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Sample records for dna synthesis clarification

  1. Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts

    SciTech Connect

    Flier, J.S.; Usher, P.; Moses, A.C.

    1986-02-01

    Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of (/sup 3/H)thymidine into DNA in these cells, the identify of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody ..cap alpha..IR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of /sup 125/I-labeled IGF-I but not /sup 125/I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. ..cap alpha..IR-3 competitively inhibits IGF-I-mediated stimulation of (/sup 3/H)thymidine incorporation into DNA. This inhibition is dependent on the concentration of ..cap alpha..IR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of < 1 ..mu..g/ml, the effect of insulin to stimulate (/sup 3/H)thymidine incorporation is not inhibited by ..cap alpha..IR-3. However, the incremental effects of higher concentrations (> 1 ..mu..g/ml) of insulin on (/sup 3/H)thymidine incorporation are inhibited by ..cap alpha..IR-3. ..cap alpha..IR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself.

  2. DNA synthesis security.

    PubMed

    Nouri, Ali; Chyba, Christopher F

    2012-01-01

    It is generally assumed that genetic engineering advances will, inevitably, facilitate the misapplication of biotechnology toward the production of biological weapons. Unexpectedly, however, some of these very advances in the areas of DNA synthesis and sequencing may enable the implementation of automated and nonintrusive safeguards to avert the illicit applications of biotechnology. In the case of DNA synthesis, automated DNA screening tools could be built into DNA synthesizers in order to block the synthesis of hazardous agents. In addition, a comprehensive safety and security regime for dual-use genetic engineering research could include nonintrusive monitoring of DNA sequencing. This is increasingly feasible as laboratories outsource this service to just a few centralized sequencing factories. The adoption of automated, nonintrusive monitoring and surveillance of the DNA synthesis and sequencing pipelines may avert many risks associated with dual-use biotechnology. Here, we describe the historical background and current challenges associated with dual-use biotechnologies and propose strategies to address these challenges.

  3. Synthesis of DNA

    DOEpatents

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  4. Biphasic DNA Synthesis in Spumaviruses

    PubMed Central

    Delelis, Olivier; Saïb, Ali; Sonigo, Pierre

    2003-01-01

    Spumaviruses are complex retroviruses whose replication cycle resembles that of hepadnaviruses, especially by a late-occurring reverse transcription step. The possible existence of an early reverse transcription as observed in other retroviruses was not documented. Using real-time quantitative PCR, we addressed directly the kinetics of DNA synthesis during spumavirus infection. An early phase of viral DNA synthesis developed until 3 h postinfection, followed by a second phase, culminating 10 h postinfection. Both phases were abolished by the reverse transcriptase inhibitor 3′-azido-3′-deoxythymidine. Similar to other retroviruses, circular forms of viral DNA harboring two long terminal repeats were mainly found in the nucleus of infected cells. Interestingly, a fraction of these circular forms were detected in the cytoplasm and in extracellular virions, a feature shared with hepadnaviruses. Combined with packaging of both viral DNA and RNA genomes in virions, early and late reverse transcription might allow spumavirus to maximize its genome replication. PMID:12829852

  5. Concepts in Biochemistry: Chemical Synthesis of DNA.

    ERIC Educational Resources Information Center

    Caruthers, Marvin H.

    1989-01-01

    Outlines the chemistry of the rapid synthesis of relatively large DNA fragments (100-200 monomers each) with yields exceeding 99 percent per coupling. DNA synthesis methodologies are outlined and a polymer-supported synthesis of DNA using deoxynucleoside phosphoramidites is described with structural formulas. (YP)

  6. Concepts in Biochemistry: Chemical Synthesis of DNA.

    ERIC Educational Resources Information Center

    Caruthers, Marvin H.

    1989-01-01

    Outlines the chemistry of the rapid synthesis of relatively large DNA fragments (100-200 monomers each) with yields exceeding 99 percent per coupling. DNA synthesis methodologies are outlined and a polymer-supported synthesis of DNA using deoxynucleoside phosphoramidites is described with structural formulas. (YP)

  7. Enzymatic initiation of DNA synthesis by yeast DNA polymerases.

    PubMed Central

    Plevani, P; Chang, L M

    1977-01-01

    Partially purified yeast RNA polymerases (RNA nucleotidyltransferases) initiate DNA synthesis by yeast DNA polymerase (DNA nucleotidyltransferase) I and to a lesser extent yeast DNA polymerase II in the replication of single-stranded DNA. The enzymatic initiation of DNA synthesis on phage fd DNA template occurs with dNTPs alone and is further stimulated by the presence of rNTPs in DNA polymerase I reactions. The presence of rNTPs has no effect on the RNA polymerase initiation of the DNA polymerase II reaction. RNA polymerases I and III are more efficient in initiation of DNA synthesis than RNA polymerase II. Analyses of the products of fd DNA replication show noncovalent linkage between the newly synthesized DNA and the template DNA, and covalent linkage between the newly synthesized RNA and DNA. PMID:325562

  8. SMFM Statement: clarification of recommendations regarding cell-free DNA aneuploidy screening.

    PubMed

    2015-12-01

    The purpose of this statement is to clarify that the Society for Maternal-Fetal Medicine (SMFM) does not recommend that cell-free DNA aneuploidy screening be offered to all pregnant women, nor does it suggest a requirement for insurance coverage for cell-free DNA screening in women at low risk of aneuploidy. However, SMFM believes, due to the ethics of patient autonomy, that the option should be available to women who request additional testing beyond what is currently recommended by professional societies. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Initiation of lymphocyte DNA synthesis.

    PubMed

    Coffman, F D; Fresa, K L; Cohen, S

    1991-01-01

    The initiation of DNA replication in T lymphocytes appears to be regulated by two distinct activities: one associated with proliferation which mediates initiation, and another associated with quiescence which blocks initiation. Activated lymphocytes and proliferating lymphoid cell lines produce an activity, termed ADR, which can initiate DNA replication in isolated, quiescent nuclei. ADR is heat-labile, has protease activity or interacts closely with a protease, and is distinct from the DNA polymerases. ADR activity is absent in quiescent lymphocytes and appears in mitogen-stimulated lymphocytes after IL-2 binding. The generation of active ADR appears to be mediated by phosphorylation of a precursor which is present in resting cells. Nuclei from mitogen-unresponsive lymphocytes fail to initiate DNA replication in response to ADR, of potential importance in the age-related decline of immunity. Quiescent lymphocytes lack ADR and synthesize an ADR-inhibitory activity. The ADR inhibitor is a heat-stable protein which suppresses the initiation of DNA synthesis, but is ineffective at suppressing elongation once DNA strand replication has begun. Nuclei from several neoplastic cell lines fail to respond to the ADR inhibitor, which may play a role in the continuous proliferation of these cells. At least one of these neoplastic cell lines produces both ADR and an inhibitory factor. These findings suggest that the regulation of proliferation is dependent on the balance between activating and inhibitory pathways.

  10. Mechanism for CCC DNA synthesis in hepadnaviruses.

    PubMed

    Sohn, Ji A; Litwin, Samuel; Seeger, Christoph

    2009-11-30

    Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC) DNA from the relaxed circular (RC) viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT), or cellular DNA repair enzymes, or both. Physical mapping of the 5' and 3' ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5' ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1) invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2) predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5' end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.

  11. Herpes Simplex Virus DNA Packaging without Measurable DNA Synthesis

    PubMed Central

    Church, Geoffrey A.; Dasgupta, Anindya; Wilson, Duncan W.

    1998-01-01

    Herpes simplex virus (HSV) type 1 DNA synthesis and packaging occur within the nuclei of infected cells; however, the extent to which the two processes are coupled remains unclear. Correct packaging is thought to be dependent upon DNA debranching or other repair processes, and such events commonly involve new DNA synthesis. Furthermore, the HSV UL15 gene product, essential for packaging, nevertheless localizes to sites of active DNA replication and may link the two events. It has previously been difficult to determine whether packaging requires concomitant DNA synthesis due to the complexity of these processes and of the viral life cycle; however, we have recently described a model system which simplifies the study of HSV assembly. Cells infected with HSV strain tsProt.A accumulate unpackaged capsids at the nonpermissive temperature of 39°C. Following release of the temperature block, these capsids proceed to package viral DNA in a single, synchronous wave. Here we report that, when DNA replication was inhibited prior to release of the temperature block, DNA packaging and later events in viral assembly nevertheless occurred at near-normal levels. We conclude that, under our conditions, HSV DNA packaging does not require detectable levels of DNA synthesis. PMID:9525593

  12. Translesion DNA Synthesis and Mutagenesis in Eukaryotes

    PubMed Central

    Sale, Julian E.

    2013-01-01

    The structural features that enable replicative DNA polymerases to synthesize DNA rapidly and accurately also limit their ability to copy damaged DNA. Direct replication of DNA damage is termed translesion synthesis (TLS), a mechanism conserved from bacteria to mammals and executed by an array of specialized DNA polymerases. This chapter examines how these translesion polymerases replicate damaged DNA and how they are regulated to balance their ability to replicate DNA lesions with the risk of undesirable mutagenesis. It also discusses how TLS is co-opted to increase the diversity of the immunoglobulin gene hypermutation and the contribution it makes to the mutations that sculpt the genome of cancer cells. PMID:23457261

  13. Mechanism for priming DNA synthesis by yeast DNA Polymerase α

    PubMed Central

    Perera, Rajika L; Torella, Rubben; Klinge, Sebastian; Kilkenny, Mairi L; Maman, Joseph D; Pellegrini, Luca

    2013-01-01

    The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001 PMID:23599895

  14. Direct electrical detection of DNA synthesis

    PubMed Central

    Pourmand, Nader; Karhanek, Miloslav; Persson, Henrik H. J.; Webb, Chris D.; Lee, Thomas H.; Zahradníková, Alexandra; Davis, Ronald W.

    2006-01-01

    Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. We report here an approach for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation. We discovered that incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface charge perturbation. This event can be detected as a transient current by a voltage-clamp amplifier. Based on current understanding of polarizable interfaces, we propose that the electrode detects proton removal from the 3′-hydroxyl group of the DNA molecule during phosphodiester bond formation. PMID:16614066

  15. Differential effect of aphidicolin on adenovirus DNA synthesis and cellular DNA synthesis.

    PubMed

    Kwant, M M; van der Vliet, P C

    1980-09-11

    There is strong evidence for a participation of DNA polymerase gamma in the replication of adenovirus (Ad) DNA. To study a possible additional role of DNA polymerase alpha we measured the effect of aphidicolin on viral DNA replication. In intact cells, aphidicolin inhibits Ad DNA synthesis weakly. The drug concentration required for 50% inhibition of Ad DNA replication was 300-400 fold higher than for a similar effect on cellular DNA synthesis. Such a differential inhibition was also observed in AGMK cells doubly infected with SV40 and the simian adenovirus SA7. No evidence was found for modification of aphidicolin in infected cells or for a change in aphidicolin sensitivity of DNA polymerase alpha after infection. The extent of inhibition of purified DNA polymerase alpha was dependent upon the dCTP concentration. The same situation was observed when DNA synthesis was studied in isolated nuclei from uninfected cells. However, in nuclei from Ad infected cells no effect of dCTP on aphidicolin sensitivity was found. These results were taken as evidence that DNA polymerase alpha does not participate in the replication of adenovirus DNA.

  16. Thymidine analogues for tracking DNA synthesis.

    PubMed

    Cavanagh, Brenton L; Walker, Tom; Norazit, Anwar; Meedeniya, Adrian C B

    2011-09-15

    Replicating cells undergo DNA synthesis in the highly regulated, S-phase of the cell cycle. Analogues of the pyrimidine deoxynucleoside thymidine may be inserted into replicating DNA, effectively tagging dividing cells allowing their characterisation. Tritiated thymidine, targeted using autoradiography was technically demanding and superseded by 5-bromo-2-deoxyuridine (BrdU) and related halogenated analogues, detected using antibodies. Their detection required the denaturation of DNA, often constraining the outcome of investigations. Despite these limitations BrdU alone has been used to target newly synthesised DNA in over 20,000 reviewed biomedical studies. A recent breakthrough in "tagging DNA synthesis" is the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). The alkyne group in EdU is readily detected using a fluorescent azide probe and copper catalysis using 'Huisgen's reaction' (1,3-dipolar cycloaddition or 'click chemistry'). This rapid, two-step biolabelling approach allows the tagging and imaging of DNA within cells whilst preserving the structural and molecular integrity of the cells. The bio-orthogonal detection of EdU allows its application in more experimental assays than previously possible with other "unnatural bases". These include physiological, anatomical and molecular biological experimentation in multiple fields including, stem cell research, cancer biology, and parasitology. The full potential of EdU and related molecules in biomedical research remains to be explored.

  17. Analytical approaches for clarification of DNA-double decker phthalocyanine binding mechanism: As an alternative anticancer chemotherapeutic

    NASA Astrophysics Data System (ADS)

    Bağda, Esra; Yabaş, Ebru; Bağda, Efkan

    2017-02-01

    In the present study a novel water soluble double-decker phthalocyanine was synthesized and calf thymus DNA interaction of the synthesized double-decker phthalocyanine was investigated. 5-(3-pyridyl)-1,3,4-oxadiazole substituted phthalonitrile 1 was prepared by a nucleophilic displacement reaction of 4-nitrophthalonitrile with 5-(3-pyridyl)-1,3,4-oxadiazole-2-thiol. Lutetium(III) double-decker phthalocyanine 2 was prepared by cyclotetramerization of compound 1. Water soluble lutetium(III) double-decker phthalocyanine 3 was prepared with quaternarization of compound 2. The synthesized double-decker phthalocyanine and calf thymus DNA interaction was investigated with UV-vis titrimetric methods, gel electrophoresis, and viscosity measurements. The fluorometric ethidium bromide replacement assay was conducted to clarify the binding mode of water soluble double-decker phthalocyanine. The thermodynamic parameters for interaction, K, ΔG0, ΔH0 and ΔS0 were calculated between the temperature ranges of 25 °C-75 °C. To the best of our knowledge, this is the first study about a double-decker phthalocyanine and DNA interaction.

  18. Analytical approaches for clarification of DNA-double decker phthalocyanine binding mechanism: As an alternative anticancer chemotherapeutic.

    PubMed

    Bağda, Esra; Yabaş, Ebru; Bağda, Efkan

    2017-02-05

    In the present study a novel water soluble double-decker phthalocyanine was synthesized and calf thymus DNA interaction of the synthesized double-decker phthalocyanine was investigated. 5-(3-pyridyl)-1,3,4-oxadiazole substituted phthalonitrile 1 was prepared by a nucleophilic displacement reaction of 4-nitrophthalonitrile with 5-(3-pyridyl)-1,3,4-oxadiazole-2-thiol. Lutetium(III) double-decker phthalocyanine 2 was prepared by cyclotetramerization of compound 1. Water soluble lutetium(III) double-decker phthalocyanine 3 was prepared with quaternarization of compound 2. The synthesized double-decker phthalocyanine and calf thymus DNA interaction was investigated with UV-vis titrimetric methods, gel electrophoresis, and viscosity measurements. The fluorometric ethidium bromide replacement assay was conducted to clarify the binding mode of water soluble double-decker phthalocyanine. The thermodynamic parameters for interaction, K, ΔG(0), ΔH(0) and ΔS(0) were calculated between the temperature ranges of 25°C-75°C. To the best of our knowledge, this is the first study about a double-decker phthalocyanine and DNA interaction.

  19. Chemically-enzymatic synthesis of photosensitive DNA.

    PubMed

    Westphal, Kinga; Zdrowowicz, Magdalena; Zylicz-Stachula, Agnieszka; Rak, Janusz

    2017-02-01

    The sensitizing propensity of radio-/photosensitizing nucleoside depends on DNA sequence surrounding a sensitizer. Therefore, in order to compare sensitizers with regard to their ability to induce a DNA damage one has to study the sequence dependence of damage yield. However, chemical synthesis of oligonucleotides labeled with sensitizing nucleosides is hindered due to the fact that a limited number of such nucleoside phosphoramidites are accessible. Here, we report on a chemically-enzymatic method, employing a DNA polymerase and ligase, that enables a modified nucleoside, in the form of its 5'-triphosphate, to be incorporated into DNA fragment in a pre-determined site. Using such a protocol two double-stranded DNA fragments - a long one, 75 base pairs (bp), and a short one, 30bp in length - were pin-point labeled with 5-bromodeoxyuridine. Four DNA polymerases together with DHPLC for the inspection of reaction progress were used to optimize the process under consideration. As an ultimate test showing that the product possessing an assumed nucleotide sequence was actually obtained, we irradiated the synthesized oligonucleotide with UVB photons and analyzed its photoreactivity with the LC-MS method. Our results prove that a general approach enabling precise labeling of DNA with any nucleoside modification processed by DNA polymerase and ligase has been worked out.

  20. Magnetic control of the DNA synthesis

    NASA Astrophysics Data System (ADS)

    Buchachenko, Anatoly L.; Orlov, Alexei P.; Kuznetsov, Dmitry A.; Breslavskaya, Natalia N.

    2013-10-01

    By using polymerases β loaded with isotopic ions 24Mg2+, 25Mg2+ and 26Mg2+ magnetic isotope effect was detected: 25Mg2+ ions with magnetic nuclei 25Mg suppress enzymatic activity by 2-3 times with respect to that of polymerases β loaded by 24Mg2+ and 26Mg2+ ions. No difference in enzymatic activity of polymerases β with 24Mg2+ and 26Mg2+ ions exists. The rate of DNA synthesis strongly depends on the magnetic field. The polymerase chain reaction is also suppressed by 25Mg2+ ions with respect to the ions with nonmagnetic nuclei. Magnetic control of the DNA synthesis may be used for medical purposes.

  1. An autoradiographic demonstration of nuclear DNA replication by DNA polymerase alpha and of mitochondrial DNA synthesis by DNA polymerase gamma.

    PubMed Central

    Geuskens, M; Hardt, N; Pedrali-Noy, G; Spadari, S

    1981-01-01

    The incorporation of thymidine into the DNA of eukaryotic cells is markedly depressed, but not completely inhibited, by aphidicolin, a highly specific inhibitor of DNA polymerase alpha. An electron microscope autoradiographic analysis of the synthesis of nuclear and mitochondrial DNA in vivo in Concanavalin A stimulated rabbit spleen lymphocytes and in Hamster cell cultures, in the absence and in the presence of aphidicolin, revealed that aphidicolin inhibits the nuclear but not the mitochondrial DNA replication. We therefore conclude that DNA polymerase alpha performs the synchronous bidirectional replication of nuclear DNA and that DNA polymerase gamma, the only DNA polymerase present in the mitochondria, performs the "strand displacement" DNA synthesis of these organelles. Images PMID:6262734

  2. Design Features of Explicit Values Clarification Methods: A Systematic Review.

    PubMed

    Witteman, Holly O; Scherer, Laura D; Gavaruzzi, Teresa; Pieterse, Arwen H; Fuhrel-Forbis, Andrea; Chipenda Dansokho, Selma; Exe, Nicole; Kahn, Valerie C; Feldman-Stewart, Deb; Col, Nananda F; Turgeon, Alexis F; Fagerlin, Angela

    2016-05-01

    Values clarification is a recommended element of patient decision aids. Many different values clarification methods exist, but there is little evidence synthesis available to guide design decisions. To describe practices in the field of explicit values clarification methods according to a taxonomy of design features. MEDLINE, all EBM Reviews, CINAHL, EMBASE, Google Scholar, manual search of reference lists, and expert contacts. Articles were included if they described 1 or more explicit values clarification methods. We extracted data about decisions addressed; use of theories, frameworks, and guidelines; and 12 design features. We identified 110 articles describing 98 explicit values clarification methods. Most of these addressed decisions in cancer or reproductive health, and half addressed a decision between just 2 options. Most used neither theory nor guidelines to structure their design. "Pros and cons" was the most common type of values clarification method. Most methods did not allow users to add their own concerns. Few methods explicitly presented tradeoffs inherent in the decision, supported an iterative process of values exploration, or showed how different options aligned with users' values. Study selection criteria and choice of elements for the taxonomy may have excluded values clarification methods or design features. Explicit values clarification methods have diverse designs but can be systematically cataloged within the structure of a taxonomy. Developers of values clarification methods should carefully consider each of the design features in this taxonomy and publish adequate descriptions of their designs. More research is needed to study the effects of different design features. © The Author(s) 2016.

  3. Neurotensin enhances estradiol induced DNA synthesis in immature rat uterus

    SciTech Connect

    Mistry, A.; Vijayan, E.

    1985-05-27

    Systemic administration of Neurotensin, a tridecapeptide, in immature rats treated with estradiol benzoate significantly enhances uterine DNA synthesis as reflected by the incorporation of /sup 3/H-thymidine. The peptide may have a direct action on the uterus. Substance P, a related peptide, had no effect on uterine DNA synthesis. 18 references, 4 tables.

  4. Clarifications, Refutations, and Resolutions

    ERIC Educational Resources Information Center

    Rabinowitz, Jan C.; And Others

    1977-01-01

    The criticisms of Rabinowitz, Mandler, and Patterson offered by Broadbent and Broadbent (AA 527 085) are discussed. A clarification of some of the results supports the original view that item recognition often involves retrieval processes similar to those of free recall. Data are presented to refute Broadbents' suggestion that a recognition test…

  5. Clarifications, Refutations, and Resolutions

    ERIC Educational Resources Information Center

    Rabinowitz, Jan C.; And Others

    1977-01-01

    The criticisms of Rabinowitz, Mandler, and Patterson offered by Broadbent and Broadbent (AA 527 085) are discussed. A clarification of some of the results supports the original view that item recognition often involves retrieval processes similar to those of free recall. Data are presented to refute Broadbents' suggestion that a recognition test…

  6. The Logic of Values Clarification

    ERIC Educational Resources Information Center

    Kazepides, A. C.

    1977-01-01

    Traces the origin of the Values Clarification movement in education in Carl Roger's clien-centered therapy and exposes its unwarranted extreme ethical stance. Examines a model episode of values clarification and shows how the theoretical confusions of the Values Clarification proponents are reflected in their actual teaching strategies. (Editor/RK)

  7. The Logic of Values Clarification

    ERIC Educational Resources Information Center

    Kazepides, A. C.

    1977-01-01

    Traces the origin of the Values Clarification movement in education in Carl Roger's clien-centered therapy and exposes its unwarranted extreme ethical stance. Examines a model episode of values clarification and shows how the theoretical confusions of the Values Clarification proponents are reflected in their actual teaching strategies. (Editor/RK)

  8. DNA Binding Peptide Directed Synthesis of Continuous DNA Nanowires for Analysis of Large DNA Molecules by Scanning Electron Microscope.

    PubMed

    Kim, Kyung-Il; Lee, Seonghyun; Jin, Xuelin; Kim, Su Ji; Jo, Kyubong; Lee, Jung Heon

    2017-01-01

    Synthesis of smooth and continuous DNA nanowires, preserving the original structure of native DNA, and allowing its analysis by scanning electron microscope (SEM), is demonstrated. Gold nanoparticles densely assembled on the DNA backbone via thiol-tagged DNA binding peptides work as seeds for metallization of DNA. This method allows whole analysis of DNA molecules with entangled 3D features. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. DNA sequencing by synthesis based on elongation delay detection

    NASA Astrophysics Data System (ADS)

    Manturov, Alexey O.; Grigoryev, Anton V.

    2015-03-01

    The one of most important problem in modern genetics, biology and medicine is determination of the primary nucleotide sequence of the DNA of living organisms (DNA sequencing). This paper describes the label-free DNA sequencing approach, based on the observation of a discrete dynamics of DNA sequence elongation phase. The proposed DNA sequencing principle are studied by numerical simulation. The numerical model for proposed label-free DNA sequencing approach is based on a cellular automaton, which can simulate the elongation stage (growth of DNA strands) and dynamics of nucleotides incorporation to rising DNA strand. The estimates for number of copied DNA sequences for required probability of nucleotide incorporation event detection and correct DNA sequence determination was obtained. The proposed approach can be applied at all known DNA sequencing devices with "sequencing by synthesis" principle of operation.

  10. DNA polymerase ι of mammals as a participant in translesion synthesis of DNA.

    PubMed

    Gening, L V

    2011-01-01

    This review describes the properties of some specialized DNA polymerases participating in translesion synthesis of DNA. Special attention is given to these properties in vivo. DNA polymerase iota (Polι) of mammals has very unusual features and is extremely error-prone. Based on available data, a hypothesis is proposed explaining how mammalian cells can explore the unusual features of DNA Polι to bypass DNA damages and to simultaneously prevent its mutagenic potential.

  11. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV.

    PubMed

    Tashjian, Tommy F; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M; Godoy, Veronica G

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB's fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability.

  12. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV

    PubMed Central

    Tashjian, Tommy F.; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M.; Godoy, Veronica G.

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB’s fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability. PMID:28298904

  13. De novo DNA synthesis using single-molecule PCR.

    PubMed

    Yehezkel, Tuval Ben; Linshiz, Gregory; Shapiro, Ehud

    2012-01-01

    The throughput of DNA reading (i.e., sequencing) has dramatically increased recently owing to the incorporation of in vitro clonal amplification. The throughput of DNA writing (i.e., synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning needs to be integrated into DNA synthesis methods. Here, we show how a new single-molecule PCR (smPCR)-based procedure can be employed as a general substitute for in vivo cloning, thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our previously described recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligonucleotides, entirely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used, in principle, in conjunction with other DNA synthesis methods as well.

  14. Inhibition of DNA synthesis in Meth A cells by chlorpromazine.

    PubMed

    Mizushima, T; Natori, S; Sekimizu, K

    1993-10-01

    We examined the influence of chlorpromazine, a phenothiazine derivative, on DNA synthesis in Meth A cells. Pulse-labelling experiments with [3H]thymidine showed that chlorpromazine inhibited DNA synthesis in cells cultured in vitro. The drug also inhibited DNA synthesis in isolated nuclei. Observation by fluorescence microscopy of fibroblastic cells stained with chlorpromazine indicated that the drug was localized in the cytoplasm and nuclear membranes, suggesting that it inhibited DNA synthesis in a manner dependent on the interaction of replication proteins with nuclear membranes. Meth A sarcomas growing in the endoderm of BALB/c mice regressed on intra-tumor injection of chlorpromazine, indicating that the drug has an anticancer action.

  15. Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis.

    PubMed

    Martin, J L; Brown, C E; Matthews-Davis, N; Reardon, J E

    1994-12-01

    Inhibition constants were determined for 16 nucleoside analog triphosphates against human DNA polymerases alpha, beta, gamma, and epsilon, and 7 nucleoside analogs were examined as inhibitors of mitochondrial DNA synthesis in human Molt-4 cells in culture. The results demonstrate no clear quantitative or qualitative correlation between inhibition of DNA polymerases, particularly mitochondrial DNA polymerase gamma, and the inhibition of mitochondrial DNA synthesis in Molt-4 cell culture. Furthermore, the data indicate that inhibition of isolated DNA polymerases may not be predictive of in vitro or in vivo toxicity. Finally, it is not clear whether inhibition of mitochondrial DNA synthesis will be an accurate predictor of the potential in vivo toxicity of antiviral nucleoside analogs.

  16. DNA polymerases and repair synthesis in NER in human cells.

    PubMed

    Lehmann, Alan R

    2011-07-15

    The late steps of nucleotide excision repair, following incisions to remove the damaged section of DNA, comprise repair synthesis and ligation. In vitro and in vivo studies have shown the size of the repaired patch to be about 30 nucleotides. In vitro studies implicated the replicative polymerases in repair synthesis, but recent in vivo data have shown that several DNA polymerases and ligases are involved in these steps in human cells. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Clarification Procedure for Gels

    NASA Technical Reports Server (NTRS)

    Barber, Patrick G.; Simpson, Norman R.

    1987-01-01

    Procedure developed to obtain transparent gels with consistencies suitable for crystal growth, by replacing sodium ions in silicate solution with potassium ions. Clarification process uses cation-exchange resin to replace sodium ions in stock solution with potassium ions, placed in 1M solution of soluble potassium salt. Slurry stirred for several hours to allow potassium ions to replace all other cations on resin. Supernatant solution decanted through filter, and beads rinsed with distilled water. Rinsing removes excess salt but leaves cation-exchange beads fully charged with potassium ions.

  18. Analytical Devices Based on Direct Synthesis of DNA on Paper.

    PubMed

    Glavan, Ana C; Niu, Jia; Chen, Zhen; Güder, Firat; Cheng, Chao-Min; Liu, David; Whitesides, George M

    2016-01-05

    This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

  19. A Clarification of Heidegger's Phenomenology

    ERIC Educational Resources Information Center

    Garza, Gilbert

    2006-01-01

    Comments on the article "Psychology and Phenomenology: A Clarification" by H. H. Kendler. This article calls for clarifications regarding its characterization of Heideggerian phenomenology. Kendler drew on his admittedly "limited understanding" (p. 322) of Heidegger and rested his critique upon a critical confusion that pervaded his…

  20. In Defense of Values Clarification.

    ERIC Educational Resources Information Center

    Kirschenbaum, Howard; And Others

    In this position paper, the authors respond to the frequent criticisms and charges that values clarification is value free, relativistic, superficial, and without a cogent theoretical or research base. The authors first examine values clarification as theory. As originally developed by Raths, values education is a theory based on the thinking of…

  1. DNA synthesis and DNA polymerase activity of herpes simplex virus type 1 temperature-sensitive mutants.

    PubMed Central

    Aron, G M; Purifoy, D J; Schaffer, P A

    1975-01-01

    Fifteen temperature-sensitive mutants of herpes simplex virus type 1 were studied with regard to the relationship between their ability to synthesize viral DNA and to induce viral DNA polymerase (DP) activity at permissive (34 C) and nonpermissive (39 C) temperatures. At 34 C, all mutants synthesized viral DNA, while at 39 C four mutants demonstrated a DNA+ phenotype, three were DNA+/-, and eight were DNA-. DNA+ mutants induced levels of DP activity similar to thhose of the wild-type virus at both temperatures, and DNA+/- mutants induced reduced levels of DP activity at 39 C but not at 34 C. Among the DNA- mutants three were DP+, two were DP+/-, and three showed reduced DP activity at 34 C with no DP activity at 39 C. DNA-, DP- mutants induced the synthesis of a temperature-sensitive DP as determined by in vivo studies. PMID:169388

  2. Most Retroviral Recombinations Occur during Minus-Strand DNA Synthesis

    PubMed Central

    Zhang, Jiayou; Tang, Ling-Yun; Li, Ting; Ma, Yan; Sapp, Christy M.

    2000-01-01

    Retroviral RNA molecules are plus, or sense in polarity, equivalent to mRNA. During reverse transcription, the first strand of the DNA molecule synthesized is minus-strand DNA. After the minus strand is polymerized, the plus-strand DNA is synthesized using the minus-strand DNA as the template. In this study, a helper cell line that contains two proviruses with two different mutated gfp genes was constructed. Recombination between the two frameshift mutant genes resulted in a functional gfp. If recombination occurs during minus-strand DNA synthesis, the plus-strand DNA will also contain the functional sequence. After the cell divides, all of its offspring will be green. However, if recombination occurs during plus-strand DNA synthesis, then only the plus-strand DNA will contain the wild-type gfp sequence and the minus-strand DNA will still carry the frameshift mutation. The double-stranded DNA containing this mismatch was subsequently integrated into the host chromosomal DNA of D17 cells, which were unable to repair the majority of mismatches within the retroviral double-strand DNA. After the cell divided, one daughter cell contained the wild-type gfp sequence and the other daughter cell contained the frameshift mutation in the gfp sequence. Under fluorescence microscopy, half the cells in the offspring were green and the other half of the cells were colorless or clear. Thus, we demonstrated that more than 98%, if not all, retroviral recombinations occurred during minus-strand DNA synthesis. PMID:10666262

  3. Nonuniform distribution of excision repair synthesis in nucleosome core DNA

    SciTech Connect

    Lan, S.Y.; Smerdon, M.J.

    1985-12-17

    We have studied the distribution in nucleosome core DNA of nucleotides incorporated by excision repair synthesis occurring immediately after UV irradiation in human cells. The differences previously observed for whole nuclei between the DNase I digestion profiles of repaired DNA (following its refolding into a nucleosome structure) and bulk DNA are obtained for isolated nucleosome core particles. Analysis of the differences obtained indicates that they could reflect a significant difference in the level of repair-incorporated nucleotides at different sites within the core DNA region. To test this possibility directly, we have used exonuclease III digestion of very homogeneous sized core particle DNA to map the distribution of repair synthesis in these regions. Results indicate that in a significant fraction of the nucleosomes the 5' and 3' ends of the core DNA are markedly enhanced in repair-incorporated nucleotides relative to the central region of the core particle. A best fit analysis indicates that a good approximation of the data is obtained for a distribution where the core DNA is uniformly labeled from the 5' end to position 62 and from position 114 to the 3' end, with the 52-base central region being devoid of repair-incorporated nucleotides. This distribution accounts for all of the quantitative differences observed previously between repaired DNA and bulk DNA following the rapid phase of nucleosome rearrangement when it is assumed that linker DNA and the core DNA ends are repaired with equal efficiency and the nucleosome structure of newly repaired DNA is identical with that of bulk chromatin. The 52-base central region that is devoid of repair synthesis contains the lowest frequency cutting sites for DNase I in vitro, as well as the only internal locations where two (rather than one) histones interact with a 10-base segment of each DNA strand.

  4. Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells.

    PubMed Central

    Nyce, J; Liu, L; Jones, P A

    1986-01-01

    Post-synthetic enzymatic hypermethylation of DNA was induced in hamster fibrosarcoma cells by the DNA synthesis inhibitors cytosine arabinoside, hydroxyurea and aphidicolin. This effect required direct inhibition of DNA polymerase alpha or reduction in deoxynucleotide pools and was not specific to a single cell type. At equivalently reduced levels of DNA synthesis, neither cycloheximide, actinomycin D nor serum deprivation affected DNA methylation in this way. The topoisomerase inhibitors nalidixic acid and novobiocin caused significant hypomethylation indicating that increased 5-mCyt content was not a necessary consequence of DNA synthesis inhibition. The induced hypermethylation occurred predominantly in that fraction of the DNA synthesized in the presence of inhibitor; was stable in the absence of drug; was most prominent in low molecular weight DNA representing sites of initiated but incomplete DNA synthesis; and occurred primarily within CpG dinucleotides, although other dinucleotides were overmethylated as well. Drug-induced CpG hypermethylation may be capable of silencing genes, an effect which may be relevant to the aberrantly expressed genes characteristic of neoplastic cells. PMID:3086840

  5. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    SciTech Connect

    Ulrich-Baker, M.G.; Wang, P.; Fitzpatrick, L.; Johnson, L.R. )

    1988-03-01

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na{sup +}-H{sup +} exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of ({sup 3}H)thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na{sup +}-H{sup +} antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity.

  6. COORDINATING DNA POLYMERASE TRAFFIC DURING HIGH AND LOW FIDELITY SYNTHESIS

    PubMed Central

    Sutton, Mark D.

    2009-01-01

    With the discovery that organisms possess multiple DNA polymerases (Pols) displaying different fidelities, processivities, and activities came the realization that mechanisms must exist to manage the actions of these diverse enzymes to prevent gratuitous mutations. Although many of the Pols encoded by most organisms are largely accurate, and participate in DNA replication and DNA repair, a sizeable fraction display a reduced fidelity, and act to catalyze potentially error-prone translesion DNA synthesis (TLS) past lesions that persist in the DNA. Striking the proper balance between use of these different enzymes during DNA replication, DNA repair, and TLS is essential for ensuring accurate duplication of the cell’s genome. This review highlights mechanisms that organisms utilize to manage the actions of their different Pols. A particular emphasis is placed on discussion of current models for how different Pols switch places with each other at the replication fork during high fidelity replication and potentially error-pone TLS. PMID:19540941

  7. Potency of carcinogens derived from covalent DNA binding and stimulation of DNA synthesis in rat liver

    SciTech Connect

    Lutz, W.K.; Buesser, M.T.; Sagelsdorff, P.

    1984-01-01

    In order to investigate the role of the stimulation of cell division for the initiation (and possibly promotion) of liver tumors by chemical carcinogens, the incorporation of radiolabelled thymidine into liver DNA was determined in male rats. Single doses of various levels of aflatoxin B1, benzidine and carbon tetrachloride (all known to be genotoxic via DNA binding) did not affect cell division, whereas several hepatocarcinogens known not to bind to DNA (alpha-HCH, clofibrate, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) gave rise to a dose-dependent stimulation of liver DNA synthesis within 24 h. An equation combining the influences of mitotic stimulation, expressed as dose required to double the control level of DNA synthesis, and DNA binding potency, expressed as the Covalent Binding Index, correlated well with the carcinogenic potency for both classes of hepatocarcinogens.

  8. Characterization of human translesion DNA synthesis across a UV-induced DNA lesion

    PubMed Central

    Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J

    2016-01-01

    Translesion DNA synthesis (TLS) during S-phase uses specialized TLS DNA polymerases to replicate a DNA lesion, allowing stringent DNA synthesis to resume beyond the offending damage. Human TLS involves the conjugation of ubiquitin to PCNA clamps encircling damaged DNA and the role of this post-translational modification is under scrutiny. A widely-accepted model purports that ubiquitinated PCNA recruits TLS polymerases such as pol η to sites of DNA damage where they may also displace a blocked replicative polymerase. We provide extensive quantitative evidence that the binding of pol η to PCNA and the ensuing TLS are both independent of PCNA ubiquitination. Rather, the unique properties of pols η and δ are attuned to promote an efficient and passive exchange of polymerases during TLS on the lagging strand. DOI: http://dx.doi.org/10.7554/eLife.19788.001 PMID:27770570

  9. Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ

    PubMed Central

    Copeland, William C.; Kasiviswanathan, Rajesh; Longley, Matthew J.

    2016-01-01

    Summary Mitochondrial DNA is replicated by the nuclear encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand crosslinks from chemotherapy agents. Although many of these lesions block DNA replication, Pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by Pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis. PMID:26530671

  10. Unscheduled synthesis of DNA and poly(ADP-ribose) in human fibroblasts following DNA damage

    SciTech Connect

    McCurry, L.S.; Jacobson, M.K.

    1981-01-01

    Unscheduled DNA synthesis has been measured in human fibroblasts under conditions of reduced rates of conversion of NAD to poly)ADP-ribose). Cells heterozygous for the xeroderma pigmentosum genotype showed normal rates of uv induced unscheduled DNA synthesis under conditions in which the rate of poly(ADP-ribose) synthesis was one-half the rate of normal cells. The addition of theophylline, a potent inhibitor of poly(ADP-ribose) polymerase, to the culture medium of normal cells blocked over 90% of the conversion of NAD to poly(ADP-ribose) following treatment with uv or N-methyl-N'-nitro-N-nitro-soguanidine but did not affect the rate of unscheduled DNA synthesis.

  11. DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis

    PubMed Central

    2015-01-01

    The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the “structure elucidation problem”: the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS’s utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 104 molecules/bead and sequencing allowed for elucidation of each compound’s synthetic history. We applied DESPS to the combinatorial synthesis of a 75 645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and

  12. DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

    PubMed

    MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

    2015-09-14

    The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR

  13. Clarification of CERCLA Entry Policy

    EPA Pesticide Factsheets

    This memorandum provides Regional Counsel with clarification on EPA’s Policy: “Entry and Continued Access Under CERCLA.” The Policy focuses on consensually gaining access for CERCLA activities at a particular location.

  14. Facile dimer synthesis for DNA-binding polyamide ligands.

    PubMed

    Wetzler, Modi; Wemmer, David E

    2010-08-06

    Pyrrole-imidazole polyamide ligands are highly sequence specific synthetic DNA-binding ligands that bind with high affinity. To counter the synthetic difficulties associated with coupling the electron-rich heterocyclic acids to the electron-deficient nucleophilic imidazole amine, a novel approach is described for synthesis of Fmoc-protected dimers for solid-phase peptide synthesis (SPPS). This method produces the dimers in high yields, is broadly applicable to other heterocyclic-containing polyamides, and results in improved ligand yields and synthesis times.

  15. Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

    PubMed

    Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

    2012-03-13

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

  16. 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

  17. The elemental role of iron in DNA synthesis and repair.

    PubMed

    Puig, Sergi; Ramos-Alonso, Lucía; Romero, Antonia María; Martínez-Pastor, María Teresa

    2017-09-07

    Iron is an essential redox element that functions as a cofactor in many metabolic pathways. Critical enzymes in DNA metabolism, including multiple DNA repair enzymes (helicases, nucleases, glycosylases, demethylases) and ribonucleotide reductase, use iron as an indispensable cofactor to function. Recent striking results have revealed that the catalytic subunit of DNA polymerases also contains conserved cysteine-rich motifs that bind iron-sulfur (Fe/S) clusters that are essential for the formation of stable and active complexes. In line with this, mitochondrial and cytoplasmic defects in Fe/S cluster biogenesis and insertion into the nuclear iron-requiring enzymes involved in DNA synthesis and repair lead to DNA damage and genome instability. Recent studies have shown that yeast cells possess multi-layered mechanisms that regulate the ribonucleotide reductase function in response to fluctuations in iron bioavailability to maintain optimal deoxyribonucleotide concentrations. Finally, a fascinating DNA charge transport model indicates how the redox active Fe/S centers present in DNA repair machinery components are critical for detecting and repairing DNA mismatches along the genome by long-range charge transfers through double-stranded DNA. These unexpected connections between iron and DNA replication and repair have to be considered to properly understand cancer, aging and other DNA-related diseases.

