Sample records for target dna strands

  1. 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.

  2. 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

  3. 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.

  4. Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

    PubMed Central

    Sheng, Gang; Zhao, Hongtu; Wang, Jiuyu; Rao, Yu; Tian, Wenwen; Swarts, Daan C.; van der Oost, John; Patel, Dinshaw J.; Wang, Yanli

    2014-01-01

    We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg2+ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand. PMID:24374628

  5. Linear nicking endonuclease-mediated strand-displacement DNA amplification.

    PubMed

    Joneja, Aric; Huang, Xiaohua

    2011-07-01

    We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Linear nicking endonuclease-mediated strand displacement DNA amplification

    PubMed Central

    Joneja, Aric; Huang, Xiaohua

    2011-01-01

    We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to five thousand nucleotides can be linearly amplified using a nicking endonuclease with seven base-pair recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length are linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly. PMID:21342654

  7. Strand displacement activated peroxidase activity of hemin for fluorescent DNA sensing.

    PubMed

    Wang, Quanbo; Xu, Nan; Gui, Zhen; Lei, Jianping; Ju, Huangxian; Yan, Feng

    2015-10-07

    To efficiently regulate the catalytic activity of the peroxidase mimic hemin, this work designs a double-stranded DNA probe containing an intermolecular dimer of hemin, whose peroxidase activity can be activated by a DNA strand displacement reaction. The double-stranded probe is prepared by annealing two strands of hemin labelled DNA oligonucleotides. Using the fluorescent oxidation product of tyramine by H2O2 as a tracing molecule, the low peroxidase activity of the hemin dimer ensures a low fluorescence background. The strand displacement reaction of the target DNA dissociates the hemin dimer and thus significantly increases the catalytic activity of hemin to produce a large amount of dityramine for fluorescence signal readout. Based on the strand displacement regulated peroxidase activity, a simple and sensitive homogeneous fluorescent DNA sensing method is proposed. The detection can conveniently be carried out in a 96-well plate within 20 min with a detection limit of 0.18 nM. This method shows high specificity, which can effectively distinguish single-base mismatched DNA from perfectly matched target DNA. The DNA strand displacement regulated catalytic activity of hemin has promising application in the determination of various DNA analytes.

  8. In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting.

    PubMed

    Chen, Xiaoyu; Janssen, Josephine M; Liu, Jin; Maggio, Ignazio; 't Jong, Anke E J; Mikkers, Harald M M; Gonçalves, Manuel A F V

    2017-09-22

    Precise genome editing involves homologous recombination between donor DNA and chromosomal sequences subjected to double-stranded DNA breaks made by programmable nucleases. Ideally, genome editing should be efficient, specific, and accurate. However, besides constituting potential translocation-initiating lesions, double-stranded DNA breaks (targeted or otherwise) are mostly repaired through unpredictable and mutagenic non-homologous recombination processes. Here, we report that the coordinated formation of paired single-stranded DNA breaks, or nicks, at donor plasmids and chromosomal target sites by RNA-guided nucleases based on CRISPR-Cas9 components, triggers seamless homology-directed gene targeting of large genetic payloads in human cells, including pluripotent stem cells. Importantly, in addition to significantly reducing the mutagenicity of the genome modification procedure, this in trans paired nicking strategy achieves multiplexed, single-step, gene targeting, and yields higher frequencies of accurately edited cells when compared to the standard double-stranded DNA break-dependent approach.CRISPR-Cas9-based gene editing involves double-strand breaks at target sequences, which are often repaired by mutagenic non-homologous end-joining. Here the authors use Cas9 nickases to generate coordinated single-strand breaks in donor and target DNA for precise homology-directed gene editing.

  9. Using DNA origami nanostructures to determine absolute cross sections for UV photon-induced DNA strand breakage.

    PubMed

    Vogel, Stefanie; Rackwitz, Jenny; Schürman, Robin; Prinz, Julia; Milosavljević, Aleksandar R; Réfrégiers, Matthieu; Giuliani, Alexandre; Bald, Ilko

    2015-11-19

    We have characterized ultraviolet (UV) photon-induced DNA strand break processes by determination of absolute cross sections for photoabsorption and for sequence-specific DNA single strand breakage induced by photons in an energy range from 6.50 to 8.94 eV. These represent the lowest-energy photons able to induce DNA strand breaks. Oligonucleotide targets are immobilized on a UV transparent substrate in controlled quantities through attachment to DNA origami templates. Photon-induced dissociation of single DNA strands is visualized and quantified using atomic force microscopy. The obtained quantum yields for strand breakage vary between 0.06 and 0.5, indicating highly efficient DNA strand breakage by UV photons, which is clearly dependent on the photon energy. Above the ionization threshold strand breakage becomes clearly the dominant form of DNA radiation damage, which is then also dependent on the nucleotide sequence.

  10. The Human DNA glycosylases NEIL1 and NEIL3 Excise Psoralen-Induced DNA-DNA Cross-Links in a Four-Stranded DNA Structure.

    PubMed

    Martin, Peter R; Couvé, Sophie; Zutterling, Caroline; Albelazi, Mustafa S; Groisman, Regina; Matkarimov, Bakhyt T; Parsons, Jason L; Elder, Rhoderick H; Saparbaev, Murat K

    2017-12-12

    Interstrand cross-links (ICLs) are highly cytotoxic DNA lesions that block DNA replication and transcription by preventing strand separation. Previously, we demonstrated that the bacterial and human DNA glycosylases Nei and NEIL1 excise unhooked psoralen-derived ICLs in three-stranded DNA via hydrolysis of the glycosidic bond between the crosslinked base and deoxyribose sugar. Furthermore, NEIL3 from Xenopus laevis has been shown to cleave psoralen- and abasic site-induced ICLs in Xenopus egg extracts. Here we report that human NEIL3 cleaves psoralen-induced DNA-DNA cross-links in three-stranded and four-stranded DNA substrates to generate unhooked DNA fragments containing either an abasic site or a psoralen-thymine monoadduct. Furthermore, while Nei and NEIL1 also cleave a psoralen-induced four-stranded DNA substrate to generate two unhooked DNA duplexes with a nick, NEIL3 targets both DNA strands in the ICL without generating single-strand breaks. The DNA substrate specificities of these Nei-like enzymes imply the occurrence of long uninterrupted three- and four-stranded crosslinked DNA-DNA structures that may originate in vivo from DNA replication fork bypass of an ICL. In conclusion, the Nei-like DNA glycosylases unhook psoralen-derived ICLs in various DNA structures via a genuine repair mechanism in which complex DNA lesions can be removed without generation of highly toxic double-strand breaks.

  11. Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

    PubMed

    Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

    2018-01-01

    DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

  12. Oxidant-induced DNA damage of target cells.

    PubMed Central

    Schraufstätter, I; Hyslop, P A; Jackson, J H; Cochrane, C G

    1988-01-01

    In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks. PMID:2843565

  13. Sequential strand displacement beacon for detection of DNA coverage on functionalized gold nanoparticles.

    PubMed

    Paliwoda, Rebecca E; Li, Feng; Reid, Michael S; Lin, Yanwen; Le, X Chris

    2014-06-17

    Functionalizing nanomaterials for diverse analytical, biomedical, and therapeutic applications requires determination of surface coverage (or density) of DNA on nanomaterials. We describe a sequential strand displacement beacon assay that is able to quantify specific DNA sequences conjugated or coconjugated onto gold nanoparticles (AuNPs). Unlike the conventional fluorescence assay that requires the target DNA to be fluorescently labeled, the sequential strand displacement beacon method is able to quantify multiple unlabeled DNA oligonucleotides using a single (universal) strand displacement beacon. This unique feature is achieved by introducing two short unlabeled DNA probes for each specific DNA sequence and by performing sequential DNA strand displacement reactions. Varying the relative amounts of the specific DNA sequences and spacing DNA sequences during their coconjugation onto AuNPs results in different densities of the specific DNA on AuNP, ranging from 90 to 230 DNA molecules per AuNP. Results obtained from our sequential strand displacement beacon assay are consistent with those obtained from the conventional fluorescence assays. However, labeling of DNA with some fluorescent dyes, e.g., tetramethylrhodamine, alters DNA density on AuNP. The strand displacement strategy overcomes this problem by obviating direct labeling of the target DNA. This method has broad potential to facilitate more efficient design and characterization of novel multifunctional materials for diverse applications.

  14. Sequence dependence of electron-induced DNA strand breakage revealed by DNA nanoarrays

    PubMed Central

    Keller, Adrian; Rackwitz, Jenny; Cauët, Emilie; Liévin, Jacques; Körzdörfer, Thomas; Rotaru, Alexandru; Gothelf, Kurt V.; Besenbacher, Flemming; Bald, Ilko

    2014-01-01

    The electronic structure of DNA is determined by its nucleotide sequence, which is for instance exploited in molecular electronics. Here we demonstrate that also the DNA strand breakage induced by low-energy electrons (18 eV) depends on the nucleotide sequence. To determine the absolute cross sections for electron induced single strand breaks in specific 13 mer oligonucleotides we used atomic force microscopy analysis of DNA origami based DNA nanoarrays. We investigated the DNA sequences 5′-TT(XYX)3TT with X = A, G, C and Y = T, BrU 5-bromouracil and found absolute strand break cross sections between 2.66 · 10−14 cm2 and 7.06 · 10−14 cm2. The highest cross section was found for 5′-TT(ATA)3TT and 5′-TT(ABrUA)3TT, respectively. BrU is a radiosensitizer, which was discussed to be used in cancer radiation therapy. The replacement of T by BrU into the investigated DNA sequences leads to a slight increase of the absolute strand break cross sections resulting in sequence-dependent enhancement factors between 1.14 and 1.66. Nevertheless, the variation of strand break cross sections due to the specific nucleotide sequence is considerably higher. Thus, the present results suggest the development of targeted radiosensitizers for cancer radiation therapy. PMID:25487346

  15. The Size of the Internal Loop in DNA Hairpins Influences Their Targeting with Partially Complementary Strands

    PubMed Central

    2015-01-01

    Targeting of noncanonical DNA structures, such as hairpin loops, may have significant diagnostic and therapeutic potential. Oligonucleotides can be used for binding to mRNA, forming a DNA/RNA hybrid duplex that inhibits translation. This kind of modulation of gene expression is called the antisense approach. In order to determine the best strategy to target a common structural motif in mRNA, we have designed a set of stem-loop DNA molecules with sequence: d(GCGCTnGTAAT5GTTACTnGCGC), where n = 1, 3, or 5, “T5” is an end loop of five thymines. We used a combination of calorimetric and spectroscopy techniques to determine the thermodynamics for the reaction of a set of hairpins containing internal loops with their respective partially complementary strands. Our aim was to determine if internal- and end-loops are promising regions for targeting with their corresponding complementary strands. Indeed, all targeting reactions were accompanied by negative changes in free energy, indicating that reactions proceed spontaneously. Further investigation showed that these negative free energy terms result from a net balance of unfavorable entropy and favorable enthalpy contributions. In particular, unfolding of hairpins and duplexes is accompanied by positive changes in heat capacity, which may be a result of exposure of hydrophobic groups to the solvent. This study provides a new method for the targeting of mRNA in order to control gene expression. PMID:25486129

  16. Streptavidin-coated magnetic beads for DNA strand separation implicate a multitude of problems during cell-SELEX.

    PubMed

    Paul, Angela; Avci-Adali, Meltem; Ziemer, Gerhard; Wendel, Hans P

    2009-09-01

    Using whole living cells as a target for SELEX (systematic evolution of ligands by exponential enrichment) experiments represents a promising method to generate cell receptor-specific aptamers. These aptamers have a huge potential in diagnostics, therapeutics, imaging, regenerative medicine, and target validation. During the SELEX for selecting DNA aptamers, one important step is the separation of 2 DNA strands to yield one of the 2 strands as single-stranded DNA aptamer. This is being done routinely by biotin labeling of the complementary DNA strand to the desired aptamer and then separating the DNA strand by using streptavidin-coated magnetic beads. After immobilization of the double-stranded DNA on these magnetic beads and alkaline denaturation, the non-biotinylated strand is being eluted and the biotinylated strand is retarded. Using Western blot analysis, we demonstrated the detachment of covalent-bonded streptavidin from the bead surface after alkaline treatment. The eluates were also contaminated with undesired biotinylated strands. Furthermore, a streptavidin-induced aggregation of target cells was demonstrated by flow cytometry and microscopic methods. Cell-specific enrichment of aptamers was not possible due to clustering and patching effects triggered by streptavidin. Therefore, the use of streptavidin-coated magnetic beads for DNA strand separation should be examined thoroughly, especially for cell-SELEX applications.

  17. Can a double stranded DNA be unzipped by pulling a single strand?: phases of adsorbed DNA.

    PubMed

    Kapri, Rajeev

    2009-04-14

    We study the unzipping of a double stranded DNA (dsDNA) by applying an external force on a single strand while leaving the other strand free. We find that the dsDNA can be unzipped to two single strands if the external force exceeds a critical value. We obtain the phase diagram, which is found to be different from the phase diagram of unzipping by pulling both the strands in opposite directions. In the presence of an attractive surface near DNA, the phase diagram gets modified drastically and shows richer surprises including a critical end point and a triple point.

  18. Synthesis of mouse centromere-targeted polyamides and physico-chemical studies of their interaction with the target double-stranded DNA.

    PubMed

    Nozeret, Karine; Bonan, Marc; Yarmoluk, Serguiy M; Novopashina, Darya S; Boutorine, Alexandre S

    2015-09-01

    Synthetic minor groove-binding pyrrole-imidazole polyamides labeled by fluorophores are promising candidates for fluorescence imaging of double-stranded DNA in isolated chromosomes or fixed and living cells. We synthesized nine hairpin and two head-to-head tandem polyamides targeting repeated sequences from mouse major satellites. Their interaction with synthetic target dsDNA has been studied by physico-chemical methods in vitro before and after coupling to various fluorophores. Great variability in affinities and fluorescence properties reveals a conclusion that these properties do not only rely on recognition rules, but also on other known and unknown structural factors. Individual testing of each probe is needed before cellular applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Programmable DNA Hydrogels Assembled from Multidomain DNA Strands.

    PubMed

    Jiang, Huiling; Pan, Victor; Vivek, Skanda; Weeks, Eric R; Ke, Yonggang

    2016-06-16

    Hydrogels are important in biological and medical applications, such as drug delivery and tissue engineering. DNA hydrogels have attracted significant attention due to the programmability and biocompatibility of the material. We developed a series of low-cost one-strand DNA hydrogels self-assembled from single-stranded DNA monomers containing multiple palindromic domains. This new hydrogel design is simple and programmable. Thermal stability, mechanical properties, and loading capacity of these one-strand DNA hydrogels can be readily regulated by simply adjusting the DNA domains. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Current-voltage characteristics of double stranded versus single stranded DNA molecules

    NASA Astrophysics Data System (ADS)

    Hartzell, B.; Chen, Hong; Heremans, J. J.; McCord, B.; Soghomonian, V.

    2004-03-01

    Investigation of DNA conductivity has focused on the native, duplex structure, with controversial results. Here, we present the influence of the double-helical structure on charge transport through lambda DNA molecules. The current-voltage (I-V) characteristics of both disulfide-labeled double stranded DNA (dsDNA) and disulfide-labeled single stranded DNA (ssDNA) were measured. The ssDNA was formed from the dsDNA using two different methods for comparison purposes: a thermal/chemical denaturation and enzymatic digestion utilizing lambda exonuclease. Resulting I-V characteristics of both the double stranded and single stranded samples were close-to-linear when measured at room temperature. However, the ssDNA samples consistently gave conductivity values about two orders of magnitude smaller in amplitude. Our results suggest an integral relationship between the native structure of DNA with its stacked base pairs and the molecule's ability to support charge transport.(NSF NIRT 0103034)

  1. Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.

    PubMed

    Liu, Yibin; Song, Chen; Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Makrigiorgos, G Mike

    2017-04-07

    Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Photochemical Acceleration of DNA Strand Displacement by Using Ultrafast DNA Photo-crosslinking.

    PubMed

    Nakamura, Shigetaka; Hashimoto, Hirokazu; Kobayashi, Satoshi; Fujimoto, Kenzo

    2017-10-18

    DNA strand displacement is an essential reaction in genetic recombination, biological processes, and DNA nanotechnology. In particular, various DNA nanodevices enable complicated calculations. However, it takes time before the output is obtained, so acceleration of DNA strand displacement is required for a rapid-response DNA nanodevice. Herein, DNA strand displacement by using DNA photo-crosslinking to accelerate this displacement is evaluated. The DNA photo-crosslinking of 3-cyanovinylcarbazole ( CNV K) was accelerated at least 20 times, showing a faster DNA strand displacement. The rate of photo-crosslinking is a key factor and the rate of DNA strand displacement is accelerated through ultrafast photo-crosslinking. The rate of DNA strand displacement was regulated by photoirradiation energy. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  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. Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template.

    PubMed

    Yuan, Quan; McHenry, Charles S

    2009-11-13

    In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

  5. 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

  6. 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

  7. Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA.

    PubMed

    Khodakov, Dmitriy A; Khodakova, Anastasia S; Huang, David M; Linacre, Adrian; Ellis, Amanda V

    2015-03-04

    Single nucleotide polymorphisms (SNPs) are a prime source of genetic diversity. Discriminating between different SNPs provides an enormous leap towards the better understanding of the uniqueness of biological systems. Here we report on a new approach for SNP discrimination using toehold-mediated DNA strand displacement. The distinctiveness of the approach is based on the combination of both 3- and 4-way branch migration mechanisms, which allows for reliable discrimination of SNPs within double-stranded DNA generated from real-life human mitochondrial DNA samples. Aside from the potential diagnostic value, the current study represents an additional way to control the strand displacement reaction rate without altering other reaction parameters and provides new insights into the influence of single nucleotide substitutions on 3- and 4-way branch migration efficiency and kinetics.

  8. Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells.

    PubMed

    Huh, Yang Hoon; Cohen, Justin; Sherley, James L

    2013-10-15

    Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs "know" the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.

  9. A strand graph semantics for DNA-based computation

    PubMed Central

    Petersen, Rasmus L.; Lakin, Matthew R.; Phillips, Andrew

    2015-01-01

    DNA nanotechnology is a promising approach for engineering computation at the nanoscale, with potential applications in biofabrication and intelligent nanomedicine. DNA strand displacement is a general strategy for implementing a broad range of nanoscale computations, including any computation that can be expressed as a chemical reaction network. Modelling and analysis of DNA strand displacement systems is an important part of the design process, prior to experimental realisation. As experimental techniques improve, it is important for modelling languages to keep pace with the complexity of structures that can be realised experimentally. In this paper we present a process calculus for modelling DNA strand displacement computations involving rich secondary structures, including DNA branches and loops. We prove that our calculus is also sufficiently expressive to model previous work on non-branching structures, and propose a mapping from our calculus to a canonical strand graph representation, in which vertices represent DNA strands, ordered sites represent domains, and edges between sites represent bonds between domains. We define interactions between strands by means of strand graph rewriting, and prove the correspondence between the process calculus and strand graph behaviours. Finally, we propose a mapping from strand graphs to an efficient implementation, which we use to perform modelling and simulation of DNA strand displacement systems with rich secondary structure. PMID:27293306

  10. Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

    PubMed

    Ge, Jia; Bai, Dong-Mei; -Geng, Xin; Hu, Ya-Lei; Cai, Qi-Yong; Xing, Ke; Zhang, Lin; Li, Zhao-Hui

    2018-01-10

    The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.

  11. Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells

    PubMed Central

    Huh, Yang Hoon; Cohen, Justin; Sherley, James L.

    2013-01-01

    Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs “know” the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency. PMID:24082118

  12. DNA-mediated strand displacement facilitates sensitive electronic detection of antibodies in human serums.

    PubMed

    Dou, Baoting; Yang, Jianmei; Shi, Kai; Yuan, Ruo; Xiang, Yun

    2016-09-15

    We describe here the development of a sensitive and convenient electronic sensor for the detection of antibodies in human serums. The sensor is constructed by self-assembly formation of a mixed monolayer containing the small molecule epitope conjugated double stranded DNA probes on gold electrode. The target antibody binds the epitope on the dsDNA probe and lowers the melting temperature of the duplex, which facilitates the displacement of the antibody-linked strand of the duplex probe by an invading methylene blue-tagged single stranded DNA (MB-ssDNA) through the strand displacement reaction and leads to the capture of many MB-ssDNA on the sensor surface. Subsequent electrochemical oxidation of the methylene blue labels results in amplified current response for sensitive monitoring of the antibodies. The antibody assay conditions are optimized and the sensor exhibits a linear range between 1.0 and 25.0nM with a detection limit of 0.67nM for the target antibody. The sensor is also selective and can be employed to detect the target antibodies in human serum samples. With the advantages of using small molecule epitope as the antibody recognition element over traditional antigen, the versatile manipulability of the DNA probes and the unique properties of the electrochemical transduction technique, the developed sensor thus hold great potential for simple and sensitive detection of different antibodies and other proteins in real samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Dynamic DNA nanotechnology using strand-displacement reactions

    NASA Astrophysics Data System (ADS)

    Zhang, David Yu; Seelig, Georg

    2011-02-01

    The specificity and predictability of Watson-Crick base pairing make DNA a powerful and versatile material for engineering at the nanoscale. This has enabled the construction of a diverse and rapidly growing set of DNA nanostructures and nanodevices through the programmed hybridization of complementary strands. Although it had initially focused on the self-assembly of static structures, DNA nanotechnology is now also becoming increasingly attractive for engineering systems with interesting dynamic properties. Various devices, including circuits, catalytic amplifiers, autonomous molecular motors and reconfigurable nanostructures, have recently been rationally designed to use DNA strand-displacement reactions, in which two strands with partial or full complementarity hybridize, displacing in the process one or more pre-hybridized strands. This mechanism allows for the kinetic control of reaction pathways. Here, we review DNA strand-displacement-based devices, and look at how this relatively simple mechanism can lead to a surprising diversity of dynamic behaviour.

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

    PubMed Central

    Hong, Ka Lok

    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

  15. Sensitive immobilization-free electrochemical DNA sensor based on isothermal circular strand displacement polymerization reaction.

    PubMed

    Xuan, Feng; Luo, Xiaoteng; Hsing, I-Ming

    2012-05-15

    A highly sensitive electrochemical DNA sensor that requires no probe immobilization has been developed based on a target recycling mechanism utilizing a DNA polymerase with a strand displacement activity. The electrochemical detection is realized by taking advantage of the difference in diffusivity between a free ferrocene-labeled peptide nucleic acid (Fc-PNA) and a Fc-PNA hybridized with a complementary DNA, while the DNA polymerase-assisted target recycling leads to signal generation and amplification. The hybridization of the target DNA opens up a stem-loop template DNA with the Fc-PNA hybridized to its extruded 5' end and allows a DNA primer to anneal and be extended by the DNA polymerase, which results in sequential displacement of the target DNA and the Fc-PNA from the template DNA. The displaced target DNA will hybridize with another template DNA, triggering another round of primer extension and strand displacement. The released Fc-PNA, due to its neutral backbone, has much higher diffusivity towards a negatively charged electrode, compared to that when it is hybridized with a negatively charged DNA. Therefore, a significantly enhanced signal of Fc can be observed. The outstanding sensitivity and simplicity make this approach a promising candidate for next-generation electrochemical DNA sensing technologies. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. AID Mediates Hypermutation by Deaminating Single Stranded DNA

    PubMed Central

    Dickerson, Sarah K.; Market, Eleonora; Besmer, Eva; Papavasiliou, F. Nina

    2003-01-01

    Activation-induced deaminase (AID) is a protein indispensable for the diversification of immunoglobulin (Ig) genes by somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion. To date, the precise role of AID in these processes has not been determined. Here we demonstrate that purified, tetrameric AID can deaminate cytidine residues in DNA, but not in RNA. Furthermore, we show that AID will bind and deaminate only single-stranded DNA, which implies a direct, functional link between hypermutation and transcription. Finally, AID does not target mutational hotspots, thus mutational targeting to specific residues must be attributed to different factors. PMID:12756266

  17. Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

    PubMed Central

    Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

    2016-01-01

    Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

  18. DNA-directed mutations. Leading and lagging strand specificity

    NASA Technical Reports Server (NTRS)

    Sinden, R. R.; Hashem, V. I.; Rosche, W. A.

    1999-01-01

    The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

  19. Invasive reaction assisted strand-displacement signal amplification for sensitive DNA detection.

    PubMed

    Zou, Bingjie; Song, Qinxin; Wang, Jianping; Liu, Yunlong; Zhou, Guohua

    2014-11-18

    A novel DNA detection assay was proposed by invasive reaction coupled with molecular beacon assisted strand-displacement signal amplification (IRASA). Target DNAs are firstly hybridized to two probes to initiate invasive reaction to produce amplified flaps. Then these flaps are further amplified by strand-displacement signal amplification. The detection limit was around 0.2 pM.

  20. Excess single-stranded DNA inhibits meiotic double-strand break repair.

    PubMed

    Johnson, Rebecca; Borde, Valérie; Neale, Matthew J; Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S H

    2007-11-01

    During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant

  1. Excess Single-Stranded DNA Inhibits Meiotic Double-Strand Break Repair

    PubMed Central

    Bishop-Bailey, Anna; North, Matthew; Harris, Sheila; Nicolas, Alain; Goldman, Alastair S. H

    2007-01-01

    During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair

  2. Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

    PubMed

    Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

    2016-03-04

    Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Connecting localized DNA strand displacement reactions

    NASA Astrophysics Data System (ADS)

    Mullor Ruiz, Ismael; Arbona, Jean-Michel; Lad, Amitkumar; Mendoza, Oscar; Aimé, Jean-Pierre; Elezgaray, Juan

    2015-07-01

    Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis). As expected, constraining distances between DNA strands leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. Appropriate design of the system, including protection and asymmetry between input and fuel strands, leads to a reproducible behaviour, at least one order of magnitude faster than the one observed under bulk conditions.Logic circuits based on DNA strand displacement reactions have been shown to be versatile enough to compute the square root of four-bit numbers. The implementation of these circuits as a set of bulk reactions faces difficulties which include leaky reactions and intrinsically slow, diffusion-limited reaction rates. In this paper, we consider simple examples of these circuits when they are attached to platforms (DNA origamis). As expected, constraining distances between DNA strands leads to faster reaction rates. However, it also induces side-effects that are not detectable in the solution-phase version of this circuitry. Appropriate design of the system, including protection and asymmetry between input and fuel strands, leads to a reproducible behaviour, at least one order of magnitude faster than the one observed under bulk conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/C5NR02434J

  4. An intercalation-locked parallel-stranded DNA tetraplex

    DOE PAGES

    Tripathi, S.; Zhang, D.; Paukstelis, P. J.

    2015-01-27

    DNA has proved to be an excellent material for nanoscale construction because complementary DNA duplexes are programmable and structurally predictable. However, in the absence of Watson–Crick pairings, DNA can be structurally more diverse. Here, we describe the crystal structures of d(ACTCGGATGAT) and the brominated derivative, d(AC BrUCGGA BrUGAT). These oligonucleotides form parallel-stranded duplexes with a crystallographically equivalent strand, resulting in the first examples of DNA crystal structures that contains four different symmetric homo base pairs. Two of the parallel-stranded duplexes are coaxially stacked in opposite directions and locked together to form a tetraplex through intercalation of the 5'-most A–A basemore » pairs between adjacent G–G pairs in the partner duplex. The intercalation region is a new type of DNA tertiary structural motif with similarities to the i-motif. 1H– 1H nuclear magnetic resonance and native gel electrophoresis confirmed the formation of a parallel-stranded duplex in solution. Finally, we modified specific nucleotide positions and added d(GAY) motifs to oligonucleotides and were readily able to obtain similar crystals. This suggests that this parallel-stranded DNA structure may be useful in the rational design of DNA crystals and nanostructures.« less

  5. Dna2 initiates resection at clean DNA double-strand breaks

    PubMed Central

    Paudyal, Sharad C.; Li, Shan; Yan, Hong; Hunter, Tony

    2017-01-01

    Abstract Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5′ strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5′ strand DNA ∼10–20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5′ strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP–MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms. PMID:28981724

  6. Translocation of double strand DNA into a biological nanopore

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

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

  7. Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks

    PubMed Central

    Merk, Karin; Breinig, Marco; Böttcher, Romy; Krebs, Stefan; Blum, Helmut; Boutros, Michael

    2017-01-01

    DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured Drosophila cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation. We validated this observation through site-specific DNA cleavage with CRISPR-cas9 followed by deep sequencing of small RNAs. DNA breaks in intron-less genes or upstream of a gene’s first intron did not efficiently trigger siRNA production. When DNA double-strand breaks were induced downstream of an intron, however, this led to robust siRNA generation. Furthermore, a downstream break slowed down splicing of the upstream intron and a detailed analysis of siRNA coverage at the targeted locus revealed that unspliced pre-mRNA contributes the sense strand to the siRNA precursor. Since splicing factors are stimulating the response but unspliced transcripts are entering the siRNA biogenesis, the spliceosome is apparently stalled in a pre-catalytic state and serves as a signaling hub. We conclude that convergent transcription at DNA breaks is stimulated by a splicing dependent control process. The resulting double-stranded RNA is converted into siRNAs that instruct the degradation of cognate mRNAs. In addition to a potential role in DNA repair, the break-induced transcription may thus be a means to cull improper RNAs from the transcriptome of Drosophila melanogaster. Since the splicing factors identified in our screen also stimulated siRNA production from high copy transgenes, it is possible that this surveillance mechanism serves in genome defense beyond DNA double-strand breaks. PMID:28628606

  8. Control of DNA strand displacement kinetics using toehold exchange.

    PubMed

    Zhang, David Yu; Winfree, Erik

    2009-12-02

    DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA molecules. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quantitatively predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.

  9. Equilibrious Strand Exchange Promoted by DNA Conformational Switching

    NASA Astrophysics Data System (ADS)

    Wu, Zhiguo; Xie, Xiao; Li, Puzhen; Zhao, Jiayi; Huang, Lili; Zhou, Xiang

    2013-01-01

    Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

  10. Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells

    PubMed Central

    Linehan, Erin K.; Schrader, Carol E.; Stavnezer, Janet

    2015-01-01

    Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID. PMID:26263206

  11. DNA Double-Strand Break Repair as Determinant of Cellular Radiosensitivity to Killing and Target in Radiation Therapy

    PubMed Central

    Mladenov, Emil; Magin, Simon; Soni, Aashish; Iliakis, George

    2013-01-01

    Radiation therapy plays an important role in the management of a wide range of cancers. Besides innovations in the physical application of radiation dose, radiation therapy is likely to benefit from novel approaches exploiting differences in radiation response between normal and tumor cells. While ionizing radiation induces a variety of DNA lesions, including base damages and single-strand breaks, the DNA double-strand break (DSB) is widely considered as the lesion responsible not only for the aimed cell killing of tumor cells, but also for the general genomic instability that leads to the development of secondary cancers among normal cells. Homologous recombination repair (HRR), non-homologous end-joining (NHEJ), and alternative NHEJ, operating as a backup, are the major pathways utilized by cells for the processing of DSBs. Therefore, their function represents a major mechanism of radiation resistance in tumor cells. HRR is also required to overcome replication stress – a potent contributor to genomic instability that fuels cancer development. HRR and alternative NHEJ show strong cell-cycle dependency and are likely to benefit from radiation therapy mediated redistribution of tumor cells throughout the cell-cycle. Moreover, the synthetic lethality phenotype documented between HRR deficiency and PARP inhibition has opened new avenues for targeted therapies. These observations make HRR a particularly intriguing target for treatments aiming to improve the efficacy of radiation therapy. Here, we briefly describe the major pathways of DSB repair and review their possible contribution to cancer cell radioresistance. Finally, we discuss promising alternatives for targeting DSB repair to improve radiation therapy and cancer treatment. PMID:23675572

  12. Dynamic protein assembly by programmable DNA strand displacement.

    PubMed

    Chen, Rebecca P; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

    2018-04-01

    Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

  13. Dynamic protein assembly by programmable DNA strand displacement

    NASA Astrophysics Data System (ADS)

    Chen, Rebecca P.; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

    2018-03-01

    Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

  14. Molecular investigation of evaporation of biodroplets containing single-strand DNA on graphene surface.

    PubMed

    Akbari, Fahimeh; Foroutan, Masumeh

    2018-02-14

    In this study, the water droplet behaviour of four different types of single-strand DNA with homogeneous base sequence on a graphene substrate during evaporation of the droplet was investigated using molecular dynamics (MD) simulation. The simulation results indicated that the evaporation depended on the DNA sequence. The observed changes can be divided into four parts: (i) vaporization mode, (ii) evaporation flux, (iii) mechanism of single-strand placement on the surface, and (iv) consideration of remaining single strands after evaporation. Our simulation observations indicated different evaporation modes for thymine biodroplets as compared to those for other biodroplets. The evaporation of the thymine biodroplets occurred with an increase in the contact angle, while that of the other biodroplets occur in a constant contact angle mode. Moreover, thymine biodroplets generate the lowest contact line compared to other single strands, and it is always placed far away from the centre of the droplets during evaporation. Investigating variations in the evaporation flux shows that thymine has the highest evaporation flux and guanine has the lowest. Moreover, during initial evaporation, the flux of evaporation increases at the triple point of the biodroplets containing thymine single strands, while it decreases in the other biodroplets. The following observation was obtained from the study of the placement of single strands on the substrate: guanine and thymine interacted slower than other single strands during evaporation with graphene, adenine single strand had a higher folding during evaporation, and guanine single strand showed the lowest end-to-end distance. The investigation of single-strand DNA after evaporation shows that adenine produces the most stable structure at the end of evaporation. In addition, cytosine is the most stretched single-strand DNA due to its lack of internal π-π stacking and hydrogen bonding. Therefore, cytosine single strand is more

  15. Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities.

    PubMed

    Wang, Guoping; Ding, Xiong; Hu, Jiumei; Wu, Wenshuai; Sun, Jingjing; Mu, Ying

    2017-10-24

    Existing isothermal nucleic acid amplification (INAA) relying on the strand displacement activity of DNA polymerase usually requires at least two primers. However, in this paper, we report an unusual isothermal multimerization and amplification (UIMA) which only needs one primer and is efficiently initiated by the strand-displacing DNA polymerases with reverse transcription activities. On electrophoresis, the products of UIMA present a cascade-shape band and they are confirmed to be multimeric DNAs with repeated target sequences. In contrast to current methods, UIMA is simple to product multimeric DNA, due to the independent of multiple primers and rolling circle structures. Through assaying the synthesized single-stranded DNA targets, UIMA performs high sensitivity and specificity, as well as the universality. In addition, a plausible mechanism of UIMA is proposed, involving short DNA bending, mismatch extension, and template slippage. UIMA is a good explanation for why nonspecific amplification easily happens in existing INAAs. As the simplest INAA till now, UIMA provides a new insight for deeply understanding INAA and opens a new avenue for thoroughly addressing nonspecific amplification.

  16. Low concentration of arsenite exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Qin Xujun; Department of Toxicology, Fourth Military Medical University, Xi'an, Shaanxi, 710032; Hudson, Laurie G.

    2008-10-01

    Epidemiological studies have associated arsenic exposure with many types of human cancers. Arsenic has also been shown to act as a co-carcinogen even at low concentrations. However, the precise mechanism of its co-carcinogenic action is unknown. Recent studies indicate that arsenic can interfere with DNA-repair processes. Poly(ADP-ribose) polymerase (PARP)-1 is a zinc-finger DNA-repair protein, which can promptly sense DNA strand breaks and initiate DNA-repair pathways. In the present study, we tested the hypothesis that low concentrations of arsenic could inhibit PAPR-1 activity and so exacerbate levels of ultraviolet radiation (UVR)-induced DNA strand breaks. HaCat cells were treated with arsenite and/ormore » UVR, and then DNA strand breaks were assessed by comet assay. Low concentrations of arsenite ({<=} 2 {mu}M) alone did not induce significant DNA strand breaks, but greatly enhanced the DNA strand breaks induced by UVR. Further studies showed that 2 {mu}M arsenite effectively inhibited PARP-1 activity. Zinc supplementation of arsenite-treated cells restored PARP-1 activity and significantly diminished the exacerbating effect of arsenite on UVR-induced DNA strand breaks. Importantly, neither arsenite treatment, nor zinc supplementation changed UVR-triggered reactive oxygen species (ROS) formation, suggesting that their effects upon UVR-induced DNA strand breaks are not through a direct free radical mechanism. Combination treatments of arsenite with PARP-1 inhibitor 3-aminobenzamide or PARP-1 siRNA demonstrate that PARP-1 is the target of arsenite. Together, these findings show that arsenite at low concentration exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity, which may represent an important mechanism underlying the co-carcinogenicity of arsenic.« less

  17. Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification.

    PubMed

    Hu, Yuhua; Xu, Xueqin; Liu, Qionghua; Wang, Ling; Lin, Zhenyu; Chen, Guonan

    2014-09-02

    A simple, ultrasensitive, and specific electrochemical biosensor was designed to determine the given DNA sequence of Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification. The target DNA (TD, the DNA sequence from the hypervarient region of 16S rDNA of Bacillus subtilis) could be detected by the differential pulse voltammetry (DPV) in a range from 0.1 fM to 20 fM with the detection limit down to 0.08 fM at the 3s(blank) level. This electrochemical biosensor exhibits high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence, which may be expected to detect single-base mismatch and single nucleotide polymorphisms (SNPs). Moreover, the applicability of the designed biosensor for detecting the given DNA sequence from Bacillus subtilis was investigated. The result obtained by electrochemical method is approximately consistent with that by a real-time quantitative polymerase chain reaction detecting system (QPCR) with SYBR Green.

  18. DNA-imprinted polymer nanoparticles with monodispersity and prescribed DNA-strand patterns

    NASA Astrophysics Data System (ADS)

    Trinh, Tuan; Liao, Chenyi; Toader, Violeta; Barłóg, Maciej; Bazzi, Hassan S.; Li, Jianing; Sleiman, Hanadi F.

    2018-02-01

    As colloidal self-assembly increasingly approaches the complexity of natural systems, an ongoing challenge is to generate non-centrosymmetric structures. For example, patchy, Janus or living crystallization particles have significantly advanced the area of polymer assembly. It has remained difficult, however, to devise polymer particles that associate in a directional manner, with controlled valency and recognition motifs. Here, we present a method to transfer DNA patterns from a DNA cage to a polymeric nanoparticle encapsulated inside the cage in three dimensions. The resulting DNA-imprinted particles (DIPs), which are 'moulded' on the inside of the DNA cage, consist of a monodisperse crosslinked polymer core with a predetermined pattern of different DNA strands covalently 'printed' on their exterior, and further assemble with programmability and directionality. The number, orientation and sequence of DNA strands grafted onto the polymeric core can be controlled during the process, and the strands are addressable independently of each other.

  19. Optimised detection of mitochondrial DNA strand breaks.

    PubMed

    Hanna, Rebecca; Crowther, Jonathan M; Bulsara, Pallav A; Wang, Xuying; Moore, David J; Birch-Machin, Mark A

    2018-05-04

    Intrinsic and extrinsic factors that induce cellular oxidative stress damage tissue integrity and promote ageing, resulting in accumulative strand breaks to the mitochondrial DNA (mtDNA) genome. Limited repair mechanisms and close proximity to superoxide generation make mtDNA a prominent biomarker of oxidative damage. Using human DNA we describe an optimised long-range qPCR methodology that sensitively detects mtDNA strand breaks relative to a suite of short mitochondrial and nuclear DNA housekeeping amplicons, which control for any variation in mtDNA copy number. An application is demonstrated by detecting 16-36-fold mtDNA damage in human skin cells induced by hydrogen peroxide and solar simulated radiation. Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  20. A Novel Computational Method to Reduce Leaky Reaction in DNA Strand Displacement.

    PubMed

    Li, Xin; Wang, Xun; Song, Tao; Lu, Wei; Chen, Zhihua; Shi, Xiaolong

    2015-01-01

    DNA strand displacement technique is widely used in DNA programming, DNA biosensors, and gene analysis. In DNA strand displacement, leaky reactions can cause DNA signals decay and detecting DNA signals fails. The mostly used method to avoid leakage is cleaning up after upstream leaky reactions, and it remains a challenge to develop reliable DNA strand displacement technique with low leakage. In this work, we address the challenge by experimentally evaluating the basic factors, including reaction time, ratio of reactants, and ion concentration to the leakage in DNA strand displacement. Specifically, fluorescent probes and a hairpin structure reporting DNA strand are designed to detect the output of DNA strand displacement, and thus can evaluate the leakage of DNA strand displacement reactions with different reaction time, ratios of reactants, and ion concentrations. From the obtained data, mathematical models for evaluating leakage are achieved by curve derivation. As a result, it is obtained that long time incubation, high concentration of fuel strand, and inappropriate amount of ion concentration can weaken leaky reactions. This contributes to a method to set proper reaction conditions to reduce leakage in DNA strand displacement.

  1. A Novel Computational Method to Reduce Leaky Reaction in DNA Strand Displacement

    PubMed Central

    Li, Xin; Wang, Xun; Song, Tao; Lu, Wei; Chen, Zhihua; Shi, Xiaolong

    2015-01-01

    DNA strand displacement technique is widely used in DNA programming, DNA biosensors, and gene analysis. In DNA strand displacement, leaky reactions can cause DNA signals decay and detecting DNA signals fails. The mostly used method to avoid leakage is cleaning up after upstream leaky reactions, and it remains a challenge to develop reliable DNA strand displacement technique with low leakage. In this work, we address the challenge by experimentally evaluating the basic factors, including reaction time, ratio of reactants, and ion concentration to the leakage in DNA strand displacement. Specifically, fluorescent probes and a hairpin structure reporting DNA strand are designed to detect the output of DNA strand displacement, and thus can evaluate the leakage of DNA strand displacement reactions with different reaction time, ratios of reactants, and ion concentrations. From the obtained data, mathematical models for evaluating leakage are achieved by curve derivation. As a result, it is obtained that long time incubation, high concentration of fuel strand, and inappropriate amount of ion concentration can weaken leaky reactions. This contributes to a method to set proper reaction conditions to reduce leakage in DNA strand displacement. PMID:26491602

  2. Yeast Pif1 Accelerates Annealing of Complementary DNA Strands

    PubMed Central

    2015-01-01

    Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of Saccharomyces cerevisiae Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg2+. Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3′-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1. PMID:25393406

  3. Yeast Pif1 accelerates annealing of complementary DNA strands.

    PubMed

    Ramanagoudr-Bhojappa, Ramanagouda; Byrd, Alicia K; Dahl, Christopher; Raney, Kevin D

    2014-12-09

    Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of Saccharomyces cerevisiae Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg(2+). Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3'-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1.

  4. Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

    PubMed Central

    Sun, Fei; Huang, Li

    2013-01-01

    Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation. PMID:23821667

  5. Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

    PubMed

    Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

    2016-08-02

    Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

  6. DNA strand displacement system running logic programs.

    PubMed

    Rodríguez-Patón, Alfonso; Sainz de Murieta, Iñaki; Sosík, Petr

    2014-01-01

    The paper presents a DNA-based computing model which is enzyme-free and autonomous, not requiring a human intervention during the computation. The model is able to perform iterated resolution steps with logical formulae in conjunctive normal form. The implementation is based on the technique of DNA strand displacement, with each clause encoded in a separate DNA molecule. Propositions are encoded assigning a strand to each proposition p, and its complementary strand to the proposition ¬p; clauses are encoded comprising different propositions in the same strand. The model allows to run logic programs composed of Horn clauses by cascading resolution steps. The potential of the model is demonstrated also by its theoretical capability of solving SAT. The resulting SAT algorithm has a linear time complexity in the number of resolution steps, whereas its spatial complexity is exponential in the number of variables of the formula. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. Strand displacement synthesis by yeast DNA polymerase ε.

    PubMed

    Ganai, Rais A; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

    2016-09-30

    DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Strand displacement synthesis by yeast DNA polymerase ε

    PubMed Central

    Ganai, Rais A.; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

    2016-01-01

    DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. PMID:27325747

  9. Stem cell identity and template DNA strand segregation.

    PubMed

    Tajbakhsh, Shahragim

    2008-12-01

    The quest for stem cell properties to distinguish their identity from that of committed daughters has led to a re-investigation of the notion that DNA strands are not equivalent, and 'immortal' DNA strands are retained in stem cells whereas newly replicated DNA strands segregate to the differentiating daughter cell during mitosis. Whether this process occurs only in stem cells, and also in all tissues, remains unclear. That individual chromosomes can be also partitioned non-randomly raises the question if this phenomenon is related to the immortal DNA hypothesis, and it underscores the need for high-resolution techniques to observe these events empirically. Although initially postulated as a mechanism to avoid DNA replication errors, alternative views including epigenetic regulation and sister chromatid silencing may provide insights into this process.

  10. Triplex-forming oligonucleotides: a third strand for DNA nanotechnology

    PubMed Central

    2018-01-01

    Abstract DNA self-assembly has proved to be a useful bottom-up strategy for the construction of user-defined nanoscale objects, lattices and devices. The design of these structures has largely relied on exploiting simple base pairing rules and the formation of double-helical domains as secondary structural elements. However, other helical forms involving specific non-canonical base-base interactions have introduced a novel paradigm into the process of engineering with DNA. The most notable of these is a three-stranded complex generated by the binding of a third strand within the duplex major groove, generating a triple-helical (‘triplex’) structure. The sequence, structural and assembly requirements that differentiate triplexes from their duplex counterparts has allowed the design of nanostructures for both dynamic and/or structural purposes, as well as a means to target non-nucleic acid components to precise locations within a nanostructure scaffold. Here, we review the properties of triplexes that have proved useful in the engineering of DNA nanostructures, with an emphasis on applications that hitherto have not been possible by duplex formation alone. PMID:29228337

  11. Autonomous parvovirus LuIII encapsidates equal amounts of plus and minus DNA strands

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bates, R.C.; Snyder, C.E.; Banerjee, P.T.

