weak to be detected. The DEB-induced cross-links were determined to be DNA-DNA ones rather than DPC through incubating the liberated DNA with proteinase K prior to unwinding and electrophoresis. However, at the highest DEB concentration tested (1000μM), a small proportion of DPC could be formed. In addition, the experiments using neutral and weakly alkaline comet assays showed that DEB did not cause double-strand breaks, but did induce single-strand breaks (SSB) and alkali-labile sites (ALS). Since SSB and ALS are repaired more rapidly than cross-links, the results suggested that DNA-DNA cross-links, rather than DPC, were probably responsible for mutagenicity/carcinogenicity of DEB.

  14. Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

    PubMed Central

    Lee, Wonbae; Gillies, John P.; Jose, Davis; Israels, Brett A.; von Hippel, Peter H.; Marcus, Andrew H.

    2016-01-01

    Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed. PMID:27694621

  15. Non-covalent assembly of meso-tetra-4-pyridyl porphine with single-stranded DNA to form nano-sized complexes with hydrophobicity-dependent DNA release and anti-tumor activity

    PubMed Central

    Ghosh, Supratim; Ucer, Kamil B.; D’Agostino, Ralph; Grant, Ken; Sirintrapun, Joseph; Thomas, Michael J.; Hantgan, Roy; Bharadwaj, Manish; Gmeiner, William H.

    2013-01-01

    DNA and porphyrin based therapeutics are important for anti-cancer treatment. The present studies demonstrate single-stranded DNA (ssDNA) assembles with meso-tetra-4-pyridyl porphine (MTP) forming porphyrin:DNA nano-complexes (PDN) that are stable in aqueous solution under physiologically relevant conditions and undergo dissociation with DNA release in hydrophobic environments, including cell membranes. PDN formation is DNA-dependent with the ratio of porphyrin:DNA being approximately two DNA nucleobases per porphyrin. PDN produce reactive oxygen species (ROS) in a light-dependent manner under conditions that favor nano-complex dissociation in the presence of hydrophobic solvents. PDN induce light-dependent cytotoxicity in vitro and anti-tumor activity towards bladder cancer xenografts in vivo. Light-dependent, PDN-mediated cell death results from ROS-mediated localized membrane damage due to lipid peroxidation with mass spectrometry indicating the generation of the lipid peroxidation products 9- and 13-hydroxy octadecanoic acid. Our results demonstrate that PDN have properties useful for therapeutic applications, including cancer treatment. PMID:23988714

  16. Protective effects of pulmonary epithelial lining fluid on oxidative stress and DNA single-strand breaks caused by ultrafine carbon black, ferrous sulphate and organic extract of diesel exhaust particles

    SciTech Connect

    Chuang, Hsiao-Chi; Cheng, Yi-Ling; Lei, Yu-Chen; Chang, Hui-Hsien; Cheng, Tsun-Jen

    2013-02-01

    Pulmonary epithelial lining fluid (ELF) is the first substance to make contact with inhaled particulate matter (PM) and interacts chemically with PM components. The objective of this study was to determine the role of ELF in oxidative stress, DNA damage and the production of proinflammatory cytokines following physicochemical exposure to PM. Ultrafine carbon black (ufCB, 15 nm; a model carbonaceous core), ferrous sulphate (FeSO{sub 4}; a model transition metal) and a diesel exhaust particle (DEP) extract (a model organic compound) were used to examine the acellular oxidative potential of synthetic ELF and non-ELF systems. We compared the effects of exposure to ufCB, FeSO{sub 4} and DEP extract on human alveolar epithelial Type II (A549) cells to determine the levels of oxidative stress, DNA single-strand breaks and interleukin-8 (IL-8) production in ELF and non-ELF systems. The effects of ufCB and FeSO{sub 4} on the acellular oxidative potential, cellular oxidative stress and DNA single-strand breakage were mitigated significantly by the addition of ELF, whereas there was no decrease following treatment with the DEP extract. There was no significant effect on IL-8 production following exposure to samples that were suspended in ELF/non-ELF systems. The results of the present study indicate that ELF plays an important role in the initial defence against PM in the pulmonary environment. Experimental components, such as ufCB and FeSO{sub 4}, induced the production of oxidative stress and led to DNA single-strand breaks, which were moderately prevented by the addition of ELF. These findings suggest that ELF plays a protective role against PM-driven oxidative stress and DNA damage. -- Highlights: ► To determine the role of ELF in ROS, DNA damage and IL-8 after exposure to PM. ► ufCB, FeSO{sub 4} and DEP extract were used to examine the protective effects of ELF. ► PM-driven oxidative stress and DNA single-strand breakage were mitigated by ELF. ► The findings

  17. In vitro conversion of MVM parvovirus single-stranded DNA to the replicative form by DNA polymerase alpha from Ehrlich ascites tumour cells.

    PubMed Central

    Faust, E A; Rankin, C D

    1982-01-01

    A partially purified preparation of DNA polymerase alpha, obtained from the cytosol of Ehrlich ascites tumour cells, has been found to catalyze the conversion of MVM parvovirus, SS DNA (5 kilobases) to RF in vitro. The reaction initiates at a natural 55 base pair hairpin which exists at the 3' terminus of MVM SS DNA. The SS leads to RF conversion is sensitive to aphidicolin, resistant to ddTTP and is promoted by purine ribonucleoside 5' triphosphates, a phenomenon which could not be explained simply by stabilization effects on the in vitro deoxynucleotide precursor pool. In the absence of rNTPs, nascent complementary strands frequently terminate prematurely at a preferred location, between 1300 and 1700 nucleotides from the initiating 3' hairpin terminus. This in vitro system, involving self-primed parvovirus DNA synthesis, provides a convenient assay for those components of the mammalian replicative DNA polymerase complex which are required for the elongation of nascent DNA chains. Images PMID:6812024

  18. Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding

    PubMed Central

    Jose, Davis; Weitzel, Steven E.; Baase, Walter A.; von Hippel, Peter H.

    2015-01-01

    Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these ‘macromolecular machines’. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3′-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2–3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3′-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and ‘DNA map’) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex. PMID:26275775

  19. The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus.

    PubMed

    Kim, Seongman; Ahn, Byung Chul; O'Callaghan, Dennis J; Kim, Seong Kee

    2012-10-25

    The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

  20. The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

    SciTech Connect

    Kim, Seongman; Chul Ahn, Byung; O'Callaghan, Dennis J.; Kim, Seong Kee

    2012-10-25

    The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealed that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.

  1. GFP-like fluorophores as DNA labels for studying DNA-protein interactions.

    PubMed

    Riedl, Jan; Ménová, Petra; Pohl, Radek; Orság, Petr; Fojta, Miroslav; Hocek, Michal

    2012-09-21

    GFP-like 3,5-difluoro-4-hydroxybenzylideneimidazolinone (FBI) and 3,5-bis(methoxy)-4-hydroxy-benzylideneimidazolinone (MBI) labels were attached to dCTP through a propargyl linker, and the resulting labeled nucleotides (dC(MBI)TP and dC(FBI)TP) were used for a facile enzymatic synthesis of oligonucleotide or DNA probes by polymerase-catalyzed primer extension. The MBI/FBI-labeled DNA probes exerted low fluorescence that was increased 2-3.2 times upon binding of a protein. The concept was demonstrated on sequence-specific binding of p53 to dsDNA and on nonspecific binding of single strand binding protein to an oligonucleotide. The FBI label was also used for a time-resolved experiment monitoring a single-nucleotide incorporation followed by primer extension by Vent(exo-) polymerase.

  2. Ortho-Stabilized (18) F-Azido Click Agents and their Application in PET Imaging with Single-Stranded DNA Aptamers.

    PubMed

    Wang, Lu; Jacobson, Orit; Avdic, Din; Rotstein, Benjamin H; Weiss, Ido D; Collier, Lee; Chen, Xiaoyuan; Vasdev, Neil; Liang, Steven H

    2015-10-19

    Azido (18) F-arenes are important and versatile building blocks for the radiolabeling of biomolecules via Huisgen cycloaddition ("click chemistry") for positron emission tomography (PET). However, routine access to such clickable agents is challenged by inefficient and/or poorly defined multistep radiochemical approaches. A high-yielding direct radiofluorination for azido (18) F-arenes was achieved through the development of an ortho-oxygen-stabilized iodonium derivative (OID). This OID strategy addresses an unmet need for a reliable azido (18) F-arene clickable agent for bioconjugation reactions. A ssDNA aptamer was radiolabeled with this agent and visualized in a xenograft mouse model of human colon cancer by PET, which demonstrates that this OID approach is a convenient and highly efficient way of labeling and tracking biomolecules.

  3. Reparation in unicellular green algae during chronic exposure to the action of mutagenic factors. II. Restoration of single-stranded DNA breaks following exposure of Chlamydomonas reinchardii to gamma-irradiation

    SciTech Connect

    Sergeeva, S.A.; Ptitsina, S.N.; Shevchenko, V.A.

    1986-12-01

    The restoration of single-stranded breaks in the DNA in different strains of unicellular green algae (chlamydomonads) during chronic exposure to the action of mutagenic factors following ..gamma..-irradiation was investigated. It was shown that the restoration of DNA breaks was most effective in the case of strain M ..gamma../sup mt/sup +//, which is resistant to radiation. Strains, that were sensitive to UV irradiation showed a similar order of DNA break restoration as the wild-type strain. Strain UVS-1 showed a higher level of restoration than the wild-type strain. The data indicated that chlamydomonads have different pathways of reparation, which lead to the restoration of breaks induced by ..gamma..-irradiation and UV-rays.

  4. Mutations in the mutY gene of Escherichia coli enhance the frequency of targeted G:C-->T:a transversions induced by a single 8-oxoguanine residue in single-stranded DNA.

    PubMed

    Moriya, M; Grollman, A P

    1993-05-01

    Oxidative damage to guanine in DNA results in the formation of 8-oxoguanine, which has been shown to induce G-->T transversions targeted to this site. The mutagenicity of this lesion was studied in several mutator strains of Escherichia coli, using single-stranded DNA containing a single 8-oxoguanine residue. The frequencies of targeted G-->T transversions increased markedly in mutY strains, while this mutagenic event was not affected in mutM or mutS strains. Introduction of a mutM mutation into a mutY strain caused a somewhat higher frequency of G-->T transversions than that in the mutY strain and the effect of a mutS mutation was marginal. We conclude that the mutY gene plays a crucial role in preventing targeted G-->T mutations derived from misreplication of the 8-oxoguanine-containing template DNA.

  5. Characterization of mucosa-associated bacterial communities of the mouse intestine by terminal restriction fragment length polymorphism: Utility of sampling strategies and methods to reduce single-stranded DNA artifacts.

    PubMed

    Costa, Estela; Puhl, Nathan J; Selinger, L Brent; Inglis, G Douglas

    2009-08-01

    Terminal restriction fragment length polymorphism (T-RFLP) is a molecular technique used for comparative analysis of microbial community structure and dynamics. We evaluated three sampling methods for recovering bacterial community DNA associated with intestinal mucosa of mice (i.e. mechanical agitation with PBS, hand washing with PBS containing Tween 80, and direct DNA extraction from mucosal plugs). In addition, the utility of two methods (i.e. Klenow fragment and mung-bean nuclease) to reduce single-stranded DNA artifacts was tested. T-RFLP analysis indicated that diverse communities of bacteria are associated with mucosa of the ileum, cecum, and descending colon of mice. Although there was no significant difference in bacterial community structure between the mechanical agitation and direct DNA extraction methods regardless of intestinal location, community diversity was reduced for the hand wash method in the colon. The use of Klenow fragment and mung-bean nuclease have been reported to eliminate single-stranded DNA artifacts (i.e. pseudo-T-restriction fragments), but neither method was beneficial for characterizing mucosa-associated bacterial communities of the mouse cecum. Our study showed that the mechanical agitation and direct plug extraction methods yielded equivalent bacterial community DNA from the mucosa of the small and large intestines of mice, but the latter method was superior for logistical reasons. We also applied a combination of different statistical approaches to analyze T-RFLP data, including statistical detection of true peaks, analysis of variance for peak number, and group significance test, which provided a quantitative improvement for the interpretation of the T-RFLP data.

  6. Evaluation of human brain damage in fire fatality by quantification of basic fibroblast growth factor (bFGF), glial fibrillary acidic protein (GFAP) and single-stranded DNA (ssDNA) immunoreactivities.