  18. Translesion Synthesis: Insights into the Selection and Switching of DNA Polymerases

    PubMed Central

    Zhao, Linlin; Washington, M. Todd

    2017-01-01

    DNA replication is constantly challenged by DNA lesions, noncanonical DNA structures and difficult-to-replicate DNA sequences. Two major strategies to rescue a stalled replication fork and to ensure continuous DNA synthesis are: (1) template switching and recombination-dependent DNA synthesis; and (2) translesion synthesis (TLS) using specialized DNA polymerases to perform nucleotide incorporation opposite DNA lesions. The former pathway is mainly error-free, and the latter is error-prone and a major source of mutagenesis. An accepted model of translesion synthesis involves DNA polymerase switching steps between a replicative DNA polymerase and one or more TLS DNA polymerases. The mechanisms that govern the selection and exchange of specialized DNA polymerases for a given DNA lesion are not well understood. In this review, recent studies concerning the mechanisms of selection and switching of DNA polymerases in eukaryotic systems are summarized. PMID:28075396

  19. Towards the Batch Synthesis of Long DNA

    DTIC Science & Technology

    2002-10-01

    MISMATCHES In a series of papers,136 the SantaLucia NN model137 of Watson - Crick paired DNA thermodynamics was successfully extended to incorporate...generally indicate a- helix coding or structural motifs for DNA incorporation into chromatin. Trifonov, E. N., “3-,!10.5-, 200- and 400-base...double-stranded DNA , is well-described by Hearst’s “weakly bending rod” model with 3.4 Å rise/bp and 13 Å radius for the helix ; its persistence length39

  20. Effects of 8-chlorodeoxyadenosine on DNA synthesis by the Klenow fragment of DNA polymerase I.

    PubMed

    Chen, Lisa S; Bahr, Michael H; Sheppard, Terry L

    2003-05-05

    8-chloro-2'-deoxyadenosine (8-Cl-dAdo) was incorporated into synthetic DNA oligonucleotides to determine its effects on DNA synthesis by the 3'-5' exonuclease-free Klenow fragment of Escherichia coli DNA Polymerase I (KF-). Single nucleotide insertion experiments were used to determine the coding potential of 8-Cl-dAdo in a DNA template. KF- inserted TTP opposite 8-Cl-dAdo in the template, but with decreased efficiency relative to natural deoxyadenosine. Running-start primer extensions with KF- resulted in polymerase pausing at 8-Cl-dAdo template sites during DNA synthesis. The 2'-deoxyribonucleoside triphosphate analogue, 8-Cl-dATP, was incorporated opposite thymidine (T) approximately two-fold less efficiently than dATP.

  1. The coordinate induction of DNA synthesis after tuber wounding

    USDA-ARS?s Scientific Manuscript database

    Tuber wounding induces a cascade of biological responses involved in processes required to heal and protect surviving plant issues. Little is known about the coordination of these processes, including essential wound-induced DNA synthesis, yet they play critical roles in maintaining marketability o...

  2. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    SciTech Connect

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K. )

    1990-11-15

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling.

  3. DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

    PubMed Central

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

    2016-01-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  4. Second-strand cDNA synthesis: classical method

    SciTech Connect

    Gubler, U.

    1987-01-01

    The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S/sub 1/ nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described.

  5. The control of lambda DNA terminase synthesis.

    PubMed Central

    Murialdo, H; Davidson, A; Chow, S; Gold, M

    1987-01-01

    Nu1 and A, the genes coding for bacteriophage lambda DNA terminase, rank among the most poorly translated genes expressed in E. coli. To understand the reason for this low level of translation the genes were cloned into plasmids and their expression measured. In addition, the wild type DNA sequences immediately preceding the genes were reduced and modified. It was found that the elements that control translation are contained in the 100 base pairs upstream from the initiation codon. Interchanging these upstream sequences with those of an efficiently translated gene dramatically increased the translation of terminase subunits. It seems unlikely that the rare codons present in the genes, and any feature of their mRNA secondary structure play a role in the control of their translation. The elimination of cos from plasmids containing Nu1 and A also resulted in an increase in terminase production. This result suggests a role for cos in the control of late gene expression. The terminase subunit overproducer strains are potentially very useful for the design of improved DNA packaging and cosmid mapping techniques. Images PMID:3029667

  6. Translesion Synthesis Past Acrolein-derived DNA Adducts by Human Mitochondrial DNA Polymerase γ*

    PubMed Central

    Kasiviswanathan, Rajesh; Minko, Irina G.; Lloyd, R. Stephen; Copeland, William C.

    2013-01-01

    Acrolein, a mutagenic aldehyde, is produced endogenously by lipid peroxidation and exogenously by combustion of organic materials, including tobacco products. Acrolein reacts with DNA bases forming exocyclic DNA adducts, such as γ-hydroxy-1,N2-propano-2′-deoxyguanosine (γ-HOPdG) and γ-hydroxy-1,N6-propano-2′-deoxyadenosine (γ-HOPdA). The bulky γ-HOPdG adduct blocks DNA synthesis by replicative polymerases but can be bypassed by translesion synthesis polymerases in the nucleus. Although acrolein-induced adducts are likely to be formed and persist in mitochondrial DNA, animal cell mitochondria lack specialized translesion DNA synthesis polymerases to tolerate these lesions. Thus, it is important to understand how pol γ, the sole mitochondrial DNA polymerase in human cells, acts on acrolein-adducted DNA. To address this question, we investigated the ability of pol γ to bypass the minor groove γ-HOPdG and major groove γ-HOPdA adducts using single nucleotide incorporation and primer extension analyses. The efficiency of pol γ-catalyzed bypass of γ-HOPdG was low, and surprisingly, pol γ preferred to incorporate purine nucleotides opposite the adduct. Pol γ also exhibited ∼2-fold lower rates of excision of the misincorporated purine nucleotides opposite γ-HOPdG compared with the corresponding nucleotides opposite dG. Extension of primers from the termini opposite γ-HOPdG was accomplished only following error-prone purine nucleotide incorporation. However, pol γ preferentially incorporated dT opposite the γ-HOPdA adduct and efficiently extended primers from the correctly paired terminus, indicating that γ-HOPdA is probably nonmutagenic. In summary, our data suggest that acrolein-induced exocyclic DNA lesions can be bypassed by mitochondrial DNA polymerase but, in the case of the minor groove γ-HOPdG adduct, at the cost of unprecedented high mutation rates. PMID:23543747

  7. Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents against Lung Cancer

    DTIC Science & Technology

    2015-12-01

    AWARD NUMBER: W81XWH-13-1-0238 TITLE: Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents against Lung Cancer PRINCIPAL...of Translesion DNA Synthesis as Therapeutic Agents against Lung Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Oxygen-rich environments can create pro-mutagenic DNA lesions such as 8-oxoguanine (8-oxo-G) that can be misreplicated during translesion DNA synthesis

  8. Using Values Clarification in Counseling Settings.

    ERIC Educational Resources Information Center

    Glaser, Barbara; Kirschenbaum, Howard

    1980-01-01

    Counselors have found values clarification useful in individual and group counseling and in personalized instruction. The counselor can take advantage of values clarification for personal and professional growth. Other techniques for values discrimination are presented in a commentary. (Author)

  9. Mediation of growth factor induced DNA synthesis and calcium mobilization by Gq and Gi2

    PubMed Central

    1993-01-01

    A newly identified subclass of the heterotrimeric GTP binding regulatory protein family, Gq, has been found to be expressed in a diverse range of cell types. We investigated the potential role of this protein in growth factor signal transduction pathways and its potential relationship to the function of other G alpha subclasses. Recent biochemical studies have suggested that Gq regulates the beta 1 isozyme of phospholipase C (PLC beta 1), an effector for some growth factors. By microinjection of inhibitory antibodies specific to distinct G alpha subunits into living cells, we have determined that G alpha q transduces bradykinin- and thrombin-stimulated intracellular calcium transients which are likely to be mediated by PLC beta 1. Moreover, we found that G alpha q function is required for the mitogenic action of both of these growth factors. These results indicate that both thrombin and bradykinin utilize Gq to couple to increases in intracellular calcium, and that Gq is a necessary component of the mitogenic action of these factors. While microinjection of antibodies against G alpha i2 did not abolish calcium transients stimulated by either of these factors, such microinjection prevented DNA synthesis in response to thrombin but not to bradykinin. These data suggest that thrombin- induced mitogenesis requires both Gq and Gi2, whereas bradykinin needs only the former. Thus, different growth factors operating upon the same cell type use overlapping yet distinct sets of G alpha subtypes in mitogenic signal transduction pathways. The direct identification of the coupling of both a pertussis toxin sensitive and insensitive G protein subtype in the mitogenic pathways utilized by thrombin offers an in vivo biochemical clarification of previous results obtained by pharmacologic studies. PMID:8458876

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

    PubMed

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

    2016-08-05

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

  11. Clarification of Workmen's Compensation Insurance.

    ERIC Educational Resources Information Center

    Shapley, Allen E.

    This document attempts to answer questions resulting from the 1972 Michigan Supreme Court Ruling relative to agricultural employees under the Workmen's Compensation Act (WCA). The sections of this paper outline a history of the WCA; employers covered; definition of "regularly employ"; clarification of "thirteen weeks";…

  12. Electrochemically directed synthesis of oligonucleotides for DNA microarray fabrication

    PubMed Central

    Egeland, Ryan D.; Southern, Edwin M.

    2005-01-01

    We demonstrate a new method for making oligonucleotide microarrays by synthesis in situ. The method uses conventional DNA synthesis chemistry with an electrochemical deblocking step. Acid is delivered to specific regions on a glass slide, thus allowing nucleotide addition only at chosen sites. The acid is produced by electrochemical oxidation controlled by an array of independent microelectrodes. Deblocking is complete in a few seconds, when competing side-product reactions are minimal. We demonstrate the successful synthesis of 17mers and discrimination of single base pair mismatched hybrids. Features generated in this study are 40 μm wide, with sharply defined edges. The synthetic technique may be applicable to fabrication of other molecular arrays. PMID:16085751

  13. Parallel DNA Synthesis on Poly(ethylene terephthalate).

    PubMed

    Holden, Matthew T; Carter, Matthew C D; Ting, Shannon K; Lynn, David M; Smith, Lloyd M

    2017-10-05

    The fabrication of DNA arrays directly on aminolyzed sheets of poly(ethylene terephthalate) (PET) is described. Array surfaces typically employ bifunctional linkers or layers of covalently attached polymers to provide substrate hydroxy groups as synthesis attachment points. An amine treatment is used here to expose hydroxy groups on films of PET. These hydroxy groups can then be used to couple phosphoramidites and initiate the array synthesis without further functionalization steps. Arrays fabricated on these substrates with a maskless array synthesizer are tolerant of the high number of chemical exposure steps required to synthesize relatively long oligonucleotides. The results might be of the greatest use to the synthetic biology community, for whom a flexible and robust substrate could enable new strategies to enhance the throughput of oligonucleotide synthesis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration.

    PubMed

    Wilson, Marenda A; Kwon, YoungHo; Xu, Yuanyuan; Chung, Woo-Hyun; Chi, Peter; Niu, Hengyao; Mayle, Ryan; Chen, Xuefeng; Malkova, Anna; Sung, Patrick; Ira, Grzegorz

    2013-10-17

    During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate DNA helicase is Pif1, an evolutionarily conserved helicase in Saccharomyces cerevisiae important for break-induced replication (BIR) as well as HR-dependent telomere maintenance in the absence of telomerase found in 10-15% of all cancers. Pif1 has a role in DNA synthesis across hard-to-replicate sites and in lagging-strand synthesis with polymerase δ (Polδ). Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half-crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Notably, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.

  15. Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

    SciTech Connect

    Heimer, Y.M.; Kol, R.; Shiloh, Y.; Riklis, E.

    1983-09-01

    A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with ..gamma.. rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.

  16. Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

    SciTech Connect

    Heimer, Y.M.; Kol, R.; Shiloh, Y.; Riklis, E.

    1983-09-01

    A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.

  17. Application of Biocatalysis to on-DNA Carbohydrate Library Synthesis.

    PubMed

    Thomas, Baptiste; Lu, Xiaojie; Birmingham, William R; Huang, Kun; Both, Peter; Reyes Martinez, Juana Elizabeth; Young, Robert J; Davie, Christopher P; Flitsch, Sabine L

    2017-05-04

    DNA-encoded libraries are increasingly used for the discovery of bioactive lead compounds in high-throughput screening programs against specific biological targets. Although a number of libraries are now available, they cover limited chemical space due to bias in ease of synthesis and the lack of chemical reactions that are compatible with DNA tagging. For example, compound libraries rarely contain complex biomolecules such as carbohydrates with high levels of functionality, stereochemistry, and hydrophilicity. By using biocatalysis in combination with chemical methods, we aimed to significantly expand chemical space and generate generic libraries with potentially better biocompatibility. For DNA-encoded libraries, biocatalysis is particularly advantageous, as it is highly selective and can be performed in aqueous environments, which is an essential feature for this split-and-mix library technology. In this work, we demonstrated the application of biocatalysis for the on-DNA synthesis of carbohydrate-based libraries by using enzymatic oxidation and glycosylation in combination with traditional organic chemistry. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. [Effect of single-stranded DNA binding proteins on template/primer-independent DNA synthesis in the presence of nicking endonuclease Nt.BspD6I].

    PubMed

    Zyrina, N V; Artiukh, R I; Svad'bina, I V; Zheleznaia, L A; Matvienko, N I

    2012-01-01

    In the presence of the Nt.BspD6I nicking endonuclease DNA polymerase Bst stimulates intensive template/primer-independent DNA synthesis. Template/primer-independent DNA synthesis could be the reason for appearing nonspecific DNA products in many DNA amplification reactions particularly in the reactions with using nicking endonucleases. Search of the modes for inhibition template/primer-independent DNA synthesis becomes an urgent task because of broadening the DNA amplification methods with using nicking endonucleases. We report here that the E. coli single-stranded DNA binding protein has no effect on the template/primer-independent DNA synthesis. In the absence of the nicking endonuclease the single-stranded DNA binding protein encoded by bacteriophage T4 gene 32 completely inhibits template/primer-independent DNA synthesis. This protein does not inhibit synthesis of specific DNA product in the presence of nicking endonuclease but remarkably decreases the amount of nonspecific products.

  19. Synchronization of mitochondrial DNA synthesis in Chinese hamster cells (line CHO) deprived of isoleucine.

    PubMed

    Ley, K D; Murphy, M M

    1973-08-01

    Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([(3)H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([(3)H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9-12 h after addition of isoleucine and virtually all [(3)H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G(1) because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

  20. Protein synthesis editing by a DNA aptamer.

    PubMed Central

    Hale, S P; Schimmel, P

    1996-01-01

    Potential errors in decoding genetic information are corrected by tRNA-dependent amino acid recognition processes manifested through editing reactions. One example is the rejection of difficult-to-discriminate misactivated amino acids by tRNA synthetases through hydrolytic reactions. Although several crystal structures of tRNA synthetases and synthetase-tRNA complexes exist, none of them have provided insight into the editing reactions. Other work suggested that editing required active amino acid acceptor hydroxyl groups at the 3' end of a tRNA effector. We describe here the isolation of a DNA aptamer that specifically induced hydrolysis of a misactivated amino acid bound to a tRNA synthetase. The aptamer had no effect on the stability of the correctly activated amino acid and was almost as efficient as the tRNA for inducing editing activity. The aptamer has no sequence similarity to that of the tRNA effector and cannot be folded into a tRNA-like structure. These and additional data show that active acceptor hydroxyl groups in a tRNA effector and a tRNA-like structure are not essential for editing. Thus, specific bases in a nucleic acid effector trigger the editing response. Images Fig. 3 Fig. 4 PMID:8610114

  1. Synthesis of damaged DNA containing the oxidative lesion 3'-oxothymidine.

    PubMed

    Bedi, Mel F; Li, Weiye; Gutwald, Taylor; Bryant-Friedrich, Amanda C

    2017-09-01

    Oxidative events that take place during regular oxygen metabolism can lead to the formation of organic or inorganic radicals. The interaction of these radicals with macromolecules in the organism and with DNA in particular is suspected to lead to apoptosis, DNA lesions and cell damage. Independent generation of DNA lesions resulting from oxidative damage is used to promote the study of their effects on biological systems. An efficient synthesis of oligodeoxyribonucleotides (ODNs) containing the oxidative damage lesion 3'-oxothymidine has been accomplished via incorporation of C3'-hydroxymethyl thymidine as its corresponding 5'-phosphoramidite. Through oxidative cleavage using sodium periodate in aqueous solution, the lesion of interest is easily generated. Due to its inherent instability it cannot be directly isolated, but must be generated in situ. 3'-Oxothymidine is a demonstrated damage product formed upon generation of the C3'-thymidinyl radical in ODN. Copyright © 2017. Published by Elsevier Ltd.

  2. Computational method and system for modeling, analyzing, and optimizing DNA amplification and synthesis

    DOEpatents

    Vandersall, Jennifer A.; Gardner, Shea N.; Clague, David S.

    2010-05-04

    A computational method and computer-based system of modeling DNA synthesis for the design and interpretation of PCR amplification, parallel DNA synthesis, and microarray chip analysis. The method and system include modules that address the bioinformatics, kinetics, and thermodynamics of DNA amplification and synthesis. Specifically, the steps of DNA selection, as well as the kinetics and thermodynamics of DNA hybridization and extensions, are addressed, which enable the optimization of the processing and the prediction of the products as a function of DNA sequence, mixing protocol, time, temperature and concentration of species.

  3. Enzymatic synthesis of organic-polymer-grafted DNA.

    PubMed

    Baccaro, Anna; Marx, Andreas

    2010-01-04

    To create bioorganic hybrid materials, interdisciplinary work in the fields of chemistry, biology and materials science is conducted. DNA block copolymers are promising hybrid materials due to the combination of properties intrinsic to both the polymer and the nucleic acid blocks. Until now, the coupling of DNA and organic polymers has been exercised post-synthetically in solution or on solid support. Herein, we report the first enzyme-catalysed synthesis of DNA-organic polymer chimeras. For this purpose, four novel 2'-deoxyuridine triphosphates carrying polymer-like moieties linked to the nucleobase were synthesised. Linear polyethylene glycol monomethyl ethers of different sizes (1) and branched polyamido dendrons with varying terminal groups (2) were chosen as building blocks. We investigated the ability of DNA polymerases to accept the copolymers in comparison to the natural substrate and showed, through primer extensions, polymerase chain reactions and rolling circle amplification, that these building blocks could serve as a surrogate for the natural thymidine. By this method, DNA hybrid materials with high molecular weight, modification density, and defined structure are accessible.

  4. Radiation hypersensitivity and radioresistant DNA synthesis in ataxia-telangiectasia

    SciTech Connect

    Painter, R.B.

    1985-11-01

    Patients with the autosomal recessive genetic disease, ataxia-telangiectasia (A-T), are cancer-prone and hypersensitive to the killing effects of ionizing radiation. In an attempt to isolate the gene(s) responsible for the hypersensitivity of A-T cells, they were transfected with normal human DNA in cosmid vectors containing a rescuable marker (G-418 resistance), and revertants to normal sensitivity were isolated and characterized. The failure of radioresistant revertants to demonstrate a reversion of the phenotype, radioresistant DNA synthesis, shows that this feature is dependent on a gene separate from the one conferring resistance to cell killing. Cells from every A-T patient thus far examined demonstrate both hypersensitivity, in terms of radiation-induced cell killing, and radioresistant DNA synthesis. The results reported here, however, show that the former is not a result of the latter, as previously proposed. Moreover, the fact that these two characteristics can be uncoupled obscures the role(s) that either of them plays in the etiology of the disease, or in the development in its other features, including cancer-proneness.

  5. Cyclosporin A inhibits DNA synthesis by epidermal Langerhans cells.

    PubMed

    Haftek, M; Urabe, A; Kanitakis, J; Dusserre, N; Thivolet, J

    Cyclosporin A, a potent immunosuppressive drug currently used in organ transplant recipients, has been shown to exert in vitro a direct antiproliferative effect on a number of cell types present in the skin, including keratinocytes, fibroblasts, and endothelial cells. Although in vitro studies suggest that cyclosporin A may interfere with the functional capacities of epidermal Langerhans cells, there is no evidence that the treatment influences the distribution or number of Langerhans cells in vivo. We used a model of normal human skin graft to "nude" mice, which is free of the human systemic control mechanisms, for studies on the DNA synthesis of human Langerhans cells under the influence of cyclosporin A. The grafted animals were given daily subcutaneous (50 mg/kg) or intraperitoneal (5, 12.5, and 25 mg/kg) drug injections during three weeks, which resulted in mean blood levels comparable to those observed in treated patients with organ transplants or psoriasis, respectively. BrdU administered during the last week of the experiment was incorporated by all cells synthesizing DNA, including those passing through S-phase. Langerhans cells were detected on deparaffinized or frozen tissue sections of xenografts with anti-CD1a and anti-HLA DR monoclonal antibodies, and the number of BrdU-positive cells was determined by double labeling. Our results indicate that the Langerhans cell DNA synthesis is impaired by therapeutic levels of cyclosporin A.

  6. HGF-induced DNA synthesis in hepatocytes is suppressed by p38.

    PubMed

    Aasrum, Monica; Brusevold, Ingvild J; Christoffersen, Thoralf; Thoresen, G Hege

    2016-12-01

    Previous studies in rat hepatocytes have shown that the MEK/ERK, PI3K/Akt and p38 pathways are all involved in the activation of DNA synthesis by EGF and that sustained activation of MEK/ERK is required. Here, we show that although HGF stimulated DNA synthesis and activated signaling in the same manner as EGF, the contribution of the signaling pathways to the induction of DNA synthesis differed. While HGF-induced DNA synthesis was dependent on MEK/ERK, with no significant contribution from PI3K/Akt, p38 suppressed HGF-induced DNA synthesis. The p38 inhibitor SB203580 increased HGF-induced DNA synthesis and enhanced the phosphorylation of ERK. In contrast, SB203580 decreased EGF-induced ERK phosphorylation. This suggests that p38 has distinct effects on DNA synthesis induced by EGF and HGF. Due to differential regulation of signaling through the MEK/ERK pathway, p38 acts as an enhancer of EGF-induced DNA synthesis and as a suppressor of HGF-induced DNA synthesis.

  7. A Comparative Analysis of Translesion DNA Synthesis Catalyzed by a High-Fidelity DNA Polymerase.

    PubMed

    Dasari, Anvesh; Deodhar, Tejal; Berdis, Anthony J

    2017-07-21

    Translesion DNA synthesis (TLS) is the ability of DNA polymerases to incorporate nucleotides opposite and beyond damaged DNA. TLS activity is an important risk factor for the initiation and progression of genetic diseases such as cancer. In this study, we evaluate the ability of a high-fidelity DNA polymerase to perform TLS with 8-oxo-guanine (8-oxo-G), a highly pro-mutagenic DNA lesion formed by reactive oxygen species. Results of kinetic studies monitoring the incorporation of modified nucleotide analogs demonstrate that the binding affinity of the incoming dNTP is controlled by the overall hydrophobicity of the nucleobase. However, the rate constant for the polymerization step is regulated by hydrogen-bonding interactions made between the incoming nucleotide with 8-oxo-G. Results generated here for replicating the miscoding 8-oxo-G are compared to those published for the replication of the non-instructional abasic site. During the replication of both lesions, binding of the nucleotide substrate is controlled by energetics associated with nucleobase desolvation, whereas the rate constant for the polymerization step is influenced by the physical nature of the DNA lesion, that is, miscoding versus non-instructional. Collectively, these studies highlight the importance of nucleobase desolvation as a key physical feature that enhances the misreplication of structurally diverse DNA lesions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

    PubMed Central

    Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe; Nielsen, Katrine E.; Højland, Torben; Wengel, Jesper; Petersen, Michael

    2011-01-01

    We report the synthesis of two C4′-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4′-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface. PMID:21062815

  9. Selective inhibition of in vitro synthesis of cancer DNA by alkaloids of beta-carboline class.

    PubMed

    Beljanski, M; Beljanski, M S

    1982-01-01

    The high template in vitro activity of native DNA from cancerous mammalian and plant tissues, compared to DNA from healthy tissues, enabled us to select substances which selectively inhibit cancer DNA synthesis. Among them, alstonine, serpentine, sempervirine and flavopereirine, all alkaloids which belong to the Beta-carboline class, distinguish cancer DNA from healthy tissue DNA inhibit DNA in vitro synthesis when native DNA from different cancerous tissues or cells is used as template. They have practically no effect on DNA from healthy tissues. The inhibitory effect of alkaloids is due to their capacity to form an 'alkaloid-cancer DNA' complex which has been characterized by use of the Sephadex column. Evidence is presented showing that these alkaloids inhibit the initiation of DNA synthesis but not chain elongation. The stimulating action caused by carcinogens during cancer DNA in vitro synthesis may be prevented and reversed by alkaloids. Furthermore, the stimulating action of steroids during in vitro synthesis of hormone target tissue DNA might be neutralized by alkaloids. However, at relatively high doses, steroids reversibly compete with alkaloids for binding sites on breast cancer DNA. This is not observed with DNA from nonhormone target tissues.

  10. Aphidicolin inhibits DNA synthesis by DNA polymerase alpha and isolated nuclei by a similar mechanism.

    PubMed Central

    Krokan, H; Wist, E; Krokan, R H

    1981-01-01

    Aphidicolin is a selective inhibitor of DNA polymerase alpha. In contrast to earlier reports, the drug was found to inhibit DNA synthesis catalyzed by DNA polymerase alpha and isolated HeLa cell nuclei by a similar mechanism. For both systems aphidicolin primarily competed with dCTP incorporation. However, the apparent Vmax for dCTP incorporation was reduced by 50-60% at relatively low concentrations of aphidicolin, thus the mechanism of inhibition is complex. Furthermore, a 2-5 fold increase in apparent Km for dTTP was observed in the presence of aphidicolin, but the apparent Km values for dATP and dGTP were essentially unaltered. This, together with additional evidence, suggested that the mechanism of action of aphidicolin involves a strong competition with dCMP incorporation, a weaker competition with dTMP incorporation and very little, if any, competition with dGMP and dAMP incorporation. PMID:6795595

  11. FANCJ promotes DNA synthesis through G-quadruplex structures.

    PubMed

    Castillo Bosch, Pau; Segura-Bayona, Sandra; Koole, Wouter; van Heteren, Jane T; Dewar, James M; Tijsterman, Marcel; Knipscheer, Puck

    2014-11-03

    Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ(-/-) cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes.

  12. FANCJ promotes DNA synthesis through G-quadruplex structures

    PubMed Central

    Castillo Bosch, Pau; Segura-Bayona, Sandra; Koole, Wouter; van Heteren, Jane T; Dewar, James M; Tijsterman, Marcel; Knipscheer, Puck

    2014-01-01

    Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mutations particularly upon replication stress or in the absence of specific helicases. To investigate how G-quadruplex structures are resolved during DNA replication, we developed a model system using ssDNA templates and Xenopus egg extracts that recapitulates eukaryotic G4 replication. Here, we show that G-quadruplex structures form a barrier for DNA replication. Nascent strand synthesis is blocked at one or two nucleotides from the G4. After transient stalling, G-quadruplexes are efficiently unwound and replicated. In contrast, depletion of the FANCJ/BRIP1 helicase causes persistent replication stalling at G-quadruplex structures, demonstrating a vital role for this helicase in resolving these structures. FANCJ performs this function independently of the classical Fanconi anemia pathway. These data provide evidence that the G4 sequence instability in FANCJ−/− cells and Fancj/dog1 deficient C. elegans is caused by replication stalling at G-quadruplexes. PMID:25193968

  13. Membrane clarification of tea extracts.

    PubMed

    Subramanian, R; Kumar, Chandini S; Sharma, Pankaj

    2014-01-01

    The ready-to-drink (RTD) tea beverages are becoming increasingly popular owing to the health benefits associated with tea polyphenols, but instability due to development of haze and formation of tea cream is a common problem encountered in the product. Membrane technology provides a scope to produce natural, additive-free RTD teas while overcoming the major disadvantages associated with the conventional decreaming methods. Approaches employing membranes for the clarification of extracts from black and green tea have been discussed together with their relative advantages and limitations. The article also outlines the concerns to be addressed in the future attempts employing membrane technology.

  14. Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis

    PubMed Central

    Guo, Caixia; Fischhaber, Paula L.; Luk-Paszyc, Margaret J.; Masuda, Yuji; Zhou, Jing; Kamiya, Kenji; Kisker, Caroline; Friedberg, Errol C.

    2003-01-01

    Polκ and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Polκ. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Polζ) and with two other Y-family polymerases, Polι and Polη. Mouse Polκ, Rev7, Polι and Polη each bind to the same ∼100 amino acid C-terminal region of Rev1. Furthermore, Rev7 competes directly with Polκ for binding to the Rev1 C-terminus. Notwith standing the physical interaction between Rev1 and Polκ, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein–protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined. PMID:14657033

  15. Potassium channel openers stimulate DNA synthesis in mouse epidermal keratinocyte and whole hair follicle cultures.

    PubMed

    Harmon, C S; Lutz, D; Ducote, J

    1993-01-01

    We have conducted studies using primary mouse epidermal keratinocyte and whole hair follicle cultures to investigate the mechanism of the hypertrichotic activity of potassium channel openers. In a time course study, the extent of stimulation of epidermal keratinocyte DNA synthesis by minoxidil increased as the rate of DNA synthesis in control cultures declined. Minoxidil stimulation of DNA synthesis in 7-day cultures required prolonged (> 1 day) exposure to the agent. Pinacidil and diazoxide also stimulated DNA synthesis in mouse epidermal keratinocyte cultures. In addition, minoxidil, pinacidil, diazoxide, and cromakalim stimulated DNA synthesis in whole-organ cultures of mouse hair follicles. These results suggest that potassium channel openers retard the loss of proliferative activity of differentiating keratinocytes and support the hypothesis that these agents stimulate hair growth through a direct effect on hair follicles.

  16. ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.

    PubMed

    Lewis, Samantha C; Uchiyama, Lauren F; Nunnari, Jodi

    2016-07-15

    Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, hundreds to thousands of mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis--events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids are necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria.

  17. ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells

    PubMed Central

    Lewis, Samantha C.; Uchiyama, Lauren F.; Nunnari, Jodi

    2017-01-01

    Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, 100–1000s mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis, events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids were necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria. PMID:27418514

  18. Study of stimulators of DNA synthesis in nerve tissue cells

    SciTech Connect

    Vitvitskii, V.N.

    1986-04-10

    Changes in proliferative activity in different regions of the brain during ontogenesis are connected with changes in the composition and properties of regulators of cell proliferation. Extracts of regions of the brain in which active cell division takes place in a given stage of development (cortex of 15- to 17-day-old embryos or cerebellum of 8- to 10-day-old rats) can stimulate the incorporation of labeled precursors into the brain cell DNA of both newborn and adult rats. Salting out at increasing ammonium sulfate concentrations, gel filtration on Sephadex, and isoelectric focusing led to the isolation of three fractions of stimulators of DNA synthesis: in acid, neutral, and alkaline pH regions. A method is described for obtaining purified preparations and for determining some physicochemical properties of the acid activator, which is a low-molecular-weight peptide capable of noticeably stimulating the incorporation of labeled precursors into the DNA of nerve tissue cells when added to an in vitro system in a concentration of the order of 1 ..mu..g/ml.

  19. Misincorporation during DNA synthesis, analyzed by gel electrophoresis.

    PubMed Central

    Hillebrand, G G; McCluskey, A H; Abbott, K A; Revich, G G; Beattie, K L

    1984-01-01

    A method has been developed for simultaneous comparison of the propensity of a DNA polymerase to misincorporate at different points on a natural template-primer. In this method elongation of a [5'-32P] primer, annealed to a bacteriophage template strand, is carried out in the presence of only three dNTPs (highly purified by HPLC). Under these conditions the rate of primer elongation (monitored by gel electrophoresis/autoradiography) is limited by the rate of misincorporation at template positions complementary to the missing dNTP. Variations in the rate of elongation (revealed by autoradiographic banding patterns) reflect variations in the propensity for misincorporation at different positions along the template. The effect on primer elongation produced by addition of a chemically modified dNTP to 'minus' reactions reveals the mispairing potential of the modified nucleotide during DNA synthesis. By use of this electrophoretic assay of misincorporation we have demonstrated that the fidelity of E. coli DNA polymerase I varies greatly at different positions along a natural template, and that BrdUTP and IodUTP can be incorporated in place of dCTP during chain elongation catalyzed by this enzyme. Images PMID:6326053

  20. A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli.

    PubMed

    Gon, Stéphanie; Camara, Johanna E; Klungsøyr, Hege K; Crooke, Elliott; Skarstad, Kirsten; Beckwith, Jon

    2006-03-08

    We present evidence for a complex regulatory interplay between the initiation of DNA replication and deoxyribonucleotide synthesis. In Escherichia coli, the ATP-bound DnaA protein initiates chromosomal replication. Upon loading of the beta-clamp subunit (DnaN) of the replicase, DnaA is inactivated as its intrinsic ATPase activity is stimulated by the protein Hda. The beta-subunit acts as a matchmaker between Hda and DnaA. Chain elongation of DNA requires a sufficient supply of deoxyribonucleotides (dNTPs), which are produced by ribonucleotide reductase (RNR). We present evidence suggesting that the molecular switch from ATP-DnaA to ADP-DnaA is a critical step coordinating DNA replication with increased deoxyribonucleotide synthesis. Characterization of dnaA and dnaN mutations that result in a constitutively high expression of RNR reveal this mechanism. We propose that the nucleotide bound state of DnaA regulates the transcription of the genes encoding ribonucleotide reductase (nrdAB). Accordingly, the conversion of ATP-DnaA to ADP-DnaA after initiation and loading of the beta-subunit DnaN would allow increased nrdAB expression, and consequently, coordinated RNR synthesis and DNA replication during the cell cycle.

  1. A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli

    PubMed Central

    Gon, Stéphanie; Camara, Johanna E; Klungsøyr, Hege K; Crooke, Elliott; Skarstad, Kirsten; Beckwith, Jon

    2006-01-01

    We present evidence for a complex regulatory interplay between the initiation of DNA replication and deoxyribonucleotide synthesis. In Escherichia coli, the ATP-bound DnaA protein initiates chromosomal replication. Upon loading of the β-clamp subunit (DnaN) of the replicase, DnaA is inactivated as its intrinsic ATPase activity is stimulated by the protein Hda. The β-subunit acts as a matchmaker between Hda and DnaA. Chain elongation of DNA requires a sufficient supply of deoxyribonucleotides (dNTPs), which are produced by ribonucleotide reductase (RNR). We present evidence suggesting that the molecular switch from ATP-DnaA to ADP-DnaA is a critical step coordinating DNA replication with increased deoxyribonucleotide synthesis. Characterization of dnaA and dnaN mutations that result in a constitutively high expression of RNR reveal this mechanism. We propose that the nucleotide bound state of DnaA regulates the transcription of the genes encoding ribonucleotide reductase (nrdAB). Accordingly, the conversion of ATP-DnaA to ADP-DnaA after initiation and loading of the β-subunit DnaN would allow increased nrdAB expression, and consequently, coordinated RNR synthesis and DNA replication during the cell cycle. PMID:16482221

  2. Coordinated leading and lagging strand DNA synthesis by using the herpes simplex virus 1 replication complex and minicircle DNA templates.

    PubMed

    Stengel, Gudrun; Kuchta, Robert D

    2011-01-01

    The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not

  3. Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase [delta

    SciTech Connect

    Swan, Michael K.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2009-09-25

    DNA polymerase {delta} (Pol {delta}) is a high-fidelity polymerase that has a central role in replication from yeast to humans. We present the crystal structure of the catalytic subunit of yeast Pol {delta} in ternary complex with a template primer and an incoming nucleotide. The structure, determined at 2.0-{angstrom} resolution, catches the enzyme in the act of replication, revealing how the polymerase and exonuclease domains are juxtaposed relative to each other and how a correct nucleotide is selected and incorporated. The structure also reveals the 'sensing' interactions near the primer terminus, which signal a switch from the polymerizing to the editing mode. Taken together, the structure provides a chemical basis for the bulk of DNA synthesis in eukaryotic cells and a framework for understanding the effects of cancer-causing mutations in Pol {delta}.

  4. Integrating Value Clarification with High School Biology

    ERIC Educational Resources Information Center

    Barman, Charles R.

    1975-01-01

    Reports on research to see if value clarification would affect student attitudes toward science and biology and improve achievement in a BSCS Yellow Version biology course. Results indicated higher achievement by the group exposed to value clarification but no significant difference in attitudes between this group and the control group. (BR)

  5. DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein

    PubMed Central

    Morin, José A.; Cerrón, Fernando; Jarillo, Javier; Beltran-Heredia, Elena; Ciesielski, Grzegorz L.; Arias-Gonzalez, J. Ricardo

    2017-01-01

    Abstract Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB–DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during ‘in situ’ DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance. PMID:28486639

  6. DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein.

    PubMed

    Morin, José A; Cerrón, Fernando; Jarillo, Javier; Beltran-Heredia, Elena; Ciesielski, Grzegorz L; Arias-Gonzalez, J Ricardo; Kaguni, Laurie S; Cao, Francisco J; Ibarra, Borja

    2017-07-07

    Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB-DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during 'in situ' DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Relationship between DNA adduct formation and unscheduled DNA synthesis (UDS) in cultured mouse epidermal keratinocytes

    SciTech Connect

    Gill, R.D.; Nettikumara, A.N.; DiGiovanni, J. ); Butterworth, B.E. )

    1991-01-01

    Primary cultures of mouse epidermal keratinocytes from SENCAR mice were treated with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (B(a)P), ({plus minus}) 7{beta}-8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (({plus minus}) anti-BPDE), and ({plus minus}) 7{beta},8{alpha}-dihydroxy-9{beta},10{beta}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (({plus minus})syn-BPDE) to examine the relationship between DNA adduct formation and the induction of unscheduled DNA synthesis (UDS). DNA adducts were measured as pmol hydrocarbon bound per mg of DNA, and UDS was quantitated autoradiographically as net grains per nucleus. A good correlation was observed between the levels of UDS detected and the amount of DNA adducts present int he cell population when comparing similar compounds within the linear dose-response range of 0.005 {mu}g/ml-0.25 {mu}g/ml. These results suggest that the present UDS assay with MEKs is a useful assay for the rapid screening of potential genotoxic agents. However, the limits of sensitivity are such that the current assay may be unable to detect a low level of DNA damage induced by some weakly genotoxic (carcinogenic) agents. In addition, while the limits of sensitivity determined in these experiments apply to the polycyclic aromatic hydrocarbon class, other classes of genotoxic compounds such as alkylating agents or crosslinking agents may exhibit different thresholds of detection.

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

    PubMed

    Seco, Elena M; Ayora, Silvia

    2017-08-21

    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. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

    SciTech Connect

    Horwitz, M.S.; Friefeld, B.R.; Keiser, H.D.

    1982-12-01

    Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60/sup 0/C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases ..cap alpha.., BETA, ..gamma.. and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.

  10. A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A

    PubMed Central

    Takahashi, Shunsuke; Kawasaki, Shohei; Miyata, Hidefumi; Kurita, Hirofumi; Mizuno, Takeshi; Matsuura, Shun-ichi; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji

    2014-01-01

    Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (−flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (−flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (−flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (−flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3′-5′exo–) was promoted by DNA stretching with buffer flow. PMID:24625741

  11. Evaluation of DNA synthesis with carbon-11-labeled 4′-thiothymidine

    PubMed Central

    Toyohara, Jun

    2016-01-01

    In the cancer research field, the preferred method for evaluating the proliferative activity of cancer cells in vivo is to measure DNA synthesis rates. The cellular proliferation rate is one of the most important cancer characteristics, and represents the gold standard of pathological diagnosis. Positron emission tomography (PET) has been used to evaluate in vivo DNA synthetic activity through visualization of enhanced nucleoside metabolism. However, methods for the quantitative measurement of DNA synthesis rates have not been fully clarified. Several groups have been engaged in research on 4′-[methyl-11C]-thiothymidine (11C-4DST) in an effort to develop a PET tracer that allows quantitative measurement of in vivo DNA synthesis rates. This mini-review summarizes the results of recent studies of the in vivo measurement of cancer DNA synthesis rates using 11C-4DST. PMID:27721942

  12. The effect of bleomycin on DNA synthesis in ataxia telangiectasia lymphoid cells

    SciTech Connect

    Cohen, M.M.; Simpson, S.J.

    1982-01-01

    Bleomycin, a radiomimetic glycopeptide, inhibits de novo DNA synthesis in ataxia telangiectasia lymphoblastoid B cells to a markedly lesser extent than in normal and xeroderma pigmentosum lymphoid cells. This observation is similar to that following ionizing radiation; however, the effect is slower following the chemical treatment. Recovery of the normal cells occurs 15-18 hours after treatment, whereas the ataxia telangiectasia lines do not attain normal levels of DNA synthesis during the entire 24-hour observation period. Similar differences were not observed following treatment with mitomycin C, a bifunctional alkylating agent, indicating a specific effect of bleomycin on DNA synthesis in ataxia telangiectasia cells. Following bleomycin treatment and preincubation with hydroxyurea, residual DNA synthesis in ataxia telangiectasia cells was similar to that in both normal and xeroderma pigmentosum lymphoid lines, suggesting that the capacity to repair the induced DNA lesion is present.

  13. Method and apparatus for synthesis of arrays of DNA probes

    DOEpatents

    Cerrina, Francesco; Sussman, Michael R.; Blattner, Frederick R.; Singh-Gasson, Sangeet; Green, Roland

    2002-04-23

    The synthesis of arrays of DNA probes sequences, polypeptides, and the like is carried out using a patterning process on an active surface of a substrate. An image is projected onto the active surface of the substrate utilizing an image former that includes a light source that provides light to a micromirror device comprising an array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two positions. Projection optics receives the light reflected from the micromirrors along an optical axis and precisely images the micromirrors onto the active surface of the substrate, which may be used to activate the surface of the substrate. The first level of bases may then be applied to the substrate, followed by development steps, and subsequent exposure of the substrate utilizing a different pattern of micromirrors, with further repeats until the elements of a two dimensional array on the substrate surface have an appropriate base bound thereto. The micromirror array can be controlled in conjunction with a DNA synthesizer supplying appropriate reagents to a flow cell containing the active substrate to control the sequencing of images presented by the micromirror array in coordination of the reagents provided to the substrate.

  14. Induction of Mitochondrial DNA Synthesis in Monkey Cells Infected by Simian Virus 40 and (or) Treated with Calf Serum

    PubMed Central

    Levine, Arnold J.