    1984-02-01

    Autonomous parvoviruses are thought to uniquely encapsidate single-stranded DNA of minus polarity. In contrast, the defective adeno-associated viruses separately encapsidate equal amounts of plus and minus DNA strands. The uniqueness of minus strand encapsidation is reexamined for the autonomous parvoviruses. Although it was found that Kilham rat virus and H-1 virus encapsidate varying but small amounts of complementary-strand DNA, it was unexpected to find that LuIII virus encapsidated equal amounts of plus and minus DNA. The extracted LuIII DNA possessed properties of double-stranded replicative-form DNA, including insensitivity to S1 endonuclease, cleavage by restriction enzymes, and conversion to unit-length, single-stranded DNAmore » when electrophoresed under denaturing conditions. However, the inability of this DNA to form single-stranded DNA circles when denatured and then renatured in the presence of formamide and the lack of double-stranded DNA circle formation after treatment with exonuclease III and reannealing shows a lack of sequence homology of the 3' and 5' termini of LuIII DNA, in contrast to adeno-associated virus DNA. Digestion of LuIII double-stranded DNA with EcoRI and HincII and separation of plus and minus DNA strands on composite agarose-acrylamide gels identified a heterogeneity present only in the plus DNA strand. These results suggest that strand specificity of viral DNA encapsidation is not a useful property for differentiation between the autonomous and defective parvoviruses. Furthermore, encapsidation by LuIII of equal amounts of complementary DNA strands in contrast to encapsidation of minus strands by H-1 virus, when propagated in the same host cell type, suggests that selection of strands for encapsidation is a virus-coded rather than host-controlled event.« less

  12. Mechanisms of double-strand-break repair during gene targeting in mammalian cells.

    PubMed Central

    Ng, P; Baker, M D

    1999-01-01

    In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism. PMID:10049929

  13. Sub-Ensemble Monitoring of DNA Strand Displacement Using Multiparameter Single-Molecule FRET.

    PubMed

    Baltierra-Jasso, Laura E; Morten, Michael J; Magennis, Steven W

    2018-03-05

    Non-enzymatic DNA strand displacement is an important mechanism in dynamic DNA nanotechnology. Here, we show that the large parameter space that is accessible by single-molecule FRET is ideal for the simultaneous monitoring of multiple reactants and products of DNA strand exchange reactions. We monitored the strand displacement from double-stranded DNA (dsDNA) by single-stranded DNA (ssDNA) at 37 °C; the data were modelled as a second-order reaction approaching equilibrium, with a rate constant of 10 m -1  s -1 . We also followed the displacement from a DNA three-way junction (3WJ) by ssDNA. The presence of three internal mismatched bases in the middle of the invading strand did not prevent displacement from the 3WJ, but reduced the second-order rate constant by about 50 %. We attribute strand exchange in the dsDNA and 3WJ to a zero-toehold pathway from the blunt-ended duplex arms. The single-molecule approach demonstrated here will be useful for studying complex DNA networks. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets.

    PubMed

    Rant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, Gerhard

    2007-10-30

    We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 x 10(8) bound targets per cm(2) sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format.

  15. Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets

    PubMed Central

    Rant, Ulrich; Arinaga, Kenji; Scherer, Simon; Pringsheim, Erika; Fujita, Shozo; Yokoyama, Naoki; Tornow, Marc; Abstreiter, Gerhard

    2007-01-01

    We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 × 108 bound targets per cm2 sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single- and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format. PMID:17951434

  16. Sequence-Specific DNA Photosplitting of Crosslinked DNAs Containing the 3-Cyanovinylcarbazole Nucleoside by Using DNA Strand Displacement.

    PubMed

    Nakamura, Shigetaka; Kawabata, Hayato; Fujimoto, Kenzo

    2016-08-17

    An oligodeoxynucleotide (ODN) containing the ultrafast reversible 3-cyanovinylcarbazole ((CNV) K) photo-crosslinker was photo-crosslinked to a complementary strand upon exposure to 366 nm irradiation and photosplit by use of 312 nm irradiation. In this paper we report that the photoreaction of (CNV) K on irradiation at 366 nm involves a photostationary state and that its reaction can be controlled by temperature. Guided by this new insight, we proposed and have now demonstrated previously unknown photosplitting of (CNV) K aided by DNA strand displacement as an alternative to heating. The photo-crosslinked double-stranded DNA (dsDNA) underwent >80 % photosplitting aided by DNA strand displacement on irradiation at 366 nm without heating. In this photosplitting based on DNA strand displacement, the relative thermal stability of the invader strand with respect to the template strands plays an important role, and an invader strand/template strand system that is more stable than the passenger strand/template strand system induces photosplitting without heating. This new strand-displacement-aided photosplitting occurred in a sequence-specific manner through irradiation at 366 nm in the presence of an invader strand. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Spectrophotometric and ultrasensitive DNA bioassay by circular-strand displacement polymerization reaction.

    PubMed

    Yu, Luxin; Wu, Wei; Chen, Junhua; Xiao, Zhuo; Ge, Chenchen; Lie, Puchang; Fang, Zhiyuan; Chen, Lingbo; Zhang, Ya; Zeng, Lingwen

    2013-12-07

    We demonstrated a new spectrophotometric DNA detection approach based on a circular strand-displacement polymerization reaction for the quantitative detection of sequence specific DNA. In this assay, the hybridization of an immobilized hairpin probe on the microtiter plate, to target DNA, results in a conformational change and leads to a stem separation. A short primer thus anneals with the open stem and triggers a polymerization reaction, allowing a cyclic reaction comprising the release of target DNA and hybridization of the target with the remaining immobilized hairpin probe. Through this cyclical process, a large number of duplex DNA complexes are produced. Finally, the biotin modified duplex DNA products can be detected via the HRP catalyzed substrate 3,3',5,5'-tetramethylbenzidine using a spectrophotometer. As a proof of concept, a short DNA sequence (20-nt) related to the South East Asia (SEA) type deletion of α-thalassemia was chosen as the model target. This proposed assay has a very high sensitivity and selectivity with a dynamic response ranging from 0.1 fM to 10 nM and the detection limit was 8 aM. It can be performed within 2 hours, and it can differentiate target SEA DNA from wild-type DNA. By substituting the hairpin probes used in the present work, this assay can be used to detect other subtypes of genetic disorders.

  18. The Effect of Basepair Mismatch on DNA Strand Displacement.

    PubMed

    Broadwater, D W Bo; Kim, Harold D

    2016-04-12

    DNA strand displacement is a key reaction in DNA homologous recombination and DNA mismatch repair and is also heavily utilized in DNA-based computation and locomotion. Despite its ubiquity in science and engineering, sequence-dependent effects of displacement kinetics have not been extensively characterized. Here, we measured toehold-mediated strand displacement kinetics using single-molecule fluorescence in the presence of a single basepair mismatch. The apparent displacement rate varied significantly when the mismatch was introduced in the invading DNA strand. The rate generally decreased as the mismatch in the invader was encountered earlier in displacement. Our data indicate that a single base pair mismatch in the invader stalls branch migration and displacement occurs via direct dissociation of the destabilized incumbent strand from the substrate strand. We combined both branch migration and direct dissociation into a model, which we term the concurrent displacement model, and used the first passage time approach to quantitatively explain the salient features of the observed relationship. We also introduce the concept of splitting probabilities to justify that the concurrent model can be simplified into a three-step sequential model in the presence of an invader mismatch. We expect our model to become a powerful tool to design DNA-based reaction schemes with broad functionality. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert

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

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

    DOE PAGES

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; ...

    2015-06-02

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

  1. A nonenzymatic DNA nanomachine for biomolecular detection by target recycling of hairpin DNA cascade amplification.

    PubMed

    Zheng, Jiao; Li, Ningxing; Li, Chunrong; Wang, Xinxin; Liu, Yucheng; Mao, Guobin; Ji, Xinghu; He, Zhike

    2018-06-01

    Synthetic enzyme-free DNA nanomachine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA nanomachine biosensor for biomolecule (such as nucleic acid, thrombin and adenosine) detection is developed by target-assisted enzyme-free hairpin DNA cascade amplifier. The whole DNA nanomachine system is constructed on gold nanoparticle which decorated with hundreds of locked hairpin substrate strands serving as DNA tracks, and the DNA nanomachine could be activated by target molecule toehold-mediated exchange on gold nanoparticle surface, resulted in the fluorescence recovery of fluorophore. The process is repeated so that each copy of the target can open multiplex fluorophore-labeled hairpin substrate strands, resulted in amplification of the fluorescence signal. Compared with the conventional biosensors of catalytic hairpin assembly (CHA) without substrate in solution, the DNA nanomachine could generate 2-3 orders of magnitude higher fluorescence signal. Furthermore, the DNA nanomachine could be used for nucleic acid, thrombin and adenosine highly sensitive specific detection based on isothermal, and homogeneous hairpin DNA cascade signal amplification in both buffer and a complicated biomatrix, and this kind of DNA nanomachine could be efficiently applied in the field of biomedical analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    PubMed

    Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

    2014-10-28

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

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

    PubMed Central

    Langston, Lance D.; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E.; Finkelstein, Jeff; Yao, Nina Y.; Indiani, Chiara; O’Donnell, Mike E.

    2014-01-01

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG–Pol ε complex and showed that it is a functional polymerase–helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes. PMID:25313033

  4. Analog Computation by DNA Strand Displacement Circuits.

    PubMed

    Song, Tianqi; Garg, Sudhanshu; Mokhtar, Reem; Bui, Hieu; Reif, John

    2016-08-19

    DNA circuits have been widely used to develop biological computing devices because of their high programmability and versatility. Here, we propose an architecture for the systematic construction of DNA circuits for analog computation based on DNA strand displacement. The elementary gates in our architecture include addition, subtraction, and multiplication gates. The input and output of these gates are analog, which means that they are directly represented by the concentrations of the input and output DNA strands, respectively, without requiring a threshold for converting to Boolean signals. We provide detailed domain designs and kinetic simulations of the gates to demonstrate their expected performance. On the basis of these gates, we describe how DNA circuits to compute polynomial functions of inputs can be built. Using Taylor Series and Newton Iteration methods, functions beyond the scope of polynomials can also be computed by DNA circuits built upon our architecture.

  5. Programmable autonomous synthesis of single-stranded DNA

    NASA Astrophysics Data System (ADS)

    Kishi, Jocelyn Y.; Schaus, Thomas E.; Gopalkrishnan, Nikhil; Xuan, Feng; Yin, Peng

    2018-02-01

    DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

  6. Programmable autonomous synthesis of single-stranded DNA.

    PubMed

    Kishi, Jocelyn Y; Schaus, Thomas E; Gopalkrishnan, Nikhil; Xuan, Feng; Yin, Peng

    2018-02-01

    DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

  7. A new photoelectrochemical biosensors based on DNA conformational changes and isothermal circular strand-displacement polymerization reaction.

    PubMed

    Zhang, Xiaoru; Xu, Yunpeng; Zhao, Yanqing; Song, Weiling

    2013-01-15

    We report a strategy for the transduction of DNA hybridization into a readily detectable photoelectrochemical signal by means of a conformational change analogous to electrochemical DNA (E-DNA) approach. To demonstrate the effect of distance change for photosensitizer to the surface of electrode on the change of photocurrent, photosensitizer Ru(bpy)(2)(dcbpy)(2+) tagged DNA stem-loop structures were self-assembled onto a nanogold modified ITO electrode. Hybridization induced a large conformational change in DNA structure, which in turn significantly altered the electron-transfer tunneling distance between the electrode and photosensitizer. The resulting change in photocurrent was proportional to the concentration of DNA in the range of 1.0×10(-10)-8.0×10(-9)M. In order to improve the sensitivity of the photoelectrochemical biosensor, an amplified detection method based on isothermal strand displacement polymerization reaction was employed. With multiple rounds of isothermal strand replication, which led to strand displacement and constituted consecutive signal amplification, a detection limit of 9.4×10(-14)M target DNA was achieved. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Single Pore Translocation of Folded, Double-Stranded, and Tetra-stranded DNA through Channel of Bacteriophage Phi29 DNA Packaging Motor

    PubMed Central

    Haque, Farzin; Wang, Shaoying; Stites, Chris; Chen, Li; Wang, Chi; Guo, Peixuan

    2015-01-01

    The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ~35-fold slower speed of translocation per base pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ~32% of channel current blockage and ~64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine. PMID:25890769

  9. Single-stranded DNA and RNA origami.

    PubMed

    Han, Dongran; Qi, Xiaodong; Myhrvold, Cameron; Wang, Bei; Dai, Mingjie; Jiang, Shuoxing; Bates, Maxwell; Liu, Yan; An, Byoungkwon; Zhang, Fei; Yan, Hao; Yin, Peng

    2017-12-15

    Self-folding of an information-carrying polymer into a defined structure is foundational to biology and offers attractive potential as a synthetic strategy. Although multicomponent self-assembly has produced complex synthetic nanostructures, unimolecular folding has seen limited progress. We describe a framework to design and synthesize a single DNA or RNA strand to self-fold into a complex yet unknotted structure that approximates an arbitrary user-prescribed shape. We experimentally construct diverse multikilobase single-stranded structures, including a ~10,000-nucleotide (nt) DNA structure and a ~6000-nt RNA structure. We demonstrate facile replication of the strand in vitro and in living cells. The work here thus establishes unimolecular folding as a general strategy for constructing complex and replicable nucleic acid nanostructures, and expands the design space and material scalability for bottom-up nanotechnology. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  10. Human DNA polymerase η accommodates RNA for strand extension.

    PubMed

    Su, Yan; Egli, Martin; Guengerich, F Peter

    2017-11-03

    Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η in vivo . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Programmable energy landscapes for kinetic control of DNA strand displacement.

    PubMed

    Machinek, Robert R F; Ouldridge, Thomas E; Haley, Natalie E C; Bath, Jonathan; Turberfield, Andrew J

    2014-11-10

    DNA is used to construct synthetic systems that sense, actuate, move and compute. The operation of many dynamic DNA devices depends on toehold-mediated strand displacement, by which one DNA strand displaces another from a duplex. Kinetic control of strand displacement is particularly important in autonomous molecular machinery and molecular computation, in which non-equilibrium systems are controlled through rates of competing processes. Here, we introduce a new method based on the creation of mismatched base pairs as kinetic barriers to strand displacement. Reaction rate constants can be tuned across three orders of magnitude by altering the position of such a defect without significantly changing the stabilities of reactants or products. By modelling reaction free-energy landscapes, we explore the mechanistic basis of this control mechanism. We also demonstrate that oxDNA, a coarse-grained model of DNA, is capable of accurately predicting and explaining the impact of mismatches on displacement kinetics.

  12. Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand

    PubMed Central

    Patel, Maha P.; Baum, Dana A.; Silverman, Scott K.

    2008-01-01

    DNA with a 5′-adenylpyrophosphoryl cap (5′-adenylated DNA; AppDNA) is an activated form of DNA that is the biochemical intermediate of the reactions catalyzed by DNA ligase, RNA ligase, polynucleotide kinase, and other nucleic acid modifying enzymes. 5′-Adenylated DNA is also useful for in vitro selection experiments. Efficient preparation of 5′-adenylated DNA is therefore desirable for several biochemical applications. Here we have developed a DNA adenylation procedure that uses T4 DNA ligase and is more reliable than a previously reported approach that used the 5′-phosphorylated donor DNA substrate to be adenylated, a DNA template, and ATP but no acceptor strand. Our improved DNA adenylation procedure uses the above components as well as an acceptor strand that has a strategically chosen C-T acceptor-template mismatch directly adjacent to the adenylation site. This mismatch permits adenylation of the donor DNA substrate but largely suppresses subsequent ligation of the donor with the acceptor, as assayed on nine different DNA substrates that collectively have all four DNA nucleotides represented at each of the first two positions. The new DNA adenylation procedure is successful using either laboratory-prepared or commercial T4 DNA ligase and works well on the preparative (2 nmol) scale for all nine of the test DNA substrates. PMID:18022669

  13. Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication

    PubMed Central

    Seco, Elena M.

    2017-01-01

    Abstract Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA ‘initiation primer’ on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. PMID:28575448

  14. SINGLE STRAND-CONTAINING REPLICATING MOLECULES OF CIRCULAR MITOCHONDRIAL DNA

    PubMed Central

    Wolstenholme, David R.; Koike, Katsuro; Cochran-Fouts, Patricia

    1973-01-01

    Mitochondrial DNAs (mtDNAs) from Chang rat solid hepatomas and Novikoff rat ascites hepatomas were examined in the electron microscope after preparation by the aqueous and by the formamide protein monolayer techniques. MtDNAs from both tumors were found to include double-forked circular molecules with a form and size suggesting they were replicative intermediates. These molecules were of two classes. In molecules of one class, all three segments were apparently totally double stranded. Molecules of the second class were distinguished by the fact that one of the segments spanning the region between the forks in which replication had occurred (the daughter segments) was either totally single stranded, or contained a single-stranded region associated with one of the forks. Daughter segments of both totally double-stranded and single strand-containing replicating molecules varied in length from about 3 to about 80% of the circular contour length of the molecule. Similar classes of replicating molecules were found in mtDNA from regenerating rat liver and chick embryos, indicating them to be normal intermediates in the replication of mtDNA All of the mtDNAs examined included partially single-stranded simple (nonforked) circular molecules. A possible scheme for the replication of mtDNA is presented, based on the different molecular forms observed PMID:4345165

  15. Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks.

    PubMed

    Chen, Yuan-Jyue; Rao, Sundipta D; Seelig, Georg

    2015-11-25

    DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

  16. DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

    PubMed Central

    Park, Sojin; Choi, Seoyun; Ahn, Byungchan

    2016-01-01

    DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents. PMID:26903030

  17. Simultaneous binding to the tracking strand, displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase

    PubMed Central

    Aarattuthodiyil, Suja; Byrd, Alicia K.; Raney, Kevin D.

    2014-01-01

    Interactions between helicases and the tracking strand of a DNA substrate are well-characterized; however, the role of the displaced strand is a less understood characteristic of DNA unwinding. Dda helicase exhibited greater processivity when unwinding a DNA fork compared to a ss/ds DNA junction substrate. The lag phase in the unwinding progress curve was reduced for the forked DNA compared to the ss/ds junction. Fewer kinetic steps were required to unwind the fork compared to the ss/ds junction, suggesting that binding to the fork leads to disruption of the duplex. DNA footprinting confirmed that interaction of Dda with a fork leads to two base pairs being disrupted whereas no disruption of base pairing was observed with the ss/ds junction. Neutralization of the phosphodiester backbone resulted in a DNA-footprinting pattern similar to that observed with the ss/ds junction, consistent with disruption of the interaction between Dda and the displaced strand. Several basic residues in the 1A domain which were previously proposed to bind to the incoming duplex DNA were replaced with alanines, resulting in apparent loss of interaction with the duplex. Taken together, these results suggest that Dda interaction with the tracking strand, displaced strand and duplex coordinates DNA unwinding. PMID:25249618

  18. 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. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  19. Molecular mechanism of DNA association with single-stranded DNA binding protein

    PubMed Central

    Maffeo, Christopher

    2017-01-01

    Abstract During DNA replication, the single-stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA) with high affinity to protect it from degradation and prevent secondary structure formation. Although SSB binds ssDNA tightly, it can be repositioned along ssDNA to follow the advancement of the replication fork. Using all-atom molecular dynamics simulations, we characterized the molecular mechanism of ssDNA association with SSB. Placed in solution, ssDNA–SSB assemblies were observed to change their structure spontaneously; such structural changes were suppressed in the crystallographic environment. Repeat simulations of the SSB–ssDNA complex under mechanical tension revealed a multitude of possible pathways for ssDNA to come off SSB punctuated by prolonged arrests at reproducible sites at the SSB surface. Ensemble simulations of spontaneous association of short ssDNA fragments with SSB detailed a three-dimensional map of local affinity to DNA; the equilibrium amount of ssDNA bound to SSB was found to depend on the electrolyte concentration but not on the presence of the acidic tips of the SSB tails. Spontaneous formation of ssDNA bulges and their diffusive motion along SSB surface was directly observed in multiple 10-µs-long simulations. Such reptation-like motion was confined by DNA binding to high-affinity spots, suggesting a two-step mechanism for SSB diffusion. PMID:29059392

  20. Hairpin Bisulfite Sequencing: Synchronous Methylation Analysis on Complementary DNA Strands of Individual Chromosomes.

    PubMed

    Giehr, Pascal; Walter, Jörn

    2018-01-01

    The accurate and quantitative detection of 5-methylcytosine is of great importance in the field of epigenetics. The method of choice is usually bisulfite sequencing because of the high resolution and the possibility to combine it with next generation sequencing. Nevertheless, also this method has its limitations. Following the bisulfite treatment DNA strands are no longer complementary such that in a subsequent PCR amplification the DNA methylation patterns information of only one of the two DNA strand is preserved. Several years ago Hairpin Bisulfite sequencing was developed as a method to obtain the pattern information on complementary DNA strands. The method requires fragmentation (usually by enzymatic cleavage) of genomic DNA followed by a covalent linking of both DNA strands through ligation of a short DNA hairpin oligonucleotide to both strands. The ligated covalently linked dsDNA products are then subjected to a conventional bisulfite treatment during which all unmodified cytosines are converted to uracils. During the treatment the DNA is denatured forming noncomplementary ssDNA circles. These circles serve as a template for a locus specific PCR to amplify chromosomal patterns of the region of interest. As a result one ends up with a linearized product, which contains the methylation information of both complementary DNA strands.

  1. Real-time study of a DNA strand displacement reaction using dual polarization interferometry.

    PubMed

    Xu, Pingping; Huang, Fujian; Liang, Haojun

    2013-03-15

    A DNA strand displacement reaction on a solid-liquid interface was investigated using dual polarization interferometry. This effective analytical technique allows the real-time, simultaneous determination of the thickness, density, and mass of a biological layer. The displacement process was examined, and the changes in thickness, density, and mass were determined. Injection of the displacement DNA resulted in an increase in density and a decrease in mass and thickness, which indicated that a portion of the target DNA was displaced from the double-stranded DNA (dsDNA). The effects of the displacement DNA concentration and toehold length on the displacement efficiency were also examined. Increasing the displacement DNA concentration and the toehold length increased the changes in mass and the displacement efficiency. At the concentration of 0.2 μM, the toeholds with 4, 5, 6, and 7 bases had displacement percentages of 24.54%, 25.99%, 30.16%, and 70.41%, respectively. At displacement DNA concentrations exceeding that of the dsDNA, the displacement percentage was not concentration-dependent. Above a certain concentration, the percentage remained stable with increasing concentration. Comparison using different toehold sequences showed that the displacement efficiency increases with increasing bonding force between the base pairs. Copyright © 2012. Published by Elsevier B.V.

  2. DNA strand-exchange patterns associated with double-strand break-induced and spontaneous mitotic crossovers in Saccharomyces cerevisiae

    PubMed Central

    2018-01-01

    Mitotic recombination can result in loss of heterozygosity and chromosomal rearrangements that shape genome structure and initiate human disease. Engineered double-strand breaks (DSBs) are a potent initiator of recombination, but whether spontaneous events initiate with the breakage of one or both DNA strands remains unclear. In the current study, a crossover (CO)-specific assay was used to compare heteroduplex DNA (hetDNA) profiles, which reflect strand exchange intermediates, associated with DSB-induced versus spontaneous events in yeast. Most DSB-induced CO products had the two-sided hetDNA predicted by the canonical DSB repair model, with a switch in hetDNA position from one product to the other at the position of the break. Approximately 40% of COs, however, had hetDNA on only one side of the initiating break. This anomaly can be explained by a modified model in which there is frequent processing of an early invasion (D-loop) intermediate prior to extension of the invading end. Finally, hetDNA tracts exhibited complexities consistent with frequent expansion of the DSB into a gap, migration of strand-exchange junctions, and template switching during gap-filling reactions. hetDNA patterns in spontaneous COs isolated in either a wild-type background or in a background with elevated levels of reactive oxygen species (tsa1Δ mutant) were similar to those associated with the DSB-induced events, suggesting that DSBs are the major instigator of spontaneous mitotic recombination in yeast. PMID:29579095

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

    NASA Astrophysics Data System (ADS)

    Zhang, Yang; Schindler, Christina; Heermann, Dieter

    2014-03-01

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

  4. Low-Energy Electron-Induced Strand Breaks in Telomere-Derived DNA Sequences-Influence of DNA Sequence and Topology.

    PubMed

    Rackwitz, Jenny; Bald, Ilko

    2018-03-26

    During cancer radiation therapy high-energy radiation is used to reduce tumour tissue. The irradiation produces a shower of secondary low-energy (<20 eV) electrons, which are able to damage DNA very efficiently by dissociative electron attachment. Recently, it was suggested that low-energy electron-induced DNA strand breaks strongly depend on the specific DNA sequence with a high sensitivity of G-rich sequences. Here, we use DNA origami platforms to expose G-rich telomere sequences to low-energy (8.8 eV) electrons to determine absolute cross sections for strand breakage and to study the influence of sequence modifications and topology of telomeric DNA on the strand breakage. We find that the telomeric DNA 5'-(TTA GGG) 2 is more sensitive to low-energy electrons than an intermixed sequence 5'-(TGT GTG A) 2 confirming the unique electronic properties resulting from G-stacking. With increasing length of the oligonucleotide (i.e., going from 5'-(GGG ATT) 2 to 5'-(GGG ATT) 4 ), both the variety of topology and the electron-induced strand break cross sections increase. Addition of K + ions decreases the strand break cross section for all sequences that are able to fold G-quadruplexes or G-intermediates, whereas the strand break cross section for the intermixed sequence remains unchanged. These results indicate that telomeric DNA is rather sensitive towards low-energy electron-induced strand breakage suggesting significant telomere shortening that can also occur during cancer radiation therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

    Wang, Wei; Liu, Juan; Sun, Lin

    2016-07-01

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

  6. Detecting the Length of Double-stranded DNA with Solid State Nanopores

    NASA Astrophysics Data System (ADS)

    Li, Jiali; Gershow, Marc; Stein, Derek; Qun, Cai; Brandin, Eric; Wang, Hui; Huang, Albert; Branton, Dan; Golovchenko, Jene

    2003-03-01

    We report on the use of nanometer scale diameter, solid-state nanopores as single molecule detectors of double stranded DNA molecules. These solid-state nanopores are fabricated in thin membranes of silicon nitride, by ion beam sculpting 1. They produce discrete electronic signals: current blockages, when an electrically biased nanopore is exposed to DNA molecules in aqueous salt solutions. We demonstrate examples of such electronic signals for 3k base pairs (bp) and 10k bp double stranded DNA molecules, which suggest that these molecules are individually translocating through the nanopore during the detection process. The translocating time for the 10k bp double stranded DNA is about 3 times longer than the 3k bp, demonstrating that a solid-state nanopore device can be used to detect the lengths of double stranded DNA molecules. Similarities and differences with signals obtained from single stranded DNA in a biological nanopores are discussed 2. 1. Li, J., Stein, D., McMullan, C., Branton, D. Aziz, M. J. and Golovchenko, J. Ion Beam Sculpting at nanometer length scales. Nature 412, 166-169 (2001). 2. Meller, A., L. Nivon, E. Brandin, Golovchenko, J. & Branton, D. Proc. Natl. Acad. Sci. USA 97, 1079-1084 (2000).

  7. Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae

    PubMed Central

    Prasai, Kanchanjunga; Robinson, Lucy C.; Scott, Rona S.; Tatchell, Kelly

    2017-01-01

    Abstract The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ− cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells. PMID:28549155

  8. Dose-Rate Effects in Breaking DNA Strands by Short Pulses of Extreme Ultraviolet Radiation.

    PubMed

    Vyšín, Luděk; Burian, Tomáš; Ukraintsev, Egor; Davídková, Marie; Grisham, Michael E; Heinbuch, Scott; Rocca, Jorge J; Juha, Libor

    2018-05-01

    In this study, we examined dose-rate effects on strand break formation in plasmid DNA induced by pulsed extreme ultraviolet (XUV) radiation. Dose delivered to the target molecule was controlled by attenuating the incident photon flux using aluminum filters as well as by changing the DNA/buffer-salt ratio in the irradiated sample. Irradiated samples were examined using agarose gel electrophoresis. Yields of single- and double-strand breaks (SSBs and DSBs) were determined as a function of the incident photon fluence. In addition, electrophoresis also revealed DNA cross-linking. Damaged DNA was inspected by means of atomic force microscopy (AFM). Both SSB and DSB yields decreased with dose rate increase. Quantum yields of SSBs at the highest photon fluence were comparable to yields of DSBs found after synchrotron irradiation. The average SSB/DSB ratio decreased only slightly at elevated dose rates. In conclusion, complex and/or clustered damages other than cross-links do not appear to be induced under the radiation conditions applied in this study.

  9. Homogenous assay for protein detection based on proximity DNA hybridization and isothermal circular strand displacement amplification reaction.

    PubMed

    Zhang, Manjun; Li, Ruimin; Ling, Liansheng

    2017-06-01

    This work proposed a homogenous fluorescence assay for proteins, based on the target-triggered proximity DNA hybridization in combination with strand displacement amplification (SDA). It benefited from target-triggered proximity DNA hybridization to specifically recognize the target and SDA making recycling signal amplification. The system included a molecular beacon (MB), an extended probe (EP), and an assistant probe (AP), which were not self-assembly in the absence of target proteins, due to the short length of the designed complementary sequence among MB, EP, and AP. Upon addition of the target proteins, EP and AP are bound to the target proteins, which induced the occurrence of proximity hybridization between MB, EP, and AP and followed by strand displacement amplification. Through the primer extension, a tripartite complex of probes and target was displaced and recycled to hybridize with another MB, and the more opened MB enabled the detection signal to amplify. Under optimum conditions, it was used for the detection of streptavidin and thrombin. Fluorescence intensity was proportional to the concentration of streptavidin and thrombin in the range of 0.2-30 and 0.2-35 nmol/L, respectively. Furthermore, this fluorescent method has a good selectivity, in which the fluorescence intensity of thrombin was ~37-fold or even larger than that of the other proteins at the same concentration. It is a new and simple method for SDA-involved target protein detection and possesses a great potential for other protein detection in the future. Graphical abstract A homogenous assay for protein detection is based on proximity DNA hybridization and strand displacement amplification reaction.

  10. C-terminal Phenylalanine of Bacteriophage T7 Single-stranded DNA-binding Protein Is Essential for Strand Displacement Synthesis by T7 DNA Polymerase at a Nick in DNA*

    PubMed Central

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C.

    2009-01-01

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5′-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations. PMID:19726688

  11. C-terminal phenylalanine of bacteriophage T7 single-stranded DNA-binding protein is essential for strand displacement synthesis by T7 DNA polymerase at a nick in DNA.

    PubMed

    Ghosh, Sharmistha; Marintcheva, Boriana; Takahashi, Masateru; Richardson, Charles C

    2009-10-30

    Single-stranded DNA-binding protein (gp2.5), encoded by gene 2.5 of bacteriophage T7, plays an essential role in DNA replication. Not only does it remove impediments of secondary structure in the DNA, it also modulates the activities of the other replication proteins. The acidic C-terminal tail of gp2.5, bearing a C-terminal phenylalanine, physically and functionally interacts with the helicase and DNA polymerase. Deletion of the phenylalanine or substitution with a nonaromatic amino acid gives rise to a dominant lethal phenotype, and the altered gp2.5 has reduced affinity for T7 DNA polymerase. Suppressors of the dominant lethal phenotype have led to the identification of mutations in gene 5 that encodes the T7 DNA polymerase. The altered residues in the polymerase are solvent-exposed and lie in regions that are adjacent to the bound DNA. gp2.5 lacking the C-terminal phenylalanine has a lower affinity for gp5-thioredoxin relative to the wild-type gp2.5, and this affinity is partially restored by the suppressor mutations in DNA polymerase. gp2.5 enables T7 DNA polymerase to catalyze strand displacement DNA synthesis at a nick in DNA. The resulting 5'-single-stranded DNA tail provides a loading site for T7 DNA helicase. gp2.5 lacking the C-terminal phenylalanine does not support this event with wild-type DNA polymerase but does to a limited extent with T7 DNA polymerase harboring the suppressor mutations.

  12. Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin.

    PubMed

    Xu, Yunying; Zhou, Wenjiao; Zhou, Ming; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2015-02-15

    Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Design and Characterization of DNA Strand-Displacement Circuits in Serum-Supplemented Cell Medium.

    PubMed

    Fern, Joshua; Schulman, Rebecca

    2017-09-15

    The functional stability and lifetimes of synthetic molecular circuits in biological environments are important for long-term, stable sensors or controllers of cell or tissue behavior. DNA-based molecular circuits, in particular DNA strand-displacement circuits, provide simple and effective biocompatible control mechanisms and sensors, but are vulnerable to digestion by nucleases present in living tissues and serum-supplemented cell culture. The stability of double-stranded and single-stranded DNA circuit components in serum-supplemented cell medium and the corresponding effect of nuclease-mediated degradation on circuit performance were characterized to determine the major routes of degradation and DNA strand-displacement circuit failure. Simple circuit design choices, such as the use of 5' toeholds within the DNA complexes used as reactants in the strand-displacement reactions and the termination of single-stranded components with DNA hairpin domains at the 3' termini, significantly increase the functional lifetime of the circuit components in the presence of nucleases. Simulations of multireaction circuits, guided by the experimentally measured operation of single-reaction circuits, enable predictive realization of multilayer and competitive-reaction circuit behavior. Together, these results provide a basic route to increased DNA circuit stability in cell culture environments.

  14. Electron attachment to DNA single strands: gas phase and aqueous solution.

    PubMed

    Gu, Jiande; Xie, Yaoming; Schaefer, Henry F

    2007-01-01

    The 2'-deoxyguanosine-3',5'-diphosphate, 2'-deoxyadenosine-3',5'-diphosphate, 2'-deoxycytidine-3',5'-diphosphate and 2'-deoxythymidine-3',5'-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3',5'-dTDP (0.17 eV) and 3',5'-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3',5'-dTDP > 3',5'-dCDP > 3',5'-dGDP > 3',5'-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3'-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3'-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3',5'-dADP(-) (0.26 eV) and 3',5'-dGDP(-) (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers

  15. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation

    PubMed Central

    Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

    2017-01-01

    The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA–DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA–DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs. PMID:28484024

  16. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation.

    PubMed

    Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

    2017-05-23

    The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves double-stranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.

  17. Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

    PubMed

    Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Song, Chen; Adalsteinsson, Viktor A; Parsons, Heather A; Lin, Nancy U; Wagle, Nikhil; Makrigiorgos, G Mike

    2017-10-01

    The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing. © 2017 American Association for Clinical

  18. Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA

    PubMed Central

    Yang, Zhiyu; Price, Nathan E.; Johnson, Kevin M.

    2017-01-01

    Abstract Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3’ddR5p) at the 3’-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3’ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3’ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. PMID:28531327

  19. Nonenzymatic Role for WRN in Preserving Nascent DNA Strands after Replication Stress

    DOE PAGES

    Su, Fengtao; Mukherjee, Shibani; Yang, Yanyong; ...

    2014-11-20

    WRN, the protein defective in Werner syndrome (WS), is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRNmore » to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals.« less

  20. Separation of 1-23-kb complementary DNA strands by urea-agarose gel electrophoresis.

    PubMed

    Hegedüs, Eva; Kókai, Endre; Kotlyar, Alexander; Dombrádi, Viktor; Szabó, Gábor

    2009-09-01

    Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea-agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of approximately 1-20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.

  1. Design and analysis of DNA strand displacement devices using probabilistic model checking

    PubMed Central

    Lakin, Matthew R.; Parker, David; Cardelli, Luca; Kwiatkowska, Marta; Phillips, Andrew

    2012-01-01

    Designing correct, robust DNA devices is difficult because of the many possibilities for unwanted interference between molecules in the system. DNA strand displacement has been proposed as a design paradigm for DNA devices, and the DNA strand displacement (DSD) programming language has been developed as a means of formally programming and analysing these devices to check for unwanted interference. We demonstrate, for the first time, the use of probabilistic verification techniques to analyse the correctness, reliability and performance of DNA devices during the design phase. We use the probabilistic model checker prism, in combination with the DSD language, to design and debug DNA strand displacement components and to investigate their kinetics. We show how our techniques can be used to identify design flaws and to evaluate the merits of contrasting design decisions, even on devices comprising relatively few inputs. We then demonstrate the use of these components to construct a DNA strand displacement device for approximate majority voting. Finally, we discuss some of the challenges and possible directions for applying these methods to more complex designs. PMID:22219398

  2. Design and Characterization of DNA Strand-Displacement Circuits in Serum-Supplemented Cell Medium

    DOE PAGES

    Fern, Joshua; Schulman, Rebecca

    2017-05-30

    The functional stability and lifetimes of synthetic molecular circuits in biological environments are important for long-term, stable sensors or controllers of cell or tissue behavior. DNA-based molecular circuits, particularly DNA strand-displacement circuits, provide simple and effective biocompatible control mechanisms and sensors, but are vulnerable to digestion by nucleases present in living tissues and serum-supplemented cell culture. The stability of double-stranded and single-stranded DNA circuit components in serum-supplemented cell medium and the corresponding effect of nuclease-mediated degradation on circuit performance were characterized to determine the major routes of degradation and DNA strand-displacement circuit failure. Simple circuit design choices, such as themore » use of 5' toeholds within the DNA complexes used as reactants in the strand-displacement reactions and the termination of single-stranded components with DNA hairpin domains at the 3' termini, significantly increase the functional lifetime of the circuit components in the presence of nucleases. Furthermore, simulations of multireaction circuits, guided by the experimentally measured operation of single-reaction circuits, enable predictive realization of multilayer and competitive-reaction circuit behavior. Altogether, these results provide a basic route to increased DNA circuit stability in cell culture environments.« less

  3. Design and Characterization of DNA Strand-Displacement Circuits in Serum-Supplemented Cell Medium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fern, Joshua; Schulman, Rebecca

    The functional stability and lifetimes of synthetic molecular circuits in biological environments are important for long-term, stable sensors or controllers of cell or tissue behavior. DNA-based molecular circuits, particularly DNA strand-displacement circuits, provide simple and effective biocompatible control mechanisms and sensors, but are vulnerable to digestion by nucleases present in living tissues and serum-supplemented cell culture. The stability of double-stranded and single-stranded DNA circuit components in serum-supplemented cell medium and the corresponding effect of nuclease-mediated degradation on circuit performance were characterized to determine the major routes of degradation and DNA strand-displacement circuit failure. Simple circuit design choices, such as themore » use of 5' toeholds within the DNA complexes used as reactants in the strand-displacement reactions and the termination of single-stranded components with DNA hairpin domains at the 3' termini, significantly increase the functional lifetime of the circuit components in the presence of nucleases. Furthermore, simulations of multireaction circuits, guided by the experimentally measured operation of single-reaction circuits, enable predictive realization of multilayer and competitive-reaction circuit behavior. Altogether, these results provide a basic route to increased DNA circuit stability in cell culture environments.« less

  4. Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae.

    PubMed

    Prasai, Kanchanjunga; Robinson, Lucy C; Scott, Rona S; Tatchell, Kelly; Harrison, Lynn

    2017-07-27

    The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ- cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. A paper-based device for double-stranded DNA detection with Zif268

    NASA Astrophysics Data System (ADS)

    Zhang, Daohong

    2017-05-01

    Here, a small analytical device was fabricated on both nitrocellulose membrane and filter paper, for the detection of biotinylated double-stranded DNA (dsDNA) from 1 nM. Zif268 was utilized for capturing the target DNA, which was a zinc finger protein that recognized only a dsDNA with specific sequence. Therefore, this detection platform could be utilized for PCR result detection, with the well-designed primers (interpolate both biotin and Zif268 binding sequence). The result of the assay could be recorded by a camera-phone, and analyzed with software. The whole assay finished within 1 hour. Due to the easy fabrication, operation and disposal of this device, this method can be employed in point-of-care detection or on-site monitoring.

  6. Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

    PubMed

    Thormar, Hans G; Gudmundsson, Bjarki; Eiriksdottir, Freyja; Kil, Siyoen; Gunnarsson, Gudmundur H; Magnusson, Magnus Karl; Hsu, Jason C; Jonsson, Jon J

    2013-04-01

    The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

  7. Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

    PubMed

    Yang, Zhiyu; Price, Nathan E; Johnson, Kevin M; Wang, Yinsheng; Gates, Kent S

    2017-06-20

    Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3'ddR5p) at the 3'-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,β-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3'ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3'ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Quantitative, non-invasive imaging of radiation-induced DNA double strand breaks in vivo

    PubMed Central

    Li, Wenrong; Li, Fang; Huang, Qian; Shen, Jingping; Wolf, Frank; He, Yujun; Liu, Xinjian; Hu, Y. Angela; Bedford, Joel. S.; Li, Chuan-Yuan

    2011-01-01

    DNA double strand breaks is a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA double strand breaks is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here we report a novel approach to image and quantify DNA double strand breaks in live mammalian cells through bi-fragment luciferase reconstitution. N- and C- terminal fragments of firefly luciferase gene were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DNA double strand breaks, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C- luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, non-invasive quantification of DNA double strand breaks in cells irradiated with x-rays and 56Fe ions. Furthermore, it allowed for the evaluation of DNA double strand breaks (DSBs) non-invasively in vivo in irradiated tumors over two weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DNA double strand break repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time. PMID:21527553

  9. Electron attachment to DNA single strands: gas phase and aqueous solution

    PubMed Central

    Gu, Jiande; Xie, Yaoming; Schaefer, Henry F.

    2007-01-01

    The 2′-deoxyguanosine-3′,5′-diphosphate, 2′-deoxyadenosine-3′,5′-diphosphate, 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3′,5′-dTDP (0.17 eV) and 3′,5′-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3′,5′-dTDP > 3′,5′-dCDP > 3′,5′-dGDP > 3′,5′-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3′-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3′-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3′,5′-dADP− (0.26 eV) and 3′,5′-dGDP− (0.32 eV) indicate that electron detachment

  10. Melting of DNA double strand after binding to geroprotective tetrapeptide.

    PubMed

    Khavinson, V Kh; Solovyov, A Yu; Shataeva, L K

    2008-11-01

    Experimental relationship between the hyperchromic effect of DNA [poly(dA-dT):poly(dA-dT)] interacting with Ala-Glu-Asp-Gly peptide is presented by a saturation isotherm. The free DNA double strand is melting (the strands separate) at 69.5 degrees C and at higher energy expenditures (enthalpy increase by 976.4 kJ/mol b.p.) in comparison with melting of the DNA-peptide complex (28 degrees C and 444.6 kJ/mol b.p.). The detected regularities of melting of duplex DNA and the thermodynamic parameters of this process indicate the natural mechanism of interaction between DNA and regulatory peptides underlying functioning of the living matter.

  11. The role of cytosine methylation on charge transport through a DNA strand

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Qi, Jianqing, E-mail: jqqi@uw.edu; Anantram, M. P., E-mail: anantmp@uw.edu; Govind, Niranjan, E-mail: niri.govind@pnnl.gov

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance throughmore » the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.« less

  12. The role of cytosine methylation on charge transport through a DNA strand

    NASA Astrophysics Data System (ADS)

    Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

    2015-09-01

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modification remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Büttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. We first analyze the effect of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and inter-strand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with the same rate. The lower conductance for the methylated strand in the experiment is suggested to be caused by the more stable structure due to the introduction of the methyl groups. We also study the role of the exchange-correlation functional and the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit.

  13. A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA.

    PubMed

    Razvi, F; Gargiulo, G; Worcel, A

    1983-08-01

    Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

  14. Immortal DNA strand cosegregation requires p53/IMPDH-dependent asymmetric self-renewal associated with adult stem cells.

    PubMed

    Rambhatla, Lakshmi; Ram-Mohan, Sumati; Cheng, Jennifer J; Sherley, James L

    2005-04-15

    Because they are long-lived and cycle continuously, adult stem cells (ASCs) are predicted as the most common precursor for cancers in adult mammalian tissues. Two unique attributes have been proposed to restrict the carcinogenic potential of ASCs. These are asymmetric self-renewal that limits their number and immortal DNA strand cosegregation that limits their accumulation of mutations due to DNA replication errors. Until recently, the molecular basis and regulation of these important ASC-specific functions were unknown. We developed engineered cultured cells that exhibit asymmetric self-renewal and immortal DNA strand cosegregation. These model cells were used to show that both ASC-specific functions are regulated by the p53 cancer gene. Previously, we proposed that IMP dehydrogenase (IMPDH) was an essential factor for p53-dependent asymmetric self-renewal. We now confirm this proposal and provide quantitative evidence that asymmetric self-renewal is acutely sensitive to even modest changes in IMPDH expression. These analyses reveal that immortal DNA strand cosegregation is also regulated by IMPDH and confirm the original implicit precept that immortal DNA strand cosegregation is specific to cells undergoing asymmetric self-renewal (i.e., ASCs). With IMPDH being the rate-determining enzyme for guanine ribonucleotide (rGNP) biosynthesis, its requirement implicates rGNPs as important regulators of ASC asymmetric self-renewal and immortal DNA strand cosegregation. An in silico analysis of global gene expression data from human cancer cell lines underscored the importance of p53-IMPDH-rGNP regulation for normal tissue cell kinetics, providing further support for the concept that ASCs are key targets for adult tissue carcinogenesis.

  15. Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

    PubMed

    Zhu, Jie; Feng, Xiaolu; 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.

  16. 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

  17. Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

    PubMed Central

    Koc, Katrina N.; Stodola, Joseph L.; Burgers, Peter M.; Galletto, Roberto

    2015-01-01

    The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo− to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities. PMID:25813050

  18. 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.

  19. Integrating DNA strand-displacement circuitry with DNA tile self-assembly

    PubMed Central

    Zhang, David Yu; Hariadi, Rizal F.; Choi, Harry M.T.; Winfree, Erik

    2013-01-01

    DNA nanotechnology has emerged as a reliable and programmable way of controlling matter at the nanoscale through the specificity of Watson–Crick base pairing, allowing both complex self-assembled structures with nanometer precision and complex reaction networks implementing digital and analog behaviors. Here we show how two well-developed frameworks, DNA tile self-assembly and DNA strand-displacement circuits, can be systematically integrated to provide programmable kinetic control of self-assembly. We demonstrate the triggered and catalytic isothermal self-assembly of DNA nanotubes over 10 μm long from precursor DNA double-crossover tiles activated by an upstream DNA catalyst network. Integrating more sophisticated control circuits and tile systems could enable precise spatial and temporal organization of dynamic molecular structures. PMID:23756381

  20. Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

    PubMed Central

    Fadrosh, Douglas W.; Andrews-Pfannkoch, Cynthia; Williamson, Shannon J.

    2011-01-01

    Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community’s genetic signature. Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using

  1. Highly sensitive "signal-on" electrochemiluminescent biosensor for the detection of DNA based on dual quenching and strand displacement reaction.

    PubMed

    Lou, Jing; Wang, Zhaoyin; Wang, Xiao; Bao, Jianchun; Tu, Wenwen; Dai, Zhihui

    2015-10-07

    A "signal-on" electrochemiluminescent DNA biosensing platform was proposed based on the dual quenching and strand displacement reaction. This novel "signal-on" detection strategy revealed its sensitivity in achieving a detection limit of 2.4 aM and its selectivity in distinguishing single nucleotide polymorphism of target DNA.

  2. Process of infection with bacteriophage phi chi 174. XL. Viral DNA replication of phi chi 174 mutants blocked in progeny single-stranded DNA synthesis.

    PubMed Central

    Fukuda, A; Sinsheimer, R L

    1976-01-01

    Mutation in several different cistrons of bacteriophage phi chi 174 blocks net progeny single-stranded DNA synthesis at the late period of infection (15). For the study of the functions of these cistrons in single-stranded DNA synthesis, asymmetric replication of replicative form DNA was examined at the late period of infection with amber mutants of these cistrons. While the normal, rapid process of asymmetric single-stranded viral DNA synthesis is blocked at the late period of these mutant infections, an asymmetric synthesis of the viral strand of replicative-form DNA is observed in this period, though at a reduced level, together with degradation of prelabeled viral strand. Some intermediate replicative-form molecules were also detected. Asymmetric synthesis of the viral strand of replicative-form DNA at the late period of phi chi infection is completely inhibited in the presence of a low concentration (35mug/ml) of chloramphenicol (which also blocks net single-stranded viral DNA synthesis). These results are discussed in terms of the possible role of the specific viral proteins for normal single-stranded DNA synthesis. PMID:1255871

  3. A novel quantitative electrochemical method to monitor DNA double-strand breaks caused by a DNA cleavage agent at a DNA sensor.