    PubMed

    Wang, Qi; Ishikawa, Takaki; Michiue, Tomomi; Zhu, Bao-Li; Maeda, Hitoshi

    2011-09-10

    Burns and inhalation of toxic gases, including carbon monoxide (CO) and cyanide, which are produced by combustion, are major factors involved in fire death. The present study immunohistochemically investigated basic fibroblast growth factor (bFGF), glial fibrillary acidic protein (GFAP) and single-stranded DNA (ssDNA) in the brains of fire fatalities (n=49) to examine the differences between fatal burns and CO intoxication, compared with those in cardiac deaths (n=24) and mechanical asphyxiation cases (n=23). In acute fire fatality, neuronal ssDNA immunopositivity in the cerebral cortex of the parietal lobe was high in both fatal burns and fatal CO intoxication, but that of the pallidum was higher for CO intoxication than for burns. The number of neurons was decreased in prolonged fire deaths, irrespective of the severity of burns or CO intoxication, but glias were increased in cases of fatal burns. Prolonged deaths due to burns had a higher glial bFGF immunopositivity in the cortex and white matter, higher and lower glial GFAP immunopositivity in the cortex and white matter, respectively, and a low neuronal ssDNA immunopositivity in the cerebral cortex and hippocampus. In prolonged deaths due to CO intoxication, however, glial bFGF and GFAP immunopositivities were low at each site, but neuronal ssDNA immunopositivity showed a higher value. These observations suggest increased cerebral neuronal ssDNA immunopositivity to be a finding of vitality in acute fire death, and a neuronal loss accompanied by active glial responses after severe burns, and a neuronal loss and progressive apoptosis without glial responses after CO intoxication to be characteristic in prolonged death.

  7. The AT-rich tract of the SV40 ori core: negative synergism and specific recognition by single stranded and duplex DNA binding proteins.

    PubMed Central

    Galli, I; Iguchi-Ariga, S M; Ariga, H

    1992-01-01

    The SV40 origin of replication comprises a run of thymine and adenine residues. Integrity of this AT-rich sequence is known to be essential for replication. We set out to study whether or not these elements can work synergistically to sustain replication. Quite surprisingly, additional copies of the AT stretch linked to a functional SV40 ori core dramatically reduce its replication in Cosl cells, probably by creating some physical block. Interestingly, the same inhibiting effect can be observed with the addition in cis of the yeast ARS consensus, which is homologous to the SV40 AT stretch. This modulation is possibly due to the action of cellular factors that recognize either of the two sequences. In fact, we demonstrate the existence of factor(s) in Cosl crude nuclear extracts that in vitro can specifically bind to either of them. Moreover, we show that these sequence-specific factor(s) (MW about 50 kDa), named SOAP, recognize both single (T-rich strand) and double stranded forms of the AT tracts. Binding to single stranded AT stretches can be specifically inhibited by the corresponding duplex form, but not vice versa. Images PMID:1321411

  8. CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.

    PubMed

    Miura, Hiromi; Gurumurthy, Channabasavaiah B; Sato, Takehito; Sato, Masahiro; Ohtsuka, Masato

    2015-08-05

    Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

  9. Models for the Binary Complex of Bacteriophage T4 Gp59 Helicase Loading Protein. GP32 Single-Stranded DNA-Binding Protein and Ternary Complex with Pseudo-Y Junction DNA

    SciTech Connect

    Hinerman, Jennifer M.; Dignam, J. David; Mueser, Timothy C.

    2012-04-05

    The bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable with that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Moreover, fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596–18607).

  10. Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein. Application of the matrix projection operator technique to analyze stopped-flow kinetics.

    PubMed

    Bujalowski, W; Jezewska, M J

    2000-01-28

    Kinetics of the Escherichia coli primary replicative helicase DnaB protein binding to a single-stranded DNA, in the presence of the ATP non-hydrolyzable analog AMP-PNP, have been performed, using the fluorescence stopped-flow technique. This is the first direct determination of the mechanism of the ssDNA recognition by a hexameric helicase. Binding of the fluorescent etheno-derivative of a ssDNA to the enzyme is characterized by a strong increase of the nucleic acid fluorescence, which provides an excellent signal to quantitatively study the mechanism of ssDNA recognition by the helicase. The kinetic experiments have been performed with a ssDNA 20-mer, depsilonA(pepsilonA)(19), that encompasses the entire, total ssDNA-binding site of the helicase and with the 10-mer depsilonA(pepsilonA)(9), which binds exclusively to the ssDNA strong subsite within the total ssDNA-binding site. Association of the DnaB helicase with the 20-mer is characterized by three relaxation times, which indicates that the binding occurs by the minimum three-step mechanism where the bimolecular binding step is followed by two isomerization steps. This mechanism is described by the equation: Helicase+ssDNAk1/(k1)<-->(k-1)(H-ssDNA)1(k2)<-->(k-2)(H-ssDNA)2 (k3)<-->(k-3)(H-ssDNA)3. The value of the bimolecular rate constant, k(1), is four to six orders of magnitude lower than the value expected for the diffusion-controlled reaction. Moreover, quantitative amplitude analysis suggests that the major conformational change of the ssDNA takes place in the formation of the (H-ssDNA)(1). These results indicate that the determined first step includes formation of the collision and an additional transition of the protein-ssDNA complex, most probably the local opening of the protein hexamer. The data indicate that the binding mechanism reflects the interactions of the ssDNA predominantly through the strong ssDNA-binding subsite. The analysis of the stopped-flow kinetics has been performed using the matrix

  11. Yellow mosaic symptom caused by the nuclear shuttle protein gene of mungbean yellow mosaic virus is associated with single-stranded DNA accumulation and mesophyll spread of the virus.

    PubMed

    Kuruba, B L; Buvani, A P; Veluthambi, K

    Mungbean yellow mosaic virus-[India:Vigna] (MYMV-[IN:Vig]), a blackgram isolate of MYMV, causes yellow mosaic disease in blackgram and mungbean. Two variable DNA-B components, KA22 and KA27, cause distinct symptoms in blackgram [V. mungo (L.) Hepper] with the same DNA-A component. KA22 + DNA-A-agroinoculated blackgram plants displayed yellow mosaic symptom and accumulated high levels of viral single-stranded (ss) DNA. KA27 + DNA-A-agroinoculated blackgram plants displayed severe stunting symptom and accumulated very low levels of viral ssDNA. However, in mungbean [V. radiata (L.) Wilczek], KA27 + DNA-A caused yellow mosaic symptom and a high level of viral ssDNA accumulated. Swapping of KA27 DNA-B with the nuclear shuttle protein gene (NSP) of KA22 DNA-B (KA27xKA22 NSP) caused yellow mosaic symptom in blackgram, suggesting that KA22 NSP is the determinant of yellow mosaic symptom. Interestingly, KA27xKA22 NSP-infected blackgram plants accumulated high levels of viral ssDNA, comparable to that of KA22 DNA-B infection, suggesting that the KA22 NSP is responsible for accumulation of high levels of viral ssDNA. MYMV distribution was studied in blackgram and mungbean plants by leaf tissue hybridization, which showed mesophyll spread of the virus in KA22-infected blackgram leaflets and in KA27-infected mungbean leaflets, both of which displayed yellow mosaic symptom. However, the virus did not accumulate in the mesophyll in the case of KA27-infected blackgram leaflets. Interestingly, the swapped KA27xKA22 NSP-infected blackgram leaflets showed mesophyll accumulation of the virus, suggesting that KA22 NSP determines its mesophyll spread.

  12. Hydration of single-stranded phosphodiester and phosphorothioate oligodeoxyribonucleotides.

    PubMed Central

    White, A P; Reeves, K K; Snyder, E; Farrell, J; Powell, J W; Mohan, V; Griffey, R H

    1996-01-01

    Infrared spectroscopy was used to identify hydration-sensitive structural differences between single- stranded phosphorothioate (PS) and phosphodiester (PO) oligodeoxyribonucleotides. Spectra were recorded in the mid-infrared region, 500-1800 cm-1, at relative humidities between 0 and 98%; the PS and PO spectra are substantially different. The hydration effects on spectral bands in these single-stranded oligodeoxyribonucleotides is markedly different from such behavior in double- and triple-stranded oligodeoxyribonucleotides. A strong absorption occurs at 656 cm-1 in the phosphorothioate sample which is completely absent from the PO spectra. Gravimetric measurements were carried out on one PS and one PO sample to monitor and confirm hydration. The calculated BET adsorption constants [Brunauer, S., Emmett, RH. and Teller, E. (1938) J. Am. Chem. Soc., 60, 309-319] are 1.2 and 1.4 water molecules per nucleotide in the first hydration layer of PS and PO respectively. While the gravimetric data indicate that the single-stranded oligodeoxyribonucleotides hydrate very similarly to duplex DNA, the mid-infrared conformational marker bands are strikingly different from those observed for duplex DNA. In particular, the Vas of the phosphate group (PO2) at 1222 cm-1 in the single-stranded PO spectra is independent of relative humidity. PMID:8774910

  13. Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors

    NASA Astrophysics Data System (ADS)

    Star, Alexander; Tu, Eugene; Niemann, Joseph; Gabriel, Jean-Christophe P.; Joiner, C. Steve; Valcke, Christian

    2006-01-01

    We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics. hemochromatosis | SNP | biosensor

  14. Molecular dynamics simulation of a tumorigenic benzo[a]pyrene metabolite bound to DNA at a single strand-double strand junction

    SciTech Connect

    Singh, S.B.; Li, B.; Shapiro, R.

    1994-12-31

    It is widely believed that cancer can be initiated when certain biochemically activated chemical substances bind to DNA to produce a covalently linked adduct. Attachment of the substance to DNA can affect the DNA shape, leading to unfortunate biological consequences. These may include replication errors, which could be among the events that start the cellular processes ultimately yielding malignant tumors. Consequently, an understanding at the molecular level of how the DNA shape is affected by such chemicals is critical to understanding the primary event in chemical carcinogenesis. If one could establish structural hallmarks that distinguish DNA bound by a malignant chemical from DNA bound by a benign one, it might ultimately be possible to employ computational tools, instead of bacterial or animal testing, to screen chemical substances for mutagenic and tumorigenic potential.

  15. Rapid screening of the heterogeneity of DNA methylation by single-strand conformation polymorphism and CE-LIF in the presence of electro-osmotic flow.

    PubMed

    Yu, Meng-Hsuan; Huang, Ya-Chi; Chang, Po-Ling

    2014-08-01

    DNA methylation is a complex event in epigenetic studies because of both the large CpG islands present upstream of the promoter region and the different distribution of DNA methylation despite similar methylation levels. For this reason, we proposed a fast, cost-effective method for the screening of DNA methylation based on SSCP and CE-LIF. In this study, the PCR products that were amplified from bisulfite-treated genomic DNA were denatured at 94°C, followed by immediate chilling in ice water to form the ssDNA. The ssDNA were separated by 1.5% poly(ethylene oxide) (Mavg 8 000 000 Da) in the presence of EOF according to the different conformations represented by their unique methylation states. This result demonstrated that four hepatocellular carcinoma cell lines represented a different heterogeneity of DNA methylation and could be distinguished by SSCP-CE. The results obtained from SSCP-CE also corresponded with those obtained from combined bisulfide restriction analysis and methylation-sensitive high-resolution melting analysis. Therefore, the proposed SSCP-CE method may potentially be used for rapid screening for determination of the heterogeneity of DNA methylation in further epigenetic studies and clinical diagnosis.

  16. Random, double- and single-strand DNA breaks can be differentiated in the method of Comet assay by the shape of the comet image.

    PubMed

    Georgieva, Milena; Zagorchev, Plamen; Miloshev, George

    2015-10-01

    Comet assay is an invaluable tool in DNA research. It is widely used to detect DNA damage as an indicator of exposure to genotoxic stress. A canonical set of parameters and specialized software programs exist for Comet assay data quantification and analysis. None of them so far has proven its potential to employ a computer-based algorithm for assessment of the shape of the comet as an indicator of the exact mechanism by which the studied genotoxins cut in the molecule of DNA. Here, we present 14 unique measurements of the comet image based on the comet morphology. Their mathematical derivation and statistical analysis allowed precise description of the shape of the comet image which in turn discriminated the cause of genotoxic stress. This algorithm led to the development of the "CometShape" software which allowed easy discrimination among different genotoxins depending on the type of DNA damage they induce.

  17. Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis.