    1971-01-01

    Infection of confluent monolayer cultures of African green monkey kidney cells with simian virus 40 results in an enhanced synthesis of nuclear and mitochondrial DNA. This is demonstrated both by an increased rate of incorporation of [3H]thymidine into mitochondrial DNA and by detection of increased amounts of mitochondrial DNA in infected cells. With monkey BSC-1 cells, where SV40 infection does not result in a stimulation of nuclear DNA synthesis, no stimulation of mitochondrial DNA synthesis is observed. SV40 infection of mouse 3T3 cells also stimulates nuclear and mitochondrial DNA synthesis. PMID:4323784

  15. CyDNA: synthesis and replication of highly Cy-dye substituted DNA by an evolved polymerase.

    PubMed

    Ramsay, Nicola; Jemth, Ann-Sofie; Brown, Anthony; Crampton, Neal; Dear, Paul; Holliger, Philipp

    2010-04-14

    DNA not only transmits genetic information but can also serve as a versatile supramolecular scaffold. Here we describe a strategy for the synthesis and replication of DNA displaying hundreds of substituents using directed evolution of polymerase function by short-patch compartmentalized self-replication (spCSR) and the widely used fluorescent dye labeled deoxinucleotide triphosphates Cy3-dCTP and Cy5-dCTP as substrates. In just two rounds of spCSR selection, we have isolated a polymerase that allows the PCR amplification of double stranded DNA fragments up to 1kb, in which all dC bases are substituted by its fluorescent dye-labeled equivalent Cy3- or Cy5-dC. The resulting "CyDNA" displays hundreds of aromatic heterocycles on the outside of the DNA helix and is brightly colored and highly fluorescent. CyDNA also exhibits significantly altered physicochemical properties compared to standard B-form DNA, including loss of silica and intercalating dye binding, resistance to cleavage by some endonucleases, an up to 40% increased apparent diameter as judged by atomic force microscopy and organic phase partitioning during phenol extraction. CyDNA also displays very bright fluorescence enabling significant signal gains in microarray and microfluidic applications. CyDNA represents a step toward a long-term goal of the encoded synthesis of DNA-based polymers of programmable and evolvable sequence and properties.

  16. One-step synthesis of silver nanoparticles, nanorods, and nanowires on the surface of DNA network.

    PubMed

    Wei, Gang; Zhou, Hualan; Liu, Zhiguo; Song, Yonghai; Wang, Li; Sun, Lanlan; Li, Zhuang

    2005-05-12

    Here, we describe a one-step synthesis of silver nanoparticles, nanorods, and nanowires on DNA network surface in the absence of surfactant. Silver ions were first adsorbed onto the DNA network and then reduced in sodium borohydride solution. Silver nanoparticles, nanorods, and nanowires were formed by controlling the size of pores of the DNA network. The diameter of the silver nanoparticles and the aspect ratio of the silver nanorods and nanowires can be controlled by adjusting the DNA concentration and reduction time.

  17. Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

    PubMed Central

    Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

    2012-01-01

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

  18. DNA binding properties of human DNA polymerase eta: implications for fidelity and polymerase switching of translesion synthesis.

    PubMed

    Kusumoto, Rika; Masutani, Chikahide; Shimmyo, Shizu; Iwai, Shigenori; Hanaoka, Fumio

    2004-12-01

    The human XPV (xeroderma pigmentosum variant) gene is responsible for the cancer-prone xeroderma pigmentosum syndrome and encodes DNA polymerase eta (pol eta), which catalyses efficient translesion synthesis past cis-syn cyclobutane thymine dimers (TT dimers) and other lesions. The fidelity of DNA synthesis by pol eta on undamaged templates is extremely low, suggesting that pol eta activity must be restricted to damaged sites on DNA. Little is known, however, about how the activity of pol eta is targeted and restricted to damaged DNA. Here we show that pol eta binds template/primer DNAs regardless of the presence of TT dimers. Rather, enhanced binding to template/primer DNAs containing TT dimers is only observed when the 3'-end of the primer is an adenosine residue situated opposite the lesion. When two nucleotides have been incorporated into the primer beyond the TT dimer position, the pol eta-template/primer DNA complex is destabilized, allowing DNA synthesis by DNA polymerases alpha or delta to resume. Our study provides mechanistic explanations for polymerase switching at TT dimer sites.

  19. DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools

    ERIC Educational Resources Information Center

    Godsell, P. M.; Balls, M.

    1973-01-01

    Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

  20. DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools

    ERIC Educational Resources Information Center

    Godsell, P. M.; Balls, M.

    1973-01-01

    Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

  1. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... of Section 508....

  2. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... of Section 508....

  3. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... of Section 508....

  4. Values Clarification: Your Job or Mine?

    ERIC Educational Resources Information Center

    McGough, Kris

    1977-01-01

    The author criticizes values clarification techniques done without parental consent in non-elective courses because they invade privacy, lead to ethical relativism, and have the potential to damage children's ego-strength. (Author/AV)

  5. Synthesis of a major mitomycin C DNA adduct via a triaminomitosene.

    PubMed

    Champeil, Elise; Paz, Manuel M; Lukasiewicz, Elaan; Kong, Wan S; Watson, Stephanie; Sapse, Anne-Marie

    2012-12-01

    We report here the synthesis of two amino precursors for the production of mitomycin C and 10-decarbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precursors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O(6)-(2-p-nitrophenylethyl)deoxyinosine followed by deprotection at the N(2) and O(6) positions.

  6. Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences

    PubMed Central

    Zhou, Xiaochuan; Cai, Shiying; Hong, Ailing; You, Qimin; Yu, Peilin; Sheng, Nijing; Srivannavit, Onnop; Muranjan, Seema; Rouillard, Jean Marie; Xia, Yongmei; Zhang, Xiaolin; Xiang, Qin; Ganesh, Renuka; Zhu, Qi; Matejko, Anna; Gulari, Erdogan; Gao, Xiaolian

    2004-01-01

    Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression. PMID:15477391

  7. Short-step chemical synthesis of DNA by use of MMTrS group for protection of 5'-hydroxyl group.

    PubMed

    Shiraishi, Miyuki; Utagawa, Eri; Ohkubo, Akihiro; Sekine, Mitsuo; Seio, Kohji

    2007-01-01

    4-methoxytrithylthio (MMTrS) group was applied for the appropriately protected four canonical nucleosides. We prepared the phosphoroamidite units by use of these nucleosides and developed the synthesis of oligodeoxynucleotides without any acidic treatment. Moreover, the new DNA synthesis protocol was applied to an automated DNA synthesizer for the synthesis of longer oligodeoxynucleotides.

  8. Nucleotide sequence of a preferred maize chloroplast genome template for in vitro DNA synthesis.

    PubMed Central

    Gold, B; Carrillo, N; Tewari, K K; Bogorad, L

    1987-01-01

    Maize chloroplast DNA sequences representing 94% of the chromosome have been surveyed for their activity as autonomously replicating sequences in yeast and as templates for DNA synthesis in vitro by a partially purified chloroplast DNA polymerase. A maize chloroplast DNA region extending over about 9 kilobase pairs is especially active as a template for the DNA synthesis reaction. Fragments from within this region are much more active than DNA from elsewhere in the chromosome and 50- to 100-fold more active than DNA of the cloning vector pBR322. The smallest of the strongly active subfragments that we have studied, the 1368-base-pair EcoRI fragment x, has been sequenced and found to contain the coding region of chloroplast ribosomal protein L16. EcoRI fragment x shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that forms a displacement loop [Wang, X.-M., Chang, C.H., Waddell, J. & Wu, M. (1984) Nucleic Acids Res. 12, 3857-3872]. Maize chloroplast DNA fragments that permit autonomous replication of DNA in yeast are not active as templates for DNA synthesis in the in vitro assay. The template active region we have identified may represent one of the origins of replication of maize chloroplast DNA. Images PMID:3025853

  9. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips.

    PubMed

    Kosuri, Sriram; Eroshenko, Nikolai; Leproust, Emily M; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M

    2010-12-01

    Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

  10. Inhibition of mouse peritoneal macrophage DNA synthesis by infection with the Arenavirus Pichinde. Interim report

    SciTech Connect

    Friedlander, A.M.; Jahrling, P.B.; Merrill, P.; Tobery, S.

    1983-01-19

    Macrophage DNA synthesis and proliferation occur during the development of cell-mediated immunity and in the early non-specific reaction to infection. Arenaviruses have a predilection for infection of cells of the reticuloendothelial system and in this study we have examined the effect of the arenavirus Pichinde on macrophage DNA synthesis. We have found that infection of mouse peritoneal macrophages with Pichinde caused a profound dose dependent inhibition of the DNA synthesis induced by macrophage growth factor/colony stimulating factor. At a multiplicity of inoculum of five there is a 75-95% inhibition of DNA synthesis. Viable virus is necessary for inhibition since Pichinde inactivated by heat or cobalt irradiation had no effect. Similarly, virus pre-treated with an antiserum to Pichinde was without inhibitory effect. Inhibition was demonstrated by measuring DNA synthesis spectrofluorometrically as well as by 3H-thymidine incorporation. The inhibition of DNA synthesis was not associated with any cytopathology. There was no evidence that the inhibition was due to soluble factors, such as prostaglandins or interferon, released by infected cells. These studies demonstrate, for the first time in vitro, a significant alteration in macrophage function caused by infection with an arenavirus. It is possible that inhibition of macrophage proliferation represents a mechanism by which some microorganisms interfere with host resistance.

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

    PubMed

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

    2016-02-01

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

  12. Ganciclovir inhibits lymphocyte proliferation by impairing DNA synthesis.

    PubMed

    Battiwalla, Minoo; Wu, Yiyuan; Bajwa, Rajinder P S; Radovic, Marija; Almyroudis, Nikolaos G; Segal, Brahm H; Wallace, Paul K; Nakamura, Ryotaro; Padmanabhan, Swaminathan; Hahn, Theresa; McCarthy, Philip L

    2007-07-01

    Cytomegalovirus (CMV) disease-related mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients has dramatically declined because of ganciclovir prophylaxis and preemptive therapeutic strategies. However, ganciclovir has not improved overall survival in randomized studies despite effectively preventing overt CMV disease. Moreover, recurrent posttransplant CMV antigenemia, associated with prolonged ganciclovir exposure, is a predictor of increased relapse of malignancy. We examined the hypothesis that ganciclovir itself may have a negative impact on immune reconstitution by testing the effect of ganciclovir on normal human lymphocytes in vitro. T-lymphocyte activation and proliferation, as measured by PHA-induced (3)H-thymidine uptake, was greatly reduced at therapeutic concentrations of ganciclovir (10 microg/mL) but not for foscarnet (300 microM/L). Moreover, ganciclovir impaired bromodeoxyuridine incorporation in proliferating lymphocytes, but did not impair lymphocyte survival or induce lymphocyte apoptosis. Collectively, these results show that ganciclovir suppresses T-lymphocyte proliferation in vitro by inhibiting DNA synthesis; with implications for T-lymphocyte function following allogeneic BMT.

  13. Sugar-oligoamides: synthesis of DNA minor groove binders.

    PubMed

    Badía, Concepción; Souard, Florence; Vicent, Cristina

    2012-12-07

    Sugar-oligoamides have been designed and synthesized as structurally simple carbohydrate-based ligands to study carbohydrate-minor groove DNA interactions. Here we report an efficient solution-phase synthetic strategy to obtain two broad families of sugar-oligoamides. The first type, structure vector A (-Py[Me]-γ-Py-Ind), has a methyl group present as a substituent on the nitrogen of pyrrole B, connected to the C terminal of the oligoamide fragment. The second type, structure vector B (-Py[(CH(2))(11)OH]-γ-Py-Ind), has an alkyl chain present on the nitrogen of pyrrole B connected to the C terminal of the oligoamide fragment and has been designed to access to di- and multivalent sugar-oligoamides. By using sequential DIPC/HOBt coupling reactions, the oligoamide fragment -Py[R]-γ-Py-Ind has been constructed. The last coupling reaction between the anomeric amino sugar and the oligoamide fragment was carried out by activating the acid derivative as a BtO- ester, which has been performed by using TFFH. The isolated esters (BtO-Py[R]-γ-Py-Ind) were coupled with selected amino sugars using DIEA in DMF. The synthesis of two different selective model vectors (vector A (1) and vector B (2)) and two types of water-soluble sugar-oligoamide ligands, with vector A structure (compounds 3-7) and with vector B structure (compound 8), was carried out.

  14. Synthesis, integration, and restriction and modification of mycoplasma virus L2 DNA

    SciTech Connect

    Dybvig, K.

    1981-01-01

    Mycoplasma virus L2 is an enveloped, nonlytic virus containing double-stranded, superhelical DNA. The L2 virion contains about 7 to 8 major proteins identified by SDS-polyacrylamide gel electrophoresis, but the virion has no discernible capsid structure. It has been suggested that the L2 virion is a DNA-protein condensation surrounded by a lipid-protein membrane. The host for mycoplasma virus L2 is Acholeplasma laidlawii. A. laidlawii has no cell wall and contains a small genome, 1 x 10/sup 9/ daltons, which is two to three times smaller than that of most bacteria. Infection of A. laidlawii by L2 is nonlytic. The studies in this thesis show that L2 DNA synthesis begins at about 1 hour of infection and lasts throughout the infection. Viral DNA synthesis is inhibited by chloramphenicol, streptomycin, and novobiocin. Packaging of L2 DNA into progeny virus is also inhibited by chloramphenicol and novobiocin. It is concluded that protein synthesis and probably DNA gyrase activity are required for L2 DNA synthesis, and for packaging of L2 DNA into progeny virus. DNA-DNA hybridization studies demonstrate that L2 DNA integrates into the host cell during infection, and subsequent to infection the cells are mycoplasma virus L2 lysogens. The viral site of integration has been roughly mapped. L2 virus is restricted and modified by A. laidlawii strains JA1 and K2. The nature of the modification in strain K2 has been elucidated. Two L2 variants containing insertions in the viral DNA were identified in these studies. Restriction endonuclease cleavage maps of these variants have been determined. DNA from L2 and another isolate of L2, MV-Lg-L 172, are compared in these studies. 74 references, 33 figures, 6 tables. (ACR)

  15. Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

    PubMed

    Christen, Matthias; Deutsch, Samuel; Christen, Beat

    2015-08-21

    Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

  16. Synthesis of DNA mimics representing HypNA-pPNA hetero-oligomers.

    PubMed

    Efimov, Vladimir A; Chakhmakhcheva, Oksana G

    2005-01-01

    The methods for the synthesis and purification of negatively charged peptide nucleic acid (PNA)-relative deoxyribonucleic acid (DNA) mimics containing alternating residues of phosphono peptide nucleic acid (pPNA) monomers and PNA-like monomers on the base of trans-4-hydroxy-L-proline are described. Examples of the chimeric oligomers hybridization with complementary DNA and ribonucleic acid fragments are demonstrated.

  17. Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study.

    PubMed Central

    Hardt, N; Pedrali-Noy, G; Focher, F; Spadari, S

    1981-01-01

    A radioautographic examination of nuclear DNA synthesis in unirradiated and u.v.-irradiated HeLa cells, in the presence and in the absence of aphidicolin, showed that aphidicolin inhibits nuclear DNA replication and has no detectable effect on DNA repair synthesis. Although the results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, they do not preclude a significant role for this enzyme in DNA repair processes. Images PLATE 1 PMID:6803764

  18. Autonomous Multistep Organic Synthesis in a Single Isothermal Solution Mediated by a DNA Walker

    PubMed Central

    He, Yu; Liu, David R.

    2010-01-01

    Multistep synthesis in the laboratory typically requires numerous reaction vessels, each containing a different set of reactants. In contrast, cells are capable of performing highly efficient and selective multistep biosynthesis under mild conditions with all reactants simultaneously present in solution. If the latter approach could be applied in the laboratory, it may improve the ease, speed, and efficiency of multistep reaction sequences. Here we show that a DNA mechanical device— a DNA walker moving along a DNA track— can be used to perform a series of amine acylation reactions in a single solution without any external intervention. The multistep products generated by this primitive ribosome mimetic are programmed by the sequence of the DNA track, are unrelated to the structure of DNA, and are formed with speeds and overall yields significantly greater than those previously achieved by multistep DNA-templated small-molecule synthesis. PMID:20935654

  19. Repair synthesis by human cell extracts in DNA damaged by cis- and trans-diamminedichloroplatinum(II).

    PubMed Central

    Hansson, J; Wood, R D

    1989-01-01

    DNA damage was induced in closed circular plasmid DNA by treatment with cis- or trans-diamminedichloroplatinum(II). These plasmids were used as substrates in reactions to give quantitative measurements of DNA repair synthesis mediated by cell free extracts from human lymphoid cell lines. Adducts induced by both drugs stimulated repair synthesis in a dose dependent manner by an ATP-requiring process. Measurements by an isopycnic gradient sedimentation method gave an upper limit for the average patch sizes in this in vitro system of around 140 nucleotides. It was estimated that up to 3% of the drug adducts induce the synthesis of a repair patch. The repair synthesis is due to repair of a small fraction of frequent drug adducts, rather than extensive repair of a rare subclass of lesions. Nonspecific DNA synthesis in undamaged plasmids, caused by exonucleolytic degradation and resynthesis, was reduced by repeated purification of intact circular forms. An extract made from cells belonging to xeroderma pigmentosum complementation group A was deficient in repair synthesis in response to the presence of cis- or trans-diamminedichloroplatinum(II) adducts in DNA. Images PMID:2554251

  20. Regulatory interactions between phospholipid synthesis and DNA replication in Caulobacter crescentus.

    PubMed Central

    Loewy, B; Marczynski, G T; Dingwall, A; Shapiro, L

    1990-01-01

    Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes. A C. crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. The first effect of withholding supplement was the cessation of synthesis of phosphatidylglycerol, a major component of the C. crescentus membrane. In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. However, after replication proceeded bidirectionally for a short time, DNA synthesis dropped to a low level. The cell cycle blocked at a distinct middivision stalked cell, and this was followed by cell death. The "glycerol-less" death of the gpsA mutant could be prevented if the cells were treated with novobiocin to prevent the initiation of DNA replication. Our observations suggest that the processivity of C. crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome. Images PMID:2211495

  1. Effect of the (+)-CC-1065-(N3-adenine)DNA adduct on in vitro DNA synthesis mediated by Escherichia coli DNA polymerase

    SciTech Connect

    Daekyu Sun; Hurley, L.H. )

    1992-03-17

    (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form an N3-adenine DNA adduct which lies in the minor groove of the DNA helix overlapping with a 5-base-pair region. As a consequence of covalent modification with (+)-CC-1065, the DNA helix bends into the minor groove and also undergoes winding and stiffening. In the studies described here, in which the authors have constructed site-directed DNA adducts on single-stranded DNA templates. They have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all show strong blocks against progression of Klenow fragment, E. coli DNA polymerase, and T4 DNA polymerase. The inhibition of bypass of drug lesions by polymerase could be partially alleviated by increasing the concentration of dNTPs and, to a small extent, by increasing polymerase levels. Klenow fragment binds equally well to a DNA template adjacent to a drug modification site and to unmodified DNA. These results taken together lead one to suspect that is primarily inhibition of base pairing around the drug modification site and not prevention of polymerase binding that leads to blockage of DNA synthesis. The winding phenomena uniquely found with these compounds may be associated with the potent biological effect known as delayed lethality.

  2. In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

    PubMed

    Saaem, Ishtiaq; Ma, Kuo-Sheng; Marchi, Alexandria N; LaBean, Thomas H; Tian, Jingdong

    2010-02-01

    Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

  3. The effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and X-irradiated Escherichia coli

    SciTech Connect

    Billen, D.

    1983-07-01

    In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to X rays. This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action. In an effort to learn more of the relationship between X-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out. The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis. However, I-, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.

  4. Effects of radioprotectors on DNA polymerase I-directed repair synthesis and DNA strand breaks in toluene-treated and x-irradiated Escherichia coli

    SciTech Connect

    Billen, D.

    1983-07-01

    In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is induced by exposure to x rays. This repair synthesis may be amplified and easily measured through inhibition of DNA ligase action. In an effort to learn more of the relationship between x-ray-induced strand breaks in cellular DNA and the extent of this repair synthesis, experiments designed to compare the influence of radioprotectors on both strand-break production and repair synthesis have been carried out. The results show that cysteamine, sodium formate, and glycerol not only protect against strand breaks but also reduce DNA polymerase I-directed repair synthesis. However, I/sup -/, an efficient hydroxyl radical scavenger, is not as effective a protective agent against strand breaks and does not measurably affect repair synthesis in our system.

  5. Fractional synthesis rates of DNA and protein in rabbit skin are not correlated.

    PubMed

    Zhang, Xiao-jun; Chinkes, David L; Wu, Zhanpin; Martini, Wenjun Z; Wolfe, Robert R

    2004-09-01

    We developed a method for measurement of skin DNA synthesis, reflecting cell division, in conscious rabbits by infusing D-[U-(13)C(6)]glucose and L-[(15)N]glycine. Cutaneous protein synthesis was simultaneously measured by infusion of L-[ring-(2)H(5)]phenylalanine. Rabbits were fitted with jugular venous and carotid arterial catheters, and were studied during the infusion of an amino acid solution (10% Travasol). The fractional synthetic rate (FSR) of DNA from the de novo nucleotide synthesis pathway, a reflection of total cell division, was 3.26 +/- 0.59%/d in whole skin and 3.08 +/- 1.86%/d in dermis (P = 0.38). The de novo base synthesis pathway accounted for 76 and 60% of the total DNA FSR in whole skin and dermis, respectively; the contribution from the base salvage pathway was 24% in whole skin and 40% in dermis. The FSR of protein in whole skin was 5.35 +/- 4.42%/d, which was greater (P < 0.05) than that in dermis (2.91 +/- 2.52%/d). The FSRs of DNA and protein were not correlated (P = 0.33), indicating that cell division and protein synthesis are likely regulated by different mechanisms. This new approach enables investigations of metabolic disorders of skin diseases and regulation of skin wound healing by distinguishing the 2 principal components of skin metabolism, which are cell division and protein synthesis.

  6. Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis.

    PubMed

    Levring, Trine B; Kongsbak, Martin; Rode, Anna K O; Woetmann, Anders; Ødum, Niels; Bonefeld, Charlotte Menné; Geisler, Carsten

    2015-09-08

    Adaptive immune responses require activation and expansion of antigen-specific T cells. Whereas early T cell activation is independent of exogenous cystine (Cys2), T cell proliferation is dependent of Cys2. However, the exact roles of Cys2 in T cell proliferation still need to be determined. The aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys2. Vice-versa, the GSH synthesis inhibitor L-buthionine-sulfoximine (BSO) and inhibition of Cys2 uptake with glutamate inhibited GSH and DNA synthesis in parallel. We further found that thioredoxin (Trx) can partly substitute for GSH during DNA synthesis. Finally, we show that GSH or Trx is required for the activity of ribonucleotide reductase (RNR), the enzyme responsible for generation of the deoxyribonucleotide DNA building blocks. In conclusion, we show that activated human T cells require exogenous Cys2 to proliferate and that this is partly explained by the fact that Cys2 is required for production of GSH, which in turn is required for optimal RNR-mediated deoxyribonucleotide synthesis and DNA replication.

  7. Iron at the cell surface controls DNA synthesis in CCl 39 cells.

    PubMed

    Alcain, F J; Löw, H; Crane, F L

    1994-08-30

    Treatment of CCl 39 cells with the impermeable iron II chelator bathophenanthroline disulfonate (BPS) inhibits both DNA synthesis and transplasma membrane electron transport. The inhibition persists when the BPS is removed, and the extract from 10(6) cells contains up to 1.28 nmoles iron II chelated to BPS. The BPS iron II chelate itself is not inhibitory. Both DNA synthesis and electron transport are restored by addition of microM iron II or iron III compounds to extracted cells. Other impermeable chelators for iron II give similar inhibition, whereas the iron III-specific Tiron or copper-specific bathocuproine sulfonate do not inhibit. The inhibition differs from the permeable iron III chelator inhibition of ribonucleotide reductase, because inhibition of DNA synthesis by the permeable chelators is reversed when chelator is removed. The response to growth factors also differs, with no impermeable chelator inhibition on 10% fetal calf serum contrasting to inhibition by permeable chelators. DNA synthesis with both activation of tyrosine kinase with EGF plus insulin or by thrombin or ceruloplasmin led to protein kinase C activation as inhibited by the impermeable chelators. It is proposed that an iron available on the cell surface is required for DNA synthesis and plasma membrane electron transport.

  8. Inhibition of DNA synthesis in CCL 39 cells by impermeable iron chelators.

    PubMed

    Alcaín, F J; Löw, H; Crane, F L

    1997-02-01

    The synthesis of DNA in CCl 39 cells is inhibited by the presence of the Fe2+ chelator bathophenanthroline disulfonate (BPS) when growth is stimulated by thrombin EGF plus insulin, but not by fetal calf serum. The presence of transferrin and Fe3+ in fetal calf serum can be the basis for lack of BPS effect with serum. The impermeable Fe3+ chelator Tiron does not, by itself, inhibit growth factor induced DNA synthesis, but it induces together with BPS inhibition on fetal calf serum induced DNA synthesis. The combined effect of BPS and Tiron is similar to inhibition of DNA synthesis by impermeable polyvalent DTPA which can chelate both Fe2+ and Fe3+ but does not inhibit ribonucleotide reductase in intact cells. Ferrous iron that bind BPS can relieve the inhibition at stoichiometric concentration. Ferric iron also prevents the inhibition even though it does not bind BPS. BPS does not inhibit DNA synthesis in HeLa cells. BPS reacts with iron from CCl 39 cells but not from HeLa cells. Data show that iron available for impermeable external chelators is in the ferrous state, and that exogenous iron should be reduced before it reverses the inhibition.

  9. The identification of translesion DNA synthesis regulators: Inhibitors in the spotlight.

    PubMed

    Bertolin, A P; Mansilla, S F; Gottifredi, V

    2015-08-01

    Over the past half-century, we have become increasingly aware of the ubiquity of DNA damage. Under the constant exposure to exogenous and endogenous genomic stress, cells must attempt to replicate damaged DNA. The encounter of replication forks with DNA lesions triggers several cellular responses, including the activation of translesion DNA synthesis (TLS), which largely depends upon specialized DNA polymerases with flexible active sites capable of accommodating bulky DNA lesions. A detrimental aspect of TLS is its intrinsic mutagenic nature, and thus the activity of the TLS polymerases must ideally be restricted to synthesis on damaged DNA templates. Despite their potential clinical importance in chemotherapy, TLS inhibitors have been difficult to identify since a direct assay designed to quantify genomic TLS events is still unavailable. Herein we discuss the methods that have been used to validate TLS inhibitors such as USP1, p21 and Spartan, highlighting research that has revealed their contribution to the control of DNA synthesis on damaged and undamaged templates. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. The identification of translesion DNA synthesis regulators: inhibitors in the spotlight

    PubMed Central

    Bertolin, AP; Mansilla, SF; Gottifredi, V

    2015-01-01

    Over the past half-century, we have become increasingly aware of the ubiquity of DNA damage. Under the constant exposure to exogenous and endogenous genomic stress, cells must attempt to replicate damaged DNA. The encounter of replication forks with DNA lesions triggers several cellular responses, including the activation of translesion DNA synthesis (TLS), which largely depends upon specialized DNA polymerases with flexible active sites capable of accommodating bulky DNA lesions. A detrimental aspect of TLS is its intrinsic mutagenic nature, and thus the activity of the TLS polymerases must ideally be restricted to synthesis on damaged DNA templates. Despite their potential clinical importance in chemotherapy, TLS inhibitors have been difficult to identify since a direct assay designed to quantify genomic TLS events is still unavailable. Herein we discuss the methods that have been used to validate TLS inhibitors such as USP1, p21 and Spartan, highlighting research that has revealed their contribution to the control of DNA synthesis on damaged and undamaged templates. PMID:26002196

  11. Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

    PubMed Central

    Decker, R S; Yamaguchi, M; Possenti, R; DePamphilis, M L

    1986-01-01

    Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended

  12. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  13. DNA-Based Synthesis and Assembly of Organized Iron Oxide Nanostructures

    NASA Astrophysics Data System (ADS)

    Khomutov, Gennady B.

    Organized bio-inorganic and hybrid bio-organic-inorganic nanostructures consisting of iron oxide nanoparticles and DNA complexes have been formed using methods based on biomineralization, interfacial and bulk phase assembly, ligand exchange and substitution, Langmuir-Blodgett technique, DNA templating and scaffolding. Interfacially formed planar DNA complexes with water-insoluble amphiphilic polycation or intercalator Langmuir monolayers were prepared and deposited on solid substrates to form immobilized DNA complexes. Those complexes were then used for the synthesis of organized DNA-based iron oxide nanostructures. Planar net-like and circular nanostructures of magnetic Fe3O4 nanoparticles were obtained via interaction of cationic colloid magnetite nanoparticles with preformed immobilized DNA/amphiphilic polycation complexes of net-like and toroidal morphologies. The processes of the generation of iron oxide nanoparticles in immobilized DNA complexes via redox synthesis with various iron sources of biological (ferritin) and artificial (FeCl3) nature have been studied. Bulk-phase complexes of magnetite nanoparticles with biomolecular ligands (DNA, spermine) were formed and studied. Novel nano-scale organized bio-inorganic nanostructures - free-floating sheet-like spermine/magnetite nanoparticle complexes and DNA/spermine/magnetite nanoparticle complexes were synthesized in bulk aqueous phase and the effect of DNA molecules on the structure of complexes was discovered.

  14. Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells.

    PubMed

    Izhar, Lior; Ziv, Omer; Cohen, Isadora S; Geacintov, Nicholas E; Livneh, Zvi

    2013-04-16

    DNA lesions can block replication forks and lead to the formation of single-stranded gaps. These replication complications are mitigated by DNA damage tolerance mechanisms, which prevent deleterious outcomes such as cell death, genomic instability, and carcinogenesis. The two main tolerance strategies are translesion DNA synthesis (TLS), in which low-fidelity DNA polymerases bypass the blocking lesion, and homology-dependent repair (HDR; postreplication repair), which is based on the homologous sister chromatid. Here we describe a unique high-resolution method for the simultaneous analysis of TLS and HDR across defined DNA lesions in mammalian genomes. The method is based on insertion of plasmids carrying defined site-specific DNA lesions into mammalian chromosomes, using phage integrase-mediated integration. Using this method we show that mammalian cells use HDR to tolerate DNA damage in their genome. Moreover, analysis of the tolerance of the UV light-induced 6-4 photoproduct, the tobacco smoke-induced benzo[a]pyrene-guanine adduct, and an artificial trimethylene insert shows that each of these three lesions is tolerated by both TLS and HDR. We also determined the specificity of nucleotide insertion opposite these lesions during TLS in human genomes. This unique method will be useful in elucidating the mechanism of DNA damage tolerance in mammalian chromosomes and their connection to pathological processes such as carcinogenesis.

  15. Liver nuclear DNA synthesis in mice following carbon tetrachloride administration or partial hepatectomy

    SciTech Connect

    Gans, J.H.; Korson, R.

    1984-02-01

    Long-term, continuous (twice per week) administration of CCl/sub 4/ to male mice resulted in a high incidence of liver nodules which appear to be resistant to the necrotizing effects of CCl/sub 4/ but showed no features of malignant neoplasia. Liver nuclear DNA synthesis was compared in mice given CCl/sub 4/ and in mice subjected to partial hepatectomy (PH). Mice were given by gavage corn oil or CCl/sub 4/ in corn oil for periods of 2 to 25 weeks and several mice were subjected to PH after 12 and 25 weeks of corn oil treatment. Mice were given (/sup 3/H)TdR during liver regeneration and newly synthesized liver nuclear DNA was isolated and separated by BND-cellulose chromatography. Greater than 85% of the labeled DNA from PH mice eluted from BND-cellulose columns as double-stranded (ds) DNA with single-stranded (ss) regions or ends and less than 15% as ds DNA. When mice were treated with CCl/sub 4/ for 8 weeks or longer a significantly greater portion of liver nuclear DNA eluted as ds DNA. Administration of HU and 5-FU with (/sup 3/H)TdR decreased (/sup 3/H)TdR incorporation into DNA to low levels incompatible with unscheduled DNA synthesis. Single doses of CCl/sub 4/ given to mice treated with corn oil for 2 to 12 weeks provided newly synthesized DNA which was primarily (>80%) ds DNA with ss regions or ends, but after 25 weeks of corn oil administration, a single dose of CCl/sub 4/ resulted in newly synthesized DNA with a greater proportion of ds DNA. The high labeling of ds DNA in mice treated with CCl/sub 4/ may have resulted from an alternate pathway of DNA synthesis catalyzed by the enzymes or enzyme complexes associated with semiconservative DNA synthesis or from proliferation of nonparenchymal cells with a rapid turn-over rate.

  16. Comparison of dna-copying fidelity during repair and semiconservative synthesis by radioactive precursor distribution analysis

    SciTech Connect

    Nemirovskii, L.E.; Vasil'ev, V.K.

    1986-04-01

    The authors compare the fidelity of DNA copying during semiconservative and reparative synthesis under normal conditions and during cortisol-induced activation of free-radical processes, by examining the distribution of radioactivity among DNA pyrimidine isopliths. Radioactivity of nucleotide material in the isopliths was measured by counting in appropriate zones of the chromatograms in toluene scintillator. The investigation shows that injury to DNA of different organs, both directly and as a result of faulty repair, leads to shortening of the pyrimidine isopliths, i.e., to changes in the primary structure of DNA. These data help to explain the simultaneously cytostatic, carcinostatic, and mutagenic action of irradiation, cortisol and hydroxyurea.

  17. Regulation of chloroplast number and DNA synthesis in higher plants. Final report, August 1995--August 1996

    SciTech Connect

    Mullet, J.E.

    1997-06-17

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focused on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The research focused on the isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  18. Pyridoxal 5'-phosphate mediated inactivation of Escherichia coli DNA polymerase I: identification of lysine-635 as an essential residue for the processive mode of DNA synthesis

    SciTech Connect

    Basu, S.; Basu, A.; Modak, M.J.

    1988-09-06

    Inactivation of Escherichia coli DNA polymerase I by pyridoxal 5'-phosphate treatment results from its reactivity at multiple lysine residues. One of these residues, lysine-758, has been shown to be located at the substrate binding site in DNA polymerase I. We now demonstrate that lysine-635 is another important target of pyridoxylation; modification of this site results in decreased rates of DNA synthesis. Addition of template-primer with or without substrate deoxynucleoside triphosphate protects lysine-635 from pyridoxylation. Analysis of the initiation versus elongation phase of DNA synthesis by lysine-635-modified enzyme revealed that elongation of the DNA chain is severely affected by the lysine-635 modification. We therefore conclude that this lysine residue plays an important role in the processive mode of DNA synthesis by E. coli DNA polymerase I.

  19. K+/H+-antiporter nigericin arrests DNA synthesis in Ehrlich ascites carcinoma cells.

    PubMed Central

    Margolis, L B; Novikova I, Y u; Rozovskaya, I A; Skulachev, V P

    1989-01-01

    Acidification of the cytoplasm of Ehrlich ascites carcinoma cells to pH 6.3 arrests DNA synthesis in these cells. Such an effect can be achieved by incubating the cells at pH 6.2 or by adding low concentrations of the K+/H+ antiporter, the antibiotic nigericin, at neutral pH. Glucose and anaerobiosis potentiate the nigericin effect. The inhibition of DNA synthesis by nigericin occurs without any significant decrease in the ATP concentration and in the mitochondrial membrane potential. The DNA synthesis inhibition is caused neither by a decrease in the intracellular [K+] nor by an increase in the intracellular [Na+] accompanying the nigericin effect (at least at low concentrations of the antibiotic). Nigericin should thus be regarded as a type of a cytostatic primarily affecting intracellular pH. PMID:2771947

  20. DNA synthesis in mouse brown adipose tissue is under. beta. -adrenergic control

    SciTech Connect

    Rehnmark, S.; Nedergaard, J. )

    1989-02-01

    The rate of DNA synthesis in mouse brown adipose tissue was followed with injections of ({sup 3}H)thymidine. Cold exposure led to a large increase in the rate of ({sup 3}H)thymidine incorporation, reaching a maximum after 8 days, after which the activity abruptly ceased. A series of norepinephrine injections was in itself able to increase ({sup 3}H)thymidine incorporation. When norepinephrine was injected in combination with the {alpha}-adrenergic antagonist phentolamine or with the {beta}-adrenergic antagonist propranolol, the stimulation was fully blocked by propranolol. It is suggested that stimulation of DNA synthesis in brown adipose tissue is a {beta}-adrenergically mediated process and that the tissue is an interesting model for studies of physiological control of DNA synthesis.

  1. Role of mucosal prostaglandins and DNA synthesis in gastric cytoprotection by luminal epidermal growth factor.

    PubMed Central

    Konturek, S J; Brzozowski, T; Piastucki, I; Dembinski, A; Radecki, T; Dembinska-Kiec, A; Zmuda, A; Gregory, H

    1981-01-01

    This study compares the effect of epidermal growth factor and prostaglandins (PGE2 or PGI2), applied topically to gastric mucosa, on gastric secretion and formation of ASA-induced gastric ulcerations in rats. Epidermal growth factor given topically in non-antisecretory doses prevented dose-dependently the formation of ASA-induced ulcers without affecting prostaglandin generation but with a significant rise in DNA synthesis in the oxyntic mucosa. The anti-ulcer effect of topical prostaglandins was also accompanied by an increase in DNA synthesis. This study indicates that topical epidermal growth factor, like PGE2 or PGI2, is cytoprotective and that this cytoprotection is not mediated by the inhibition of gastric secretion or prostaglandin formation but related to the increase in DNA synthesis in oxyntic mucosa. PMID:7030877

  2. K+/H+-antiporter Nigericin Arrests DNA Synthesis in Ehrlich Ascites Carcinoma Cells

    NASA Astrophysics Data System (ADS)

    Margolis, Leonid B.; Novikova, Irina Yu.; Rozovskaya, Irina A.; Skulachev, Vladimir P.

    1989-09-01

    Acidification of the cytoplasm of Ehrlich ascites carcinoma cells to pH 6.3 arrests DNA synthesis in these cells. Such an effect can be achieved by incubating the cells at pH 6.2 or by adding low concentrations of the K+/H+ antiporter, the antibiotic nigericin, at neutral pH. Glucose and anaerobiosis potentiate the nigericin effect. The inhibition of DNA synthesis by nigericin occurs without any significant decrease in the ATP concentration and in the mitochondrial membrane potential. The DNA synthesis inhibition is caused neither by a decrease in the intracellular [K+] nor by an increase in the intracellular [Na+] accompanying the nigericin effect (at least at low concentrations of the antibiotic). Nigericin should thus be regarded as a type of a cytostatic primarily affecting intracellular pH.

  3. Mechanism and dynamics of translesion DNA synthesis catalyzed by the Escherichia coli Klenow fragment.

    PubMed

    Sheriff, Asim; Motea, Edward; Lee, Irene; Berdis, Anthony J

    2008-08-19

    Translesion DNA synthesis represents the ability of a DNA polymerase to incorporate and extend beyond damaged DNA. In this report, the mechanism and dynamics by which the Escherichia coli Klenow fragment performs translesion DNA synthesis during the misreplication of an abasic site were investigated using a series of natural and non-natural nucleotides. Like most other high-fidelity DNA polymerases, the Klenow fragment follows the "A-rule" of translesion DNA synthesis by preferentially incorporating dATP opposite the noninstructional lesion. However, several 5-substituted indolyl nucleotides lacking classical hydrogen-bonding groups are incorporated approximately 100-fold more efficiently than the natural nucleotide. In general, analogues that contain large substituent groups in conjunction with significant pi-electron density display the highest catalytic efficiencies ( k cat/ K m) for incorporation. While the measured K m values depend upon the size and pi-electron density of the incoming nucleotide, k cat values are surprisingly independent of both biophysical features. As expected, the efficiency by which these non-natural nucleotides are incorporated opposite templating nucleobases is significantly reduced. This reduction reflects minimal increases in K m values coupled with large decreases in k cat values. The kinetic data obtained with the Klenow fragment are compared to that of the high-fidelity bacteriophage T4 DNA polymerase and reveal distinct differences in the dynamics by which these non-natural nucleotides are incorporated opposite an abasic site. These biophysical differences argue against a unified mechanism of translesion DNA synthesis and suggest that polymerases employ different catalytic strategies during the misreplication of damaged DNA.

  4. The Mechanism and Dynamics of Translesion DNA Synthesis Catalyzed by the Escherichia coli Klenow fragment

    PubMed Central

    Sheriff, Asim; Motea, Edward; Lee, Irene; Berdis, Anthony J.

    2009-01-01

    Translesion DNA synthesis represents the ability of a DNA polymerase to incorporate and extend beyond damaged DNA. In this report, the mechanism and dynamics by which the Escherichia coli Klenow fragment performs translesion DNA synthesis during the misreplication of an abasic site were investigated using a series of natural and non-natural nucleotides. Like most other high fidelity DNA polymerases, the Klenow fragment follows the “A-rule” of translesion DNA synthesis by preferentially incorporating dATP opposite the non-instructional lesion. However, several 5-substituted indolyl nucleotides lacking classical hydrogen-bonding groups are incorporated ~100-fold more efficiently than the natural nucleotide. In general, analogs that contain large substituent groups in conjunction with significant π-electron density display the highest catalytic efficiencies (kcat/Km) forincorporation. While the measured Km values depend upon the size and π-electron density of the incoming nucleotide, kcat values are surprisingly independent of both biophysical features. As expected, the efficiency by which these non-natural nucleotides are incorporated opposite templating nucleobases is significantly reduced. This reduction reflects minimal increases in Km values coupled with large decreases in kcat. The kinetic data obtained with the Klenow fragment are compared to that of the high fidelity bacteriophage T4 DNA polymerase and reveal distinct differences in the dynamics by which these non-natural nucleotides are incorporated opposite an abasic site. These biophysical differences argue against a unified mechanism of translesion DNA synthesis and suggest that polymerases employ different catalytic strategies during the misreplication of damaged DNA. PMID:18652487

  5. The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7.

    PubMed

    Zhang, Huidong; Lee, Seung-Joo; Richardson, Charles C

    2012-07-06

    DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.

  6. Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle.