    PubMed

    Banasiak, Anna; Cassidy, John; Colleran, John

    2018-06-01

    To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create a DNA monolayer at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to a DNA cleavage agent. The nuclease properties of a model DNA cleavage agent, copper bis-phenanthroline ([Cu II (phen) 2 ] 2+ ), that can cleave DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21% after subjecting the DNA sensor to a nuclease assay containing [Cu II (phen) 2 ] 2+ , a reductant and an oxidant. This percentage indicates that 6 base pairs were cleaved in the nuclease assay from the immobilised 30 base pair strands. The DNA cleavage can be also induced electrochemically in the absence of a chemical reductant. [Cu II (phen) 2 ] 2+ intercalates between DNA base pairs and, on application of a suitable potential, can be reduced to [Cu I (phen) 2 ] + , with dissolved oxygen acting as the required oxidant. This reduction process is facilitated through DNA strands via long-range electron transfer, resulting in DNA cleavage of 23%. The control measurements for both chemically and electrochemically induced cleavage revealed that DNA strand breaks did not occur under experimental conditions in the absence of [Cu II (phen) 2 ] 2+ . Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

    PubMed

    Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

    2006-02-01

    During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

  5. Oncogenic ras-driven cancer cell vesiculation leads to emission of double-stranded DNA capable of interacting with target cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Audemard, Eric

    2014-08-22

    Highlights: • Oncogenic H-ras stimulates emission of extracellular vesicles containing double-stranded DNA. • Vesicle-associated extracellular DNA contains mutant N-ras sequences. • Vesicles mediate intercellular transfer of mutant H-ras DNA to normal fibroblasts where it remains for several weeks. • Fibroblasts exposed to vesicles containing H-ras DNA exhibit increased proliferation. - Abstract: Cell free DNA is often regarded as a source of genetic cancer biomarkers, but the related mechanisms of DNA release, composition and biological activity remain unclear. Here we show that rat epithelial cell transformation by the human H-ras oncogene leads to an increase in production of small, exosomal-like extracellularmore » vesicles by viable cancer cells. These EVs contain chromatin-associated double-stranded DNA fragments covering the entire host genome, including full-length H-ras. Oncogenic N-ras and SV40LT sequences were also found in EVs emitted from spontaneous mouse brain tumor cells. Disruption of acidic sphingomyelinase and the p53/Rb pathway did not block emission of EV-related oncogenic DNA. Exposure of non-transformed RAT-1 cells to EVs containing mutant H-ras DNA led to the uptake and retention of this material for an extended (30 days) but transient period of time, and stimulated cell proliferation. Thus, our study suggests that H-ras-mediated transformation stimulates vesicular emission of this histone-bound oncogene, which may interact with non-transformed cells.« less

  6. Mammalian DNA single-strand break repair: an X-ra(y)ted affair.

    PubMed

    Caldecott, K W

    2001-05-01

    The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein-protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented. Copyright 2001 John Wiley & Sons, Inc.

  7. 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

  8. Binary electrokinetic separation of target DNA from background DNA primers.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    James, Conrad D.; Derzon, Mark Steven

    2005-10-01

    This report contains the summary of LDRD project 91312, titled ''Binary Electrokinetic Separation of Target DNA from Background DNA Primers''. This work is the first product of a collaboration with Columbia University and the Northeast BioDefense Center of Excellence. In conjunction with Ian Lipkin's lab, we are developing a technique to reduce false positive events, due to the detection of unhybridized reporter molecules, in a sensitive and multiplexed detection scheme for nucleic acids developed by the Lipkin lab. This is the most significant problem in the operation of their capability. As they are developing the tools for rapidly detecting themore » entire panel of hemorrhagic fevers this technology will immediately serve an important national need. The goal of this work was to attempt to separate nucleic acid from a preprocessed sample. We demonstrated the preconcentration of kilobase-pair length double-stranded DNA targets, and observed little preconcentration of 60 base-pair length single-stranded DNA probes. These objectives were accomplished in microdevice formats that are compatible with larger detection systems for sample pre-processing. Combined with Columbia's expertise, this technology would enable a unique, fast, and potentially compact method for detecting/identifying genetically-modified organisms and multiplexed rapid nucleic acid identification. Another competing approach is the DARPA funded IRIS Pharmaceutical TIGER platform which requires many hours for operation, and an 800k$ piece of equipment that fills a room. The Columbia/SNL system could provide a result in 30 minutes, at the cost of a few thousand dollars for the platform, and would be the size of a shoebox or smaller.« less

  9. 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

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

    PubMed

    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.

  11. Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model.

    PubMed

    Noguchi, Yasunori; Yuan, Zuanning; Bai, Lin; Schneider, Sarah; Zhao, Gongpu; Stillman, Bruce; Speck, Christian; Li, Huilin

    2017-11-07

    During replication initiation, the core component of the helicase-the Mcm2-7 hexamer-is loaded on origin DNA as a double hexamer (DH). The two ring-shaped hexamers are staggered, leading to a kinked axial channel. How the origin DNA interacts with the axial channel is not understood, but the interaction could provide key insights into Mcm2-7 function and regulation. Here, we report the cryo-EM structure of the Mcm2-7 DH on dsDNA and show that the DNA is zigzagged inside the central channel. Several of the Mcm subunit DNA-binding loops, such as the oligosaccharide-oligonucleotide loops, helix 2 insertion loops, and presensor 1 (PS1) loops, are well defined, and many of them interact extensively with the DNA. The PS1 loops of Mcm 3, 4, 6, and 7, but not 2 and 5, engage the lagging strand with an approximate step size of one base per subunit. Staggered coupling of the two opposing hexamers positions the DNA right in front of the two Mcm2-Mcm5 gates, with each strand being pressed against one gate. The architecture suggests that lagging-strand extrusion initiates in the middle of the DH that is composed of the zinc finger domains of both hexamers. To convert the Mcm2-7 DH structure into the Mcm2-7 hexamer structure found in the active helicase, the N-tier ring of the Mcm2-7 hexamer in the DH-dsDNA needs to tilt and shift laterally. We suggest that these N-tier ring movements cause the DNA strand separation and lagging-strand extrusion. Copyright © 2017 the Author(s). Published by PNAS.

  12. Portable and sensitive quantitative detection of DNA based on personal glucose meters and isothermal circular strand-displacement polymerization reaction.

    PubMed

    Xu, Xue-tao; Liang, Kai-yi; Zeng, Jia-ying

    2015-02-15

    A portable and sensitive quantitative DNA detection method based on personal glucose meters and isothermal circular strand-displacement polymerization reaction was developed. The target DNA triggered target recycling process, which opened capture DNA. The released target then found another capture DNA to trigger another polymerization cycle, which was repeated for many rounds, resulting in the multiplication of the DNA-invertase conjugation on the surface of Streptavidin-MNBs. The DNA-invertase was used to catalyze the hydrolysis of sucrose into glucose for PGM readout. There was a liner relationship between the signal of PGM and the concentration of target DNA in the range of 5.0 to 1000 fM, which is lower than some DNA detection method. In addition, the method exhibited excellent sequence selectivity and there was almost no effect of biological complex to the detection performance, which suggested our method can be successfully applied to DNA detection in real biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. A major role of DNA polymerase δ in replication of both the leading and lagging DNA strands

    PubMed Central

    Prakash, Louise; Prakash, Satya

    2015-01-01

    SUMMARY Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis. PMID:26145172

  14. Targeting DNA double strand break repair with hyperthermia and DNA-PKcs inhibition to enhance the effect of radiation treatment.

    PubMed

    van Oorschot, Bregje; Granata, Giovanna; Di Franco, Simone; Ten Cate, Rosemarie; Rodermond, Hans M; Todaro, Matilde; Medema, Jan Paul; Franken, Nicolaas A P

    2016-10-04

    Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

  15. The Human L1 Element Causes DNA Double-Strand Breaks in Breast Cancer

    DTIC Science & Technology

    2006-08-01

    cancer is complex. However, defects in DNA repair genes in the double-strand break repair pathway are cancer predisposing. My lab has characterized...a new potentially important source of double-strand breaks (DSBs) in human cells and are interested in characterizing which DNA repair genes act on...this particular source of DNA damage. Selfish DNA accounts for 45% of the human genome. We have recently demonstrated that one particular selfish

  16. Signal-off Electrochemiluminescence Biosensor Based on Phi29 DNA Polymerase Mediated Strand Displacement Amplification for MicroRNA Detection.

    PubMed

    Chen, Anyi; Gui, Guo-Feng; Zhuo, Ying; Chai, Ya-Qin; Xiang, Yun; Yuan, Ruo

    2015-06-16

    A target induced cycling strand displacement amplification (SDA) mediated by phi29 DNA polymerase (phi29) was first investigated and applied in a signal-off electrochemiluminescence (ECL) biosensor for microRNA (miRNA) detection. Herein, the target miRNA triggered the phi29-mediated SDA which could produce amounts of single-stranded DNA (assistant probe) with accurate and comprehensive nucleotide sequence. Then, the assistant probe hybridized with the capture probe and the ferrocene-labeled probe (Fc-probe) to form a ternary "Y" structure for ECL signal quenching by ferrocene. Therefore, the ECL intensity would decrease with increasing concentration of the target miRNA, and the sensitivity of biosensor would be promoted on account of the efficient signal amplification of the target induced cycling reaction. Besides, a self-enhanced Ru(II) ECL system was designed to obtain a stable and strong initial signal to further improve the sensitivity. The ECL assay for miRNA-21 detection is developed with excellent sensitivity of a concentration variation from 10 aM to 1.0 pM and limit of detection down to 3.3 aM.

  17. Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

    PubMed

    Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

    2015-09-15

    MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. The Role of Cytosine Methylation on Charge Transport through a DNA Strand

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Qi, Jianqing; Govind, Niranjan; Anantram, M. P.

    Cytosine methylation has been found to play a crucial role in various biological processes, including a number of human diseases. The detection of this small modifi-cation remains challenging. In this work, we computationally explore the possibility of detecting methylated DNA strands through direct electrical conductance measurements. Using density functional theory and the Landauer-Buttiker method, we study the electronic properties and charge transport through an eight base-pair methylated DNA strand and its native counterpart. Specifically, we compare the results generated with the widely used B3LYP exchange-correlation (XC) functional and CAM-B3LYP based tuned range-separated hybrid density functional. We first analyze the effectmore » of cytosine methylation on the tight-binding parameters of two DNA strands and then model the transmission of the electrons and conductance through the strands both with and without decoherence. We find that with both functionals, the main difference of the tight-binding parameters between the native DNA and the methylated DNA lies in the on-site energies of (methylated) cytosine bases. The intra- and interstrand hopping integrals between two nearest neighboring guanine base and (methylated) cytosine base also change with the addition of the methyl groups. Our calculations show that in the phase-coherent limit, the transmission of the methylated strand is close to the native strand when the energy is nearby the highest occupied molecular orbital (HOMO) level and larger than the native strand by 5 times in the bandgap. The trend in transmission also holds in the presence of the decoherence with both functionals. We also study the effect of contact coupling by choosing coupling strengths ranging from weak to strong coupling limit. Our results suggest that the effect of the two different functionals is to alter the on-site energies of the DNA bases at the HOMO level, while the transport properties don't depend much on the two

  19. DNA strand scission induced by adriamycin and aclacinomycin A.

    PubMed

    Someya, A; Tanaka, N

    1979-08-01

    The binding of adriamycin and aclacinomycin A with PM2 DNA, and the consequent cleavage of DNA have been demonstrated by agarose gel electrophoresis, using an ethidium bromide assay. Adriamycin was observed to induce a single strand scission of DNA in the presence of a reducing agent, but aclacinomycin A caused much less degree of DNA breaks. The DNA cleavage was enhanced by Cu2+ and Fe2+, but not significantly by Ni2+, Zn2+, Mg2+ and Ca2+, suggesting that reduction and auto-oxidation of the quinone moiety and H2O2 production participate in the DNA-cutting effect. The DNA degradation was dependent upon concentrations of the anthracyclines and CuCl2. The degree of DNA cleavage at 0.04 mM adriamycin was similar to that at 0.4 mM aclacinomycin A in the presence of 1 mM NADPH and 0.4 mM CuCl2. DNA was degraded to small fragments at 0.4 mM adriamycin and 0.2 mM CuCl2. The anthracycline-induced DNA cleavage was stimulated by H2O2, but partially inhibited by potassium iodide, superoxide dismutase, catalase and nitrogen gas atmosphere. The results suggested that both free radical of anthracycline quinones and hydroxyl radical directly react with DNA strands.

  20. Cosegregation of chromosomes containing immortal DNA strands in cells that cycle with asymmetric stem cell kinetics.

    PubMed

    Merok, Joshua R; Lansita, Janice A; Tunstead, James R; Sherley, James L

    2002-12-01

    A long-standing intriguing hypothesis in cancer biology is that adult stem cells avoid mutations from DNA replication errors by a unique pattern of chromosome segregation. At each asymmetric cell division, adult stem cells have been postulated to selectively retain a set of chromosomes that contain old template DNA strands (i.e., "immortal DNA strands"). Using cultured cells that cycle with asymmetric cell kinetics, we confirmed both the existence of immortal DNA strands and the cosegregation of chromosomes that bear them. Our findings also lead us to propose a role for immortal DNA strands in tissue aging as well as cancer.

  1. A universal molecular translator for non-nucleic acid targets that enables dynamic DNA assemblies and logic operations.

    PubMed

    Tang, Wei; Hu, Shichao; Wang, Huaming; Zhao, Yan; Li, Na; Liu, Feng

    2014-11-28

    A universal molecular translator based on the target-triggered DNA strand displacement was developed, which was able to convert various kinds of non-nucleic acid targets into a unique output DNA. This translation strategy was successfully applied in directing dynamic DNA assemblies and in realizing three-input logic gate operations.

  2. Preparation of Single-Stranded Bacteriophage M13 DNA by Precipitation with Polyethylene Glycol.

    PubMed

    Green, Michael R; Sambrook, Joseph

    2017-11-01

    Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected bacteria into the surrounding medium. Several methods are available to purify the polymorphic filamentous particles. In this protocol, the particles are concentrated by precipitation with polyethylene glycol (PEG) in the presence of high salt. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. The resulting preparation is pure enough to be used as a template for DNA sequencing. A yield of 5-10 µg of single-stranded DNA/mL of infected cells may be expected from recombinant bacteriophages bearing inserts of 300-1000 nt. © 2017 Cold Spring Harbor Laboratory Press.

  3. PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Hui; Gao, Pu; Rajashankar, Kanagalaghatta R.

    C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering upmore » of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a “locked” conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.« less

  4. Contribution of gold nanoparticles to the catalytic DNA strand displacement in leakage reduction and signal amplification.

    PubMed

    Wang, Bei; Zhou, Xiang; Yao, Dongbao; Sun, Xianbao; He, Miao; Wang, Xiaojing; Yin, Xue; Liang, Haojun

    2017-10-03

    A new model using a gold nanoparticle (AuNP)-DNA system to constrain leakage and improve efficiency of catalytic toehold-mediated strand displacement reactions was outlined. A 10-bp spacer on AuNPs and fourfold amount of fuels were determined for good performance of this model with an optimized toehold strategy. After the reaction at 25 °C for 10 h, a 258 pM target could be identified, which is a remarkable improvement compared with the traditional AuNP-DNA system without fuel. Moreover, this model was also studied to differentiate specific single nucleotide polymorphism on target with superior selection factors. This model may help by introducing a proposition of target detection to guide further investigation.

  5. Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

    PubMed Central

    Fornander, Louise H.; Renodon-Cornière, Axelle; Kuwabara, Naoyuki; Ito, Kentaro; Tsutsui, Yasuhiro; Shimizu, Toshiyuki; Iwasaki, Hiroshi; Nordén, Bengt; Takahashi, Masayuki

    2014-01-01

    The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction. PMID:24304898

  6. Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament.

    PubMed

    Fornander, Louise H; Renodon-Cornière, Axelle; Kuwabara, Naoyuki; Ito, Kentaro; Tsutsui, Yasuhiro; Shimizu, Toshiyuki; Iwasaki, Hiroshi; Nordén, Bengt; Takahashi, Masayuki

    2014-02-01

    The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

  7. 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.

  8. Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

    PubMed

    Kukiełka, E; Cederbaum, A I

    1995-04-15

    Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

  9. Trifluorothymidine exhibits potent antitumor activity via the induction of DNA double-strand breaks.

    PubMed

    Suzuki, Norihiko; Nakagawa, Fumio; Nukatsuka, Mamoru; Fukushima, Masakazu

    2011-05-01

    TAS-102 is an oral anticancer drug composed of trifluorothymidine (TFT) and TPI (an inhibitor of thymidine phosphorylase that strongly inhibits the biodegradation of TFT). Similar to 5-fluorouracil (5FU) and 5-fluoro-2'-deoxyuridine (FdUrd), TFT also inhibits thymidylate synthase (TS), a rate-limiting enzyme of DNA biosynthesis, and is incorporated into DNA. TFT exhibits an anticancer effect on colorectal cancer cells that have acquired 5FU and/or FdUrd resistance as a result of the overexpression of TS. Therefore, we examined the mode of action of TFT-induced DNA damage after its incorporation into DNA. When HeLa cells were treated with TFT, the number of ring-open aldehyde forms at apurinic/apyrimidinic sites increased in a dose-dependent manner, although we previously reported that no detectable excisions of TFT paired to adenine were observed using uracil DNA glycosylases, thymine DNA glycosylase or methyl-CpG binding domain 4 and HeLa whole cell extracts. To investigate the functional mechanism of TFT-induced DNA damage, we measured the phosphorylation of ATR, ATM, BRCA2, chk1 and chk2 in nuclear extracts of HeLa cells after 0, 24, 48 or 72 h of exposure to an IC(50) concentration of TFT, FdUrd or 5FU using Western blot analysis or an enzyme-linked immunosorbent assay (ELISA). Unlike FdUrd and 5FU, TFT resulted in an earlier phosphorylation of ATR and chk1 proteins after only 24 h of exposure, while phosphorylated ATM, BRCA2 and chk2 proteins were detected after more than 48 h of exposure to TFT. These results suggest that TFT causes single-strand breaks followed by double-strand breaks in the DNA of TFT-treated cells. TFT (as TAS-102) showed a more potent antitumor activity than oral 5FU on CO-3 colon cancer xenografts in mice, and such antitumor potency was supported by the increased number of double-strand breaks occurring after single-strand breaks in the DNA of the TFT-treated tumors. These results suggest that TFT causes single-strand breaks after its

  10. Crystal Structure of a CRISPR RNA-guided Surveillance Complex Bound to a ssDNA Target

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mulepati, Sabin; Heroux, Annie; Bailey, Scott

    In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of proteinmore » subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.« less

  11. Repair of DNA Strand Breaks in a Minichromosome In Vivo: Kinetics, Modeling, and Effects of Inhibitors

    PubMed Central

    Kumala, Slawomir; Fujarewicz, Krzysztof; Jayaraju, Dheekollu; Rzeszowska-Wolny, Joanna; Hancock, Ronald

    2013-01-01

    To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ∼170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20–30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair. PMID:23382828

  12. Target-regulated proximity hybridization with three-way DNA junction for in situ enhanced electronic detection of marine biotoxin based on isothermal cycling signal amplification strategy.

    PubMed

    Liu, Bingqian; Chen, Jinfeng; Wei, Qiaohua; Zhang, Bing; Zhang, Lan; Tang, Dianping

    2015-07-15

    A new signal amplification strategy based on target-regulated DNA proximity hybridization (TRPH) reaction accompanying formation of three-way DNA junction was designed for electronic detection of Microcystin-LR (MC-LR used in this case), coupling with junction-induced isothermal cycling signal amplification. Initially, a sandwiched-type immunoreaction was carried out in a low-cost PCR tube between anti-MC-LR mAb1 antibody-labeled DNA1 (mAb1-DNA1) and anti-MC-LR mAb2-labeled DNA2 (mAb2-DNA2) in the presence of target to form a three-way DNA junction. Then, the junction could undergo an unbiased strand displacement reaction on an h-like DNA nanostructure-modified electrode (labeled with methylene blue redox tag on the short DNA strand), thereby resulting in the dissociation of methylene blue-labeled signal DNA from the electrode. The newly formed double-stranded DNA could be cleaved again by exonuclease III, and the released three-way DNA junction retriggered the strand-displacement reaction with h-like DNA nanostructures for junction recycling. During the strand-displacement reaction, numerous methylene blue-labeled DNA strands were far away from the electrode, thus decreasing the detectable electrochemical signal within the applied potentials. Under optimal conditions, the TRPH-based immunosensing system exhibited good electrochemical responses for detecting target MC-LR at a concentration as low as 1.0ngkg(-1) (1.0ppt). Additionally, the precision, reproducibility, specificity and method accuracy were also investigated with acceptable results. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Design and Analysis of Compact DNA Strand Displacement Circuits for Analog Computation Using Autocatalytic Amplifiers.

    PubMed

    Song, Tianqi; Garg, Sudhanshu; Mokhtar, Reem; Bui, Hieu; Reif, John

    2018-01-19

    A main goal in DNA computing is to build DNA circuits to compute designated functions using a minimal number of DNA strands. Here, we propose a novel architecture to build compact DNA strand displacement circuits to compute a broad scope of functions in an analog fashion. A circuit by this architecture is composed of three autocatalytic amplifiers, and the amplifiers interact to perform computation. We show DNA circuits to compute functions sqrt(x), ln(x) and exp(x) for x in tunable ranges with simulation results. A key innovation in our architecture, inspired by Napier's use of logarithm transforms to compute square roots on a slide rule, is to make use of autocatalytic amplifiers to do logarithmic and exponential transforms in concentration and time. In particular, we convert from the input that is encoded by the initial concentration of the input DNA strand, to time, and then back again to the output encoded by the concentration of the output DNA strand at equilibrium. This combined use of strand-concentration and time encoding of computational values may have impact on other forms of molecular computation.

  14. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    PubMed Central

    Jette, Nicholas; Lees-Miller, Susan P.

    2015-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

  15. Winding single-molecule double-stranded DNA on a nanometer-sized reel

    PubMed Central

    You, Huijuan; Iino, Ryota; Watanabe, Rikiya; Noji, Hiroyuki

    2012-01-01

    A molecular system of a nanometer-sized reel was developed from F1–ATPase, a rotary motor protein. By combination with magnetic tweezers and optical tweezers, single-molecule double-stranded DNA (dsDNA) was wound around the molecular reel. The bending stiffness of dsDNA was determined from the winding tension (0.9–6.0 pN) and the diameter of the wound loop (21.4–8.5 nm). Our results were in good agreement with the conventional worm-like chain model and a persistence length of 54 ± 9 nm was estimated. This molecular reel system offers a new platform for single-molecule study of micromechanics of sharply bent DNA molecules and is expected to be applicable to the elucidation of the molecular mechanism of DNA-associating proteins on sharply bent DNA strands. PMID:22772992

  16. Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

    PubMed

    He, Quan; Shumate, Christie K; White, Mark A; Molineux, Ian J; Yin, Y Whitney

    2013-11-01

    Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB. © 2013. Published by Elsevier B.V.

  17. DNA translocation by human uracil DNA glycosylase: the case of single-stranded DNA and clustered uracils.

    PubMed

    Schonhoft, Joseph D; Stivers, James T

    2013-04-16

    Human uracil DNA glycosylase (hUNG) plays a central role in DNA repair and programmed mutagenesis of Ig genes, requiring it to act on sparsely or densely spaced uracil bases located in a variety of contexts, including U/A and U/G base pairs, and potentially uracils within single-stranded DNA (ssDNA). An interesting question is whether the facilitated search mode of hUNG, which includes both DNA sliding and hopping, changes in these different contexts. Here we find that hUNG uses an enhanced local search mode when it acts on uracils in ssDNA, and also, in a context where uracils are densely clustered in duplex DNA. In the context of ssDNA, hUNG performs an enhanced local search by sliding with a mean sliding length larger than that of double-stranded DNA (dsDNA). In the context of duplex DNA, insertion of high-affinity abasic product sites between two uracil lesions serves to significantly extend the apparent sliding length on dsDNA from 4 to 20 bp and, in some cases, leads to directionally biased 3' → 5' sliding. The presence of intervening abasic product sites mimics the situation where hUNG acts iteratively on densely spaced uracils. The findings suggest that intervening product sites serve to increase the amount of time the enzyme remains associated with DNA as compared to nonspecific DNA, which in turn increases the likelihood of sliding as opposed to falling off the DNA. These findings illustrate how the search mechanism of hUNG is not predetermined but, instead, depends on the context in which the uracils are located.

  18. Minimalist Approach to Complexity: Templating the Assembly of DNA Tile Structures with Sequentially Grown Input Strands.

    PubMed

    Lau, Kai Lin; Sleiman, Hanadi F

    2016-07-26

    Given its highly predictable self-assembly properties, DNA has proven to be an excellent template toward the design of functional materials. Prominent examples include the remarkable complexity provided by DNA origami and single-stranded tile (SST) assemblies, which require hundreds of unique component strands. However, in many cases, the majority of the DNA assembly is purely structural, and only a small "working area" needs to be aperiodic. On the other hand, extended lattices formed by DNA tile motifs require only a few strands; but they suffer from lack of size control and limited periodic patterning. To overcome these limitations, we adopt a templation strategy, where an input strand of DNA dictates the size and patterning of resultant DNA tile structures. To prepare these templating input strands, a sequential growth technique developed in our lab is used, whereby extended DNA strands of defined sequence and length may be generated simply by controlling their order of addition. With these, we demonstrate the periodic patterning of size-controlled double-crossover (DX) and triple-crossover (TX) tile structures, as well as intentionally designed aperiodicity of a DX tile structure. As such, we are able to prepare size-controlled DNA structures featuring aperiodicity only where necessary with exceptional economy and efficiency.

  19. MO-AB-BRA-04: Radiation Measurements with a DNA Double-Strand-Break Dosimeter

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Obeidat, M; Cline, K; Stathakis, S

    Purpose: Many types of dosimeters are used to measure radiation, but none of them directly measures the biological effect of this dose. The purpose here is to create a dosimeter that can measure the probability of double-strand breaks (DSB) for DNA, which is directly related to the biological effect of radiation. Methods: The dosimeter has DNA strands, which are labeled on one end with biotin and on the other with fluorescein. The biotin attaches these strands to magnetic beads. We suspended the DNA dosimeter in phosphate-buffered saline (PBS) as it matches the internal environment of the body. We placed smallmore » volumes (50µL) of the DNA dosimeter into tubes and irradiated these samples in a water-equivalent plastic phantom with several doses (three samples per dose). After irradiating the samples, a magnet was placed against the tubes. The fluorescein attached to broken DNA strands was extracted (called the supernatant) and placed into a different tube. The fluorescein on the unbroken strands remained attached to the beads in the tube and was re-suspended with 50µL of PBS. A fluorescence reader was used to measure the fluorescence for both the re-suspended beads and supernatant. To prove that we are measuring DSB, we tested dosimeter response with two different lengths of attached DNA strands (1 and 4 kilo-base pair). Results: The probability of DSB at the dose levels of 5, 10, 25, and 50 Gy were 0.05, 0.08, 0.12, and 0.19, respectively, while the coefficients of variation were 0.14, 0.07, 0.02, and 0.01, respectively. The 4 kilo-base-pair dosimeter produced 5.3 times the response of the 1 kilo-base-pair dosimeter. Conclusion: The DNA dosimeter yields a measurable response to dose that scales with the DNA strand length. The goal now is to refine the dosimeter fabrication to reproducibly create a low coefficient of variation for the lower doses. This work was supported in part by Yarmouk University (Irbid, Jordan) and CPRIT (RP140105)« less

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

    PubMed

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

    2016-01-01

    The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor, acting by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in double-stranded DNA damage, such as ionizing radiation and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases the sensitivity of lymphoma cells to ionizing radiation. In the current study, we show that additional peptides derived from Vif, A3G, and APOBEC3F, which contain the LYYF motif, inhibit deamination activity. Each residue in the Vif25-39 sequence moderately contributes to the inhibitory effect, whereas replacing a single residue in the LYYF motif completely abrogates inhibition of deamination. Treatment of A3G-expressing lymphoma cells exposed to ionizing radiation with the new inhibitory peptides reduces double-strand break repair after irradiation. Incubation of cultured irradiated lymphoma cells with peptides that inhibit double-strand break repair halts their propagation. These results suggest that A3G may be a potential therapeutic target that is amenable to peptide and peptidomimetic inhibition. © 2015 FEBS.

  1. 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

  2. Determination and analysis of site-specific 125I decay-induced DNA double-strand break end-group structures.

    PubMed

    Datta, Kamal; Weinfeld, Michael; Neumann, Ronald D; Winters, Thomas A

    2007-02-01

    End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cell's ability to repair DSBs. The 3'-end groups of strand breaks caused by gamma radiation, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3'-phosphoglycolate and 3'-phosphate, with 5'-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by 125I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by 125I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3'-hydroxyl (3'-OH) and 3'-phosphate (3'-P) end groups and 5'-ends (> or = 42%) terminated by phosphate. A 32P postlabeling assay failed to detect 3'-phosphoglycolate in a restriction fragment terminated by the 125I-induced DNA double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer.

  3. An amplified graphene oxide-based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling for bioassays.

    PubMed

    Hu, Kun; Liu, Jinwen; Chen, Jia; Huang, Yong; Zhao, Shulin; Tian, Jianniao; Zhang, Guohai

    2013-04-15

    An amplified graphene oxide (GO) based fluorescence aptasensor based on target-triggered aptamer hairpin switch and strand-displacement polymerization recycling is developed for bioassays. The dye-labeled single-strand DNA (aptamer hairpin) was adsorbed on the surface of GO, which result in the fluorescence quenching of dye, and exhibiting minimal background fluorescence. Upon the target, primer and polymerase, the stem of the aptamer hairpin was opened, and binds with the primer to triggers the circular target strand-displacement polymerization reaction, which produces huge amounts of duplex helixes DNA and lead to strong fluorescence emission due to shielding of nucelobases within its double-helix structure. During the polymerization reaction, the primer was extended, and target was displaced. And the displaced target recognizes and hybridizes with another hairpin probe, triggering the next round of polymerization reaction, and the circle process induces fluorescence signal amplification for the detection of analyte. To test the feasibility of the aptasensor systems, interferon-gamma (IFN-γ) was employed as a model analyte. A detection limit as low as 1.5 fM is obtained based on the GO aptasensor with a linear range of three orders of magnitude. The present method was successfully applied for the detection of IFN-γ in human plasma. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. A novel nonenzymatic cascade amplification for ultrasensitive photoelectrochemical DNA sensing based on target driven to initiate cyclic assembly of hairpins.

    PubMed

    Wen, Guangming; Dong, Wenxia; Liu, Bin; Li, Zhongping; Fan, Lifang

    2018-05-29

    A novel cascade photoelectrochemical (PEC) signal amplification biosensing tactics was developed for DNA detection based on a target-driven DNA association to induce cyclic hairpin assembly. In the circulatory system there are two ssDNA (A and B) and two hairpins (C and D). The hybridization of these ssDNA led to the formation of an A-target-B structure. The close proximity of their toehold and branch-migration regions was able to induce the cyclic hairpin assembly. Afterwards, the assembly result further causes the separation of a double-stranded probe DNA (Q:F) to switch the PEC signal via toehold-mediated strand replacement. As such, the signal stranded DNA-CdS QDs (F) as the signal tag was released in the presence of the target DNA. The signal DNA-CdS QDs was then coated to F-doped tin oxide (FTO) electrode leading to the "signal-on" PEC signal. The designed biosensing strategy showed a low detection limit of 21.3 pM for target DNA and a broad linear range from 50 pM to 100 nM. This signal amplification PEC sensing method exhibited a potential application to detect protein molecules, RNA or metal ions via changing the sequence of A and B recognition. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Neurological disorders associated with DNA strand-break processing enzymes

    PubMed Central

    Jiang, Bingcheng; Glover, J.N. Mark

    2016-01-01

    The termini of DNA strand breaks induced by reactive oxygen species or by abortive DNA metabolic intermediates require processing to enable subsequent gap filling and ligation to proceed. The three proteins, tyrosyl DNA-phosphodiesterase 1 (TDP1), aprataxin (APTX) and polynucleotide kinase/phosphatase (PNKP) each act on a discrete set of modified strand-break termini. Recently, a series of neurodegenerative and neurodevelopmental disorders have been associated with mutations in the genes coding for these proteins. Mutations in TDP1 and APTX have been linked to Spinocerebellar ataxia with axonal neuropathy (SCAN1) and Ataxia-ocular motor apraxia 1 (AOA1), respectively, while mutations in PNKP are considered to be responsible for Microcephaly with seizures (MCSZ) and Ataxia-ocular motor apraxia 4 (AOA4). Here we present an overview of the mechanisms of these proteins and how their impairment may give rise to their respective disorders. PMID:27470939

  6. DNA strand-displacement-induced fluorescence enhancement for highly sensitive and selective assay of multiple microRNA in cancer cells.

    PubMed

    Wu, Ping; Tu, Yunqiu; Qian, Yingdan; Zhang, Hui; Cai, Chenxin

    2014-01-28

    We report a new strategy for evaluating multiple miRNA expressions in cancer cells based on DNA strand-displacement-induced fluorescence enhancement. This assay has the ability to discriminate the target from even single-base mismatched sequences or other miRNAs.

  7. Strand-invading linear probe combined with unmodified PNA.

    PubMed

    Asanuma, Hiroyuki; Niwa, Rie; Akahane, Mariko; Murayama, Keiji; Kashida, Hiromu; Kamiya, Yukiko

    2016-09-15

    Efficient strand invasion by a linear probe to fluorescently label double-stranded DNA has been implemented by employing a probe and unmodified PNA. As a fluorophore, we utilized ethynylperylene. Multiple ethynylperylene residues were incorporated into the DNA probe via a d-threoninol scaffold. The ethynylperylene did not significantly disrupt hybridization with complementary DNA. The linear probe self-quenched in the absence of target DNA and did not hybridize with PNA. A gel-shift assay revealed that linear probe and PNA combination invaded the central region of double-stranded DNA upon heat-shock treatment to form a double duplex. To further suppress the background emission and increase the stability of the probe/DNA duplex, a probe containing anthraquinones as well as ethynylperylene was synthesized. This probe and PNA invader pair detected an internal sequence in a double-stranded DNA with high sensitivity when heat shock treatment was used. The probe and PNA pair was able to invade at the terminus of a long double-stranded DNA at 40°C at 100mM NaCl concentration. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Trifluorothymidine exhibits potent antitumor activity via the induction of DNA double-strand breaks

    PubMed Central

    SUZUKI, NORIHIKO; NAKAGAWA, FUMIO; NUKATSUKA, MAMORU; FUKUSHIMA, MASAKAZU

    2011-01-01

    TAS-102 is an oral anticancer drug composed of trifluorothymidine (TFT) and TPI (an inhibitor of thymidine phosphorylase that strongly inhibits the biodegradation of TFT). Similar to 5-fluorouracil (5FU) and 5-fluoro-2′-deoxyuridine (FdUrd), TFT also inhibits thymidylate synthase (TS), a rate-limiting enzyme of DNA biosynthesis, and is incorporated into DNA. TFT exhibits an anticancer effect on colorectal cancer cells that have acquired 5FU and/or FdUrd resistance as a result of the overexpression of TS. Therefore, we examined the mode of action of TFT-induced DNA damage after its incorporation into DNA. When HeLa cells were treated with TFT, the number of ring-open aldehyde forms at apurinic/apyrimidinic sites increased in a dose-dependent manner, although we previously reported that no detectable excisions of TFT paired to adenine were observed using uracil DNA glycosylases, thymine DNA glycosylase or methyl-CpG binding domain 4 and HeLa whole cell extracts. To investigate the functional mechanism of TFT-induced DNA damage, we measured the phosphorylation of ATR, ATM, BRCA2, chk1 and chk2 in nuclear extracts of HeLa cells after 0, 24, 48 or 72 h of exposure to an IC50 concentration of TFT, FdUrd or 5FU using Western blot analysis or an enzyme-linked immunosorbent assay (ELISA). Unlike FdUrd and 5FU, TFT resulted in an earlier phosphorylation of ATR and chk1 proteins after only 24 h of exposure, while phosphorylated ATM, BRCA2 and chk2 proteins were detected after more than 48 h of exposure to TFT. These results suggest that TFT causes single-strand breaks followed by double-strand breaks in the DNA of TFT-treated cells. TFT (as TAS-102) showed a more potent antitumor activity than oral 5FU on CO-3 colon cancer xenografts in mice, and such antitumor potency was supported by the increased number of double-strand breaks occurring after single-strand breaks in the DNA of the TFT-treated tumors. These results suggest that TFT causes single-strand breaks after its

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

    PubMed Central

    Calderwood, Stuart K.

    2016-01-01

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

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

    PubMed

    Calderwood, Stuart K

    2016-05-26

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

  11. Compiler-aided systematic construction of large-scale DNA strand displacement circuits using unpurified components

    NASA Astrophysics Data System (ADS)

    Thubagere, Anupama J.; Thachuk, Chris; Berleant, Joseph; Johnson, Robert F.; Ardelean, Diana A.; Cherry, Kevin M.; Qian, Lulu

    2017-02-01

    Biochemical circuits made of rationally designed DNA molecules are proofs of concept for embedding control within complex molecular environments. They hold promise for transforming the current technologies in chemistry, biology, medicine and material science by introducing programmable and responsive behaviour to diverse molecular systems. As the transformative power of a technology depends on its accessibility, two main challenges are an automated design process and simple experimental procedures. Here we demonstrate the use of circuit design software, combined with the use of unpurified strands and simplified experimental procedures, for creating a complex DNA strand displacement circuit that consists of 78 distinct species. We develop a systematic procedure for overcoming the challenges involved in using unpurified DNA strands. We also develop a model that takes synthesis errors into consideration and semi-quantitatively reproduces the experimental data. Our methods now enable even novice researchers to successfully design and construct complex DNA strand displacement circuits.

  12. DNA strand breaks signal the induction of DNA double-strand break repair in Saccharomyces cerevisiae.

    PubMed

    Singh, Rakesh Kumar; Krishna, Malini

    2005-12-01

    Genotoxic stress induces a checkpoint signaling cascade to generate a stress response. Saccharomyces cerevisiae shows an altered radiation response under different type of stress. Although the induction of repair has been implicated in enhanced survival after exposure to the challenging stress, the nature of the signal remains poorly understood. This study demonstrates that low doses of gamma radiation and bleomycin induce RAD52-dependent recombination repair pathway in the wild-type strain D-261. Prior exposure of cells to DNA-damaging agents (gamma radiation or bleomycin) equips them better for the subsequent damage caused by challenging doses. However, exposure to UV light, which does not cause strand breaks, was ineffective. This was confirmed by PFGE studies. This indicates that the strand breaks probably serve as the signal for induction of the recombination repair pathway while pyrimidine dimers do not. The nature of the induced repair was investigated by mutation scoring in special strain D-7, which showed that the induced repair is essentially error free.

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

    PubMed

    Awate, Sanket; Brosh, Robert M

    2017-06-08

    Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

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

    PubMed Central

    Awate, Sanket; Brosh, Robert M.

    2017-01-01

    Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies. PMID:28594346

  15. Isolation and characterization of naturally occurring hairpin structures in single-stranded DNA of coliphage M13

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Niyogi, S.K.; Mitra, S.

    With precise conditions of digestion with single-strand-specific nucleases, namely, endonuclease S1 of Aspergillus oryzae and exonuclease I of Escherichia coli, nuclease-resistant DNA cores can be obtained reproducibly from single-stranded M13 DNA. The DNA cores are composed almost exclusively of two sizes (60 and 44 nucleotides long). These have high (G + C)-contents relative to that of intact M13 DNA, and arise from restricted regions of the M13 genome. The resistance of these fragments to single-strand-specific nucleases and their nondenaturability strongly suggest the presence of double-stranded segments in these core pieces. That the core pieces are only partially double-stranded is shownmore » by their lack of complete base complementarity and their pattern of elution from hydroxyapatite.« less

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Poeschla, Eric, E-mail: poeschla.eric@mayo.edu

    Central initiation of plus strand synthesis is a conserved feature of lentiviruses and certain other retroelements. This complication of the standard reverse transcription mechanism produces a transient “central DNA flap” in the viral cDNA, which has been proposed to mediate its subsequent nuclear import. This model has assumed that the important feature is the flapped DNA structure itself rather than the process that produces it. Recently, an alternative kinetic model was proposed. It posits that central plus strand synthesis functions to accelerate conversion to the double-stranded state, thereby helping HIV-1 to evade single-strand DNA-targeting antiviral restrictions such as APOBEC3 proteins,more » and perhaps to avoid innate immune sensor mechanisms. The model is consistent with evidence that lentiviruses must often synthesize their cDNAs when dNTP concentrations are limiting and with data linking reverse transcription and uncoating. There may be additional kinetic advantages for the artificial genomes of lentiviral gene therapy vectors. - Highlights: • Two main functional models for HIV central plus strand synthesis have been proposed. • In one, a transient central DNA flap in the viral cDNA mediates HIV-1 nuclear import. • In the other, multiple kinetic consequences are emphasized. • One is defense against APOBEC3G, which deaminates single-stranded DNA. • Future questions pertain to antiviral restriction, uncoating and nuclear import.« less

  17. DNA logic gate based on metallo-toehold strand displacement.

    PubMed

    Deng, Wei; Xu, Huaguo; Ding, Wei; Liang, Haojun

    2014-01-01

    DNA is increasingly being used as an ideal material for the construction of nanoscale structures, circuits, and machines. Toehold-mediated DNA strand displacement reactions play a very important role in these enzyme-free constructions. In this study, the concept of metallo-toehold was utilized to further develop a mechanism for strand displacement driven by Ag+ ions, in which the intercalation of cytosine-cytosine mismatched base pairs on the toeholds provides additional control by varying of the concentration of Ag+ ions. The characteristics of displacement reaction in response to different concentration of Ag+ ions are investigated by fluorescence spectral and non-denaturing polyacrylamide gel electrophoresis. The reaction can successfully occur when the concentration of Ag+ ions is suitabe; excess Ag+ ions block the reaction. Furthermore, the displacement reaction can be tuned and controlled most efficiently under the condition of two C:C mismatched base pairs placed on the six-nt toehold. Based on our research, a mechanism was developed to construct Boolean logic gate AND and OR by employing strand displacement reaction as a tool, Ag+ and Hg2+ as input.

  18. Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.

    PubMed

    Swarts, Daan C; van der Oost, John; Jinek, Martin

    2017-04-20

    The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Reliable method for generating double-stranded DNA vectors containing site-specific base modifications.

    PubMed

    Brégeon, Damien; Doetsch, Paul W

    2004-11-01

    Cells of all living organisms are continuously exposed to physical and chemical agents that damage DNA and alter the integrity of their genomes. Despite the relatively high efficiency of the different repair pathways, some lesions remain in DNA when it is replicated or transcribed. Lesion bypass by DNA and RNA polymerases has been the subject of numerous investigations. However, knowledge of the in vivo mechanism of transcription lesion bypass is very limited because no robust methodology is available. Here we describe a protocol based on the synthesis of a complementary strand of a circular, single-stranded DNA molecule, which allows for the production of large amounts of double-stranded DNA containing a lesion at a specific position in a transcribed sequence. Such constructs can subsequently be used for lesion bypass studies in vivo by RNA polymerase and to ascertain how these events can be affected by the genetic background of the cells.

  20. On the biophysics and kinetics of toehold-mediated DNA strand displacement

    PubMed Central

    Srinivas, Niranjan; Ouldridge, Thomas E.; Šulc, Petr; Schaeffer, Joseph M.; Yurke, Bernard; Louis, Ard A.; Doye, Jonathan P. K.; Winfree, Erik

    2013-01-01

    Dynamic DNA nanotechnology often uses toehold-mediated strand displacement for controlling reaction kinetics. Although the dependence of strand displacement kinetics on toehold length has been experimentally characterized and phenomenologically modeled, detailed biophysical understanding has remained elusive. Here, we study strand displacement at multiple levels of detail, using an intuitive model of a random walk on a 1D energy landscape, a secondary structure kinetics model with single base-pair steps and a coarse-grained molecular model that incorporates 3D geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Two factors explain the dependence of strand displacement kinetics on toehold length: (i) the physical process by which a single step of branch migration occurs is significantly slower than the fraying of a single base pair and (ii) initiating branch migration incurs a thermodynamic penalty, not captured by state-of-the-art nearest neighbor models of DNA, due to the additional overhang it engenders at the junction. Our findings are consistent with previously measured or inferred rates for hybridization, fraying and branch migration, and they provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems. PMID:24019238

  1. On the biophysics and kinetics of toehold-mediated DNA strand displacement.

    PubMed

    Srinivas, Niranjan; Ouldridge, Thomas E; Sulc, Petr; Schaeffer, Joseph M; Yurke, Bernard; Louis, Ard A; Doye, Jonathan P K; Winfree, Erik

    2013-12-01

    Dynamic DNA nanotechnology often uses toehold-mediated strand displacement for controlling reaction kinetics. Although the dependence of strand displacement kinetics on toehold length has been experimentally characterized and phenomenologically modeled, detailed biophysical understanding has remained elusive. Here, we study strand displacement at multiple levels of detail, using an intuitive model of a random walk on a 1D energy landscape, a secondary structure kinetics model with single base-pair steps and a coarse-grained molecular model that incorporates 3D geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Two factors explain the dependence of strand displacement kinetics on toehold length: (i) the physical process by which a single step of branch migration occurs is significantly slower than the fraying of a single base pair and (ii) initiating branch migration incurs a thermodynamic penalty, not captured by state-of-the-art nearest neighbor models of DNA, due to the additional overhang it engenders at the junction. Our findings are consistent with previously measured or inferred rates for hybridization, fraying and branch migration, and they provide a biophysical explanation of strand displacement kinetics. Our work paves the way for accurate modeling of strand displacement cascades, which would facilitate the simulation and construction of more complex molecular systems.

  2. Enhancing the response rate of strand displacement-based electrochemical aptamer sensors using bivalent binding aptamer-cDNA probes.