    PubMed

    Glavac, Damjan; Potocnik, Uros; Podpecnik, Darja; Zizek, Teofil; Smerkolj, Sava; Ravnik-Glavac, Metka

    2002-04-01

    We have studied 57 different mutations within three beta-globin gene promoter fragments with sizes 52 bp, 77 bp, and 193 bp by fluorescent capillary electrophoresis CE-SSCP analysis. For each mutation and wild type, energetically most-favorable predicted secondary structures were calculated for sense and antisense strands using the MFOLD DNA-folding algorithm in order to investigate if any correlation exists between predicted DNA structures and actual CE migration time shifts. The overall CE-SSCP detection rate was 100% for all mutations in three studied DNA fragments. For shorter 52 bp and 77 bp DNA fragments we obtained a positive correlation between the migration time shifts and difference in free energy values of predicted secondary structures at all temperatures. For longer 193 bp beta-globin gene fragments with 46 mutations MFOLD predicted different secondary structures for 89% of mutated strands at 25 degrees C and 40 degrees C. However, the magnitude of the mobility shifts did not necessarily correlate with their secondary structures and free energy values except for the sense strand at 40 degrees C where this correlation was statistically significant (r = 0.312, p = 0.033). Results of this study provided more direct insight into the mechanism of CE-SSCP and showed that MFOLD prediction could be helpful in making decisions about the running temperatures and in prediction of CE-SSCP data patterns, especially for shorter (50-100 bp) DNA fragments.

  18. Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining.

    PubMed

    Grabarz, Anastazja; Barascu, Aurélia; Guirouilh-Barbat, Josée; Lopez, Bernard S

    2012-01-01

    A DNA double strand break (DSB) is a highly toxic lesion, which can generate genetic instability and profound genome rearrangements. However, DSBs are required to generate diversity during physiological processes such as meiosis or the establishment of the immune repertoire. Thus, the precise regulation of a complex network of processes is necessary for the maintenance of genomic stability, allowing genetic diversity but protecting against genetic instability and its consequences on oncogenesis. Two main strategies are employed for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by single-stranded DNA (ssDNA) resection and requires sequence homology with an intact partner, while NHEJ requires neither resection at initiation nor a homologous partner. Thus, resection is an pivotal step at DSB repair initiation, driving the choice of the DSB repair pathway employed. However, an alternative end-joining (A-EJ) pathway, which is highly mutagenic, has recently been described; A-EJ is initiated by ssDNA resection but does not require a homologous partner. The choice of the appropriate DSB repair system, for instance according the cell cycle stage, is essential for genome stability maintenance. In this context, controlling the initial events of DSB repair is thus an essential step that may be irreversible, and the wrong decision should lead to dramatic consequences. Here, we first present the main DSB repair mechanisms and then discuss the importance of the choice of the appropriate DSB repair pathway according to the cell cycle phase. In a third section, we present the early steps of DSB repair i.e., DSB signaling, chromatin remodeling, and the regulation of ssDNA resection. In the last part, we discuss the competition between the different DSB repair mechanisms. Finally, we conclude with the importance of the fine tuning of this network for genome stability maintenance and for tumor protection in fine.

  19. Requirements for bypass of UV-induced lesions in single-stranded DNA of bacteriophage phi X174 in Salmonella typhimurium.

    PubMed Central

    Slater, S C; Maurer, R

    1991-01-01

    According to the current model for mutagenic bypass of UV-induced lesions, efficient bypass requires three proteins: activated RecA (RecA*) and either activated UmuD (UmuD') and UmuC or their plasmid-encoded analogues, MucA' and MucB. RecA* aids synthesis of UmuD' and UmuC (and MucA'/MucB) at two levels: by inactivation of the LexA transcriptional repressor of these genes and by cleavage of UmuD (and MucA) to produce the active fragments, UmuD' (MucA'). A third role for RecA is revealed when these two roles are otherwise satisfied in a suitably engineered strain. An often-suggested possible role for RecA in bypass is inhibition of editing by the epsilon subunit of DNA polymerase III. Here, by demonstrating that elimination of epsilon by deletion of its gene, dnaQ, does not relieve the requirement for the third function of RecA, we show that RecA must perform some function other than, or in addition to, inhibition of epsilon. We also show that elimination of epsilon does not relieve the requirement for either Muc protein. Moreover, we observed reactivation of irradiated phi X174 in unirradiated cells expressing MucA' and MucB. This finding makes it unlikely that the additional role of recA involves derepression of an unidentified gene or cleavage of an unidentified protein and makes it more likely that RecA participates directly in bypass. PMID:1847514

  20. Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α-L-LNA Adenine Monomers: High-affinity Targeting of Single-Stranded DNA

    PubMed Central

    Andersen, Nicolai K.; Anderson, Brooke A.; Wengel, Jesper

    2014-01-01

    Development of conformationally restricted nucleotide building blocks continues to attract considerable interest due to their successful use within antisense, antigene and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples hereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity and enhanced enzymatic stability relative to unmodified strands. Here, we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-L-LNA adenine monomers W–Z. The synthesis of target phosphoramidites 1–4 initiates from pentafuranose 5, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine intermediate 9, which after a series of non-trivial protecting group manipulations affords key intermediate 15. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation gives targets 1–4 in ~1–3% overall yield over eleven steps from 5. ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNA of considerable interest for DNA-targeting applications. PMID:24304240

  1. Requirements for bypass of UV-induced lesions in single-stranded DNA of bacteriophage phi X174 in Salmonella typhimurium

    SciTech Connect

    Slater, S.C.; Maurer, R. )

    1991-02-15

    According to the current model for mutagenic bypass of UV-induced lesions, efficient bypass requires three proteins: activated RecA (RecA*) and either activated UmuD (UmuD') and UmuC or their plasmid-encoded analogues, MucA' and MucB. RecA* aids synthesis of UmuD' and UmuC (and MucA'/MucB) at two levels: by inactivation of the LexA transcriptional repressor of these genes and by cleavage of UmuD (and MucA) to produce the active fragments, UmuD' (MucA'). A third role for RecA is revealed when these two roles are otherwise satisfied in a suitably engineered strain. An often-suggested possible role for RecA in bypass is inhibition of editing by the epsilon subunit of DNA polymerase III. Here, by demonstrating that elimination of epsilon by deletion of its gene, dnaQ, does not relieve the requirement for the third function of RecA, we show that RecA must perform some function other than, or in addition to, inhibition of epsilon. We also show that elimination of epsilon does not relieve the requirement for either Muc protein. Moreover, we observed reactivation of irradiated phi X174 in unirradiated cells expressing MucA' and MucB. This finding makes it unlikely that the additional role of recA involves derepression of an unidentified gene or cleavage of an unidentified protein and makes it more likely that RecA participates directly in bypass.

  2. Fecal virome of healthy chickens reveals a large diversity of the eukaryote viral community, including novel circular single-stranded DNA viruses.

    PubMed

    Lima, Diane; Cibulski, Samuel Paulo; Finkler, Fabrine; Teixeira, Thais; Varela, Ana Paula; Cerva, Cristine; Loiko, Márcia; Scheffer, Camila; Dos Santos, Helton; Mayer, Fabiana; Roehe, Paulo

    2017-01-18

    This study is focused on the identification of the fecal virome of healthy chickens raised in high-density, export-driven poultry farms in Brazil. Following high-throughput sequencing, a total of 7,743 de novo-assembled contigs were constructed and compared with known nucleotide/amino acid sequences from the GenBank database. Analyses with BLASTx revealed that 279 contigs (4%) were related to sequences of eukaryotic viruses. Viral genome sequences (total or partial) indicative of members of recognized viral families, including Adenoviridae, Caliciviridae, Circoviridae, Parvoviridae, Picobirnaviridae, Picornaviridae and Reoviridae, were identified, some of those representing novel genotypes. In addition, a range of circular replication-associated protein encoding (CRESS) DNA viruses, were also identified. The characterization of the fecal virome of healthy chickens described here provides not only a description of the viruses encountered in such niche but should also represent a baseline for future studies comparing viral populations in healthy and diseased chicken flocks. Moreover, it may also be relevant for human health, since chickens represent a significant proportion of the animal protein consumed worldwide.

  3. Labeling nuclear DNA using DAPI.

    PubMed

    Chazotte, Brad

    2011-01-01

    A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, propidium iodide, and acridine orange. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. It is believed that DAPI associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. Cells must be permeabilized and/or fixed for DAPI to enter the cell and to bind DNA. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA. This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture.

  4. Detection of SERS active labelled DNA based on surface affinity to silver nanoparticles.

    PubMed

    Harper, Mhairi M; Dougan, Jennifer A; Shand, Neil C; Graham, Duncan; Faulds, Karen

    2012-05-07

    Developments in specific DNA detection assays have been shown to be increasingly beneficial for molecular diagnostics and biological research. Many approaches use optical spectroscopy as an assay detection method and, owing to the sensitivity and molecular specificity offered, surface enhanced Raman scattering (SERS) spectroscopy has become a competitively exploited technique. This study utilises SERS to demonstrate differences in affinity of dye labelled DNA through differences in electrostatic interactions with silver nanoparticles. Results show clear differences in the SERS intensity obtained from single stranded DNA, double stranded DNA and a free dye label and demonstrate surface attraction is driven through electrostatic charges on the nucleotides and not the SERS dye. It has been further demonstrated that, through optimisation of experimental conditions and careful consideration of sequence composition, a DNA detection method with increased sample discrimination at lower DNA concentrations can be achieved.

  5. Simultaneous labeling of single- and double-strand DNA breaks by DNA breakage detection-FISH (DBD-FISH).

    PubMed

    Fernández, José Luis; Cajigal, Dioleyda; Gosálvez, Jaime

    2011-01-01

    DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) permits simultaneous and selective labeling of single- and double-strand DNA breaks in individual cells, either in the whole genome or within specific DNA sequences. In this technique, cells are embedded into agarose microgels, lysed and subjected to electrophoresis under nondenaturing conditions. Subsequently, the produced "comets" are exposed to a controlled denaturation step which transforms DNA breaks into single-stranded DNA regions, detected by hybridization with whole genome fluorescent probes or the probes to specific DNA sequences. This makes possible a targeted analysis of various chromatin areas for the presence of DNA breaks. The migration length of the DBD-FISH signal is proportional to the number of double strand breaks, whereas its fluorescence intensity depends on numbers of single-strand breaks.The detailed protocol for detection of two types of DNA breaks produced by ionizing radiation is presented. The technique can be used to determine intragenomic and intercellular heterogeneity in the induction and repair of DNA damage.

  6. Label-free DNA hybridization detection by various spectroscopy methods using triphenylmethane dyes as a probe

    NASA Astrophysics Data System (ADS)

    Tu, Jiaojiao; Cai, Changqun; Ma, Ying; Luo, Lin; Weng, Chao; Chen, Xiaoming

    2012-12-01

    A new assay is developed for direct detection of DNA hybridization using triphenylmethane dye as a probe. It is based on various spectroscopic methods including resonance light scattering (RLS), circular dichroism (CD), ultraviolet spectra and fluorescence spectra, as well as atomic force microscopy (AFM), six triphenylmethane dyes interact with double strand DNA (dsDNA) and single strand DNA (ssDNA) were investigated, respectively. The interaction results in amplified resonance light scattering signals and enables the detection of hybridization without the need for labeling DNA. Mechanism investigations have shown that groove binding occurs between dsDNA and these triphenylmethane dyes, which depends on G-C sequences of dsDNA and the molecular volumes of triphenylmethane dyes. Our present approaches display the advantages of simple and fast, accurate and reliable, and the artificial samples were determined with satisfactory results.

  7. One-dimensional TRFLP-SSCP is an effective DNA fingerprinting strategy for soil Archaea that is able to simultaneously differentiate broad taxonomic clades based on terminal fragment length polymorphisms and closely related sequences based on single stranded conformation polymorphisms.

    PubMed

    Swanson, Colby A; Sliwinski, Marek K

    2013-09-01

    DNA fingerprinting methods provide a means to rapidly compare microbial assemblages from environmental samples without the need to first cultivate species in the laboratory. The profiles generated by these techniques are able to identify statistically significant temporal and spatial patterns, correlations to environmental gradients, and biological variability to estimate the number of replicates for clone libraries or next generation sequencing (NGS) surveys. Here we describe an improved DNA fingerprinting technique that combines terminal restriction fragment length polymorphisms (TRFLP) and single stranded conformation polymorphisms (SSCP) so that both can be used to profile a sample simultaneously rather than requiring two sequential steps as in traditional two-dimensional (2-D) gel electrophoresis. For the purpose of profiling Archaeal 16S rRNA genes from soil, the dynamic range of this combined 1-D TRFLP-SSCP approach was superior to TRFLP and SSCP. 1-D TRFLP-SSCP was able to distinguish broad taxonomic clades with genetic distances greater than 10%, such as Euryarchaeota and the Thaumarchaeal clades g_Ca. Nitrososphaera (formerly 1.1b) and o_NRP-J (formerly 1.1c) better than SSCP. In addition, 1-D TRFLP-SSCP was able to simultaneously distinguish closely related clades within a genus such as s_SCA1145 and s_SCA1170 better than TRFLP. We also tested the utility of 1-D TRFLP-SSCP fingerprinting of environmental assemblages by comparing this method to the generation of a 16S rRNA clone library of soil Archaea from a restored Tallgrass prairie. This study shows 1-D TRFLP-SSCP fingerprinting provides a rapid and phylogenetically informative screen of Archaeal 16S rRNA genes in soil samples.