    PubMed

    Hedglin, Mark; Benkovic, Stephen J

    2017-06-28

    During S-phase, minor DNA damage may be overcome by DNA damage tolerance (DDT) pathways that bypass such obstacles, postponing repair of the offending damage to complete the cell cycle and maintain cell survival. In translesion DNA synthesis (TLS), specialized DNA polymerases replicate the damaged DNA, allowing stringent DNA synthesis by a replicative polymerase to resume beyond the offending damage. Dysregulation of this DDT pathway in human cells leads to increased mutation rates that may contribute to the onset of cancer. Furthermore, TLS affords human cancer cells the ability to counteract chemotherapeutic agents that elicit cell death by damaging DNA in actively replicating cells. Currently, it is unclear how this critical pathway unfolds, in particular, where and when TLS occurs on each template strand. Given the semidiscontinuous nature of DNA replication, it is likely that TLS on the leading and lagging strand templates is unique for each strand. Since the discovery of DDT in the late 1960s, most studies on TLS in eukaryotes have focused on DNA lesions resulting from ultraviolet (UV) radiation exposure. In this review, we revisit these and other related studies to dissect the step-by-step intricacies of this complex process, provide our current understanding of TLS on leading and lagging strand templates, and propose testable hypotheses to gain further insights.

  7. A cell-free system for DNA repair synthesis using purified enzymes from the Novikoff hepatoma

    SciTech Connect

    Small, P.K.

    1988-01-01

    Novikoff DNA polymerase-{beta} and Novikoff DNase V have been used in a cell-free DNA excision repair system for UV-irradiated substrates to determine their DNA repair capabilities. The repair system was shown to depend upon UV-irradiated DNA, incision by phage T4 UV-endonuclease, excision by DNase V and synthesis by DNA polymerase-{beta}; ligation was not included. Highly purified calf thymus DNA was UV-irradiated at 500-750 J/m{sup 2} and incised by T4 UV-endonuclease. The repair system was used to follow the purification of DNase V and DNA polymerase-{beta}. For increased specificity, the parameters of UV-irradiation, incision, excision and synthesis were confirmed on highly supercoiled, covalently closed, phage PM2 DNA. Optimal DNA and Mg{sup 2+} concentrations were determined for the repair assay, which was shown to be linear with respect to time. Excision of the 3{prime}-apyrimidinic site and the 5{prime}-pyrimidine dimer by bidirectional DNase V, presumed to occur from the above experiments, was studied more thoroughly using lightly UV-irradiated ({sup 3}H)poly(dT)poly (dA), labeled in both the base and the sugar, and incised with T4 UV-endonuclease.

  8. DNA synthesis in periportal and perivenous hepatocytes of intact and hepatectomized young mice.

    PubMed

    Fernández-Blanco, A; Inda, A M; Errecalde, A L

    2015-01-01

    DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

  9. Values Clarification: Essential for Leadership Learning

    ERIC Educational Resources Information Center

    Fritz, Mackenzie R.; Guthrie, Kathy L.

    2017-01-01

    Values clarification is a dynamic process in which people come to understand what they individually view as important in their lives by placing a name or label to what one values. This process commonly occurs during the traditional college years and is a critical component of leadership education. This qualitative study examined how junior-level…

  10. Bias and Obfuscation in Kendler's (2005) "Clarification"

    ERIC Educational Resources Information Center

    Schneider, Kirk J.

    2006-01-01

    This paper comments on the article "Psychology and Phenomenology: A Clarification" by H. H. Kendler. In this article, Kendler misrepresented contemporary existential-humanistic psychology and conventional (or natural) scientific psychology. With regard to the former, he presented a confused, unwittingly biased, and all-too-stereotypic picture.…

  11. Values Clarification: Essential for Leadership Learning

    ERIC Educational Resources Information Center

    Fritz, Mackenzie R.; Guthrie, Kathy L.

    2017-01-01

    Values clarification is a dynamic process in which people come to understand what they individually view as important in their lives by placing a name or label to what one values. This process commonly occurs during the traditional college years and is a critical component of leadership education. This qualitative study examined how junior-level…

  12. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... have access to and use of information and data that is comparable to individuals without...

  13. 48 CFR 339.201 - Clarification.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING ACQUISITION OF INFORMATION TECHNOLOGY Electronic and Information Technology 339.201 Clarification. FAR Subpart 39.2, Electronic and Information Technology, requires Federal agencies to ensure that... have access to and use of information and data that is comparable to individuals without...

  14. Stimulation of DNA synthesis in cultured rat alveolar type II cells

    SciTech Connect

    Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

    1985-01-01

    Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated /sup 3/H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of /sup 3/H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture.

  15. Stimulation of adrenal DNA synthesis in cadmium-treated male rats

    SciTech Connect

    Nishiyama, S.; Nakamura, K.

    1984-07-01

    Cadmium chloride (CdCl2) at a dose of 1 mg/kg body wt was injected into male rats of the Wistar strain, weighing 250 g on the average, twice a day (12-hr intervals) for 7 consecutive days. DNA and RNA contents and (/sup 3/H)-thymidine and (/sup 3/H)-uridine incorporation into the acid-insoluble fraction significantly increased in the adrenals of rats treated with Cd for 2 and 7 consecutive days. Adrenal protein content and weight also significantly increased. These results indicate that continued treatment with Cd stimulates DNA and RNA synthesis in the adrenal cortex, which in turn results in the increase of the total protein contents of the adrenal gland and subsequently in the enlargement of the gland. Serum adrenocorticotrophin (ACTH) and insulin levels in Cd-treated rats were not higher than control levels, suggesting that the stimulation of DNA synthesis in the adrenals of Cd-treated rats is due to factor(s) other than serum ACTH and insulin. Treatment with Cd inhibited DNA synthesis in cultured adrenocortical cells at concentrations of 10(-4) to 10(-8) M, suggesting that Cd does not directly stimulate DNA synthesis in the adrenal gland in vivo. Although the adrenal gland became enlarged, the total adrenal corticosterone content decreased significantly. The decrease of total adrenal corticosterone content may be due to the fall in serum ACTH level of Cd-treated rats.

  16. EGFR Modulates DNA Synthesis and Repair through Tyr Phosphorylation of Histone H4

    PubMed Central

    Chou, Ruey-Hwang; Wang, Ying-Nai; Hsieh, Yi-Hsien; Li, Long-Yuan; Xia, Weiya; Chang, Wei-Chao; Chang, Ling-Chu; Cheng, Chien-Chia; Lai, Chien-Chen; Hsu, Jennifer L.; Chang, Wei-Jung; Chiang, Shu-Ya; Lee, Hong-Jen; Liao, Hsin-Wei; Chuang, Pei-Huan; Chen, Hui-Yu; Wang, Hung-Ling; Kuo, Sheng-Chu; Chen, Chung-Hsuan; Yu, Yung-Luen; Hung, Mien-Chie

    2014-01-01

    Summary Posttranslational modifications of histones play fundamental roles in many biological functions. Specifically, histone H4-K20 methylation is critical in DNA synthesis and repair. However, little is known about how these functions are regulated by the upstream stimuli. Here, we identify a tyrosine phosphorylation site at Y72 of histone H4, which facilitates recruitment of histone methyltransferases (HMTases), SET8 and SUV4-20H, to enhance its K20 methylation, thereby promoting DNA synthesis and repair. Phosphorylation-defective histone H4 mutant is deficient in K20 methylation, leading to reduced DNA synthesis, delayed cell cycle progression, and decreased DNA repair ability. Disrupting the interaction between epidermal growth factor receptor (EGFR) and histone H4 by Y72 peptide significantly reduced tumor growth. Furthermore, EGFR expression clinically correlates with histone H4-Y72 phosphorylation, H4-K20 mono-methylation, and the Ki-67 proliferation marker. These findings uncover a mechanism by which EGFR transduces signal to chromatin to regulate DNA synthesis and repair. PMID:25073158

  17. Post-synthesis DNA Modifications Using a trans-Cyclooctene Click Handle

    PubMed Central

    Wang, Ke; Wang, Danzhu; Ji, Kaili; Chen, Weixuan; Zheng, Yueqin; Dai, Chaofeng

    2015-01-01

    Post-synthesis DNA modification is a very useful method for DNA functionalization. This is achieved by using a modified NTP, which has a handle for further modifications, replacing the corresponding natural NTP in polymerase-catalyzed DNA synthesis. Subsequently, the handle can be used for further functionalization after PCR, preferably through a very fast reaction. Herein we describe polymerase-mediated incorporation of trans-cyclooctene modified thymidine triphosphate (TCO-TTP). Subsequently, the trans-cyclooctene group was reacted with a tetrazine tethered to other functional groups through a very fast click reaction. The utility of this DNA functionalization method was demonstrated with the incorporation of a boronic acid group and a fluorophore. The same approach was also successfully used in modifying a known aptamer for fluorescent labelling applications. PMID:25407744

  18. [Species and tissue differences of reparative DNA synthesis in embryonic cell cultures treated with carcinogens].

    PubMed

    Budunova, I V; Belitskiĭ, G A

    1982-01-01

    DNA repair synthesis (RS) was studied in embryonic cell cultures exposed to different carcinogenic factors: UV-light, N-methyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, aflatoxin BI and 7,12-dimethylbenz(a)anthracene. DNA RS level was shown to be higher in human liver cells than in murine ones. Tissue-dependent differences in DNA RS of cells damaged by carcinogens were found, too. RS-activity was higher in human, mouse and rat fibroblast cultures than in liver cultures of the same species. RS level in human kidney cultures was similar to that in human fibroblasts. The said differences should be taken into account in the evaluation of the results of testing of chemical agents for carcinogenicity, using their ability to cause DNA repair synthesis.

  19. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

    PubMed Central

    Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-01-01

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

  20. Capture of a third Mg²⁺ is essential for catalyzing DNA synthesis.

    PubMed

    Gao, Yang; Yang, Wei

    2016-06-10

    It is generally assumed that an enzyme-substrate (ES) complex contains all components necessary for catalysis and that conversion to products occurs by rearrangement of atoms, protons, and electrons. However, we find that DNA synthesis does not occur in a fully assembled DNA polymerase-DNA-deoxynucleoside triphosphate complex with two canonical metal ions bound. Using time-resolved x-ray crystallography, we show that the phosphoryltransfer reaction takes place only after the ES complex captures a third divalent cation that is not coordinated by the enzyme. Binding of the third cation is incompatible with the basal ES complex and requires thermal activation of the ES for entry. It is likely that the third cation provides the ultimate boost over the energy barrier to catalysis of DNA synthesis.

  1. Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique.

    PubMed

    Knobloch, Jürgen; Kunz, Werner; Grevelding, Christoph G

    2002-12-01

    The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.

  2. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    PubMed

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.

  3. DNA gyrase (GyrB)/topoisomerase IV (ParE) inhibitors: synthesis and antibacterial activity.

    PubMed

    East, Stephen P; White, Clara Bantry; Barker, Oliver; Barker, Stephanie; Bennett, James; Brown, David; Boyd, E Andrew; Brennan, Christopher; Chowdhury, Chandana; Collins, Ian; Convers-Reignier, Emmanuelle; Dymock, Brian W; Fletcher, Rowena; Haydon, David J; Gardiner, Mihaly; Hatcher, Stuart; Ingram, Peter; Lancett, Paul; Mortenson, Paul; Papadopoulos, Konstantinos; Smee, Carol; Thomaides-Brears, Helena B; Tye, Heather; Workman, James; Czaplewski, Lloyd G

    2009-02-01

    The synthesis and antibacterial activities of three chemotypes of DNA supercoiling inhibitors based on imidazolo[1,2-a]pyridine and [1,2,4]triazolo[1,5-a]pyridine scaffolds that target the ATPase subunits of DNA gyrase and topoisomerase IV (GyrB/ParE) is reported. The most potent scaffold was selected for optimization leading to a series with potent Gram-positive antibacterial activity and a low resistance frequency.

  4. Stimulation of DNA and Collagen Synthesis by Autologous Growth Factor in Cultured Fetal Rat Calvaria

    NASA Astrophysics Data System (ADS)

    Canalis, Ernesto; Peck, William A.; Raisz, Lawrence G.

    1980-11-01

    Conditioned medium derived from organ or cell cultures prepared from 19- to 21-day fetal rat calvaria stimulated the incorporation of [3H]proline into collagen and of [3H]thymidine into DNA in organ cultures of the same tissue. Addition of cortisol enhanced the effect on collagen but not on DNA synthesis. These effects appeared to be due to a nondialyzable and heat-stable growth factor.

  5. [Analysis of effectiveness of cDNA synthesis, induced using complementary primers and primers containing a noncomplementary base matrix].

    PubMed

    D'iachenko, L B; Chenchik, A A; Khaspekov, G L; Tatarenko, A O; Bibilashvili, R Sh

    1994-01-01

    We have studied the efficiency of DNA synthesis catalyzed by M-MLV reverse transcriptase or Thermus aquaticus DNA polymerase for primers (4-17 nucleotides long) either completely matched or possessing a single mismatched base pair at all possible positions in the primer. It has been shown that DNA synthesis efficiency depends not only on the position of mismatched base pair but on the length and primary structure of the primer. The enzyme, template, and primer concentrations determine the relative level of mismatched DNA synthesis.

  6. Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5'-flaps.

    PubMed

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

    2015-04-30

    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.

  7. Porous silicon microparticles as an alternative support for solid phase DNA synthesis

    NASA Astrophysics Data System (ADS)

    McInnes, Steven; Graney, Sean; Khung, Yit-lung; Voelcker, Nicolas H.

    2006-01-01

    Current methods to produce short DNA strands (oligonucleotides) involve the stepwise coupling of phosphoramidites onto a solid support, typically controlled pore glass. The full-length oligonucleotide is then cleaved from the solid support using a suitable aqueous or organic base and the oligonucleotide is subsequently separated from the spent support. This final step, albeit seemingly easy, invariably leads to increased production costs due to increased synthesis time and reduced yields. This paper describes the preparation of a dissolvable support for DNA synthesis based on porous silicon (pSi). Initially it was thought that the pSi support would undergo dissolution by hydrolysis upon cleavage of the freshly synthesised oligonucleotide strands with ammonium hydroxide. The ability to dissolve the solid support after completion of the synthesis cycle would eliminate the separation step required in current DNA synthesis protocols, leading to simpler and faster synthesis as well as increased yields, however it was found that the functionalisation of the pSi imparted a stability that impeded the dissolution. This strategy may also find applications for drug delivery where the controlled release of carrier-immobilised short antisense DNA is desired. The approach taken involves the fabrication of porous silicon (pSi) microparticles and films. Subsequently, the pSi is oxidised and functionalised with a dimethoxytrityl protected propanediol to facilitate the stepwise solid phase synthesis of DNA oligonucleotides. The functionalisation of the pSi is monitored by diffuse reflectance infrared spectroscopy and the successful trityl labelling of the pSi is detected by UV-Vis spectroscopy after release of the dimethoxytrityl cation in the presence of trichloroacetic acid (TCA). Oligonucleotide yields can be quantified by UV-Vis spectroscopy.

  8. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    PubMed

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  9. A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases.

    PubMed

    Sun, Fei; Huang, Li

    2016-07-01

    Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase (PolB), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of PolB and PolD from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPfA1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both PolB and PolD were efficient in strand displacement. HPfA1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPfA1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.

  10. Studies on the mechanism of action of azinomycin B: definition of regioselectivity and sequence selectivity of DNA cross-link formation and clarification of the role of the naphthoate.

    PubMed

    Coleman, Robert S; Perez, Ronelito J; Burk, Christopher H; Navarro, Antonio

    2002-11-06

    Evaluation of the sequence selectivity, noncovalent association, and orientation of the DNA cross-linking agent azinomycin B on its duplex DNA receptor is described. A strong correlation between sequence nucleophilicity and cross-linking yield was observed, and steric effects due to the thymine C5-methyl group were identified. Detailed studies on the role of the azinomycin naphthoate using viscometry, fluorescence contact energy transfer, and DNA unwinding assays point to a nonintercalative binding mode for this group. A kinetic assay for agent regioselectivity was used to determine the orientation of binding and covalent cross-link formation.

  11. Incorporation of dA opposite N3-ethylthymidine terminates in vitro DNA synthesis

    SciTech Connect

    Bhanot, O.S.; Grevatt, P.C.; Donahue, J.M.; Gabrielides, C.N.; Solomon, J.J. )

    1990-11-01

    N3-Ethylthymidine (N3-Et-dT) was site specifically incorporated into a 17-nucleotide oligomer to investigate the significance of DNA ethylation at the central hydrogen-bonding site (N3) of thymine. The 5'-(dimethoxytrityl)-protected N3-Et-dT was converted to the corresponding 3'-phosphoramidite and used to incorporate N3-Et-dT at a single site in the oligonucleotide during synthesis by the phospite triester method. The purified N3-Et-dT-containing oligomer was ligated to a second 17-mer to yield a 34-nucleotide template with N3-Et-dT present at position 26 from the 3'-end. The template DNA, which corresponds to a specific sequence at gene G of bacteriophase {var phi}X174, was used to study the specificity of nucleotide incorporation opposite N3-Et-dT. At 10 {mu}M dNTP and 5 mM Mg{sup 2{plus}}, N3-Et-dT blocked DNA synthesis by Escherichia coli polymerase I (Klenow fragment): 96{percent} immediately 3' to N3-Et-dT and 4{percent} after incorporation of a nucleotide opposite N3-Et-dT (incorporation-dependent blocked product). DNA replication past the lesion (postlesion synthesis) was negligible. Incorporation opposite N3-Et-dT increased with increased dNTP concentrations, reaching 35{percent} at 200 {mu}M. Postlesion synthesis remained negligible. DNA sequencing of the incorporation-dependent blocked product revealed that dA is incorporated opposite N3-Et-dT consistent with the A rule in mutagenesis. Formation of the N3-Et-dT{center dot}dA base pair at the 3'-end of the growing chain terminated DNA synthesis. These results implicate N3-Et-dT as a potentially cytotoxic lesion produced by ethylating agents.

  12. Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras

    PubMed Central

    Roviello, G. N.; Gröschel, S.; Pedone, C.

    2009-01-01

    Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the α-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy. PMID:19629638

  13. In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis.

    PubMed

    Wong, T W; Clayton, D A

    1985-10-01

    Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light-strand replication.

  14. Effects of starvation and hormones on DNA synthesis in silk gland cells of the silkworm, Bombyx mori.

    PubMed

    Li, Yao-Feng; Chen, Xiang-Yun; Zhang, Chun-Dong; Tang, Xiao-Fang; Wang, La; Liu, Tai-Hang; Pan, Min-Hui; Lu, Cheng

    2016-08-01

    Silk gland cells of silkworm larvae undergo multiple cycles of endomitosis for the synthesis of silk proteins during the spinning phase. In this paper, we analyzed the endomitotic DNA synthesis of silk gland cells during larval development, and found that it was a periodic fluctuation, increasing during the vigorous feeding phase and being gradually inhibited in the next molting phase. That means it might be activated by a self-regulating process after molting. The expression levels of cyclin E, cdt1 and pcna were consistent with these developmental changes. Moreover, we further examined whether these changes in endomitotic DNA synthesis resulted from feeding or hormonal stimulation. The results showed that DNA synthesis could be inhibited by starvation and re-activated by re-feeding, and therefore appears to be dependent on nutrition. DNA synthesis was suppressed by in vivo treatment with 20-hydroxyecdysone (20E). However, there was no effect on DNA synthesis by in vitro 20E treatment or by either in vivo or in vitro juvenile hormone treatment. The levels of Akt and 4E-BP phosphorylation in the silk glands were also reduced by starvation and in vivo treatment with 20E. These results indicate that the activation of endomitotic DNA synthesis during the intermolt stages is related to feeding and DNA synthesis is inhibited indirectly by 20E. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  15. Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

    PubMed Central

    Han, Chunhua; Wani, Gulzar; Zhao, Ran; Qian, Jiang; Sharma, Nidhi; He, Jinshan; Zhu, Qianzheng; Wang, Qi-En; Wani, Altaf A

    2015-01-01

    Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER. PMID:25483071

  16. Synthesis and properties of 4′-ThioDNA: unexpected RNA-like behavior of 4′-ThioDNA

    PubMed Central

    Inoue, Naonori; Minakawa, Noriaki; Matsuda, Akira

    2006-01-01

    The synthesis and properties of fully modified 4′-thioDNAs, oligonucleotides consisting of 2′-deoxy-4′-thionucleosides, were examined. In addition to the known literature properties (preferable hybridization with RNA and resistance to endonuclease hydrolysis), we also observed higher resistance of 4′-thioDNA to 3′-exonuclease cleavage. Furthermore, we found that fully modified 4′-thioDNAs behaved like RNA molecules in their hybridization properties and structural aspect, at least in the case of the 4′-thioDNA duplex. This observation was confirmed by experiments using groove binders, in which a 4′-thioDNA duplex interacts with an RNA major groove binder, lividomycin A, but not with DNA groove binders, to give an increase in its thermal stability. Since a 4′-thioDNA duplex competitively inhibited the hydrolysis of an RNA duplex by RNase V1, it was not only the physical properties but also this biological data suggested that a 4′-thioDNA duplex has an RNA-like structure. PMID:16855286

  17. Microwave synthesis of electrically conductive gold nanowires on DNA scaffolds.

    PubMed

    Kundu, Subrata; Liang, Hong

    2008-09-02

    Biological molecules, in particular DNA, have shown great potential to be used as interconnects of nanodevices and computational elements. In this research, we synthesized electrically conductive gold nanowires for the first time exploiting an electroless and microwave heating method for 120-180 s. Our results indicate that DNA serves as a reducing and nonspecific capping agent for the growth of nanowires. The current voltage ( I- V) characteristics of the Au nanowires are continuous, exhibiting Ohmic behavior having low contact resistance with the gold electrodes. The nanowires have a diameter of 10-15 nm in solution and of 20-30 nm in immobilized DNA with resistivity comparable to pure metals. The method is highly selective with deposition confined to the DNA itself. The nanowires we fabricated can be used as building blocks for functional nanodevices, sensors, and optoelectronics.

  18. Strong inhibition of replicative DNA synthesis in the developing rat cerebral cortex and glioma cells by roscovitine.

    PubMed

    Yakisich, Juan Sebastian; Vita, Marina Fernanda; Siden, Ake; Tasat, Deborah Ruth; Cruz, Mabel

    2010-06-01

    The effects of the cyclin-dependent kinase inhibitors roscovitine and olomoucine on DNA synthesis rate during normal rat brain development were studied by using short time (90 min) incubation. Both purine analogues at 100 microM concentration decreased the DNA synthesis of rat cerebral cortex in an age-dependent manner. The maximum inhibitory effect (approximately 90% for roscovitine, approximately 60% for olomoucine) occurred in rats of 2-13 days postnatal age. In adult rats (> 60 days postnatal age), the effect of both purine analogues was low. Roscovitine even at 200 microM concentration did not inhibit the fraction of DNA synthesis insensitive to hydroxyurea (unscheduled DNA synthesis (UDS)). In addition, in the RG2 rat glioma model, roscovitine produced a strong inhibition of DNA synthesis in glioma cells when compared to adult normal tissue. Since in adult rat brain more than 60% of DNA synthesis is related to DNA repair, usually measured as UDS, our results indicate that roscovitine strongly blocks ongoing DNA synthesis connected with replicative processes.

  19. Design, synthesis, and characterization of nucleosomes containing site-specific DNA damage.

    PubMed

    Taylor, John-Stephen

    2015-12-01

    How DNA damaged is formed, recognized, and repaired in chromatin is an area of intense study. To better understand the structure activity relationships of damaged chromatin, mono and dinucleosomes containing site-specific damage have been prepared and studied. This review will focus on the design, synthesis, and characterization of model systems of damaged chromatin for structural, physical, and enzymatic studies.

  20. Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

    SciTech Connect

    Gabrielson, E.W.; Gerwin, B.I.; Harris, C.C.; Roberts, A.B.; Sporn, M.B.; Lechner, J.F.

    1988-08-01

    Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.

  1. Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus suspension cultures

    Treesearch

    R. Minocha; S.C. Minocha; A. Komamine; W.C. Shortle

    1991-01-01

    Various inhibitors of polyamine biosynthesis were used to study the role of polyamines in DNA synthesis and cell division in suspension cultures of Catharanthus roseus (L) G. Don. Arginine decarboxylase (ADC; EC 4.1.1.19) was the major enzyme responsible for putrescine production. DL α-difluoromethylarginine inhibited ADC activity, cellular...

  2. Design, synthesis, and structure-activity relationship studies of new phenolic DNA gyrase inhibitors.

    PubMed

    Lübbers, Thomas; Angehrn, Peter; Gmünder, Hans; Herzig, Silvia

    2007-08-15

    Starting from a biased needle screening hit 3a, we report herein the design and synthesis of a series of novel 2,3-dihydroisoindol-1-ones structurally related to cyclothialidine 2 with DNA gyrase inhibitory activity. In this series, some compounds exhibited promising antibacterial activity against gram-positive bacterial strains.

  3. Molecular Recognition of DNA. Synthesis of Novel Bases for Triple Helix Formation

    DTIC Science & Technology

    1991-01-01

    to the purine strand in the major groove of the Watson - Crick double helical DNA (TAT, C+GC triplets). Purine oligonucleotides bind antiparallel to...R&T Code 4135018 S MAy 05 199411 "Molecular Recognition of DNA . Synthesis of Novel Bases for Triple Helix Formation" Peter B. Dervan cv _California...035 T"IQA""D PART I A) Completed work (1988-91) Triple Helix Formation by Oligonucleotides on DNA Extended to the Physiological pH Range. T. J. Povsic

  4. Synthesis and characterization of DNA nano-meso-microspheres as drug delivery carriers for intratumoral chemotherapy

    NASA Astrophysics Data System (ADS)

    Enriquez Schumacher, Iris Vanessa

    Conventional cancer chemotherapy results in systemic toxicity which severely limits effectiveness and often adversely affects patient quality of life. There is a need to find new drugs and delivery methods for less toxic therapy. Previous studies concerning DNA complexing with chemotherapy drugs suggest unique opportunities for DNA as a mesosphere drug carrier. The overall objective of this research was devoted to the synthesis and evaluation of novel DNA-drug nano-mesospheres designed for localized chemotherapy via intratumoral injection. My research presents DNA nano-meso-microspheres (DNA-MS) that were prepared using a modified steric stabilization method originally developed in this lab for the preparation of albumin MS. DNA-MS were prepared with glutaraldehyde covalent crosslinking (genipin crosslinking was attempted) through the DNA base pairs. In addition, novel crosslinking of DNA-MS was demonstrated using chromium, gadolinium, or iron cations through the DNA phosphate groups. Covalent and ionic crosslinked DNA-MS syntheses yielded smooth and spherical particle morphologies with multimodal size distributions. Optimized DNA-MS syntheses produced particles with narrow and normal size distributions in the 50nm to 5mum diameter size range. In aqueous dispersions approximately 200% swelling was observed with dispersion stability for more than 48 hours. Typical process conditions included a 1550rpm initial mixing speed and particle filtration through 20mum filters to facilitate preparation. DNA-MS were in situ loaded during synthesis for the first time with mitoxantrone, 5-fluorouracil, and methotrexate. DNA-MS drug incorporation was 12%(w/w) for mitoxantrone, 9%(w/w) for methotrexate, and 5%(w/w) for 5-fluorouracil. In vitro drug release into phosphate buffered saline was observed for over 35 days by minimum sink release testing. The effect of gadolinium crosslink concentration on mitoxantrone release was evaluated at molar equivalences in the range of 20% to

  5. Synthesis of the tellurium-derivatized phosphoramidites and their incorporation into DNA oligonucleotides.

    PubMed

    Jiang, Sibo; Sheng, Jia; Huang, Zhen

    2011-12-01

    In this unit, an efficient method for the synthesis of 2'-tellerium-modified phosphoramidite and its incorporation into oligonucleotide are presented. We choose 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine nucleosides (S.1, S.2) as starting materials due to their easy preparation. The 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine can be converted to the corresponding 2'-tellerium-derivatized nucleosides by treating with the telluride nucleophiles. Subsequently, the 2'-Te-nucleosides can be transformed into 3'-phosphoramidites, which are the building blocks for DNA/RNA synthesis. The DNA synthesis, purification, and applications of oligonucleotides containing 2'-Te-U or 2'-Te-T are described in the protocol.

  6. Kinetics of incorporation of O/sup 6/-methyldeoxyguanosine monophosphate during in vitro DNA synthesis

    SciTech Connect

    Snow, E.T.; Foote, R.S.; Mitra, S.

    1984-09-11

    O/sup 6/-Methyldeoxyguanosine triphosphate (m/sup 6/dGTP), known to be produced in vivo by methylation of deoxyguanosine triphosphate with simple methylating mutagens, is utilized by prokaryotic DNA polymerases during in vitro replication of synthetic and natural DNA template-primers. A study of the kinetic behavior of m/sup 6/dGTP during DNA replication in vitro and of its effect on DNA replication indicates that m/sup 6/dGTP acts as an analogue of dATP with K/sub m//sup app/ of about 6 ..mu..M for Escherichia coli DNA polymerase I (Klenow fragment) compared to the K/sub m//sup app/ of about 0.8 ..mu..M for dATP. m/sup 6/dGTP is not incorporated in the complete absence of dATP (a competitive inhibitor). m/sup 6/dGTP also inhibits in vitro DNA synthesis. Different DNA polymerases behave differently in utilization and turnover of m/sup 6/dGTP. T4 DNA polymerase shows stronger discrimination against m/sup 6/dGMP incorporation than either T5 DNA polymerase or E. coli DNA polymerase I. The possibility that m/sup 6/dGTP is unlikely to contribute significantly to in vivo mutation is discussed.

  7. DNA-directed in vitro synthesis of proteins involved in bacterial transcription and translation.

    PubMed Central

    Zarucki-Schulz, T; Jerez, C; Goldberg, G; Kung, H F; Huang, K H; Brot, N; Weissbach, H

    1979-01-01

    The in vitro synthesis of elongation factor (EF)-Tu (tufB), the beta beta' subunits of RNA polymerase, ribosomal proteins L10 and L12 directed by DNA from the transducing phage lambda rifd 18, EF-Tu (tufA), EF-G, and the alpha subunit of RNA polymerase directed by DNA from the transducing phage lambda fus3 has been investigated in a crude and a partially defined protein-synthesizing system. Proteins L10 and L12 are synthesized in the partially defined system almost as well as in the crude system. However, the synthesis of EF-Tu, EF-G, and the alpha and beta beta' subunits of RNA polymerase is far less efficient in the partially defined system. An active fraction that stimulates the synthesis of these latter proteins has been obtained by fractionation of a high-speed supernatant on DEAE-cellulose. Because previous studies showed that this fraction (1 M DEAE salt eluate) contains a protein, called L factor, that stimulates beta-galactosidase synthesis in vitro, L factor was tested for activity. Although L factor stimulates the synthesis of the beta beta' subunits, it has little or no effect on the in vitro synthesis of the other products studied. In the present experiments, the ratio of L12/L10 and of EF-Tu (tufA)/EF-G formed is 4-6. These values are consistent with in vivo results. Images PMID:160561

  8. Co-opting the Fanconi anemia genomic stability pathway enables herpesvirus DNA synthesis and productive growth.

    PubMed

    Karttunen, Heidi; Savas, Jeffrey N; McKinney, Caleb; Chen, Yu-Hung; Yates, John R; Hukkanen, Veijo; Huang, Tony T; Mohr, Ian

    2014-07-03

    DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.

  9. Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

    PubMed Central

    Karttunen, Heidi; Savas, Jeffrey N.; McKinney, Caleb; Chen, Yu-Hung; Yates, John R.; Hukkanen, Veijo; Huang, Tony T.; Mohr, Ian

    2015-01-01

    SUMMARY DNA damage associated with viral DNA synthesis can result in double strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi Anemia (FA) genomic stability pathway is exploited by HSV1 to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV1-infected cells resulted in monoubiquitination of FA effector proteins, FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments and FANCI-D2 interacted with a multi-subunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, while HSV1 productive growth was impaired in monoubiquitination-defective FA patient cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for non-homologous end-joining (NHEJ). This identifies the FA-pathway as a new cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral lifecycle. PMID:24954902

  10. In vivo effects of T-2 mycotoxin on synthesis of proteins and DNA in rat tissues

    SciTech Connect

    Thompson, W.L.; Wannemacher, R.W. Jr. )

    1990-09-15

    Rats were given an ip injection of T-2 mycotoxin (T-2), the T-2 metabolite, T-2 tetraol (tetraol), or cycloheximide. Serum, liver, heart, kidney, spleen, muscle, and intestine were collected at 3, 6, and 9 hr postinjection after a 2-hr pulse at each time with (14C)leucine and (3H)thymidine. Protein and DNA synthesis levels in rats were determined by dual-label counting of the acid-precipitable fraction of tissue homogenates. Rats given a lethal dose of T-2, tetraol, or cycloheximide died between 14 and 20 hr. Maximum inhibition of protein synthesis at the earliest time period was observed in additional rats given the same lethal dose of the three treatments and continued for the duration of the study (9 hr). With sublethal doses of T-2 or tetraol, the same early decrease in protein synthesis was observed but, in most of the tissues, recovery was seen with time. In the T-2-treated rats. DNA synthesis in the six tissues studied was also suppressed, although to a lesser degree. With sublethal doses, complete recovery of DNA synthesis took place in four of the six tissues by 9 hr after toxin exposure. The appearance of newly translated serum proteins did not occur in the animals treated with T-2 mycotoxin or cycloheximide, as evidenced by total and PCA-soluble serum levels of labeled leucine. An increase in tissue-pool levels of free leucine and thymidine in response to T-2 mycotoxin was also noted. T-2 mycotoxin, its metabolite, T-2 tetraol, and cycloheximide cause a rapid inhibition of protein and DNA synthesis in all tissue types studied. These results are compared with the responses seen in in vitro studies.

  11. Alternative solutions and new scenarios for translesion DNA synthesis by human PrimPol.

    PubMed

    Martínez-Jiménez, María I; García-Gómez, Sara; Bebenek, Katarzyna; Sastre-Moreno, Guillermo; Calvo, Patricia A; Díaz-Talavera, Alberto; Kunkel, Thomas A; Blanco, Luis

    2015-05-01

    PrimPol is a recently described DNA polymerase that has the virtue of initiating DNA synthesis. In addition of being a sensu stricto DNA primase, PrimPol's polymerase activity has a large capacity to tolerate different kind of lesions. The different strategies used by PrimPol for DNA damage tolerance are based on its capacity to "read" certain lesions, to skip unreadable lesions, and as an ultimate solution, to restart DNA synthesis beyond the lesion thus acting as a TLS primase. This lesion bypass potential, revised in this article, is strengthened by the preferential use of moderate concentrations of manganese ions as the preferred metal activator. We show here that PrimPol is able to extend RNA primers with ribonucleotides, even when bypassing 8oxoG lesions, suggesting a potential new scenario for PrimPol as a TLS polymerase assisting transcription. We also show that PrimPol displays a high degree of versatility to accept or induce distortions of both primer and template strands, creating alternative alignments based on microhomology that would serve to skip unreadable lesions and to connect separate strands. In good agreement, PrimPol is highly prone to generate indels at short nucleotide repeats. Finally, an evolutionary view of the relationship between translesion synthesis and primase functions is briefly discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. DNA (deoxyribonucleic acid) synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes

    SciTech Connect

    Naish, S.J.; Perreault, S.D.; Zirkin, B.R.

    1987-01-01

    The authors investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in /sup 3/H-thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. The authors conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.

  13. A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips

    PubMed Central

    Kosuri, Sriram; Eroshenko, Nikolai; LeProust, Emily; Super, Michael; Way, Jeffrey; Li, Jin Billy; Church, George M.

    2010-01-01

    Development of cheap, high-throughput, and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology1. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis2. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude3,4,5, yet efforts to scale their use have been largely unsuccessful due to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols, and enzymatic error correction to develop a highly parallel gene synthesis platform. We tested our platform by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ~35 kilo-basepairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ~2.5 megabases of DNA, which is at least 50 times larger than previously published attempts. PMID:21113165

  14. Detection and quantification of DNA strand breaks using the ROPS (random oligonucleotide primed synthesis) assay.

    PubMed

    Boyko, Alex; Kovalchuk, Igor

    2010-01-01

    DNA double strand breaks (DSBs) arise from spontaneous DNA damage due to metabolic activities or from direct and indirect damaging effects of stress. DSBs are also formed transiently during such processes as replication, transcription, and DNA repair. The level of DSBs positively correlates with the activities of homologous and nonhomologous DNA repair pathways, which in turn inversely correlate with methylation levels and chromatin structure. Thus, measurement of strand breaks can provide an informative picture of genome stability of a given cell. The use of random oligonucleotide-primed synthesis for the analysis of DSB levels is described. Applications of the assay for quantitative detection of 3'OH, 3'P, or DNA strand breaks at a cleavage site of the deoxyribose residue are discussed.

  15. RecA interacts with Klenow and enhances fidelity of DNA synthesis in vitro.

    PubMed

    Karthikeyan, G; Lakshmikant, G S; Wagle, M D; Krishnamoorthy, G; Rao, B J

    1999-08-01

    To understand the molecular basis of RecA-mediated DNA-repair, we tested the replicative fidelity of the large fragment of Pol I (Klenow) in RecA-DNA complexes in vitro. Klenow synthesis was error-prone in naked DNA substrates but essentially error-free in RecA coated complexes. Escherichia coli SSB, causes no such improvement in Klenow fidelity. RecA filaments promote better exonucleolytic proofreading by Klenow than on naked DNA substrates at select sites when replication is "stalled" due to a missing dNTP. Addition of RecA to pyrene sulfonylchloride-labeled Klenow resulted in a specific increase in steady-state fluorescence anisotropy and a concomitant decrease in fluorescence lifetime. These observations suggest the possibility of a direct interaction between RecA and Klenow even in the absence of DNA which may mediate the observed improvement in Klenow fidelity.

  16. Translesion Synthesis of 2′-Deoxyguanosine Lesions by Eukaryotic DNA Polymerases

    PubMed Central

    2016-01-01

    With the discovery of translesion synthesis DNA polymerases, great strides have been made in the last two decades in understanding the mode of replication of various DNA lesions in prokaryotes and eukaryotes. A database search indicated that approximately 2000 articles on this topic have been published in this period. This includes research involving genetic and structural studies as well as in vitro experiments using purified DNA polymerases and accessory proteins. It is a daunting task to comprehend this exciting and rapidly emerging area of research. Even so, as the majority of DNA damage occurs at 2′-deoxyguanosine residues, this perspective attempts to summarize a subset of this field, focusing on the most relevant eukaryotic DNA polymerases responsible for their bypass. PMID:27760288

  17. Synthesis and evaluation of new spacers for use as dsDNA endcaps

    PubMed Central

    Ng, Pei-Sze; Laing, Brian M.; Balasundarum, Ganesan; Pingle, Maneesh; Friedman, Alan; Bergstrom, Donald E.

    2010-01-01

    A series of aliphatic and aromatic spacer molecules designed to cap the ends of DNA duplexes have been synthesized. The spacers were converted into dimethoxytrityl protected phosphoramidites as synthons for oligonucleotides synthesis. The effect of the spacers on the stability of short DNA duplexes was assessed by melting temperature studies. Endcaps containing amide groups were found to be less stabilizing than the hexaethylene glycol spacer. Endcaps containing either a terthiophene or a naphthalene tetracarboxylic acid dimide were found to be significantly more stabilizing. The former showed a preference for stacking above an A•T base pair. Spacers containing only methylene (-CH2-) and amide (-CONH-) groups interact weakly with DNA and consequently may be optimal for applications that require minimal influence on DNA structure but require a way to hold the ends of double-stranded DNA together. PMID:20715857

  18. Synthesis and evaluation of new spacers for use as dsDNA end-caps.

    PubMed

    Ng, Pei-Sze; Laing, Brian M; Balasundarum, Ganesan; Pingle, Maneesh; Friedman, Alan; Bergstrom, Donald E

    2010-08-18

    A series of aliphatic and aromatic spacer molecules designed to cap the ends of DNA duplexes have been synthesized. The spacers were converted into dimethoxytrityl-protected phosphoramidites as synthons for oligonucleotides synthesis. The effect of the spacers on the stability of short DNA duplexes was assessed by melting temperature studies. End-caps containing amide groups were found to be less stabilizing than the hexaethylene glycol spacer. End-caps containing either a terthiophene or a naphthalene tetracarboxylic acid diimide were found to be significantly more stabilizing. The former showed a preference for stacking above an A*T base pair. Spacers containing only methylene (-CH(2)-) and amide (-CONH-) groups interact weakly with DNA and consequently may be optimal for applications that require minimal influence on DNA structure but require a way to hold the ends of double-stranded DNA together.

  19. DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present.

    PubMed

    Chen, Cheng-Yao

    2014-01-01

    Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger's dideoxy chain-terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ϕ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ϕ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

  20. Action of cytochalasin D on DNA synthesis in cells in culture

    SciTech Connect

    Glushankova, N.A.

    1986-10-01

    To solve the problem of the effect of changes in the actin cytoskeleton on DNA replication during the action of cytochalasins, the effect of long-term incubation of normal cells with cytochalasin D (CCD), which selectively destroys the microfilament system but does not affect transport of sugars, was investigated. Incorporation of labeled thymidine into mononuclear and binuclear cells in the presence of CCD and after its removal by rinsing also was studied separately. To investigate DNA synthesis the method of autoradiography with /sup 3/H-thymidine was used. A culture of mouse fibroblasts of the BALB/3T3 line and a secondary culture of fibroblasts obtained by trypsinization of mouse embryos (MEF) were used. On incubation of MEF and 3T3 cells, gradual inhibition of DNA synthesis is observed. The results obtained indicate that structural changes in the active cytoskeleton can abruptly and reversibly disturb passage of the normal cell through the cycle.

  1. Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA.

    PubMed

    Bullard, Whitney; Cliffe, Laura; Wang, Pengcheng; Wang, Yinsheng; Sabatini, Robert

    2015-12-01

    Telomeric DNA of trypanosomatids possesses a modified thymine base, called base J, that is synthesized in a two-step process; the base is hydroxylated by a thymidine hydroxylase forming hydroxymethyluracil (hmU) and a glucose moiety is then attached by the J-associated glucosyltransferase (JGT). To examine the importance of JGT in modifiying specific thymine in DNA, we used a Leishmania episome system to demonstrate that the telomeric repeat (GGGTTA) stimulates J synthesis in vivo while mutant telomeric sequences (GGGTTT, GGGATT, and GGGAAA) do not. Utilizing an in vitro GT assay we find that JGT can glycosylate hmU within any sequence with no significant change in Km or kcat, even mutant telomeric sequences that are unable to be J-modified in vivo. The data suggests that JGT possesses no DNA sequence specificity in vitro, lending support to the hypothesis that the specificity of base J synthesis is not at the level of the JGT reaction.