    PubMed

    Zhang, Ziping; Tao, Cancan; Yin, Jungang; Wang, Yunhui; Li, Yanshen

    2018-04-30

    Electrochemical aptamer (EA) sensors based on aptamer-cDNA duplex probes (cDNA: complementary DNA) and target induced strand displacement (TISD) recognition are sensitive, selective and capable of detecting a wide variety of target analytes. While substantial research efforts have focused on engineering of new signaling mechanisms for the improvement of sensor sensitivity, little attention was paid to the enhancement of sensor response rate. Typically, the previous TISD based EA sensors exhibited relatively long response times larger than 30min, which mainly resulted from the suboptimal aptamer-cDNA probe structure in which most of aptamer bases were paired to the cDNA bases. In an effort to improve the response rate of this type of sensors, we report here the rational engineering of a quickly responsive and sensitive aptamer-cDNA probe by employing the conception of bivalent interaction in supramolecular chemistry. We design a bivalent cDNA strand through linking two short monovalent cDNA sequences, and it is simultaneously hybridized to two electrode-immobilized aptamer probes to form a bivalent binding (BB) aptamer-cDNA probe. This class of BB probe possesses the advantages of less aptamer bases paired to the cDNA bases for quick response rate and good structural stability for high sensor sensitivity. By use of the rationally designed BB aptamer-cDNA probe, a TISD based EA sensor against ATP with significantly enhanced response rate (with a displacement equilibrium time of 4min) and high sensitivity was successfully constructed. We believe that our BB probe conception will help guide future designs and applications of TISD based EA sensors. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Ultrasensitive electrochemical DNA detection based on dual amplification of circular strand-displacement polymerase reaction and hybridization chain reaction.

    PubMed

    Wang, Cui; Zhou, Hui; Zhu, Wenping; Li, Hongbo; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

    2013-09-15

    We developed a novel electrochemical strategy for ultrasensitive DNA detection using a dual amplification strategy based on the circular strand-displacement polymerase reaction (CSDPR) and the hybridization chain reaction (HCR). In this assay, hybridization of hairpin-shaped capture DNA to target DNA resulted in a conformational change of the capture DNA with a concomitant exposure of its stem. The primer was then hybridized with the exposed stem and triggered a polymerization reaction, allowing a cyclic reaction comprising release of target DNA, hybridization of target with remaining capture DNA, polymerization initiated by the primer. Furthermore, the free part of the primer propagated a chain reaction of hybridization events between two DNA hairpin probes with biotin labels, enabling an electrochemical reading using the streptavidin-alkaline phosphatase. The proposed biosensor showed to have very high sensitivity and selectivity with a dynamic response range through 10fM to 1nM, and the detect limit was as low as 8fM. The proposed strategy could have the potential for molecular diagnostics in complex biological systems. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. SU-E-T-05: Comparing DNA Strand Break Yields for Photons under Different Irradiation Conditions with Geant4-DNA.

    PubMed

    Pater, P; Bernal, M; Naqa, I El; Seuntjens, J

    2012-06-01

    To validate and scrutinize published DNA strand break data with Geant4-DNA and a probabilistic model. To study the impact of source size, electronic equilibrium and secondary electron tracking cutoff on direct relative biological effectiveness (DRBE). Geant4 (v4.9.5) was used to simulate a cylindrical region of interest (ROI) with r = 15 nm and length = 1.05 mm, in a slab of liquid water of 1.06 g/cm 3 density. The ROI was irradiated with mono-energetic photons, with a uniformly distributed volumetric isotropic source (0.28, 1.5 keV) or a plane beam (0.662, 1.25 MeV), of variable size. Electrons were tracked down to 50 or 10 eV, with G4-DNA processes and energy transfer greater than 10.79 eV was scored. Based on volume ratios, each scored event had a 0.0388 probability of happening on either DNA helix (break). Clusters of at least one break on each DNA helix within 3.4 nm were found using a DBSCAN algorithm and categorized as double strand breaks (DSB). All other events were categorized as single strand breaks (SSB). Geant4-DNA is able to reproduce strand break yields previously published. Homogeneous irradiation conditions should be present throughout the ROI for DRBE comparisons. SSB yields seem slightly dependent on the primary photon energy. DRBEs show a significant increasing trend for lower energy incident photons. A lower electron cutoff produces higher SSB yields, but decreases the SSB/DSB yields ratio. The probabilistic and geometrical DNA models can predict equivalent results. Using Geant4, we were able to reproduce previously published results on the direct strand break yields of photon and study the importance of irradiation conditions. We also show an ascending trend for DRBE with lower incident photon energies. A probabilistic model coupled with track structure analysis can be used to simulate strand break yields. NSERC, CIHR. © 2012 American Association of Physicists in Medicine.

  5. Highly sensitive and selective microRNA detection based on DNA-bio-bar-code and enzyme-assisted strand cycle exponential signal amplification.

    PubMed

    Dong, Haifeng; Meng, Xiangdan; Dai, Wenhao; Cao, Yu; Lu, Huiting; Zhou, Shufeng; Zhang, Xueji

    2015-04-21

    Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNP-DNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

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

    PubMed Central

    Rao, Venigalla B.; Feiss, Michael

    2016-01-01

    Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead’s portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL’s N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage φ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of packaging and ejection of the motor. Current focus is on the structures of packaging complexes and the dynamics of TerL during DNA packaging, endonuclease regulation, and motor mechanics. PMID:26958920

  7. Solving probability reasoning based on DNA strand displacement and probability modules.

    PubMed

    Zhang, Qiang; Wang, Xiaobiao; Wang, Xiaojun; Zhou, Changjun

    2017-12-01

    In computation biology, DNA strand displacement technology is used to simulate the computation process and has shown strong computing ability. Most researchers use it to solve logic problems, but it is only rarely used in probabilistic reasoning. To process probabilistic reasoning, a conditional probability derivation model and total probability model based on DNA strand displacement were established in this paper. The models were assessed through the game "read your mind." It has been shown to enable the application of probabilistic reasoning in genetic diagnosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

    NASA Astrophysics Data System (ADS)

    Zhang, Z.; Birkedal, V.; Gothelf, K. V.

    2016-05-01

    The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection.

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

    PubMed

    Ribeck, Noah; Saleh, Omar A

    2013-01-01

    The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

  10. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target

    PubMed Central

    Mulepati, Sabin; Héroux, Annie; Bailey, Scott

    2015-01-01

    In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kDa complex is called Cascade. Here we report the 3.03Å crystal structure of Cascade bound to a single-stranded DNA target. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This non-canonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding. PMID:25123481

  11. Differential Targeting of Unpaired Bases within Duplex DNA by the Natural Compound Clerocidin: A Valuable Tool to Dissect DNA Secondary Structure

    PubMed Central

    Nadai, Matteo; Palù, Giorgio; Palumbo, Manlio; Richter, Sara N.

    2012-01-01

    Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures. PMID:23285245

  12. Differential targeting of unpaired bases within duplex DNA by the natural compound clerocidin: a valuable tool to dissect DNA secondary structure.

    PubMed

    Nadai, Matteo; Palù, Giorgio; Palumbo, Manlio; Richter, Sara N

    2012-01-01

    Non-canonical DNA structures have been postulated to mediate protein-nucleic acid interactions and to function as intermediates in the generation of frame-shift mutations when errors in DNA replication occur, which result in a variety of diseases and cancers. Compounds capable of binding to non-canonical DNA conformations may thus have significant diagnostic and therapeutic potential. Clerocidin is a natural diterpenoid which has been shown to selectively react with single-stranded bases without targeting the double helix. Here we performed a comprehensive analysis on several non-canonical DNA secondary structures, namely mismatches, nicks, bulges, hairpins, with sequence variations in both the single-stranded region and the double-stranded flanking segment. By analysis of clerocidin reactivity, we were able to identify the exposed reactive residues which provided information on both the secondary structure and the accessibility of the non-paired sites. Mismatches longer than 1 base were necessary to be reached by clerocidin reactive groups, while 1-base nicks were promptly targeted by clerocidin; in hairpins, clerocidin reactivity increased with the length of the hairpin loop, while, interestingly, reactivity towards bulges reached a maximum in 3-base-long bulges and declined in longer bulges. Electrophoretic mobility shift analysis demonstrated that bulges longer than 3 bases (i.e. 5- and 7-bases) folded or stacked on the duplex region therefore being less accessible by the compound. Clerocidin thus represents a new valuable diagnostic tool to dissect DNA secondary structures.

  13. Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility*

    PubMed Central

    Buechner, Claudia N.; Heil, Korbinian; Michels, Gudrun; Carell, Thomas; Kisker, Caroline; Tessmer, Ingrid

    2014-01-01

    Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5′-3′ helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a “final” verification state, which may then trigger the recruitment of further NER proteins. PMID:24338567

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Neto, J.L. Siqueira; Instituto de Biologia, UNICAMP, Campinas, SP; Lira, C.B.B.

    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 ismore » 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.« less

  15. Artificial and Solar UV Radiation Induces Strand Breaks and Cyclobutane Pyrimidine Dimers in Bacillus subtilis Spore DNA

    PubMed Central

    Slieman, Tony A.; Nicholson, Wayne L.

    2000-01-01

    The loss of stratospheric ozone and the accompanying increase in solar UV flux have led to concerns regarding decreases in global microbial productivity. Central to understanding this process is determining the types and amounts of DNA damage in microbes caused by solar UV irradiation. While UV irradiation of dormant Bacillus subtilis endospores results mainly in formation of the “spore photoproduct” 5-thyminyl-5,6-dihydrothymine, genetic evidence indicates that an additional DNA photoproduct(s) may be formed in spores exposed to solar UV-B and UV-A radiation (Y. Xue and W. L. Nicholson, Appl. Environ. Microbiol. 62:2221–2227, 1996). We examined the occurrence of double-strand breaks, single-strand breaks, cyclobutane pyrimidine dimers, and apurinic-apyrimidinic sites in spore DNA under several UV irradiation conditions by using enzymatic probes and neutral or alkaline agarose gel electrophoresis. DNA from spores irradiated with artificial 254-nm UV-C radiation accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, while DNA from spores exposed to artificial UV-B radiation (wavelengths, 290 to 310 nm) accumulated only cyclobutane pyrimidine dimers. DNA from spores exposed to full-spectrum sunlight (UV-B and UV-A radiation) accumulated single-strand breaks, double-strand breaks, and cyclobutane pyrimidine dimers, whereas DNA from spores exposed to sunlight from which the UV-B component had been removed with a filter (“UV-A sunlight”) accumulated only single-strand breaks and double-strand breaks. Apurinic-apyrimidinic sites were not detected in spore DNA under any of the irradiation conditions used. Our data indicate that there is a complex spectrum of UV photoproducts in DNA of bacterial spores exposed to solar UV irradiation in the environment. PMID:10618224

  16. Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin-biotin recognition.

    PubMed

    Borovok, Natalia; Iram, Natalie; Zikich, Dragoslav; Ghabboun, Jamal; Livshits, Gideon I; Porath, Danny; Kotlyar, Alexander B

    2008-09-01

    We describe a method for the preparation of novel long (hundreds of nanometers), uniform, inter-molecular G4-DNA molecules composed of four parallel G-strands. The only long continuous G4-DNA reported so far are intra-molecular structures made of a single G-strand. To enable a tetra-molecular assembly of the G-strands we developed a novel approach based on avidin-biotin biological recognition. The steps of the G4-DNA production include: (i) Enzymatic synthesis of long poly(dG)-poly(dC) molecules with biotinylated poly(dG)-strand; (ii) Formation of a complex between avidin-tetramer and four biotinylated poly(dG)-poly(dC) molecules; (iii) Separation of the poly(dC) strands from the poly(dG)-strands, which are connected to the avidin; (iv) Assembly of the four G-strands attached to the avidin into tetra-molecular G4-DNA. The average contour length of the formed structures, as measured by AFM, is equal to that of the initial poly(dG)-poly(dC) molecules, suggesting a tetra-molecular mechanism of the G-strands assembly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA molecules having similar contour length. The CD spectra of the tetra- and mono-molecular G4-DNA are markedly different, suggesting different structural organization of these two types of molecules. The tetra-molecular G4-DNA nanostructures showed clear electrical polarizability. This suggests that they may be useful for molecular electronics.

  17. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    PubMed Central

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification. PMID:26729209

  18. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    NASA Astrophysics Data System (ADS)

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

  19. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification.

    PubMed

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-05

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

  20. Magnetic-field-induced DNA strand breaks in brain cells of the rat.

    PubMed Central

    Lai, Henry; Singh, Narendra P

    2004-01-01

    In previous research, we found that rats acutely (2 hr) exposed to a 60-Hz sinusoidal magnetic field at intensities of 0.1-0.5 millitesla (mT) showed increases in DNA single- and double-strand breaks in their brain cells. Further research showed that these effects could be blocked by pretreating the rats with the free radical scavengers melatonin and N-tert-butyl-alpha-phenylnitrone, suggesting the involvement of free radicals. In the present study, effects of magnetic field exposure on brain cell DNA in the rat were further investigated. Exposure to a 60-Hz magnetic field at 0.01 mT for 24 hr caused a significant increase in DNA single- and double-strand breaks. Prolonging the exposure to 48 hr caused a larger increase. This indicates that the effect is cumulative. In addition, treatment with Trolox (a vitamin E analog) or 7-nitroindazole (a nitric oxide synthase inhibitor) blocked magnetic-field-induced DNA strand breaks. These data further support a role of free radicals on the effects of magnetic fields. Treatment with the iron chelator deferiprone also blocked the effects of magnetic fields on brain cell DNA, suggesting the involvement of iron. Acute magnetic field exposure increased apoptosis and necrosis of brain cells in the rat. We hypothesize that exposure to a 60-Hz magnetic field initiates an iron-mediated process (e.g., the Fenton reaction) that increases free radical formation in brain cells, leading to DNA strand breaks and cell death. This hypothesis could have an important implication for the possible health effects associated with exposure to extremely low-frequency magnetic fields in the public and occupational environments. PMID:15121512

  1. Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

    PubMed

    Son, Kyung-No; Liang, Zhiguo; Lipton, Howard L

    2015-09-01

    Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN

  2. Extended Minus-Strand DNA as Template for R-U5-Mediated Second-Strand Transfer in Recombinational Rescue of Primer Binding Site-Modified Retroviral Vectors

    PubMed Central

    Mikkelsen, Jacob Giehm; Lund, Anders H.; Dybkær, Karen; Duch, Mogens; Pedersen, Finn Skou

    1998-01-01

    We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed. PMID:9499117

  3. TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus.

    PubMed

    Wu, Yong; Gao, Tieli; Wang, Xiaolin; Hu, Youjin; Hu, Xuyun; Hu, Zhiqing; Pang, Jialun; Li, Zhuo; Xue, Jinfeng; Feng, Mai; Wu, Lingqian; Liang, Desheng

    2014-03-28

    Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4 kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy.

    PubMed

    Murthy, Vaibhav; Dacus, Dalton; Gamez, Monica; Hu, Changkun; Wendel, Sebastian O; Snow, Jazmine; Kahn, Andrew; Walterhouse, Stephen H; Wallace, Nicholas A

    2018-06-08

    The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as

  5. Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase.

    PubMed

    Chan, Pan F; Germe, Thomas; Bax, Benjamin D; Huang, Jianzhong; Thalji, Reema K; Bacqué, Eric; Checchia, Anna; Chen, Dongzhao; Cui, Haifeng; Ding, Xiao; Ingraham, Karen; McCloskey, Lynn; Raha, Kaushik; Srikannathasan, Velupillai; Maxwell, Anthony; Stavenger, Robert A

    2017-05-30

    A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

  6. Host-Guest Recognition-Assisted Electrochemical Release: Its Reusable Sensing Application Based on DNA Cross Configuration-Fueled Target Cycling and Strand Displacement Reaction Amplification.

    PubMed

    Chang, Yuanyuan; Zhuo, Ying; Chai, Yaqin; Yuan, Ruo

    2017-08-15

    In this work, an elegantly designed host-guest recognition-assisted electrochemical release was established and applied in a reusable electrochemical biosensor for the detection of microRNA-182-5p (miRNA-182-5p), a prostate cancer biomarker in prostate cancer, based on the DNA cross configuration-fueled target cycling and strand displacement reaction (SDR) amplification. With such a design, the single target miRNA input could be converted to large numbers of single-stranded DNA (S1-Trp and S2-Trp) output, which could be trapped by cucurbit[8]uril methyl viologen (CB-8-MV 2+ ) based on the host-guest recognition, significantly enhancing the sensitivity for miRNA detection. Moreover, the nucleic acids products obtained from the process of cycling amplification could be utilized sufficiently, avoiding the waste and saving the experiment cost. Impressively, by resetting a settled voltage, the proposed biosensor could release S1-Trp and S2-Trp from the electrode surface, attributing that the guest ion methyl viologen (MV 2+ ) was reduced to MV +· under this settled voltage and formed a more-stable CB-8-MV +· -MV +· complex. Once O 2 was introduced in this system, MV +· could be oxidized to MV 2+ , generating the complex of CB-8-MV 2+ for capturing S1-Trp and S2-Trp again in only 5 min. As a result, the simple and fast regeneration of biosensor for target detection was realized on the base of electrochemical redox-driven assembly and release, overcoming the challenges of time-consuming, burdensome operations and expensive experimental cost in traditional reusable biosensors and updating the construction method for a reusable bisensor. Furthermore, the biosensor could be reused for more than 10 times with a regeneration rate of 93.20%-102.24%. After all, the conception of this work provides a novel thought for the construction of effective reusable biosensor to detect miRNA and other biomarkers and has great potential application in the area requiring the release of

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

    Ricchetti, M; Fairhead, C; Dujon, B

    1999-11-04

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

  9. Probe and method for DNA detection

    DOEpatents

    Yeh, Hsin-Chih; Werner, James Henry; Sharma, Jaswinder Kumar; Martinez, Jennifer Suzanne

    2013-07-02

    A hybridization probe containing two linear strands of DNA lights up upon hybridization to a target DNA using silver nanoclusters that have been templated onto one of the DNA strands. Hybridization induces proximity between the nanoclusters on one strand and an overhang on the other strand, which results in enhanced fluorescence emission from the nanoclusters.

  10. Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance

    PubMed Central

    Sedman, Tiina; Gaidutšik, Ilja; Villemson, Karin; Hou, YingJian; Sedman, Juhan

    2014-01-01

    Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability. PMID:25389272

  11. Responsive hairpin DNA aptamer switch to program the strand displacement reaction for the enhanced electrochemical assay of ATP.

    PubMed

    Wang, Li; Fang, Li; Liu, Shufeng

    2015-09-07

    A responsive hairpin DNA aptamer switch was ingeniously designed for enzyme-free, sensitive and selective electrochemical detection of ATP. It takes full advantage of the target-triggered liberation effect of the toehold region and the concomitant proximity effect with the branch-migration region to execute the toehold-mediated strand displacement reaction on the electrode surface.

  12. Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

    PubMed Central

    Langston, Lance; O’Donnell, Mike

    2017-01-01

    Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand. DOI: http://dx.doi.org/10.7554/eLife.23449.001 PMID:28346143

  13. The occurrence of antibodies against single-stranded DNA in the sera of patients with acute and chronic leukaemia.

    PubMed Central

    Izui, S; Lambert, P H; Carpentier, N; Miescher, P A

    1976-01-01

    One hundred and seventy-five sera from thirty-three patients with acute myeloid leukaemia, forty-two patients with chronic myeloid leukaemia and twelve patients with acute lymphatic leukaemia were examined by a radioimmunological technique for the presence of antibodies against single-stranded and double-stranded DNA. The levels of single-stranded DNA binding activity was significantly higher in all three types of leukaemia compared to those of healthy controls. In contrast, none of these sera exhibited a positive reaction with double-stranded DNA. In some cases the level of serum anti-DNA antibodies increased after the decrease of the leucocyte count. The presence of anti-DNA antibodies in leukaemic patients may have some biological significance. PMID:780020

  14. Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale

    PubMed Central

    Yu, Xiong; Egelman, Edward H.

    2010-01-01

    Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single stranded DNA with ∼ 9 subunits per turn in contrast to the filaments formed on double stranded DNA with ∼ 6.4 subunits per turn, and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy (STEM) that the Dmc1 filament formed on single stranded DNA has a mass per unit length expected from ∼ 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions, and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or STEM to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. PMID:20600108

  15. RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication.

    PubMed

    Ruff, Patrick; Donnianni, Roberto A; Glancy, Eleanor; Oh, Julyun; Symington, Lorraine S

    2016-12-20

    DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal

    PubMed Central

    Fuller, James R; Rice, Phoebe A

    2017-01-01

    The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal. DOI: http://dx.doi.org/10.7554/eLife.21777.001 PMID:28177285

  17. Scaling up digital circuit computation with DNA strand displacement cascades.

    PubMed

    Qian, Lulu; Winfree, Erik

    2011-06-03

    To construct sophisticated biochemical circuits from scratch, one needs to understand how simple the building blocks can be and how robustly such circuits can scale up. Using a simple DNA reaction mechanism based on a reversible strand displacement process, we experimentally demonstrated several digital logic circuits, culminating in a four-bit square-root circuit that comprises 130 DNA strands. These multilayer circuits include thresholding and catalysis within every logical operation to perform digital signal restoration, which enables fast and reliable function in large circuits with roughly constant switching time and linear signal propagation delays. The design naturally incorporates other crucial elements for large-scale circuitry, such as general debugging tools, parallel circuit preparation, and an abstraction hierarchy supported by an automated circuit compiler.

  18. A Role for Single-Stranded Exonucleases in the Use of DNA as a Nutrient▿

    PubMed Central

    Palchevskiy, Vyacheslav; Finkel, Steven E.

    2009-01-01

    Nutritional competence is the ability of bacterial cells to utilize exogenous double-stranded DNA molecules as a nutrient source. We previously identified several genes in Escherichia coli that are important for this process and proposed a model, based on models of natural competence and transformation in bacteria, where it is assumed that single-stranded DNA (ssDNA) is degraded following entry into the cytoplasm. Since E. coli has several exonucleases, we determined whether they play a role in the long-term survival and the catabolism of DNA as a nutrient. We show here that mutants lacking either ExoI, ExoVII, ExoX, or RecJ are viable during all phases of the bacterial life cycle yet cannot compete with wild-type cells during long-term stationary-phase incubation. We also show that nuclease mutants, alone or in combination, are defective in DNA catabolism, with the exception of the ExoX− single mutant. The ExoX− mutant consumes double-stranded DNA better than wild-type cells, possibly implying the presence of two pathways in E. coli for the processing of ssDNA as it enters the cytoplasm. PMID:19329645

  19. The high stability of the triple helices formed between short purine oligonucleotides and SIV/HIV-2 vpx genes is determined by the targeted DNA structure.

    PubMed Central

    Svinarchuk, F; Monnot, M; Merle, A; Malvy, C; Fermandjian, S

    1995-01-01

    In our previous works we have shown that the oligonucleotides 5'-GGGGAGGGGGAGG-3' and 5'-GGAGGGGGAGGGG-3' give very stable and specific triplexes with their target double stranded DNAs [Svinarchuk, F., Bertrand, J.-R. and Malvy, C. (1994) Nucleic Acids Res., 22, 3742-3747; Svinarchuk, F., Paoletti, J. and Malvy, C. (1995) J. Biol. Chem., 270, 14 068-14,071]. The target for the invariable part of these oligonucleotides, 5'-GGAGGGGGAGG-3', is found in a highly conserved 20 bp long purine/pyrimidine tract of the vpx gene of the SIV and HIV-2 viruses and could be a target for oligonucleotide directed antivirus therapy. Here were report on the ability of four purine oligonucleotides with different lengths (11-, 14-, 17- and 20-mer) to form triplexes with the purine/pyrimidine stretch of the vpx gene. Triplex formation was tested by joint dimethyl sulfate (DMS) footprint, gel-retardation assay, circular dichroism (CD) and UV-melting studies. Dimethyl sulfate footprint studies revealed the antiparallel orientation of the third strand to the purine strand of the Watson-Crick duplex. However, the protection of the guanines at the ends of the target sequence decreased as the length of the third strand oligonucleotide increased. Melting temperature studies provided profiles with only one transition for all of the triplexes. The melting temperatures of the triplexes were found to be the same as for the targeted duplex in the case of the 11- and 14-mer third strands while for the 17- and 20-mer third strands the melting temperature of the triplexes were correspondingly 4 and 8 degrees C higher than for the duplex. Heating and cooling melting curves were reversible for all of the tested triplexes except one with the 20-mer third strand oligonucleotide. Circular dichroism spectra showed the ability of the target DNA to adopt an A-like DNA conformation. Upon triplex formation the A-DNA form becomes even more pronounced. This effect depends on the length of the third strand

  20. Structural Insights into the HIV-1 Minus-strand Strong-stop DNA*

    PubMed Central

    Chen, Yingying; Maskri, Ouerdia; Chaminade, Françoise; René, Brigitte; Benkaroun, Jessica; Godet, Julien; Mély, Yves; Mauffret, Olivier; Fossé, Philippe

    2016-01-01

    An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3′-end of the genomic RNA with the complementary r region at the 3′-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex). PMID:26668324

  1. Temperature-dependent conformations of exciton-coupled Cy3 dimers in double-stranded DNA

    NASA Astrophysics Data System (ADS)

    Kringle, Loni; Sawaya, Nicolas P. D.; Widom, Julia; Adams, Carson; Raymer, Michael G.; Aspuru-Guzik, Alán; Marcus, Andrew H.

    2018-02-01

    Understanding the properties of electronically interacting molecular chromophores, which involve internally coupled electronic-vibrational motions, is important to the spectroscopy of many biologically relevant systems. Here we apply linear absorption, circular dichroism, and two-dimensional fluorescence spectroscopy to study the polarized collective excitations of excitonically coupled cyanine dimers (Cy3)2 that are rigidly positioned within the opposing sugar-phosphate backbones of the double-stranded region of a double-stranded (ds)-single-stranded (ss) DNA fork construct. We show that the exciton-coupling strength of the (Cy3)2-DNA construct can be systematically varied with temperature below the ds-ss DNA denaturation transition. We interpret spectroscopic measurements in terms of the Holstein vibronic dimer model, from which we obtain information about the local conformation of the (Cy3)2 dimer, as well as the degree of static disorder experienced by the Cy3 monomer and the (Cy3)2 dimer probe locally within their respective DNA duplex environments. The properties of the (Cy3)2-DNA construct we determine suggest that it may be employed as a useful model system to test fundamental concepts of protein-DNA interactions and the role of electronic-vibrational coherence in electronic energy migration within exciton-coupled bio-molecular arrays.

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

    PubMed Central

    Nadassy, Katalin; Tomás-Oliveira, Isabel; Alberts, Ian; Janin, Joël; Wodak, Shoshana J.

    2001-01-01

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

  3. Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling.

    PubMed

    Choi, Jun-Hyuk; Lindsey-Boltz, Laura A; Kemp, Michael; Mason, Aaron C; Wold, Marc S; Sancar, Aziz

    2010-08-03

    ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.

  4. DNA double strand break induction in yeast.

    PubMed

    Kiefer, J; Egenolf, R; Ikpeme, S E

    2002-01-01

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

  5. Toehold-mediated strand displacement reaction triggered isothermal DNA amplification for highly sensitive and selective fluorescent detection of single-base mutation.

    PubMed

    Zhu, Jing; Ding, Yongshun; Liu, Xingti; Wang, Lei; Jiang, Wei

    2014-09-15

    Highly sensitive and selective detection strategy for single-base mutations is essential for risk assessment of malignancy and disease prognosis. In this work, a fluorescent detection method for single-base mutation was proposed based on high selectivity of toehold-mediated strand displacement reaction (TSDR) and powerful signal amplification capability of isothermal DNA amplification. A discrimination probe was specially designed with a stem-loop structure and an overhanging toehold domain. Hybridization between the toehold domain and the perfect matched target initiated the TSDR along with the unfolding of the discrimination probe. Subsequently, the target sequence acted as a primer to initiate the polymerization and nicking reactions, which released a great abundant of short sequences. Finally, the released strands were annealed with the reporter probe, launching another polymerization and nicking reaction to produce lots of G-quadruplex DNA, which could bind the N-methyl mesoporphyrin IX to yield an enhanced fluorescence response. However, when there was even a single base mismatch in the target DNA, the TSDR was suppressed and so subsequent isothermal DNA amplification and fluorescence response process could not occur. The proposed approach has been successfully implemented for the identification of the single-base mutant sequences in the human KRAS gene with a detection limit of 1.8 pM. Furthermore, a recovery of 90% was obtained when detecting the target sequence in spiked HeLa cells lysate, demonstrating the feasibility of this detection strategy for single-base mutations in biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. The impact of base stacking on the conformations and electrostatics of single-stranded DNA.

    PubMed

    Plumridge, Alex; Meisburger, Steve P; Andresen, Kurt; Pollack, Lois

    2017-04-20

    Single-stranded DNA (ssDNA) is notable for its interactions with ssDNA binding proteins (SSBs) during fundamentally important biological processes including DNA repair and replication. Previous work has begun to characterize the conformational and electrostatic properties of ssDNA in association with SSBs. However, the conformational distributions of free ssDNA have been difficult to determine. To capture the vast array of ssDNA conformations in solution, we pair small angle X-ray scattering with novel ensemble fitting methods, obtaining key parameters such as the size, shape and stacking character of strands with different sequences. Complementary ion counting measurements using inductively coupled plasma atomic emission spectroscopy are employed to determine the composition of the ion atmosphere at physiological ionic strength. Applying this combined approach to poly dA and poly dT, we find that the global properties of these sequences are very similar, despite having vastly different propensities for single-stranded helical stacking. These results suggest that a relatively simple mechanism for the binding of ssDNA to non-specific SSBs may be at play, which explains the disparity in binding affinities observed for these systems. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Analysis of strand transfer and template switching mechanisms of DNA gap repair by homologous recombination in Escherichia coli: predominance of strand transfer.

    PubMed

    Izhar, Lior; Goldsmith, Moshe; Dahan, Ronny; Geacintov, Nicholas; Lloyd, Robert G; Livneh, Zvi

    2008-09-12

    Daughter strand gaps formed upon interruption of replication at DNA lesions in Escherichia coli can be repaired by either translesion DNA synthesis or homologous recombination (HR) repair. Using a plasmid-based assay system that enables discrimination between strand transfer and template switching (information copying) modes of HR gap repair, we found that approximately 80% of strand gaps were repaired by physical strand transfer from the donor, whereas approximately 20% appear to be repaired by template switching. HR gap repair operated on both small and bulky lesions and largely depended on RecA and RecF but not on the RecBCD nuclease. In addition, we found that HR was mildly reduced in cells lacking the RuvABC and RecG proteins involved in resolution of Holliday junctions. These results, obtained for the first time under conditions that detect the two HR gap repair mechanisms, provide in vivo high-resolution molecular evidence for the predominance of the strand transfer mechanism in HR gap repair. A small but significant portion of HR gap repair appears to occur via a template switching mechanism.

  8. A DNA Nanotube-Peptide biocomplex for mRNA Detection and Its Application in Cancer Diagnosis and Targeted Therapy.

    PubMed

    Ji, Xiaoting; Lv, Haoyuan; Guo, Jiayi; Ding, Caifeng; Luo, Xiliang

    2018-04-25

    A biocomplex of DNA nanotube-peptide, consisting of six concatenated DNA strands, three lock DNA strands and a cell-penetrating peptide was developed herein. The barrel structured DNA nanotube-peptide was successfully applied as a co-drug delivery system for targeting cancer therapy. The mucin 1 proteins (MUC-1) aptamer which is part of DNA nanotube can specially recognize MUC-1 protein on the surface of MCF-7 cells. Cyclo (Arg-Gly-Asp-D-phe-Lys) (cRGD), as a cell-penetrating peptide, facilitates recruitment and uptake of targeting drugs by binding to integrin receptors (αvβ3) of cytomembrane surface. Anti-cancer drug doxorubicin (DOX) and paclitaxel (PTX) were loaded into the capsulated DNA nanotube-peptide (CDNP), which was used as co-drug cargo models. The as-prepared biocomplex can be utilized not only to deliver drug but also to achieve the anticancer effect in vivo. The experimental results suggested that the treatment efficacy of co-drug delivery platform (CDNP/DOX/PTX) was better than single-drug delivery platform (CDNP/DOX or CDNP/PTX). This system that was composed by DNA strands and peptide has good biocompatibility and biodegradability. Furthermore, the system can readily achieve detection of target mRNA of MCF-7 cell in vitro. The detection limits of mRNA are 9.7×10-8 M and 1.8×10-8 M by using CDNP/DOX and CDNP/PTX-FITC as a probe, respectively. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale.

    PubMed

    Yu, Xiong; Egelman, Edward H

    2010-08-20

    Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single-stranded DNA with approximately 9 subunits per turn in contrast to the filaments formed on double-stranded DNA with approximately 6.4 subunits per turn and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy that the Dmc1 filament formed on single-stranded DNA has a mass per unit length expected from approximately 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or scanning transmission electron microscopy, to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  10. Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

    PubMed

    Fornander, Louise H; Frykholm, Karolin; Reymer, Anna; Renodon-Cornière, Axelle; Takahashi, Masayuki; Nordén, Bengt

    2012-06-01

    Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

  11. [Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA].

    PubMed

    Jia, Xiao-Wei; Zhang, Guo-Hui; Shi, Hai-Yan

    2012-12-01

    Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription. We express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction. The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15. kod-ssb may in future be used to enhance DNA and cDNA amplification.

  12. Human FAN1 promotes strand incision in 5'-flapped DNA complexed with RPA.

    PubMed

    Takahashi, Daisuke; Sato, Koichi; Hirayama, Emiko; Takata, Minoru; Kurumizaka, Hitoshi

    2015-09-01

    Fanconi anaemia (FA) is a human infantile recessive disorder. Seventeen FA causal proteins cooperatively function in the DNA interstrand crosslink (ICL) repair pathway. Dual DNA strand incisions around the crosslink are critical steps in ICL repair. FA-associated nuclease 1 (FAN1) is a DNA structure-specific endonuclease that is considered to be involved in DNA incision at the stalled replication fork. Replication protein A (RPA) rapidly assembles on the single-stranded DNA region of the stalled fork. However, the effect of RPA on the FAN1-mediated DNA incision has not been determined. In this study, we purified human FAN1, as a bacterially expressed recombinant protein. FAN1 exhibited robust endonuclease activity with 5'-flapped DNA, which is formed at the stalled replication fork. We found that FAN1 efficiently promoted DNA incision at the proper site of RPA-coated 5'-flapped DNA. Therefore, FAN1 possesses the ability to promote the ICL repair of 5'-flapped DNA covered by RPA. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  13. Investigating actinomycin D binding to G-quadruplex, i-motif and double-stranded DNA in 27-nt segment of c-MYC gene promoter.

    PubMed

    Niknezhad, Zhila; Hassani, Leila; Norouzi, Davood

    2016-01-01

    c-MYC DNA is an attractive target for drug design, especially for cancer chemotherapy. Around 90% of c-MYC transcription is controlled by NHE III1, whose 27-nt purine-rich strand has the ability to form G-quadruplex structure. In this investigation, interaction of ActD with 27-nt G-rich strand (G/c-MYC) and its equimolar mixture with the complementary sequence, (GC/c-MYC) as well as related C-rich oligonucleotide (C/c-MYC) was evaluated. Molecular dynamic simulations showed that phenoxazine and lactone rings of ActD come close to the outer G-tetrad nucleotides indicating that ActD binds through end-stacking to the quadruplex DNA. RMSD and RMSF revealed that fluctuation of the quadruplex DNA increases upon interaction with the drug. The results of spectrophotometry and spectrofluorometry indicated that ActD most probably binds to the c-MYC quadruplex and duplex DNA via end-stacking and intercalation, respectively and polarity of ActD environment decreases due to the interaction. It was also found that binding of ActD to the GC-rich DNA is stronger than the two other forms of DNA. Circular dichroism results showed that the type of the three forms of DNA structures doesn't change, but their compactness alters due to their interaction with ActD. Finally, it can be concluded that ActD binds differently to double stranded DNA, quadruplex DNA and i-motif. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages

    PubMed Central

    Greinert, R.; Volkmer, B.; Henning, S.; Breitbart, E. W.; Greulich, K. O.; Cardoso, M. C.; Rapp, Alexander

    2012-01-01

    UVA (320–400 nm) represents the main spectral component of solar UV radiation, induces pre-mutagenic DNA lesions and is classified as Class I carcinogen. Recently, discussion arose whether UVA induces DNA double-strand breaks (dsbs). Only few reports link the induction of dsbs to UVA exposure and the underlying mechanisms are poorly understood. Using the Comet-assay and γH2AX as markers for dsb formation, we demonstrate the dose-dependent dsb induction by UVA in G1-synchronized human keratinocytes (HaCaT) and primary human skin fibroblasts. The number of γH2AX foci increases when a UVA dose is applied in fractions (split dose), with a 2-h recovery period between fractions. The presence of the anti-oxidant Naringin reduces dsb formation significantly. Using an FPG-modified Comet-assay as well as warm and cold repair incubation, we show that dsbs arise partially during repair of bi-stranded, oxidative, clustered DNA lesions. We also demonstrate that on stretched chromatin fibres, 8-oxo-G and abasic sites occur in clusters. This suggests a replication-independent formation of UVA-induced dsbs through clustered single-strand breaks via locally generated reactive oxygen species. Since UVA is the main component of solar UV exposure and is used for artificial UV exposure, our results shine new light on the aetiology of skin cancer. PMID:22941639

  15. Fluorescence Resonance Energy Transfer-Based Photonic Circuits Using Single-Stranded Tile Self-Assembly and DNA Strand Displacement.

    PubMed

    Zhang, Xuncai; Ying, Niu; Shen, Chaonan; Cui, Guangzhao

    2017-02-01

    Structural DNA nanotechnology has great potential in the fabrication of complicated nanostructures and devices capable of bio-sensing and logic function. A variety of nanostructures with desired shapes have been created in the past few decades. But the application of nanostructures remains to be fully studied. Here, we present a novel biological information processing system constructed on a self-assembled, spatially addressable single-stranded tile (SST) nanostructure as DNA nano-manipulation platform that created by SST self-assembly technology. Utilizing DNA strand displacement technology, the fluorescent dye that is pre-assembled in the nano-manipulation platform is transferred from the original position to the destination, which can achieve photonic logic circuits by FRET signal cascades, including logic AND, OR, and NOT gates. And this transfer process is successfully validated by visual DSD software. The transfer process proposed in this study may provide a novel method to design complicated biological information processing system constructed on a SST nanostructure, and can be further used to develop intelligent delivery of drug molecules in vivo.

  16. Dielectric dispersion for short double-strand DNA.

    PubMed

    Omori, Shinji; Katsumoto, Yoichi; Yasuda, Akio; Asami, Koji

    2006-05-01

    A complex dielectric constant for double-strand DNA molecules with a length of not greater than 120 base pairs in an aqueous solution containing 30 mM NaCl was systematically measured as a function of chain length in such a way that experimental uncertainties associated with the molecular-weight distribution of specimens were virtually excluded. In contrast to the past experimental and theoretical studies for much longer DNA molecules, both the molar specific dielectric increment and the relaxation time are proportional to the chain length. These scaling rules cannot be accounted for by any theory so far proposed that gives analytical expressions for those two quantities in the long-chain limit.

  17. The role of differing probe and target strand lengths in DNA microarrays investigated via Monte Carlo molecular simulation

    NASA Astrophysics Data System (ADS)

    Rivard, Brea R.; Cooper, Sarah J.; Stubbs, John M.

    2018-02-01

    DNA duplexes consisting of a 25mer together with shorter complementary sequences were studied over a range of temperature and surface binding motifs using a coarse-grained two-site nucleotide model. Results were analyzed in terms of hydrogen bonding interactions and structural characteristics and indicate that hybridization is most stable when furthest from the surface binding site. Strand elongation and straightening near the bound end are found to be correlated to duplex destabilization.

  18. A conserved MCM single-stranded DNA binding element is essential for replication initiation.

    PubMed

    Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

    2014-04-01

    The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.

  19. A conserved MCM single-stranded DNA binding element is essential for replication initiation

    PubMed Central

    Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

    2014-01-01

    The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001 PMID:24692448

  20. Asymmetric segregation of template DNA strands in basal-like human breast cancer cell lines

    PubMed Central

    2013-01-01

    Background and methods Stem or progenitor cells from healthy tissues have the capacity to co-segregate their template DNA strands during mitosis. Here, we set out to test whether breast cancer cell lines also possess the ability to asymmetrically segregate their template DNA strands via non-random chromosome co-segregation, and whether this ability correlates with certain properties attributed to breast cancer stem cells (CSCs). We quantified the frequency of asymmetric segregation of template DNA strands in 12 human breast cancer cell lines, and correlated the frequency to molecular subtype, CD44+/CD24-/lo phenotype, and invasion/migration ability. We tested if co-culture with human mesenchymal stem cells, which are known to increase self-renewal, can alter the frequency of asymmetric segregation of template DNA in breast cancer. Results We found a positive correlation between asymmetric segregation of template DNA and the breast cancer basal-like and claudin-low subtypes. There was an inverse correlation between asymmetric segregation of template DNA and Her2 expression. Breast cancer samples with evidence of asymmetric segregation of template DNA had significantly increased invasion and borderline significantly increased migration abilities. Samples with high CD44+/CD24-/lo surface expression were more likely to harbor a consistent population of cells that asymmetrically segregated its template DNA; however, symmetric self-renewal was enriched in the CD44+/CD24-/lo population. Co-culturing breast cancer cells with human mesenchymal stem cells expanded the breast CSC pool and decreased the frequency of asymmetric segregation of template DNA. Conclusions Breast cancer cells within the basal-like subtype can asymmetrically segregate their template DNA strands through non-random chromosome segregation. The frequency of asymmetric segregation of template DNA can be modulated by external factors that influence expansion or self-renewal of CSC populations. Future

  1. Implementing Nonlinear Feedback Controllers Using DNA Strand Displacement Reactions.

    PubMed

    Sawlekar, Rucha; Montefusco, Francesco; Kulkarni, Vishwesh V; Bates, Declan G

    2016-07-01

    We show how an important class of nonlinear feedback controllers can be designed using idealized abstract chemical reactions and implemented via DNA strand displacement (DSD) reactions. Exploiting chemical reaction networks (CRNs) as a programming language for the design of complex circuits and networks, we show how a set of unimolecular and bimolecular reactions can be used to realize input-output dynamics that produce a nonlinear quasi sliding mode (QSM) feedback controller. The kinetics of the required chemical reactions can then be implemented as enzyme-free, enthalpy/entropy driven DNA reactions using a toehold mediated strand displacement mechanism via Watson-Crick base pairing and branch migration. We demonstrate that the closed loop response of the nonlinear QSM controller outperforms a traditional linear controller by facilitating much faster tracking response dynamics without introducing overshoots in the transient response. The resulting controller is highly modular and is less affected by retroactivity effects than standard linear designs.

  2. Cisplatin enhances the formation of DNA single- and double-strand breaks by hydrated electrons and hydroxyl radicals.

    PubMed

    Rezaee, Mohammad; Sanche, Léon; Hunting, Darel J

    2013-03-01

    The synergistic interaction of cisplatin with ionizing radiation is the clinical rationale for the treatment of several cancers including head and neck, cervical and lung cancer. The underlying molecular mechanism of the synergy has not yet been identified, although both DNA damage and repair processes are likely involved. Here, we investigate the indirect effect of γ rays on strand break formation in a supercoiled plasmid DNA (pGEM-3Zf-) covalently modified by cisplatin. The yields of single- and double-strand breaks were determined by irradiation of DNA and cisplatin/DNA samples with (60)Co γ rays under four different scavenging conditions to examine the involvement of hydrated electrons and hydroxyl radicals in inducing the DNA damage. At 5 mM tris in an N2 atmosphere, the presence of an average of two cisplatins per plasmid increased the yields of single- and double-strand breaks by factors of 1.9 and 2.2, respectively, relative to the irradiated unmodified DNA samples. Given that each plasmid of 3,200 base pairs contained an average of two cisplatins, this represents an increase in radiosensitivity of 3,200-fold on a per base pair basis. When hydrated electrons were scavenged by saturating the samples with N2O, these enhancement factors decreased to 1.5 and 1.2, respectively, for single- and double-strand breaks. When hydroxyl radicals were scavenged using 200 mM tris, the respective enhancement factors were 1.2 and 1.6 for single- and double-strand breaks, respectively. Furthermore, no enhancement in DNA damage by cisplatin was observed after scavenging both hydroxyl radicals and hydrated electrons. These findings show that hydrated electrons can induce both single- and double-strand breaks in the platinated DNA, but not in unmodified DNA. In addition, cisplatin modification is clearly an extremely efficient means of increasing the formation of both single- and double-strand breaks by the hydrated electrons and hydroxyl radicals created by ionizing

  3. RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

    PubMed

    Liu, Tina Y; Iavarone, Anthony T; Doudna, Jennifer A

    2017-01-01

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag) of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

  4. 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.

  5. Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

    PubMed Central

    Bolderson, Emma; Tomimatsu, Nozomi; Richard, Derek J.; Boucher, Didier; Kumar, Rakesh; Pandita, Tej K.; Burma, Sandeep; Khanna, Kum Kum

    2010-01-01

    DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability. PMID:20019063

  6. Annealing of Complementary DNA Sequences During Double-Strand Break Repair in Drosophila Is Mediated by the Ortholog of SMARCAL1.

    PubMed

    Holsclaw, Julie Korda; Sekelsky, Jeff

    2017-05-01

    DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology-directed repair strategies, with the predominant form of homology-directed repair being synthesis-dependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development. SDSA has also been proposed to be the primary mechanism for integration of large insertions during genome editing with CRISPR/Cas9. Despite the central role for SDSA in genome stability, little is known about the defining step: annealing. We hypothesized that annealing during SDSA is performed by the annealing helicase SMARCAL1, which can anneal RPA-coated single DNA strands during replication-associated DNA damage repair. We used unique genetic tools in Drosophila melanogaster to test whether the fly ortholog of SMARCAL1, Marcal1, mediates annealing during SDSA. Repair that requires annealing is significantly reduced in Marcal1 null mutants in both synthesis-dependent and synthesis-independent (single-strand annealing) assays. Elimination of the ATP-binding activity of Marcal1 also reduced annealing-dependent repair, suggesting that the annealing activity requires translocation along DNA. Unlike the null mutant, however, the ATP-binding defect mutant showed reduced end joining, shedding light on the interaction between SDSA and end-joining pathways. Copyright © 2017 by the Genetics Society of America.

  7. SAMHD1 Sheds Moonlight on DNA Double-Strand Break Repair.

    PubMed

    Cabello-Lobato, Maria Jose; Wang, Siyue; Schmidt, Christine Katrin

    2017-12-01

    SAMHD1 (sterile α motif and histidine (H) aspartate (D) domain-containing protein 1) is known for its antiviral activity of hydrolysing deoxynucleotides required for virus replication. Daddacha et al. identify a hydrolase-independent, moonlighting function of SAMHD1 that facilitates homologous recombination of DNA double-strand breaks (DSBs) by promoting recruitment of C-terminal binding protein interacting protein (CTIP), a DNA-end resection factor, to damaged DNA. These findings could benefit anticancer treatment. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Divalent counterion-induced condensation of triple-strand DNA.