  8. Label-free DNA sensor by boron-doped diamond electrode using an ac impedimetric approach.

    PubMed

    Weng, Jian; Zhang, Jianfeng; Li, Hui; Sun, Liping; Lin, Chenghong; Zhang, Qiqing

    2008-09-15

    An electrochemical biosensor using a boron-doped diamond (BDD) electrode is described for differentiating between gene sequences according to DNA hybridization events using an ac impedimetric approach. BDD electrodes were dipped into a 1% solution of polyethylenimine (PEI) to adsorb a thin layer of positively charged PEI on the surface of BDD, then PEI-modified BDD electrodes were used to immobilize negatively charged single-stranded PCR fragments from Exon 7 of human p53 gene. Alternating current impedimetric measurements were first performed on these systems in phosphate buffered saline (PBS) and then upon exposure to single-stranded DNA (ssDNA). When the ssDNA-immobilized BDD electrode and solution ssDNA were completely complementary, a large drop in impedance was measured. Complementary DNA could be clearly detected at concentrations down to 10 (-19) g mL (-1) at a fixed frequency (10 Hz). Higher concentrations of DNA gave faster hybridization with saturation occurring at levels above 1.0 pg mL (-1.) Responses were much lower upon exposure to noncDNA, even at higher concentrations. The results show it is possible to directly detect target DNA at a fixed frequency and without additional labeling.

  9. Molybdenum disulfide (MoS2) nanoflakes as inherently electroactive labels for DNA hybridization detection.

    PubMed

    Loo, Adeline Huiling; Bonanni, Alessandra; Ambrosi, Adriano; Pumera, Martin

    2014-10-21

    The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide nanomaterials for sensing and biosensing purposes represents an upcoming research area which holds great promise. Hence, our findings are anticipated to have significant contributions towards the fabrication of future DNA biosensors.

  10. Electrochemical DNA biosensor for detection of porcine oligonucleotides using ruthenium(II) complex as intercalator label redox

    NASA Astrophysics Data System (ADS)

    Halid, Nurul Izni Abdullah; Hasbullah, Siti Aishah; Ahmad, Haslina; Heng, Lee Yook; Karim, Nurul Huda Abd; Harun, Siti Norain

    2014-09-01

    A DNA biosensor detection of oligonucleotides via the interactions of porcine DNA with redox active complex based on the electrochemical transduction is described. A ruthenium(II) complex, [Ru(bpy)2(PIP)]2+, (bpy = 2,2'bipyridine, PIP = 2-phenylimidazo[4,5-f[[1,10-phenanthroline]) as DNA label has been synthesized and characterized by 1H NMR and mass spectra. The study was carried out by covalent bonding immobilization of porcine aminated DNA probes sequences on screen printed electrode (SPE) modified with succinimide-acrylic microspheres and [Ru(bpy)2(PIP)]2+ was used as electrochemical redox intercalator label to detect DNA hybridization event. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the ruthenium (II) complex was active. The results indicate that the interaction of [Ru(bpy)2(PIP)]2+ with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.

  11. Single strand transposition at the host replication fork

    PubMed Central

    Lavatine, Laure; He, Susu; Caumont-Sarcos, Anne; Guynet, Catherine; Marty, Brigitte; Chandler, Mick; Ton-Hoang, Bao

    2016-01-01

    Members of the IS200/IS605 insertion sequence family differ fundamentally from classical IS essentially by their specific single-strand (ss) transposition mechanism, orchestrated by the Y1 transposase, TnpA, a small HuH enzyme which recognizes and processes ss DNA substrates. Transposition occurs by the ‘peel and paste’ pathway composed of two steps: precise excision of the top strand as a circular ss DNA intermediate; and subsequent integration into a specific ssDNA target. Transposition of family members was experimentally shown or suggested by in silico high-throughput analysis to be intimately coupled to the lagging strand template of the replication fork. In this study, we investigated factors involved in replication fork targeting and analysed DNA-binding properties of the transposase which can assist localization of ss DNA substrates on the replication fork. We showed that TnpA interacts with the β sliding clamp, DnaN and recognizes DNA which mimics replication fork structures. We also showed that dsDNA can facilitate TnpA targeting ssDNA substrates. We analysed the effect of Ssb and RecA proteins on TnpA activity in vitro and showed that while RecA does not show a notable effect, Ssb inhibits integration. Finally we discuss the way(s) in which integration may be directed into ssDNA at the replication fork. PMID:27466393

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

    PubMed

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

    2016-03-16

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

  13. Labeling-free fluorescent detection of DNA hybridization through FRET from pyrene excimer to DNA intercalator SYBR green I.

    PubMed

    Zhou, Ruyi; Xu, Chen; Dong, Jie; Wang, Guojie

    2015-03-15

    A novel labeling-free fluorescence complex probe has been developed for DNA hybridization detection based on fluorescence resonance energy transfer (FRET) mechanism from pyrene excimer of pyrene-functionalized poly [2-(N, N-dimethylamino) ethyl methacrylate] (PFP) to SYBR Green I (SG, a specific intercalator of double-stranded DNA) in a cost-effective, rapid and simple manner. The complex probe consists of the positively charged PFP, SG and negatively charged single-stranded DNA (ssDNA). Upon adding a complementary strand to the complex probe solution, double-stranded DNA (dsDNA) was formed, followed by the intercalation of SG into dsDNA. The pyrene excimer emission was overlapped with the absorption of SG very well and the electrostatic interactions between PFP and dsDNA kept them in close proximity, enabling efficient FRET from pyrene excimer to SG. The fluorescence of SG in the duplex DNA resulting from FRET can be successfully applied to detect DNA hybridization with high sensitivity for a very low detection limit of 10nM and excellent selectivity for detection of single base pair mismatch.

  14. Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition.

    PubMed

    Morozov, V; Wawrousek, E F

    2008-03-01

    Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs alone, or in combination with other factors, to introduce a point mutation into the alphaB-crystallin gene in one-celled mouse embryos. We found that co-injection of ssDNA along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative nonhomologous end-joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been previously reported.

  15. An electrochemical DNA biosensor based on Oracet Blue as a label for detection of Helicobacter pylori.

    PubMed

    Hajihosseini, Saeedeh; Nasirizadeh, Navid; Hejazi, Mohammad Saeid; Yaghmaei, Parichereh

    2016-10-01

    An innovative method of a DNA electrochemical biosensor based on Oracet Blue (OB) as an electroactive label and gold electrode (AuE) for detection of Helicobacter pylori, was offered. A single-stranded DNA probe with a thiol modification was covalently immobilized on the surface of the AuE by forming an Au-S bond. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization by measuring the electrochemical signals of reduction of the OB binding to double-stranded DNA (ds-DNA). Our results showed that OB-based DNA biosensor has a decent potential for detection of single-base mismatch in target DNA. Selectivity of the proposed DNA biosensor was further confirmed in the presence of non-complementary and complementary DNA strands. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 0.3nmolL(-1) to 240.0nmolL(-1), and the detection limit was 0.17nmolL(-1), whit a promising reproducibility and repeatability.

  16. Cellular Site in Bacillus subtilis of a Nuclease Which Preferentially Degrades Single-Stranded Nucleic Acids

    PubMed Central

    Birnboim, H. C.

    1966-01-01

    Birnboim, H. C. (Albert Einstein College of Medicine, New York, N.Y.). Cellular site in Bacillus subtilis of a nuclease which preferentially degrades single-stranded nucleic acids. J. Bacteriol. 91:1004–1011. 1966.—A nuclease, identified by a marked preference for single-stranded nucleic acids, has been demonstrated in extracts of Bacillus subtilis. The enzyme was associated with the cell wall-membrane fraction of mechanically disrupted cells and was released from cells which had been converted to protoplasts by lysozyme. The nuclease activity prepared by the latter procedure was found to be activated and solubilized by treatment with trypsin. The enzyme had about 2% activity on native deoxyribonucleic acid (DNA) as compared with denatured DNA. By use of CsCl analytical density gradient ultracentrifugation, this preparation was shown to degrade denatured DNA selectively in mixtures of native and denatured DNA. PMID:4956329

  17. Single-strand conformation polymorphism analysis using capillary array electrophoresis for large-scale mutation detection.

    PubMed

    Larsen, Lars Allan; Jespersgaard, Cathrine; Andersen, Paal Skytt

    2007-01-01

    This protocol describes capillary array electrophoresis single-strand conformation polymorphism (CAE-SSCP), a screening method for detection of unknown and previously identified mutations. The method detects 98% of mutations in a sample material and can be applied to any organism where the goal is to determine genetic variation. This protocol describes how to screen for mutations in 192 singleplex or up to 768 multiplex samples over 3 days. The protocol is based on the principle of sequence-specific mobility of single-stranded DNA in a native polymer, and covers all stages in the procedure, from initial DNA purification to final CAE-SSCP data analysis, as follows: DNA is purified, followed by PCR amplification using fluorescent primers. After PCR amplification, double-stranded DNA is heat-denatured to separate the strands and subsequently cooled on ice to avoid reannealing. Finally, samples are analyzed by capillary electrophoresis and appropriate analysis software.

  18. Molybdenum disulfide (MoS2) nanoflakes as inherently electroactive labels for DNA hybridization detection

    NASA Astrophysics Data System (ADS)

    Loo, Adeline Huiling; Bonanni, Alessandra; Ambrosi, Adriano; Pumera, Martin

    2014-09-01

    The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide nanomaterials for sensing and biosensing purposes represents an upcoming research area which holds great promise. Hence, our findings are anticipated to have significant contributions towards the fabrication of future DNA biosensors.The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide

  19. Multiple tag labeling method for DNA sequencing

    DOEpatents

    Mathies, R.A.; Huang, X.C.; Quesada, M.A.

    1995-07-25

    A DNA sequencing method is described which uses single lane or channel electrophoresis. Sequencing fragments are separated in the lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radioisotope labels. 5 figs.

  20. Multiple tag labeling method for DNA sequencing

    DOEpatents

    Mathies, Richard A.; Huang, Xiaohua C.; Quesada, Mark A.

    1995-01-01

    A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

  1. The genetics of amphibian decline: population substructure and molecular differentiation in the Yosemite toad, Bufo canorus (Anura, Bufonidae) based on single-strand conformation polymorphism analysis (SSCP) and mitochondrial DNA sequence data

    USGS Publications Warehouse

    Shaffer, H. Bradley; Fellers, Gary M.; Magee, Allison; Voss, S. Randal

    2000-01-01

    We present a comprehensive survey of genetic variation across the range of the narrowly distributed endemic Yosemite toad Bufo canorus, a declining amphibian restricted to the Sierra Nevada of California. Based on 322 bp of mitochondrial cytochrome b sequence data, we found limited support for the monophyly of B. canorus and its closely related congener B. exsul to the exclusion of the widespread western toad B. boreas. However, B. exsul was always phylogenetically nested within B. canorus, suggesting that the latter may not be monophyletic. SSCP (single-strand conformation polymorphism) analysis of 372 individual B. canorus from 28 localities in Yosemite and Kings Canyon National Parks revealed no shared haplotypes among these two regions and lead us to interpret these two parks as distinct management units for B. canorus. Within Yosemite, we found significant genetic substructure both at the level of major drainages and among breeding ponds. Kings Canyon samples show a different pattern, with substantial variation among breeding sites, but no substructure among drainages. Across the range of B. canorus as well as among Yosemite ponds, we found an isolation-by-distance pattern suggestive of a stepping stone model of migration. However, in Kings Canyon we found no hint of such a pattern, suggesting that movement patterns of toads may be quite different in these nearby parklands. Our data imply that management for B. canorus should focus at the individual pond level, and effective management may necessitate reintroductions if local extirpations occur. A brief review of other pond-breeding anurans suggests that highly structured populations are often the case, and thus that our results for B. canorus may be general for other species of frogs and toads.