  2. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    SciTech Connect

    Jones, R.A.

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  3. Site Specific Synthesis and in-situ Immobilization of Fluorescent Silver Nanoclusters on DNA Nanoscaffolds Using Tollens Reaction

    SciTech Connect

    Pal, Suchetan; Varghese, R.; Deng, Z.; Zhao, Z.; Kumar, A.; Yan, Hao; Liu, Yan

    2011-04-06

    DNA strands with specific sequences and covalently attached sugar moieties were used for the site-specific incorporation of the sugar units on a DNA origami scaffold. This approach enabled the subsequent site-specific synthesis and in situ immobilization of fluorescent Ag clusters at predefined positions on the DNA nanoscaffold by treatment with the Tollens reagent.

  4. The OPERA hypothesis: assumptions and clarifications.

    PubMed

    Patel, Aniruddh D

    2012-04-01

    Recent research suggests that musical training enhances the neural encoding of speech. Why would musical training have this effect? The OPERA hypothesis proposes an answer on the basis of the idea that musical training demands greater precision in certain aspects of auditory processing than does ordinary speech perception. This paper presents two assumptions underlying this idea, as well as two clarifications, and suggests directions for future research.

  5. Risk Communication, Values Clarification, and Vaccination Decisions.

    PubMed

    Witteman, Holly O; Chipenda Dansokho, Selma; Exe, Nicole; Dupuis, Audrey; Provencher, Thierry; Zikmund-Fisher, Brian J

    2015-10-01

    Many health-related decisions require choosing between two options, each with risks and benefits. When presented with such tradeoffs, people often make choices that fail to align with scientific evidence or with their own values. This study tested whether risk communication and values clarification methods could help parents and guardians make evidence-based, values-congruent decisions about children's influenza vaccinations. In 2013-2014 we conducted an online 2×2 factorial experiment in which a diverse sample of U.S. parents and guardians (n = 407) were randomly assigned to view either standard information about influenza vaccines or risk communication using absolute and incremental risk formats. Participants were then either presented or not presented with an interactive values clarification interface with constrained sliders and dynamic visual feedback. Participants randomized to the risk communication condition combined with the values clarification interface were more likely to indicate intentions to vaccinate (β = 2.10, t(399) = 2.63, p < 0.01). The effect was particularly notable among participants who had previously demonstrated less interest in having their children vaccinated against influenza (β = -2.14, t(399) = -2.06, p < 0.05). When assessing vaccination status reported by participants who agreed to participate in a follow-up study six months later (n = 116), vaccination intentions significantly predicted vaccination status (OR = 1.66, 95%CI (1.13, 2.44), p < 0.05) and rates of informed choice (OR = 1.51, 95%CI (1.07, 2.13), p < 0.012), although there were no direct effects of experimental factors on vaccination rates. Qualitative analysis suggested that logistical barriers impeded immunization rates. Risk communication and values clarification methods may contribute to increased vaccination intentions, which may, in turn, predict vaccination status if logistical barriers are also addressed.

  6. Design and Synthesis of Triangulated DNA Origami Trusses.

    PubMed

    Matthies, Michael; Agarwal, Nayan P; Schmidt, Thorsten L

    2016-03-09

    DNA nanotechnology offers unique control over matter on the nanoscale. Here, we extend the DNA origami method to cover a range of wireframe truss structures composed of equilateral triangles, which use less material per volume than standard multiple-helix bundles. From a flat truss design, we folded tetrahedral, octahedral, or irregular dodecahedral trusses by exchanging few connector strands. Other than standard origami designs, the trusses can be folded in low-salt buffers that make them compatible with cell culture buffers. The structures also have defined cavities that may in the future be used to precisely position functional elements such as metallic nanoparticles or enzymes. Our graph routing program and a simple design pipeline will enable other laboratories to make use of this valuable and potent new construction principle for DNA-based nanoengineering.

  7. Synthesis of Sindbis virus complementary DNA by avian myeloblastosis virus RNA-directed DNA polymerase.

    PubMed

    Yuferov, V; Grandgenett, D P; Bondurant, M; Riggin, C; Tigges, M

    1978-07-24

    Sindbis virus 42 S RNA was efficiently transcribed into complementary DNA (CDNA) by avian myeloblastosis virus alphabeta DNA polymerase using oligo- (dT) or single-stranded calf thymus DNA as primers. Both of the Sindbis virus cDNA products were able to protect 60% of 125I-labeled Sindbis virus RNA, at near equal weight ratios, from RNAase A and T1 digestion. Using hybridization kinetics, the Crt 1/2 value for hybridization of the calf thymus-primed cDNA product with excess Sindbis RNA was determined to be 1.8 9 10-2 mol . s . 1-1. Thes data demonstrate that the Sindbis virus cDNA products are relatively uniform representations of Sindbis virus RNA sequences.

  8. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    DOEpatents

    Gardner, Shea N; Mariella, Jr., Raymond P; Christian, Allen T; Young, Jennifer A; Clague, David S

    2013-06-25

    A method of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths.

  9. The Effects of Arsenic Trioxide on DNA Synthesis and Genotoxicity in Human Colon Cancer Cells

    PubMed Central

    Stevens, Jacqueline J.; Graham, Barbara; Walker, Alice M.; Tchounwou, Paul B.; Rogers, Christian

    2010-01-01

    Colon cancer is the third leading cause of cancer-related deaths worldwide. Recent studies in our laboratory have demonstrated that arsenic trioxide is cytotoxic in human colon cancer (HT-29), lung (A549) and breast (MCF-7) carcinoma cells. The purpose of the present study is to investigate the effects of arsenic trioxide on DNA synthesis and the possible genotoxic effects on human colon cancer cells. HT-29 cells were cultured according to standard protocol, followed by exposure to various doses (0, 2, 4, 6, 8, 10, and 12 μg/mL) of arsenic trioxide for 24 h. The proliferative response (DNA synthesis) to arsenic trioxide was assessed by [3H]thymidine incorporation. The genotoxic effects of arsenic-induced DNA damage in a human colon cancer cell line was evaluated by the alkaline single cell gel electrophoresis. Results indicated that arsenic trioxide affected DNA synthesis in HT-29 cells in a biphasic manner; showing a slight but not significant increase in cell proliferation at lower levels of exposure (2, 4 and 6 μg/mL) followed by a significant inhibition of cell proliferation at higher doses (i.e., 8 and 10 μg/mL). The study also confirmed that arsenic trioxide exposure caused genotoxicity as revealed by the significant increase in DNA damage, comet tail-lengths, and tail moment when compared to non-exposed cells. Results of the [3H]thymidine incorporation assay and comet assay revealed that exposure to arsenic trioxide affected DNA synthesis and exhibited genotoxic effects in human colon cancer cells. PMID:20623008

  10. Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR

    PubMed Central

    Nußbaumer, Felix; Juen, Michael Andreas; Gasser, Catherina; Kremser, Johannes; Müller, Thomas; Tollinger, Martin

    2017-01-01

    Abstract We report the synthesis of atom-specifically 13C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-13C-modified pyrimidine and 8-13C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-13C-2′deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min−1. Finally, we used 13C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s−1 and with a backward rate constant of 10.6 s−1.

  11. Homologous Recombination and Translesion DNA Synthesis Play Critical Roles on Tolerating DNA Damage Caused by Trace Levels of Hexavalent Chromium

    PubMed Central

    Chen, Youjun; Zhou, Yi-Hui; Neo, Dayna; Clement, Jean; Takata, Minoru; Takeda, Shunichi; Sale, Julian; Wright, Fred A.; Swenberg, James A.; Nakamura, Jun

    2016-01-01

    Contamination of potentially carcinogenic hexavalent chromium (Cr(VI)) in the drinking water is a major public health concern worldwide. However, little information is available regarding the biological effects of a nanomoler amount of Cr(VI). Here, we investigated the genotoxic effects of Cr(VI) at nanomoler levels and their repair pathways. We found that DNA damage response analyzed based on differential toxicity of isogenic cells deficient in various DNA repair proteins is observed after a three-day incubation with K2CrO4 in REV1-deficient DT40 cells at 19.2 μg/L or higher as well as in TK6 cells deficient in polymerase delta subunit 3 (POLD3) at 9.8 μg/L or higher. The genotoxicity of Cr(VI) decreased ~3000 times when the incubation time was reduced from three days to ten minutes. TK mutation rate also significantly decreased from 6 day to 1 day exposure to Cr(VI). The DNA damage response analysis suggest that DNA repair pathways, including the homologous recombination and REV1- and POLD3-mediated error-prone translesion synthesis pathways, are critical for the cells to tolerate to DNA damage caused by trace amount of Cr(VI). PMID:27907204

  12. Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

    PubMed Central

    2011-01-01

    Background Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate. Results Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast. Conclusions Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies. PMID:22152062

  13. Synthesis and characterization of DNA minor groove binding alkylating agents.

    PubMed

    Iyer, Prema; Srinivasan, Ajay; Singh, Sreelekha K; Mascara, Gerard P; Zayitova, Sevara; Sidone, Brian; Fouquerel, Elise; Svilar, David; Sobol, Robert W; Bobola, Michael S; Silber, John R; Gold, Barry

    2013-01-18

    Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

  14. Synthesis and Characterization of DNA Minor Groove Binding Alkylating Agents

    PubMed Central

    Iyer, Prema; Srinivasan, Ajay; Singh, Sreelekha K.; Mascara, Gerard P.; Zayitova, Sevara; Sidone, Brian; Fouquerel, Elise; Svilar, David; Sobol, Robert W.; Bobola, Michael S.; Silber, John R.; Gold, Barry

    2012-01-01

    Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases the N-terminus was appended with a O-methyl sulfonate ester while the C-terminus group was varied with non-polar and polar sidechains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) vs. major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is > 10-fold higher than the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells over-expressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization. PMID:23234400

  15. Inhibition of Mn(2+)-induced error-prone DNA synthesis with Cd(2+) and Zn(2+).

    PubMed

    Gening, L V; Lakhin, A V; Stelmashook, E V; Isaev, N K; Tarantul, V Z

    2013-10-01

    Bivalent metal cations are key components in the reaction of DNA synthesis. They are necessary for all DNA polymerases, being involved as cofactors in catalytic mechanisms of nucleotide polymerization. It is also known that in the presence of Mn2+ the accuracy of DNA synthesis is considerably decreased. The findings of this work show that Cd2+ and Zn2+ selectively inhibit the Mn2+-induced error-prone DNA polymerase activity in extracts of cells from human and mouse tissues. Moreover, these cations in low concentrations also can efficiently inhibit the activity of homogeneous preparations of DNA polymerase iota (Pol ι), which is mainly responsible for the Mn2+-induced error-prone DNA polymerase activity in cell extracts. Using a primary culture of granular cells from postnatal rat cerebellum, we show that low concentrations of Cd2+ significantly increase cell survival in the presence of toxic Mn2+ doses. Thus, we have shown that in some cases low concentrations of Cd2+ can display a positive influence on cells, whereas it is widely acknowledged that this metal is not a necessary microelement and is toxic for organisms.

  16. Cell cycle specific distribution of killin: evidence for negative regulation of both DNA and RNA synthesis.

    PubMed

    Qiao, Man; Luo, Dan; Kuang, Yi; Feng, Haiyan; Luo, Guangping; Liang, Peng

    2015-01-01

    p53 tumor-suppressor gene is a master transcription factor which controls cell cycle progression and apoptosis. killin was discovered as one of the p53 target genes implicated in S-phase control coupled to cell death. Due to its extreme proximity to pten tumor-suppressor gene on human chromosome 10, changes in epigenetic modification of killin have also been linked to Cowden syndrome as well as other human cancers. Previous studies revealed that Killin is a high-affinity DNA-binding protein with preference to single-stranded DNA, and it inhibits DNA synthesis in vitro and in vivo. Here, co-localization studies of RFP-Killin with either GFP-PCNA or endogenous single-stranded DNA binding protein RPA during S-phase show that Killin always adopts a mutually exclusive punctuated nuclear expression pattern with the 2 accessory proteins in DNA replication. In contrast, when cells are not in S-phase, RFP-Killin largely congregates in the nucleolus where rRNA transcription normally occurs. Both of these cell cycle specific localization patterns of RFP-Killin are stable under high salt condition, consistent with Killin being tightly associated with nucleic acids within cell nuclei. Together, these cell biological results provide a molecular basis for Killin in competitively inhibiting the formation of DNA replication forks during S-phase, as well as potentially negatively regulate RNA synthesis during other cell cycle phases.

  17. Single-Molecule Measurements of Synthesis by DNA Polymerase with Base-Pair Resolution

    NASA Astrophysics Data System (ADS)

    Christian, Thomas; Romano, Louis; Rueda, David

    2010-03-01

    The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3'-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule F"orster resonance energy transfer intensity drops in discrete steps to values consistent with single nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer-template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change following the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the pre-insertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.

  18. Synthesis of DNA Oligodeoxynucleotides Containing Site-Specific 1,3-Butadiene- Deoxyadenosine Lesions

    PubMed Central

    Wickramaratne, Susith; Seiler, Christopher L.

    2016-01-01

    Post-oligomerization synthesis is a useful technique for preparing site-specifically modified DNA oligomers. This approach involves site-specific incorporation of inherently reactive halogenated nucleobases into DNA strands using standard solid phase synthesis, followed by post-oligomerization nucleophilic aromatic substitution (SNAr) reactions with carcinogen-derived synthons. In these reactions, the inherent reactivities of DNA and carcinogen-derived species are reversed: the modified DNA nucleobase acts as an electrophile, while the carcinogen-derived species acts as a nucleophile. In the present protocol, we describe the use of the post-oligomerization approach to prepare DNA strands containing site- and stereospecific N6-adenine and N1, N6-adenine adducts induced by epoxide metabolites of the known human and animal carcinogen, 1,3-butadiene (BD). The resulting oligomers containing site specific, structurally defined DNA adducts can be used in structural and biological studies to reveal the roles of specific BD adducts in carcinogenesis and mutagenesis. PMID:26344227

  19. The nexus of vitamin homeostasis and DNA synthesis and modification in mammalian brain.

    PubMed

    Spector, Reynold; Johanson, Conrad E

    2014-01-10

    The purpose of this review is to discuss the implications of the 2009 discovery of the sixth deoxyribonucleoside (dN) [5-hydroxymethyldeoxycytidine (hmdC)] in DNA which is the most abundant in neurons. The concurrent discovery of the three ten-eleven translocation enzymes (TET) which not only synthesize but also oxidize hmdC in DNA, prior to glycosylase removal and base excision repair, helps explain many heretofore unexplained phenomena in brain including: 1) the high concentration of ascorbic acid (AA) in neurons since AA is a cofactor for the TET enzymes, 2) the requirement for reduced folates and the dN synthetic enzymes in brain, 3) continued DNA synthesis in non-dividing neurons to repair the dynamic formation/removal of hmdC, and 4) the heretofore unexplained mechanism to remove 5-methyldeoxycytidine, the fifth nucleoside, from DNA. In these processes, we also describe the important role of choroid plexus and CSF in supporting vitamin homeostasis in brain: especially for AA and folates, for hmdC synthesis and removal, and methylated deoxycytidine (mdC) removal from DNA in brain. The nexus linking AA and folates to methylation, hydroxymethylation, and demethylation of DNA is pivotal to understanding not only brain development but also the subsequent function.

  20. The nexus of vitamin homeostasis and DNA synthesis and modification in mammalian brain

    PubMed Central

    2014-01-01

    The purpose of this review is to discuss the implications of the 2009 discovery of the sixth deoxyribonucleoside (dN) [5-hydroxymethyldeoxycytidine (hmdC)] in DNA which is the most abundant in neurons. The concurrent discovery of the three ten-eleven translocation enzymes (TET) which not only synthesize but also oxidize hmdC in DNA, prior to glycosylase removal and base excision repair, helps explain many heretofore unexplained phenomena in brain including: 1) the high concentration of ascorbic acid (AA) in neurons since AA is a cofactor for the TET enzymes, 2) the requirement for reduced folates and the dN synthetic enzymes in brain, 3) continued DNA synthesis in non-dividing neurons to repair the dynamic formation/removal of hmdC, and 4) the heretofore unexplained mechanism to remove 5-methyldeoxycytidine, the fifth nucleoside, from DNA. In these processes, we also describe the important role of choroid plexus and CSF in supporting vitamin homeostasis in brain: especially for AA and folates, for hmdC synthesis and removal, and methylated deoxycytidine (mdC) removal from DNA in brain. The nexus linking AA and folates to methylation, hydroxymethylation, and demethylation of DNA is pivotal to understanding not only brain development but also the subsequent function. PMID:24410751

  1. Entamoeba invadens: dynamics of DNA synthesis during differentiation from trophozoite to cyst.

    PubMed

    Singh, Nishant; Bhattacharya, Sudha; Paul, Jaishree

    2011-02-01

    The DNA dynamics which mediate conversion of uni-nucleate trophozoite into quadrinucleate cyst in Entamoeba histolytica is not well understood. Here, we have addressed this question in Entamoeba invadens (a model system for encystation) through a detailed time course study of the differentiation process. We combined flow cytometric analysis with the change in rate of thymidine incorporation and the number of nuclei per cell. Our data shows that during encystment the cell population passes through three phases: (1) Early phase (0-8h); of rapid DNA synthesis which may correspond to completion of ongoing DNA replication. Bi-nucleated cells increase with concomitant drop in uni-nucleated cells. (2) Commitment phase (8-24h); in which DNA synthesis rate slows down. Possibly new rounds of replication are initiated which proceed slowly, followed by mitosis at 20 h. After this the number of bi- and uni-nucleated cells gradually decline and the tri- and tetra-nucleated cells begin to increase. (3) Consolidation phase (24-72 h); in which the rate of DNA synthesis shows a small increase till 32 h and then begins to decline. The G2/M peak reappears at 48 h, showing that more rounds of DNA replication may be getting completed, followed by nuclear division. By 72 h the encystment is virtually complete. The bi-nucleated stage could be an intermediate both in the conversion of trophozoite to cyst and back. Our study provides a comprehensive view of DNA dynamics during encystation and excystation of E. invadens. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Structural Basis of High-Fidelity DNA Synthesis by Yeast DNA Polymerase δ

    SciTech Connect

    Swan, M.; Johnson, R; Prakash, L; Prakash, S; Aggarwal, A

    2009-01-01

    DNA polymerase ? (Pol ?) has a crucial role in eukaryotic replication. Now the crystal structure of the yeast DNA Pol ? catalytic subunit in complex with template primer and incoming nucleotide is presented at 2.0-A resolution, providing insight into its high fidelity and a framework to understand the effects of mutations involved in tumorigenesis.

  3. Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma.

    PubMed Central

    Kollek, R; Goulian, M

    1981-01-01

    The initial event in the replication cycle of parvovirus H-1 is conversion of the single-stranded linear viral DNA to the double-stranded linear replicative form. We describe here detection of an activity in uninfected cell extracts that carries out this reaction. The activity was purified and identified as DNA polymerase gamma. Images PMID:6947222

  4. [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.

  5. In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

    PubMed

    Reff, M E; Davidson, R L

    1979-01-01

    Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase.

  6. In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

    PubMed Central

    Reff, M E; Davidson, R L

    1979-01-01

    Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase. PMID:284320

  7. Coordinated Leading and Lagging Strand DNA Synthesis by Using the Herpes Simplex Virus 1 Replication Complex and Minicircle DNA Templates ▿

    PubMed Central

    Stengel, Gudrun; Kuchta, Robert D.

    2011-01-01

    The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not

  8. Functional effects of cis-thymine glycol lesions on DNA synthesis in vitro

    SciTech Connect

    Clark, J.M.; Beardsley, G.P.

    1987-08-25

    Thymine glycol, a DNA lesion produced by ionizing radiation, has been introduced site specifically at high frequency into a synthetic oligonucleotide by chemical oxidation of the single thymine residue within the sequence. The lesion-containing template was then annealed to a complementary synthetic primer and used to study the effects of cis-thymine glycol lesions on DNA polymerase function in vitro. Synthesis by polymerase I (Klenow fragment), T4 DNA polymerase, and polymerase ..cap alpha../sub 2/ was arrested quantitatively at the site of the lesion. AMV reverse transcriptase was less inhibited and was able to synthesize past a significant fraction of the lesions. Changing of the template base immediately 5' to thymine glycol from A to C did not significantly alter the pattern of synthesis arrest for any of the polymerases. The correct nucleotide, dAMP, was inserted opposite the lesion more than 90% of the time by all four polymerases, suggesting that thymine glycol forms a reasonably stable base pair with adenine. However, the 3'-5' exonuclease activity of polymerase I removed a 3'-terminal dAMP residue more rapidly from an A-thymine glycol base pair than from an A-T base pair. These results suggest that increased nucleotide turnover at the site of the lesion contributes to the inhibitory effects of thymine glycol lesions on DNA synthesis in vitro, at least for polymerases such as polymerase I that have intrinsic or associated editing exonuclease functions.

  9. DNA Origami Rotaxanes: Tailored Synthesis and Controlled Structure Switching.

    PubMed

    Powell, John T; Akhuetie-Oni, Benjamin O; Zhang, Zhao; Lin, Chenxiang

    2016-09-12

    Mechanically interlocked supramolecular assemblies are appealing building blocks for creating functional nanodevices. Herein, we describe the multistep assembly of large DNA origami rotaxanes that are capable of programmable structural switching. We validated the topology and structural integrity of these rotaxanes by analyzing the intermediate and final products of various assembly routes by electrophoresis and electron microscopy. We further analyzed two structure-switching behaviors of our rotaxanes, which are both mediated by DNA hybridization. In the first mechanism, the translational motion of the macrocycle can be triggered or halted at either terminus. In the second mechanism, the macrocycle can be elongated after completion of the rotaxane assembly, giving rise to a unique structure that is otherwise difficult to access.

  10. Endotoxin or cytokines attenuate ozone-induced DNA synthesis in rat nasal transitional epithelium

    SciTech Connect

    Hotchkiss, J.A.; Harkema, J.R. )

    1992-06-01

    Pretreatment of rats with endotoxin (E), a potent inducer of tumor necrosis factor alpha (TNF), and interleukin 1 beta (IL 1), or a combination of TNF and IL1, has been shown to increase levels of lung antioxidant enzymes and protect against pulmonary toxicity associated with hyperoxia. Inhalation of ozone (O3) induces cell injury, followed by increased DNA synthesis, cell proliferation, and secretory cell metaplasia in rat nasal transitional epithelium (NTE). This study was designed to test the effects of E, TNF, and IL1 pretreatment on acute O3-induced NTE cell injury as measured by changes in NTE cell DNA synthesis. Rats were exposed to either 0.8 ppm O3 or air for 6 hr in whole-body inhalation chambers. Immediately before exposure, rats in each group were injected intraperitoneally (ip) with either saline alone or saline containing E, TNF, IL1, or both TNF and IL1. Eighteen hours postexposure, rats were injected ip with bromodeoxyuridine to label cells undergoing DNA synthesis and were euthanized 2 hr later. NTE was processed for light microscopy and immunochemically stained to identify cells that had incorporated BrdU into nuclear DNA. The number of BrdU-labeled NTE nuclei per millimeter of basal lamina was quantitated. There were no significant differences in the number of BrdU-labeled NTE nuclei in air-exposed rats that were injected with E, TNF, IL1, or TNF/IL1 compared with those in saline-injected, air-exposed controls. Rats that were injected with saline and exposed to O3 had approximately 10 times the number of BrdU-labeled NTE nuclei than saline-injected, air-exposed control rats. O3 exposure also induced a significant increase in labeled nuclei in rats that were pretreated with TNF alone. In contrast, pretreatment with E, IL1, or TNF/IL1 attenuated the O3-induced increase in NTE DNA synthesis.

  11. Efficient synthesis of supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

    SciTech Connect

    Kodadek, T.; Gamper, H.

    1988-05-03

    The authors report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, they report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. They demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.

  12. Design, synthesis and selection of DNA-encoded small-molecule libraries.

    PubMed

    Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

    2009-09-01

    Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

  13. Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting Nucleopeptides.

    PubMed

    Mercurio, Maria Emilia; Tomassi, Stefano; Gaglione, Maria; Russo, Rosita; Chambery, Angela; Lama, Stefania; Stiuso, Paola; Cosconati, Sandro; Novellino, Ettore; Di Maro, Salvatore; Messere, Anna

    2016-12-02

    Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears nucleobases. They can recognize DNA and RNA targets modulating their biological functions. To date, the lack of an effective strategy for the synthesis of nucleopeptides prevents their evaluation for biological and biomedical applications. Herein, we describe an unprecedented approach that enables the synthesis of cationic both homo and heterosequence nucleopeptides wholly on solid support with high yield and purity. Spectroscopic studies indicate advantageous properties of the nucleopeptides in terms of binding, thermodynamic stability and sequence specific recognition. Biostability assay and laser scanning confocal microscopy analyses reveal that the nucleopeptides feature acceptable serum stability and ability to cross the cell membrane.

  14. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    DOEpatents

    Gardner, Shea N [San Leandro, CA; Mariella, Jr., Raymond P.; Christian, Allen T [Tracy, CA; Young, Jennifer A [Berkeley, CA; Clague, David S [Livermore, CA

    2011-01-18

    A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

  15. 5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

    NASA Technical Reports Server (NTRS)

    Frank, Natia L.; Meade, Thomas J.

    2003-01-01

    Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.

  16. DNA Dispose, but Subjects Decide. Learning and the Extended Synthesis.

    PubMed

    Lindholm, Markus

    Adaptation by means of natural selection depends on the ability of populations to maintain variation in heritable traits. According to the Modern Synthesis this variation is sustained by mutations and genetic drift. Epigenetics, evodevo, niche construction and cultural factors have more recently been shown to contribute to heritable variation, however, leading an increasing number of biologists to call for an extended view of speciation and evolution. An additional common feature across the animal kingdom is learning, defined as the ability to change behavior according to novel experiences or skills. Learning constitutes an additional source for phenotypic variation, and change in behavior may induce long lasting shifts in fitness, and hence favor evolutionary novelties. Based on published studies, I demonstrate how learning about food, mate choice and habitats has contributed substantially to speciation in the canonical story of Darwin's finches on the Galapagos Islands. Learning cannot be reduced to genetics, because it demands decisions, which requires a subject. Evolutionary novelties may hence emerge both from shifts in allelic frequencies and from shifts in learned, subject driven behavior. The existence of two principally different sources of variation also prevents the Modern Synthesis from self-referring explanations.

  17. Mechanism of ultraviolet-induced mutagenesis: Extent and fidelity of in vitro DNA synthesis on irradiated templates*

    PubMed Central

    Villani, Giuseppe; Boiteux, Serge; Radman, Miroslav

    1978-01-01

    The effect of UV irradiation on the extent and fidelity of DNA synthesis in vitro was studied by using homopolymers and primed single-stranded ϕX174 phage DNA as substrates. Unfractionated and fractionated cell-free extracts from Escherichia coli pol+ and polA1 mutants as well as purified DNA polymerase I were used as sources of enzymatic activity. (DNA polymerases, as used here, refer to deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7.) The extent of inhibition of DNA synthesis on UV-irradiated ϕX174 DNA suggested that pyrimidine dimers act as an absolute block for chain elongation by DNA polymerases I and III. Experiments with an irradiated poly(dC) template failed to detect incorporation of noncomplementary bases due to pyrimidine dimers. A large increase in the turnover of nucleoside triphosphates to free monophosphates during synthesis by DNA polymerase I on irradiated ϕX174 DNA has been observed. We propose that this nucleotide turnover is due to idling by DNA polymerase (i.e., incorporation and subsequent excision of nucleotides opposite UV photolesions, by the 3′→5′ “proofreading” exonuclease) thus preventing replication past pyrimidine dimers and the potentially mutagenic event that should result. In support of this hypothesis, DNA synthesis by DNA polymerase from avian myeloblastosis virus and by mammalian DNA polymerase α, both of which are devoid of any exonuclease activity, was found to be only partially inhibited, but not blocked, by UV irradiation of the template and accompanied by an increased incorporation of noncomplementary nucleotides. It is suggested that UV mutagenesis in bacteria requires an induced modification of the cellular DNA replication machinery, possibly an inhibition of the 3′→5′ exonuclease activity associated with DNA polymerases. PMID:356043

  18. Naked Eye DNA detection: Synthesis, characterization and DNA binding studies of a novel azo-guanidine

    NASA Astrophysics Data System (ADS)

    Jamil, Muhammad; Altaf, Ataf Ali; Badshah, Amin; Shafiqullah; Ahmad, Ibrar; Zubair, Muhammad; Kemal, Saqib; Ali, Muhammad Irshad

    2013-03-01

    A novel class of azo-guanidine compounds is introduced in this article. The novel compound 2-(2-hydroxyphenyl)-1-(phenylamino)-3-(phenylimino)guanidine (AG) was synthesized and well characterized by using different analytical instrumental techniques like elemental analysis, FTIR, 1H and 13C NMR, UV-Visible spectroscopy and cyclic voltammetry. The new compound was found interacting with DNA and shows clear color change in the solution. The AG-DNA complex was qualitatively and quantitatively characterized with UV-Visible spectroscopy and cyclic voltammetry. Electrostatic mode of interaction, clear color change and moderate binding constant (Kb = 104) indicate its potential use as DNA staining agent.

  19. [DNA reduplication cycle during chromosome polytenization in the salivary gland cells of Chironomus thummi larvae. III. The determination of the duration of the DNA synthesis period].

    PubMed

    Gundrina, L I; Sherudilo, A I; Mitina, R L

    1984-08-01

    The duration of DNA synthesis in the salivary gland cells of Chironomus thummi larvae of the IV instar was determined by means of autoradiography and cytophotometry. Cells of different levels of ploidy differ in the duration of their DNA synthesis period. The tS of 2(10)c and 2(11)c cells was equal to 17 and 22 hours, respectively. The doubling of DNA content of the chironomid salivary gland cells leads to a 1.3 time increase in the duration of S-phase.

  20. Synthesis and DNA interaction of a mixed proflavine-phenanthroline Tröger base.

    PubMed

    Baldeyrou, Brigitte; Tardy, Christelle; Bailly, Christian; Colson, Pierre; Houssier, Claude; Charmantray, Franck; Demeunynck, Martine

    2002-04-01

    We report the synthesis of an asymmetric Tröger base containing the two well characterised DNA binding chromophores, proflavine and phenanthroline. The mode of interaction of the hybrid molecule was investigated by circular and linear dichroism experiments and a biochemical assay using DNA topoisomerase I. The data are compatible with a model in which the proflavine moiety intercalates between DNA base pairs and the phenanthroline ring occupies the DNA groove. DNase I cleavage experiments were carried out to investigate the sequence preference of the hybrid ligand and a well resolved footprint was detected at a site encompassing two adjacent 5'-GTC.5-GAC triplets. The sequence preference of the asymmetric molecule is compared to that of the symmetric analogues.

  1. Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis

    PubMed Central

    Ortega, Janice; Li, Jessie Y.; Lee, Sanghee; Tong, Dan; Gu, Liya; Li, Guo-Min

    2015-01-01

    Proliferating cell nuclear antigen (PCNA) plays essential roles in eukaryotic cells during DNA replication, DNA mismatch repair (MMR), and other events at the replication fork. Earlier studies show that PCNA is regulated by posttranslational modifications, including phosphorylation of tyrosine 211 (Y211) by the epidermal growth factor receptor (EGFR). However, the functional significance of Y211-phosphorylated PCNA remains unknown. Here, we show that PCNA phosphorylation by EGFR alters its interaction with mismatch-recognition proteins MutSα and MutSβ and interferes with PCNA-dependent activation of MutLα endonuclease, thereby inhibiting MMR at the initiation step. Evidence is also provided that Y211-phosphorylated PCNA induces nucleotide misincorporation during DNA synthesis. These findings reveal a novel mechanism by which Y211-phosphorylated PCNA promotes cancer development and progression via facilitating error-prone DNA replication and suppressing the MMR function. PMID:25825764

  2. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  3. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailing description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  4. The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

    PubMed Central

    Hirota, Kouji; Yoshikiyo, Kazunori; Guilbaud, Guillaume; Tsurimoto, Toshiki; Murai, Junko; Tsuda, Masataka; Phillips, Lara G.; Narita, Takeo; Nishihara, Kana; Kobayashi, Kaori; Yamada, Kouich; Nakamura, Jun; Pommier, Yves; Lehmann, Alan; Sale, Julian E.; Takeda, Shunichi

    2015-01-01

    The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases. PMID:25628356

  5. Mutagenicity of a Model DNA-Peptide Cross-Link in Human Cells: Roles of Translesion Synthesis DNA Polymerases.

    PubMed

    Pande, Paritosh; Ji, Shaofei; Mukherjee, Shivam; Schärer, Orlando D; Tretyakova, Natalia Y; Basu, Ashis K

    2017-02-20

    DNA-protein cross-links are formed upon exposure of cellular DNA to various agents, including antitumor drugs, UV light, transition metals, and reactive oxygen species. They are thought to contribute to cancer, aging, and neurodegenerative diseases. It has been proposed that DNA-protein cross-links formed in cells are subject to proteolytic degradation to the corresponding DNA-peptide cross-links (DpCs). To investigate the effects of DpCs on DNA replication, we have constructed plasmid DNA containing a 10-mer Myc peptide covalently linked to C7 of 7-deaza-dG, a hydrolytically stable mimic of N7-dG lesions. Following transfection in human embryonic kidney cells (HEK 293T), progeny plasmids were recovered and sequenced. Translesion synthesis (TLS) past DpC was 76% compared to that of the unmodified control. The DpC induced 20% targeted G → A and G → T plus 15% semitargeted mutations, notably at a guanine (G5) five bases 3' to the lesion site. Proteolytic digestion of the DpC reduced the mutation frequency considerably, indicating that the covalently attached 10-mer peptide was responsible for the observed mutations. TLS efficiency and targeted mutations were reduced upon siRNA knockdown of pol η, pol κ, or pol ζ, indicating that they participate in error-prone bypass of the DpC lesion. However, the semitargeted mutation at G5 was only reduced upon knockdown of pol ζ, suggesting its critical role in this type of mutations. Our results indicate that DpCs formed at the N7 position of guanine can induce both targeted and semitargeted mutations in human cells and that the TLS polymerases play a critical role in their error-prone bypass.

  6. The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells.

    PubMed

    Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

    Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD>KO>WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N'-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA. Copyright © 2016 Elsevier

  7. Synthesis, photochemical properties and DNA binding studies of dna cleaving agents based on chiral dipyridine dihydrodioxins salts

    NASA Astrophysics Data System (ADS)

    Shamaev, Alexei

    activated by UV-light. The mechanism of o-quinone release and intramolecular ET was studied in detail by methods of Ultrafast Transient Absortion Spectroscopy and supported by high-level quantum mechanical calculations. The binding properties of chiral intercalators based on PDHD to various DNA oligonucleotides were studied by various methods and DNA cleavage properties indicating strong binding and cleaving ability of the synthesized PDHDs. Also, a new method for synthesis of cyclohexa[e]pyrenes which possibly capable of intramolecular ET and electron transfer-oxidative stress (ET-OS) DNA cleavage was developed and partially accomplished.

  8. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

    PubMed

    Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

    2017-01-05

    The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Recent Advances in the Synthesis and Functions of Reconfigurable Interlocked DNA Nanostructures.

    PubMed

    Lu, Chun-Hua; Cecconello, Alessandro; Willner, Itamar

    2016-04-27

    Interlocked circular DNA nanostructures, e.g., catenanes or rotaxanes, provide functional materials within the area of DNA nanotechnology. Specifically, the triggered reversible reconfiguration of the catenane or rotaxane structures provides a means to yield new DNA switches and to use them as dynamic scaffolds for controlling chemical functions and positioning functional cargoes. The synthesis of two-ring catenanes and their switchable reconfiguration by pH, metal ions, or fuel/anti-fuel stimuli are presented, and the functions of these systems, as pendulum or rotor devices or as switchable catalysts, are described. Also, the synthesis of three-, five-, and seven-ring catenanes is presented, and their switchable reconfiguration using fuel/anti-fuel strands is addressed. Implementation of the dynamically reconfigured catenane structures for the programmed organization of Au nanoparticle (NP) assemblies, which allows the plasmonic control of the fluorescence properties of Au NP/fluorophore loads associated with the scaffold, and for the operation of logic gates is discussed. Interlocked DNA rotaxanes and their different synthetic approaches are presented, and their switchable reconfiguration by means of fuel/anti-fuel strands or photonic stimuli is described. Specifically, the use of the rotaxane as a scaffold to organize Au NP assemblies, and the control of the fluorescence properties with Au NP/fluorophore hybrids loaded on the rotaxane scaffold, are introduced. The future prospectives and challenges in the field of interlocked DNA nanostructures and the possible applications are discussed.

  10. Design and Synthesis of Efficient Fluorescent Dyes for Incorporation into DNA Backbone and Biomolecule Detection

    PubMed Central

    Wang, Wei; Li, Alexander D. Q.

    2008-01-01

    We report here the design and synthesis of a series of π-conjugated fluorescent dyes with D-A-D (D: donor; A: Acceptor), D-π-D, A-π-A, and D-π-A for applications as the signaling motif in biological-synthetic hybrid foldamers for DNA detection. Horner-Wadsworth-Emmons (HWE) reaction and Knoevenagel condensation were demonstrated as the optimum ways for construction of long π-conjugated systems. Such rod-like chromophores have distinct advantages, as their fluorescence properties are not quenched by the presence of DNA. To be incorporated into the backbone of DNA, the chromophores need to be reasonably soluble in organic solvent for solid-phase synthesis, and therefore a strategy of using flexible tetra(ethylene glycol) (TEG) linkers at either end of these rod-like dyes were developed. The presence of TEG facilitates the protection of the chain-growing hydroxyl group with DMTrCl (dimethoxy trityl chloride) as well as the activation of the coupling step with phosphoramidite chemistry on an automated DNA synthesizer. To form fluorescence resonance energy transfer (FRET) pairs, six synthetic chromophores with blue to red fluorescence have been developed and those with orthogonal fluorescent emission were chosen for incorporation into DNA-chromophore hybrid foldamers. PMID:17508711

  11. Changes in gastrointestinal DNA synthesis produced by acute and chronic ethanol consumption in the rat: a biochemical study.

    PubMed

    Seitz, H K; Czygan, P; Kienapfel, H; Veith, S; Schmidt-Gayk, H; Kommerell, B

    1983-02-01

    The effect of acute and chronic ethanol administration on DNA synthesis in the gastrointestinal tract of the rat was investigated. Acute intragastric ethanol administration (3 g/kg; 50%) decreased significantly in vivo DNA synthesis when measured 1 hour after alcohol application in the stomach and in the upper small intestine, whereas acute intravenous ethanol administration had no significant effect. In contrast, chronic ethanol ingestion resulted in a significant increase of in vivo and in vitro DNA synthesis in the upper gastrointestinal tract. In addition, even a more enhanced stimulation of DNA synthesis after chronic ethanol consumption was found in isolated intestinal cells. These results indicate an inhibition of gastrointestinal cell regeneration directly after the oral application of ethanol. The enhanced cellular regenerativity observed after chronic ethanol consumption may be secondary to the ethanol induced damage of the gastrointestinal tract.

  12. On-Flow Synthesis of Co-Polymerizable Oligo-Microspheres and Application in ssDNA Amplification

    PubMed Central

    Lee, Se Hee; Lee, Jae Ha; Lee, Ho Won; Kim, Yang-Hoon; Jeong, Ok Chan; Ahn, Ji-Young

    2016-01-01

    We fabricated droplet-based microfluidic platform for copolymerizable microspheres with acrydite modified DNA probe. The copolymerizable 3-D polyacrylamide microspheres were successfully produced from microcontinuous-flow synthesis with on-channel solidification. DNA copolymerization activity, surface presentation and thermostability were assessed by using fluorescent labeled complementary probe. The binding performance was only visible on the surface area of oligo-microspheres. We show that the resulting oligo-microspheres can be directly integrated into a streamlined microsphere-PCR protocol for amplifying ssDNA. Our microspheres could be utilized as a potential material for ssDNA analysis such as DNA microarray and automatic DNA SELEX process. PMID:27447941

  13. Synthesis of DNA in excised watermelon cotyledons grown in water and benzyladenine.

    PubMed

    Galli, M G

    1984-03-01

    Excised watermelon cotyledons were grown in water and benzyladenine, which greatly promotes growth, breakdown of reserves and development of organelles. In order to investigate the involvement of DNA synthesis in these benzyladenine-induced effects, [(3)H]thymidine was applied continuously (for 3 d) or administered briefly (5 h) to excised cotyledons at various stages of development. Autoradiographic analysis of squashed and sectioned cotyledons showed that both the cytoplasm (mainly in the region of the plastids) and most of the nuclei were labelled. Both types of labelling were promoted by benzyladenine treatment. The highest percentage of labelled nuclei was found in the early stages of growth (first day after excision of cotyledons), long before the burst of enzymatic activities involved in the germination processes. The possible meaning of the increase of nuclear DNA, apart from the normal replicative synthesis preceding cell division, is discussed.

  14. Iron chelators hydroxyurea and bathophenanthroline disulfonate inhibit DNA synthesis by different pathways.

    PubMed

    Alcaín, F J; Löw, H; Crane, F L; Navas, P

    1994-09-01

    We previously showed that thrombin-stimulated DNA synthesis in CCL 39 cells was inhibited by hydroxyurea (HU) and bathophenanthroline disulfonate (BPS) (Proc. Natl. Acad. Sci. USA, in press). A clear difference exists between these two inhibitors. Inhibition mediated by HU was immediate and must be present in the culture medium. BPS was equally effective when it was present in the medium or after preincubation, but it required at least 12 h to achieve maximal effect. The permeable form 1,10 phenanthroline had the same inhibitory effect in short-term incubations that BPS. Moreover, 1,10 phenanthroline was cytotoxic in long-term incubations indicating that the site of BPS inhibition was outside the cell. Further, long-term incubations with HU did not affect the ability of the cell to reinitiate DNA synthesis after removal of the chelator.

  15. [Synthesis of biflavones and their interaction with DNA].