    PubMed

    Qiu, Xiangyun; Parsegian, V Adrian; Rau, Donald C

    2010-12-14

    Understanding and manipulation of the forces assembling DNA/RNA helices have broad implications for biology, medicine, and physics. One subject of significance is the attractive force between dsDNA mediated by polycations of valence ≥ 3. Despite extensive studies, the physical origin of the "like-charge attraction" remains unsettled among competing theories. Here we show that triple-strand DNA (tsDNA), a more highly charged helix than dsDNA, is precipitated by alkaline-earth divalent cations that are unable to condense dsDNA. We further show that our observation is general by examining several cations (Mg(2+), Ba(2+), and Ca(2+)) and two distinct tsDNA constructs. Cation-condensed tsDNA forms ordered hexagonal arrays that redissolve upon adding monovalent salts. Forces between tsDNA helices, measured by osmotic stress, follow the form of hydration forces observed with condensed dsDNA. Probing a well-defined system of point-like cations and tsDNAs with more evenly spaced helical charges, the counterintuitive observation that the more highly charged tsDNA (vs. dsDNA) is condensed by cations of lower valence provides new insights into theories of polyelectrolytes and the biological and pathological roles of tsDNA. Cations and tsDNAs also hold promise as a model system for future studies of DNA-DNA interactions and electrostatic interactions in general.

  9. Single nucleotide-level mapping of DNA double-strand breaks in human HEK293T cells.

    PubMed

    Pope, Bernard J; Mahmood, Khalid; Jung, Chol-Hee; Georgeson, Peter; Park, Daniel J

    2017-03-01

    Constitutional biological processes involve the generation of DNA double-strand breaks (DSBs). The production of such breaks and their subsequent resolution are also highly relevant to neurodegenerative diseases and cancer, in which extensive DNA fragmentation has been described Stephens et al. (2011), Blondet et al. (2001). Tchurikov et al. Tchurikov et al. (2011, 2013) have reported previously that frequent sites of DSBs occur in chromosomal domains involved in the co-ordinated expression of genes. This group report that hot spots of DSBs in human HEK293T cells often coincide with H3K4me3 marks, associated with active transcription Kravatsky et al. (2015) and that frequent sites of DNA double-strand breakage are likely to be relevant to cancer genomics Tchurikov et al. (2013, 2016) . Recently, they applied a RAFT (rapid amplification of forum termini) protocol that selects for blunt-ended DSB sites and mapped these to the human genome within defined co-ordinate 'windows'. In this paper, we re-analyse public RAFT data to derive sites of DSBs at the single-nucleotide level across the built genome for human HEK293T cells (https://figshare.com/s/35220b2b79eaaaf64ed8). This refined mapping, combined with accessory ENCODE data tracks and ribosomal DNA-related sequence annotations, will likely be of value for the design of clinically relevant targeted assays such as those for cancer susceptibility, diagnosis, treatment-matching and prognostication.

  10. Induction of single- and double-strand breaks in plasmid DNA by monoenergetic alpha-particles with energies below the Bragg-maximum.

    PubMed

    Scholz, V; Weidner, J; Köhnlein, W; Frekers, D; Wörtche, H J

    1997-01-01

    The yield of single-strand breaks (ssb) and double-strand breaks (dsb) produced by alpha-particles at the end of their track in DNA-films was determined experimentally. Helium nuclei were accelerated to 600 keV in the 400 kV ion accelerator and scattered at a carbon target. The elastically scattered alpha-particles with energies of 344 keV and 485 keV were used to irradiate supercircular plasmid DNA in vacuo. For the dosimetry of the alpha-particles a surface barrier detector was used and the energy distribution of the alpha-particles determined. The energy loss of the particles in the DNA-layer was calculated. DNA samples were separated into the three conformational isomers using agarose gel electrophoresis. After fluorochromation the number of ssb and dsb per plasmid DNA molecule was established from the band intensities assuming the validity of Poisson statistics. Linear dose effect correlations were found for ssb and dsb per plasmid molecule. In the case of 344 keV-alpha-particles the yield of dsb was (8.6 +/- 0.9) x 10(-11) breaks/Gy x dalton. The ratio of ssb/dsb was 0.5 +/- 0.2. This is at least a factor of six larger than the ratio found in experiments with higher energy alpha-particles and from model calculations. Similar experiments with protons yielded a relative biological effectiveness (rbe) value of 2.8 for the induction of double-strand breaks by track end alpha-particles.

  11. 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

  12. Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

    PubMed Central

    Talhaoui, Ibtissam; Lebedeva, Natalia A.; Zarkovic, Gabriella; Saint-Pierre, Christine; Kutuzov, Mikhail M.; Sukhanova, Maria V.; Matkarimov, Bakhyt T.; Gasparutto, Didier; Saparbaev, Murat K.; Lavrik, Olga I.; Ishchenko, Alexander A.

    2016-01-01

    Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3′-cordycepin, 5′- and 3′-phosphate and also to 5′-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5′-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2′-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2′,1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1′ of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs. PMID:27471034

  13. Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

    PubMed

    Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

    2009-05-01

    Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

  14. Arranging eukaryotic nuclear DNA polymerases for replication: Specific interactions with accessory proteins arrange Pols α, δ, and ϵ in the replisome for leading-strand and lagging-strand DNA replication.

    PubMed

    Kunkel, Thomas A; Burgers, Peter M J

    2017-08-01

    Biochemical and cryo-electron microscopy studies have just been published revealing interactions among proteins of the yeast replisome that are important for highly coordinated synthesis of the two DNA strands of the nuclear genome. These studies reveal key interactions important for arranging DNA polymerases α, δ, and ϵ for leading and lagging strand replication. The CMG (Mcm2-7, Cdc45, GINS) helicase is central to this interaction network. These are but the latest examples of elegant studies performed in the recent past that lead to a much better understanding of how the eukaryotic replication fork achieves efficient DNA replication that is accurate enough to prevent diseases yet allows evolution. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

  15. Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats.

    PubMed

    Gabsalilow, Lilia; Schierling, Benno; Friedhoff, Peter; Pingoud, Alfred; Wende, Wolfgang

    2013-04-01

    Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.

  16. Lipophilic oligonucleotides spontaneously insert into lipid membranes, bind complementary DNA strands, and sequester into lipid-disordered domains.

    PubMed

    Bunge, Andreas; Kurz, Anke; Windeck, Anne-Kathrin; Korte, Thomas; Flasche, Wolfgang; Liebscher, Jürgen; Herrmann, Andreas; Huster, Daniel

    2007-04-10

    For the development of surface functionalized bilayers, we have synthesized lipophilic oligonucleotides to combine the molecular recognition mechanism of nucleic acids and the self-assembly characteristics of lipids in planar membranes. A lipophilic oligonucleotide consisting of 21 thymidine units and two lipophilic nucleotides with an alpha-tocopherol moiety as a lipophilic anchor was synthesized using solid-phase methods with a phosphoramadite strategy. The interaction of the water soluble lipophilic oligonucleotide with vesicular lipid membranes and its capability to bind complementary DNA strands was studied using complementary methods such as NMR, EPR, DSC, fluorescence spectroscopy, and fluorescence microscopy. This oligonucleotide inserted stably into preformed membranes from the aqueous phase. Thereby, no significant perturbation of the lipid bilayer and its stability was observed. However, the non-lipidated end of the oligonucleotide is exposed to the aqueous environment, is relatively mobile, and is free to interact with complementary DNA strands. Binding of the complementary single-stranded DNA molecules is fast and accomplished by the formation of Watson-Crick base pairs, which was confirmed by 1H NMR chemical shift analysis and fluorescence resonance energy transfer. The molecular structure of the membrane bound DNA double helix is very similar to the free double-stranded DNA. Further, the membrane bound DNA double strands also undergo regular melting. Finally, in raft-like membrane mixtures, the lipophilic oligonucleotide was shown to preferentially sequester into liquid-disordered membrane domains.

  17. 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

  18. Comparison of direct DNA strand breaks induced by low energy electrons with different inelastic cross sections

    NASA Astrophysics Data System (ADS)

    Li, Jun-Li; Li, Chun-Yan; Qiu, Rui; Yan, Cong-Chong; Xie, Wen-Zhang; Zeng, Zhi; Tung, Chuan-Jong

    2013-09-01

    In order to study the influence of inelastic cross sections on the simulation of direct DNA strand breaks induced by low energy electrons, six different sets of inelastic cross section data were calculated and loaded into the Geant4-DNA code to calculate the DNA strand break yields under the same conditions. The six sets of the inelastic cross sections were calculated by applying the dielectric function method of Emfietzoglou's optical-data treatments, with two different optical datasets and three different dispersion models, using the same Born corrections. Results show that the inelastic cross sections have a notable influence on the direct DNA strand break yields. The yields simulated with the inelastic cross sections based on Hayashi's optical data are greater than those based on Heller's optical data. The discrepancies are about 30-45% for the single strand break yields and 45-80% for the double strand break yields. Among the yields simulated with cross sections of the three different dispersion models, generally the greatest are those of the extended-Drude dispersion model, the second are those of the extended-oscillator-Drude dispersion model, and the last are those of the Ashley's δ-oscillator dispersion model. For the single strand break yields, the differences between the first two are very little and the differences between the last two are about 6-57%. For the double strand break yields, the biggest difference between the first two can be about 90% and the differences between the last two are about 17-70%.

  19. Preparation of Double-Stranded (Replicative Form) Bacteriophage M13 DNA.

    PubMed

    Green, Michael R; Sambrook, Joseph

    2017-11-01

    The double-stranded, closed-circular, replicative form (RF) of M13 DNA is present in high copy numbers in infected cells, and its physical characteristics are essentially identical to those of closed-circular plasmid DNAs. Any of the methods commonly used to purify plasmid DNA can therefore be used to isolate M13 RF DNA. This protocol describes the isolation of M13 RF DNA by alkaline lysis from small volumes (1-2 mL) of infected bacterial cultures. The yield of DNA (1-4 mg, depending on the size of the M13 clone) is more than enough for most purposes in molecular cloning. However, should more DNA be needed, the procedure can easily be scaled up. © 2017 Cold Spring Harbor Laboratory Press.

  20. Effects of Homology Length in the Repeat Region on Minus-Strand DNA Transfer and Retroviral Replication

    PubMed Central

    Dang, Que; Hu, Wei-Shau

    2001-01-01

    Homology between the two repeat (R) regions in the retroviral genome mediates minus-strand DNA transfer during reverse transcription. We sought to define the effects of R homology lengths on minus-strand DNA transfer. We generated five murine leukemia virus (MLV)-based vectors that contained identical sequences but different lengths of the 3′ R (3, 6, 12, 24 and 69 nucleotides [nt]); 69 nt is the full-length MLV R. After one round of replication, viral titers from the vector with a full-length downstream R were compared with viral titers generated from the other four vectors with reduced R lengths. Viral titers generated from vectors with R lengths reduced to one-third (24 nt) or one-sixth (12 nt) that of the wild type were not significantly affected; however, viral titers generated from vectors with only 3- or 6-nt homology in the R region were significantly lower. Because expression and packaging of the RNA were similar among all the vectors, the differences in the viral titers most likely reflected the impact of the homology lengths on the efficiency of minus-strand DNA transfer. The molecular nature of minus-strand DNA transfer was characterized in 63 proviruses. Precise R-to-R transfer was observed in most proviruses generated from vectors with 12-, 24-, or 69-nt homology in R, whereas aberrant transfers were predominantly used to generate proviruses from vectors with 3- or 6-nt homology. Reverse transcription using RNA transcribed from an upstream promoter, termed read-in RNA transcripts, resulted in most of the aberrant transfers. These data demonstrate that minus-strand DNA transfer is homology driven and a minimum homology length is required for accurate and efficient minus-strand DNA transfer. PMID:11134294

  1. Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

    PubMed

    Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M; Högberg, Björn

    2013-07-01

    Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

  2. Off-Target Effects of Drugs that Disrupt Human Mitochondrial DNA Maintenance

    PubMed Central

    Young, Matthew J.

    2017-01-01

    Nucleoside reverse transcriptase inhibitors (NRTIs) were the first drugs used to treat human immunodeficiency virus (HIV) the cause of acquired immunodeficiency syndrome. Development of severe mitochondrial toxicity has been well documented in patients infected with HIV and administered NRTIs. In vitro biochemical experiments have demonstrated that the replicative mitochondrial DNA (mtDNA) polymerase gamma, Polg, is a sensitive target for inhibition by metabolically active forms of NRTIs, nucleotide reverse transcriptase inhibitors (NtRTIs). Once incorporated into newly synthesized daughter strands NtRTIs block further DNA polymerization reactions. Human cell culture and animal studies have demonstrated that cell lines and mice exposed to NRTIs display mtDNA depletion. Further complicating NRTI off-target effects on mtDNA maintenance, two additional DNA polymerases, Pol beta and PrimPol, were recently reported to localize to mitochondria as well as the nucleus. Similar to Polg, in vitro work has demonstrated both Pol beta and PrimPol incorporate NtRTIs into nascent DNA. Cell culture and biochemical experiments have also demonstrated that antiviral ribonucleoside drugs developed to treat hepatitis C infection act as off-target substrates for POLRMT, the mitochondrial RNA polymerase and primase. Accompanying the above-mentioned topics, this review examines: (1) mtDNA maintenance in human health and disease, (2) reports of DNA polymerases theta and zeta (Rev3) localizing to mitochondria, and (3) additional drugs with off-target effects on mitochondrial function. Lastly, mtDNA damage may induce cell death; therefore, the possibility of utilizing compounds that disrupt mtDNA maintenance to kill cancer cells is discussed. PMID:29214156

  3. High sensitive and direct fluorescence detection of single viral DNA sequences by integration of double strand probes onto microgels particles.

    PubMed

    Aliberti, A; Cusano, A M; Battista, E; Causa, F; Netti, P A

    2016-02-21

    A novel class of probes for fluorescence detection was developed and combined to microgel particles for a high sensitive fluorescence detection of nucleic acids. A double strand probe with an optimized fluorescent-quencher couple was designed for the detection of different lengths of nucleic acids (39 nt and 100 nt). Such probe proved efficient in target detection in different contests and specific even in presence of serum proteins. The conjugation of double strand probes onto polymeric microgels allows for a sensitive detection of DNA sequences from HIV, HCV and SARS corona viruses with a LOD of 1.4 fM, 3.7 fM and 1.4 fM, respectively, and with a dynamic range of 10(-9)-10(-15) M. Such combination enhances the sensitivity of the detection of almost five orders of magnitude when compared to the only probe. The proposed platform based on the integration of innovative double strand probe into microgels particles represents an attractive alternative to conventional sensitive DNA detection technologies that rely on amplifications methods.

  4. Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair

    DOE PAGES

    Andres, Sara N.; Appel, C. Denise; Westmoreland, James W.; ...

    2015-01-12

    Ctp1 (also known as CtIP or Sae2) collaborates with Mre11-Rad50-Nbs1 to initiate repair of DNA double-strand breaks (DSBs), but its functions remain enigmatic. In this paper, we report that tetrameric Schizosaccharomyces pombe Ctp1 contains multivalent DNA-binding and DNA-bridging activities. Through structural and biophysical analyses of the Ctp1 tetramer, we define the salient features of Ctp1 architecture: an N-terminal interlocking tetrameric helical dimer-of-dimers (THDD) domain and a central intrinsically disordered region (IDR) linked to C-terminal 'RHR' DNA-interaction motifs. The THDD, IDR and RHR are required for Ctp1 DNA-bridging activity in vitro, and both the THDD and RHR are required for efficientmore » DSB repair in S. pombe. Finally, our results establish non-nucleolytic roles of Ctp1 in binding and coordination of DSB-repair intermediates and suggest that ablation of human CtIP DNA binding by truncating mutations underlie the CtIP-linked Seckel and Jawad syndromes.« less

  5. Direct Comparison of Amino Acid and Salt Interactions with Double-Stranded and Single-Stranded DNA from Explicit-Solvent Molecular Dynamics Simulations.

    PubMed

    Andrews, Casey T; Campbell, Brady A; Elcock, Adrian H

    2017-04-11

    Given the ubiquitous nature of protein-DNA interactions, it is important to understand the interaction thermodynamics of individual amino acid side chains for DNA. One way to assess these preferences is to perform molecular dynamics (MD) simulations. Here we report MD simulations of 20 amino acid side chain analogs interacting simultaneously with both a 70-base-pair double-stranded DNA and with a 70-nucleotide single-stranded DNA. The relative preferences of the amino acid side chains for dsDNA and ssDNA match well with values deduced from crystallographic analyses of protein-DNA complexes. The estimated apparent free energies of interaction for ssDNA, on the other hand, correlate well with previous simulation values reported for interactions with isolated nucleobases, and with experimental values reported for interactions with guanosine. Comparisons of the interactions with dsDNA and ssDNA indicate that, with the exception of the positively charged side chains, all types of amino acid side chain interact more favorably with ssDNA, with intercalation of aromatic and aliphatic side chains being especially notable. Analysis of the data on a base-by-base basis indicates that positively charged side chains, as well as sodium ions, preferentially bind to cytosine in ssDNA, and that negatively charged side chains, and chloride ions, preferentially bind to guanine in ssDNA. These latter observations provide a novel explanation for the lower salt dependence of DNA duplex stability in GC-rich sequences relative to AT-rich sequences.

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

    PubMed

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

    2014-05-28

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

  7. Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction.

    PubMed

    Guo, Qiuping; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Li, Wei; Tang, Hongxing; Li, Huimin

    2009-02-01

    Here we have developed a sensitive DNA amplified detection method based on isothermal strand-displacement polymerization reaction. This method takes advantage of both the hybridization property of DNA and the strand-displacement property of polymerase. Importantly, we demonstrate that our method produces a circular polymerization reaction activated by the target, which essentially allows it to self-detect. Functionally, this DNA system consists of a hairpin fluorescence probe, a short primer and polymerase. Upon recognition and hybridization with the target ssDNA, the stem of the hairpin probe is opened, after which the opened probe anneals with the primer and triggers the polymerization reaction. During this process of the polymerization reaction, a complementary DNA is synthesized and the hybridized target is displaced. Finally, the displaced target recognizes and hybridizes with another probe, triggering the next round of polymerization reaction, reaching a target detection limit of 6.4 x 10(-15) M.

  8. Colorimetric detection of UV light-induced single-strand DNA breaks using gold nanoparticles.

    PubMed

    Kim, Joong Hyun; Chung, Chan Ho; Chung, Bong Hyun

    2013-02-21

    We developed a colorimetric method to specifically detect single-strand DNA breaks using gold nanoparticles. In our assay, broken DNA cannot stabilize gold nanoparticles to prevent salt-induced aggregation as good as intact DNA can, and this effect can be easily observed with the naked eye as a red-to-purple color change.

  9. Single-strand DNA binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs

    PubMed Central

    Pandita, Raj K.; Chow, Tracy T.; Udayakumar, Durga; Bain, Amanda L.; Cubeddu, Liza; Hunt, Clayton R.; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E.; Khanna, Kum Kum; Shay, Jerry W.; Pandita, Tej K.

    2015-01-01

    Proliferating mammalian stem and cancer cells express telomerase (TERT) in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA binding protein SSB1, which has a critical role in DNA double-strand break repair. Here we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacted with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduced TERT interaction with telomeres and lead to G-overhang loss. While SSB1 was recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relied upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. PMID:25589350

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

    PubMed

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

    2016-03-24

    For the first time, the thermodynamics are described for the formation of double-stranded DNA (ds-DNA)-single-walled carbon nanotube (SWNT) hybrids. This treatment is applied to the exchange reaction of sodium cholate (SC) molecules on SWNTs and the ds-DNAs d(A)20 -d(T)20 and nuclear factor (NF)-κB decoy. UV/Vis/near-IR spectroscopy with temperature variations was used for analyzing the exchange reaction on the SWNTs with four different chiralities: (n,m)=(8,3), (6,5), (7,5), and (8,6). Single-stranded DNAs (ss-DNAs), including d(A)20 and d(T)20, are also used for comparison. The d(A)20-d(T)20 shows a drastic change in its thermodynamic parameters around the melting temperature (Tm ) of the DNA oligomer. No such Tm dependency was measured, owing to high Tm in the NF-κB decoy DNA and no Tm in the ss-DNA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

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

  12. Topoisomerase VI senses and exploits both DNA crossings and bends to facilitate strand passage

    PubMed Central

    Wendorff, Timothy J

    2018-01-01

    Type II topoisomerases manage DNA supercoiling and aid chromosome segregation using a complex, ATP-dependent duplex strand passage mechanism. Type IIB topoisomerases and their homologs support both archaeal/plant viability and meiotic recombination. Topo VI, a prototypical type IIB topoisomerase, comprises two Top6A and two Top6B protomers; how these subunits cooperate to engage two DNA segments and link ATP turnover to DNA transport is poorly understood. Using multiple biochemical approaches, we show that Top6B, which harbors the ATPase activity of topo VI, recognizes and exploits the DNA crossings present in supercoiled DNA to stimulate subunit dimerization by ATP. Top6B self-association in turn induces extensive DNA bending, which is needed to support duplex cleavage by Top6A. Our observations explain how topo VI tightly coordinates DNA crossover recognition and ATP binding with strand scission, providing useful insights into the operation of type IIB topoisomerases and related meiotic recombination and GHKL ATPase machineries. PMID:29595473

  13. Nucleosomes Suppress the Formation of Double-strand DNA Breaks during Attempted Base Excision Repair of Clustered Oxidative Damages*

    PubMed Central

    Cannan, Wendy J.; Tsang, Betty P.; Wallace, Susan S.; Pederson, David S.

    2014-01-01

    Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. PMID:24891506

  14. Structural Reorganization and the Cooperative Binding of Single-stranded Telomere DNA in Sterkiella nova*

    PubMed Central

    Buczek, Pawel; Horvath, Martin P.

    2009-01-01

    In Sterkiella nova, α and β telomere proteins bind cooperatively with single-stranded DNA to form a ternary α·β·DNA complex. Association of telomere protein subunits is DNA-dependent, and α-β association enhances DNA affinity. To further understand the molecular basis for binding cooperativity, we characterized several possible stepwise assembly pathways using isothermal titration calorimetry. In one path, α and DNA first form a stable α·DNA complex followed by addition of β in a second step. Binding energy accumulates with nearly equal free energy of association for each of these steps. Heat capacity is nonetheless dramatically different with ΔCp = −305 ± 3 cal mol−1 K−1 for α binding with DNA and ΔCp = −2010 ± 20 cal mol−1 K−1 for addition of β to complete the α·β·DNA complex. By examining alternate routes including titration of single-stranded DNA with a preformed α·β complex, a significant portion of binding energy and heat capacity could be assigned to structural reorganization involving protein-protein interactions and repositioning of the DNA. Structural reorganization probably affords a mechanism to regulate high affinity binding of telomere single-stranded DNA with important implications for telomere biology. Regulation of telomere complex dissociation is thought to involve post-translational modifications in the lysine-rich C-terminal portion of β. We observed no difference in binding energetics or crystal structure when comparing complexes prepared with full-length β or a C-terminally truncated form, supporting interesting parallels between the intrinsically disordered regions of histones and this portion of β. PMID:17082188

  15. cgDNAweb: a web interface to the cgDNA sequence-dependent coarse-grain model of double-stranded DNA.

    PubMed

    De Bruin, Lennart; Maddocks, John H

    2018-06-14

    The sequence-dependent statistical mechanical properties of fragments of double-stranded DNA is believed to be pertinent to its biological function at length scales from a few base pairs (or bp) to a few hundreds of bp, e.g. indirect read-out protein binding sites, nucleosome positioning sequences, phased A-tracts, etc. In turn, the equilibrium statistical mechanics behaviour of DNA depends upon its ground state configuration, or minimum free energy shape, as well as on its fluctuations as governed by its stiffness (in an appropriate sense). We here present cgDNAweb, which provides browser-based interactive visualization of the sequence-dependent ground states of double-stranded DNA molecules, as predicted by the underlying cgDNA coarse-grain rigid-base model of fragments with arbitrary sequence. The cgDNAweb interface is specifically designed to facilitate comparison between ground state shapes of different sequences. The server is freely available at cgDNAweb.epfl.ch with no login requirement.

  16. DNA repair targeted therapy: the past or future of cancer treatment?

    PubMed Central

    Gavande, Navnath S.; VanderVere-Carozza, Pamela S.; Hinshaw, Hilary D.; Jalal, Shadia I.; Sears, Catherine R.; Pawelczak, Katherine S.; Turchi, John J.

    2016-01-01

    The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small, modest changes in structure including mismatched bases and simple methylation events to oxidized bases, intra- and interstrand DNA crosslinks, DNA double strand breaks and protein-DNA adducts. Pathways required for the repair of these lesions include mismatch repair, base excision repair, nucleotide excision repair, and the homology directed repair/Fanconi anemia pathway. Each of these pathways contributes to genetic stability, and mutations in genes encoding proteins involved in these pathways have been demonstrated to promote genetic instability and cancer. In fact, it has been suggested all cancers display defects in DNA repair. It has also been demonstrated that the ability of cancer cells to repair therapeutically induced DNA damage impacts therapeutic efficacy. This has led to targeting DNA repair pathways and proteins to develop anti-cancer agents that will increase sensitivity to traditional chemotherapeutics. While initial studies languished and were plagued by a lack of specificity and a defined mechanism of action, more recent approaches to exploit synthetic lethal interaction and develop high affinity chemical inhibitors have proven considerably more effective. In this review we will highlight recent advances and discuss previous failures in targeting DNA repair to pave the way for future DNA repair targeted agents and their use in cancer therapy. PMID:26896565

  17. RNA from the 5' end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site.

    PubMed

    Christensen, Shawn M; Ye, Junqiang; Eickbush, Thomas H

    2006-11-21

    Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the element's RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5' end of the R2 element. In the absence of this 5' RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3' untranslated region of the R2 transcript is present. In the presence of the 320-nt 5' RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5' RNA is removed from this subunit. We postulate that the removal of the 5' RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

  18. 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.

  19. A homogeneous nucleic acid hybridization assay based on strand displacement.

    PubMed Central

    Vary, C P

    1987-01-01

    A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement. Images PMID:3309890

  20. DNA strand displacement reaction for programmable release of biomolecules.

    PubMed

    Ramezani, Hamid; Jed Harrison, D

    2015-05-14

    Sample cleanup is a major processing step in many analytical assays. Here, we propose an approach to capture-and-release of analytes based on the DNA strand displacement reaction (SDR) and demonstrate its application to a fluoroimmunoassay on beads for a thyroid cancer biomarker, thyroglobulin. The SDR-based cleanup showed no interference from matrix molecules in serum.

  1. Discrimination of three types of homopolymers in single-stranded DNA with solid-state nanopores through external control of the DNA motion.

    PubMed

    Akahori, Rena; Yanagi, Itaru; Goto, Yusuke; Harada, Kunio; Yokoi, Takahide; Takeda, Ken-Ichi

    2017-08-22

    To achieve DNA sequencing with solid-state nanopores, the speed of the DNA in the nanopore must be controlled to obtain sequence-specific signals. In this study, we fabricated a nanopore-sensing system equipped with a DNA motion controller. DNA strands were immobilized on a Si probe, and approach of this probe to the nanopore vicinity could be controlled using a piezo actuator and stepper motor. The area of the Si probe was larger than the area of the membrane, which meant that the immobilized DNA could enter the nanopore without the need for the probe to scan to determine the location of the nanopore in the membrane. We demonstrated that a single-stranded DNA could be inserted into and removed from a nanopore in our experimental system. The number of different ionic-current levels observed while DNA remained in the nanopore corresponded to the number of different types of homopolymers in the DNA.

  2. Two-Tailed Comet Assay (2T-Comet): Simultaneous Detection of DNA Single and Double Strand Breaks.

    PubMed

    Cortés-Gutiérrez, Elva I; Fernández, José Luis; Dávila-Rodríguez, Martha I; López-Fernández, Carmen; Gosálvez, Jaime

    2017-01-01

    A modification of the original comet assay was developed for the simultaneous evaluation of DNA single strand breaks (SSBs) and double strand breaks (DSBs) in human spermatozoa. The two-dimensional perpendicular tail comet assay (2T-comet) combines non-denaturing and denaturant conditions to the same sperm nucleoid. In this case, the species-specific deproteinized sperm is first subjected to an electrophoretic field under non-denaturing conditions to mobilize isolated free discrete DNA fragments produced from DSBs; this is then followed by a second electrophoresis running perpendicular to the first one but under alkaline conditions to produce DNA denaturation, exposing SSBs on the same linear DNA chain or DNA fragments flanked by DSBs. This procedure results in a two dimensional comet tail emerging from the core where two types of original DNA affected molecule can be simultaneously discriminated. The 2T-comet is a fast, sensitive, and reliable procedure to distinguish between single and double strand DNA damage within the same cell. It is an innovative method for assessing sperm DNA integrity, which has important implications for human fertility and andrological pathology. This technique may be adapted to assess different DNA break types in other species and other cell types.

  3. TH-CD-201-11: Optimizing the Response and Cost of a DNA Double-Strand Break Dosimeter

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Obeidat, M; Cline, K; Stathakis, S

    Purpose: A DNA double-strand break (DSB) dosimeter was developed to measure the biological effect of radiation. The goal here is to refine the fabrication method of this dosimeter to reproducibly create a low coefficient of variation (CoV) and reduce the cost for the dosimeter. Methods: Our dosimeter consists of 4 kilo-base pair DNA strands (labeled on one end with biotin and on the other with fluorescein) attached to streptavidin magnetic beads. The final step of the DNA dosimeter fabrication is to suspend these attached beads in phosphate-buffered saline (PBS). The amount of PBS used to suspend the attached beads andmore » the relative volume of the DNA strands to the beads both affect the CoV and dosimeter cost. We diluted the beads attached with DNA in different volumes of PBS (100, 200, and 400 µL) to create different concentrations of the DNA dosimeter. Then we irradiated these dosimeters (50 µL samples) in a water-equivalent plastic phantom at 25 and 50 Gy (three samples per dose) and calculated the CoV for each dosimeter concentration. Also, we used different masses of DNA strands (1, 2, 8, 16, 24, and 32 µg) to attach to the same volume of magnetic beads (100 µL) to explore how this affects the cost of the dosimeter. Results: The lowest CoV was produced for the highest concentration of dosimeter (100 µL of PBS), which created CoV of 2.0 and 1.0% for 25 and 50 Gy, respectively. We found that the lowest production cost for the dosimeter occurs by attaching 16 µg of DNA strands with 100 µL of beads. Conclusion: : We optimized the fabrication of the DNA dosimeter to produce low CoV and cost, but we still need to explore ways to further improve the dosimeter for use at lower doses. This work was supported in part by Yarmouk University (Irbid, Jordan) and CPRIT (RP140105)« less

  4. Zinc Chromate Induces Chromosome Instability and DNA Double Strand Breaks in Human Lung Cells

    PubMed Central

    Xie, Hong; Holmes, Amie L.; Young, Jamie L.; Qin, Qin; Joyce, Kellie; Pelsue, Stephen C.; Peng, Cheng; Wise, Sandra S.; Jeevarajan, Antony S.; Wallace, William T.; Hammond, Dianne; Wise, John Pierce

    2014-01-01

    Hexavalent chromium Cr(VI) is a respiratory toxicant and carcinogen, with solubility playing an important role in its carcinogenic potential. Zinc chromate, a water insoluble or ‘particulate’ Cr(VI) compound, has been shown to be carcinogenic in epidemiology studies and to induce tumors in experimental animals, but its genotoxicity is poorly understood. Our study shows that zinc chromate induced concentration-dependent increases in cytotoxicity, chromosome damage and DNA double strand breaks in human lung cells. In response to zinc chromate-induced breaks, MRE11 expression was increased and ATM and ATR were phosphorylated, indicating that the DNA double strand break repair system was initiated in the cells. In addition, our data show that zinc chromate-induced double strand breaks were only observed in the G2/M phase population, with no significant amount of double strand breaks observed in G1 and S phase cells. These data will aid in understanding the mechanisms of zinc chromate toxicity and carcinogenesis. PMID:19027772

  5. Dewar Lesion Formation in Single- and Double-Stranded DNA is Quenched by Neighboring Bases.

    PubMed

    Bucher, Dominik B; Pilles, Bert M; Carell, Thomas; Zinth, Wolfgang

    2015-07-16

    UV-induced Dewar lesion formation is investigated in single- and double-stranded oligonucleotides with ultrafast vibrational spectroscopy. The quantum yield for the conversion of the (6-4) lesion to the Dewar isomer in DNA strands is reduced by a factor of 4 in comparison to model dinucleotides. Time resolved spectroscopy reveals a fast process in the excited state with spectral characteristics of bases which are adjacent to the excited (6-4) lesion. These kinetic components have large amplitudes and indicate that an additional quenching channel acts in the stranded DNA systems and reduces the Dewar formation yield. Presumably relaxation evolves via a charge transfer to the neighboring guanine and the paired cytosine participates in a double-stranded oligomer. Changes in the decay of the relaxed excited electronic state of the (6-4) chromophore point to modifications in the excited state energy landscape which may lead to an additional reduction of the Dewar formation yield.

  6. Dietary choline deficiency causes DNA strand breaks and alters epigenetic marks on DNA and histones.

    PubMed

    Zeisel, Steven H

    2012-05-01

    Dietary choline is an important modulator of gene expression (via epigenetic marks) and of DNA integrity. Choline was discovered to be an essential nutrient for some humans approximately one decade ago. This requirement is diminished in young women because estrogen drives endogenous synthesis of phosphatidylcholine, from which choline can be derived. Almost half of women have a single nucleotide polymorphism that abrogates estrogen-induction of endogenous synthesis, and these women require dietary choline just as do men. In the US, dietary intake of choline is marginal. Choline deficiency in people is associated with liver and muscle dysfunction and damage, with apoptosis, and with increased DNA strand breaks. Several mechanisms explain these modifications to DNA. Choline deficiency increases leakage of reactive oxygen species from mitochondria consequent to altered mitochondrial membrane composition and enhanced fatty acid oxidation. Choline deficiency impairs folate metabolism, resulting in decreased thymidylate synthesis and increased uracil misincorporation into DNA, with strand breaks resulting during error-prone repair attempts. Choline deficiency alters DNA methylation, which alters gene expression for critical genes involved in DNA mismatch repair, resulting in increased mutation rates. Any dietary deficiency which increases mutation rates should be associated with increased risk of cancers, and this is the case for choline deficiency. In rodent models, diets low in choline and methyl-groups result in spontaneous hepatocarcinomas. In human epidemiological studies, there are interesting data that suggest that this also may be the case for humans, especially those with SNPs that increase the dietary requirement for choline. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Dietary Choline Deficiency causes DNA Strand Breaks and Alters Epigenetic Marks on DNA and Histones

    PubMed Central

    Zeisel, Steven H.

    2011-01-01

    Dietary choline is an important modulator of gene expression (via epigenetic marks) and of DNA integrity. Choline was discovered to be an essential nutrient for some humans approximately one decade ago. This requirement is diminished in young women because estrogen drives endogenous synthesis of phosphatidylcholine, from which choline can be derived. Almost half of women have a single nucleotide polymorphism that abrogates estrogen-induction of endogenous synthesis, and these women require dietary choline just as do men. In the US, dietary intake of choline is marginal. Choline deficiency in people is associated with liver and muscle dysfunction and damage, with apoptosis, and with increased DNA strand breaks. Several mechanisms explain these modifications to DNA. Choline deficiency increases leakage of reactive oxygen species from mitochondria consequent to altered mitochondrial membrane composition and enhanced fatty acid oxidation. Choline deficiency impairs folate metabolism, resulting in decreased thymidylate synthesis and increased uracil misincorporation into DNA, with strand breaks resulting during error-prone repair attempts. Choline deficiency alters DNA methylation, which alters gene expression for critical genes involved in DNA mismatch repair, resulting in increased mutation rates. Any dietary deficiency which increases mutation rates should be associated with increased risk of cancers, and this is the case for choline deficiency. In rodent models, diets low in choline and methyl-groups result in spontaneous hepatocarcinomas. In human epidemiological studies, there are interesting data that suggest that this also may be the case for humans, especially those with SNPs that increase the dietary requirement for choline. PMID:22041500

  8. How a short double-stranded DNA bends

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  9. Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks

    PubMed Central

    Belotserkovskii, Boris P.; Neil, Alexander J.; Saleh, Syed Shayon; Shin, Jane Hae Soo; Mirkin, Sergei M.; Hanawalt, Philip C.

    2013-01-01

    The ability of DNA to adopt non-canonical structures can affect transcription and has broad implications for genome functioning. We have recently reported that guanine-rich (G-rich) homopurine-homopyrimidine sequences cause significant blockage of transcription in vitro in a strictly orientation-dependent manner: when the G-rich strand serves as the non-template strand [Belotserkovskii et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences., Proc. Natl Acad. Sci. USA, 107, 12816–12821]. We have now systematically studied the effect of the sequence composition and single-stranded breaks on this blockage. Although substitution of guanine by any other base reduced the blockage, cytosine and thymine reduced the blockage more significantly than adenine substitutions, affirming the importance of both G-richness and the homopurine-homopyrimidine character of the sequence for this effect. A single-strand break in the non-template strand adjacent to the G-rich stretch dramatically increased the blockage. Breaks in the non-template strand result in much weaker blockage signals extending downstream from the break even in the absence of the G-rich stretch. Our combined data support the notion that transcription blockage at homopurine-homopyrimidine sequences is caused by R-loop formation. PMID:23275544

  10. The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein.

    PubMed

    Gabus, C; Auxilien, S; Péchoux, C; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W; Nandi, P; Darlix, J L

    2001-04-06

    The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand transfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo. Copyright 2001 Academic Press.

  11. Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.

    PubMed

    Scalabrin, Matteo; Quintieri, Luigi; Palumbo, Manlio; Riccardi Sirtori, Federico; Gatto, Barbara

    2017-02-20

    The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.

  12. Correlated Template-Switching Events during Minus-Strand DNA Synthesis: a Mechanism for High Negative Interference during Retroviral Recombination

    PubMed Central

    Anderson, Jeffrey A.; Teufel, Ronald J.; Yin, Philip D.; Hu, Wei-Shau

    1998-01-01

    Two models for the mechanism of retroviral recombination have been proposed: forced copy choice (minus-strand recombination) and strand displacement-assimilation (plus-strand recombination). Each minus-strand recombination event results in one template switch, whereas each plus-strand recombination event results in two template switches. Recombinant proviruses with one and more than one template switches were previously observed. Recombinants with one template switch were generated by minus-strand recombination, while recombinants containing more than one template switch may have been generated by plus-strand recombination or by correlated minus-strand recombination. We recently observed that retroviral recombination exhibits high negative interference whereby the frequency of recombinants containing multiple template-switching events is higher than expected. To delineate the mechanism that generates recombinants with more than one template switch, we devised a system that permits only minus-strand recombination. Two highly homologous vectors, WH204 and WH221, containing eight different restriction site markers were used. The primer binding site (PBS) of WH221 was deleted; although reverse transcription cannot initiate from WH221 RNA, it can serve as a template for DNA synthesis in heterozygotic virions. After one round of retroviral replication, the structures of the recombinant proviruses were examined. Recombinants containing two, three, four, and five template switches were observed at 1.4-, 10-, 65-, and 50-fold-higher frequencies, respectively, than expected. This indicates that minus-strand recombination events are correlated and can generate proviruses with multiple template switches efficiently. The frequencies of recombinants containing multiple template switches were similar to those observed in the previous system, which allowed both minus- and plus-strand recombination. Thus, the previously reported high negative interference during retroviral

  13. Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA.

    PubMed

    Piette, J; Calberg-Bacq, C M; Lopez, M; van de Vorst, A

    1984-04-05

    Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.

  14. Defective double-strand DNA break repair and chromosomal translocations by MYC overexpression.

    PubMed

    Karlsson, Asa; Deb-Basu, Debabrita; Cherry, Athena; Turner, Stephanie; Ford, James; Felsher, Dean W

    2003-08-19

    DNA repair mechanisms are essential for the maintenance of genomic integrity. Disruption of gene products responsible for DNA repair can result in chromosomal damage. Improperly repaired chromosomal damage can result in the loss of chromosomes or the generation of chromosomal deletions or translocations, which can lead to tumorigenesis. The MYC protooncogene is a transcription factor whose overexpression is frequently associated with human neoplasia. MYC has not been previously implicated in a role in DNA repair. Here we report that the overexpression of MYC disrupts the repair of double-strand DNA breaks, resulting in a several-magnitude increase in chromosomal breaks and translocations. We found that MYC inhibited the repair of gamma irradiation DNA breaks in normal human cells and blocked the repair of a single double-strand break engineered to occur in an immortal cell line. By spectral karyotypic analysis, we found that MYC even within one cell division cycle resulted in a several-magnitude increase in the frequency of chromosomal breaks and translocations in normal human cells. Hence, MYC overexpression may be a previously undescribed example of a dominant mutator that may fuel tumorigenesis by inducing chromosomal damage.

  15. Nucleosomes suppress the formation of double-strand DNA breaks during attempted base excision repair of clustered oxidative damages.

    PubMed

    Cannan, Wendy J; Tsang, Betty P; Wallace, Susan S; Pederson, David S

    2014-07-18

    Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. In-stem labelling allows visualization of DNA strand displacements by distinct fluorescent colour change.

    PubMed

    Barrois, Sebastian; Wagenknecht, Hans-Achim

    2013-05-21

    The combination of thiazole orange (TO) and thiazole red (TR) as an internal pair of fluorescent DNA base surrogates ("DNA traffic lights") allows us to follow at least two consecutive DNA strand displacements in real time through a distinct fluorescence colour change from green to red and vice versa.

  17. Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction

    NASA Astrophysics Data System (ADS)

    Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

    2014-01-01

    DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag+-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science.

  18. Target-Catalyzed DNA Four-Way Junctions for CRET Imaging of MicroRNA, Concatenated Logic Operations, and Self-Assembly of DNA Nanohydrogels for Targeted Drug Delivery.

    PubMed

    Bi, Sai; Xiu, Bao; Ye, Jiayan; Dong, Ying

    2015-10-21

    Here we report a target-catalyzed DNA four-way junction (DNA-4WJ) on the basis of toehold-mediated DNA strand displacement reaction (TM-SDR), which is readily applied in enzyme-free amplified chemiluminescence resonance energy transfer (CRET) imaging of microRNA. In this system, the introduction of target microRNA-let-7a (miR-let-7a) activates a cascade of assembly steps with four DNA hairpins, followed by a disassembly step in which the target microRNA is displaced and released from DNA-4WJ to catalyze the self-assembly of additional branched junctions. As a result, G-quadruplex subunit sequences and fluorophore fluorescein amidite (FAM) are encoded in DNA-4WJ in a close proximity, stimulating a CRET process in the presence of hemin/K(+) to form horseradish peroxidase (HRP)-mimicking DNAzyme that catalyzes the generation of luminol/H2O2 chemiluminescence (CL), which further transfers to FAM. The background signal is easily reduced using magnetic graphene oxide (MGO) to remove unreacted species through magnetic separation, which makes a great contribution to improve the detection sensitivity and achieves a detection limit as low as 6.9 fM microRNA-let-7a (miR-let-7a). In addition, four-input concatenated logic circuits with an automatic reset function have been successfully constructed relying on the architecture of the proposed DNA-4WJ. More importantly, DNA nanohydrogels are self-assembled using DNA-4WJs as building units after centrifugation, which are driven by liquid crystallization and dense packaging of building units. Moreover, the DNA nanohydrogels are readily functionalized by incorporating with aptamers, bioimaging agents, and drug loading sites, which thus are served as efficient nanocarriers for targeted drug delivery and cancer therapy with high loading capacity and excellent biocompatibility.

  19. DNA Meter: Energy Tunable, Quantitative Hybridization Assay

    PubMed Central

    Braunlin, William; Völker, Jens; Plum, G. Eric; Breslauer, Kenneth J.

    2015-01-01

    We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop-containing competitor strands (C*) that hybridize to a probe strand (P). Such initial “pre-binding” of a probe strand modulates its effective “availability” for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T). We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a “tuning fork,” since the C* strand exhibits branch points (forks) at the duplex-bulge interfaces within the complex. By varying the loop to create families of such “tuning forks,” one can construct C*P “energy ladders” capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a “DNA Meter.” Here we present data that establish proof-of-principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity. PMID:23529692

  20. Targeting Extracellular DNA to Deliver IGF-1 to the Injured Heart

    NASA Astrophysics Data System (ADS)

    Khan, Raffay S.; Martinez, Mario D.; Sy, Jay C.; Pendergrass, Karl D.; Che, Pao-Lin; Brown, Milton E.; Cabigas, E. Bernadette; Dasari, Madhuri; Murthy, Niren; Davis, Michael E.

    2014-03-01

    There is a great need for the development of therapeutic strategies that can target biomolecules to damaged myocardium. Necrosis of myocardium during a myocardial infarction (MI) is characterized by extracellular release of DNA, which can serve as a potential target for ischemic tissue. Hoechst, a histological stain that binds to double-stranded DNA can be conjugated to a variety of molecules. Insulin-like growth factor-1 (IGF-1), a small protein/polypeptide with a short circulating-half life is cardioprotective following MI but its clinical use is limited by poor delivery, as intra-myocardial injections have poor retention and chronic systemic presence has adverse side effects. Here, we present a novel delivery vehicle for IGF-1, via its conjugation to Hoechst for targeting infarcted tissue. Using a mouse model of ischemia-reperfusion, we demonstrate that intravenous delivery of Hoechst-IGF-1 results in activation of Akt, a downstream target of IGF-1 and protects from cardiac fibrosis and dysfunction following MI.

  1. Single-strand DNA-binding protein SSB1 facilitates TERT recruitment to telomeres and maintains telomere G-overhangs.

    PubMed

    Pandita, Raj K; Chow, Tracy T; Udayakumar, Durga; Bain, Amanda L; Cubeddu, Liza; Hunt, Clayton R; Shi, Wei; Horikoshi, Nobuo; Zhao, Yong; Wright, Woodring E; Khanna, Kum Kum; Shay, Jerry W; Pandita, Tej K

    2015-03-01

    Proliferating mammalian stem and cancer cells express telomerase [telomerase reverse transcriptase (TERT)] in an effort to extend chromosomal G-overhangs and maintain telomere ends. Telomerase-expressing cells also have higher levels of the single-stranded DNA-binding protein SSB1, which has a critical role in DNA double-strand break (DSB) repair. Here, we report that SSB1 binds specifically to G-strand telomeric DNA in vitro and associates with telomeres in vivo. SSB1 interacts with the TERT catalytic subunit and regulates its interaction with telomeres. Deletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss. Although SSB1 is recruited to DSB sites, we found no corresponding change in TERT levels at these sites, implying that SSB1-TERT interaction relies upon a specific chromatin structure or context. Our findings offer an explanation for how telomerase is recruited to telomeres to facilitate G-strand DNA extension, a critical step in maintaining telomere ends and cell viability in all cancer cells. Cancer Res; 75(5); 858-69. ©2015 AACR. ©2015 American Association for Cancer Research.

  2. Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

    PubMed

    Kumar, S; Peng, X; Daley, J; Yang, L; Shen, J; Nguyen, N; Bae, G; Niu, H; Peng, Y; Hsieh, H-J; Wang, L; Rao, C; Stephan, C C; Sung, P; Ira, G; Peng, G

    2017-04-17

    Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting

  3. Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

    NASA Technical Reports Server (NTRS)

    Winters, T. A.; Russell, P. S.; Kohli, M.; Dar, M. E.; Neumann, R. D.; Jorgensen, T. J.