  2. A novel single-stranded RNA virus in Nesidiocoris tenuis.

    PubMed

    Xu, Pengjun; Song, Xueru; Yang, Xianming; Tang, Zhaoqi; Ren, Guangwei; Lu, Yanhui

    2017-04-01

    The complete genome sequence of a novel single-stranded RNA virus in Nesidiocoris tenuis was determined by RNA-seq and rapid amplification of cDNA ends (RACE) methodologies and was named N. tenuis virus 1. The genomic RNA was 3970 nucleotides (nt) in length and contained two putative open reading frames (ORFs). ORF1 encoded a polypeptide with 283 amino acids containing a viral (superfamily 1) RNA helicase (Hel) domain, and ORF2 encoded a polypeptide with 294 amino acids containing an RNA-dependent RNA polymerase (RdRP) domain. Phylogenetic analysis using the deduced amino acid sequences indicated that the N. tenuis virus 1 clustered with Blackford virus; however, the low bootstrap values and unique genomic structure suggested that the virus is a prototype of a new type of unclassified viruses. The prevalence of N. tenuis virus 1 infection in field populations of N. tenuis differed between three locations, with 28.32% of the 113 sampled individuals testing positive for the virus.

  3. Single-strand conformation polymorphism analysis for differentiating phytoplasma strains.

    PubMed

    Musić, Martina Seruga; Skorić, Dijana

    2013-01-01

    Single-strand conformation polymorphism (SSCP) analysis is a sensitive and rapid technique for detecting DNA polymorphisms and mutations in PCR-amplified fragments. Due to its technical simplicity, it is widely used as a screening tool in various investigations, ranging from clinical diagnosis of human hereditary diseases to the characterization of microbial communities. This method can also be used successfully on phytoplasmas as a tool for the detection of molecular variability in conserved housekeeping genes such as 16S rRNA and tuf, as well as in more variable genes, revealing the presence of polymorphisms undetected by routine RFLP analyses. The reliability of SSCP has been confirmed by multiple alignments and phylogenetic analyses of representative sequences showing different SSCP profiles. However, it is not broadly applied in phytoplasma research yet. The technique provides an inexpensive, convenient, and sensitive method for determining sequence variation and to differentiate phytoplasma strains, and is particularly suitable for epidemiological studies or as a fast screening, typing tool when dealing with a large number of field samples.

  4. High resolution single strand conformation polymorphism analysis using formamide and ethidium bromide staining.

    PubMed Central

    Xie, T; Ho, S L; Ma, O C

    1997-01-01

    Single strand conformation polymorphism (SSCP) analysis using ethidium bromide can be improved by adding formamide as the denaturant. This gives higher resolution than previous SSCP methods; it had 100% sensitivity in the discrimination of 14 PCR samples from two different genes, even for a long fragment close to the upper limit of 250 base pairs. This modified procedure is a rapid, simple, safe, and yet highly sensitive method for detecting structural differences in DNA fragments. Images PMID:9497922

  5. Murine protein which binds preferentially to oligo-C-rich single-stranded nucleic acids.

    PubMed Central

    Goller, M; Funke, B; Gehe-Becker, C; Kröger, B; Lottspeich, F; Horak, I

    1994-01-01

    Two single-stranded nucleic acid binding proteins mCBP and mCTBP were identified by means of their binding to a potential recombination hotspot in LTRs of mouse retro-transposons. Both are nuclear proteins of 35 and 55 kDa respectively. mCBP binds preferentially to oligo dC, mCTBP to oligo dCdT. mCBP was purified and its cDNA was isolated and sequenced. Images PMID:8208614

  6. Understanding Single-Stranded Telomere End Binding by an Essential Protein

    DTIC Science & Technology

    2000-08-01

    disclosed outside the government, (b) used by the Government for manufacture or, in the case of computer software documentation, for preparing the same or...similar computer software , or (c) used by a party other than the Government, except that the Government may release or disclose technical data to...Consulting Merck Research Labs. Pfizer Red Storm Software Zymogenetics 8 Biochemical Investigation of a Sequence-Specific, Single-Stranded DNA Binding

  7. [Comparative study of single strand conformation polymorphism of 4.5S RNA gene in enterobacteria].

    PubMed

    Huang, Y; Gong, L; Zhang, L; Li, S; Zhu, S

    1994-04-01

    A recently developed technique, non-isotopic single strand conformation polymorphism analysis (PCR-SSCP), was applied to study the conserved feature of 4.5S RNA gene in enterobacteria. The 4.5S RNA gene was amplified by the polymerase chain reaction, using the template DNA extracted respectively from five strains of Escherichia coli and three strains of different genera in Enterobacteriaceae, i.e. Proteus vulgaris, Serratia marcescens and Enterobacter aerogenes. The PCR products were then carried out SSCP analysis. The experimental results showed that there seemed to be no detectable differences in the size and single strand conformation of 4.5S RNA genes from above strains, except the negative strand conformation of Enterobacter aerogenes. Thus it can be seen that the secondary structures of 4.5S RNA gene in enterobacteria are quite conservative.

  8. Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods

    PubMed Central

    Hadwiger, Philipp; Wagner, Ernst; Lamb, Don C.

    2017-01-01

    Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between

  9. A Single-Stranded Junction Modulates Nanosecond Motional Ordering of the Substrate Recognition Duplex of a Group I Ribozyme

    PubMed Central

    Nguyen, Phuong; Shi, Xuesong; Sigurdsson, Snorri Th.; Herschlag, Daniel

    2013-01-01

    Rigid spinning: Site-directed spin-labeling studies using a rigid nitroxide spin label (Ç) reveal that both length and sequence of a single-stranded junction (J1/2) modulate nanosecond motional ordering of the substrate-recognition duplex (P1) of the 120 kD group I ribozyme. The studies demonstrate an approach for experimental measurements of nanosecond dynamics in high-molecular-weight RNA complexes. PMID:23900919

  10. Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.

    PubMed

    Nikiforov, Theo T; Roman, Steven

    2015-05-15

    Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).

  11. A human systemic lupus erythematosus-related anti-cardiolipin/single-stranded DNA autoantibody is encoded by a somatically mutated variant of the developmentally restricted 51P1 V[sub H] gene

    SciTech Connect

    Van Es, J.H.; Aanstoot, H.; Gmelig-Meyling, F.H.J.; Derksen, R.H.W.M.; Logtenberg, T. )

    1992-09-15

    The authors report the Ig H and L chain V region sequences from the cDNAs encoding a monoclonal human IgG anti-cardiolipin/ssDNA autoantibody (R149) derived from a patient with active SLE. Comparison with the germ-line V-gene repertoire of this patient revealed that R149 likely arose as a consequence of an Ag-driven selection process. The Ag-binding portions of the V regions were characterized by a high number of arginine residues, a property that has been associated with anti-dsDNA autoantibodies from lupus-prone mice and patients with SLE. The V[sub H] gene encoding autoantibody R149 was a somatically mutated variant of the 51P1 gene segment, which is frequently associated with the restricted fetal B cell repertoire, malignant CD5 B cells, and natural antibodies. These data suggest that in SLE patients a common antigenic stimulus may evoke anti-DNA and anti-cardiolipin autoantibodies and provide further evidence that a small set of developmentally restricted V[sub H] genes can give rise to disease-associated autoantibodies through Ag-selected somatic mutations. 42 refs., 5 figs.

  12. Surface-modified ZnSe waveguides for label-free infrared attenuated total reflection detection of DNA hybridization.

    PubMed

    Riccardi, Carla S; Hess, Dennis W; Mizaikoff, Boris

    2011-12-07

    This communication presents a novel label-free biosensing method to monitor DNA hybridization via infrared attenuated total reflection (IR-ATR) spectroscopy using surface-modified ZnSe waveguides. Well-defined carboxyl-terminated monolayers were formed at H-terminated ZnSe by direct photochemical activation. Chemical activation of the acidic function was obtained by using succinimide/carbodiimide linkers. The sequential surface modification reactions were characterized by XPS and IR-ATR spectroscopy. Finally, a single stranded DNA probe with a C6-NH(2) 5' modifier was coupled to the ester-terminated surface via peptide bonding, and the hybridization of the immobilized DNA sequence with its complementary strand was directly evaluated by IR-ATR spectroscopy in the mid-infrared (MIR) spectral regime (3-20 μm) without requiring an additional label. A shift of the vibrational modes corresponding to the phosphodiester and deoxyribose structures of the DNA backbone was observed. Hence, this approach substantiates a novel strategy for label-free DNA detection utilizing mid-infrared spectroscopy as the optical sensing platform.

  13. Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    PubMed Central

    Gu, Maxwell; Berrido, Andrea; Gonzalez, Walter G.; Miksovska, Jaroslava; Chambers, Jeremy W.; Leng, Fenfei

    2016-01-01

    DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. In this paper we report the synthesis of a type of unique fluorescence-labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 carrying a 42 nt. AT sequence with fluorescein and dabcyl labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905. Since the fluorescence intensity of pAB1_FL905 is dependent on its supercoiling status, pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high-throughput screening assays to identify inhibitors that target various DNA topoisomerases. PMID:27796331

  14. Electrochemical DNA biosensor for detection of porcine oligonucleotides using ruthenium(II) complex as intercalator label redox

    SciTech Connect

    Halid, Nurul Izni Abdullah; Hasbullah, Siti Aishah; Heng, Lee Yook; Karim, Nurul Huda Abd; Ahmad, Haslina; Harun, Siti Norain

    2014-09-03

    A DNA biosensor detection of oligonucleotides via the interactions of porcine DNA with redox active complex based on the electrochemical transduction is described. A ruthenium(II) complex, [Ru(bpy){sub 2}(PIP)]{sup 2+}, (bpy = 2,2′bipyridine, PIP = 2-phenylimidazo[4,5-f[[1,10-phenanthroline]) as DNA label has been synthesized and characterized by 1H NMR and mass spectra. The study was carried out by covalent bonding immobilization of porcine aminated DNA probes sequences on screen printed electrode (SPE) modified with succinimide-acrylic microspheres and [Ru(bpy){sub 2}(PIP)]{sup 2+} was used as electrochemical redox intercalator label to detect DNA hybridization event. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the ruthenium (II) complex was active. The results indicate that the interaction of [Ru(bpy){sub 2}(PIP)]{sup 2+} with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.

  15. Graphene-Assisted Label-Free Homogeneous Electrochemical Biosensing Strategy based on Aptamer-Switched Bidirectional DNA Polymerization.

    PubMed

    Wang, Wenxiao; Ge, Lei; Sun, Ximei; Hou, Ting; Li, Feng

    2015-12-30

    In this contribution, taking the discrimination ability of graphene over single-stranded (ss) DNA/double-stranded (ds) DNA in combination with the electrochemical impedance transducer, we developed a novel label-free homogeneous electrochemical biosensor using graphene-modified glassy carbon electrode (GCE) as the sensing platform. To convert the specific aptamer-target recognition into ultrasensitive electrochemical signal output, a novel aptamer-switched bidirectional DNA polymerization (BDP) strategy, capable of both target recycling and exponential signal amplification, was compatibly developed in this study. In this strategy, all the designed DNA structures could be adsorbed on the graphene/GCE and, thus, serve as the electrochemical impedance signal reporter, while the target acts as a trigger of this BDP reaction, in which these designed DNA structures are bound together and, then, converted to long dsDNA duplex. The distinct difference in electrochemical impedance spectroscopy between the designed structures and generated long dsDNA duplex on the graphene/GCE allows label-free and homogeneous detection of target down to femto-gram level. The target can be displaced from aptamer through the polymerization to initiate the next recognition-polymerization cycle. Herein, the design and signaling principle of aptamer-switched BDP amplification system were elucidated, and the working conditions were optimized. This method not only provides a universal platform for electrochemical biosensing but also shows great potential in biological process researches and clinic diagnostics.

  16. Fluorescence "turn-on" determination of H2O2 using multilayer porous SiO2/NGQDs and PdAu mimetics enzymatic/oxidative cleavage of single-stranded DNA.