    PubMed

    Zhang, Zun-Ting; Gao, Run-Li; Zhuang, Su-Kai

    2009-08-01

    To explore new biflavones, 7-hydroxy-8-hydroxymethyl-4'-methoxyisoflavone (1), (5, 7-dihydroxyflavone-8-yl)-(7'-hydroxy-4"-methoxyisoflavone-8'-yl)methane (2), bis(7-hydroxy-4'-methoxyflavone-8-yl) methane (3), bis(3', 5'-diisopropyl-7, 4'-dihydroxy-isoflavone-8-yl)methane (4), and bis(7-hydroxy-isoflavone-8-yl) methane (5) were designed and synthesized from chrysin, formononetin, 7, 4'-dihydroxy-3', 5'-diisopropyl-isoflavone and 7-hydroxy-isoflavone. Their structures were identified with IR, 1H NMR, 13C NMR and elemental analysis. The binding of 1-5 with DNA was studied with fluorescent spectroscopy. Compounds 2-5 showed higher binding affinity with DNA than 1. According to the Stern-Volmer equation, the binding constants of 2, 3 were determined at 35 degrees C and 25 degrees C respectively, they were Kq2 (25 degrees C) = 1.95 x 10(4) Lx mol(-1) and Kq2 (35 degrees C) = 1.67 x 10(4) L x mol(-1); Kq3 (25 degrees C) = 1.89 x 10(4) L x mol(-1) and Kq3 (35 degrees C) = 1.58 x 10(4) L x mol(-1). The quenching mechanism of 2, 3 was suggested as static quenching.

  16. [Synthesis of chrysin derivatives and their interaction with DNA].

    PubMed

    Zhang, Zun-Ting; Chen, Li-Li

    2007-05-01

    Using chrysin as a leading compound, intermediate 5, 7-dihydroxy-6, 8-bis (hydroxymethyl) flavone (1) was synthesized by hydroxymethylation. The intermediate reacted with different alcohols to afford 5, 7-dihydroxy-6, 8-bis ( methoxymethyl) flavone (2), 6, 8-bis (ethoxymethyl) -5, 7dihydroxyflavone (3), 6, 8-bis-(butoxymethyl)-5, 7-dihydroxyflavone (4), 6, 8-bis (pentyloxymethyl) -5,7-dihydroxy flavone (5) and 6, 8-bis-(ethoxymethyl) -5-hydroxy-7-methoxyflavone (6). These compounds were characterized by IR, 1H NMR, 13C NMR and element analysis. The crystal structure of 6 was determined by X-ray crystal diffraction. The interaction of the derivatives with CT-DNA was studied by fluorescent spectroscopy. According to the Stern-Volmer equation, the quenching constants of the compounds 1 - 4 were measured, separately, they were K(q1) = 9.71 x 10(3) L x mol(-1), K(q2) = 2.25 x 10(4) L x mol(-1), K(q3) = 1.03 x 10(4) L x mol(-1) and K(q4) = 7.96 x 10(3) L x mol(-1). Compounds 1-4 showed higher binding affinity with DNA than chrysin did. The results provided the experimental basis for developing a more effective flavonoid and worthing further thoroughly study.

  17. Clarification of values in counselling and psychotherapy.

    PubMed

    Mickleburgh, W E

    1992-09-01

    When an individual acts contrary to personal values, then there is dissonance, with consequences of guilt, anxiety, despair, or alienation. If unresolved and of sufficient strength these feelings may manifest in mental illness. Thus, clarification of values and resolution of value conflict are relevant to counselling and psychotherapy. A framework for the systematic examination of values in therapy is described. Values are classified as personal, social, environmental and sensate. In therapy, goals may be matched for congruence between personal values and overt behaviour. Processes which erode values and ways to maintain integrity of personal value systems are considered.

  18. Clarification on the Clarification on the April 8th Memorandum from H.E.W.

    ERIC Educational Resources Information Center

    Journal of the National Association for Bilingual Education, 1976

    1976-01-01

    The clarification of the April 8th memorandum is that "the Lau remedies are minimum requirements and that in cases depending on student language dominance, grade level, and academic achievement, a bilingual program 'is' the remedy, and the only educationally sound way of ensuring effective participation in the instructional program." (NQ)

  19. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs): Synthesis and DNA Binding

    PubMed Central

    Kirillova, Yuliya; Boyarskaya, Nataliya; Dezhenkov, Andrey; Tankevich, Mariya; Prokhorov, Ivan; Varizhuk, Anna; Eremin, Sergei; Esipov, Dmitry; Smirnov, Igor; Pozmogova, Galina

    2015-01-01

    New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA. PMID:26469337

  20. The Foundry: the DNA synthesis and construction Foundry at Imperial College

    PubMed Central

    Chambers, Stephen; Kitney, Richard; Freemont, Paul

    2016-01-01

    The establishment of a DNA synthesis and construction foundry at Imperial College in London heralds a new chapter in the development of synthetic biology to meet new global challenges. The Foundry employs the latest technology to make the process of engineering biology easier, faster and scalable. The integration of advanced software, automation and analytics allows the rapid design, build and testing of engineered organisms. PMID:27284027

  1. Identification of polymerase and processivity inhibitors of vaccinia DNA synthesis using a stepwise screening approach

    PubMed Central

    Silverman, Janice Elaine Y.; Ciustea, Mihai; Shudofsky, Abigail M. Druck; Bender, Florent; Shoemaker, Robert H.; Ricciardi, Robert P.

    2008-01-01

    Nearly all DNA polymerases require processivity factors to ensure continuous incorporation of nucleotides. Processivity factors are specific for their cognate DNA polymerases. For this reason, the vaccinia DNA polymerase (E9) and the proteins associated with processivity (A20 and D4) are excellent therapeutic targets. In this study, we show the utility of stepwise rapid plate assays that i) screen for compounds that block vaccinia DNA synthesis, ii) eliminate trivial inhibitors, e.g. DNA intercalators, and iii) distinguish whether inhibitors are specific for blocking DNA polymerase activity or processivity. The sequential plate screening of 2,222 compounds from the NCI Diversity Set library yielded a DNA polymerase inhibitor (NSC 55636) and a processivity inhibitor (NSC 123526) that were capable of reducing vaccinia viral plaques with minimal cellular cytotoxicity. These compounds are predicted to block cellular infection by the smallpox virus, variola, based on the very high sequence identity between A20, D4 and E9 of vaccinia and the corresponding proteins of variola. PMID:18621425

  2. Two different mechanisms are involved for the bleomycin-induced DNA repair synthesis in permeabilized HeLa cells.

    PubMed

    Park, I S; Koh, H Y; Park, J K; Park, S D

    1989-11-15

    Bleomycin-induced DNA repair synthesis in the permeabilized HeLa cells was sensitive to aphidicolin, an inhibitor of DNA polymerase alpha and delta, and to dideoxythymidine triphosphate (ddTTP), a specific inhibitor of DNA polymerase beta. Upon combined treatment with these inhibitors, the DNA repair synthesis was inhibited to an even higher degree. This indicated that the aphidicolin- and ddTTP-sensitive DNA repair syntheses may occur by independent mechanisms. The structure of incomplete repair patches being accumulated in the presence of these inhibitors was investigated by digestion of DNA with exonuclease III after incubation with Klenow fragment and T4 DNA ligase. The results have suggested that the patch accumulating in the presence of aphidicolin is a single-stranded gap made by excision enzyme(s), whereas that accumulating in the presence of ddTTP may be generated by strand displacement.

  3. Quercetin-Iron Complex: Synthesis, Characterization, Antioxidant, DNA Binding, DNA Cleavage, and Antibacterial Activity Studies.

    PubMed

    Raza, Aun; Xu, Xiuquan; Xia, Li; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-11-01

    Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand.

  4. Rutin-Nickel Complex: Synthesis, Characterization, Antioxidant, DNA Binding, and DNA Cleavage Activities.

    PubMed

    Raza, Aun; Bano, Shumaila; Xu, Xiuquan; Zhang, Rong Xian; Khalid, Haider; Iqbal, Furqan Muhammad; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-12-17

    The rutin-nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV-visible spectroscopy, IR, mass spectrometry, (1)H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.

  5. Gab1 mediates neurite outgrowth, DNA synthesis, and survival in PC12 cells.

    PubMed

    Korhonen, J M; Saïd, F A; Wong, A J; Kaplan, D R

    1999-12-24

    The Gab1-docking protein has been shown to regulate phosphatidylinositol 3-kinase PI3K activity and potentiate nerve growth factor (NGF)-induced survival in PC12 cells. Here, we investigated the potential of Gab1 to induce neurite outgrowth and DNA synthesis, two other important aspects of NGF-induced neuronal differentiation of PC12 cells and NGF-independent survival. We generated a recombinant adenovirus encoding hemagglutinin (HA)-epitope-tagged Gab1 and expressed this protein in PC12 cells. HA-Gab1 was constitutively tyrosine-phosphorylated in PC12 cells and induced the phosphorylation of Akt/protein kinase B and p44/42 mitogen-activated protein kinase. HA-Gab1-stimulated a 10-fold increase in neurite outgrowth in the absence of NGF and a 5-fold increase in NGF-induced neurite outgrowth. HA-Gab1 also stimulated DNA synthesis and caused NGF-independent survival in PC12 cells. Finally, we found that HA-Gab1-induced neuritogenesis was completely suppressed by pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activity and 50% suppressed by inhibition of PI3K activity. In contrast, HA-Gab1-stimulated cell survival was efficiently suppressed only by inhibition of both PI3K and MEK activities. These results indicate that Gab1 is capable of mediating differentiation, DNA synthesis, and cell survival and uses both PI3K and MEK signaling pathways to achieve its effects.

  6. Iron reverses impermeable chelator inhibition of DNA synthesis in CCl 39 cells.

    PubMed

    Alcain, F J; Löw, H; Crane, F L

    1994-08-16

    Treatment of Chinese hamster lung fibroblasts (CCl 39 cells) with the impermeable iron(II) chelator bathophenanthroline disulfonate (BPS) inhibits DNA synthesis when cell growth is initiated with growth factors including epidermal growth factor plus insulin, thrombin, or ceruloplasmin, but not with 10% fetal calf serum. The BPS treatment inhibits transplasma membrane electron transport. The treatment leads to release of iron from the cells as determined by BPS iron(II) complex formation over 90 min. Growth factor stimulation of DNA synthesis and electron transport are restored by addition of di- or trivalent iron to the cells in the form of ferric ammonium citrate, ferrous ammonium sulfate, or diferric transferrin. The effect with BPS differs from the inhibition of growth by hydroxyurea, which acts on the ribonucleotide reductase, or diethylenetriaminepentaacetic acid, which is another impermeable chelating agent, in that these agents inhibit growth in 10% fetal calf serum. The BPS effect is consistent with removal of iron from a site on the cell surface that controls DNA synthesis.

  7. Helium-neon laser irradiation enhances DNA synthesis in a human neuroblastoma cell line

    NASA Astrophysics Data System (ADS)

    Condon, Michael R.; Gump, Frank; Wu, Wen-hsien

    1993-07-01

    To gain further insight into the mechanism of cell photostimulation by laser light ((lambda) equals 632.8 nm), DNA synthesis was measured in the human neuroblastoma cell line BE(2)-C. Cells were irradiated at high density to establish the characteristics of cellular energy into S- phase in response to laser stimulation. BE(2)-C cells after release from a quiescent, growth arrested state exhibited increased incorporation of isotope 12 hours after replating at subconfluent density in the presence of 2.5% fetal bovine serum (FBS) and [3H] thymidine. In contrast, cells replated under the same conditions, but stimulated with 15% FBS exhibited a time lag of approximately 16 hours in apparent DNA synthesis. These results were not corroborated by flow cytometry. Laser irradiation did not affect the fraction of cells entering S-phase. It therefore appears that the stimulatory effect of He-Ne laser irradiation on BE(2)-C cells is to enhance DNA synthesis while not altering the G1-S transition rate.

  8. Iron Reverses Impermeable Chelator Inhibition of DNA Synthesis in CCl39 Cells

    NASA Astrophysics Data System (ADS)

    Alcain, Francisco J.; Low, Hans; Crane, Frederick L.

    1994-08-01

    Treatment of Chinese hamster lung fibro-blasts (CCl 39 cells) with the impermeable iron(II) chelator bathophenanthroline disulfonate (BPS) inhibits DNA synthesis when cell growth is initiated with growth factors including epidermal growth factor plus insulin, thrombin, or ceruloplasmin, but not with 10% fetal calf serum. The BPS treatment inhibits transplasma membrane electron transport. The treatment leads to release of iron from the cells as determined by BPS iron(II) complex formation over 90 min. Growth factor stimulation of DNA synthesis and electron transport are restored by addition of di- or trivalent iron to the cells in the form of ferric ammonium citrate, ferrous ammonium sulfate, or diferric transferrin. The effect with BPS differs from the inhibition of growth by hydroxyurea, which acts on the ribonucleotide reductase, or diethylenetriaminepentaacetic acid, which is another impermeable chelating agent, in that these agents inhibit growth in 10% fetal calf serum. The BPS effect is consistent with removal of iron from a site on the cell surface that controls DNA synthesis.

  9. Synthesis of linear polyethylenimine derivatives for DNA transfection.

    PubMed

    Brissault, Blandine; Kichler, Antoine; Guis, Christine; Leborgne, Christian; Danos, Olivier; Cheradame, Hervé

    2003-01-01

    A series of linear polymers containing varying amounts of ethylenimine or N-propylethylenimine units were synthesized by hydrolysis and/or reduction of polyethyloxazolines. The pK(a)s of the polyamines were determined potentiometrically. Gel mobility shift assay showed that the efficiency of DNA complexation was related to the fraction of amino groups that are protonated at neutral pH. The effects of cationic charge density and molar weight of the polymers on the transfection efficiency were evaluated on HepG2 cells. The results obtained with different copolymers show that the transfection efficiency primarily depends on the fraction of ethylenimine units included in the polymer albeit the molar weight is also of importance. On the basis of the results obtained with poly(N-propylethylenimines), we also demonstrate that the high transfection efficiency of polyethylenimines does not solely rely on their capacity to capture protons which are transferred into the endo-lysosomes during acidification.

  10. Involvement of translesion synthesis DNA polymerases in DNA interstrand crosslink repair.

    PubMed

    Roy, Upasana; Schärer, Orlando D

    2016-08-01

    DNA interstrand crosslinks (ICLs) covalently join the two strands of a DNA duplex and block essential processes such as DNA replication and transcription. Several important anti-tumor drugs such as cisplatin and nitrogen mustards exert their cytotoxicity by forming ICLs. However, multiple complex pathways repair ICLs and these are thought to contribute to the development of resistance towards ICL-inducing agents. While the understanding of many aspects of ICL repair is still rudimentary, studies in recent years have provided significant insights into the pathways of ICL repair. In this perspective we review the recent advances made in elucidating the mechanisms of ICL repair with a focus on the role of TLS polymerases. We describe the emerging models for how these enzymes contribute to and are regulated in ICL repair, discuss the key open questions and examine the implications for this pathway in anti-cancer therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. DNA repair after ultraviolet irradiation of ICR 2A frog cells: pyrimidine dimers are long acting blocks to nascent DNA synthesis

    SciTech Connect

    Rosenstein, B.S.; Setlow, R.B.

    1980-08-01

    The ability of ICR 2A frog cells to repair DNA damage induced by ultraviolet irradiation was examined. These cells are capable of photoreactivation but are nearly totally deficient in excision repair. They have the ability to convert the small molecular weight DNA made after irradiation into large molecules but do not show an enhancement in this process when the UV dose is delivered in two separate exposures separated by a 3- or 24-h incubation. Total DNA synthesis is depressed and low molecular weight DNA continues to be synthesized during pulse-labeling as long as 48 h after irradiation. The effects of pyrimidine dimer removal through exposure of UV irradiated cells to photoreactivating light indicate that dimers act as the critical lesions blocking DNA synthesis.

  12. Roles of the Envelope Proteins in the Amplification of Covalently Closed Circular DNA and Completion of Synthesis of the Plus-Strand DNA in Hepatitis B Virus ▿

    PubMed Central

    Lentz, Thomas B.; Loeb, Daniel D.

    2011-01-01

    Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion. PMID:21900164

  13. Concept clarification of neonatal neurobehavioural organization.

    PubMed

    Bell, Aleeca F; Lucas, Ruth; White-Traut, Rosemary C

    2008-03-01

    This paper is a report of a concept analysis of neonatal neurobehavioural organization for healthy full-term infants. The neonatal period is an opportune time for researchers and clinicians to assess and intervene for optimal neurobehavioural organization. Yet there is inconsistency and lack of clarity in a scientifically grounded definition of neonatal neurobehavioural organization. Clarification of the concept will strengthen research findings that influence practice for optimal infant development. A concept analysis of the literature between 1939 and 2007 (n = 57) was conducted using Penrod and Hupcey's principle-based concept analysis and Morse's concept clarification. The concept analysis within and across multiple disciplines revealed: (1) a view of the concept as a holistic phenomenon with multiple dimensions; (2) no agreement on the ideal instrument to operationally define the concept; and (3) consistency in implied meaning, but great variability in terminology. Neonatal neurobehavioural organization was defined as the ability of the neonate to use goal-directed states of consciousness, in reciprocal interaction with the caregiving environment, to facilitate the emergence of differentiating, hierarchical, and coordinated neurobehavioural systems, with ever-increasing resiliency and capacity to learn from complex stimuli. A clear conceptual definition will help the international community to communicate effectively within and between disciplines and to apply evidence-based research findings. It will encourage the development of valid and reliable instruments to capture the concept's multiple dimensions and direct attention to the infant's experience, which sculpts early neurobehavioural organization.

  14. The persistence in the liver of residual duck hepatitis B virus covalently closed circular DNA is not dependent upon new viral DNA synthesis.

    PubMed

    Reaiche, Georget Y; Le Mire, Marc F; Mason, William S; Jilbert, Allison R

    2010-10-25

    Residual hepatitis B virus (HBV) DNA can be detected following the resolution of acute HBV infection. Our previous work using duck hepatitis B virus (DHBV) infected ducks, indicated that ~80% of residual DHBV DNA in the liver is in the covalently closed circular DNA (cccDNA) form, suggesting that viral DNA synthesis is suppressed. The current study asked more directly if maintenance of residual DHBV cccDNA is dependent upon ongoing viral DNA synthesis. Ducks that recovered from acute DHBV infection were divided into 2 groups and treated with the antiviral drug, Entecavir (ETV), or placebo. No major differences in the stability of cccDNA or levels of residual cccDNA were observed in liver biopsy tissues taken 95 days apart from ETV treated and placebo control ducks. The data suggest that residual DHBV cccDNA is highly stable and present in a cell population with a rate of turnover similar to normal, uninfected hepatocytes. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Local injection of insulin-zinc stimulates DNA synthesis in skin donor site wound.

    PubMed

    Zhang, Xiao-jun; Chinkes, David L; Sadagopa Ramanujam, V M; Wolfe, Robert R

    2007-01-01

    Our group has reported the ability of local insulin-zinc injection to accelerate skin wound healing. This experiment was conducted to investigate the metabolic mechanisms of locally injected insulin-zinc in accelerating wound healing. A partial thickness skin donor site wound was created on the back, and indwelling catheters were placed in the carotid artery and jugular vein in anesthetized rabbits. On day 7 after injury, the wound was either injected with 0.2 U of insulin-zinc, 0.4 microg zinc (the amount of zinc in 0.2 U of insulin-zinc), or not injected; stable isotope tracers were infused for measurement of wound DNA synthetic rate as a reflection of cell proliferation. Wound protein synthetic and breakdown rates were also measured. The local insulin-zinc injection increased wound insulin concentration from below 5 pmol/L to 1,260+/-780 pmol/L with minor changes in blood glucose concentration that did not require exogenous glucose replacement. In the insulin-zinc-injected wound, the total DNA synthetic rate was increased by approximately 50% (p<0.05 vs. control). In the zinc-injected wound, whereas total DNA synthetic rate was increased (p<0.05 vs. control), net protein deposition (synthesis-breakdown) was less (p<0.05) than those in the control and insulin-zinc groups. We conclude that local insulin-zinc injection stimulates wound DNA synthesis, which would be expected to accelerate wound reepithelialization.

  16. Inhibitor of DNA synthesis is present in normal chicken serum

    SciTech Connect

    Franklin, R.A.; Davila, D.R.; Westly, H.J.; Kelley, K.W.

    1986-03-05

    The authors have found that heat-inactivated serum (57/sup 0/C for 1 hour) from normal chickens reduces the proliferation of mitogen-stimulated chicken and murine splenocytes as well as some transformed mammalian lymphoblastoid cell lines. Greater than a 50% reduction in /sup 3/H-thymidine incorporation was observed when concanavalin A (Con A)-activated chicken splenocytes that were cultured in the presence of 10% autologous or heterologous serum were compared to mitogen-stimulated cells cultured in the absence of serum. Normal chicken serum (10%) also caused greater than 95% suppression of /sup 3/H-thymidine incorporation by bovine (EBL-1 and BL-3) and gibbon ape (MLA 144) transformed lymphoblastoid cell lines. The only cell line tested that was not inhibited by chicken serum was an IL-2-dependent, murine cell line. Chicken serum also inhibited both /sup 3/H-thymidine incorporation and IL-2 synthesis by Con A-activated murine splenocytes. Suppression was caused by actions other than cytotoxicity because viability of chicken splenocytes was unaffected by increasing levels of chicken serum. Furthermore, dialyzed serum retained its activity, which suggested that thymidine in the serum was not inhibiting uptake of radiolabeled thymidine. Suppressive activity was not due to adrenal glucocorticoids circulating in plasma because neither physiologic nor pharmacologic doses of corticosterone had inhibitory effects on mitogen-stimulated chicken splenocytes. These data demonstrate that an endogenous factor that is found in normal chicken serum inhibits proliferation of T-cells from chickens and mice as well as some transformed mammalian lymphoblastoid cell lines.

  17. Mining Enzyme Diversity of Transcriptome Libraries through DNA Synthesis for Benzylisoquinoline Alkaloid Pathway Optimization in Yeast.

    PubMed

    Narcross, Lauren; Bourgeois, Leanne; Fossati, Elena; Burton, Euan; Martin, Vincent J J

    2016-12-16

    The ever-increasing quantity of data deposited to GenBank is a valuable resource for mining new enzyme activities. Falling costs of DNA synthesis enables metabolic engineers to take advantage of this resource for identifying superior or novel enzymes for pathway optimization. Previously, we reported synthesis of the benzylisoquinoline alkaloid dihydrosanguinarine in yeast from norlaudanosoline at a molar conversion of 1.5%. Molar conversion could be improved by reduction of the side-product N-methylcheilanthifoline, a key bottleneck in dihydrosanguinarine biosynthesis. Two pathway enzymes, an N-methyltransferase and a cytochrome P450 of the CYP719A subfamily, were implicated in the synthesis of the side-product. Here, we conducted an extensive screen to identify enzyme homologues whose coexpression reduces side-product synthesis. Phylogenetic trees were generated from multiple sources of sequence data to identify a library of candidate enzymes that were purchased codon-optimized and precloned into expression vectors designed to facilitate high-throughput analysis of gene expression as well as activity assay. Simple in vivo assays were sufficient to guide the selection of superior enzyme homologues that ablated the synthesis of the side-product, and improved molar conversion of norlaudanosoline to dihydrosanguinarine to 10%.

  18. Synthesis, Characterization, and DNA Binding Profile of a Macrocyclic β-Sheet Analogue of ARC Protein

    PubMed Central

    2015-01-01

    ARC repressor (apoptosis repressor with caspase recruitment domain) is a protein which binds selectively to a specific sequence of DNA. In humans, ARC is primarily expressed in striated muscle tissue, which normally does not undergo rapid cell turnover. This suggests that ARC may play a protective role in the prevention against Duchenne Muscular Dystrophy and several types of tumors. In this Letter we report the synthesis, characterization, and conformational analysis of a β-sheet ARC repressor mimetic, based on the amino acid sequence of the β-sheet domain in the ARC protein. The ability of this β-sheet macrocycle to bind to double-stranded DNA was also evaluated using spectroscopic methods. Our data show that the synthetic peptide has a defined conformation and is able to bind DNA with reasonable affinity. These initial results lay the groundwork for the design of novel β-sheets folded peptides as valuable substitutes of transcription factor proteins in drug therapy. PMID:26713108

  19. Green synthesis of gold nanoparticles for staining human cervical cancer cells and DNA binding assay.

    PubMed

    De, Swati; Kundu, Rikta; Ghorai, Atanu; Mandal, Ranju Prasad; Ghosh, Utpal

    2014-11-01

    Gold nanoparticles have been functionalized by non-ionic surfactants (polysorbates) used in pharmaceutical formulations. This results in the formation of more well-dispersed gold nanoparticles (GNPs) than the GNPs formed in neat water. The synthesized GNPs show good temporal stability. The synthesis conditions are mild and environmentally benign. The GNPs can bind to ct-DNA and displace bound dye molecules. The DNA-binding assay is significant as it preliminarily indicated that DNA-GNP conjugates can be formed. Such conjugates are extremely promising for applications in nanobiotechnology. The GNPs can also stain the human cervical cancer (HeLa) cells over a wide concentration range while remaining non-cytotoxic, thus providing a non invasive cell staining method. This result is very promising as we observe staining of HeLa cells at very low GNP concentrations (1 μM) while the cell viability is retained even at 10-fold higher GNP concentrations.

  20. Ordered replication of DNA sequences: synthesis of mouse satellite and adjacent main band sequences.

    PubMed

    Dooley, D C; Ozer, H L

    1979-03-01

    The replication of mouse satellite DNA was delayed when synchronized 3T3 cells were exposed to low concentrations of hydroxyurea during S phase, It appears that the onset of satellite replication is not a time dependent event, but instead requires that a certain amount of main band DNA be synthesized first. Using hydroxyapatite chromatography and S1 nuclease digestion, a procedure was developed to quantitate the synthesis of both satellite and neighboring main band sequences. The replication kinetics of satellite determined by this method agree with previous estimates. Main band sequences adjacent to satellite appear to replicate in concert with satellite DNA. The results are discussed and related to the limitations of the techniques utilized.

  1. A Transcriptional Repressor ZBTB1 Promotes Chromatin Remodeling and Translesion DNA Synthesis

    PubMed Central

    Kim, Hyungjin; Dejsuphong, Donniphat; Adelmant, Guillaume; Ceccaldi, Raphael; Yang, Kailin; Marto, Jarrod A.; D’Andrea, Alan D.

    2014-01-01

    SUMMARY Timely DNA replication across damaged DNA is critical for maintaining genomic integrity. Translesion DNA synthesis (TLS) allows bypass of DNA lesions using error-prone TLS polymerases. The E3 ligase RAD18 is necessary for PCNA monoubiquitination and TLS polymerase recruitment; however, the regulatory steps upstream of RAD18 activation are less understood. Here, we show that the UBZ4 domain-containing transcriptional repressor ZBTB1 is a critical upstream regulator of TLS. The UBZ4 motif is required for PCNA monoubiquitination and survival after UV damage. ZBTB1 associates with KAP-1, a transcriptional repressor whose phosphorylation relaxes chromatin after DNA damage. ZBTB1 depletion impairs formation of phospho-KAP-1 at UV damage sites and reduces RAD18 recruitment. Furthermore, phosphorylation of KAP-1 is necessary for efficient PCNA modification. We propose that ZBTB1 is required for PCNA monoubiquitination, by localizing phospho-KAP-1 to chromatin and enhancing RAD18 accessibility. Collectively, our study implicates a new ubiquitin-binding protein in orchestrating chromatin remodeling during DNA repair. PMID:24657165

  2. Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

    SciTech Connect

    Lovrinovic, Marina; Niemeyer, Christof M. . E-mail: christof.niemeyer@uni-dortmund.de

    2005-09-30

    We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.

  3. In vitro synthesis of large peptide molecules using glucosylated single-stranded bacteriophage T4D DNA template.

    PubMed Central

    Hulen, C; Legault-Demare, J

    1975-01-01

    Denatured Bacteriophage T4D DNA is able to stimulate aminoacid incorporation into TCA-precipitable material in an in vitro protein synthesis system according to base DNA sequences. Newly synthesized polypeptides remain associated with ribosomes and have a molecular weight in range of 15,000 to 45,000 Daltons. PMID:1052527

  4. Stimulation of Endomitotic DNA Synthesis and Cell Elongation by Gibberellic Acid in Epicotyls Grown from Gamma-irradiated Pea Seeds 1

    PubMed Central

    Callebaut, Alfons; Van Oostveldt, Patrick; Van Parijs, Roger

    1980-01-01

    Large doses of γ-irradiation, given to air-dried pea seeds, inhibit the endomitotic DNA synthesis in pea epicotyls during germination in darkness. The cortex cells of the etiolated epicotyls reach only the 4 C DNA level, whereas cortex cells of unirradiated seeds reach the 8 C DNA level. Epicotyl elongation and cell elongation are also reduced. Application of gibberellic acid restores the endomitotic DNA synthesis and the cell elongation in epicotyls of irradiated seeds. The cortex cells reach again the 8 C DNA level in darkness. The results suggest that γ-irradiation blocks endomitotic DNA synthesis and cell elongation by lowering the concentration of endogenous gibberellins. PMID:16661127

  5. 76 FR 28031 - Clarification of Statement of Policy

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-13

    ... CORPORATION Clarification of Statement of Policy AGENCY: Federal Deposit Insurance Corporation (FDIC). ACTION: Clarification of Statement of Policy for Section 19 of the Federal Deposit Insurance Act. SUMMARY: The FDIC originally promulgated the Statement of Policy for Section 19 of the Federal Deposit Insurance Act (SOP) in...

  6. Teaching Values in the Schools: Clarification or Indoctrination?

    ERIC Educational Resources Information Center

    Baer, Richard A., Jr.

    1982-01-01

    Notes that both liberals and conservatives have criticized values clarification courses on the grounds that they assume only one correct position regarding values: ethical relativism. Maintains that values clarification fails to live up to its own claims of openness and neutrality. (Author/JM)

  7. Steric and electrostatic effects in DNA synthesis by the SOS-induced DNA polymerases II and IV of Escherichia coli.

    PubMed

    Silverman, Adam P; Jiang, Qingfei; Goodman, Myron F; Kool, Eric T

    2007-12-04

    The SOS-induced DNA polymerases II and IV (pol II and pol IV, respectively) of Escherichia coli play important roles in processing lesions that occur in genomic DNA. Here we study how electrostatic and steric effects play different roles in influencing the efficiency and fidelity of DNA synthesis by these two enzymes. These effects were probed by the use of nonpolar shape analogues of thymidine, in which substituted toluenes replace the polar thymine base. We compared thymine with nonpolar analogues to evaluate the importance of hydrogen bonding in the polymerase active sites, while we used comparisons among a set of variably sized thymine analogues to measure the role of steric effects in the two enzymes. Steady-state kinetics measurements were carried out to evaluate activities for nucleotide insertion and extension. The results showed that both enzymes inserted nucleotides opposite nonpolar template bases with moderate to low efficiency, suggesting that both polymerases benefit from hydrogen bonding or other electrostatic effects involving the template base. Surprisingly, however, pol II inserted nonpolar nucleotide (dNTP) analogues into a primer strand with high (wild-type) efficiency, while pol IV handled them with an extremely low efficiency. Base pair extension studies showed that both enzymes bypass non-hydrogen-bonding template bases with moderately low efficiency, suggesting a possible beneficial role of minor groove hydrogen bonding interactions at the N-1 position. Measurement of the two polymerases' sensitivity to steric size changes showed that both enzymes were relatively flexible, yielding only small kinetic differences with increases or decreases in nucleotide size. Comparisons are made to recent data for DNA pol I (Klenow fragment), the archaeal polymerase Dpo4, and human pol kappa.

  8. The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery.

    PubMed

    O'Reilly, Rachel K; Turberfield, Andrew J; Wilks, Thomas R

    2017-09-15

    Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify "successful" structures. The largest libraries made by conventional synthesis are currently of the order of 10(8) distinct molecules. To put this in context, there are 10(13) ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required. DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a

  9. Structure of a mutant form of proliferating cell nuclear antigen that blocks translesion DNA synthesis

    PubMed Central

    Freudenthal, Bret D.; Ramaswamy, S.; Hingorani, Manju M.; Washington, M. Todd

    2009-01-01

    Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein that functions as a sliding clamp during DNA replication. Several mutant forms of PCNA that block translesion DNA synthesis have been identified in genetic studies in yeast. One such mutant protein (encoded by the rev6-1 allele) is a glycine to serine substitution at residue 178, located at the subunit interface of PCNA. To better understand how this substitution interferes with translesion synthesis, we have determined the X-ray crystal structure of the G178S PCNA mutant protein. This substitution has little effect on the structure of the domain in which the substitution occurs. Instead, significant, local structural changes are observed in the adjacent subunit. The most notable difference between mutant and wild-type structures is in a single, extended loop (comprising amino acid residues 105-110), which we call loop J. In the mutant protein structure, loop J adopts a very different conformation in which the atoms of the protein backbone have moved by as much as 6.5 Å from their positions in the wild-type structure. To better understand the functional consequences of this structural change, we have examined the ability of this mutant protein to stimulate nucleotide incorporation by DNA polymerase eta (pol η). Steady state kinetic studies show that while wild-type PCNA stimulates incorporation by pol η opposite an abasic site, the mutant PCNA protein actually inhibits incorporation opposite this DNA lesion. These results show that the position of loop J in PCNA plays an essential role in facilitating translesion synthesis. PMID:19053247

  10. Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.

    PubMed

    Kim, Jinsook; Song, Insil; Jo, Ara; Shin, Joo-Ho; Cho, Hana; Eoff, Robert L; Guengerich, F Peter; Choi, Jeong-Yun

    2014-10-20

    DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The Δ1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The Δ1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1-25 variant, was ∼7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ι, whereas the Δ1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences

  11. Temporal and topographic changes in DNA synthesis after induced follicular atresia

    SciTech Connect

    Greenwald, G.S. )

    1989-07-01

    Hamsters were hypophysectomized on the morning of estrus (Day 1) and injected immediately with 30 IU pregnant mare's serum (PMS). This was followed on Day 4 by the injection of an antiserum to PMS (PMS-AS) that initiated follicular atresia (Time zero). From 0 to 72 h after PMS-AS, the animals were injected with (3H)thymidine and killed 4 h later. One ovary was saved for autoradiography and histology; from the other ovary, 5-10 large antral follicles were dissected and pooled, and incorporation into DNA was determined by scintillation counting. DNA synthesis dropped sharply between 12 and 18 h, coinciding with a fall in labeling index of the cumulus oophorus and thecal endothelial cells and a sharp fall in thecal vascularity. In contrast, for the mural granulosa cells bordering on the antral cavity, labeling index dropped sharply between 8 and 12 h when thecal vascularity was still high. The earliest sign of atresia was evident by 4 h in cumulus cells when, paradoxically, DNA synthesis was still high. It took 3 days for atresia of the antral follicles to progress to advanced stages, as evidenced by pseudo-pronuclei in the free floating ovum, further erosion of the mural granulosa, and minimal DNA/follicle. However, the theca still retained its histological integrity and contained no pyknotic cells. Although by 48 h the granulosal compartment was in disarray (DNA/follicle significantly different from earlier values), the egg was still viable, as judged by maximal fluorescence after the addition of fluoroscein diacetate.

  12. Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions

    PubMed Central

    Tsaalbi-Shtylik, Anastasia; Ferrás, Cristina; Pauw, Bea; Hendriks, Giel; Temviriyanukul, Piya; Carlée, Leone; Calléja, Fabienne; van Hees, Sandrine; Akagi, Jun-Ichi; Iwai, Shigenori; Hanaoka, Fumio; Jansen, Jacob G.

    2015-01-01

    In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa–Atr–Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects. PMID:25869665

  13. DNA Binding and Recognition of a CC Mismatch in a DNA Duplex by Water-Soluble Peptidocalix[4]arenes: Synthesis and Applications.

    PubMed

    Alavijeh, Nahid S; Zadmard, Reza; Balalaie, Saeed; Alavijeh, Mohammad S; Soltani, Nima

    2016-10-07

    Water-soluble peptidocalix[4]arenes were synthesized by the introduction of arginine-rich narrow groove-binding residues at lower rims through solid-phase synthesis. The study of binding of these water-soluble bidentate ligands to well-matched and mismatched DNA duplexes by fluorescent titrations, ethidium bromide (EB) displacement assays, DNA-melting experiments, and circular dichroism (CD) analysis revealed a sequence-dependent groove-binding mechanism.

  14. Toward a designed genetic system with biochemical function: polymerase synthesis of single and multiple size-expanded DNA base pairs.

    PubMed

    Lu, Haige; Krueger, Andrew T; Gao, Jianmin; Liu, Haibo; Kool, Eric T

    2010-06-21

    The development of alternative architectures for genetic information-encoding systems offers the possibility of new biotechnological tools as well as basic insights into the function of the natural system. In order to examine the potential of benzo-expanded DNA (xDNA) to encode and transfer biochemical information, we carried out a study of the processing of single xDNA pairs by DNA Polymerase I Klenow fragment (Kf, an A-family sterically rigid enzyme) and by the Sulfolobus solfataricus polymerase Dpo4 (a flexible Y-family polymerase). Steady-state kinetics were measured and compared for enzymatic synthesis of the four correct xDNA pairs and twelve mismatched pairs, by incorporation of dNTPs opposite single xDNA bases. Results showed that, like Kf, Dpo4 in most cases selected the correctly paired partner for each xDNA base, but with efficiency lowered by the enlarged pair size. We also evaluated kinetics for extension by these polymerases beyond xDNA pairs and mismatches, and for exonuclease editing by the Klenow exo+ polymerase. Interestingly, the two enzymes were markedly different: Dpo4 extended pairs with relatively high efficiencies (within 18-200-fold of natural DNA), whereas Kf essentially failed at extension. The favorable extension by Dpo4 was tested further by stepwise synthesis of up to four successive xDNA pairs on an xDNA template.

  15. Synthesis and base pairing properties of DNA-RNA heteroduplex containing 5-hydroxyuridine.

    PubMed

    Cui, Song; Kim, Yong-Hoon; Jin, Cheng-Hao; Kim, Sang Kook; Rhee, Man-Hee; Kwon, Oh-Shin; Moon, Byung Jo

    2009-06-30

    5-Hydroxyuridine (5-OHU) is a major lesion of uridine and cytosine produced in RNA by various chemical oxidants. To elucidate its biochemical and biophysical effects on RNA replication, the site-specifically modified oligoribonucleotides containing 5-OHU were synthesized with C5-hydroxy-5'-O-DMTr-2'-TBDMS-uridine phosphoramidite using automated solid phase synthesis. The base-pairing properties of nucleotides opposite 5-OHU in 24 mer oligoribonulcleotides with dNTP were studied using three reverse transcriptases (Super-Script(TM)II-, AMV-, MMLV-RT) in cDNA synthesis. Adenine as well as guanine was incorporated preferentially by all reverse transcriptases. In the UV-melting temperature experiment, the results from the relative stabilities of the base pairs were A : 5-OHU > G : 5-OHU > T : 5-OHU approximately C : 5-OHU. Circular Dichroism (CD) studies showed that DNA-RNA containing 5-OHU heteroduplexes exhibit a similar conformation between the A-type RNA and B-type DNA. These results suggest that 5-OHU from oxidative damage was mainly influenced by adenine mismatch.

  16. Restriction of gut-derived endotoxin impairs DNA synthesis for liver regeneration.

    PubMed

    Cornell, R P

    1985-11-01

    The influence of restricting gut-derived endotoxin availability on liver regeneration after partial hepatectomy was evaluated. Partial hepatectomy was performed by 67% liver resection of ether-anesthetized rats. Liver regeneration was quantified after partial hepatectomy by [3H]thymidine incorporation into hepatic DNA; endotoxemia due to absorption of endogenous endotoxin from the gut into the portal circulation was determined by qualitative lysate assay of perchloric acid-extracted plasma samples, and plasma levels of the hepatotrophic factors insulin and glucagon were measured by radioimmunoassay. Treatments to restrict gut-derived endotoxin included chronic gavage with neomycin and cefazolin for gut sterilization, chronic gavage with cholestyramine to bind endotoxin within the gut, subcutaneous administration of polymyxin B to neutralize the lipid A portion of circulating endotoxin, intraperitoneal induction of endotoxin tolerance by progressively higher doses of endotoxin, and experimentation with isolator-reared defined flora Fisher rats that were Gram-negative bacteria deficient and therefore endotoxin deficient. All treatments to restrict endogenous endotoxin impaired DNA synthesis in regenerating livers particularly 21 h posthepatectomy when replication was increasing most rapidly in normal rats. We hypothesize that impairment of DNA synthesis after partial hepatectomy in endotoxin-restricted animals was due to the observed lack of normal systemic endotoxemic as well as hyperinsulinemic and hyperglucagonemic responses to 67% liver resection.

  17. Timing of initiation of macronuclear DNA synthesis is set during the preceding cell cycle in Paramecium tetraurelia: analysis of the effects of abrupt changes in nutrient level

    SciTech Connect

    Ching, A.S.L.; Berger, J.D.

    1986-11-01

    In many eukaryotic organisms, initiation of DNA synthesis is associated with a major control point within the cell cycle and reflects the commitment of the cell to the DNA replication-division portion of the cell cycle. In paramecium, the timing of DNA synthesis initiation is established prior to fission during the preceding cell cycle. DNA synthesis normally starts at 0.25 in the cell cycle. When dividing cells are subjected to abrupt nutrient shift-up by transfer from a chemostat culture to medium with excess food, or shift-down from a well-fed culture to exhausted medium, DNA synthesis initiation in the post-shift cell cycle occurs at 0.25 of the parental cell cycle and not at either 0.25 in the post-shift cell cycle or at 0.25 in the equilibrium cell cycle produced under the post-shift conditions. The long delay prior to initiation of DNA synthesis following nutritional shift-up is not a consequence of continued slow growth because the rate of protein synthesis increases rapidly to the normal level after shift-up. Analysis of the relation between increase in cell mass and initiation of DNA synthesis following nutritional shifts indicates that increase in cell mass, per se, is neither a necessary nor a sufficient condition for initiation of DNA synthesis, in spite of the strong association between accumulation of cell mass and initiation of DNA synthesis in cells growing under steady-state conditions.

  18. Photoaugmentation in the hairless mouse: a study using ornithine decarboxylase activity and alteration of DNA synthesis as markers of epidermal response

    SciTech Connect

    Gange, R.W.; Mendelson, I.R.