    1999-01-01

    Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

  4. Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.

    PubMed

    Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui; Falconieri, Veronica; Rao, Prashant; Merk, Alan; Eng, Edward T; Raczkowski, Ashleigh M; Fox, Tara; Earl, Lesley A; Patel, Dinshaw J; Subramaniam, Sriram

    2017-10-05

    Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition. Published by Elsevier Inc.

  5. RNA polymerase gate loop guides the nontemplate DNA strand in transcription complexes.

    PubMed

    NandyMazumdar, Monali; Nedialkov, Yuri; Svetlov, Dmitri; Sevostyanova, Anastasia; Belogurov, Georgiy A; Artsimovitch, Irina

    2016-12-27

    Upon RNA polymerase (RNAP) binding to a promoter, the σ factor initiates DNA strand separation and captures the melted nontemplate DNA, whereas the core enzyme establishes interactions with the duplex DNA in front of the active site that stabilize initiation complexes and persist throughout elongation. Among many core RNAP elements that participate in these interactions, the β' clamp domain plays the most prominent role. In this work, we investigate the role of the β gate loop, a conserved and essential structural element that lies across the DNA channel from the clamp, in transcription regulation. The gate loop was proposed to control DNA loading during initiation and to interact with NusG-like proteins to lock RNAP in a closed, processive state during elongation. We show that the removal of the gate loop has large effects on promoter complexes, trapping an unstable intermediate in which the RNAP contacts with the nontemplate strand discriminator region and the downstream duplex DNA are not yet fully established. We find that although RNAP lacking the gate loop displays moderate defects in pausing, transcript cleavage, and termination, it is fully responsive to the transcription elongation factor NusG. Together with the structural data, our results support a model in which the gate loop, acting in concert with initiation or elongation factors, guides the nontemplate DNA in transcription complexes, thereby modulating their regulatory properties.

  6. 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.

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

    NASA Technical Reports Server (NTRS)

    Kohli, M.; Jorgensen, T. J.

    1999-01-01

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

  8. Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

    PubMed Central

    Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

    2013-01-01

    Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

  9. Spatial distribution and yield of DNA double-strand breaks induced by 3-7 MeV helium ions in human fibroblasts

    NASA Technical Reports Server (NTRS)

    Rydberg, Bjorn; Heilbronn, Lawrence; Holley, William R.; Lobrich, Markus; Zeitlin, Cary; Chatterjee, Aloke; Cooper, Priscilla K.

    2002-01-01

    Accelerated helium ions with mean energies at the target location of 3-7 MeV were used to simulate alpha-particle radiation from radon daughters. The experimental setup and calibration procedure allowed determination of the helium-ion energy distribution and dose in the nuclei of irradiated cells. Using this system, the induction of DNA double-strand breaks and their spatial distributions along DNA were studied in irradiated human fibroblasts. It was found that the apparent number of double-strand breaks as measured by a standard pulsed-field gel assay (FAR assay) decreased with increasing LET in the range 67-120 keV/microm (corresponding to the energy of 7-3 MeV). On the other hand, the generation of small and intermediate-size DNA fragments (0.1-100 kbp) increased with LET, indicating an increased intratrack long-range clustering of breaks. The fragment size distribution was measured in several size classes down to the smallest class of 0.1-2 kbp. When the clustering was taken into account, the actual number of DNA double-strand breaks (separated by at least 0.1 kbp) could be calculated and was found to be in the range 0.010-0.012 breaks/Mbp Gy(-1). This is two- to threefold higher than the apparent yield obtained by the FAR assay. The measured yield of double-strand breaks as a function of LET is compared with theoretical Monte Carlo calculations that simulate the track structure of energy depositions from helium ions as they interact with the 30-nm chromatin fiber. When the calculation is performed to include fragments larger than 0.1 kbp (to correspond to the experimental measurements), there is good agreement between experiment and theory.

  10. 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 Central

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

    2013-01-01

    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. PMID:23430749

  11. Signal-on fluorescence biosensor for microRNA-21 detection based on DNA strand displacement reaction and Mg2+-dependent DNAzyme cleavage.

    PubMed

    Yin, Huan-Shun; Li, Bing-Chen; Zhou, Yun-Lei; Wang, Hai-Yan; Wang, Ming-Hui; Ai, Shi-Yun

    2017-10-15

    MicroRNAs have been involved into many biological processes and are regarded as disease biomarkers. Simple, rapid, sensitive and selective method for microRNA detection is crucial for early diagnosis and therapy of diseases. In this work, sensitive fluorescence assay was developed for microRNA-21 detection based on DNA polymerase induced strand displacement amplification reaction, Mg 2+ -dependent DNAzyme catalysis reaction, and magnetic separation. In the presence of target microRNA-21, amounts of trigger DNA could be produced with DNA polymerase induced strand displacement amplification reaction, and the trigger DNA could be further hybridized with signal DNA, which was labeled with biotin and AMCA dye. After introduction of Mg 2+ , trigger DNA could form DNAzyme to cleave signal DNA. After magnetic separation, the DNA fragment with AMCA dye could give fluorescence signal, which was related to microRNA-21 concentration. Based on the two efficient signal amplifications, the developed method showed high detection sensitivity with low detection limit of 0.27fM (3σ). In addition, this fluorescence strategy also possessed excellent detection specificity, and could be applied to analyze microRNA-21 expression level in serum of cancer patient. According to the obtained results, the developed fluorescence method might be a promising detection platform for microRNA-21 quantitative analysis in biomedical research and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Kinetic and thermodynamic hysteresis imposed by intercalation of proflavine in ferrocene-modified double-stranded DNA.

    PubMed

    Gebala, Magdalena; La Mantia, Fabio; Schuhmann, Wolfgang

    2013-07-22

    Surface-confined immobilized redox species often do not show the expected zero peak separation in slow-scan cyclic voltammograms. This phenomenon is frequently associated to experimental drawbacks and hence neglected. However, a nonzero peak separation, which is common to many electrochemical systems with high structural flexibility, can be rationally assigned to a thermodynamic hysteresis. To study this phenomenon, a surface-confined redox species was used. Specifically, a DNA strand which is tagged with ferrocene (Fc) moieties at its 5' end and its complementary capture probe is thiolated at the 3' end was self-assembled in a monolayer at a Au electrode with the Fc moieties being located at the bottom plane of the double-stranded DNA (dsDNA). The DNA-bound Fc undergoes rapid electron transfer with the electrode surface as evaluated by fast scan cyclic voltammetry. The electron transfer is sensitive to the ion transport along the DNA strands, a phenomenon which is modulated upon specific intercalation of proflavine into surface-bound dsDNA. The electron transfer rate of the Fc(0/+) redox process is influenced by the cationic permselectivity of the DNA monolayer. In addition to the kinetic hindrance, a thermodynamic effect correlated with changes in the activity coefficients of the Fc(0/+) moieties near the gold-dsDNA interface is observed and discussed as source of the observed hysteresis causing the non-zero peak separation in the voltammograms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Crystal structure of a four-stranded intercalated DNA: d(C4)

    NASA Technical Reports Server (NTRS)

    Chen, L.; Cai, L.; Zhang, X.; Rich, A.

    1994-01-01

    The crystal structure of d(C4) solved at 2.3-A resolution reveals a four-stranded molecule composed of two interdigitated or intercalated duplexes. The duplexes are held together by hemiprotonated cytosine-cytosine base pairs and are parallel stranded, but the two duplexes point in opposite directions. The molecule has a slow right-handed twist of 12.4 degrees between covalently linked cytosine base pairs, and the base stacking distance is 3.1 A. This is in general agreement with the NMR studies. A biological role for DNA in this conformation is suggested.

  14. Single-cell template strand sequencing by Strand-seq enables the characterization of individual homologs.

    PubMed

    Sanders, Ashley D; Falconer, Ester; Hills, Mark; Spierings, Diana C J; Lansdorp, Peter M

    2017-06-01

    The ability to distinguish between genome sequences of homologous chromosomes in single cells is important for studies of copy-neutral genomic rearrangements (such as inversions and translocations), building chromosome-length haplotypes, refining genome assemblies, mapping sister chromatid exchange events and exploring cellular heterogeneity. Strand-seq is a single-cell sequencing technology that resolves the individual homologs within a cell by restricting sequence analysis to the DNA template strands used during DNA replication. This protocol, which takes up to 4 d to complete, relies on the directionality of DNA, in which each single strand of a DNA molecule is distinguished based on its 5'-3' orientation. Culturing cells in a thymidine analog for one round of cell division labels nascent DNA strands, allowing for their selective removal during genomic library construction. To preserve directionality of template strands, genomic preamplification is bypassed and labeled nascent strands are nicked and not amplified during library preparation. Each single-cell library is multiplexed for pooling and sequencing, and the resulting sequence data are aligned, mapping to either the minus or plus strand of the reference genome, to assign template strand states for each chromosome in the cell. The major adaptations to conventional single-cell sequencing protocols include harvesting of daughter cells after a single round of BrdU incorporation, bypassing of whole-genome amplification, and removal of the BrdU + strand during Strand-seq library preparation. By sequencing just template strands, the structure and identity of each homolog are preserved.

  15. Construction of a microfluidic chip, using dried-down reagents, for LATE-PCR amplification and detection of single-stranded DNA.

    PubMed

    Jia, Yanwei; Mak, Pui-In; Massey, Conner; Martins, Rui P; Wangh, Lawrence J

    2013-12-07

    LATE-PCR is an advanced form of non-symmetric PCR that efficiently generates single-stranded DNA which can readily be characterized at the end of amplification by hybridization to low-temperature fluorescent probes. We demonstrate here for the first time that monoplex and duplex LATE-PCR amplification and probe target hybridization can be carried out in double layered PDMS microfluidics chips containing dried reagents. Addition of a set of reagents during dry down overcomes the common problem of single-stranded oligonucleotide binding to PDMS. These proof-of-principle results open the way to construction of inexpensive point-of-care devices that take full advantage of the analytical power of assays built using LATE-PCR and low-temperature probes.

  16. Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

    PubMed

    Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

    2015-03-31

    Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

  17. Detection of DNA damage based on metal-mediated molecular beacon and DNA strands displacement reaction.

    PubMed

    Xiong, Yanxiang; Wei, Min; Wei, Wei; Yin, Lihong; Pu, Yuepu; Liu, Songqin

    2014-01-24

    DNA hairpin structure probes are usually designed by forming intra-molecular duplex based on Watson-Crick hydrogen bonds. In this paper, a molecular beacon based on silver ions-mediated cytosine-Ag(+)-cytosine base pairs was used to detect DNA. The inherent characteristic of the metal ligation facilitated the design of functional probe and the adjustment of its binding strength compared to traditional DNA hairpin structure probes, which make it be used to detect DNA in a simple, rapid and easy way with the help of DNA strands displacement reaction. The method was sensitive and also possesses the good specificity to differentiate the single base mismatched DNA from the complementary DNA. It was also successfully applied to study the damage effect of classic genotoxicity chemicals such as styrene oxide and sodium arsenite on DNA, which was significant in food science, environmental science and pharmaceutical science. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, Huamin; Smith, Lloyd M.

    1997-01-01

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support.

  19. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, H.; Smith, L.M.

    1997-01-07

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support. 3 figs.

  20. Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.

    PubMed

    Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

    2016-06-21

    Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson-Crick base-pairing even in the 3'-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5' base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.

  1. 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.

  2. Detection of Strand Cleavage And Oxidation Damage Using Model DNA Molecules Captured in a Nanoscale Pore

    NASA Technical Reports Server (NTRS)

    Vercoutere, W.; Solbrig, A.; DeGuzman, V.; Deamer, D.; Akeson, M.

    2003-01-01

    We use a biological nano-scale pore to distinguish among individual DNA hairpins that differ by a single site of oxidation or a nick in the sugar-phosphate backbone. In earlier work we showed that the protein ion channel alpha-hemolysin can be used as a detector to distinguish single-stranded from double-stranded DNA, single base pair and single nucleotide differences. This resolution is in part a result of sensitivity to structural changes that influence the molecular dynamics of nucleotides within DNA. The strand cleavage products we examined here included a 5-base-pair (5-bp) hairpin with a 5-prime five-nucleotide overhang, and a complementary five-nucleotide oligomer. These produced predictable shoulder-spike and rapid near-full blockade signatures, respectively. When combined, strand annealing was monitored in real time. The residual current level dropped to a lower discrete level in the shoulder-spike blockade signatures, and the duration lengthened. However, these blockade signatures had a shorter duration than the unmodified l0bp hairpin. To test the pore sensitivity to nucleotide oxidation, we examined a 9-bp hairpin with a terminal 8-oxo-deoxyguanosine (8-oxo-dG), or a penultimate 8-oxo-dG. Each produced blockade signatures that differed from the otherwise identical control 9bp hairpins. This study showed that DNA structure is modified sufficiently by strand cleavage or oxidation damage at a single site to alter in a predictable manner the ionic current blockade signatures produced. This technique improves the ability to assess damage to DNA, and can provide a simple means to help characterize the risks of radiation exposure. It may also provide a method to test radiation protection.

  3. Photosensitization by iodinated DNA minor groove binding ligands: Evaluation of DNA double-strand break induction and repair.

    PubMed

    Briggs, Benjamin; Ververis, Katherine; Rodd, Annabelle L; Foong, Laura J L; Silva, Fernando M Da; Karagiannis, Tom C

    2011-05-03

    Iodinated DNA minor groove binding bibenzimidazoles represent a unique class of UVA photosensitizer and their extreme photopotency has been previously characterized. Earlier studies have included a comparison of three isomers, referred to as ortho-, meta- and para-iodoHoechst, which differ only in the location of the iodine substituent in the phenyl ring of the bibenzimidazole. DNA breakage and clonogenic survival studies in human erythroleukemic K562 cells have highlighted the higher photo-efficiency of the ortho-isomer (subsequently designated UV(A)Sens) compared to the meta- and para-isomers. In this study, the aim was to compare the induction and repair of DNA double-strand breaks induced by the three isomers in K562 cells. Further, we examined the effects of the prototypical broad-spectrum histone deacetylase inhibitor, Trichostatin A, on ortho-iodoHoechst/UVA-induced double-strand breaks in K562 cells. Using γH2AX as a molecular marker of the DNA lesions, our findings indicate a disparity in the induction and particularly, in the repair kinetics of double-strand breaks for the three isomers. The accumulation of γH2AX foci induced by the meta- and para-isomers returned to background levels within 24 and 48 h, respectively; the number of γH2AX foci induced by ortho-iodoHoechst remained elevated even after incubation for 96 h post-irradiation. These findings provide further evidence that the extreme photopotency of ortho-iodoHoechst is due to not only to the high quantum yield of dehalogenation, but also to the severity of the DNA lesions which are not readily repaired. Finally, our findings which indicate that Trichostatin A has a remarkable potentiating effect on ortho-iodoHoechst/UVA-induced DNA lesions are encouraging, particularly in the context of cutaneous T-cell lymphoma, for which a histone deacetylase inhibitor is already approved for therapy. This finding prompts further evaluation of the potential of combination therapies. Copyright © 2011

  4. 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

  5. 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.

  6. Targeting the centriolar replication factor STIL synergizes with DNA damaging agents for treatment of ovarian cancer.

    PubMed

    Rabinowicz, Noa; Mangala, Lingegowda S; Brown, Kevin R; Checa-Rodriguez, Cintia; Castiel, Asher; Moskovich, Oren; Zarfati, Giulia; Trakhtenbrot, Luba; Levy-Barda, Adva; Jiang, Dahai; Rodriguez-Aguayo, Cristian; Pradeep, Sunila; van Praag, Yael; Lopez-Berestein, Gabriel; David, Ahuvit; Novikov, Ilya; Huertas, Pablo; Rottapel, Robert; Sood, Anil K; Izraeli, Shai

    2017-04-18

    Advanced ovarian cancer is an incurable disease. Thus, novel therapies are required. We wished to identify new therapeutic targets for ovarian cancer. ShRNA screen performed in 42 ovarian cancer cell lines identified the centriolar replication factor STIL as an essential gene for ovarian cancer cells. This was verified in-vivo in orthotopic human ovarian cancer mouse models. STIL depletion by administration of siRNA in neutral liposomes resulted in robust anti-tumor effect that was further enhanced in combination with cisplatin. Consistent with this finding, STIL depletion enhanced the extent of DNA double strand breaks caused by DNA damaging agents. This was associated with centrosomal depletion, ongoing genomic instability and enhanced formation of micronuclei. Interestingly, the ongoing DNA damage was not associated with reduced DNA repair. Indeed, we observed that depletion of STIL enhanced canonical homologous recombination repair and increased BRCA1 and RAD51 foci in response to DNA double strand breaks. Thus, inhibition of STIL significantly enhances the efficacy of DNA damaging chemotherapeutic drugs in treatment of ovarian cancer.

  7. Targeting the centriolar replication factor STIL synergizes with DNA damaging agents for treatment of ovarian cancer

    PubMed Central

    Rabinowicz, Noa; Mangala, Lingegowda S.; Brown, Kevin R.; Checa-Rodriguez, Cintia; Castiel, Asher; Moskovich, Oren; Zarfati, Giulia; Trakhtenbrot, Luba; Levy-Barda, Adva; Jiang, Dahai; Rodriguez-Aguayo, Cristian; Pradeep, Sunila; van Praag, Yael; Lopez-Berestein, Gabriel; David, Ahuvit; Novikov, Ilya; Huertas, Pablo; Rottapel, Robert; Sood, Anil K.; Izraeli, Shai

    2017-01-01

    Advanced ovarian cancer is an incurable disease. Thus, novel therapies are required. We wished to identify new therapeutic targets for ovarian cancer. ShRNA screen performed in 42 ovarian cancer cell lines identified the centriolar replication factor STIL as an essential gene for ovarian cancer cells. This was verified in-vivo in orthotopic human ovarian cancer mouse models. STIL depletion by administration of siRNA in neutral liposomes resulted in robust anti-tumor effect that was further enhanced in combination with cisplatin. Consistent with this finding, STIL depletion enhanced the extent of DNA double strand breaks caused by DNA damaging agents. This was associated with centrosomal depletion, ongoing genomic instability and enhanced formation of micronuclei. Interestingly, the ongoing DNA damage was not associated with reduced DNA repair. Indeed, we observed that depletion of STIL enhanced canonical homologous recombination repair and increased BRCA1 and RAD51 foci in response to DNA double strand breaks. Thus, inhibition of STIL significantly enhances the efficacy of DNA damaging chemotherapeutic drugs in treatment of ovarian cancer. PMID:28423708

  8. Target-triggering multiple-cycle signal amplification strategy for ultrasensitive detection of DNA based on QCM and SPR.

    PubMed

    Song, Weiling; Yin, Wenshuo; Sun, Wenbo; Guo, Xiaoyan; He, Peng; Yang, Xiaoyan; Zhang, Xiaoru

    2018-04-24

    Detection of ultralow concentrations of nucleic acid sequences is a central challenge in the early diagnosis of genetic diseases. Herein, we developed a target-triggering cascade multiple cycle amplification for ultrasensitive DNA detection using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). It was based on the exonuclease Ⅲ (Exo Ⅲ)-assisted signal amplification and the hybridization chain reaction (HCR). The streptavidin-coated Au-NPs (Au-NPs-SA) were assembled on the HCR products as recognition element. Upon sensing of target DNA, the duplex DNA probe triggered the Exo Ⅲ cleavage process, accompanied by generating a new secondary target DNA and releasing target DNA. The released target DNA and the secondary target DNA were recycled. Simultaneously, numerous single strands were liberated and acted as the trigger of HCR to generate further signal amplification, resulting in the immobilization of abundant Au-NPs-SA on the gold substrate. The QCM sensor results were found to be comparable to that achieved using a SPR sensor platform. This method exhibited a high sensitivity toward target DNA with a detection limit of 0.70 fM. The high sensitivity and specificity make this method a great potential for detecting DNA with trace amounts in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Transcriptome analysis by strand-specific sequencing of complementary DNA

    PubMed Central

    Parkhomchuk, Dmitri; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Banaru, Maria; Hallen, Linda; Krobitsch, Sylvia; Lehrach, Hans; Soldatov, Alexey

    2009-01-01

    High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online. PMID:19620212

  10. Transcriptome analysis by strand-specific sequencing of complementary DNA.

    PubMed

    Parkhomchuk, Dmitri; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Banaru, Maria; Hallen, Linda; Krobitsch, Sylvia; Lehrach, Hans; Soldatov, Alexey

    2009-10-01

    High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.

  11. Synthetic lethality in DNA repair network: A novel avenue in targeted cancer therapy and combination therapeutics.

    PubMed

    Bhattacharjee, Sonali; Nandi, Saikat

    2017-12-01

    Synthetic lethality refers to a lethal phenotype that results from the simultaneous disruptions of two genes, while the disruption of either gene alone is viable. Many DNA double strand break repair (DSBR) genes have synthetic lethal relationships with oncogenes and tumor suppressor genes, which can be exploited for targeted cancer therapy, an approach referred to as combination therapy. DNA double-strand breaks (DSBs) are one of the most toxic lesions to a cell and can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). HR and NHEJ genes are particularly attractive targets for cancer therapy because these genes have altered expression patterns in cancer cells when compared with normal cells and these genetic abnormalities can be targeted for selectively killing cancer cells. Here, we review recent advances in the development of small molecule inhibitors against HR and NHEJ genes to induce synthetic lethality and address the future directions and clinical relevance of this approach. © 2017 IUBMB Life, 69(12):929-937, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  12. DNA "nano-claw": logic-based autonomous cancer targeting and therapy.

    PubMed

    You, Mingxu; Peng, Lu; Shao, Na; Zhang, Liqin; Qiu, Liping; Cui, Cheng; Tan, Weihong

    2014-01-29

    Cell types, both healthy and diseased, can be classified by inventories of their cell-surface markers. Programmable analysis of multiple markers would enable clinicians to develop a comprehensive disease profile, leading to more accurate diagnosis and intervention. As a first step to accomplish this, we have designed a DNA-based device, called "Nano-Claw". Combining the special structure-switching properties of DNA aptamers with toehold-mediated strand displacement reactions, this claw is capable of performing autonomous logic-based analysis of multiple cancer cell-surface markers and, in response, producing a diagnostic signal and/or targeted photodynamic therapy. We anticipate that this design can be widely applied in facilitating basic biomedical research, accurate disease diagnosis, and effective therapy.

  13. Cyclic perylene diimide: Selective ligand for tetraplex DNA binding over double stranded DNA.

    PubMed

    Vasimalla, Suresh; Sato, Shinobu; Takenaka, Fuminori; Kurose, Yui; Takenaka, Shigeori

    2017-12-15

    Synthesized cyclic perylene diimide, cPDI, showed the binding constant of 6.3 × 10 6  M -1 with binding number of n = 2 with TA-core as a tetraplex DNA in 50 mM Tris-HCl buffer (pH = 7.4) containing 100 mM KCl using Schatchard analysis and showed a higher preference for tetraplex DNA than for double stranded DNA with over 10 3 times. CD spectra showed that TA-core induced its antiparallel conformation upon addition of cPDI in the absence or presence of K + or Na + ions. The cPDI inhibits the telomerase activity with IC 50 of 0.3 µM using TRAP assay which is potential anti-cancer drug with low side effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Size fractionation of double-stranded DNA by precipitation with polyethylene glycol

    PubMed Central

    Lis, John T.; Schleif, Robert

    1975-01-01

    We show that DNA molecules of differing molecular mass are separable by selective precipitation with polyethylene glycol (PEG†). Higher molecular mass DNA precipitates at lower PEG concentrations than lower molecular mass DNA. Double-stranded DNA can be fractionated at least in the range of 3 × 107 to 1 × 105 daltons. The effects of PEG concentration, sodium chloride concentration, DNA concentration, pH, divalent ions, precipitation time, and centrifugal force have been determined. These studies show PEG precipitation offers a size fractionation method for DNA which is convenient, of high capacity, and applicable over a wide range of conditions. However, resolution is not high and separation of two species approaches 100% only if they differ in molecular mass by at least a factor of two. Images PMID:236548

  15. Single molecule characterization of DNA binding and strand displacement reactions on lithographic DNA origami microarrays.

    PubMed

    Scheible, Max B; Pardatscher, Günther; Kuzyk, Anton; Simmel, Friedrich C

    2014-03-12

    The combination of molecular self-assembly based on the DNA origami technique with lithographic patterning enables the creation of hierarchically ordered nanosystems, in which single molecules are positioned at precise locations on multiple length scales. Based on a hybrid assembly protocol utilizing DNA self-assembly and electron-beam lithography on transparent glass substrates, we here demonstrate a DNA origami microarray, which is compatible with the requirements of single molecule fluorescence and super-resolution microscopy. The spatial arrangement allows for a simple and reliable identification of single molecule events and facilitates automated read-out and data analysis. As a specific application, we utilize the microarray to characterize the performance of DNA strand displacement reactions localized on the DNA origami structures. We find considerable variability within the array, which results both from structural variations and stochastic reaction dynamics prevalent at the single molecule level.

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

    PubMed Central

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

    2006-01-01

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

  17. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.

    PubMed

    Hu, Jiazhi; Meyers, Robin M; Dong, Junchao; Panchakshari, Rohit A; Alt, Frederick W; Frock, Richard L

    2016-05-01

    Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.

  18. Enhanced DNA Sensing via Catalytic Aggregation of Gold Nanoparticles

    PubMed Central

    Huttanus, Herbert M.; Graugnard, Elton; Yurke, Bernard; Knowlton, William B.; Kuang, Wan; Hughes, William L.; Lee, Jeunghoon

    2014-01-01

    A catalytic colorimetric detection scheme that incorporates a DNA-based hybridization chain reaction into gold nanoparticles was designed and tested. While direct aggregation forms an inter-particle linkage from only ones target DNA strand, the catalytic aggregation forms multiple linkages from a single target DNA strand. Gold nanoparticles were functionalized with thiol-modified DNA strands capable of undergoing hybridization chain reactions. The changes in their absorption spectra were measured at different times and target concentrations and compared against direct aggregation. Catalytic aggregation showed a multifold increase in sensitivity at low target concentrations when compared to direct aggregation. Gel electrophoresis was performed to compare DNA hybridization reactions in catalytic and direct aggregation schemes, and the product formation was confirmed in the catalytic aggregation scheme at low levels of target concentrations. The catalytic aggregation scheme also showed high target specificity. This application of a DNA reaction network to gold nanoparticle-based colorimetric detection enables highly-sensitive, field-deployable, colorimetric readout systems capable of detecting a variety of biomolecules. PMID:23891867

  19. Programmable Regulation of DNA Conjugation to Gold Nanoparticles via Strand Displacement.

    PubMed

    Zhang, Cheng; Wu, Ranfeng; Li, Yifan; Zhang, Qiang; Yang, Jing

    2017-10-31

    Methods for conjugating DNA to gold nanoparticles (AuNPs) have recently attracted considerable attention. The ability to control such conjugation in a programmable way is of great interest. Here, we have developed a logic-based method for manipulating the conjugation of thiolated DNA species to AuNPs via cascading DNA strand displacement. Using this method, several logic-based operation systems are established and up to three kinds of DNA signals are introduced at the same time. In addition, a more sensitive catalytic logic-based operation is also achieved based on an entropy-driven process. In the experiment, all of the DNA/AuNPs conjugation results are verified by agrose gel. This strategy promises great potential for automatically conjugating DNA stands onto label-free gold nanoparticles and can be extended to constructing DNA/nanoparticle devices for applications in diagnostics, biosensing, and molecular robotics.

  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. Adaptation of the TdT assay for semi-quantitative flow cytometric detection of DNA strand breaks

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bromidge, T.J.; Howe, D.J.; Johnson, S.A.

    The enzyme Terminal Deoxynucleotidyl Transferase (TdT) is a DNA polymerase which can be used to label DNA strand breaks by the incorporation of a labelled nucleotide followed by a fluorescent detection step. The amount of label incorporated can then be assessed by flow cytometry. The mechanism of action of TdT, however, will allow the addition of varying numbers of nucleotides to the free 3{prime} termini produced by DNA strand breaks. The substitution of Digoxigenin (DIG){trademark} labelled dideoxynucleotides for labelled deoxy-nucleotides in the TdT assay will limit the addition of label to a DNA break to a single nucleotide, thus ensuringmore » a direct relationship between an increase in DNA strand breaks and an increase in fluorescence. We have used this adaptation of the TdT assay to evaluate DNA damage incurred in lymphocytes, from patients with Chronic Lymphocytic Leukemia (CLL), on exposure to UV irradiation and apoptosis-inducing drugs, fludarabine and 2-Chloro-2{prime}-deoxyadenosine (2-CdA). This technique may give a good indication of the susceptibility of CLL patients to apoptosis inducing drugs, and hence an indication of the likely response to these therapies. 7 refs., 2 figs., 2 tabs.« less

  2. Force-induced rupture of double-stranded DNA in the absence and presence of covalently bonded anti-tumor drugs: Insights from molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Upadhyaya, Anurag; Nath, Shesh; Kumar, Sanjay

    2018-06-01

    DNA intra-strand cross-link (ICL) agents are widely used in the treatment of cancer. ICLs are thought to form a link between the same strand (intra-strand) or complimentary strand (inter-strand) and thereby increase the stability of DNA, which forbids the processes like replication and transcription. As a result, cell death occurs. In this work, we have studied the enhanced stability of a double stranded DNA in the presence of ICLs and compared our findings with the results obtained in the absence of these links. Using atomistic simulations with explicit solvent, a force is applied along and perpendicular to the direction of the helix and we measured the rupture force and the unzipping force of DNA-ICL complexes. Our results show that the rupture and the unzipping forces increase significantly in the presence of these links. The ICLs bind to the minor groove of DNA, which enhance the DNA stabilisation. Such information may be used to design alternative drugs that can stall replication and transcription that are critical to a growing number of anticancer drug discovery efforts.

  3. Triple Helix Formation in a Topologically Controlled DNA Nanosystem.

    PubMed

    Yamagata, Yutaro; Emura, Tomoko; Hidaka, Kumi; Sugiyama, Hiroshi; Endo, Masayuki

    2016-04-11

    In the present study, we demonstrate single-molecule imaging of triple helix formation in DNA nanostructures. The binding of the single-molecule third strand to double-stranded DNA in a DNA origami frame was examined using two different types of triplet base pairs. The target DNA strand and the third strand were incorporated into the DNA frame, and the binding of the third strand was controlled by the formation of Watson-Crick base pairing. Triple helix formation was monitored by observing the structural changes in the incorporated DNA strands. It was also examined using a photocaged third strand wherein the binding of the third strand was directly observed using high-speed atomic force microscopy during photoirradiation. We found that the binding of the third strand could be controlled by regulating duplex formation and the uncaging of the photocaged strands in the designed nanospace. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes.

    PubMed

    Zhu, X Q; Gasser, R B

    1998-06-01

    In this study, we assessed single-strand conformation polymorphism (SSCP)-based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS-2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP-based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS-2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.

  5. Chemo-mechanical pushing of proteins along single-stranded DNA.

    PubMed

    Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M

    2016-05-31

    Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

  6. What Controls the "Off/On Switch" in the Toehold-Mediated Strand Displacement Reaction on DNA Conjugated Gold Nanoparticles?

    PubMed

    Yao, Dongbao; Wang, Bei; Xiao, Shiyan; Song, Tingjie; Huang, Fujian; Liang, Haojun

    2015-06-30

    In DNA dynamic nanotechnology, a toehold-mediated DNA strand-displacement reaction has demonstrated its capability in building complex autonomous system. In most cases, the reaction is performed in pure DNA solution that is essentially a one-phase system. In the present work, we systematically investigated the reaction in a heterogeneous media, in which the strand that implements a displacing action is conjugated on gold nanoparticles. By monitoring the kinetics of spherical nucleic acid (SNA) assembly driven by toehold-mediated strand displacement reaction, we observed significant differences, i.e., the abrupt jump in behavior of an "off/on switch", in the reaction rate when the invading toehold was extended to eight bases from seven bases. These phenomena are attributed to the effect of steric hindrance arising from the high density of invading strand conjugated to AuNPs. Based on these studies, an INHIBIT logic gate presenting good selectivity was developed.

  7. Genetic recombination induced by DNA double-strand break in bacteriophage T4: nature of the left/right bias.

    PubMed

    Shcherbakov, Victor P; Shcherbakova, Tamara; Plugina, Lidiya; Sizova, Svetlana; Kudryashova, Elena; Granovsky, Igor

    2008-06-01

    The experimental system combining double-strand breaks (DSBs), produced site-specifically by SegC endonuclease, with the famous advantages of the bacteriophage T4 rII mutant recombination analysis was used here to elucidate the origin of the recombination bias on two sides of the DSB, especially pronounced in gene 39 (topoisomerase II) and gene 59 (41-helicase loader) mutants. Three sources were found to contribute to the bias: (1) the SegC endonuclease may remain bound to the end of the broken DNA and thus protect it from exonuclease degradation; (2) in heteroduplex heterozygotes (HHs), arising as the recombinant products in the left-hand crosses, the transcribed strands are of rII mutant phenotype, so they, in contrast to the right-hand HHs, do not produce plaques on the lawn of the lambda-lysogenic host; and (3) the intrinsic polarity of T4 chromosome, reflected in transcription, may be a cause for discrimination of promoter-proximal and promoter-distal DNA sequences. It is shown that the apparent recombination bias does not imply one-sidedness of the DSB repair but just reflects a different depth of the end processing. It is inferred that the cause, underlying the "intrinsic" bias, might be interference between strand exchange and transcription. Topoisomerase and helicase functions are necessary to turn the process in favor of strand exchange. The idea is substantiated that the double-stranded to single-stranded DNA transition edge (not ss-DNA tip) serves as an actual recombinogenic element.

  8. Effect of Escherichia coli DNA binding protein on the transcription of single-stranded phage M13 DNA by Escherichia coli RNA polymerase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Niyogi, S.K.; Ratrie, H. III; Datta, A.K.

    E. coli DNA binding protein strongly inhibits the transcription of single-stranded rather than double-stranded phage M13 DNA by E. coli RNA polymerase. This inhibition cannot be significantly overcome by increasing the concentration of RNA polymerase. Nor does the order of addition of binding protein affect its inhibitory property: inhibition is evident whether binding protein is added before or after the formation of the RNA polymerase--DNA complex. Inhibition is also observed if binding protein is added at various times after initiation of RNA synthesis. Maximal inhibition occurs at a binding protein-to-DNA ratio (w/w) of about 8:1. This corresponds to one bindingmore » protein molecule covering about 30 nucleotides, in good agreement with values obtained by physical measurements.« less

  9. Programming chemical kinetics: engineering dynamic reaction networks with DNA strand displacement

    NASA Astrophysics Data System (ADS)

    Srinivas, Niranjan

    Over the last century, the silicon revolution has enabled us to build faster, smaller and more sophisticated computers. Today, these computers control phones, cars, satellites, assembly lines, and other electromechanical devices. Just as electrical wiring controls electromechanical devices, living organisms employ "chemical wiring" to make decisions about their environment and control physical processes. Currently, the big difference between these two substrates is that while we have the abstractions, design principles, verification and fabrication techniques in place for programming with silicon, we have no comparable understanding or expertise for programming chemistry. In this thesis we take a small step towards the goal of learning how to systematically engineer prescribed non-equilibrium dynamical behaviors in chemical systems. We use the formalism of chemical reaction networks (CRNs), combined with mass-action kinetics, as our programming language for specifying dynamical behaviors. Leveraging the tools of nucleic acid nanotechnology (introduced in Chapter 1), we employ synthetic DNA molecules as our molecular architecture and toehold-mediated DNA strand displacement as our reaction primitive. Abstraction, modular design and systematic fabrication can work only with well-understood and quantitatively characterized tools. Therefore, we embark on a detailed study of the "device physics" of DNA strand displacement (Chapter 2). We present a unified view of strand displacement biophysics and kinetics by studying the process at multiple levels of detail, using an intuitive model of a random walk on a 1-dimensional energy landscape, a secondary structure kinetics model with single base-pair steps, and a coarse-grained molecular model that incorporates three-dimensional geometric and steric effects. Further, we experimentally investigate the thermodynamics of three-way branch migration. Our findings are consistent with previously measured or inferred rates for

  10. Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein.

    PubMed

    Liu, Jie; Berger, Christopher L; Morrical, Scott W

    2013-11-12

    Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA binding protein (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a prenucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly at physiologically relevant ionic strengths and Gp32 concentrations. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX-ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components (recombinase, mediator, and SSB) on native ssDNA.

  11. Long repeating (TTAGGG)n single stranded DNA self-condenses into compact beaded filaments stabilized by G-quadruplex formation.

    PubMed

    Kar, Anirban; Jones, Nathan; Arat, N Özlem; Fishel, Richard; Griffith, Jack

    2018-04-19

    Conformations adopted by long stretches of single stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo. This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ~150 nt, and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3')n as well as the interrogation of its structure by electron microscopy (EM) and single molecule magnetic tweezers (smMT). This repeat is of particular interest as it contains a run of 3 contiguous guanine residues capable of forming G quartets as ssDNA. Fluorescent-dye exclusion assays confirmed that this G-strand ssDNA forms ubiquitous G-quadruplex folds. EM revealed thick bead-like filaments that condensed the DNA ~12 fold. The bead-like structures were 5 nm and 8 nm in diameter and linked by thin filaments. The G-strand ssDNA displayed initial stability to smMT force extension that ultimately released in steps that were multiples ~28 nm at forces between 6-12 pN; well below the >20 pN required to unravel G-quadruplexes. Most smMT steps were consistent with the disruption of the beads seen by EM. Binding by RAD51 distinctively altered the force extension properties of the G-strand ssDNA, suggesting a stochastic G-quadruplex-dependent condensation model that is discussed. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  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. The RNA surveillance protein SMG1 activates p53 in response to DNA double-strand breaks but not exogenously oxidized mRNA

    PubMed Central

    Gewandter, Jennifer S; Bambara, Robert A

    2011-01-01

    DNA damage, stalled replication forks, errors in mRNA splicing and availability of nutrients activate specific phosphatidylinositiol-3-kinase-like kinases (PIKKs) that in turn phosphorylate downstream targets such as p53 on serine 15. While the PIKK proteins ATM and ATR respond to specific DNA lesions, SMG1 responds to errors in mRNA splicing and when cells are exposed to genotoxic stress. Yet, whether genotoxic stress activates SMG1 through specific types of DNA lesions or RNA damage remains poorly understood. Here, we demonstrate that siRNA oligonucleotides targeting the mRNA surveillance proteins SMG1, Upf1, Upf2 or the PIKK protein ATM attenuated p53 (ser15) phosphorylation in cells damaged by high oxygen (hyperoxia), a model of persistent oxidative stress that damages nucleotides. In contrast, loss of SMG1 or ATM, but not Upf1 or Upf2 reduced p53 (ser15) phosphorylation in response to DNA double strand breaks produced by expression of the endonuclease I-PpoI. To determine whether SMG1-dependent activation of p53 was in response to oxidative mRNA damage, mRNA encoding green fluorescence protein (GFP) transcribed in vitro was oxidized by Fenton chemistry and transfected into cells. Although oxidation of GFP mRNA resulted in dose-dependent fragmentation of the mRNA and reduced expression of GFP, it did not stimulate p53 or the p53-target gene p21. These findings establish SMG1 activates p53 in response to DNA double strand breaks independent of the RNA surveillance proteins Upf1 or Upf2; however, these proteins can stimulate p53 in response to oxidative stress but not necessarily oxidized RNA. PMID:21701263

  14. 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.

  15. Elongated unique DNA strand deposition on microstructured substrate by receding meniscus assembly and capillary force

    PubMed Central

    Charlot, B.; Bardin, F.; Sanchez, N.; Roux, P.; Teixeira, S.; Schwob, E.

    2014-01-01

    Ordered deposition of elongated DNA molecules was achieved by the forced dewetting of a DNA solution droplet over a microstructured substrate. This technique allows trapping, uncoiling, and deposition of DNA fragments without the need of a physicochemical anchoring of the molecule and results in the combing of double stranded DNA from the edge of microwells on a polydimethylsiloxane (PDMS) substrate. The technique involves scanning a droplet of DNA solution caught between a movable blade and a PDMS substrate containing an array of microwells. The deposition and elongation appears when the receding meniscus dewets microwells, the latter acting here as a perturbation in the dewetting line forcing the water film to break locally. Thus, DNA molecules can be deposited in an ordered manner and elongated conformation based solely on a physical phenomenon, allowing uncoiled DNA molecules to be observed in all their length. However, the exact mechanism that governs the deposition of DNA strands is not well understood. This paper is an analysis of the physical phenomenon occurring in the deposition process and is based on observations made with the use of high frame/second rate video microscopy. PMID:24753724

  16. 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.

  17. Perspectives on the combination of radiotherapy and targeted therapy with DNA repair inhibitors in the treatment of pancreatic cancer

    PubMed Central

    Yang, Shih-Hung; Kuo, Ting-Chun; Wu, Hsu; Guo, Jhe-Cyuan; Hsu, Chiun; Hsu, Chih-Hung; Tien, Yu-Wen; Yeh, Kun-Huei; Cheng, Ann-Lii; Kuo, Sung-Hsin

    2016-01-01

    Pancreatic cancer is highly lethal. Current research that combines radiation with targeted therapy may dramatically improve prognosis. Cancerous cells are characterized by unstable genomes and activation of DNA repair pathways, which are indicated by increased phosphorylation of numerous factors, including H2AX, ATM, ATR, Chk1, Chk2, DNA-PKcs, Rad51, and Ku70/Ku80 heterodimers. Radiotherapy causes DNA damage. Cancer cells can be made more sensitive to the effects of radiation (radiosensitization) through inhibition of DNA repair pathways. The synergistic effects, of two or more combined non-lethal treatments, led to co-administration of chemotherapy and radiosensitization in BRCA-defective cells and patients, with promising results. ATM/Chk2 and ATR/Chk1 pathways are principal regulators of cell cycle arrest, following DNA double-strand or single-strand breaks. DNA double-stranded breaks activate DNA-dependent protein kinase, catalytic subunit (DNA-PKcs). It forms a holoenzyme with Ku70/Ku80 heterodimers, called DNA-PK, which catalyzes the joining of nonhomologous ends. This is the primary repair pathway utilized in human cells after exposure to ionizing radiation. Radiosensitization, induced by inhibitors of ATM, ATR, Chk1, Chk2, Wee1, PP2A, or DNA-PK, has been demonstrated in preclinical pancreatic cancer studies. Clinical trials are underway. Development of agents that inhibit DNA repair pathways to be clinically used in combination with radiotherapy is warranted for the treatment of pancreatic cancer. PMID:27621574

  18. Chemo-mechanical pushing of proteins along single-stranded DNA

    PubMed Central

    Sokoloski, Joshua E.; Kozlov, Alexander G.; Galletto, Roberto; Lohman, Timothy M.

    2016-01-01

    Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3′-Cy3–labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5′ to 3′ ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5′ to 3′) pushing of the SSB toward the 3′ ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3′ to 5′) direction, are observed with EcRep and EcUvrD (both 3′ to 5′ ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA. PMID:27185951

  19. Target capture during Mos1 transposition.

    PubMed

    Pflieger, Aude; Jaillet, Jerôme; Petit, Agnès; Augé-Gouillou, Corinne; Renault, Sylvaine

    2014-01-03

    DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide.

  20. Target Capture during Mos1 Transposition*

    PubMed Central

    Pflieger, Aude; Jaillet, Jerôme; Petit, Agnès; Augé-Gouillou, Corinne; Renault, Sylvaine

    2014-01-01

    DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide. PMID:24269942

  1. Concentration-Dependent Exchange of Replication Protein A on Single-Stranded DNA Revealed by Single-Molecule Imaging

    PubMed Central

    Gibb, Bryan; Ye, Ling F.; Gergoudis, Stephanie C.; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick; Greene, Eric C.

    2014-01-01

    Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein necessary for all aspects of DNA metabolism involving an ssDNA intermediate, including DNA replication, repair, recombination, DNA damage response and checkpoint activation, and telomere maintenance [1], [2], [3]. The role of RPA in most of these reactions is to protect the ssDNA until it can be delivered to downstream enzymes. Therefore a crucial feature of RPA is that it must bind very tightly to ssDNA, but must also be easily displaced from ssDNA to allow other proteins to gain access to the substrate. Here we use total internal reflection fluorescence microscopy and nanofabricated DNA curtains to visualize the behavior of Saccharomyces cerevisiae RPA on individual strands of ssDNA in real-time. Our results show that RPA remains bound to ssDNA for long periods of time when free protein is absent from solution. In contrast, RPA rapidly dissociates from ssDNA when free RPA is present in solution allowing rapid exchange between the free and bound states. In addition, the S. cerevisiae DNA recombinase Rad51 and E. coli single-stranded binding protein (SSB) also promote removal of RPA from ssDNA. These results reveal an unanticipated exchange between bound and free RPA suggesting a binding mechanism that can confer exceptionally slow off rates, yet also enables rapid displacement through a direct exchange mechanism that is reliant upon the presence of free ssDNA-binding proteins in solution. Our results indicate that RPA undergoes constant microscopic dissociation under all conditions, but this is only manifested as macroscopic dissociation (i.e. exchange) when free proteins are present in solution, and this effect is due to mass action. We propose that the dissociation of RPA from ssDNA involves a partially dissociated intermediate, which exposes a small section of ssDNA allowing other proteins to access to the DNA. PMID:24498402

  2. Twin target self-amplification-based DNA machine for highly sensitive detection of cancer-related gene.

    PubMed

    Xu, Huo; Jiang, Yifan; Liu, Dengyou; Liu, Kai; Zhang, Yafeng; Yu, Suhong; Shen, Zhifa; Wu, Zai-Sheng

    2018-06-29

    The sensitive detection of cancer-related genes is of great significance for early diagnosis and treatment of human cancers, and previous isothermal amplification sensing systems were often based on the reuse of target DNA, the amplification of enzymatic products and the accumulation of reporting probes. However, no reporting probes are able to be transformed into target species and in turn initiate the signal of other probes. Herein we reported a simple, isothermal and highly sensitive homogeneous assay system for tumor suppressor p53 gene detection based on a new autonomous DNA machine, where the signaling probe, molecular beacon (MB), was able to execute the function similar to target DNA besides providing the common signal. In the presence of target p53 gene, the operation of DNA machine can be initiated, and cyclical nucleic acid strand-displacement polymerization (CNDP) and nicking/polymerization cyclical amplification (NPCA) occur, during which the MB was opened by target species and cleaved by restriction endonuclease. In turn, the cleaved fragments could activate the next signaling process as target DNA did. According to the functional similarity, the cleaved fragment was called twin target, and the corresponding fashion to amplify the signal was named twin target self-amplification. Utilizing this newly-proposed DNA machine, the target DNA could be detected down to 0.1 pM with a wide dynamic range (6 orders of magnitude) and single-base mismatched targets were discriminated, indicating a very high assay sensitivity and good specificity. In addition, the DNA machine was not only used to screen the p53 gene in complex biological matrix but also was capable of practically detecting genomic DNA p53 extracted from A549 cell line. This indicates that the proposed DNA machine holds the potential application in biomedical research and early clinical diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Evidence for a Single-Stranded Adenovirus-Associated Virus Genome: Isolation and Separation of Complementary Single Strands

    PubMed Central

    Berns, K. I.; Rose, J. A.