    PubMed

    Liang, Linlin; Lan, Feifei; Li, Li; Su, Min; Ge, Shenguang; Yu, Jinghua; Liu, Haiyun; Yan, Mei

    2016-08-15

    A 3D microfluidic paper-based fluorescence analytical device with hollow channels based on the turn-on switching of a resonance energy transfer triggered by the •OH induced cleavage of a DNA strand was successfully constructed. And this fluorescent nanoplatform was first designed to achieve in situ and real-time determination of H2O2 released from cancer cells to obtain an accurate determination. With optimal conditions, the proposed method displayed excellent analytical performance for the detection of H2O2 ranging from 0.3 to 1.0mM with a detection limit of 0.1nM. The favorable performances of this sensor were due to the peroxidase-like activity of nitrogen-doped graphene quantum dots (multilayer porous SiO2 act as stabilizer to load more nitrogen-doped graphene quantum dots for signal amplification) and folic acid-pPdAu/GO (which also could act as an efficient fluorescence quencher and a recognition element of cancer cells by folic acid). It was worth noting that it could be used for visually determined the flux of H2O2 from the cells. Therefore, the developed biosensor holds potential for ultrasensitive quantitative analysis of H2O2 and supplies valuable information for diabetes mellitus research and clinical diagnosis.

  17. Solid-State, Dye-Labeled DNA Detects Volatile Compounds in the Vapor Phase

    PubMed Central

    White, Joel; Truesdell, Kathleen; Williams, Lloyd B; AtKisson, Mary S; Kauer, John S

    2008-01-01

    This paper demonstrates a previously unreported property of deoxyribonucleic acid—the ability of dye-labeled, solid-state DNA dried onto a surface to detect odors delivered in the vapor phase by changes in fluorescence. This property is useful for engineering systems to detect volatiles and provides a way for artificial sensors to emulate the way cross-reactive olfactory receptors respond to and encode single odorous compounds and mixtures. Recent studies show that the vertebrate olfactory receptor repertoire arises from an unusually large gene family and that the receptor types that have been tested so far show variable breadths of response. In designing biomimetic artificial noses, the challenge has been to generate a similarly large sensor repertoire that can be manufactured with exact chemical precision and reproducibility and that has the requisite combinatorial complexity to detect odors in the real world. Here we describe an approach for generating and screening large, diverse libraries of defined sensors using single-stranded, fluorescent dye–labeled DNA that has been dried onto a substrate and pulsed with brief exposures to different odors. These new solid-state DNA-based sensors are sensitive and show differential, sequence-dependent responses. Furthermore, we show that large DNA-based sensor libraries can be rapidly screened for odor response diversity using standard high-throughput microarray methods. These observations describe new properties of DNA and provide a generalized approach for producing explicitly tailored sensor arrays that can be rationally chosen for the detection of target volatiles with different chemical structures that include biologically derived odors, toxic chemicals, and explosives. PMID:18215112

  18. A human NDP-kinase B specifically binds single-stranded poly-pyrimidine sequences.

    PubMed Central

    Hildebrandt, M; Lacombe, M L; Mesnildrey, S; Véron, M

    1995-01-01

    Recently, a DNA binding protein 'PUF' was purified that binds to a poly-pyrimidine rich element in the human c-myc promoter. Cloning of the corresponding gene surprisingly identified this putative transcription factor as isoform B of the enzyme nucleoside diphosphate kinase (NDPK-B) [Postel et al. (1993) Science, 261, 478-480], the product of the potential metastasis suppressor gene nm23-H2. Using different recombinant NDP kinases, we demonstrate by electrophoretic mobility shift analysis (EMSA) that the NDP kinase DNA binding properties are predominantly observed with human isoform B. Unlike typical DNA binding proteins that are involved in transcriptional regulation, binding occurs to single-stranded DNA rather than to a double-stranded oligonucleotide. As a consequence, complexes of single-stranded DNA and NDPK-B are generated from double-stranded oligonucleotide hybrids in an ATP independent manner. In addition to the c-myc element, NDPK-B is binding in vitro to a variety of poly-pyrimidine rich sequences including dC or dT homo-oligomers, (CT)n dinucleotide repeats, the initiator region of the Adenovirus major late promoter and even poly-pyrimidine rich RNAs. The possible consequences of these findings in understanding the multiple roles of NDP kinase are discussed. Images PMID:7479028

  19. Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

    PubMed Central

    Murgha, Yusuf E.; Rouillard, Jean-Marie; Gulari, Erdogan

    2014-01-01

    Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification. PMID:24733454

  20. Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.

    PubMed

    Murgha, Yusuf E; Rouillard, Jean-Marie; Gulari, Erdogan

    2014-01-01

    Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

  1. DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

    PubMed

    Ye, Yu-Dan; Xia, Li; Xu, Dang-Dang; Xing, Xiao-Jing; Pang, Dai-Wen; Tang, Hong-Wu

    2016-11-15

    Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity.

  2. PCR synthesis of double stranded DNA labeled with 5-bromouridine. A step towards finding a bromonucleoside for clinical trials.

    PubMed

    Michalska, Barbara; Sobolewski, Ireneusz; Polska, Katarzyna; Zielonka, Justyna; Zylicz-Stachula, Agnieszka; Skowron, Piotr; Rak, Janusz

    2011-12-05

    Incorporation of 5-bromouridine (5BrdU) into DNA makes it sensitive to UV and ionizing radiation, which opens up a prospective route for the clinical usage of 5-bromouridine and other halonucleosides. In the present work the polymerase chain reaction (PCR) protocol, which enables a long DNA fragment (resembling DNA synthesized in the cell in the presence of halonucleosides) to be completely substituted with 5BrdU, was optimized. Using HPLC coupled to enzymatic digestion, it was demonstrated that the actual amounts of native nucleosides and 5BrdU correspond very well to those calculated from the sequence of PCR products. The synthesized DNA is photosensitive to photons of 300nm. HPLC analysis demonstrated that the photolysis of labeled PCR products leads to a significant decrease in the 5BrdU signal and the simultaneous occurrence of a uridine peak. Agarose and polyacrylamide gel electrophoresis suggest that single strand breaks and cross-links are formed as a result of UV irradiation. The PCR protocol described in the current paper may be employed for labeling DNA not only with BrdU but also with other halonucleosides.

  3. Genetic analysis of Trichinella populations by 'cold' single-strand conformation polymorphism analysis.

    PubMed

    Gasser, Robin B; Hu, Min; El-Osta, Youssef Abs; Zarlenga, Dante S; Pozio, Edoardo

    2005-09-05

    A non-isotopic single-strand conformation polymorphism ('cold' SSCP) technique has been assessed for the analysis of sequence variability in the expansion segment 5 (ES5) of domain IV and the D3 domain of nuclear ribosomal DNA within and/or among isolates and individual muscle (first-stage) larvae representing all currently recognized species/genotypes of Trichinella. Data are consistent with the ability of cold SSCP to identify intra-specific as well as inter-specific variability among Trichinella genotypes. The cold SSCP approach should be applicable to a range of other genetic markers for comparative studies of Trichinella populations globally.

  4. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    PubMed

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    (99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99m)Tc-labeled HYNIC-DAPI compound with that of (99m)Tc pertechnetate ((99m)TcO4(-)). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by (99m)TcO4(-) (0.51), and the number of DSBs increased fivefold in the (99m)Tc-HYNIC-DAPI-treated sample compared with the (99m)TcO4(-) treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99m)TcO4(-) treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99m)Tc-HYNIC-DAPI-treated samples. These results indicated that (99m)Tc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99m)Tc-labeled compound with DNA. In contrast to these results, (99m)TcO4(-) induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99m)Tc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by

  5. 99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

    PubMed Central

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, 99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a 99mTc-labeled HYNIC-DAPI compound with that of 99mTc pertechnetate (99mTcO4−). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by 99mTcO4− (0.51), and the number of DSBs increased fivefold in the 99mTc-HYNIC-DAPI-treated sample compared with the 99mTcO4− treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the 99mTcO4– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the 99mTc-HYNIC-DAPI-treated samples. These results indicated that 99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the 99mTc-labeled compound with DNA. In contrast to these results, 99mTcO4− induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of 99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of

  6. Gel-based nonradioactive single-strand conformational polymorphism and mutation detection: limitations and solutions.

    PubMed

    Gupta, Vibhuti; Arora, Reetakshi; Gochhait, Sailesh; Bairwa, Narendra K; Bamezai, Rameshwar N K

    2014-01-01

    Single-strand conformation polymorphism (SSCP) for screening mutations/single-nucleotide polymorphisms (SNPs) is a simple, cost-effective technique, saving an expensive exercise of sequencing each and every polymerase chain reaction product and assisting in choosing only the amplicons of interest with expected mutations. The principle of detection of small changes in DNA sequences is based on changes in single-strand DNA conformations. The changes in electrophoretic mobility that SSCP detects are sequence dependent. The limitations faced in SSCP range from routine polyacrylamide gel electrophoresis (PAGE) problems to the problems of resolving mutant DNA bands. Both these problems can be solved by controlling PAGE conditions and by varying physical and environmental conditions such as pH, temperature, voltage, gel type and percentage, addition of additives or denaturants, and others. Despite much upgrading of the technology for mutation detection, SSCP remains the method of choice to analyze mutations and SNPs in order to understand genomic variations, both spontaneous and induced, and the genetic basis of diseases.

  7. [Application of DNA labeling technology in forensic botany].

    PubMed

    Znang, Xian; Li, Jing-Lin; Zhang, Xiang-Yu

    2008-12-01

    Forensic botany is a study of judicial plant evidence. Recently, researches on DNA labeling technology have been a mainstream of forensic botany. The article systematically reviews various types of DNA labeling techniques in forensic botany with enumerated practical cases, as well as the potential forensic application of each individual technique. The advantages of the DNA labeling technology over traditional morphological taxonomic methods are also summarized.

  8. Single-strand conformation polymorphism (SSCP) analysis of the molecular pathology of hemophilia B.

    PubMed

    David, D; Rosa, H A; Pemberton, S; Diniz, M J; Campos, M; Lavinha, J

    1993-01-01

    In the present study, we report the application of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis to the screening of seven functionally important factor IX gene (FIX) regions (total length 2.66 kb) in 9 unrelated haemophilia B patients of Portuguese or African origin. In eight of the patients an altered migration pattern of single-stranded DNA was observed. Direct sequencing of the relevant DNA fragments unveiled the following sequence alterations: two novel mutations, namely FIXBarcelos Thr-380-Pro and FIXLousada 9bp insertion at position 31,309 or 31,318; five mutations previously reported in other ethnic groups (FIXPorto Arg-145-His, FIXLuanda Gly-207-Arg, FIXPenafiel Arg-248-Gln, FIXSesimbra Arg-333-Gln, FIXCascais Arg-333-Stop); and a normal variant, G-->T transvertion at position 6,596 in intron 2. We propose hypothetical models for the generation of the 9 bp duplication (FIXLousada). We have performed molecular modeling studies in order to predict the structure of the variant FIX molecules.

  9. Recombineering: highly efficient in vivo genetic engineering using single-strand oligos.

    PubMed

    Sawitzke, James A; Thomason, Lynn C; Bubunenko, Mikhail; Li, Xintian; Costantino, Nina; Court, Donald L

    2013-01-01

    Recombineering provides the ability to make rapid, precise, and inexpensive genetic alterations to any DNA sequence, either in the chromosome or cloned onto a vector that replicates in E. coli (or other recombineering-proficient bacteria), and to do so in a highly efficient manner. Complicated genetic constructs that are impossible to make with in vitro genetic engineering can be created in days with recombineering. Recombineering with single-strand DNA (ssDNA) can be used to create single or multiple clustered point mutations, small or large (up to 10kb) deletions, and small (10-20 base) insertions such as sequence tags. Using optimized conditions, point mutations can be made with such high frequencies that they can be found without selection. This technology excels at creating both directed and random mutations.