    1981-01-01

    Photoaugmentation is the potentiation of UVB-induced cutaneous erythema by UV irradiation. We have examined other cutaneous responses to UVB irradiation-the 4 hr depression of DNA synthesis, the 48 hr stimulation of DNA synthesis, and the induction of ornithine decarboxylase (ODC), to determine whether these were also susceptible to augmentation by UVA, which does not cause these responses when administered alone. No photoaugmentation of DNA synthesis, stimulation or ODC induction occurred. The early depression of DNA synthesis was slightly augmented for this did not consistently reach significance.

  19. 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

  20. Impairment of HIV-1 cDNA synthesis by DBR1 knockdown.

    PubMed

    Galvis, Alvaro E; Fisher, Hugh E; Nitta, Takayuki; Fan, Hung; Camerini, David

    2014-06-01

    Previous studies showed that short hairpin RNA (shRNA) knockdown of the RNA lariat debranching enzyme (DBR1) led to a decrease in the production of HIV-1 cDNA. To further characterize this effect, DBR1 shRNA was introduced into GHOST-R5X4 cells, followed by infection at a multiplicity near unity with HIV-1 or an HIV-1-derived vector. DNA and RNA were isolated from whole cells and from cytoplasmic and nuclear fractions at different times postinfection. Inhibition of DBR1 had little or no effect on the formation of minus-strand strong-stop cDNA but caused a significant reduction in the formation of intermediate and full-length cDNA. Moreover, minus-strand strong-stop DNA rapidly accumulated in the cytoplasm in the first 2 h of infection but shifted to the nuclear fraction by 6 h postinfection. Regardless of DBR1 inhibition, greater than 95% of intermediate-length and full-length HIV-1 cDNA was found in the nuclear fraction at all time points. Thus, under these experimental conditions, HIV-1 cDNA synthesis was initiated in the cytoplasm and completed in the nucleus or perinuclear region of the infected cell. When nuclear import of the HIV-1 reverse transcription complex was blocked by expressing a truncated form of the mRNA cleavage and polyadenylation factor CPSF6, the completion of HIV-1 vector cDNA synthesis was detected in the cytoplasm, where it was not inhibited by DBR1 knockdown. Refinement of the cell fractionation procedure indicated that the completion of reverse transcription occurred both within nuclei and in the perinuclear region. Taken together the results indicate that in infections at a multiplicity near 1, HIV-1 reverse transcription is completed in the nucleus or perinuclear region of the infected cell, where it is dependent on DBR1. When nuclear transport is inhibited, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. Thus, there are at least two mechanisms of HIV-1 reverse transcription that require different

  1. Nucleotides with altered hydrogen bonding capacities impede human DNA polymerase η by reducing synthesis in the presence of the major cisplatin DNA adduct.

    PubMed

    Nilforoushan, Arman; Furrer, Antonia; Wyss, Laura A; van Loon, Barbara; Sturla, Shana J

    2015-04-15

    Human DNA polymerase η (hPol η) contributes to anticancer drug resistance by catalyzing the replicative bypass of DNA adducts formed by the widely used chemotherapeutic agent cis-diamminedichloroplatinum (cisplatin). A chemical basis for overcoming bypass-associated resistance requires greater knowledge of how small molecules influence the hPol η-catalyzed bypass of DNA adducts. In this study, we demonstrated how synthetic nucleoside triphosphates act as hPol η substrates and characterized their influence on hPol η-mediated DNA synthesis over unmodified and platinated DNA. The single nucleotide incorporation efficiency of the altered nucleotides varied by more than 10-fold and the higher incorporation rates appeared to be attributable to the presence of an additional hydrogen bond between incoming dNTP and templating base. Finally, full-length DNA synthesis in the presence of increasing concentrations of synthetic nucleotides reduced the amount of DNA product independent of the template, representing the first example of hPol η inhibition in the presence of a platinated DNA template.

  2. Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination.

    PubMed

    Ho, Jenny; Huang, Yi; Danquah, Michael K; Wang, Huanting; Forde, Gareth M

    2010-03-18

    DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(D,L-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 microm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

  3. Nicotine inhibits collagen synthesis and alkaline phosphatase activity, but stimulates DNA synthesis in osteoblast-like cells

    SciTech Connect

    Ramp, W.K.; Lenz, L.G.; Galvin, R.J. )

    1991-05-01

    Use of smokeless tobacco is associated with various oral lesions including periodontal damage and alveolar bone loss. This study was performed to test the effects of nicotine on bone-forming cells at concentrations that occur in the saliva of smokeless tobacco users. Confluent cultures of osteoblast-like cells isolated from chick embryo calvariae were incubated for 2 days with nicotine added to the culture medium (25-600 micrograms/ml). Nicotine inhibited alkaline phosphatase in the cell layer and released to the medium, whereas glycolysis (as indexed by lactate production) was unaffected or slightly elevated. The effects on medium and cell layer alkaline phosphatase were concentration dependent with maximal inhibition occurring at 600 micrograms nicotine/ml. Nicotine essentially did not affect the noncollagenous protein content of the cell layer, but did inhibit collagen synthesis (hydroxylation of ({sup 3}H)proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of ({sup 3}H)hydroxyproline to the medium was also decreased in a dose-dependent manner, as was the collagenase-digestible protein for both the medium and cell layer. In contrast, DNA synthesis (incorporation of ({sup 3}H)thymidine) was more than doubled by the alkaloid, whereas total DNA content was slightly inhibited at 600 micrograms/ml, suggesting stimulated cell turnover. Morphologic changes occurred in nicotine-treated cells including rounding up, detachment, and the occurrence of numerous large vacuoles. These results suggest that steps to reduce the salivary concentration of nicotine in smokeless tobacco users might diminish damaging effects of this product on alveolar bone.

  4. Therapeutic touch affects DNA synthesis and mineralization of human osteoblasts in culture.

    PubMed

    Jhaveri, Ankur; Walsh, Stephen J; Wang, Yatzen; McCarthy, MaryBeth; Gronowicz, Gloria

    2008-11-01

    Complementary and alternative medicine (CAM) techniques are commonly used in hospitals and private medical facilities; however, the effectiveness of many of these practices has not been thoroughly studied in a scientific manner. Developed by Dr. Dolores Krieger and Dora Kunz, Therapeutic Touch is one of these CAM practices and is a highly disciplined five-step process by which a practitioner can generate energy through their hands to promote healing. There are numerous clinical studies on the effects of TT but few in vitro studies. Our purpose was to determine if Therapeutic Touch had any effect on osteoblast proliferation, differentiation, and mineralization in vitro. TT was performed twice a week for 10 min each on human osteoblasts (HOBs) and on an osteosarcoma-derived cell line, SaOs-2. No significant differences were found in DNA synthesis, assayed by [(3)H]-thymidine incorporation at 1 or 2 weeks for SaOs-2 or 1 week for HOBs. However, after four TT treatments in 2 weeks, TT significantly (p = 0.03) increased HOB DNA synthesis compared to controls. Immunocytochemistry for Proliferating Cell Nuclear Antigen (PCNA) confirmed these data. At 2 weeks in differentiation medium, TT significantly increased mineralization in HOBs (p = 0.016) and decreased mineralization in SaOs-2 (p = 0.0007), compared to controls. Additionally, Northern blot analysis indicated a TT-induced increase in mRNA expression for Type I collagen, bone sialoprotein, and alkaline phosphatase in HOBs and a decrease of these bone markers in SaOs-2 cells. In conclusion, Therapeutic Touch appears to increase human osteoblast DNA synthesis, differentiation and mineralization, and decrease differentiation and mineralization in a human osteosarcoma-derived cell line. (c) 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  5. Assessment of DNA synthesis in Islet-1{sup +} cells in the adult murine heart

    SciTech Connect

    Weinberger, Florian Mehrkens, Dennis Starbatty, Jutta Nicol, Philipp Eschenhagen, Thomas

    2015-01-02

    Highlights: • Islet-1 was expressed in the adult heart. • Islet-1-positive cells did not proliferate in the adult heart. • Sinoatrial node cells did not proliferate in the adult heart. - Abstract: Rationale: Islet-1 positive (Islet-1{sup +}) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1{sup +} cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine ({sup 3}H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of {sup 3}H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1{sup +} cells. Whereas Islet{sup −} non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1{sup +} cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.

  6. Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis.

    PubMed

    Maciąg-Dorszyńska, Monika; Węgrzyn, Grzegorz; Guzow-Krzemińska, Beata

    2014-04-01

    Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus.

  7. Enhanced unscheduled DNA synthesis in UV-irradiated human skin explants treated with T4N5 liposomes

    SciTech Connect

    Yarosh, D.B.; Kibitel, J.T.; Green, L.A.; Spinowitz, A. )

    1991-07-01

    Epidermal keratinocytes cultured from explants of skin cancer patients, including biopsies from xeroderma pigmentosum patients, were ultraviolet light-irradiated and DNA repair synthesis was measured. Repair capacity was much lower in xeroderma pigmentosum patients than in normal patients. The extent of DNA repair replication did not decline with the age of the normal patient. Treatment with T4N5 liposomes containing a DNA repair enzyme enhanced repair synthesis in both normal and xeroderma pigmentosum keratinocytes in an irradiation- and liposome-dose dependent manner. These results provide no evidence that aging people or skin cancer patients are predisposed to cutaneous malignancy by a DNA repair deficiency, but do demonstrate that T4N5 liposomes enhance DNA repair in the keratinocytes of the susceptible xeroderma pigmentosum and skin cancer population.

  8. Enhanced GSH synthesis by Bisphenol A exposure promoted DNA methylation process in the testes of adult rare minnow Gobiocypris rarus.

    PubMed

    Yuan, Cong; Zhang, Yingying; Liu, Yan; Zhang, Ting; Wang, Zaizhao

    2016-09-01

    DNA methylation is a commonly studied epigenetic modification. The mechanism of BPA on DNA methylation is poorly understood. The present study aims to explore whether GSH synthesis affects DNA methylation in the testes of adult male rare minnow Gobiocypris rarus in response to Bisphenol A (BPA). Male G. rarus was exposed to 1, 15 and 225μgL(-1) BPA for 7 days. The levels of global DNA methylation, hydrogen peroxide (H2O2) and glutathione (GSH) in the testes were analyzed. Meanwhile, the levels of enzymes involved in DNA methylation and de novo GSH synthesis, and the substrate contents for GSH production were measured. Furthermore, gene expression profiles of the corresponding genes of all studied enzymes were analyzed. Results indicated that BPA at 15 and 225μgL(-1) caused hypermethylation of global DNA in the testes. The 15μgL(-1) BPA resulted in significant decrease of ten-eleven translocation proteins (TETs) while 225μgL(-1) BPA caused significant increase of DNA methyltransferase proteins (DNMTs). Moreover, 225μgL(-1) BPA caused significant increase of H2O2 and GSH levels, and the de novo GSH synthesis was enhanced. These results indicated that the significant decrease of the level of TETs may be sufficient to cause the DNA hypermethylation by 15μgL(-1) BPA. However, the significantly increased of DNMTs contributed to the significant increase of DNA methylation levels by 225μgL(-1) BPA. Moreover, the elevated de novo GSH synthesis may promote the DNA methylation process. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Fabrication of polyurethane molecular stamps for the synthesis of DNA microarray

    NASA Astrophysics Data System (ADS)

    Liu, Zhengchun; He, Quanguo; Xiao, Pengfeng; He, Nongyao; Lu, Zuhong; Bo, Liang

    2001-10-01

    Polyurethane based on polypropylene glycol (PPG) and Toluene diisocyanate (TDI) using 3,3'-dichloride-4,4'- methylenedianiline (MOCA) as the crosslinker is presented for the first time to fabricate molecular stamps (PU stamps) for the synthesis of DNA microarray with contact procedure. The predictability of the process is achieved by utilizing commercially available starting materials. SEM analysis of the morphology of PU stamps and master showed that PU elastometer could replicate subtly the motherboard's patterns with high fidelity. It was proved from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, which guarantee the well-distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays confirmed polyurethane is an excellent material for molecular stamps.

  10. Synthesis and antiproliferative activity of some new DNA-targeted alkylating pyrroloquinolines.

    PubMed

    Ferlin, M G; Dalla Via, L; Gia, O M

    2004-02-15

    Two novel DNA-direct alkylating agents, consisting of aniline mustard linked to an angular 3H-pyrrolo[3,2-f]quinoline nucleus, were synthetized and assayed for their in vitro antiproliferative activity. Simple convergent synthesis, consisting of separate preparation of 9-chloro-3H-pyrrolo[3,2-f]quinoline and p-amino-aniline derivatives, and following their linkage by substitution reactions 8a, b and 10, yielded the corresponding diol derivatives 7b and 9. Biological properties were evaluated with respect to cell growth inhibition, ability to form cross-links with DNA, and capacity to give rise to a molecular complex with the macromolecule for 7b, 8b, 9 and 10.

  11. Synthesis, Optical Properties, and DNA Interaction of New Diquats Based on Triazolopyridines and Triazoloquinolines.

    PubMed

    Llabres-Campaner, Pedro J; Guijarro, Lluís; Giarratano, Claudia; Ballesteros-Garrido, Rafael; Zaragozá, Ramón J; Aurell, M José; García-España, Enrique; Ballesteros, Rafael; Abarca, Belén

    2017-09-18

    New diquat derivatives based on [1,2,3]triazolo[1,5-a]pyridine and [1,2,3]triazolo[1,5-a]quinoline have been synthesized in excellent yields. To evaluate the effect of the alkyl bridge length, ethane and propane dibromo alkane substrates were used for their synthesis. Theoretical calculations predicted a very small energetic barrier between the two possible enantiomers P (Ra ) and M (Sa ), which makes them very difficult to resolve. Thermal denaturation studies, UV/Visible spectroscopy, and fluorescence titrations with ct-DNA evidenced the intercalation of the quinoline derivatives in DNA. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Novel designed enediynes: molecular design, chemical synthesis, mode of cycloaromatization and guanine-specific DNA cleavage.

    PubMed

    Toshima, K; Ohta, K; Kano, T; Nakamura, T; Nakata, M; Kinoshita, M; Matsumura, S

    1996-01-01

    The molecular design and chemical synthesis of novel enediyne molecules related to the neocarzinostatin chromophore (1), and their chemical and DNA cleaving properties are described. The 10-membered enediyne triols 16-18 were effectively synthesized from xylitol (10) in a short step, and found to be quite stable when handled at room temperature. The representative and acylated enediyne 16 was cycloaromatized by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in cyclohexa-1,4-diene-benzene to give the benzenoid product 21 through a radical pathway. On the other hand, the enediyne 16 was cycloaromatized by diethylamine in dimethyl sulfoxide-Tris-HCl, pH 8.5 buffer to afford another benzenoid product 22 as a diethylamine adduct through a polar pathway. Furthermore, the enediynes 16-18 were found to exhibit guanine-specific DNA cleavage under weakly basic conditions with no additive.

  13. A chemical method for fast and sensitive detection of DNA synthesis in vivo.

    PubMed

    Salic, Adrian; Mitchison, Timothy J

    2008-02-19

    We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction ("click" chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.

  14. N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis.

    PubMed

    Taggart, David J; Dayeh, Daniel M; Fredrickson, Saul W; Suo, Zucai

    2014-10-01

    The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5'-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated -1 or -2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of -2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of -1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non

  15. Acrylamide exposure induces a delayed unscheduled DNA synthesis in germ cells of male mice that is correlated with the temporal pattern of adduct formation in testis DNA

    SciTech Connect

    Sega, G.A.; Generoso, E.E.; Brimer, P.A. )

    1990-01-01

    A study of meiotic and postmeiotic germ-cell-stage sensitivity of male mice to induction of unscheduled DNA synthesis (UDS) by acrylamide showed that DNA repair could be detected in early spermatocytes (after the last scheduled DNA synthesis) through about mid-spermatid stages. No DNA repair could be detected in later stages. The maximum UDS response was observed 6 hr after i.p. exposure and was about 5 times greater than the response measured immediately after treatment. This is the longest delay between chemical treatment and maximum UDS response yet observed in mouse germ cells. There was a linear relationship between the UDS response and acrylamide exposure from 7.8 to 125 mg/kg. By using 14C-labeled acrylamide it was determined that the temporal pattern of adduct formation in testes DNA paralleled that of the UDS response, with maximum binding occurring 4 to 6 hr after exposure. In contrast, the temporal pattern of adduct formation in liver DNA showed maximum binding within 1 to 2 hr after exposure and was an order of magnitude greater than that found for the testis DNA.

  16. Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome.

    PubMed

    Baranovskiy, Andrey G; Babayeva, Nigar D; Zhang, Yinbo; Gu, Jianyou; Suwa, Yoshiaki; Pavlov, Youri I; Tahirov, Tahir H

    2016-05-06

    The human primosome, a 340-kilodalton complex of primase and DNA polymerase α (Polα), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases δ and ϵ. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Polα, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis.

    PubMed

    Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J

    2016-03-29

    In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion.

  18. Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis

    PubMed Central

    Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J.

    2016-01-01

    In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion. PMID:26976599

  19. Requirement of Rad5 for DNA Polymerase ζ-Dependent Translesion Synthesis in Saccharomyces cerevisiae

    PubMed Central

    Pagès, Vincent; Bresson, Anne; Acharya, Narottam; Prakash, Satya; Fuchs, Robert P.; Prakash, Louise

    2008-01-01

    In yeast, Rad6–Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases η or ζ or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities—a ubiquitin ligase that promotes Mms2–Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polζ. Using duplex plasmids carrying different site-specific DNA lesions—an abasic site, a cis–syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct—we show that Rad5 is needed for Polζ-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS. PMID:18757916

  20. Synthesis, characterization, and photoactivated DNA cleavage by copper (II)/cobalt (II) mediated macrocyclic complexes.

    PubMed

    Naik, H R Prakash; Naik, H S Bhojya; Aravinda, T; Lamani, D S

    2010-01-01

    We report the synthesis of new photonuclease consisting of two Co(II)/Cu(II) complexes of macrocyclic fused quinoline. Metal complexes are [MLX(2)], type where M = Co(II) (5), Cu(II) (6), and X = Cl, and are well characterized by elemental analysis, Fourier transform infrared spectroscopy, (1)H-NMR and electronic spectra. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of Cu(II), and more so than that of cobalt. The chemistry of ternary and binary Co(II) complexes showing efficient light induced (360 nm) DNA cleavage activity is summarized. The role of the metal in photoinduced DNA cleavage reactions is explored by designing complex molecules having macrocyclic structure. The mechanistic pathways are found to be concentration dependent on Co(II)/Cu(II) complexes and the photoexcitation energy photoredox chemistry. Highly effective DNA cleavage ability of 6 is attributed to the effective cooperation of the metal moiety.

  1. Design and synthesis of fluorescent substrates for human tyrosyl-DNA phosphodiesterase I

    PubMed Central

    Rideout, Marc C.; Raymond, Amy C.; Burgin, Alex B.

    2004-01-01

    Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that acts upon protein–DNA covalent complexes. Tdp1 hydrolyzes 3′-phosphotyrosyl bonds to generate 3′-phosphate DNA and free tyrosine in vitro. Mutations in Tdp1 have been linked to patients with spinocerebellar ataxia, and over-expression of Tdp1 results in resistance to known anti-cancer compounds. Tdp1 has been shown to be involved in double-strand break repair in yeast, and Tdp1 has also been implicated in single-strand break repair in mammalian cells. Despite the biological importance of this enzyme and the possibility that Tdp1 may be a molecular target for new anti-cancer drugs, there are very few assays available for screening inhibitor libraries or for characterizing Tdp1 function, especially under pre-steady-state conditions. Here, we report the design and synthesis of a fluorescence-based assay using oligonucleotide and nucleotide substrates containing 3′-(4-methylumbelliferone)-phosphate. These substrates are efficiently cleaved by Tdp1, generating the fluorescent 4-methylumbelliferone reporter molecule. The kinetic characteristics determined for Tdp1 using this assay are in agreement with the previously published values, and this fluorescence-based assay is validated using the standard gel-based methods. This sensitive assay is ideal for kinetic analysis of Tdp1 function and for high-throughput screening of Tdp1 inhibitory molecules. PMID:15333697

  2. Functional Role of NBS1 in Radiation Damage Response and Translesion DNA Synthesis.

    PubMed

    Saito, Yuichiro; Komatsu, Kenshi

    2015-08-20

    Nijmegen breakage syndrome (NBS) is a recessive genetic disorder characterized by increased sensitivity to ionizing radiation (IR) and a high frequency of malignancies. NBS1, a product of the mutated gene in NBS, contains several protein interaction domains in the N-terminus and C-terminus. The C-terminus of NBS1 is essential for interactions with MRE11, a homologous recombination repair nuclease, and ATM, a key player in signal transduction after the generation of DNA double-strand breaks (DSBs), which is induced by IR. Moreover, NBS1 regulates chromatin remodeling during DSB repair by histone H2B ubiquitination through binding to RNF20 at the C-terminus. Thus, NBS1 is considered as the first protein to be recruited to DSB sites, wherein it acts as a sensor or mediator of DSB damage responses. In addition to DSB response, we showed that NBS1 initiates Polη-dependent translesion DNA synthesis by recruiting RAD18 through its binding at the NBS1 C-terminus after UV exposure, and it also functions after the generation of interstrand crosslink DNA damage. Thus, NBS1 has multifunctional roles in response to DNA damage from a variety of genotoxic agents, including IR.

  3. Design, synthesis and DNA-binding study of some novel morpholine linked thiazolidinone derivatives

    NASA Astrophysics Data System (ADS)

    War, Javeed Ahmad; Srivastava, Santosh Kumar; Srivastava, Savitri Devi

    2017-02-01

    The emergence of multiple drug resistance amongst bacterial strains resulted in many clinical drugs to be ineffective. Being vulnerable to bacterial infections any lack in the development of new antimicrobial drugs could pose a serious threat to public health. Here we report design and synthesis of a novel class of morpholine linked thiazolidinone hybrid molecules. The compounds were characterized by FT-IR, NMR and HRMS techniques. Susceptibility tests showed that most of the synthesized molecules were highly active against multiple bacterial strains. Compound 3f displayed MIC values which were better than the standard drug for most of the tested strains. DNA being a well defined target for many antimicrobial drugs was probed as possible target for these synthetic molecules. DNA-binding study of 3f with sm-DNA was probed through UV-vis absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. The studies revealed that compound 3f has strong affinity towards DNA and binds at the minor groove. The docking studies revealed that the compound 3f shows preferential binding towards A/T residues.

  4. Depletion of Kupffer cells modulates ethanol-induced hepatocyte DNA synthesis in C57Bl/6 mice.

    PubMed

    Owumi, Solomon E; Corthals, Stacy M; Uwaifo, Anthony O; Kamendulis, Lisa M; Klaunig, James E

    2014-08-01

    Kupffer cells (KCs) are important in hepatic homeostasis and responses to xenobiotics. KCs are activated on interaction with endotoxin, releasing cytokines, and reactive oxygen species normally associated with increased gene expression, cellular growth, or hepatic injury. Ethanol-induced endotoxemia is one means of KC activation. We propose that KC depletion attenuates the effect of EtOH-induced endotoxemia to impact the hepatic growth response. Hepatic DNA synthesis was examined in KC competent (KC+) or KC-depleted (KC-) C57BL/6 mice fed EtOH-containing diet in the presence or absence of polyphenol-60 antioxidant. KC depletion was assessed by F4/80 antigen, and DNA synthesis was assessed by 5-bromo-2'-deoxyuridine incorporation. Tumor necrosis factor alpha (TNF-α) messenger RNA released was quantified by RT-PCR/electrophoresis. ERK1/2 phosphorylation was evaluated by Western blotting, and Nrf2 and CYP2E1protein were also assayed. Apoptosis and hepatic injury were examined by the Tunnel assay and hepatic transaminases in serum, respectively. Hepatic transaminases in serum (AST and ALT) were within normal range. Over 90% of KC was depleted by clodronate treatment. KC depletion decreased TNF-α mRNA release, ERK1/2 phosphorylation, and hepatocyte DNA synthesis. KC depletion is associated with increased numbers of apoptotic cells bodies in KC- mice. Antioxidant treatment decreased DNA synthesis, Nrf2, and CYP2E1 protein expression in EtOH-consuming mice. Our data indicate that upon ethanol exposure, KC participates in hepatic DNA synthesis and growth responses. Collectively, these observations suggest that KC depletion attenuates the downstream effect of ethanol-induced endotoxemia by reduced cytokine and reactive oxygen species production with its concomitant effect on MAPK-signaling pathway on hepatocyte DNA synthesis.

  5. Phospholipase C-gamma 1 can induce DNA synthesis by a mechanism independent of its lipase activity.

    PubMed Central

    Smith, M R; Liu, Y L; Matthews, N T; Rhee, S G; Sung, W K; Kung, H F

    1994-01-01

    Inositol phospholipid-specific phospholipase C (PLC) is involved in several signaling pathways leading to cellular growth and differentiation. Our previous studies reported the induction of DNA synthesis in quiescent NIH 3T3 cells after microinjection of PLC and the inhibition of serum- or Ras-stimulated DNA synthesis by a mixture of monoclonal antibodies to PLC-gamma 1. In the course of our investigation of anti-PLC-gamma 1 monoclonal antibodies, we found that each antibody exerts different inhibitory effects on the phosphatidylinositol-hydrolyzing activity of PLC-gamma 1 and that the inhibition of enzymatic activity does not correlate with the inhibition of DNA synthesis observed in the microinjection assay. PLC-gamma 1 with defective enzymatic activity was synthesized by substituting phenylalanine for histidine within the PLC-gamma 1 catalytic domain at amino acids 335 and 380, and mutant enzymes were expressed using a vaccinia expression system. The mutant enzymes were purified and microinjected into quiescent NIH 3T3 cells to evaluate their mitogenic activity. A moderate induction of DNA synthesis occurred after injection of mutant PLC-gamma 1. This mitogenic activity was inhibited by an antibody (alpha E 8-4) that does not significantly inhibit PLC-gamma 1 enzyme activity, which indicates that something else has to be inhibited. Furthermore, the partial induction of DNA synthesis observed with mutant PLC-gamma 1 was increased to levels seen with wild-type PLC-gamma 1 by coinjection of mutant PLC-gamma 1 with two second messengers, diacylglycerol and inositol trisphosphate. These results suggest that the mitogenic activity of PLC-gamma 1 does not exclusively result from the enzymatic activity of the lipase and that another activity inherent to the PLC-gamma 1 molecule can also induce DNA synthesis in quiescent cells. Images PMID:8022819

  6. Arachidonic acid stimulates DNA synthesis in brown preadipocytes through the activation of protein kinase C and MAPK.

    PubMed

    Garcia, Bibian; Martinez-de-Mena, Raquel; Obregon, Maria-Jesus

    2012-10-01

    Arachidonic acid (AA) is a polyunsaturated fatty acid that stimulates the proliferation of many cellular types. We studied the mitogenic potential of AA in rat brown preadipocytes in culture and the signaling pathways involved. AA is a potent mitogen which induces 4-fold DNA synthesis in brown preadipocytes. The AA mitogenic effect increases by NE addition. AA also increases the mitogenic action of different growth factor combinations. Other unsaturated and saturated fatty acids do not stimulate DNA synthesis to the same extent as AA. We analyzed the role of PKC and MEK/MAPK signaling pathways. PKC inhibition by bisindolilmaleimide I (BIS) abolishes AA and phorbol ester stimulation of DNA synthesis and reduces the mitogenic activity of different growth factors in brown preadipocytes. Brown preadipocytes in culture express PKC α, δ, ε and ζ isoforms. Pretreatment with high doses of the phorbol ester PDBu, induces downregulation of PKCs ε and δ and reproduces the effect of BIS indicating that AA-dependent induction of DNA synthesis requires PKC activity. AA also activates MEK/MAPK pathway and the inhibition of MEK activity inhibits AA stimulation of DNA synthesis and brown adipocyte proliferation. Inhibition of PKC δ by rottlerin abolishes AA-dependent stimulation of DNA synthesis and MAPK activation, whereas PKC ε inhibition does not produce any effect. In conclusion, our results identify AA as a potent mitogen for brown adipocytes and demonstrate the involvement of the PDBu-sensitive PKC δ isoform and MEK/MAPK pathway in AA-induced proliferation of brown adipocytes. Increased proliferative activity might increase the thermogenic capacity of brown fat. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

    PubMed Central

    Hirota, Kouji; Tsuda, Masataka; Mohiuddin; Tsurimoto, Toshiki; Cohen, Isadora S.; Livneh, Zvi; Kobayashi, Kaori; Narita, Takeo; Nishihara, Kana; Murai, Junko; Iwai, Shigenori; Guilbaud, Guillaume; Sale, Julian E.; Takeda, Shunichi

    2016-01-01

    The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass. PMID:27185888

  8. Adaptation and validation of DNA synthesis detection by fluorescent dye derivatization for high-throughput screening.

    PubMed

    Ranall, Max V; Gabrielli, Brian G; Gonda, Thomas J

    2010-05-01

    Cellular proliferation is fundamental to organism development, tissue renewal, and diverse disease states such as cancer. In vitro measurement of proliferation by high-throughput screening allows rapid characterization of the effects of small-molecule or genetic treatments on primary and established cell lines. Current assays that directly measure the cell cycle are not amenable to high-throughput processing and analysis. Here we report the adaptation of the chemical method for detecting DNA synthesis by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into both high-throughput liquid handling and high-content imaging analysis. We demonstrate that chemical detection of EdU incorporation is effective for high-resolution analysis and quantitation of DNA synthesis by high-content imaging. To validate this assay platform we used treatments of MCF10A cells with media supplements and pharmacological inhibitors that are known to affect cell proliferation. Treatments with specific kinase inhibitors indicate that EGF and serum stimulation employs both the mitogen extracellular kinase (MEK)/extracellular-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K)/AKT signaling networks. As described here, this method is fast, reliable, and inexpensive and yields robust data that can be easily interpreted.

  9. Promotion of role clarification in the Health Care Team Challenge.

    PubMed

    Hudson, C C; Gauvin, S; Tabanfar, R; Poffenroth, A M; Lee, J S; O'Riordan, A L

    2017-01-31

    Interprofessional collaboration has consistently been associated with positive client-care outcomes. Role clarification is one facet of interprofessional collaboration that is thought to be crucial for effective interprofessional team functioning. Given the positive outcomes associated with interprofessional collaboration, educators have begun to integrate formal interprofessional education events into healthcare curricula. The Health Care Team Challenge (HCTC) is a collaborative competition designed to promote interprofessional competencies among students in healthcare fields. The current study empirically investigated whether this event promoted role clarification among participants. Sixteen participants in five healthcare professions (occupational therapy, physiotherapy, clinical psychology, nursing, and medicine) completed two questionnaires to assess role clarification before and after participating in this event. Results indicate that participants' understanding of their own and other professions' roles improved after participating in this team activity. These results suggest that the HCTC is effective in promoting role clarification and collaboration among healthcare students.

  10. 76 FR 64813 - Electronic Prescriptions for Controlled Substances Clarification

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-19

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF JUSTICE Drug... Substances Clarification AGENCY: Drug Enforcement Administration (DEA), Department of Justice. ] ACTION.... FOR FURTHER INFORMATION, CONTACT: Imelda L. Paredes, Office of Diversion Control, Drug Enforcement...

  11. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12 Inmate request for record clarification. Where the inmate believes that his or her FBI...

  12. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12 Inmate request for record clarification. Where the inmate believes that his or her FBI...

  13. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12 Inmate request for record clarification. Where the inmate believes that his or her FBI...

  14. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12 Inmate request for record clarification. Where the inmate believes that his or her FBI...

  15. A Critique of Values: Clarification in Drug Education.

    ERIC Educational Resources Information Center

    Chng, Chwee Lye

    1980-01-01

    Values clarification is widely used in drug education programs. Criticisms are offered on the role of content in valuing, the position of ethical relativism, the danger of indoctrination, and the social pressure to conform. (Author)

  16. Protein, RNA, and DNA synthesis in cultures of skin fibroblasts from healthy subjects and patients with rheumatic diseases

    SciTech Connect

    Abakumova, O.Y.; Kutsenko, N.G.; Panasyuk, A.F.

    1985-07-01

    To study the mechanism of the lasting disturbance of fibroblast function, protein, RNA and DNA synthesis was investigated in skin fibroblasts from patients with rheumatoid arthritis (RA) and systemic scleroderma (SS). The labeled precursors used to analyze synthesis of protein, RNA, and DNA were /sup 14/C-protein hydrolysate, (/sup 14/C)uridine, and (/sup 14/C) thymidine. Stimulation was determined by measuring incorporation of (/sup 14/C)proline into fibroblast proteins. During analysis of stability of fast-labeled RNA tests were carried out to discover whether all measurable radioactivity belonged to RNA molecules.

  17. Inhibition of DNA synthesis by carvacrol in mouse myoblast cells bearing a human N-RAS oncogene.

    PubMed

    Zeytinoglu, H; Incesu, Z; Baser, K H C

    2003-05-01

    Monoterpenes are dietary components found in the essential oils of a wide variety of plants. A number of these monoterpenes have antitumor activity. We have investigated the effects of carvacrol obtained by fractional distillation of Origanum onites L. essential oil, on DNA synthesis of N-ras transformed myoblast cells, CO25. Incubation of the cells with different doses of carvacrol prevented DNA synthesis in the growth medium and ras-activating medium, which contains dexamethasone. This result demonstrates that carvacrol inhibits growth of myoblast cells even after activation of mutated N-ras oncogene, suggesting the possibility that carvacrol may find application in cancer therapy.

  18. DNA Methyltransferase protein synthesis is reduced in CXXC finger protein 1-deficient embryonic stem cells.

    PubMed

    Butler, Jill S; Palam, Lakshmi R; Tate, Courtney M; Sanford, Jeremy R; Wek, Ronald C; Skalnik, David G

    2009-05-01

    CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is required for embryogenesis. CFP1 is also a component of the Setd1A and Setd1B histone H3K4 methyltransferase complexes. Murine embryonic stem (ES) cells lacking CFP1 fail to differentiate, and exhibit a 70% reduction in global genomic cytosine methylation and a 50% reduction in DNA methyltransferase (DNMT1) protein and activity. This study investigated the underlying mechanism for reduced DNMT1 expression in CFP1-deficient ES cells. DNMT1 transcript levels were significantly elevated in ES cells lacking CFP1, despite the observed reduction in DNMT1 protein levels. To address the posttranscriptional mechanisms by which CFP1 regulates DNMT1 protein activity, pulse/chase analyses were carried out, demonstrating a modest reduction in DNMT1 protein half-life in CFP1-deficient ES cells. Additionally, global protein synthesis was decreased in ES cells lacking CFP1, contributing to a reduction in the synthesis of DNMT1 protein. ES cells lacking CFP1 were found to contain elevated levels of phosphorylated eIF2alpha, and an accompanying reduction in translation initiation as revealed by a lower level of polyribosomes. These results reveal a novel role for CFP1 in the regulation of translation initiation, and indicate that loss of CFP1 function leads to decreased DNMT1 protein synthesis and half-life.

  19. Nurse attitudes toward childbirth: a concept clarification.

    PubMed

    Levine, Martha S; Lowe, Nancy K

    2014-01-01

    To clarify the concept of "nurse attitudes toward childbirth." It has been suggested that the international trend of escalating cesarean birth rates can be attributed to attitudes that perceive childbirth as an illness. Nurses' attitudes about childbirth direct their nursing care and may influence patient outcomes like cesarean birth. However, the concept "nurse attitudes toward childbirth" must be clarified to inform future research. An English-language literature review, from 1990 to present, was performed using CINAHL, PubMed, and Ovid. Norris's model of concept clarification was used. Although the nursing literature poorly defined "nurse attitudes" and rarely used a conceptual framework, the discipline of psychology has been refining this concept for over 40 years. Psychologists have established that attitude can predict behavior as demonstrated through testing of the theory of planned behavior. Various types of "nurse attitudes toward childbirth" were identified through our literature review, and five central beliefs were noted. This resulted in the development of a preliminary model using theory of planned behavior as a foundation. Finally, potential research hypotheses were generated. This paper clarifies "nurse attitudes toward childbirth" and supports its use for research. Nurse scholars have demonstrated that labor and delivery nurses do have individual attitudes toward childbirth, and the measurement of these attitudes may predict nursing care intentions and behavior. This concept is appropriate, important, and may be used as a means of exploring relationships between nursing care and the rising primary cesarean birth rate. © 2013 Wiley Periodicals, Inc.

  20. Application of the ICRP clarification of the tritium metabolic model.

    PubMed

    Potter, Charles A

    2004-10-01

    In 2001, the International Commission on Radiological Protection published a clarification to the model for tritium metabolism. This clarification described the use of the gastrointestinal tract model, respiratory tract model, and transfer compartment in calculations of tritium metabolism. This information was used to derive intake retention fractions for tritium and tritium compounds including tritiated water, organically bound tritium, and tritides. In addition, dose coefficients were derived for tritide compounds including general categories described by the ICRP and some specific compounds.

  1. An integrative model links multiple inputs and signaling pathways to the onset of DNA synthesis in hepatocytes

    PubMed Central

    Huard, Jérémy; Mueller, Stephanie; Gilles, Ernst D; Klingmüller, Ursula; Klamt, Steffen

    2012-01-01

    During liver regeneration, quiescent hepatocytes re-enter the cell cycle to proliferate and compensate for lost tissue. Multiple signals including hepatocyte growth factor, epidermal growth factor, tumor necrosis factor α, interleukin-6, insulin and transforming growth factor β orchestrate these responses and are integrated during the G1 phase of the cell cycle. To investigate how these inputs influence DNA synthesis as a measure for proliferation, we established a large-scale integrated logical model connecting multiple signaling pathways and the cell cycle. We constructed our model based upon established literature knowledge, and successively improved and validated its structure using hepatocyte-specific literature as well as experimental DNA synthesis data. Model analyses showed that activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways was sufficient and necessary for triggering DNA synthesis. In addition, we identified key species in these pathways that mediate DNA replication. Our model predicted oncogenic mutations that were compared with the COSMIC database, and proposed intervention targets to block hepatocyte growth factor-induced DNA synthesis, which we validated experimentally. Our integrative approach demonstrates that, despite the complexity and size of the underlying interlaced network, logical modeling enables an integrative understanding of signaling-controlled proliferation at the cellular level, and thus can provide intervention strategies for distinct perturbation scenarios at various regulatory levels. PMID:22443451

  2. Induction of unscheduled DNA synthesis in suspensions of rat hepatocytes by an environmental toxicant, 3,3'4,4'-tetrachloroazobenzene.

    PubMed

    Hsia, M T; Kreamer, B L

    1979-04-01

    Unscheduled DNA synthesis was induced by 3,3'4,4'-tetrachloroazobenzene (TCAB)) in freshly isolated suspensions of rat hepatocytes. A dose-dependent response was demonstrated. Hepatocellular DNA was obtained after the chloroform-isoamyl alchohol-phenol extraction of the isolated nuclei. The induction of unscheduled DNA synthesis was measured by the incorporation of [3H]-thymidine in the presence of hydroxyurea as determined by the scintillation counting assay. DNA repair data obtained in this study on benzo[a]pyrene and methyl methanesulfonate are comparable to a previous report using primary cultures of hepatocytes and cesium chloride gradients. Hence, the present method offers promise as a rapid and sensitive screen for chemical carcinogens.

  3. Synergistic template-free synthesis of dsDNA by Thermococcus nautili primase PolpTN2, DNA polymerase PolB, and pTN2 helicase.

    PubMed

    Béguin, Pierre; Gill, Sukhvinder; Charpin, Nicole; Forterre, Patrick

    2015-01-01

    A combination of three enzymes from the hyperthermophilic archaeon Thermococcus nautili, DNA primase PolpTN2, DNA polymerase PolB, and pTN2 DNA helicase, was found to synthesize up to 300-400 ng/µl dsDNA from deoxynucleotide triphosphates in less than 30 min in the absence of added template DNA and oligonucleotide primer. The reaction did not occur below 64 °C. No synthesis was observed if PolpTN2 or PolB were left out; helicase was not essential but accelerated the reaction. The DNA synthesized consisted of highly reiterated palindromic sequences reaching up to more that 10 kb. Sequence analysis of three independent reaction products synthesized at different temperatures showed that the palindromes shared a common pentanucleotide core, suggesting that random nucleic acid fragments were not responsible for priming the reaction. When enzymes were added sequentially, preincubation with primase plus helicase followed by PolB led to a shorter delay before the onset of the reaction as compared to preincubation with PolB plus helicase followed by primase. This suggests that the primase generates seeds that are subsequently amplified and elongated in synergy with PolB by a mechanism involving hairpin formation and slippage synthesis.

  4. Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis.

    PubMed

    Gilbert, M Thomas P; Binladen, Jonas; Miller, Webb; Wiuf, Carsten; Willerslev, Eske; Poinar, Hendrik; Carlson, John E; Leebens-Mack, James H; Schuster, Stephan C

    2007-01-01

    Although ancient DNA (aDNA) miscoding lesions have been studied since the earliest days of the field, their nature remains a source of debate. A variety of conflicting hypotheses exist about which miscoding lesions constitute true aDNA damage as opposed to PCR polymerase amplification error. Furthermore, considerable disagreement and speculation exists on which specific damage events underlie observed miscoding lesions. The root of the problem is that it has previously been difficult to assemble sufficient data to test the hypotheses, and near-impossible to accurately determine the specific strand of origin of observed damage events. With the advent of emulsion-based clonal amplification (emPCR) and the sequencing-by-synthesis technology this has changed. In this paper we demonstrate how data produced on the Roche GS20 genome sequencer can determine miscoding lesion strands of origin, and subsequently be interpreted to enable characterization of the aDNA damage behind the observed phenotypes. Through comparative analyses on 390,965 bp of modern chloroplast and 131,474 bp of ancient woolly mammoth GS20 sequence data we conclusively demonstrate that in this sample at least, a permafrost preserved specimen, Type 2 (cytosine-->thymine/guanine-->adenine) miscoding lesions represent the overwhelming majority of damage-derived miscoding lesions. Additionally, we show that an as yet unidentified guanine-->adenine analogue modification, not the conventionally argued cytosine-->uracil deamination, underpins a significant proportion of Type 2 damage. How widespread these implications are for aDNA will become apparent as future studies analyse data recovered from a wider range of substrates.