    1970-01-01

    Single-stranded adenovirus-associated virus type 2 deoxyribonucleic acid (AAV-2 DNA) has been isolated from the virion after enzymatic pretreatment of the particles by heating at 53 C for 1 hr in 0.015 m NaCl plus 0.0015 m sodium citrate in the presence of 1% sodium dodecyl sulfate. Double-stranded AAV-2 DNA present as a marker is not denatured by this treatment. AAV-2 single-stranded DNA is composed of two complementary species which can be separated in neutral CsCl when 5-bromodeoxyuridine has been substituted for thymidine in the DNA. The present report is the first documented instance of the separation of complementary strands of an animal virus DNA. PMID:5429749

  4. Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices.

    PubMed

    Giuffrida, Maria Chiara; Zanoli, Laura Maria; D'Agata, Roberta; Finotti, Alessia; Gambari, Roberto; Spoto, Giuseppe

    2015-02-01

    Nucleic-acid amplification is a crucial step in nucleic-acid-sequence-detection assays. The use of digital microfluidic devices to miniaturize amplification techniques reduces the required sample volume and the analysis time and offers new possibilities for process automation and integration in a single device. The recently introduced droplet polymerase-chain-reaction (PCR) amplification methods require repeated cycles of two or three temperature-dependent steps during the amplification of the nucleic-acid target sequence. In contrast, low-temperature isothermal-amplification methods have no need for thermal cycling, thus requiring simplified microfluidic-device features. Here, the combined use of digital microfluidics and molecular-beacon (MB)-assisted isothermal circular-strand-displacement polymerization (ICSDP) to detect microRNA-210 sequences is described. MicroRNA-210 has been described as the most consistently and predominantly upregulated hypoxia-inducible factor. The nmol L(-1)-pmol L(-1) detection capabilities of the method were first tested by targeting single-stranded DNA sequences from the genetically modified Roundup Ready soybean. The ability of the droplet-ICSDP method to discriminate between full-matched, single-mismatched, and unrelated sequences was also investigated. The detection of a range of nmol L(-1)-pmol L(-1) microRNA-210 solutions compartmentalized in nanoliter-sized droplets was performed, establishing the ability of the method to detect as little as 10(-18) mol of microRNA target sequences compartmentalized in 20 nL droplets. The suitability of the method for biological samples was tested by detecting microRNA-210 from transfected K562 cells.

  5. Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute

    PubMed Central

    Miyoshi, Tomohiro; Ito, Kosuke; Murakami, Ryo; Uchiumi, Toshio

    2016-01-01

    Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson–Crick base-pairing even in the 3′-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5′ base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region. PMID:27325485

  6. Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents.

    PubMed

    Zaboikin, Michail; Zaboikina, Tatiana; Freter, Carl; Srinivasakumar, Narasimhachar

    2017-01-01

    Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that

  7. Selection and Characterization of Single Stranded DNA Aptamers for the Hormone Abscisic Acid

    PubMed Central

    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

    2013-01-01

    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. PMID:23971905

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

    PubMed Central

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

    2015-01-01

    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. PMID:25903123

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.

    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 sitemore » 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.« less

  10. 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

  11. 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.

  12. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. 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

  14. Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae.

    PubMed

    Vernal, Javier; Serpa, Viviane I; Tavares, Carolina; Souza, Emanuel M; Pedrosa, Fábio O; Terenzi, Hernán

    2007-05-01

    An open reading frame encoding a protein similar in size and sequence to the Escherichia coli single-stranded DNA binding protein (SSB protein) was identified in the Herbaspirillum seropedicae genome. This open reading frame was cloned into the expression plasmid pET14b. The SSB protein from H. seropedicae, named Hs_SSB, was overexpressed in E. coli strain BL21(DE3) and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein. The apparent molecular mass of the native Hs_SSB was estimated by gel filtration, suggesting that the native protein is a tetramer made up of four similar subunits. The purified protein binds to single-stranded DNA (ssDNA) in a similar manner to other SSB proteins. The production of this recombinant protein in good yield opens up the possibility of obtaining its 3D-structure and will help further investigations into DNA metabolism.

  15. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, C.; Heilmann, J.; Rink, H.

    The lens epithelium is the initiation site for the development of radiation induced cataracts. While in the cortex and nucleus radiation interacts with proteins, experimental results from cultured lenses and lens epithelial cells demonstrate mutagenic and cytotoxic effects in the epithelium. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the radiation's relative biological effectiveness (RBE), because cosmic rays differ significantly from X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiations. Irradiations were performed either with 300 kV X-rays or at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. For strand break measurements hydroxyapatite chromatography of alka-line unwound DNA (overall strand breaks) and non-denaturing filter elution technique (double strand breaks) were applied. Experiments showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV/μm more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of

  16. An alternative mechanism for radioprotection by dimethyl sulfoxide; possible facilitation of DNA double-strand break repair.

    PubMed

    Kashino, Genro; Liu, Yong; Suzuki, Minoru; Masunaga, Shin-ichiro; Kinashi, Yuko; Ono, Koji; Tano, Keizo; Watanabe, Masami

    2010-01-01

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

  17. A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification.

    PubMed

    Cheng, Wei; Zhang, Wei; Yan, Yurong; Shen, Bo; Zhu, Dan; Lei, Pinhua; Ding, Shijia

    2014-12-15

    A novel electrochemical biosensing strategy was developed for ultrasensitive and specific detection of target DNA using a cascade signal amplification based on molecular beacon (MB) mediated circular strand displacement (CSD), rolling circle amplification (RCA), biotin-strepavidin system, and enzymatic amplification. The target DNA hybridized with the loop portion of MB probe immobilized on the gold electrode and triggered the CSD, leading to multiple biotin-tagged DNA duplex. Furthermore, via biotin-streptavidin interaction, the RCA was implemented, producing long massive tandem-repeat DNA sequences for binding numerous biotinylated detection probes. This enabled an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor showed very high sensitivity and selectivity with a dynamic response range from 1 fM to 100 pM. The proposed strategy could have the potential for applying in clinical molecular diagnostics and environmental monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Repairing the sickle cell mutation. I. Specific covalent binding of a photoreactive third strand to the mutated base pair.

    PubMed

    Broitman, S; Amosova, O; Dolinnaya, N G; Fresco, J R

    1999-07-30

    A DNA third strand with a 3'-psoralen substituent was designed to form a triplex with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue "hook" complementary to an overhang of a linear duplex target was added to the 5'-end of the third strand via a T(4) linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.

  19. Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair.

    PubMed

    Ihara, Makoto; Takeshita, Satoshi; Okaichi, Kumio; Okumura, Yutaka; Ohnishi, Takeo

    2014-03-01

    From the role of double strand DNA dependent protein kinase (DNA-PKcs) activity of non-homologous end joining (NHEJ) repair for DNA double strand breaks (DSBs), we aim to define possible associations between thermo-sensitisation and the enzyme activities in X-ray irradiated cells. DNA-PKcs deficient mouse, Chinese hamster and human cultured cells were compared to the parental wild-type cells. The radiosensitivities, the number of DSBs and DNA-PKcs activities after heat-treatment were measured. Both DNA-PKcs deficient cells and the wild-type cells showed increased radiosensitivities after heat-treatment. The wild-type cells have two repair processes; fast repair and slow repair. In contrast, DNA-PKcs deficient cells have only the slow repair process. The fast repair component apparently disappeared by heat-treatment in the wild-type cells. In both cell types, additional heat exposure enhanced radiosensitivities. Although DNA-PKcs activity was depressed by heat, the inactivated DNA-PKcs activity recovered during an incubation at 37 °C. DSB repair efficiency was dependent on the reactivation of DNA-PKcs activity. It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.

  20. HTLV-I Tax Increases Genetic Instability by Inducing DNA Double Strand Breaks during DNA Replication and Switching Repair to NHEJ

    PubMed Central

    Baydoun, Hicham H.; Bai, Xue Tao; Shelton, Shary; Nicot, Christophe

    2012-01-01

    Background Appropriate responses to damaged DNA are indispensible for preserving genome stability and preventing cancer. Tumor viruses often target DNA repair machinery to achieve transformation. The Human T-cell leukemia virus type I (HTLV-I) is the only known transforming human retrovirus and the etiological agent of Adult T-cell Leukemia (ATLL). Although HTLV-I-transformed leukemic cells have numerous genetic lesions, the precise role of the viral tax gene in this process is not fully understood. Results Our results show a novel function of HTLV-I oncoprotein Tax as an inducer of genomic DNA double strand breaks (DDSB) during DNA replication. We also found that Tax acts as a potent inhibitor of homologous recombination (HR) DNA repair through the activation of the NF-kB pathway. These results were confirmed using HTLV-I molecular clones expressing Tax at physiological levels in a natural context. We further found that HTLV-I- and Tax-transformed cells are not more susceptible to DNA damaging agents and repair DNA lesions at a rate similar to that of normal cells. Finally, we demonstrated that during S phase, Tax-associated DDSB are preferentially repaired using the error-prone non-homologous end joining (NHEJ) pathway. Conclusions This study provides new insights in Tax effects on DNA repair and genome instability. Although it may not be self sufficient, the creation of DNA breaks and subsequent abnormal use of the non-conservative NHEJ DNA repair during the S phase in HTLV-I-infected Tax-expressing cells may cooperate with other factors to increase genetic and genome instability and favor transformation. PMID:22916124

  1. Multifunctional DNA-gold nanoparticles for targeted doxorubicin delivery.

    PubMed

    Alexander, Colleen M; Hamner, Kristen L; Maye, Mathew M; Dabrowiak, James C

    2014-07-16

    In this report we describe the synthesis, characterization, and cytotoxic properties of DNA-capped gold nanoparticles having attached folic acid (FA), a thermoresponsive polymer (p), and/or poly(ethylene glycol) (PEG) oligomers that could be used to deliver the anticancer drug doxorubicin (DOX) in chemotherapy. The FA-DNA oligomer used in the construction of the delivery vehicle was synthesized through the reaction of the isolated folic acid N-hydroxysuccinimide ester with the amino-DNA and the conjugated DNA product was purified using high performance liquid chromatography (HPLC). This approach ultimately allowed control of the amount of FA attached to the surface of the delivery vehicle. Cytotoxicity studies using SK-N-SH neuroblastoma cells with drug loaded delivery vehicles were carried out using a variety of exposure times (1-48 h) and recovery times (1-72 h), and in order to access the effects of varying amounts of attached FA, in culture media deficient in FA. DOX loaded delivery vehicles having 50% of the DNA strands with attached FA were more cytotoxic than when all of the strands contained FA. Since FA stimulates cell growth, the reduced cytotoxicity of vehicles fully covered with FA suggests that the stimulatory effects of FA can more than compensate for the cytotoxic effects of the drug on the cell population. While attachment of hexa-ethylene glycol PEG(18) to the surface of the delivery vehicle had no effect on cytotoxicity, 100% FA plus the thermoresponsive polymer resulted in IC50 = 0.48 ± 0.01 for an exposure time of 24 h and a recovery time of 1 h, which is an order of magnitude more cytotoxic than free DOX. Confocal microscopic studies using fluorescence detection showed that SK-N-SH neuroblastoma cells exposed to DOX-loaded vehicles have drug accumulation inside the cell and, in the case of vehicles with attached FA and thermoresponsive polymer, the drug appears more concentrated. Since the biological target of DOX is DNA, the latter

  2. Ligand induced stabilization of the melting temperature of the HSV-1 single-strand DNA binding protein using the thermal shift assay.

    PubMed

    Rupesh, Kanchi Ravi; Smith, Aaron; Boehmer, Paul E

    2014-11-28

    We have adapted the thermal shift assay to measure the ligand binding properties of the herpes simplex virus-1 single-strand DNA binding protein, ICP8. By measuring SYPRO Orange fluorescence in microtiter plates using a fluorescence-enabled thermal cycler, we have quantified the effects of oligonucleotide ligands on the melting temperature of ICP8. We found that single-stranded oligomers raise the melting temperature of ICP8 in a length- and concentration-dependent manner, ranging from 1°C for (dT)5 to a maximum of 9°C with oligomers ⩾10 nucleotides, with an apparent Kd of <1μM for (dT)20. Specifically, the results indicate that ICP8 is capable of interacting with oligomers as short as 5 nucleotides. Moreover, the observed increases in melting temperature of up to 9°C, indicates that single-strand DNA binding significantly stabilizes the structure of ICP8. This assay may be applied to investigate the ligand binding proteins of other single-strand DNA binding proteins and used as a high-throughput screen to identify compounds with therapeutic potential that inhibit single-strand DNA binding. As proof of concept, the single-strand DNA binding agent ciprofloxacin reduces the ligand induced stabilization of the melting temperature of ICP8 in a dose-dependent manner. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

    PubMed

    Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

    2016-07-29

    Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species.

  4. Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection

    PubMed Central

    Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

    2016-01-01

    Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world’s attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg2+ ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species. PMID:27483277

  5. Cdc45-induced loading of human RPA onto single-stranded DNA

    PubMed Central

    Tessmer, Ingrid; Prus, Piotr; Schlott, Bernhard; Pospiech, Helmut

    2017-01-01

    Abstract Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA. Pull-down assays and surface plasmon resonance studies revealed that Cdc45-bound RPA complexed with ssDNA in the 8–10 nucleotide binding mode, but dissociated when RPA covered a 30-mer. Real-time analysis of RPA-ssDNA binding demonstrated that Cdc45 catalytically loaded RPA onto ssDNA. This placement reaction required physical contacts of Cdc45 with the RPA70A subdomain. Our results imply that Cdc45 controlled stabilization of the 8-nt RPA binding mode, the subsequent RPA transition into 30-mer mode and facilitated an ordered binding to ssDNA. We propose that a Cdc45-mediated loading guarantees a seamless deposition of RPA on newly emerging ssDNA at the nascent replication fork. PMID:28100698

  6. Cdc45-induced loading of human RPA onto single-stranded DNA.

    PubMed

    Szambowska, Anna; Tessmer, Ingrid; Prus, Piotr; Schlott, Bernhard; Pospiech, Helmut; Grosse, Frank

    2017-04-07

    Cell division cycle protein 45 (Cdc45) is an essential component of the eukaryotic replicative DNA helicase. We found that human Cdc45 forms a complex with the single-stranded DNA (ssDNA) binding protein RPA. Moreover, it actively loads RPA onto nascent ssDNA. Pull-down assays and surface plasmon resonance studies revealed that Cdc45-bound RPA complexed with ssDNA in the 8-10 nucleotide binding mode, but dissociated when RPA covered a 30-mer. Real-time analysis of RPA-ssDNA binding demonstrated that Cdc45 catalytically loaded RPA onto ssDNA. This placement reaction required physical contacts of Cdc45 with the RPA70A subdomain. Our results imply that Cdc45 controlled stabilization of the 8-nt RPA binding mode, the subsequent RPA transition into 30-mer mode and facilitated an ordered binding to ssDNA. We propose that a Cdc45-mediated loading guarantees a seamless deposition of RPA on newly emerging ssDNA at the nascent replication fork. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Asymmetric Regulation of Bipolar Single-stranded DNA Translocation by the Two Motors within Escherichia coli RecBCD Helicase*

    PubMed Central

    Xie, Fuqian; Wu, Colin G.; Weiland, Elizabeth; Lohman, Timothy M.

    2013-01-01

    Repair of double-stranded DNA breaks in Escherichia coli is initiated by the RecBCD helicase that possesses two superfamily-1 motors, RecB (3′ to 5′ translocase) and RecD (5′ to 3′ translocase), that operate on the complementary DNA strands to unwind duplex DNA. However, it is not known whether the RecB and RecD motors act independently or are functionally coupled. Here we show by directly monitoring ATP-driven single-stranded DNA translocation of RecBCD that the 5′ to 3′ rate is always faster than the 3′ to 5′ rate on DNA without a crossover hotspot instigator site and that the translocation rates are coupled asymmetrically. That is, RecB regulates both 3′ to 5′ and 5′ to 3′ translocation, whereas RecD only regulates 5′ to 3′ translocation. We show that the recently identified RecBC secondary translocase activity functions within RecBCD and that this contributes to the coupling. This coupling has implications for how RecBCD activity is regulated after it recognizes a crossover hotspot instigator sequence during DNA unwinding. PMID:23192341

  8. The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mak, Amanda Nga-Sze; Bradley, Philip; Cernadas, Raul A.

    2012-02-10

    DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides via unique repeat-variable diresidues (RVDs). The crystal structure of PthXo1 bound to its DNA target was determined by high-throughput computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second makes a base-specific contact to the DNA sense strand.more » Two degenerate amino-terminal repeats also interact with the DNA. Containing several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector-DNA recognition.« less

  9. A novel carbohydrate derived compound FCP5 causes DNA strand breaks and oxidative modifications of DNA bases in cancer cells.

    PubMed

    Czubatka, Anna; Sarnik, Joanna; Lucent, Del; Blasiak, Janusz; Witczak, Zbigniew J; Poplawski, Tomasz

    2015-02-05

    1,5-Anhydro-6-deoxy-methane-sulfamido-D-glucitol (FCP5) is a functionalized carbohydrate containing functional groups that render it potentially therapeutically useful. According to our concept of 'functional carb-pharmacophores' (FCPs) incorporation of the methanesulfonamido pharmacophore to 1,5 glucitol could create a therapeutically useful compound. Our previous studies revealed that FCP5 was cytotoxic to cancer cells. Therefore, in this work we assessed the cytotoxic mechanisms of FCP5 in four cancer cell lines - HeLa, LoVo, A549 and MCF-7, with particular focus on DNA damage and repair. A broad spectrum of methods, including comet assay with modifications, DNA repair enzyme assay, plasmid relaxation assay, and DNA fragmentation assay, were used. We also checked the potential for FCP5 to induce apoptosis. The results show that FCP5 can induce DNA strand breaks as well as oxidative modifications of DNA bases. DNA lesions induced by FCP5 were not entirely repaired in HeLa cells and DNA repair kinetics differs from other cell lines. Results from molecular docking and plasmid relaxation assay suggest that FCP5 binds to the major groove of DNA with a preference for adenosine-thymine base pair sequences and directly induces DNA strand breaks. Thus, FCP5 may represent a novel lead for the design of new major groove-binding compounds. The results also confirmed the validity of functional carb-pharmacophores as a new source of innovative drugs. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

    Kurian, P; Dunston, G; Lindesay, J

    2016-02-21

    Macroscopic quantum effects in living systems have been studied widely in pursuit of fundamental explanations for biological energy transport and sensing. While it is known that type II endonucleases, the largest class of restriction enzymes, induce DNA double-strand breaks by attacking phosphodiester bonds, the mechanism by which simultaneous cutting is coordinated between the catalytic centers remains unclear. We propose a quantum mechanical model for collective electronic behavior in the DNA helix, where dipole-dipole oscillations are quantized through boundary conditions imposed by the enzyme. Zero-point modes of coherent oscillations would provide the energy required for double-strand breakage. Such quanta may be preserved in the presence of thermal noise by the enzyme's displacement of water surrounding the DNA recognition sequence. The enzyme thus serves as a decoherence shield. Palindromic mirror symmetry of the enzyme-DNA complex should conserve parity, because symmetric bond-breaking ceases when the symmetry of the complex is violated or when physiological parameters are perturbed from optima. Persistent correlations in DNA across longer spatial separations-a possible signature of quantum entanglement-may be explained by such a mechanism. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    Kurian, P.; Dunston, G.; Lindesay, J.

    2015-01-01

    Macroscopic quantum effects in living systems have been studied widely in pursuit of fundamental explanations for biological energy transport and sensing. While it is known that type II endonucleases, the largest class of restriction enzymes, induce DNA double-strand breaks by attacking phosphodiester bonds, the mechanism by which simultaneous cutting is coordinated between the catalytic centers remains unclear. We propose a quantum mechanical model for collective electronic behavior in the DNA helix, where dipole-dipole oscillations are quantized through boundary conditions imposed by the enzyme. Zero-point modes of coherent oscillations would provide the energy required for double-strand breakage. Such quanta may be preserved in the presence of thermal noise by the enzyme’s displacement of water surrounding the DNA recognition sequence. The enzyme thus serves as a decoherence shield. Palindromic mirror symmetry of the enzyme-DNA complex should conserve parity, because symmetric bond-breaking ceases when the symmetry of the complex is violated or when physiological parameters are perturbed from optima. Persistent correlations in DNA across longer spatial separations—a possible signature of quantum entanglement—may be explained by such a mechanism. PMID:26682627

  12. Development of Single-Stranded DNA Aptamers for Specific Bisphenol A Detection

    PubMed Central

    Jo, Minjoung; Ahn, Ji-Young; Lee, Joohyung; Lee, Seram; Hong, Sun Woo; Yoo, Jae-Wook; Kang, Jeehye; Dua, Pooja

    2011-01-01

    The development of reagents with high affinity and specificity to small molecules is crucial for the high-throughput detection of chemical compounds, such as toxicants or pollutants. Aptamers are short and single-stranded (ss) oligonucleotides able to recognize target molecules with high affinity. Here, we report the selection of ssDNA aptamers that bind to Bisphenol A (BPA), an environmental hormone. Using SELEX process, we isolated high affinity aptamers to BPA from a 1015 random library of 60 mer ssDNAs. The selected aptamers bound specifically to BPA, but not to structurally similar molecules, such as Bisphenol B with one methyl group difference, or 4,4′-Bisphenol with 2 methyl groups difference. Using these aptamers, we developed an aptamer-based sol–gel biochip and detected BPA dissolved in water. This novel BPA aptamer-based detection can be further applied to the universal and high-specificity detection of small molecules. PMID:21413891

  13. p73 coordinates with Δ133p53 to promote DNA double-strand break repair.

    PubMed

    Gong, Hongjian; Zhang, Yuxi; Jiang, Kunpeng; Ye, Shengfan; Chen, Shuming; Zhang, Qinghe; Peng, Jinrong; Chen, Jun

    2018-03-06

    Tumour repressor p53 isoform Δ133p53 is a target gene of p53 and an antagonist of p53-mediated apoptotic activity. We recently demonstrated that Δ133p53 promotes DNA double-strand break (DSB) repair by upregulating transcription of the repair genes RAD51, LIG4 and RAD52 in a p53-independent manner. However, Δ133p53 lacks the transactivation domain of full-length p53, and the mechanism by which it exerts transcriptional activity independently of full-length p53 remains unclear. In this report, we describe the accumulation of high levels of both Δ133p53 and p73 (a p53 family member) at 24 h post γ-irradiation (hpi). Δ133p53 can form a complex with p73 upon γ-irradiation. The co-expression of Δ133p53 and p73, but not either protein alone, can significantly promote DNA DSB repair mechanisms, including homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). p73 and Δ133p53 act synergistically to promote the expression of RAD51, LIG4 and RAD52 by joining together to bind to region containing a Δ133p53-responsive element (RE) and a p73-RE in the promoters of all three repair genes. In addition to its accumulation at 24 hpi, p73 protein expression also peaks at 4 hpi. The depletion of p73 not only reduces early-stage apoptotic frequency (4-6 hpi), but also significantly increases later-stage DNA DSB accumulation (48 hpi), leading to cell cycle arrest in the G2 phase and, ultimately, cell senescence. In summary, the apoptotic regulator p73 also coordinates with Δ133p53 to promote DNA DSB repair, and the loss of function of p73 in DNA DSB repair may underlie spontaneous and carcinogen-induced tumorigenesis in p73 knockout mice.

  14. Channel Size Conversion of Phi29 DNA-Packaging Nanomotor for Discrimination of Single- and Double-Stranded Nucleic Acids

    PubMed Central

    Geng, Jia; Wang, Shaoying; Fang, Huaming; Guo, Peixuan

    2013-01-01

    Nanopores have been utilized to detect the conformation and dynamics of polymers, including DNA and RNA. Biological pores are extremely reproducible at the atomic level with uniform channel sizes. The channel of the bacterial virus phi29 DNA packaging motor is a natural conduit for the transportation of double-stranded DNA (dsDNA), and has the largest diameter among the well-studied biological channels. The larger channel facilitates translocation of dsDNA, and offers more space for further channel modification and conjugation. Interestingly, the relatively large wild type channel, which translocates dsDNA, cannot detect single-stranded nucleic acids (ssDNA or ssRNA) under the current experimental conditions. Herein, we reengineered this motor channel by removing the internal loop segment of the channel. The modification resulted in two classes of channels. One class was the same size as the wild type channel, while the other class had a cross-sectional area about 60% of the wild type. This smaller channel was able to detect the real-time translocation of single stranded nucleic acids at single-molecule level. While the wild type connector exhibited a one-way traffic property with respect to dsDNA translocation, the loop deleted connector was able to translocate ssDNA and ssRNA with equal competencies from both termini. This finding of size alterations in reengineered motor channels expands the potential application of the phi29 DNA packaging motor in nanomedicine, nanobiotechnology, and high-throughput single pore DNA sequencing. PMID:23488809

  15. Translocation of single-stranded DNA through single-walled carbon nanotubes.

    PubMed

    Liu, Haitao; He, Jin; Tang, Jinyao; Liu, Hao; Pang, Pei; Cao, Di; Krstic, Predrag; Joseph, Sony; Lindsay, Stuart; Nuckolls, Colin

    2010-01-01

    We report the fabrication of devices in which one single-walled carbon nanotube spans a barrier between two fluid reservoirs, enabling direct electrical measurement of ion transport through the tube. A fraction of the tubes pass anomalously high ionic currents. Electrophoretic transport of small single-stranded DNA oligomers through these tubes is marked by large transient increases in ion current and was confirmed by polymerase chain reaction analysis. Each current pulse contains about 10(7) charges, an enormous amplification of the translocated charge. Carbon nanotubes simplify the construction of nanopores, permit new types of electrical measurements, and may open avenues for control of DNA translocation.

  16. Programmable and multiparameter DNA-based logic platform for cancer recognition and targeted therapy.

    PubMed

    You, Mingxu; Zhu, Guizhi; Chen, Tao; Donovan, Michael J; Tan, Weihong

    2015-01-21

    The specific inventory of molecules on diseased cell surfaces (e.g., cancer cells) provides clinicians an opportunity for accurate diagnosis and intervention. With the discovery of panels of cancer markers, carrying out analyses of multiple cell-surface markers is conceivable. As a trial to accomplish this, we have recently designed a DNA-based device that is capable of performing autonomous logic-based analysis of two or three cancer cell-surface markers. Combining the specific target-recognition properties of DNA aptamers with toehold-mediated strand displacement reactions, multicellular marker-based cancer analysis can be realized based on modular AND, OR, and NOT Boolean logic gates. Specifically, we report here a general approach for assembling these modular logic gates to execute programmable and higher-order profiling of multiple coexisting cell-surface markers, including several found on cancer cells, with the capacity to report a diagnostic signal and/or deliver targeted photodynamic therapy. The success of this strategy demonstrates the potential of DNA nanotechnology in facilitating targeted disease diagnosis and effective therapy.

  17. Programmable and Multiparameter DNA-Based Logic Platform For Cancer Recognition and Targeted Therapy

    PubMed Central

    2014-01-01

    The specific inventory of molecules on diseased cell surfaces (e.g., cancer cells) provides clinicians an opportunity for accurate diagnosis and intervention. With the discovery of panels of cancer markers, carrying out analyses of multiple cell-surface markers is conceivable. As a trial to accomplish this, we have recently designed a DNA-based device that is capable of performing autonomous logic-based analysis of two or three cancer cell-surface markers. Combining the specific target-recognition properties of DNA aptamers with toehold-mediated strand displacement reactions, multicellular marker-based cancer analysis can be realized based on modular AND, OR, and NOT Boolean logic gates. Specifically, we report here a general approach for assembling these modular logic gates to execute programmable and higher-order profiling of multiple coexisting cell-surface markers, including several found on cancer cells, with the capacity to report a diagnostic signal and/or deliver targeted photodynamic therapy. The success of this strategy demonstrates the potential of DNA nanotechnology in facilitating targeted disease diagnosis and effective therapy. PMID:25361164

  18. DNA strand breakage and lipid peroxidation after exposure to welding fumes in vivo.

    PubMed

    Chuang, Cheng-Hung; Huang, Chong-En; Chen, Hsiu-Ling

    2010-01-01

    A remarkable number of complex aerosols are generated from welding processes. The objective of this study was to compare DNA damage and lipid peroxidation in plasma and in lung and in liver tissue of rats exposed to welding fumes in an exposure chamber with those of control animals. Three air samples from the chamber were also collected to assess the exposure dose for each exposure (total samplings = 18). Eight male Sprague-Dawley rats were exposed to welding fumes at a concentration of 1540.76 mg/m(3) for 10 min/day six times on day 1, day 3, day 7, day 15, day 30 and day 40. Lung, liver and kidney injury was measured following exposure, as well as in unexposed control rats (n = 4 at the beginning of the study). DNA strand breakage [tail moment (TMOM)] in exposed animals showed significant differences at day 1, day 4, day 7 and day 15 relative to the levels in control animals. Malondialdehyde (MDA, a lipid peroxidation product) levels increased gradually post-welding to 0.4 microM at 7 days. MDA and TMOM both reached maximum levels 7 days after the first exposure. At the start, an increasing trend in DNA strand breakage was more obvious than increases in MDA levels; MDA seemed to reflect long-term effects of exposure to welding fumes since it increased after 7 days and was sustained to 40 days in vivo. Significant differences in both MDA levels and DNA strand breakage were seen in lung, liver and kidney 40 days after the first fume inhalation. We conclude that acute exposure of rats to welding fumes causes noticeable oxidative damage and lipid peroxidation effects and that DNA damage may recover after long and repeat exposure. More chronic inhalation and low-dose studies are needed in order to further assess the effects of inhalation of welding fumes on DNA and to elucidate the possible causal mechanisms associated with the biologically damaging effects of welding fumes.

  19. Three-input majority logic gate and multiple input logic circuit based on DNA strand displacement.

    PubMed

    Li, Wei; Yang, Yang; Yan, Hao; Liu, Yan

    2013-06-12

    In biomolecular programming, the properties of biomolecules such as proteins and nucleic acids are harnessed for computational purposes. The field has gained considerable attention due to the possibility of exploiting the massive parallelism that is inherent in natural systems to solve computational problems. DNA has already been used to build complex molecular circuits, where the basic building blocks are logic gates that produce single outputs from one or more logical inputs. We designed and experimentally realized a three-input majority gate based on DNA strand displacement. One of the key features of a three-input majority gate is that the three inputs have equal priority, and the output will be true if any of the two inputs are true. Our design consists of a central, circular DNA strand with three unique domains between which are identical joint sequences. Before inputs are introduced to the system, each domain and half of each joint is protected by one complementary ssDNA that displays a toehold for subsequent displacement by the corresponding input. With this design the relationship between any two domains is analogous to the relationship between inputs in a majority gate. Displacing two or more of the protection strands will expose at least one complete joint and return a true output; displacing none or only one of the protection strands will not expose a complete joint and will return a false output. Further, we designed and realized a complex five-input logic gate based on the majority gate described here. By controlling two of the five inputs the complex gate can realize every combination of OR and AND gates of the other three inputs.

  20. Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines.

    PubMed

    Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E; Krishnan, Aswini R; Tsui, Tzuhan; Aguilera, Joseph A; Advani, Sunil; Crotty Alexander, Laura E; Brumund, Kevin T; Wang-Rodriguez, Jessica; Ongkeko, Weg M

    2016-01-01

    Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 h to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines

    PubMed Central

    Yu, Vicky; Rahimy, Mehran; Korrapati, Avinaash; Xuan, Yinan; Zou, Angela E.; Krishnan, Aswini R.; Tsui, Tzuhan; Aguilera, Joseph A.; Advani, Sunil; Crotty Alexander, Laura E.; Brumund, Kevin T.; Wang-Rodriguez, Jessica

    2016-01-01

    Objectives Evaluate the cytotoxicity and genotoxicity of short- and long-term e-cigarette vapor exposure on a panel of normal epithelial and head and neck squamous cell carcinoma (HNSCC) cell lines. Materials and Methods HaCaT, UMSCC10B, and HN30 were treated with nicotine-containing and nicotine-free vapor extract from two popular e-cigarette brands for periods ranging from 48 hours to 8 weeks. Cytotoxicity was assessed using Annexin V flow cytometric analysis, trypan blue exclusion, and clonogenic assays. Genotoxicity in the form of DNA strand breaks was quantified using the neutral comet assay and γ-H2AX immunostaining. Results E-cigarette-exposed cells showed significantly reduced cell viability and clonogenic survival, along with increased rates of apoptosis and necrosis, regardless of e-cigarette vapor nicotine content. They also exhibited significantly increased comet tail length and accumulation of γ-H2AX foci, demonstrating increased DNA strand breaks. Conclusion E-cigarette vapor, both with and without nicotine, is cytotoxic to epithelial cell lines and is a DNA strand break-inducing agent. Further assessment of the potential carcinogenic effects of e-cigarette vapor is urgently needed. PMID:26547127

  2. DNA mismatch-specific targeting and hypersensitivity of mismatch-repair-deficient cells to bulky rhodium(III) intercalators

    PubMed Central

    Hart, Jonathan R.; Glebov, Oleg; Ernst, Russell J.; Kirsch, Ilan R.; Barton, Jacqueline K.

    2006-01-01

    Mismatch repair (MMR) is critical to maintaining the integrity of the genome, and deficiencies in MMR are correlated with cancerous transformations. Bulky rhodium intercalators target DNA base mismatches with high specificity. Here we describe the application of bulky rhodium intercalators to inhibit cellular proliferation differentially in MMR-deficient cells compared with cells that are MMR-proficient. Preferential inhibition by the rhodium complexes associated with MMR deficiency is seen both in a human colon cancer cell line and in normal mouse fibroblast cells; the inhibition of cellular proliferation depends strictly on the MMR deficiency of the cell. Furthermore, our assay of cellular proliferation is found to correlate with DNA mismatch targeting by the bulky metallointercalators. It is the Δ-isomer that is active both in targeting base mismatches and in inhibiting DNA synthesis. Additionally, the rhodium intercalators promote strand cleavage at the mismatch site with photoactivation, and we observe that the cellular response is enhanced with photoactivation. Targeting DNA mismatches may therefore provide a cell-selective strategy for chemotherapeutic design. PMID:17030786

  3. Molecular Genetic Characterization of Mutagenesis Using a Highly Sensitive Single-Stranded DNA Reporter System in Budding Yeast.

    PubMed

    Chan, Kin

    2018-01-01

    Mutations are permanent alterations to the coding content of DNA. They are starting material for the Darwinian evolution of species by natural selection, which has yielded an amazing diversity of life on Earth. Mutations can also be the fundamental basis of serious human maladies, most notably cancers. In this chapter, I describe a highly sensitive reporter system for the molecular genetic analysis of mutagenesis, featuring controlled generation of long stretches of single-stranded DNA in budding yeast cells. This system is ~100- to ~1000-fold more susceptible to mutation than conventional double-stranded DNA reporters, and is well suited for generating large mutational datasets to investigate the properties of mutagens.

  4. [Single-molecule detection and characterization of DNA replication based on DNA origami].

    PubMed

    Wang, Qi; Fan, Youjie; Li, Bin

    2014-08-01

    To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

  5. Quantitative analysis and prediction of G-quadruplex forming sequences in double-stranded DNA

    PubMed Central

    Kim, Minji; Kreig, Alex; Lee, Chun-Ying; Rube, H. Tomas; Calvert, Jacob; Song, Jun S.; Myong, Sua

    2016-01-01

    Abstract G-quadruplex (GQ) is a four-stranded DNA structure that can be formed in guanine-rich sequences. GQ structures have been proposed to regulate diverse biological processes including transcription, replication, translation and telomere maintenance. Recent studies have demonstrated the existence of GQ DNA in live mammalian cells and a significant number of potential GQ forming sequences in the human genome. We present a systematic and quantitative analysis of GQ folding propensity on a large set of 438 GQ forming sequences in double-stranded DNA by integrating fluorescence measurement, single-molecule imaging and computational modeling. We find that short minimum loop length and the thymine base are two main factors that lead to high GQ folding propensity. Linear and Gaussian process regression models further validate that the GQ folding potential can be predicted with high accuracy based on the loop length distribution and the nucleotide content of the loop sequences. Our study provides important new parameters that can inform the evaluation and classification of putative GQ sequences in the human genome. PMID:27095201

  6. IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

    PubMed

    Liu, Mingming; Ba, Zhaoqing; Costa-Nunes, Pedro; Wei, Wei; Li, Lanxia; Kong, Fansi; Li, Yan; Chai, Jijie; Pontes, Olga; Qi, Yijun

    2017-03-01

    Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair. © 2017 American Society of Plant Biologists. All rights reserved.

  7. Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

    PubMed

    Villalobos, Michael J; Betti, Christopher J; Vaughan, Andrew T M

    2006-01-01

    Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location.

  8. Label-Free Sensitive Detection of DNA Methyltransferase by Target-Induced Hyperbranched Amplification with Zero Background Signal.

    PubMed

    Zhang, Yan; Wang, Xin-Yan; Zhang, Qianyi; Zhang, Chun-Yang

    2017-11-21

    DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-l-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments, and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH 2 -modified 3' end for the prevention of nonspecific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3'-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3'-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10 -6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.

  9. A cascade autocatalytic strand displacement amplification and hybridization chain reaction event for label-free and ultrasensitive electrochemical nucleic acid biosensing.

    PubMed

    Chen, Zhiqiang; Liu, Ying; Xin, Chen; Zhao, Jikuan; Liu, Shufeng

    2018-08-15

    Herein, an autocatalytic strand displacement amplification (ASDA) strategy was proposed for the first time, which was further ingeniously coupled with hybridization chain reaction (HCR) event for the isothermal, label-free and multiple amplification toward nucleic acid detection. During the ASDA module, the target recognition opens the immobilized hairpin probe (IP) and initiates the annealing of the auxiliary DNA strand (AS) with the opened IP for the successive polymerization and nicking reaction in the presence of DNA polymerase and nicking endonuclease. This induces the target recycling and generation of a large amount of intermediate DNA sequences, which can be used as target analogy to execute the autocatalytic strand displacement amplification. Simultaneously, the introduced AS strand can propagate the HCR between two hairpins (H1 and H2) to form a linear DNA concatamer with cytosine (C)-rich loop region, which can facilitate the in-situ synthesis of silver nanoclusters (AgNCs) as electrochemical tags for further amplification toward target responses. With current cascade ASDA and HCR strategy, the detection of target DNA could be achieved with a low detection limit of about 0.16 fM and a good selectivity. The developed biosensor also exhibits the distinct advantages of flexibility and simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. RNA-dependent RNA targeting by CRISPR-Cas9

    PubMed Central

    Strutt, Steven C; Torrez, Rachel M; Kaya, Emine; Negrete, Oscar A

    2018-01-01

    Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo. We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. These results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications. PMID:29303478

  11. Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase.

    PubMed

    Pierrat, Olivier A; Maxwell, Anthony

    2005-03-22

    Microcin B17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.

  12. Histone H3 and the histone acetyltransferase Hat1p contribute to DNA double-strand break repair.

    PubMed

    Qin, Song; Parthun, Mark R

    2002-12-01

    The modification of newly synthesized histones H3 and H4 by type B histone acetyltransferases has been proposed to play a role in the process of chromatin assembly. The type B histone acetyltransferase Hat1p and specific lysine residues in the histone H3 NH(2)-terminal tail (primarily lysine 14) are redundantly required for telomeric silencing. As many gene products, including other factors involved in chromatin assembly, have been found to participate in both telomeric silencing and DNA damage repair, we tested whether mutations in HAT1 and the histone H3 tail were also sensitive to DNA-damaging agents. Indeed, mutations both in specific lysine residues in the histone H3 tail and in HAT1 resulted in sensitivity to methyl methanesulfonate. The DNA damage sensitivity of the histone H3 and HAT1 mutants was specific for DNA double-strand breaks, as these mutants were sensitive to the induction of an exogenous restriction endonuclease, EcoRI, but not to UV irradiation. While histone H3 mutations had minor effects on nonhomologous end joining, the primary defect in the histone H3 and HAT1 mutants was in the recombinational repair of DNA double-strand breaks. Epistasis analysis indicates that the histone H3 and HAT1 mutants may influence DNA double-strand break repair through Asf1p-dependent chromatin assembly.

  13. INDUCTION OF DNA STRAND BREAKS BY TRIHALOMETHANES IN PRIMARY HUMAN LUNG EPITHELIAL CELLS

    EPA Science Inventory


    Abstract

    Trihalomethanes (TEMs) are disinfection by-products and suspected human carcinogens present in chlorinated drinking water. Previous studies have shown that many THMs induce sister chromatid exchanges and DNA strand breaks in human peripheral blood lymphocyte...

  14. Conjugated Polymers/DNA Hybrid Materials for Protein Inactivation.

    PubMed

    Zhao, Likun; Zhang, Jiangyan; Xu, Huiming; Geng, Hao; Cheng, Yongqiang

    2016-09-07

    Chromophore-assisted light inactivation (CALI) is a powerful tool for analyzing protein functions due to the high degree of spatial and temporal resolution. In this work, we demonstrate a CALI approach based on conjugated polymers (CPs)/DNA hybrid material for protein inactivation. The target protein is conjugated with single-stranded DNA in advance. Single-stranded DNA can form CPs/DNA hybrid material with cationic CPs via electrostatic and hydrophobic interactions. Through the formation of CPs/DNA hybrid material, the target protein that is conjugated with DNA is brought into close proximity to CPs. Under irradiation, CPs harvest light and generate reactive oxygen species (ROS), resulting in the inactivation of the adjacent target protein. This approach can efficiently inactivate any target protein which is conjugated with DNA and has good specificity and universality, providing a new strategy for studies of protein function and adjustment of protein activity.

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

    PubMed Central

    Muniyappa, Kalappa; Yan, Jie

    2013-01-01

    The RecA filament formed on double-stranded (ds) DNA is proposed to be a functional state analogous to that generated during the process of DNA strand exchange. RecA polymerization and de-polymerization on dsDNA is governed by multiple physiological factors. However, a comprehensive understanding of how these factors regulate the processes of polymerization and de-polymerization of RecA filament on dsDNA is still evolving. Here, we investigate the effects of temperature, pH, tensile force, and DNA ends (in particular ssDNA overhang) on the polymerization and de-polymerization dynamics of the E. coli RecA filament at a single-molecule level. Our results identified the optimal conditions that permitted spontaneous RecA nucleation and polymerization, as well as conditions that could maintain the stability of a preformed RecA filament. Further examination at a nano-meter spatial resolution, by stretching short DNA constructs, revealed a striking dynamic RecA polymerization and de-polymerization induced saw-tooth pattern in DNA extension fluctuation. In addition, we show that RecA does not polymerize on S-DNA, a recently identified novel base-paired elongated DNA structure that was previously proposed to be a possible binding substrate for RecA. Overall, our studies have helped to resolve several previous single-molecule studies that reported contradictory and inconsistent results on RecA nucleation, polymerization and stability. Furthermore, our findings also provide insights into the regulatory mechanisms of RecA filament formation and stability in vivo. PMID:23825559

  16. Quantitative Viral Community DNA Analysis Reveals the Dominance of Single-Stranded DNA Viruses in Offshore Upper Bathyal Sediment from Tohoku, Japan

    PubMed Central

    Yoshida, Mitsuhiro; Mochizuki, Tomohiro; Urayama, Syun-Ichi; Yoshida-Takashima, Yukari; Nishi, Shinro; Hirai, Miho; Nomaki, Hidetaka; Takaki, Yoshihiro; Nunoura, Takuro; Takai, Ken

    2018-01-01

    Previous studies on marine environmental virology have primarily focused on double-stranded DNA (dsDNA) viruses; however, it has recently been suggested that single-stranded DNA (ssDNA) viruses are more abundant in marine ecosystems. In this study, we performed a quantitative viral community DNA analysis to estimate the relative abundance and composition of both ssDNA and dsDNA viruses in offshore upper bathyal sediment from Tohoku, Japan (water depth = 500 m). The estimated dsDNA viral abundance ranged from 3 × 106 to 5 × 106 genome copies per cm3 sediment, showing values similar to the range of fluorescence-based direct virus counts. In contrast, the estimated ssDNA viral abundance ranged from 1 × 108 to 3 × 109 genome copies per cm3 sediment, thus providing an estimation that the ssDNA viral populations represent 96.3–99.8% of the benthic total DNA viral assemblages. In the ssDNA viral metagenome, most of the identified viral sequences were associated with ssDNA viral families such as Circoviridae and Microviridae. The principle components analysis of the ssDNA viral sequence components from the sedimentary ssDNA viral metagenomic libraries found that the different depth viral communities at the study site all exhibited similar profiles compared with deep-sea sediment ones at other reference sites. Our results suggested that deep-sea benthic ssDNA viruses have been significantly underestimated by conventional direct virus counts and that their contributions to deep-sea benthic microbial mortality and geochemical cycles should be further addressed by such a new quantitative approach. PMID:29467725

  17. Target-induced Catalytic Assembly of Y-Shaped DNA and Its Application for In Situ Imaging of MicroRNAs.

    PubMed

    Xue, Chang; Zhang, Shu-Xin; Ouyang, Chang-He; Chang, Dingran; Salena, Bruno J; Li, Yingfu; Wu, Zai-Sheng

    2018-06-14

    DNA is a highly programmable material that can be configured into unique high-order structures, such as DNA branched junctions containing multiple helical arms converging at a center. Herein we show that DNA programmability can deliver in situ growth of a 3-way junction-based DNA structure (denoted Y-shaped DNA) with the use of three hairpin-shaped DNA molecules as precursors, a specific microRNA target as a recyclable trigger, and a DNA polymerase as a driver. We demonstrate that the Y-shaped configuration comes with the benefit of restricted freedom of movement in confined cellular environment, which makes the approach ideally suited for in situ imaging of small RNA targets, such as microRNAs. Comparative analysis illustrates that the proposed imaging technique is superior to both the classic fluorescence in situ hybridization (FISH) method and an analogous amplified imaging method via programmed growth of a double-stranded DNA (rather than Y-shaped DNA) product. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    1998-01-01

    Most genetic regulatory mechanisms involve protein–DNA interactions. In these processes, the classical Watson–Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein–DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA–protein interactions. PMID:9770480

  20. Push back to respond better: regulatory inhibition of the DNA double-strand break response.

    PubMed

    Panier, Stephanie; Durocher, Daniel

    2013-10-01

    Single DNA lesions such as DNA double-strand breaks (DSBs) can cause cell death or trigger genome rearrangements that have oncogenic potential, and so the pathways that mend and signal DNA damage must be highly sensitive but, at the same time, selective and reversible. When initiated, boundaries must be set to restrict the DSB response to the site of the lesion. The integration of positive and, crucially, negative control points involving post-translational modifications such as phosphorylation, ubiquitylation and acetylation is key for building fast, effective responses to DNA damage and for mitigating the impact of DNA lesions on genome integrity.

  1. Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

    PubMed

    Thomason, Lynn C; Costantino, Nina; Court, Donald L

    2016-09-13

    Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single-strand

  2. Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV.