  10. The development of a silica nanoparticle-based label-free DNA biosensor

    NASA Astrophysics Data System (ADS)

    Kell, Arnold J.; Pagé, Lilianne; Tan, Sophie; Charlebois, Isabelle; Boissinot, Maurice; Leclerc, Mario; Simard, Benoit

    2011-09-01

    A silica nanoparticle-based DNA biosensor capable of detecting Bacillus anthracis bacteria through the use of unlabelled ss-oligonucleotides has been developed. The biosensor makes use of the optical changes that accompany a nanoparticle-immobilized cationic conjugated polymer (polythiophene) interacting with single-stranded vs. hybridized oligonucleotides, where a fluorescence signal appears only when hybridized DNA is present (i.e. only when the ss-oligonucleotide interacting with the polymer has hybridized with its complement). In order to enhance the sensitivity of the biosensor, two different nanoparticle architectures were developed and used to elucidate how the presence of neighboring fluorophores on the nanoparticle surface affects Förster-resonant energy transfer (FRET) between the polythiophene/oligonucleotide complex (FRET donor) and the fluorophores (FRET acceptors). We demonstrate that the silica nanoparticle-based FRET platform lowers the limit of detection at least 10-fold in comparison to the polythiophene itself, and allows the detection of ~2 × 10-12 moles of ss-oligonucleotide in a 100 μL sample with a standard fluorimeter (i.e. has a limit of detection of ~2 nM ssDNA). Such nanoparticle-based biosensor platforms are beneficial because of the robustness and stability inherent to their covalent assembly and they provide a valuable new tool that may allow for the sensitive, label-free detection (the target DNA that produces the fluorescence signal is unlabelled) without the use of polymerase chain reaction.A silica nanoparticle-based DNA biosensor capable of detecting Bacillus anthracis bacteria through the use of unlabelled ss-oligonucleotides has been developed. The biosensor makes use of the optical changes that accompany a nanoparticle-immobilized cationic conjugated polymer (polythiophene) interacting with single-stranded vs. hybridized oligonucleotides, where a fluorescence signal appears only when hybridized DNA is present (i.e. only when

  11. Singlet oxygen induces predominantly G to T transversions on a single-stranded shuttle vector replicated in monkey cells.

    PubMed

    Ribeiro, D T; De Oliveira, R C; Di Mascio, P; Menck, C F

    1994-08-01

    To elucidate the mechanisms of mutagenesis by singlet oxygen DNA damage in mammalian cells, a SV40-derived single-stranded shuttle vector was exposed to the water soluble endoperoxide 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). The damaged vector was transfected into monkey COS7 cells and the plasmid progeny exhibited up to 10 fold increase on the mutation frequency in the supF target gene, when compared to untreated vector. The sequence in the supF locus of such mutants revealed that singlet oxygen-induced mutagenesis in single-stranded vector is significantly different from spontaneous mutagenesis. Among the base substitutions, most of the mutations involved deoxyguanosines, being G to T transversions the predominant type of change. The data indicate that mutagenesis by singlet oxygen in mammalian cells may be generated by an error prone bypass of damaged deoxyguanosines at the template DNA.

  12. Electrophoresis of end-labeled DNA: Theory and experiment

    NASA Astrophysics Data System (ADS)

    Lau, Henry W.; Archer, Lynden A.

    2010-03-01

    The dynamic behavior of end-labeled DNA during free-solution electrophoresis is investigated using a simple dumbbell model for the labeled DNA. We study the effect of the applied field, label size, and chain stiffness on DNA conformation and electrophoretic mobility. High applied fields are predicted to magnify the size-dependence of mobility and to yield a nonmonotonic dependence of electrophoretic mobility on applied field. The effectiveness of leveraging label size and DNA chain stiffness for improving resolution is also discussed in the context of DNA deformation. To evaluate the most salient model predictions, we use capillary electrophoresis experiments to characterize the size- and field-dependent mobility of dsDNA fragments (300 bp-2 kbp) end-functionalized with streptavidin. Our experimental results are found to be in generally good accord with expectations based on the dumb-bell model. We discuss implications of these findings for fast, size-based separation of DNA in free solution.

  13. Lack of induction of single-strand breaks in mammalian cells by sodium azide and its proximal mutagen

    SciTech Connect

    Arenaz, P.; Nilan, R.A.; Kleinhofs, A.

    1981-01-01

    The mutagenicity of sodium azide in both higher plants and bacteria is well documented. However, in mammalian cells, research on the effects of azide on gene mutations has produced conflicting results. Furthermore, no research has been conducted on the effects of azide and its proximal mutagen (mutagenic metabolite) on single-strand breaks. The experiments herein were designed to overcome this lack of information on azide mutagenicity and to evaluate the potential hazard of azide exposure to man. Chinese hamster V/sub 79/ cells were treated with either azide or its proximal mutagen(s) for 2 h or 6 h respectively and analyzed by alkaline elution for single-strand breaks. The data showed that there was no effect of either azide or the proximal mutagen(s) on single-strand DNA breaks nor was there any indication that azide or the proximal mutagen(s) induced DNA-protein crosslinks. The data suggest that neither azide nor its proximal mutagen(s) interact directly with DNA and suggest that the methods may be applied to any specified population and dose scenario.

  14. Simultaneous detection and semiquantification of DNA damage in normal and apoptotic cells: triple-immunofluorescent labeling using DAPI, antibodies, and TUNEL.

    PubMed

    Agrawal, Anant; Godar, Dianne E

    2012-07-01

    We developed a triple-labeling immunofluorescence technique that simultaneously identifies total DNA (DAPI), DNA damage (antibodies), and dead cells [terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells] and a method that semiquantifies DNA damage in paraffin-embedded tissues. Using this technique in combination with our analysis method, scientists can now simultaneously detect and compare the relative amounts of DNA damage of almost any kind (except single-strand and double-strand breaks), using indirect fluorescent antibody labeling, in both normal and dying cells of different tissues. Simultaneous labeling of DNA damage and dead or TUNEL-positive cells can reduce processing costs and analysis time, and can lead to discoveries concerning how cells die from different DNA damages. We used increasing doses of UV (290 to 400 nm) radiation to create DNA damage in the form of cyclobutane pyrimidine dimers and 6-4 photoproducts that kill some of the cells in 3-dimensional tissue-engineered skin and vaginal samples. We describe a protocol that reliably detects and semiquantifies DNA damage in both normal and apoptotic cells. We show this triple-labeling immunofluorescence technique and analysis method yields linear UV dose response curves for damage to DNA bases that allows semiquantification of cyclobutane pyrimidine dimers and calculation of its repair rate (T=1 and 24 h), whereas TUNEL allows quantification of the number of apoptotic cells. Scientists can now create beautiful fluorescent pictures that simultaneously detect DNA damage in both normal and apoptotic cells to assess and semiquantify the damage to understand better how different insults lead to the cell's demise.

  15. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    PubMed Central

    Ren, Xiaomei; El-Sagheer, Afaf H.; Brown, Tom

    2016-01-01

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX. PMID:26819406

  16. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.

    PubMed

    Ren, Xiaomei; El-Sagheer, Afaf H; Brown, Tom

    2016-05-05

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.

  17. A fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp.

    PubMed

    Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

    2011-06-01

    The present study aimed to establish a fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp. Based on the sequences of the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA, we designed a set of genus-specific primers for the amplification of Fasciola ITS-2, with an estimated size of 140 bp. These primers were labelled by fluorescence dyes, and the PCR products were analyzed by capillary electrophoresis under non-denaturing conditions (F-PCR-SSCP). Capillary electrophoresis analysis of the fluorescence-labelled DNA fragments displayed three different peak profiles that allowed the accurate identification of Fasciola species: one single peak specific for either Fasciola hepatica or Fasciola gigantica and a doublet peak corresponding to the "intermediate" Fasciola. Validation of our novel method was performed using Fasciola specimens from different host animals from China, Spain, Nigeria, and Egypt. This F-PCR-SSCP assay provides a rapid, simple, and robust tool for the identification and differentiation between Fasciola spp.

  18. Phosphorylated 5-ethynyl-2'-deoxyuridine for advanced DNA labeling.

    PubMed

    Seo, Siyoong; Onizuka, Kazumitsu; Nishioka, Chieko; Takahashi, Eiki; Tsuneda, Satoshi; Abe, Hiroshi; Ito, Yoshihiro

    2015-04-21

    The representative DNA-labeling agent 5-ethynyl-2'-deoxyuridine (EdU) was chemically modified to improve its function. Chemical monophosphorylation was expected to enhance the efficiency of the substrate in DNA polymerization by circumventing the enzymatic monophosphorylation step that consumes energy. In addition, to enhance cell permeability, the phosphates were protected with bis-pivaloyloxymethyl that is stable in buffer and plasma, and degradable inside various cell types. The phosphorylated EdU (PEdU) was less toxic than EdU, and had the same or a slightly higher DNA-labeling ability in vitro. PEdU was also successfully applied to DNA labeling in vivo. In conclusion, PEdU can be used as a less toxic DNA-labeling agent for studies that require long-term cell survival or very sensitive cell lines.

  19. DNA Hybridization: Nonradioactive Labeling Now Available for the Laboratory.

    ERIC Educational Resources Information Center

    Freeman, Lenore Gardner

    1984-01-01

    The advantages of DNA hybridization procedures for classroom and clinical use can now be realized by the recent development of nonradioactive DNA labeling/detection procedures. These methods (which are described) can replace the use of isotopes in standard DNA hybridization procedures. (JN)

  20. Capillary Electrophoresis Single-Strand Conformational Polymorphisms as a Method to Differentiate Algal Species.

    PubMed

    Jernigan, Alice; Hestekin, Christa

    2015-01-01

    Capillary electrophoresis single-strand conformational polymorphism (CE-SSCP) was explored as a fast and inexpensive method to differentiate both prokaryotic (blue-green) and eukaryotic (green and brown) algae. A selection of two blue-green algae (Nostoc muscorum and Anabaena inaequalis), five green algae (Chlorella vulgaris, Oedogonium foveolatum, Mougeotia sp., Scenedesmus quadricauda, and Ulothrix fimbriata), and one brown algae (Ectocarpus sp.) were examined and CE-SSCP electropherogram "fingerprints" were compared to each other for two variable regions of either the 16S or 18S rDNA gene. The electropherogram patterns were remarkably stable and consistent for each particular species. The patterns were unique to each species, although some common features were observed between the different types of algae. CE-SSCP could be a useful method for monitoring changes in an algae species over time as potential shifts in species occurred.

  1. Capillary Electrophoresis Single-Strand Conformational Polymorphisms as a Method to Differentiate Algal Species

    PubMed Central

    Jernigan, Alice; Hestekin, Christa

    2015-01-01

    Capillary electrophoresis single-strand conformational polymorphism (CE-SSCP) was explored as a fast and inexpensive method to differentiate both prokaryotic (blue-green) and eukaryotic (green and brown) algae. A selection of two blue-green algae (Nostoc muscorum and Anabaena inaequalis), five green algae (Chlorella vulgaris, Oedogonium foveolatum, Mougeotia sp., Scenedesmus quadricauda, and Ulothrix fimbriata), and one brown algae (Ectocarpus sp.) were examined and CE-SSCP electropherogram “fingerprints” were compared to each other for two variable regions of either the 16S or 18S rDNA gene. The electropherogram patterns were remarkably stable and consistent for each particular species. The patterns were unique to each species, although some common features were observed between the different types of algae. CE-SSCP could be a useful method for monitoring changes in an algae species over time as potential shifts in species occurred. PMID:26101693

  2. Data mining cDNAs reveals three new single stranded RNA viruses in Nasonia (Hymenoptera: Pteromalidae).

    PubMed

    Oliveira, D C S G; Hunter, W B; Ng, J; Desjardins, C A; Dang, P M; Werren, J H

    2010-02-01

    We report three novel small RNA viruses uncovered from cDNA libraries from parasitoid wasps in the genus Nasonia. The genome of this kind of virus is a positive-sense single-stranded RNA with a 3' poly(A), which facilitates cloning from cDNAs. Two of the viruses, NvitV-1 and NvitV-2, possess a RNA-dependent RNA polymerase that associates them with the family Iflaviridae of the order Picornavirales. A third virus, NvitV-3, is most similar to the Nora virus from Drosophila. A reverse transcription-PCR method developed for NvitV-1 indicates that it is a persistent commensal infection of Nasonia.

  3. Single-strand conformation polymorphism (SSCP) analysis of HLA-DRB1*1101-06.

    PubMed

    Mora, B; Petronzelli, F; Grillo, R; Ferrante, P; Mazzilli, M C

    1996-10-01

    Single-strand conformation polymorphism (SSCP) has been developed as a method for detecting the presence of mutations in a segment of DNA. We applied it to the subtyping of the DR11 group of alleles. The SSCP patterns of DRB1-DR52 group-specific products were defined in cell lines representing the DRB1*1101-06 alleles, using non-denaturing acrylamide gel electrophoresis and silver staining. Only one set of gel electrophoresis conditions was able to discriminate the DR11 alleles tested. The protocol was validated in an analysis of 105 DR11-positive individuals previously typed by oligonucleotides probing. The study demonstrates the suitability of the SSCP technique to define the DRB1*1101-06 alleles, the technique being particularly valuable in confirming and extending the oligotyping of DRB1-DR52 heterozygous samples.

  4. Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs.