    PubMed

    Xiao, Qing; Min, Taishan; Ma, Shuangping; Hu, Lingna; Chen, Hongyan; Lu, Daru

    2018-04-18

    Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13-14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6-19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.

  3. The Mechanism of Double-Strand DNA Break Repair by the Nonhomologous DNA End Joining Pathway

    PubMed Central

    Lieber, Michael R.

    2011-01-01

    Double-strand DNA breaks are common events in eukaryotic cells, and there are two major pathways for repairing them: homologous recombination and nonhomologous DNA end joining (NHEJ). The diverse causes of DSBs result in a diverse chemistry of DNA ends that must be repaired. Across NHEJ evolution, the enzymes of the NHEJ pathway exhibit a remarkable degree of structural tolerance in the range of DNA end substrate configurations upon which they can act. In vertebrate cells, the nuclease, polymerases and ligase of NHEJ are the most mechanistically flexible and multifunctional enzymes in each of their classes. Unlike repair pathways for more defined lesions, NHEJ repair enzymes act iteratively, act in any order, and can function independently of one another at each of the two DNA ends being joined. NHEJ is critical not only for the repair of pathologic DSBs as in chromosomal translocations, but also for the repair of physiologic DSBs created during V(D)J recombination and class switch recombination. Therefore, patients lacking normal NHEJ are not only sensitive to ionizing radiation, but also severely immunodeficient. PMID:20192759

  4. Double stranded nucleic acid biochips

    DOEpatents

    Chernov, Boris; Golova, Julia

    2006-05-23

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

  5. Xrcc1-dependent and Ku-dependent DNA double-strand break repair kinetics in Arabidopsis plants.

    PubMed

    Charbonnel, Cyril; Gallego, Maria E; White, Charles I

    2010-10-01

    Double-strand breakage (DSB) of DNA involves loss of information on the two strands of the DNA fibre and thus cannot be repaired by simple copying of the complementary strand which is possible with single-strand DNA damage. Homologous recombination (HR) can precisely repair DSB using another copy of the genome as template and non-homologous recombination (NHR) permits repair of DSB with little or no dependence on DNA sequence homology. In addition to the well-characterised Ku-dependent non-homologous end-joining (NHEJ) pathway, much recent attention has been focused on Ku-independent NHR. The complex interrelationships and regulation of NHR pathways remain poorly understood, even more so in the case of plants, and we present here an analysis of Ku-dependent and Ku-independent repair of DSB in Arabidopsis thaliana. We have characterised an Arabidopsis xrcc1 mutant and developed quantitative analysis of the kinetics of appearance and loss of γ-H2AX foci as a tool to measure DSB repair in dividing root tip cells of γ-irradiated plants in vivo. This approach has permitted determination of DSB repair kinetics in planta following a short pulse of γ-irradiation, establishing the existence of a Ku-independent, Xrcc1-dependent DSB repair pathway. Furthermore, our data show a role for Ku80 during the first minutes post-irradiation and that Xrcc1 also plays such a role, but only in the absence of Ku. The importance of Xrcc1 is, however, clearly visible at later times in the presence of Ku, showing that alternative end-joining plays an important role in DSB repair even in the presence of active NHEJ. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

  6. Mapping yeast origins of replication via single-stranded DNA detection.

    PubMed

    Feng, Wenyi; Raghuraman, M K; Brewer, Bonita J

    2007-02-01

    Studies in th Saccharomyces cerevisiae have provided a framework for understanding how eukaryotic cells replicate their chromosomal DNA to ensure faithful transmission of genetic information to their daughter cells. In particular, S. cerevisiae is the first eukaryote to have its origins of replication mapped on a genomic scale, by three independent groups using three different microarray-based approaches. Here we describe a new technique of origin mapping via detection of single-stranded DNA in yeast. This method not only identified the majority of previously discovered origins, but also detected new ones. We have also shown that this technique can identify origins in Schizosaccharomyces pombe, illustrating the utility of this method for origin mapping in other eukaryotes.

  7. Sequence selective capture, release and analysis of DNA using a magnetic microbead-assisted toehold-mediated DNA strand displacement reaction.

    PubMed

    Khodakov, Dmitriy A; Khodakova, Anastasia S; Linacre, Adrian; Ellis, Amanda V

    2014-07-21

    This paper reports on the modification of magnetic beads with oligonucleotide capture probes with a specially designed pendant toehold (overhang) aimed specifically to capture double-stranded PCR products. After capture, the PCR products were selectively released from the magnetic beads by means of a toehold-mediated strand displacement reaction using short artificial oligonucleotide triggers and analysed using capillary electrophoresis. The approach was successfully shown on two genes widely used in human DNA genotyping, namely human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) and amelogenin.

  8. Analysis of Duck Hepatitis B Virus Reverse Transcription Indicates a Common Mechanism for the Two Template Switches during Plus-Strand DNA Synthesis

    PubMed Central

    Havert, Michael B.; Ji, Lin; Loeb, Daniel D.

    2002-01-01

    The synthesis of the hepadnavirus relaxed circular DNA genome requires two template switches, primer translocation and circularization, during plus-strand DNA synthesis. Repeated sequences serve as donor and acceptor templates for these template switches, with direct repeat 1 (DR1) and DR2 for primer translocation and 5′r and 3′r for circularization. These donor and acceptor sequences are at, or near, the ends of the minus-strand DNA. Analysis of plus-strand DNA synthesis of duck hepatitis B virus (DHBV) has indicated that there are at least three other cis-acting sequences that make contributions during the synthesis of relaxed circular DNA. These sequences, 5E, M, and 3E, are located near the 5′ end, the middle, and the 3′ end of minus-strand DNA, respectively. The mechanism by which these sequences contribute to the synthesis of plus-strand DNA was unclear. Our aim was to better understand the mechanism by which 5E and M act. We localized the DHBV 5E element to a short sequence of approximately 30 nucleotides that is 100 nucleotides 3′ of DR2 on minus-strand DNA. We found that the new 5E mutants were partially defective for primer translocation/utilization at DR2. They were also invariably defective for circularization. In addition, examination of several new DHBV M variants indicated that they too were defective for primer translocation/utilization and circularization. Thus, this analysis indicated that 5E and M play roles in both primer translocation/utilization and circularization. In conjunction with earlier findings that 3E functions in both template switches, our findings indicate that the processes of primer translocation and circularization share a common underlying mechanism. PMID:11861843

  9. p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

    PubMed

    Gong, Lu; Gong, Hongjian; Pan, Xiao; Chang, Changqing; Ou, Zhao; Ye, Shengfan; Yin, Le; Yang, Lina; Tao, Ting; Zhang, Zhenhai; Liu, Cong; Lane, David P; Peng, Jinrong; Chen, Jun

    2015-03-01

    The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

  10. Flavonoids with DNA strand-scission activity from Rhus javanica var. roxburghiana.

    PubMed

    Lin, Chun-Nan; Chen, Hui-Ling; Yen, Ming-Hong

    2008-01-01

    The flavonoids isolated from the stems of Rhus javanica var. roxburghiana, taxifolin (1), fisetin (2), fustin (3), 3,7,4'-trihydroxyflavanone (4) and 3,7,4'-trihydroxyflavone (5) caused breakage of supercoiled plasmid pBR322 DNA in the presence of Cu(II). Cu(I) was shown to be an essential intermediate by using the Cu(I)-specific sequestering reagent neocuproine. The Cu(II)-mediated DNA scissions induced by 1, 2, 3 and 5 were inhibited by the addition of catalase and exhibited DNA strand break by the addition of KI and superoxide dimutase (SOD), while in the Cu(II)-mediated DNA scissions induced by 4 was inhibited by the addition of KI, SOD, and catalase. It is concluded that 1, 2, 3, and 5 can induce H2O2 and superoxide anion, while 4 can induce OH* and H2O2 and subsequent oxidative damage of DNA in the presence of Cu(II).

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

    PubMed

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

    2006-11-10

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

  12. An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

    PubMed Central

    Wendelsdorf, Katherine V.; Song, Zhuo; Cao, Yang; Samuels, David C.

    2009-01-01

    Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication. PMID:19132079

  13. Molecular dynamics study of some non-hydrogen-bonding base pair DNA strands

    NASA Astrophysics Data System (ADS)

    Tiwari, Rakesh K.; Ojha, Rajendra P.; Tiwari, Gargi; Pandey, Vishnudatt; Mall, Vijaysree

    2018-05-01

    In order to elucidate the structural activity of hydrophobic modified DNA, the DMMO2-D5SICS, base pair is introduced as a constituent in different set of 12-mer and 14-mer DNA sequences for the molecular dynamics (MD) simulation in explicit water solvent. AMBER 14 force field was employed for each set of duplex during the 200ns production-dynamics simulation in orthogonal-box-water solvent by the Particle-Mesh-Ewald (PME) method in infinite periodic boundary conditions (PBC) to determine conformational parameters of the complex. The force-field parameters of modified base-pair were calculated by Gaussian-code using Hartree-Fock /ab-initio methodology. RMSD Results reveal that the conformation of the duplex is sequence dependent and the binding energy of the complex depends on the position of the modified base-pair in the nucleic acid strand. We found that non-bonding energy had a significant contribution to stabilising such type of duplex in comparison to electrostatic energy. The distortion produced within strands by such type of base-pair was local and destabilised the duplex integrity near to substitution, moreover the binding energy of duplex depends on the position of substitution of hydrophobic base-pair and the DNA sequence and strongly supports the corresponding experimental study.

  14. Addition of DNA to CrVI and Cytochrome b5 Containing Proteoliposomes Leads to Generation of DNA Strand Breaks and CrIII Complexes

    PubMed Central

    Borthiry, Griselda R.; Antholine, William E.; Myers, Judith M.; Myers, Charles R.

    2009-01-01

    Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b5 and NADPH :P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of CrVI (hexavalent chromium, as CrO42− ), and the generation of CrV, superoxide (O2·−) , and hydroxyl radical (HO˙). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O2. CrV is formed under both conditions, so the breaks are not mediated directly by CrV. The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO˙ from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO42− , and plasmid DNA, intense EPR signals at g = 5.7and g = 5.0 were observed. These signals are attributed to specific CrIII complexes with large zero field splitting (ZFS). Without DNA, the signals in the g = 5 region were weak. The large ZFS signals were not seen, when CrIIICl3 was incubated with DNA, suggesting that the CrIII–DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g = 2 is attributed to CrIII complexes with a small ZFS. This g = 2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific CrIII complexes that depend on the presence of plasmid DNA and the manner in which the CrIII is formed. PMID:18729091

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

    NASA Technical Reports Server (NTRS)

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

    2014-01-01

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

  16. Cascade of chromosomal rearrangements caused by a heterogeneous T-DNA integration supports the double-stranded break repair model for T-DNA integration.

    PubMed

    Hu, Yufei; Chen, Zhiyu; Zhuang, Chuxiong; Huang, Jilei

    2017-06-01

    Transferred DNA (T-DNA) from Agrobacterium tumefaciens can be integrated into the plant genome. The double-stranded break repair (DSBR) pathway is a major model for T-DNA integration. From this model, we expect that two ends of a T-DNA molecule would invade into a single DNA double-stranded break (DSB) or independent DSBs in the plant genome. We call the later phenomenon a heterogeneous T-DNA integration, which has never been observed. In this work, we demonstrated it in an Arabidopsis T-DNA insertion mutant seb19. To resolve the chromosomal structural changes caused by T-DNA integration at both the nucleotide and chromosome levels, we performed inverse PCR, genome resequencing, fluorescence in situ hybridization and linkage analysis. We found, in seb19, a single T-DNA connected two different chromosomal loci and caused complex chromosomal rearrangements. The specific break-junction pattern in seb19 is consistent with the result of heterogeneous T-DNA integration but not of recombination between two T-DNA insertions. We demonstrated that, in seb19, heterogeneous T-DNA integration evoked a cascade of incorrect repair of seven DSBs on chromosomes 4 and 5, and then produced translocation, inversion, duplication and deletion. Heterogeneous T-DNA integration supports the DSBR model and suggests that two ends of a T-DNA molecule could be integrated into the plant genome independently. Our results also show a new origin of chromosomal abnormalities. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  17. Single-stranded DNA condensed with poly-L-lysine results in nanometric particles that are significantly smaller, more stable in physiological ionic strength fluids and afford higher efficiency of gene delivery than their double-stranded counterparts.

    PubMed

    Molas, M; Bartrons, R; Perales, J C

    2002-08-15

    Nonviral gene transfer vectors have been actively studied in the past years in order to obtain structural entities with minimum size and defined shape. The final size of a gene transfer vector, which is compacted into unimolecular complexes, is directly proportional to the mass of the nucleic acid to be compacted. Thus, the purpose of this study was to assess the possibility of producing ssDNA vectors and their biophysical and biological characterization. We have obtained ssDNA/poly-L-lysine complexes that are significantly smaller than their double-stranded counterparts. We have also identified a lesser aggregative behavior of compacted single-stranded vs. double-stranded DNA vectors in the presence of physiological NaCl concentrations. Expression of compacted ssDNA is observed in hepatoma cell lines. Moreover, we have successfully delivered galactosylated ssDNA complexes into cells that express the asialoglycoprotein receptor via receptor-mediated endocytosis. The reduced size and biophysical behavior of ssDNA vectors may provide an advantage for transfection of eukaryotic cells.

  18. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, C.; Heilmann, J.; Rink, H.

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV μ -1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non

  19. Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

    PubMed

    Baumstark-Khan, C; Heilmann, J; Rink, H

    2003-01-01

    The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of

  20. Electron attachment-induced DNA single-strand breaks at the pyrimidine sites

    PubMed Central

    Gu, Jiande; Wang, Jing; Leszczynski, Jerzy

    2010-01-01

    To elucidate the contribution of pyrimidine in DNA strand breaks caused by low-energy electrons (LEEs), theoretical investigations of the LEE attachment-induced C3′–O3′, and C5′–O5′ σ bond as well as N-glycosidic bond breaking of 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate were performed using the B3LYP/DZP++ approach. The base-centered radical anions are electronically stable enough to assure that either the C–O or glycosidic bond breaking processes might compete with the electron detachment and yield corresponding radical fragments and anions. In the gas phase, the computed glycosidic bond breaking activation energy (24.1 kcal/mol) excludes the base release pathway. The low-energy barrier for the C3′–O3′ σ bond cleavage process (∼6.0 kcal/mol for both cytidine and thymidine) suggests that this reaction pathway is the most favorable one as compared to other possible pathways. On the other hand, the relatively low activation energy barrier (∼14 kcal/mol) for the C5′–O5′ σ bond cleavage process indicates that this bond breaking pathway could be possible, especially when the incident electrons have relatively high energy (a few electronvolts). The presence of the polarizable medium greatly increases the activation energies of either C–O σ bond cleavage processes or the N-glycosidic bond breaking process. The only possible pathway that dominates the LEE-induced DNA single strands in the presence of the polarizable surroundings (such as in an aqueous solution) is the C3′–O3′ σ bond cleavage (the relatively low activation energy barrier, ∼13.4 kcal/mol, has been predicted through a polarizable continuum model investigation). The qualitative agreement between the ratio for the bond breaks of C5′–O5′, C3′–O3′ and N-glycosidic bonds observed in the experiment of oligonucleotide tetramer CGAT and the theoretical sequence of the bond breaking reaction pathways have been found

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

    PubMed

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

    1994-08-11

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

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

    PubMed Central

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

    2006-01-01

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

  3. Dissimilar kinetic behavior of electrically manipulated single- and double-stranded DNA tethered to a gold surface.

    PubMed

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

    2006-05-15

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

  4. DNA-PKcs, a novel functional target of acriflavine, mediates acriflavine's p53-dependent synergistic anti-tumor efficiency with melphalan.

    PubMed

    Cao, Ji; Lin, Guanyu; Gong, Yanling; Pan, Peichen; Ma, Yaxi; Huang, Ping; Ying, Meidan; Hou, Tingjun; He, Qiaojun; Yang, Bo

    2016-12-01

    Acriflavine (ACF), a known antibacterial drug, has recently been recognized as a suitable candidate for cancer chemotherapy. However, the molecular target of ACF is not fully understood, which limits its application in cancer therapy. In this study, we established a structure-specific probe-based pull-down approach to comprehensively profile the potential target of ACF, and we identified DNA dependent protein kinase catalytic subunit (DNA-PKcs) as the direct target of ACF. Since DNA-PKcs facilitates the repair process following DNA double-strand breaks, we further developed a drug combination strategy that combined ACF with the bifunctional alkylating agent melphalan, which exerted a p53-dependent synergistic efficacy against human cancer cells both in vitro and in vivo. With these findings, our study demonstrated that structure-specific probe-based pull-down approaches can be used to identify new functional target of drug, and provided novel opportunities for the development of ACF-based antitumor chemotherapies. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

    PubMed

    Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

    2016-06-15

    The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Lighting Up the Thioflavin T by Parallel-Stranded TG(GA) n DNA Homoduplexes.

    PubMed

    Zhu, Jinbo; Yan, Zhiqiang; Zhou, Weijun; Liu, Chuanbo; Wang, Jin; Wang, Erkang

    2018-06-22

    Thioflavin T (ThT) was once regarded to be a specific fluorescent probe for the human telomeric G-quadruplex, but more other kinds of DNA were found that can also bind to ThT in recent years. Herein, we focus on G-rich parallel-stranded DNA and utilize fluorescence, absorbance, circular dichroism, and surface plasmon resonance spectroscopy to investigate its interaction with ThT. Pyrene label and molecular modeling are applied to unveil the binding mechanism. We find a new class of non-G-quadruplex G-rich parallel-stranded ( ps) DNA with the sequence of TG(GA) n can bind to ThT and increase the fluorescence with an enhancement ability superior to G-quadruplex. The optimal binding specificity for ThT is conferred by two parts. The first part is composed of two bases TG at the 5' end, which is a critical domain and plays an important role in the formation of the binding site for ThT. The second part is the rest alternative d(GA) bases, which forms the ps homoduplex and cooperates with the TG bases at the 5' end to bind the ThT.

  7. In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

    PubMed Central

    Ding, Wei; Bishop, Michelle E.; Lyn-Cook, Lascelles E.; Davis, Kelly J.; Manjanatha, Mugimane G.

    2016-01-01

    Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals. PMID:27166647

  8. In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver.

    PubMed

    Ding, Wei; Bishop, Michelle E; Lyn-Cook, Lascelles E; Davis, Kelly J; Manjanatha, Mugimane G

    2016-05-04

    Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

  9. Stabilised DNA secondary structures with increasing transcription localise hypermutable bases for somatic hypermutation in IGHV3-23.

    PubMed

    Duvvuri, Bhargavi; Duvvuri, Venkata R; Wu, Jianhong; Wu, Gillian E

    2012-07-01

    Somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID) is a transcription-coupled mechanism most responsible for generating high affinity antibodies. An issue remaining enigmatic in SHM is how AID is preferentially targeted during transcription to hypermutable bases in its substrates (WRC motifs) on both DNA strands. AID targets only single stranded DNA. By modelling the dynamical behaviour of IGHV3-23 DNA, a commonly used human variable gene segment, we observed that hypermutable bases on the non-transcribed strand are paired whereas those on transcribed strand are mostly unpaired. Hypermutable bases (both paired and unpaired) are made accessible to AID in stabilised secondary structures formed with increasing transcription levels. This observation provides a rationale for the hypermutable bases on both the strands of DNA being targeted to a similar extent despite having differences in unpairedness. We propose that increasing transcription and RNAP II stalling resulting in the formation and stabilisation of stem-loop structures with AID hotspots in negatively supercoiled region can localise the hypermutable bases of both strands of DNA, to AID-mediated SHM.

  10. Opposing roles for DNA structure-specific proteins Rad1, Msh2, Msh3, and Sgs1 in yeast gene targeting.

    PubMed

    Langston, Lance D; Symington, Lorraine S

    2005-06-15

    Targeted gene replacement (TGR) in yeast and mammalian cells is initiated by the two free ends of the linear targeting molecule, which invade their respective homologous sequences in the chromosome, leading to replacement of the targeted locus with a selectable gene from the targeting DNA. To study the postinvasion steps in recombination, we examined the effects of DNA structure-specific proteins on TGR frequency and heteroduplex DNA formation. In strains deleted of RAD1, MSH2, or MSH3, we find that the frequency of TGR is reduced and the mechanism of TGR is altered while the reverse is true for deletion of SGS1, suggesting that Rad1 and Msh2:Msh3 facilitate TGR while Sgs1 opposes it. The altered mechanism of TGR in the absence of Msh2:Msh3 and Rad1 reveals a separate role for these proteins in suppressing an alternate gene replacement pathway in which incorporation of both homology regions from a single strand of targeting DNA into heteroduplex with the targeted locus creates a mismatch between the selectable gene on the targeting DNA and the targeted gene in the chromosome.

  11. Effects of (-)-epigallocatechin gallate (EGCG) on DNA strand breaks as evaluated by single-cell gel electrophoresis (SCG) in human lymphocytes.

    PubMed

    Yuquan, L; Takeshita, T; Morimoto, K

    2001-01-01

    (-)-Epigallocatechin gallate (EGCG), a catechin polyphenol component, is the main ingredient of green tea extract. Although the anti-carcinogenic and cancer inhibitory effects of EGCG have been widely reported, its genotoxicity is not clear and seldom reported. In this study, we examined the effects of EGCG on DNA strand breaks in the isolated lymphocytes and whole blood lymphocytes obtained from two smoking subjects and a nonsmoking healthy subject using a single cell gel electrophoresis (SCG) assay. The results showed that after 2 hrs of treating the isolated lymphocytes from the smokers, EGCG induced a significant, increase in DNA strand breaks at concentrations from 2.5×10(-5) M to 2.0×10(-4) M, while after 2 hrs of treating the whole blood obtained from the same smokers, EGCG suppressed the DNA strand breaks in the lymphocytes at concentrations of 1.0×10(-4) M and 2.0×10(-4) M. A similar suppressive result was also shown in the whole blood lymphocytes from the nonsmoker at nearly the same concentrations, while at concentrations of 1.0×10(-3) M or 2.0×10(-3) M, EGCG induced a significant increase in DNA strand breaks in the whole blood lymphocytes from the nonsmoker. This result suggests that EGCG is not only inhibitory against DNA strand breaks in whole blood, but also genotoxic to the isolated or whole blood lymphocytes at high concentrations. Thus, more research is needed to comprehensively assess the effects of EGCG on genetic materials.

  12. Strand exchange of telomeric DNA catalyzed by the Werner syndrome protein (WRN) is specifically stimulated by TRF2

    PubMed Central

    Edwards, Deanna N.; Orren, David K.; Machwe, Amrita

    2014-01-01

    Werner syndrome (WS), caused by loss of function of the RecQ helicase WRN, is a hereditary disease characterized by premature aging and elevated cancer incidence. WRN has DNA binding, exonuclease, ATPase, helicase and strand annealing activities, suggesting possible roles in recombination-related processes. Evidence indicates that WRN deficiency causes telomeric abnormalities that likely underlie early onset of aging phenotypes in WS. Furthermore, TRF2, a protein essential for telomere protection, interacts with WRN and influences its basic helicase and exonuclease activities. However, these studies provided little insight into WRN's specific function at telomeres. Here, we explored the possibility that WRN and TRF2 cooperate during telomeric recombination processes. Our results indicate that TRF2, through its interactions with both WRN and telomeric DNA, stimulates WRN-mediated strand exchange specifically between telomeric substrates; TRF2's basic domain is particularly important for this stimulation. Although TRF1 binds telomeric DNA with similar affinity, it has minimal effects on WRN-mediated strand exchange of telomeric DNA. Moreover, TRF2 is displaced from telomeric DNA by WRN, independent of its ATPase and helicase activities. Together, these results suggest that TRF2 and WRN act coordinately during telomeric recombination processes, consistent with certain telomeric abnormalities associated with alteration of WRN function. PMID:24880691

  13. Sequence-Dependent Elasticity and Electrostatics of Single-Stranded DNA: Signatures of Base-Stacking

    PubMed Central

    McIntosh, Dustin B.; Duggan, Gina; Gouil, Quentin; Saleh, Omar A.

    2014-01-01

    Base-stacking is a key factor in the energetics that determines nucleic acid structure. We measure the tensile response of single-stranded DNA as a function of sequence and monovalent salt concentration to examine the effects of base-stacking on the mechanical and thermodynamic properties of single-stranded DNA. By comparing the elastic response of highly stacked poly(dA) and that of a polypyrimidine sequence with minimal stacking, we find that base-stacking in poly(dA) significantly enhances the polymer’s rigidity. 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 mean-field predictions. Further, we provide a model-independent estimate of the free energy difference between stacked poly(dA) and unstacked polypyrimidine, finding it to be ∼−0.25 kBT/base and nearly constant over three orders of magnitude in salt concentration. PMID:24507606

  14. Gener: a minimal programming module for chemical controllers based on DNA strand displacement

    PubMed Central

    Kahramanoğulları, Ozan; Cardelli, Luca

    2015-01-01

    Summary: Gener is a development module for programming chemical controllers based on DNA strand displacement. Gener is developed with the aim of providing a simple interface that minimizes the opportunities for programming errors: Gener allows the user to test the computations of the DNA programs based on a simple two-domain strand displacement algebra, the minimal available so far. The tool allows the user to perform stepwise computations with respect to the rules of the algebra as well as exhaustive search of the computation space with different options for exploration and visualization. Gener can be used in combination with existing tools, and in particular, its programs can be exported to Microsoft Research’s DSD tool as well as to LaTeX. Availability and implementation: Gener is available for download at the Cosbi website at http://www.cosbi.eu/research/prototypes/gener as a windows executable that can be run on Mac OS X and Linux by using Mono. Contact: ozan@cosbi.eu PMID:25957353

  15. Gener: a minimal programming module for chemical controllers based on DNA strand displacement.

    PubMed

    Kahramanoğulları, Ozan; Cardelli, Luca

    2015-09-01

    : Gener is a development module for programming chemical controllers based on DNA strand displacement. Gener is developed with the aim of providing a simple interface that minimizes the opportunities for programming errors: Gener allows the user to test the computations of the DNA programs based on a simple two-domain strand displacement algebra, the minimal available so far. The tool allows the user to perform stepwise computations with respect to the rules of the algebra as well as exhaustive search of the computation space with different options for exploration and visualization. Gener can be used in combination with existing tools, and in particular, its programs can be exported to Microsoft Research's DSD tool as well as to LaTeX. Gener is available for download at the Cosbi website at http://www.cosbi.eu/research/prototypes/gener as a windows executable that can be run on Mac OS X and Linux by using Mono. ozan@cosbi.eu. © The Author 2015. Published by Oxford University Press.

  16. RNA-dependent RNA targeting by CRISPR-Cas9

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine

    Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

  17. RNA-dependent RNA targeting by CRISPR-Cas9

    DOE PAGES

    Strutt, Steven C.; Torrez, Rachel M.; Kaya, Emine; ...

    2018-01-05

    Double-stranded DNA (dsDNA) binding and cleavage by Cas9 is a hallmark of type II CRISPR-Cas bacterial adaptive immunity. All known Cas9 enzymes are thought to recognize DNA exclusively as a natural substrate, providing protection against DNA phage and plasmids. Here, we show that Cas9 enzymes from both subtypes II-A and II-C can recognize and cleave single-stranded RNA (ssRNA) by an RNA-guided mechanism that is independent of a protospacer-adjacent motif (PAM) sequence in the target RNA. RNA-guided RNA cleavage is programmable and site-specific, and we find that this activity can be exploited to reduce infection by single-stranded RNA phage in vivo.more » We also demonstrate that Cas9 can direct PAM-independent repression of gene expression in bacteria. In conclusion, these results indicate that a subset of Cas9 enzymes have the ability to act on both DNA and RNA target sequences, and suggest the potential for use in programmable RNA targeting applications.« less

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

    PubMed Central

    SOLANKY, DIPESH; HAYDEL, SHELLEY E.

    2012-01-01

    This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which primarily associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays. PMID:22940101

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

    PubMed Central

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-01-01

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

  20. In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

    PubMed

    Martini, Laura; Meyer, Adam J; Ellefson, Jared W; Milligan, John N; Forlin, Michele; Ellington, Andrew D; Mansy, Sheref S

    2015-10-16

    An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

  1. PNA binding to the non-template DNA strand interferes with transcription, suggesting a blockage mechanism mediated by R-loop formation.

    PubMed

    Belotserkovskii, Boris P; Hanawalt, Philip C

    2015-11-01

    Peptide Nucleic Acids (PNAs) are artificial DNA mimics with superior nucleic acid binding capabilities. T7 RNA polymerase (T7 RNAP) transcription upon encountering PNA bound to the non-template DNA strand was studied in vitro. A characteristic pattern of blockage signals was observed, extending downstream from the PNA binding site, similar to that produced by G-rich homopurine-homopyrimidine (hPu-hPy) sequences and likely caused by R-loop formation. Since blocked transcription complexes in association with stable R-loops may interfere with replication and in some cases trigger apoptosis, targeted R-loop formation might be employed to inactivate selected cells, such as those in tumors, based upon their unique complement of expressed genes. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    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

  4. 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

  5. Strand Invasion Based Amplification (SIBA®): a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

    PubMed

    Hoser, Mark J; Mansukoski, Hannu K; Morrical, Scott W; Eboigbodin, Kevin E

    2014-01-01

    Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

  6. Biotechnological mass production of DNA origami

    NASA Astrophysics Data System (ADS)

    Praetorius, Florian; Kick, Benjamin; Behler, Karl L.; Honemann, Maximilian N.; Weuster-Botz, Dirk; Dietz, Hendrik

    2017-12-01

    DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising ‘cassettes’, with each cassette comprising two Zn2+-dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in

  7. Biotechnological mass production of DNA origami.

    PubMed

    Praetorius, Florian; Kick, Benjamin; Behler, Karl L; Honemann, Maximilian N; Weuster-Botz, Dirk; Dietz, Hendrik

    2017-12-06

    DNA nanotechnology, in particular DNA origami, enables the bottom-up self-assembly of micrometre-scale, three-dimensional structures with nanometre-precise features. These structures are customizable in that they can be site-specifically functionalized or constructed to exhibit machine-like or logic-gating behaviour. Their use has been limited to applications that require only small amounts of material (of the order of micrograms), owing to the limitations of current production methods. But many proposed applications, for example as therapeutic agents or in complex materials, could be realized if more material could be used. In DNA origami, a nanostructure is assembled from a very long single-stranded scaffold molecule held in place by many short single-stranded staple oligonucleotides. Only the bacteriophage-derived scaffold molecules are amenable to scalable and efficient mass production; the shorter staple strands are obtained through costly solid-phase synthesis or enzymatic processes. Here we show that single strands of DNA of virtually arbitrary length and with virtually arbitrary sequences can be produced in a scalable and cost-efficient manner by using bacteriophages to generate single-stranded precursor DNA that contains target strand sequences interleaved with self-excising 'cassettes', with each cassette comprising two Zn 2+ -dependent DNA-cleaving DNA enzymes. We produce all of the necessary single strands of DNA for several DNA origami using shaker-flask cultures, and demonstrate end-to-end production of macroscopic amounts of a DNA origami nanorod in a litre-scale stirred-tank bioreactor. Our method is compatible with existing DNA origami design frameworks and retains the modularity and addressability of DNA origami objects that are necessary for implementing custom modifications using functional groups. With all of the production and purification steps amenable to scaling, we expect that our method will expand the scope of DNA nanotechnology in many

  8. IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration.

    PubMed

    Stewart, Barry J; Wardle, Simon J; Haniford, David B

    2002-08-15

    The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This strand transfer transpososome (STT) does appear to support an intermolecular disintegration reaction, but only at a very low level. Strikingly, assembly of the STT is not dependent on IHF, a host protein that is required for de novo assembly of all previously characterized Tn10 transpososomes. We suggest that disintegration substrates are able to form both transposon end and target type contacts with transposase because of their enhanced conformational flexibility. This probably allows the conformation of DNA within the complex that prevents the destructive disintegration reaction, and is responsible for relaxing the DNA sequence requirements for STT formation relative to other Tn10 transpososomes.

  9. IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration

    PubMed Central

    Stewart, Barry J.; Wardle, Simon J.; Haniford, David B.

    2002-01-01

    The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This strand transfer transpososome (STT) does appear to support an intermolecular disintegration reaction, but only at a very low level. Strikingly, assembly of the STT is not dependent on IHF, a host protein that is required for de novo assembly of all previously characterized Tn10 transpososomes. We suggest that disintegration substrates are able to form both transposon end and target type contacts with transposase because of their enhanced conformational flexibility. This probably allows the conformation of DNA within the complex that prevents the destructive disintegration reaction, and is responsible for relaxing the DNA sequence requirements for STT formation relative to other Tn10 transpososomes. PMID:12169640

  10. Double-stranded DNA organization in bacteriophage heads: an alternative toroid-based model.

    PubMed Central

    Hud, N V

    1995-01-01

    Studies of the organization of double-stranded DNA within bacteriophage heads during the past four decades have produced a wealth of data. However, despite the presentation of numerous models, the true organization of DNA within phage heads remains unresolved. The observations of toroidal DNA structures in electron micrographs of phage lysates have long been cited as support for the organization of DNA in a spool-like fashion. This particular model, like all other models, has not been found to be consistent will all available data. Recently we proposed that DNA within toroidal condensates produced in vitro is organized in a manner significantly different from that suggested by the spool model. This new toroid model has allowed the development of an alternative model for DNA organization within bacteriophage heads that is consistent with a wide range of biophysical data. Here we propose that bacteriophage DNA is packaged in a toroid that is folded into a highly compact structure. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:8534805

  11. Identification and molecular characterization of a novel circular single-stranded DNA virus associated with yerba mate in Argentina.

    PubMed

    Bejerman, Nicolás; de Breuil, Soledad; Nome, Claudia

    2018-06-06

    A single-stranded DNA (ssDNA) virus was detected in Yerba mate samples showing chlorotic linear patterns, chlorotic rings and vein yellowing. The full-genome sequences of six different isolates of this ssDNA circular virus were obtained, which share > 99% sequence identity with each other. The newly identified virus has been tentatively named as yerba mate-associated circular DNA virus (YMaCV). The 2707 nt-long viral genome has two and three open reading frame on its complementary and virion-sense strands, respectively. The coat protein is more similar to that of mastreviruses (44% identity), whereas the replication-associated protein of YMaCV is more similar (49% identity) to that encoded by a recently described, unclassified ssDNA virus isolated on trees in Brazil. This is the first report of a circular DNA virus associated with yerba mate. Its unique genome organization and phylogenetic relationships indicates that YMaCV represents a distinct evolutionary lineage within the ssDNA viruses and therefore this virus should be classified as a member of a new species within an unassigned genus or family.

  12. APOBEC3B cytidine deaminase targets the non-transcribed strand of tRNA genes in yeast.

    PubMed

    Saini, Natalie; Roberts, Steven A; Sterling, Joan F; Malc, Ewa P; Mieczkowski, Piotr A; Gordenin, Dmitry A

    2017-05-01

    Variations in mutation rates across the genome have been demonstrated both in model organisms and in cancers. This phenomenon is largely driven by the damage specificity of diverse mutagens and the differences in DNA repair efficiency in given genomic contexts. Here, we demonstrate that the single-strand DNA-specific cytidine deaminase APOBEC3B (A3B) damages tRNA genes at a 1000-fold higher efficiency than other non-tRNA genomic regions in budding yeast. We found that A3B-induced lesions in tRNA genes were predominantly located on the non-transcribed strand, while no transcriptional strand bias was observed in protein coding genes. Furthermore, tRNA gene mutations were exacerbated in cells where RNaseH expression was completely abolished (Δrnh1Δrnh35). These data suggest a transcription-dependent mechanism for A3B-induced tRNA gene hypermutation. Interestingly, in strains proficient in DNA repair, only 1% of the abasic sites formed upon excision of A3B-deaminated cytosines were not repaired leading to mutations in tRNA genes, while 18% of these lesions failed to be repaired in the remainder of the genome. A3B-induced mutagenesis in tRNA genes was found to be efficiently suppressed by the redundant activities of both base excision repair (BER) and the error-free DNA damage bypass pathway. On the other hand, deficiencies in BER did not have a profound effect on A3B-induced mutations in CAN1, the reporter for protein coding genes. We hypothesize that differences in the mechanisms underlying ssDNA formation at tRNA genes and other genomic loci are the key determinants of the choice of the repair pathways and consequently the efficiency of DNA damage repair in these regions. Overall, our results indicate that tRNA genes are highly susceptible to ssDNA-specific DNA damaging agents. However, increased DNA repair efficacy in tRNA genes can prevent their hypermutation and maintain both genome and proteome homeostasis. Published by Elsevier B.V.

  13. Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction

    NASA Astrophysics Data System (ADS)

    Park, Yeonkyung; Lee, Chang Yeol; Kang, Shinyoung; Kim, Hansol; Park, Ki Soo; Park, Hyun Gyu

    2018-02-01

    In this work, we developed a novel, label-free, and enzyme-free strategy for the colorimetric detection of microRNA (miRNA), which relies on a target-catalyzed toehold-mediated strand displacement (TMSD) reaction. The system employs a detection probe that specifically binds to the target miRNA and sequentially releases a catalyst strand (CS) intended to trigger the subsequent TMSD reaction. Thus, the presence of target miRNA releases the CS that mediates the formation of an active G-quadruplex DNAzyme which is initially caged and inactivated by a blocker strand. In addition, a fuel strand that is supplemented for the recycling of the CS promotes another TMSD reaction, consequently generating a large number of active G-quadruplex DNAzymes. As a result, a distinct colorimetric signal is produced by the ABTS oxidation promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Based on this novel strategy, we successfully detected miR-141, a promising biomarker for human prostate cancer, with high selectivity. The diagnostic capability of this system was also demonstrated by reliably determining target miR-141 in human serum, showing its great potential towards real clinical applications. Importantly, the proposed approach is composed of separate target recognition and signal transduction modules. Thus, it could be extended to analyze different target miRNAs by simply redesigning the detection probe while keeping the same signal transduction module as a universal signal amplification unit, which was successfully demonstrated by analyzing another target miRNA, let-7d.

  14. Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction.

    PubMed

    Park, Yeonkyung; Lee, Chang Yeol; Kang, Shinyoung; Kim, Hansol; Park, Ki Soo; Park, Hyun Gyu

    2018-02-23

    In this work, we developed a novel, label-free, and enzyme-free strategy for the colorimetric detection of microRNA (miRNA), which relies on a target-catalyzed toehold-mediated strand displacement (TMSD) reaction. The system employs a detection probe that specifically binds to the target miRNA and sequentially releases a catalyst strand (CS) intended to trigger the subsequent TMSD reaction. Thus, the presence of target miRNA releases the CS that mediates the formation of an active G-quadruplex DNAzyme which is initially caged and inactivated by a blocker strand. In addition, a fuel strand that is supplemented for the recycling of the CS promotes another TMSD reaction, consequently generating a large number of active G-quadruplex DNAzymes. As a result, a distinct colorimetric signal is produced by the ABTS oxidation promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Based on this novel strategy, we successfully detected miR-141, a promising biomarker for human prostate cancer, with high selectivity. The diagnostic capability of this system was also demonstrated by reliably determining target miR-141 in human serum, showing its great potential towards real clinical applications. Importantly, the proposed approach is composed of separate target recognition and signal transduction modules. Thus, it could be extended to analyze different target miRNAs by simply redesigning the detection probe while keeping the same signal transduction module as a universal signal amplification unit, which was successfully demonstrated by analyzing another target miRNA, let-7d.

  15. Efficiencies of induction of DNA double strand breaks in solution by photoabsorption at phosphorus and platinum.

    PubMed

    Maeda, Munetoshi; Kobayashi, Katsumi; Hieda, Kotaro

    2004-01-01

    This paper aims at determining and comparing the cross sections and quantum yields for DNA strand break induction by the Auger effect at the K-shell of phosphorus and at the LIII-shell of platinum. Using synchrotron radiation, free and Pt-bound pBR322 plasmid DNA were irradiated in solution with monochromatic X-rays, the energies of which were 2.153 and 2.147 keV, corresponding to "on" and "below" the phosphorus K-shell photoabsorption, and 11.566 and 11.542 keV for "above" and "below" the L(III)-shell photoabsorption of platinum, respectively. To suppress indirect effects by hydroxyl radicals, DMSO (1M) was used as a scavenger. The inner-shell photoabsorption of phosphorus and of platinum significantly increased the induction of DNA double strand breaks (DSB), whereas it had little effect on single strand break (SSB) induction. The quantum yields for the induction of DSB were calculated to be 0.017 and 1.13, in the case of phosphorus and platinum, respectively. CONCLSIONS: The value of the quantum yield for the DSB induction of platinum was about 66-fold larger than that for the phosphorus. These results clearly demonstrate that the quantum yield of DSB depends upon the magnitude of the Auger cascade.

  16. CdS nanowires formed by chemical synthesis using conjugated single-stranded DNA molecules

    NASA Astrophysics Data System (ADS)

    Sarangi, S. N.; Sahu, S. N.; Nozaki, S.

    2018-03-01

    CdS nanowires were successfully grown by chemical synthesis using two conjugated single-stranded (ss) DNA molecules, poly G (30) and poly C (30), as templates. During the early stage of the synthesis with the DNA molecules, the Cd 2+ interacts with Poly G and Poly C and produces the (Cd 2+)-Poly GC complex. As the growth proceeds, it results in nanowires. The structural analysis by grazing angle x-ray diffraction and transmission electron microscopy confirmed the zinc-blende CdS nanowires with the growth direction of <220>. Although the nanowires are well surface-passivated with the DNA molecules, the photoluminescence quenching was caused by the electron transfer from the nanowires to the DNA molecules. The quenching can be used to detect and label the DNAs.

  17. Absolute cross-sections for DNA strand breaks and crosslinks induced by low energy electrons

    PubMed Central

    Chen, Wenzhuang; Chen, Shiliang; Dong, Yanfang; Cloutier, Pierre; Sanche, Léon

    2016-01-01

    Absolute cross sections (CSs) for the interaction of low energy electrons with condensed macromolecules are essential parameters to accurately model ionizing radiation induced reactions. To determine CSs for various conformational DNA damage induced by 2–20 eV electrons, we investigated the influence of the attenuation length (AL) and penetration factor (f) using a mathematical model. Solid films of super-coiled plasmid DNA with thicknesses of 10, 15 and 20 nm were irradiated with 4.6, 5.6, 9.6 and 14.6 eV electrons. DNA conformational changes were quantified by gel electrophoresis, and the respective yields were extrapolated from exposure–response curves. The absolute CS, AL and f values were generated by applying the model developed by Rezaee et al. The values of AL were found to lie between 11 and 16 nm with the maximum at 14.6 eV. The absolute CSs for the loss of the supercoiled (LS) configuration and production of crosslinks (CL), single strand breaks (SSB) and double strand breaks (DSB) induced by 4.6, 5.6, 9.6 and 14.6 eV electrons are obtained. The CSs for SSB are smaller, but similar to those for LS, indicating that SSB are the main conformational damage. The CSs for DSB and CL are about one order of magnitude smaller than those of LS and SSB. The value of f is found to be independent of electron energy, which allows extending the absolute CSs for these types of damage within the range 2–20 eV, from previous measurements of effective CSs. When comparison is possible, the absolute CSs are found to be in good agreement with those obtained from previous similar studies with double-stranded DNA. The high values of the absolute CSs of 4.6 and 9.6 eV provide quantitative evidence for the high efficiency of low energy electrons to induce DNA damage via the formation of transient anions. PMID:27878170

  18. Absolute cross-sections for DNA strand breaks and crosslinks induced by low energy electrons.

    PubMed

    Chen, Wenzhuang; Chen, Shiliang; Dong, Yanfang; Cloutier, Pierre; Zheng, Yi; Sanche, Léon

    2016-12-07

    Absolute cross sections (CSs) for the interaction of low energy electrons with condensed macromolecules are essential parameters to accurately model ionizing radiation induced reactions. To determine CSs for various conformational DNA damage induced by 2-20 eV electrons, we investigated the influence of the attenuation length (AL) and penetration factor (f) using a mathematical model. Solid films of supercoiled plasmid DNA with thicknesses of 10, 15 and 20 nm were irradiated with 4.6, 5.6, 9.6 and 14.6 eV electrons. DNA conformational changes were quantified by gel electrophoresis, and the respective yields were extrapolated from exposure-response curves. The absolute CS, AL and f values were generated by applying the model developed by Rezaee et al. The values of AL were found to lie between 11 and 16 nm with the maximum at 14.6 eV. The absolute CSs for the loss of the supercoiled (LS) configuration and production of crosslinks (CL), single strand breaks (SSB) and double strand breaks (DSB) induced by 4.6, 5.6, 9.6 and 14.6 eV electrons are obtained. The CSs for SSB are smaller, but similar to those for LS, indicating that SSB are the main conformational damage. The CSs for DSB and CL are about one order of magnitude smaller than those of LS and SSB. The value of f is found to be independent of electron energy, which allows extending the absolute CSs for these types of damage within the range 2-20 eV, from previous measurements of effective CSs. When comparison is possible, the absolute CSs are found to be in good agreement with those obtained from previous similar studies with double-stranded DNA. The high values of the absolute CSs of 4.6 and 9.6 eV provide quantitative evidence for the high efficiency of low energy electrons to induce DNA damage via the formation of transient anions.

  19. Manipulation of double-stranded DNA melting by force

    NASA Astrophysics Data System (ADS)

    Singh, Amit Raj; Granek, Rony

    2017-09-01

    By integrating elasticity—as described by the Gaussian network model—with bond binding energies that distinguish between different base-pair identities and stacking configurations, we study the force induced melting of a double-stranded DNA (dsDNA). Our approach is a generalization of our previous study of thermal dsDNA denaturation [J. Chem. Phys. 145, 144101 (2016), 10.1063/1.4964285] to that induced by force at finite temperatures. It allows us to obtain semimicroscopic information about the opening of the chain, such as whether the dsDNA opens from one of the ends or from the interior, forming an internal bubble. We study different types of force manipulation: (i) "end unzipping," with force acting at a single end base pair perpendicular to the helix, (ii) "midunzipping," with force acting at a middle base pair perpendicular to the helix, and (iii) "end shearing," where the force acts at opposite ends along the helix. By monitoring the free-energy landscape and probability distribution of intermediate denaturation states, we show that different dominant intermediate states are stabilized depending on the type of force manipulation used. In particular, the bubble state of the sequence L60B36, which we have previously found to be a stable state during thermal denaturation, is absent for end unzipping and end shearing, whereas very similar bubbles are stabilized by midunzipping, or when the force location is near the middle of the chain. Ours results offer a simple tool for stabilizing bubbles and loops using force manipulations at different temperatures, and may implicate on the mechanism in which DNA enzymes or motors open regions of the chain.

  20. Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hata, Kuniki; Advanced Science Research Center, Japan Atomic Energy Agency, 2-4 Shirakatashirane, Tokai-mura, Naka-gun, Ibaraki 319-1195; Urushibara, Ayumi

    Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield ofmore » DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.« less