    PubMed

    Wang, Mugui; Wei, Chuchu; Ye, Xiufen; Liu, Jianping; Zhang, Cuicui; Chen, Hao; Zhang, Xiaobo; Tu, Jumin

    2015-04-15

    We developed an effective protocol for generating high-purity heteroduplexes via annealing single-stranded DNAs (ssDNAs) derived from plasmid DNA by asymmetric polymerase chain reaction (A-PCR). With the addition of dimethyl sulfoxide, a one-step A-PCR procedure can generate ssDNAs stably at a range of reaction temperatures. Several annealing buffers can anneal two ssDNAs into heteroduplexes effectively. We further developed a simple strategy to create d(GATC) hemimethylated heteroduplexes by annealing fully methylated homoduplexes in the presence of excessive unmethylated ssDNAs. The constructed heteroduplexes have been well tested as substrates for mismatch repair in Escherichia coli and, thus, can be used in various biotechnology applications.

  5. Analysis of population structures of viral isolates using single-strand conformation polymorphism method.

    PubMed

    Delaunay, Agnès; Rolland, Mathieu; Jacquot, Emmanuel

    2009-01-01

    The analysis of viral populations requires the use of techniques that describe characteristics of individuals. The single-strand conformation polymorphism (SSCP) makes possible the identification of genetic differences between viral sequences and constitutes an alternative to the expensive and time-consuming cloning and sequencing strategies. Applied to small genomic regions (from 100 to 500 bases in length), SSCP patterns could describe, under appropriate experimental conditions, single nucleotide variations in the studied sequence. The different steps of a complete SSCP procedure, from sampling to pattern analysis (including nucleic acid extraction, RT-PCR amplification, double-stranded DNA quantification, polyacrylamide gel preparation, electrophoresis conditions, and staining procedures), are described using a region (500 bases) of the barley yellow dwarfvirus-PAV (BYDV-PAV, Luteovirus) genome as molecular target.

  6. TDP1 facilitates chromosomal single-strand break repair in neurons and is neuroprotective in vivo.

    PubMed

    Katyal, Sachin; el-Khamisy, Sherif F; Russell, Helen R; Li, Yang; Ju, Limei; Caldecott, Keith W; McKinnon, Peter J

    2007-11-14

    Defective Tyrosyl-DNA phosphodiesterase 1 (TDP1) can cause spinocerebellar ataxia with axonal neuropathy (SCAN1), a neurodegenerative syndrome associated with marked cerebellar atrophy and peripheral neuropathy. Although SCAN1 lymphoblastoid cells show pronounced defects in the repair of chromosomal single-strand breaks (SSBs), it is unknown if this DNA repair activity is important for neurons or for preventing neurodegeneration. Therefore, we generated Tdp1-/- mice to assess the role of Tdp1 in the nervous system. Using both in vitro and in vivo assays, we found that cerebellar neurons or primary astrocytes derived from Tdp1-/- mice display an inability to rapidly repair DNA SSBs associated with Top1-DNA complexes or oxidative damage. Moreover, loss of Tdp1 resulted in age-dependent and progressive cerebellar atrophy. Tdp1-/- mice treated with topotecan, a drug that increases levels of Top1-DNA complexes, also demonstrated significant loss of intestinal and hematopoietic progenitor cells. These data indicate that TDP1 is required for neural homeostasis, and reveal a widespread requisite for TDP1 function in response to acutely elevated levels of Top1-associated DNA strand breaks.

  7. TDP1 facilitates chromosomal single-strand break repair in neurons and is neuroprotective in vivo

    PubMed Central

    Katyal, Sachin; El-Khamisy, Sherif F; Russell, Helen R; Li, Yang; Ju, Limei; Caldecott, Keith W; McKinnon, Peter J

    2007-01-01

    Defective Tyrosyl-DNA phosphodiesterase 1 (TDP1) can cause spinocerebellar ataxia with axonal neuropathy (SCAN1), a neurodegenerative syndrome associated with marked cerebellar atrophy and peripheral neuropathy. Although SCAN1 lymphoblastoid cells show pronounced defects in the repair of chromosomal single-strand breaks (SSBs), it is unknown if this DNA repair activity is important for neurons or for preventing neurodegeneration. Therefore, we generated Tdp1−/− mice to assess the role of Tdp1 in the nervous system. Using both in vitro and in vivo assays, we found that cerebellar neurons or primary astrocytes derived from Tdp1−/− mice display an inability to rapidly repair DNA SSBs associated with Top1–DNA complexes or oxidative damage. Moreover, loss of Tdp1 resulted in age-dependent and progressive cerebellar atrophy. Tdp1−/− mice treated with topotecan, a drug that increases levels of Top1–DNA complexes, also demonstrated significant loss of intestinal and hematopoietic progenitor cells. These data indicate that TDP1 is required for neural homeostasis, and reveal a widespread requisite for TDP1 function in response to acutely elevated levels of Top1-associated DNA strand breaks. PMID:17914460

  8. Preparation of (32)P-end-labeled DNA fragments for performing DNA-binding experiments.

    PubMed

    Carey, Michael F; Peterson, Craig L; Smale, Stephen T

    2013-05-01

    The generation of a uniquely (32)P-end-labeled DNA fragment is essential for DNA-binding experiments such as DNase I footprinting and ethylation interference. We describe here a protocol for end-labeling a restriction fragment. For a plasmid DNA bearing a region containing the binding site of interest, cleaving with a single restriction endonuclease generates a 5' overhang containing a phosphate. This is generally necessary for both common forms of fragment end-labeling: phosphorylation with polynucleotide kinase and "filling in the end" with DNA polymerases (e.g., Klenow fragment). For the phosphorylation reaction, as described here, the phosphate is removed with calf intestinal phosphatase or bacterial alkaline phosphatase, and the resulting free 5'-OH is phosphorylated with polynucleotide kinase and [γ-(32)P]ATP. This generates a plasmid labeled at each end with γ-(32)P. The molar amount of plasmid DNA must be below the amount of ATP added to the reaction and the ATP must be of sufficiently high specific activity to generate a fragment labeled to the extent necessary for many DNA-binding experiments. To generate a uniquely end-labeled DNA fragment, the labeled plasmid is heat-treated to inactivate any remaining kinase and recleaved with a second endonuclease, releasing a short DNA fragment and a longer vector fragment. The DNA fragment is purified from the labeled vector on a 5%-8% native polyacrylamide gel. The preparation and labeling of DNA restriction fragments typically takes 1-2 d.

  9. Gold surface supported spherical liposome-gold nano-particle nano-composite for label free DNA sensing.

    PubMed

    Bhuvana, M; Narayanan, J Shankara; Dharuman, V; Teng, W; Hahn, J H; Jayakumar, K

    2013-03-15

    Immobilization of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposome-gold nano-particle (DOPE-AuNP) nano-composite covalently on 3-mercaptopropionic acid (MPA) on gold surface is demonstrated for the first time for electrochemical label free DNA sensing. Spherical nature of the DOPE on the MPA monolayer is confirmed by the appearance of sigmoidal voltammetric profile, characteristic behavior of linear diffusion, for the MPA-DOPE in presence of [Fe(CN)(6)](3-/4-) and [Ru(NH(3))(6)](3+) redox probes. The DOPE liposome vesicle fusion is prevented by electroless deposition of AuNP on the hydrophilic amine head groups of the DOPE. Immobilization of single stranded DNA (ssDNA) is made via simple gold-thiol linkage for DNA hybridization sensing in the presence of [Fe(CN)(6)](3-/4-). The sensor discriminates the hybridized (complementary target hybridized), un-hybridized (non-complementary target hybridized) and single base mismatch target hybridized surfaces sensitively and selectively without signal amplification. The lowest target DNA concentration detected is 0.1×10(-12)M. Cyclic voltammetry (CV), electrochemical impedance (EIS), differential pulse voltammetry (DPV) and quartz crystal microbalance (QCM) techniques are used for DNA sensing on DOPE-AuNP nano-composite. Transmission Electron Microscopy (TEM), Fourier Transform Infrared Spectroscopy (FTIR), Atomic Force Microscopy (AFM), Dynamic Light Scattering (DLS) and Ultraviolet-Visible (UV) spectroscopic techniques are used to understand the interactions between the DOPE, AuNP and ssDNA. The results indicate the presence of an intact and well defined spherical DOPE-AuNP nano-composite on the gold surface. The method could be applied for fabrication of the surface based liposome-AuNP-DNA composite for cell transfection studies at reduced reagents and costs.

  10. Microchannel DNA Sequencing by End-Labelled Free Solution Electrophoresis

    SciTech Connect

    Barron, A.

    2005-09-29

    The further development of End-Labeled Free-Solution Electrophoresis will greatly simplify DNA separation and sequencing on microfluidic devices. The development and optimization of drag-tags is critical to the success of this research.

  11. Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

    PubMed

    Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B

    2015-01-01

    Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

  12. Fiber optofluidic biosensor for the label-free detection of DNA hybridization and methylation based on an in-line tunable mode coupler.

    PubMed

    Gao, Ran; Lu, Dan-Feng; Cheng, Jin; Jiang, Yi; Jiang, Lan; Xu, Jian-Dong; Qi, Zhi-Mei

    2016-12-15

    An optical fiber optofluidic biosensor for the detection of DNA hybridization and methylation has been proposed and experimentally demonstrated. An in-line fiber Michelson interferometer was formed in the photonic crystal fiber. A micrhole in the collapsed region, which combined the tunable mode coupler and optofluidic channel, was fabricated by using femtosecond laser micromachining. The mode field diameter of the guided light is changed with the refractive index in the optofluidic channel, which results in the tunable coupling ratio. Label-free detections of the DNA hybridization and methylation have been experimentally demonstrated. The probe single stranded DNA (ssDNA) was bound with the surface of the optofluidic channel through the Poly-l-lysine layer, and the hybridization between a short 22-mer probe ssDNA and a complementary target ssDNA was carried out and detected by interrogating the fringe visibility of the reflection spectrum. Then, the DNA methylation was also detected through the binding between the methylated DNA and the 5-methylcytosine (5-mC) monoclonal antibody. The experiments results demonstrate that the limit of detection of 5nM is achieved, establishing the tunable mode coupler as a sensitive and versatile biosensor. The sensitive optical fiber optofluidic biosensor possesses high specificity and low temperature cross-sensitivity.

  13. Gating of single-layer graphene with single-stranded deoxyribonucleic acids.

    PubMed

    Lin, Jian; Teweldebrhan, Desalegne; Ashraf, Khalid; Liu, Guanxiong; Jing, Xiaoye; Yan, Zhong; Li, Rong; Ozkan, Mihri; Lake, Roger K; Balandin, Alexander A; Ozkan, Cengiz S

    2010-05-21

    Patterning of biomolecules on graphene layers could provide new avenues to modulate their electrical properties for novel electronic devices. Single-stranded deoxyribonucleic acids (ssDNAs) are found to act as negative-potential gating agents that increase the hole density in single-layer graphene. Current-voltage measurements of the hybrid ssDNA/graphene system indicate a shift in the Dirac point and "intrinsic" conductance after ssDNA is patterned. The effect of ssDNA is to increase the hole density in the graphene layer, which is calculated to be on the order of 1.8 x 10(12) cm(-2). This increased density is consistent with the Raman frequency shifts in the G-peak and 2D band positions and the corresponding changes in the G-peak full width at half maximum. Ab initio calculations using density functional theory rule out significant charge transfer or modification of the graphene band structure in the presence of ssDNA fragments.

  14. Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies.

    PubMed

    Rombouts, K; Braeckmans, K; Remaut, K

    2016-02-17

    Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this Review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular mRNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs.

  15. Developing an electrochemical deoxyribonucleic acid (DNA) biosensor on the basis of human interleukine-2 gene using an electroactive label.

    PubMed

    Pournaghi-Azar, M H; Hejazi, M S; Alipour, E

    2006-06-16

    Development of an electrochemical DNA biosensor based on a human interleukine-2 (IL-2) gene probe, using a pencil graphite electrode (PGE) as transducer and methylene blue (MB) as electroactive label is described. The sensor relies on the immobilization of a 20-mer single stranded oligonucleotide probe (hIL-2) related to the IL-2 gene on the electrode. The hybridization between the probe and its complementary sequence (chIL-2) as the target was studied by square wave voltammetry (SWV) of MB accumulated on the PGE. In this approach the extent of hybridization is evaluated on the basis of the difference between SWV signals of MB accumulated on the probe-PGE and MB accumulated on the probe-target-PGE. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Some experimental variables affecting the performance of the biosensor including: polishing of PGE, its electrochemical activation conditions (i.e.,