  5. DNA damage and metallothionein synthesis in human hepatoma cells (HepG2) exposed to cadmium.

    PubMed

    Fatur, T; Tusek, M; Falnoga, I; Scancar, J; Lah, T T; Filipic, M

    2002-08-01

    Cadmium is an important heavy metal environmental toxicant, which is classified as a human carcinogen. The comet assay was used to evaluate the levels of DNA damage in a metabolically competent HepG2 cell line after treatment with low, non-cytotoxic and physiologically relevant concentrations of cadmium, alone and in combination with the dietary mutagen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and with the environmental mutagen benzo[a]pyrene (B(a)P). After exposure of the cells to 10, 100 and 1000 nM CdCl(2), a dose- and time-dependent increase of DNA damage was detected. Maximal damage was found after 12 h of treatment, but declined with further incubation with CdCl(2). The increased synthesis of metallothioneins on exposure to CdCl(2) up to 12 h suggests that they are responsible for the adaptation of HepG2 cells to the DNA damaging effects of CdCl(2). Co-treatment of the cells with CdCl(2) (10-1000 nM) and IQ (300 microM) induced a dose-dependent increase of DNA damage compared to cells treated with IQ alone. Co-genotoxic activity was also observed by increased formation of micronuclei in cells exposed to IQ and 1000 nM CdCl(2); at this concentration, CdCl(2) alone also induced micronuclei in HepG2 cells. Our results support the hypothesis that direct and indirect mechanisms are involved in cadmium-induced DNA damage.

  6. Enhancement of major histocompatibility class I protein synthesis by DNA damage in cultured human fibroblasts and keratinocytes

    SciTech Connect

    Lambert, M.E.; Ronai, Z.A.; Weinstein, I.B.; Garrels, J.I.

    1989-02-01

    Exposure of primary human fibroblasts or simian virus 40-transformed human keratinocytes to several different classes of DNA damage, including UV light C (254 nm), resulted in a rapid increase in the expression of human major histocompatibility class I (MHC-I) proteins. MHC-I induction was also detected after exposure to low doses of the protein synthesis inhibitor cycloheximide, suggesting that MHC-I induction by DNA damage may be a component in a derepressible cellular SOS pathway.

  7. [Clarification on publications concerning the synthesis of acetylsalicylic acid].

    PubMed

    Lafont, O

    1996-01-01

    Charles Frédéric Gerhardt (1816-1856) mentioned in his Traité de chimie Organique (1854) a publication, in French (realized in 1852 but published in 1853) entitled "Researches on anhydrous organic acids" in which, was reported the reaction of sodium salicylate with acetyl chloride. He thought that the reaction product was an acid anhydride, but obtained really crude acetylsalicylic acid. Later on, but also in 1853, a publication in german, by the same author related the same experiments. Surprisingly only the second publication has been mentioned in most of the historical studies on the subject. Acetyl salicylic acid was identified and synthesised in 1859 by von Gilm by another method and the product obtained by Gerhardt was identified to it in 1869.

  8. Nascent DNA processing by RecJ favors lesion repair over translesion synthesis at arrested replication forks in Escherichia coli.

    PubMed

    Courcelle, Charmain T; Chow, Kin-Hoe; Casey, Andrew; Courcelle, Justin

    2006-06-13

    DNA lesions that arrest replication can lead to rearrangements, mutations, or lethality when not processed accurately. After UV-induced DNA damage in Escherichia coli, RecA and several recF pathway proteins are thought to process arrested replication forks and ensure that replication resumes accurately. Here, we show that the RecJ nuclease and RecQ helicase, which partially degrade the nascent DNA at blocked replication forks, are required for the rapid recovery of DNA synthesis and prevent the potentially mutagenic bypass of UV lesions. In the absence of RecJ, or to a lesser extent RecQ, the recovery of replication is significantly delayed, and both the recovery and cell survival become dependent on translesion synthesis by polymerase V. The RecJ-mediated processing is proposed to restore the region containing the lesion to a form that allows repair enzymes to remove the blocking lesion and DNA synthesis to resume. In the absence of nascent DNA processing, polymerase V can synthesize past the lesion to prevent lethality, although this occurs with slower kinetics and a higher frequency of mutagenesis.

  9. Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo

    SciTech Connect

    Heintz, N.H.; Stillman, B.W.

    1988-05-01

    A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4/sup 0/C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases.

  10. Tim-Tipin dysfunction creates an indispensible reliance on the ATR-Chk1 pathway for continued DNA synthesis.

    PubMed

    Smith, Kevin D; Fu, Michael A; Brown, Eric J

    2009-10-05

    The Tim (Timeless)-Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim-Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR-Chk1 pathway engagement. We now demonstrate that Tim-Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR-Chk1 pathway in Tim-Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim-Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim-Tipin dysfunction. Together, these experiments indicate that the Tim-Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR-Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

  11. A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses.

    PubMed

    Hasegawa, Mayu; Iwai, Shigenori; Kuraoka, Isao

    2010-06-17

    Individuals with inherited xeroderma pigmentosum (XP) disorder and Cockayne syndrome (CS) are deficient in nucleotide excision repair and experience hypersensitivity to sunlight. Although there are several diagnostic assays for these disorders, the recovery of RNA synthesis (RRS) assay that can discriminate between XP cells and CS cells is very laborious. Here, we report on a novel non-radioisotope RRS assay that uses bromouridine (a uridine analog) as an alternative to (3)H-uridine. This assay can easily detect RNA polymerase I transcription in nucleoli and RNA polymerase II transcription in nuclei. The non-RI RSS assay also can rapidly detect normal RRS activity in HeLa cells. Thus, this assay is useful as a novel and easy technique for CS diagnosis. Because RRS is thought to be related to transcription-coupled DNA repair, which is triggered by the blockage of transcriptional machinery by DNA lesions, this assay may be of use for analysis of DNA repair, transcription, and/or genetic toxicity. Copyright 2010 Elsevier B.V. All rights reserved.

  12. Synthesis of full length and truncated microcin B17 analogues as DNA gyrase poisons.

    PubMed

    Thompson, Robert E; Collin, Frédéric; Maxwell, Anthony; Jolliffe, Katrina A; Payne, Richard J

    2014-03-14

    Microcin B17 (MccB17) is a post-translationally modified peptide containing thiazole and oxazole heterocycles that interrupt the peptide backbone. MccB17 is capable of poisoning DNA gyrase through stabilization of the gyrase-DNA cleavage complex and has therefore attracted significant attention. Using a combination of Fmoc-strategy solid-phase peptide synthesis and solution-phase fragment assembly we have prepared a library of full-length and truncated MccB17 analogues to investigate key structural requirements for gyrase-poisoning activity. Synthetic peptides lacking the glycine-rich N-terminal portion of the full-length sequence showed strong stabilization of the gyrase-DNA cleavage complex with increased potency relative to the full-length sequences. This truncation, however, led to a decrease in antibacterial activity of these analogues relative to their full-length counterparts indicating a potential role of the N-terminal region of the natural product for cellular uptake.

  13. [Autoradiographic investigations on the effect of city smog extract on DNA synthesis and cell cycle of mammalian cells in vitro. I. Effect of city smog extract on DNA synthesis of kidney- and embryonic cells of the golden hamster in vitro (author's transl)].

    PubMed

    Krampitz, G; Seemayer, N

    1979-05-01

    We analysed the effect of city smog extract from Düsseldorf on DNA synthesis of mammalian cells in vitro. Airborne dust was extracted with aceton and thereafter transferred to dimethylsulfoxide. Dosage was calculated according to the benzo(a)pyrene content. We used logarithmically growing cultures of hamster kidney and embryonic cells. DNA synthesis was determined autoradiographically by incorporation of 3H-Thymidine. We found that city smog extract exerted a dose-dependent cytotoxic effect leading to a decrease of DNA synthesizing cells. High concentrations of city smog extract induced cell necrosis and suppressed DNA synthesis completely. Moderate doses of extract caused a dose-dependent, but temporary cessation of DNA synthesis. Cells resumed DNA synthesis after a certain delay. Low concentrations of city smog extract induced no detectable effects. Inhibition of DNA synthesis was evident already one hour after addition of extract. Therefore a direct effect on DNA metabolism could be supposed. Furthermore, exposed cultures demonstrated a delay in entrance of new cells into the DNA synthesis. Alterations in DNA synthesis could be of great importance for carcinogenesis, especially if we take in consideration the content of carcinogens in city smog extract.

  14. Unscheduled deoxyribonucleic acid (DNA) synthesis assays for toxicological studies. May 1977-March 1990 (A Bibliography from the NTIS data base). Report for May 1977-March 1990

    SciTech Connect

    Not Available

    1990-04-01

    This bibliography contains citations concerning the unscheduled DNA synthesis (UDS) assay for toxicological studies. UDS assays provide very sensitive measures of damage to DNA by detecting induction of DNA synthesis in non-S-phase cells. UDS toxicological studies analyzing gamma radiation, drugs, pesticides, nerve gas, jet engine fuels, ultraviolet light, chlorated organic compounds, and aromatic compounds are discussed. UDS studies using both human and animal tissue cultures are described. (Contains 57 citations fully indexed and including a title list.)

  15. Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction.

    PubMed

    Zhang, Yinbo; Baranovskiy, Andrey G; Tahirov, Emin T; Tahirov, Tahir H; Pavlov, Youri I

    2016-07-01

    DNA polymerases (pols) are sophisticated protein machines operating in the replication, repair and recombination of genetic material in the complex environment of the cell. DNA pol reactions require at least two divalent metal ions for the phosphodiester bond formation. We explore two understudied roles of metals in pol transactions with emphasis on polα, a crucial enzyme in the initiation of DNA synthesis. We present evidence that the combination of many factors, including the structure of the template/primer, the identity of the metal, the metal turnover in the pol active site, and the influence of the concentration of nucleoside triphosphates, affect DNA pol synthesis. On the poly-dT70 template, the increase of Mg(2+) concentration within the range typically used for pol reactions led to the severe loss of the ability of pol to extend DNA primers and led to a decline in DNA product sizes when extending RNA primers, simulating the effect of "counting" of the number of nucleotides in nascent primers by polα. We suggest that a high Mg(2+) concentration promotes the dynamic formation of unconventional DNA structure(s), thus limiting the apparent processivity of the enzyme. Next, we found that Zn(2+) supported robust polα reactions when the concentration of nucleotides was above the concentration of ions; however, there was only one nucleotide incorporation by the Klenow fragment of DNA pol I. Zn(2+) drastically inhibited polα, but had no effect on Klenow, when Mg(2+) was also present. It is possible that Zn(2+) perturbs metal-mediated transactions in pol active site, for example affecting the step of pyrophosphate removal at the end of each pol cycle necessary for continuation of polymerization. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. UAP56 is a novel interacting partner of Bcr in regulating vascular smooth muscle cell DNA synthesis

    SciTech Connect

    Sahni, Abha; Wang, Nadan; Alexis, Jeffrey D.

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer UAP56 is an important regulator of DNA synthesis in vascular smooth muscle cells. Black-Right-Pointing-Pointer UAP56 binds to Bcr. Black-Right-Pointing-Pointer Interaction between Bcr and UAP56 is critical for Bcr induced DNA synthesis. -- Abstract: Bcr is a serine/threonine kinase that is a critical regulator of vascular smooth muscle cell inflammation and proliferation. We have previously demonstrated that Bcr acts in part via phosphorylation and inhibition of PPAR{gamma}. We have identified the RNA helicase UAP56 as another substrate of Bcr. In this report we demonstrate that knockdown of UAP56 blocks Bcr induced DNA synthesis in vascular smooth muscle cells (VSMC). We also found that over expression of Bcr increased the expression of cyclin E and decreased the expression of p27. Knockdown of UAP56 reversed the effect of Bcr on cyclin E and p27 expression. Furthermore, we found that Bcr binds to UAP56 and demonstrate that binding of UAP56 to Bcr is critical for Bcr induced DNA synthesis in VSMC. Our data identify UAP56 as an important binding partner of Bcr and a novel target for inhibiting vascular smooth muscle cell proliferation.

  17. SYNTHESIS, IN VITRO METABOLISM, MUTAGENICITY, AND DNA-ADDUCTION OF NAPHTHO[1,2-E]PYRENE

    EPA Science Inventory

    SYNTHESIS, IN V1TRO METABOLISM, MUTAGENICITY , AND DNA-ADDUCnON OF NAPHTHO[l ,2-e ]PYRENE

    Literature data, although limited, underscore the contribution of C24HI4 polycyclic aromatic hydrocarbons to the biological activity of the extracts of complex environmental samples....

  18. SYNTHESIS, IN VITRO METABOLISM, MUTAGENICITY, AND DNA-ADDUCTION OF NAPHTHO[1,2-E]PYRENE

    EPA Science Inventory

    SYNTHESIS, IN V1TRO METABOLISM, MUTAGENICITY , AND DNA-ADDUCnON OF NAPHTHO[l ,2-e ]PYRENE

    Literature data, although limited, underscore the contribution of C24HI4 polycyclic aromatic hydrocarbons to the biological activity of the extracts of complex environmental samples....

  19. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

    1999-01-01

    Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

  20. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: the importance of the A structure

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; Politis, P. K.; Van Aerschot, A.; Busson, R.; Herdewijn, P.; Orgel, L. E.; Bada, J. L. (Principal Investigator); Dolan, M. (Principal Investigator)

    1999-01-01

    Hexitol nucleic acid (HNA) is an analogue of DNA containing the standard nucleoside bases, but with a phosphorylated 1,5-anhydrohexitol backbone. HNA oligomers form duplexes having the nucleic acid A structure with complementary DNA or RNA oligomers. The HNA decacytidylate oligomer is an efficient template for the oligomerization of the 5'-phosphoroimidazolides of guanosine or deoxyguanosine. Comparison of the oligomerization efficiencies on HNA, RNA, and DNA decacytidylate templates under various conditions suggests strongly that only nucleic acid double helices with the A structure support efficient template-directed synthesis when 5'-phosphoroimidazolides of nucleosides are used as substrates.

  1. Inhibition of poly(ADP-ribose) synthesis may affect DNA repair prior to ligation.

    PubMed

    Ireland, C M; Stewart, B W

    1987-01-01

    The effects of modification of poly(ADP-ribosyl)ation reactions have been examined in normal (F107) and ataxia telangiectasia (AT23) fibroblasts following damage by methyl methanesulphonate (MMS) and u.v. light. The technique of benzoylated DEAE (BD)-cellulose chromatography was utilized to estimate both the extent and nature of the damage to DNA induced by these agents and to examine the effects of an inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide (3AB), on these parameters. Single strand breakage, determined by nucleoid sedimentation, and levels of poly ADP(ribose) synthesis were monitored. Increase in the proportion of DNA containing single-stranded regions, as measured by stepwise elution from BD-cellulose, was observed following MMS damage in both cell types. In the presence of 3AB, a further accumulation of DNA containing single-stranded regions occurred, with the effect being more prominent in AT23 fibroblasts. U.v. light damage did not induce increased binding to BD-cellulose in normal cells, and the increase observed in AT23 cells was much less than that seen following alkylation damage. Examination of the nature of single-stranded damage by caffeine gradient elution from BD-cellulose following MMS treatment revealed discrete structural lesions, which were enhanced in the presence of 3AB. A similar effect was exerted by arabinofuranosyl cytosine. The behaviour of these intermediates, which could be associated with repair, was not in accord with the suggestion that 3AB inhibits only the ligation stage of the repair process. Our results suggest that specific intermediate stages in DNA repair are sensitive to 3AB, and it seems likely that these stages occur prior to ligation.

  2. Synthesis and spectroscopic studies of the aminoglycoside (neomycin)--perylene conjugate binding to human telomeric DNA.

    PubMed

    Xue, Liang; Ranjan, Nihar; Arya, Dev P

    2011-04-12

    Synthesis of a novel perylene-neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5'-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA-RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV-vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand-quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4.

  3. Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases

    SciTech Connect

    Moore, P.D.; Bose, K.K.; Rabkin, S.D.; Strauss, B.S.

    1981-01-01

    In vitro DNA synthesis on a phi X174 template primed with a restriction fragment and catalyzed by the Escherichia coli DNA polymerase I large (Klenow) fragment (pol I) terminates at the nucleotide preceding a site that has been altered by ultraviolet irradiation or treatment with N-acetylaminofluorene. Termination on ultraviolet-irradiated templates is similar when synthesis is catalyzed by E. coli DNA polymerase III holoenzyme (pol III), phage T4DNA polymerase a polymerase ..cap alpha.. from human lymphoma cells, or avian myeloblastosis virus reverse transcriptase. 3' ..-->.. 5' exonuclease activity cannot be detected in the reverse transcriptase and DNA polymerase ..cap alpha.. preparations. On N-acetylaminofluorene templates, pol I, pol III, and T4 polymerase reactions terminate immediately preceding the lesion, whereas reverse transcriptase-catalyzed reactions and, at some positions in the sequence, polymerase ..cap alpha..-catalyzed reactions terminate at the site of the lesion. Substitution of Mn/sup 2 +/ for Mg/sup 2 +/ changes the pattern of pol I-catalyzed termination sites. The data sugest that termination is a complicated process that does not depend exclusively on the 3' ..-->.. 5' exonuclease activity associated with many polymerases.

  4. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    PubMed Central

    Maddocks, Oliver D.K.; Labuschagne, Christiaan F.; Adams, Peter D.; Vousden, Karen H.

    2016-01-01

    Summary Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

  5. The structure-based design, synthesis and biological evaluation of DNA-binding bisintercalating bisanthrapyrazole anticancer compounds

    PubMed Central

    Hasinoff, Brian B.; Liang, Hong; Wu, Xing; Guziec, Lynn J.; Guziec, Frank S.; Marshall, Kyle; Yalowich, Jack C.

    2008-01-01

    Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. In order to find stronger DNA binding and more potent cytotoxic compounds, a series of ester-coupled bisanthrapyrazole derivatives of 7-chloro-2-[2-[(2-hydroxyethyl)methylamino]ethyl]anthra[1,9-cd]pyrazol-6(2H)-one (AP9) were designed and evaluated by molecular docking techniques. Because the anthrapyrazoles are unable to be reductively activated like doxorubicin and other anthracyclines, they should not be cardiotoxic like the anthracyclines. Based on the docking scores of a series of bisanthrapyrazoles with different numbers of methylene linkers (n) that were docked into an X-ray structure of double-stranded DNA, five bisanthrapyrazoles (n = 1 to 5) were selected for synthesis and physical and biological evaluation. The synthesized compounds were evaluated for DNA binding and bisintercalation by measuring the DNA melting temperature increase, for growth inhibitory effects on the human erythroleukemic K562 cell line, and for DNA topoisomerase IIα-mediated cleavage of DNA and inhibition of DNA topoisomerase IIα decatenation activities. The results suggest that the bisanthrapyrazoles with n = 2 to 5 formed bisintercalation complexes with DNA. In conclusion, a novel group of bisintercalating anthrapyrazole compounds have been designed, synthesized and biologically evaluated as possible anticancer agents. PMID:18258442

  6. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis.

    PubMed

    Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy; Bhowmick, Rahul; Hickson, Ian D; Kanemaki, Masato T

    2017-04-15

    DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8-9 complex, a paralog of the MCM2-7 replicative helicase. We show that MCM8-9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8-9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8-9 as an alternative replicative helicase. © 2017 Natsume et al.; Published by Cold Spring Harbor Laboratory Press.

  7. The RNA accordion model for template positioning by telomerase RNA during telomeric DNA synthesis

    PubMed Central

    Berman, Andrea J.; Akiyama, Benjamin M.; Stone, Michael D.; Cech, Thomas R.

    2011-01-01

    Telomerase is a ribonucleoprotein (RNP) enzyme that maintains the ends of linear eukaryotic chromosomes and whose activation is a hallmark of 90% of all cancers. This RNP minimally contains a reverse transcriptase protein subunit (TERT) that catalyzes telomeric DNA synthesis and an RNA subunit (TER) that has templating, architectural and protein-scaffolding roles. Telomerase is unique among polymerases in that it synthesizes multiple copies of the template on the 3′ end of a primer following a single binding event, a process known as repeat addition processivity (RAP). Using biochemical assays and single-molecule Förster resonance energy transfer (smFRET) experiments on Tetrahymena thermophila telomerase, we now directly demonstrate that TER contributes to template positioning within the active site and to the template translocation required for RAP. We propose that the single-stranded RNA elements flanking the template act as a molecular accordion, undergoing reciprocal extension and compaction during telomerase translocation. PMID:22101935

  8. A role for PCNA2 in translesion synthesis by Arabidopsis thaliana DNA polymerase eta.

    PubMed

    Kunz, Bernard A

    2008-10-01

    Eukaryotic DNA polymerase eta (Poleta) confers ultraviolet (UV) resistance by catalyzing translesion synthesis (TLS) past UV photoproducts. Poleta has been studied extensively in budding yeast and mammalian cells, where its interaction with monoubiquitylated proliferating cell nuclear antigen (PCNA) is necessary for its biological activity. Recently, in collaboration with other investigators, our laboratory demonstrated that Arabidopsis thaliana Poleta is required for UV resistance in plants. Furthermore, the purified enzyme can perform TLS opposite a cyclobutane pyrimidine dimer and interacts with PCNA. Intriguingly, the biological activity of Poleta in a heterologous yeast assay depends on co-expression with Arabidopsis PCNA2 and Poleta sequences implicated in binding PCNA or ubiquitin. We suggest that interaction of Arabidopsis Poleta with ubiquitylated PCNA2 is required for TLS past UV photoproducts by Poleta.

  9. Quantification of histoautoradiographic evidence of DNA repair synthesis in the liver.

    PubMed

    Hochmann, J; Stambergová, H

    1988-01-01

    Histoautoradiography was used to detect dimethylnitrosamine-induced 3H-thymidine incorporation in vivo into G phase hepatocytes. A description of the standard procedure for counting the grains as well as the mode of mathematical evaluation is presented. The results exhibited higher sensitivity than those in the investigation of the DNA repair synthesis by means of a scintillation counter using the method of detection of hydroxyurea-resistant incorporation of 3H-thymidine. Thus it was possible to simplify the investigation by lowering the number of evaluated cells. A suitable compromise between precision and laboriousness will probably be achieved by counting 20 hepatocytes per animal. In case that there are striking differences between the experimental and the control group, a qualitative conclusion may be drawn even without counting the grains.

  10. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression.

    PubMed Central

    Kim, S Y; Byrn, R; Groopman, J; Baltimore, D

    1989-01-01

    The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes. Images PMID:2760980

  11. An Integrated System for DNA Sequencing by Synthesis Using Novel Nucleotide Analogues

    PubMed Central

    Guo, Jia; Yu, Lin; Turro, Nicholas J.; Ju, Jingyue

    2010-01-01

    Conspectus The Human Genome Project has concluded, but its successful completion has increased, rather than decreased, the need for high-throughput DNA sequencing technologies. The possibility of clinically screening a full genome for an individual's mutations offers tremendous benefits, both for pursuing personalized medicine as well as uncovering the genomic contributions to diseases. The Sanger sequencing method—although enormously productive for more than 30 years—requires an electrophoretic separation step that, unfortunately, remains a key technical obstacle for achieving economically acceptable full-genome results. Alternative sequencing approaches thus focus on innovations that can reduce costs. The DNA sequencing by synthesis (SBS) approach has shown great promise as a new sequencing platform, with particular progress reported recently. The general fluorescent SBS approach involves (i) incorporation of nucleotide analogs bearing fluorescent reporters, (ii) identification of the incorporated nucleotide by its fluorescent emissions, and (iii) cleavage of the fluorophore, along with the reinitiation of the polymerase reaction for continuing sequence determination. In this Account, we review the construction of a DNA-immobilized chip and the development of novel nucleotide reporters for the SBS sequencing platform. Click chemistry, with its high selectivity and coupling efficiency, was explored for surface immobilization of DNA. The first generation (G-1) modified nucleotides for SBS feature a small chemical moiety capping the 3′-OH and a fluorophore tethered to the base through a chemically cleavable linker; the design ensures that the nucleotide reporters are good substrates for the polymerase. The 3′-capping moiety and the fluorophore on the DNA extension products, generated by the incorporation of the G-1 modified nucleotides, are cleaved simultaneously to reinitiate the polymerase reaction. The sequence of a DNA template immobilized on a surface

  12. Repeated allergen exposure of sensitized Brown-Norway rats induces airway cell DNA synthesis and remodelling.

    PubMed

    Salmon, M; Walsh, D A; Koto, H; Barnes, P J; Chung, K F

    1999-09-01

    Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall. This study assessed the effect of repeated allergen exposure on ASM and epithelial cell deoxyribonucleic acid (DNA) synthesis, cell recruitment and airway wall pathology. Brown-Norway rats were sensitized and then exposed to ovalbumin or saline aerosol every 3 days on six occasions. After the final exposure, rats were administered twice daily for 7 days with the DNA S-phase marker bromodeoxyuridine (BrdU). Using a triple immunohistochemical staining technique, BrdU incorporation into ASM and epithelium was quantified employing computer-assisted image analysis. There were >3-fold mean increases in BrdU incorporation into ASM from 1.3% of cells (95% confidence interval (CI) 1.0-1.6) in saline controls to 4.7% (95% CI 2.6-6.7) after allergen exposure (p<0.001), and in airway epithelium, from 1.3 (95% CI 0.6-2.0) BrdU-positive cells x mm basement membrane(-1) in saline controls to 4.9 (95% CI 3.0-6.7) after allergen exposure (p<0.001). There was increased subepithelial collagen deposition and mucus secretion along with a significant eosinophil and lymphocyte recruitment to the airways. Increased rates of deoxyribonucleic acid synthesis in both airway smooth muscle and epithelial cells along with changes to the airway wall pathology may precede the establishment of smooth muscle thickening and airway remodelling after repeated allergen exposure in rats. This model seems to be appropriate for studying structural changes within the airways as observed in asthma.

  13. Synthesis and Crystal Structure Study of 2’-Se-Adenosine-Derivatized DNA

    SciTech Connect

    Sheng, J.; Salon, J; Gan, J; Huang, Z

    2010-01-01

    The selenium derivatization of nucleic acids is a novel and promising strategy for 3D structure determination of nucleic acids. Selenium can serve as an excellent anomalous scattering center to solve the phase problem, which is one of the two major bottlenecks in macromolecule X-ray crystallography. The other major bottleneck is crystallization. It has been demonstrated that the incorporated selenium functionality at the 2'-positions of the nucleosides and nucleotides is stable and does not cause significant structure perturbation. Furthermore, it was observed that the 2'-Se-derivatization could facilitate crystallization of oligonucleotides with fast crystal growth and high diffraction quality. Herein, we describe a convenient synthesis of the 2'-Se-adenosine phosphoramidite, and report the first synthesis and X-ray crystal structure determination of the DNA containing the 2'-Se-A derivatization. The 3D structure of 2'-Se-A-DNA decamer [5'-GTACGCGT(2'-Se-A)C-3']{sub 2} was determined at 1.75 {angstrom} resolution, the 2'-Se-functionality points to the minor groove, and the Se-modified and native structures are virtually identical. Moreover, we have observed that the 2'-Se-A modification can greatly facilitate the crystal growth with high diffraction quality. In conjunction with the crystallization facilitation by the 2'-Se-U and 2'-Se-T, this novel observation on the 2'-Se-A functionality suggests that the 2'-Se moiety is sole responsible for the crystallization facilitation and the identity of nucleobases does not influence the crystal growth significantly.

  14. Bisindolylmaleimide protein-kinase-C inhibitors delay the decline in DNA synthesis in mouse hair follicle organ cultures.

    PubMed

    Harmon, C S; Nevins, T D; Ducote, J; Lutz, D

    1997-01-01

    We have used a series of bisindolylmaleimide selective protein-kinase C (PKC) inhibitors to investigate the role of this enzyme in the regulation of cell proliferation in mouse hair follicle organ cultures. Mouse whisker follicles were isolated by microdissection, and rates of DNA synthesis during culture were determined from 3H-thymidine incorporation. The bisindolylmaleimides Ro 31-7549, Ro 31-8161, Ro 31-8425 and Ro 31-8830 inhibit isolated brain PKC with IC50 values of 8-80 nM, are > 60-fold less potent against protein kinase A, and inhibit PKC-mediated protein phosphorylation in platelets with IC50 values in the range 0.25-4.4 microM. These PKC inhibitors were found to increase levels of mouse hair follicle DNA synthesis, with EC50 values in the range 1-4 microM and maximal levels in the range 151-197% of control. Ro 31-7549 had an IC50 value 50-fold lower than that of minoxidil, while the maximal level of DNA synthesis for the PKC inhibitor was 86% higher. Incubation of mouse hair follicles with Ro 31-7549 resulted in a delay of approximately 24 h in the onset of decline in follicular DNA synthesis rates. Ro 31-6045 and Ro 31-7208, bisindolylmaleimides without activity in the platelet PKC assay, did not affect mouse hair follicle DNA synthesis rates. Taken together, these findings show that PKC mediates, at least in part, the rapid loss of proliferative activity that occurs in mouse whisker follicles in culture, and provide further evidence that PKC plays a role as a negative proliferative signal in hair follicles.

  15. Both hypoxanthine and adenosine stimulate DNA synthesis independently in serum-starved L cells treated with platelet protein.

    PubMed Central

    Klenow, H; Flodgaard, H

    1983-01-01

    The effect of human platelet factors and purine derivatives on DNA synthesis has been investigated in mouse fibroblast-like L cells whose growth was arrested by serum starvation. When such cells were exposed to diluted platelet extract (e.g., 35 micrograms of protein per ml), a stimulatory effect on net DNA synthesis was observed. This effect was almost abolished by dialysis of the extract. The stimulation was, however, recovered by supplementing the diluted and dialyzed extract with hypoxanthine or adenosine. Similar phenomena were observed in pulse-labeling experiments performed with [3H]thymidine. In this case, however, there was a marginal stimulatory effect of adenosine or hypoxanthine alone. When the cells were treated with saturating concentrations of pure platelet-derived growth factor (PDGF), a stimulatory effect on pulse labeling was again obtained by the simultaneous presence of hypoxanthine or adenosine. In serum-starved cells of a mutant line of L cells deficient in hypoxanthine phosphoribosyltransferase, there was, however, no stimulatory effect on pulse labeling by hypoxanthine when it was added alone or together with either PDGF or diluted dialyzed platelet extract. It is suggested that the stimulation of DNA synthesis by the purine derivatives in the presence of a certain type of platelet proteins, probably involving PDGF, may be explained by their function as precursors for a purine ribonucleotide pool that is specifically related to DNA synthesis. Treatment of serum-starved L cells with high concentrations of dialyzed platelet extract (e.g., 240 micrograms of protein per ml) showed that platelets contain an additional type of factor that may substitute for the requirement of adenosine or hypoxanthine for DNA synthesis to take place. It is suggested that the effect of this type of factor may be to activate the catabolic activity of the purine salvage pathway. PMID:6584864

  16. Nascent DNA synthesis during homologous recombination is synergistically promoted by the rad51 recombinase and DNA homology.

    PubMed

    Mundia, Maureen M; Desai, Vatsal; Magwood, Alissa C; Baker, Mark D

    2014-05-01

    In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (3' extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 3' extension for homology lengths of 500 bp to 2 kb. At values below ∼500 bp, the frequency of 3' extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 3' extension. Overexpression of wild-type Rad51 stimulated the frequency of 3' extension by ∼3-fold for homology lengths <900 bp, but when homology was >2 kb, 3' extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 3' extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 3' extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 3' extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1-8, rescues the 3' extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 3' extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization.

  17. Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

    PubMed Central

    Mundia, Maureen M.; Desai, Vatsal; Magwood, Alissa C.; Baker, Mark D.

    2014-01-01

    In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (3′ extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 3′ extension for homology lengths of 500 bp to 2 kb. At values below ∼500 bp, the frequency of 3′ extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 3′ extension. Overexpression of wild-type Rad51 stimulated the frequency of 3′ extension by ∼3-fold for homology lengths <900 bp, but when homology was >2 kb, 3′ extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 3′ extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 3′ extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 3′ extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1–8, rescues the 3′ extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 3′ extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization. PMID:24583581

  18. Participation of translesion synthesis DNA polymerases in the maintenance of chromosome integrity in yeast Saccharomyces cerevisiae.

    PubMed

    Kochenova, O V; Soshkina, J V; Stepchenkova, E I; Inge-Vechtomov, S G; Shcherbakova, P V

    2011-01-01

    We employed a genetic assay based on illegitimate hybridization of heterothallic Saccharomyces cerevisiae strains (the α-test) to analyze the consequences for genome stability of inactivating translesion synthesis (TLS) DNA polymerases. The α-test is the only assay that measures the frequency of different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material simultaneously. All these events are manifested as illegitimate hybridization and can be distinguished by genetic analysis of the hybrids and cytoductants. We studied the effect of Polζ, Polη, and Rev1 deficiency on the genome stability in the absence of genotoxic treatment and in UV-irradiated cells. We show that, in spite of the increased percent of accurately repaired primary lesions, chromosome fragility, rearrangements, and loss occur in the absence of Polζ and Polη. Our findings contribute to further refinement of the current models of translesion synthesis and the organization of eukaryotic replication fork.

  19. Highly Parallel Method for Synthesis of DNA Repeats Enables Discovery of “Smart” Protein Polymers

    PubMed Central

    Amiram, Miriam; Quiroz, Felipe Garcia; Callahan, Daniel J.; Chilkoti, Ashutosh

    2010-01-01

    Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap-extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of a glucagon-like peptide-1 analog. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover “smart” biopolymers that exhibit fully reversible thermally responsive behavior. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a “one-pot” parallel format. PMID:21258353

  20. Long conducting polymer nanonecklaces with a `beads-on-a-string' morphology: DNA nanotube-template synthesis and electrical properties

    NASA Astrophysics Data System (ADS)

    Chen, Guofang; Mao, Chengde

    2016-05-01

    Complex and functional nanostructures are always desired. Herein, we present the synthesis of novel long conducting polymer nanonecklaces with a `beads-on-a-string' morphology by the DNA nanotube-template approach and in situ oxidative polymerization of the 3-methylthiophene monomer with FeCl3 as the oxidant/catalyst. The length of the nanonecklaces is up to 60 μm, and the polymer beads of around 20-25 nm in diameter are closely packed along the axis of the DNA nanotube template with a density of ca. 45 particles per μm. The formation of porous DNA nanotubes impregnated with FeCl3 was also demonstrated as intermediate nanostructures. The mechanisms for the formation of both the porous DNA nanotubes and the conducting polymer nanonecklaces are discussed in detail. The as-synthesized polymer/DNA nanonecklaces exhibit good electrical properties.Complex and functional nanostructures are always desired. Herein, we present the synthesis of novel long conducting polymer nanonecklaces with a `beads-on-a-string' morphology by the DNA nanotube-template approach and in situ oxidative polymerization of the 3-methylthiophene monomer with FeCl3 as the oxidant/catalyst. The length of the nanonecklaces is up to 60 μm, and the polymer beads of around 20-25 nm in diameter are closely packed along the axis of the DNA nanotube template with a density of ca. 45 particles per μm. The formation of porous DNA nanotubes impregnated with FeCl3 was also demonstrated as intermediate nanostructures. The mechanisms for the formation of both the porous DNA nanotubes and the conducting polymer nanonecklaces are discussed in detail. The as-synthesized polymer/DNA nanonecklaces exhibit good electrical properties. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01603k

  1. Inhibition of semiconservative DNA synthesis in ICR 2A frog cells exposed to monochromatic uv wavelengths (252-313 nm) and photoreactivating light

    SciTech Connect

    Rosenstein, B.S.

    1982-06-01

    Exposure of ICR 2A frog cells to monochromatic uv wavelengths in the range 252-313 nm caused an inhibition of semiconservative DNA synthesis which was partially relieved in cells receiving a post irradiation treatment with photoreactivating light (>350 nm). Hence pyrimidine dimers acted as lesions blocking DNA synthesis in uv-irradiated cells based upon the specificity of photoreactivating enzyme for the light-dependent monomerization of dimers in DNA. Compared with the shorter wavelengths tested, however, this recovery of DNA synthesis was not as great in cells exposed to 302-nm radiation and was nearly absent in 313-nm-irradiated cells up to 12 hr after treatment. These results suggest that nondimer photoproducts also play an important role in causing DNA synthesis inhibition in cells exposed to wavelengths greater than 300 nm.

  2. Bacterial Obg proteins: GTPases at the nexus of protein and DNA synthesis.

    PubMed

    Kint, Cyrielle; Verstraeten, Natalie; Hofkens, Johan; Fauvart, Maarten; Michiels, Jan

    2014-08-01

    Obg proteins (also known as ObgE, YhbZ and CgtA) are conserved P-loop GTPases, essential for growth in bacteria. Like other GTPases, Obg proteins cycle between a GTP-bound ON and a GDP-bound OFF state, thereby controlling cellular processes. Interestingly, the in vitro biochemical properties of Obg proteins suggest that they act as sensors for the cellular GDP/GTP pools and adjust their activity according to the cellular energy status. Obg proteins have been attributed a host of cellular functions, including roles in essential cellular processes (DNA replication, ribosome maturation) and roles in different stress adaptation pathways (stringent response, sporulation, general stress response). This review summarizes the current knowledge on Obg activity and function. Furthermore, we present a model that integrates the different functions of Obg by assigning it a fundamental role in cellular physiology, at the hub of protein and DNA synthesis. In particular, we believe that Obg proteins might provide a connection between different global pathways in order to fine-tune cellular processes in response to a given energy status.

  3. PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis

    PubMed Central

    Kumar, Shiv; Tao, Chuanjuan; Chien, Minchen; Hellner, Brittney; Balijepalli, Arvind; Robertson, Joseph W. F.; Li, Zengmin; Russo, James J.; Reiner, Joseph E.; Kasianowicz, John J.; Ju, Jingyue

    2012-01-01

    We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5′-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2′-deoxyguanosine-5′-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform. PMID:23002425

  4. PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis.

    PubMed

    Kumar, Shiv; Tao, Chuanjuan; Chien, Minchen; Hellner, Brittney; Balijepalli, Arvind; Robertson, Joseph W F; Li, Zengmin; Russo, James J; Reiner, Joseph E; Kasianowicz, John J; Ju, Jingyue

    2012-01-01

    We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5'-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.

  5. Thermodynamics of translesion synthesis across a major DNA adduct of antitumor oxaliplatin: differential scanning calorimetric study.

    PubMed

    Florian, Jakub; Brabec, Viktor

    2012-02-06

    Differential scanning calorimetry (DSC) was used to measure the thermodynamic changes associated with translesion synthesis across major lesion induced in DNA by antitumor oxaliplatin [1,2-d(GG) intrastrand cross-link]. Insertion of matched nucleotides dC at the primer terminus (across unique 3'- or 5'-dG in the unplatinated template) and subsequent extensions resulted in an incremental increase in thermodynamic parameters. In contrast, incorporation of dC opposite either platinated dG in the intrastrand cross-link formed in the template strand and subsequent extensions by one nucleotide resulted only in little changes in thermodynamics. A similar thermodynamic delay was observed for a control template primer containing a dG:dT mismatch across 3'- or 5'-dG in the template and subsequent Watson-Crick primer extensions. The thermodynamic scarcity generated by either the lesion or mismatches was not localized but extended to the 5'-downstream sites, which may be connected with the phenomenon termed "short-term memory" of replication errors retained by some DNA polymerases responding to DNA damages or mismatches. Interestingly, formation of the 1,2-d(GG) intrastrand cross-link of oxaliplatin altered the overall DSC profiles of the dG:dT mismatch template/primers only in a very small extent. While addition of matched nucleotide dC across either dG in the template strand was thermodynamically favored over the presence of a mismatched dT (ΔΔG(0)(310) was 7.6 or 6.8 kJ mol(-1), ΔΔH was 14 or 49 kJ mol(-1)), no such thermodynamic advantage was observed with the 1,2-d(GG) intrastrand cross-link of oxaliplatin at these positions (ΔΔG(0)(310) was 2.8 or -0.3 kJ mol(-1), ΔΔH was 4 or 9 kJ mol(-1)). The equilibrium thermodynamic data also provide insight into the processes associated with misincorporation of incorrect nucleotides during replication bypass across major cross-links of antitumor oxaliplatin. On the other hand, besides thermodynamic effects also kinetic

  6. Synthesis, Acetylation, and Phosphorylation of Histone IV and Its Binding to DNA During Spermatogenesis in Trout*

    PubMed Central

    Louie, Andrew J.; Dixon, Gordon H.

    1972-01-01

    During spermatogenesis in trout testis, histone IV is extensively modified by acetylation and phosphorylation. To examine the relationship of synthesis of histone IV to its modification, histone IV labeled with [3H]aminoacids and inorganic [32P]phosphate was prepared from testis cells by acid extraction and column chromatography. Purified histone IV was resolved by starch gel electrophoresis into 10 bands, of which nine are modified by acetylation and/or phosphorylation. In the first 4 hr of labeling, the diacetyl-histone IV band showed the highest proportion of [3H]aminoacid label. After 12 hr of incorporation, more label was found in the triacetyl and tetraacetyl bands. A significant amount of amino-acid label in the two major bands (the unsubstituted and monoacetyl bands) of histone IV was not seen until 16 hr of incubation. From 1 to 12 days, the proportion of label in the unsubstituted and monoacetylated bands increased, while that in the tetra-, tri-, and monoacetyl bands decreased. Very little [3H]aminoacid was found in the phosphorylated bands of histone IV in the first 12 hr. However, after 16 hr about 20% of the total 3H was found in the phosphorylated bands. The proportion increased to 33% and remained at this level between 1 and 8 days, but, by 16 days, had decreased to 12% of the total. These data suggest that an “obligatory” acetylation of recently synthesized histone IV is involved in the correct binding of newly synthesized histone IV to DNA. We propose that ε-amino acetylation of lysyl residues 5, 8, 12, and 16 neutralizes their positive charges and allows the NH2-terminal region of histone IV to assume the correct conformation (in this case, an α-helix), and fit into the major groove of DNA. Deacetylation then “locks” histone IV to DNA by ionic linkages. The biological significance of phosphorylation of histone IV is not known. Images PMID:4505675

  7. Glucocorticoid suppression of human lymphocyte DNA synthesis. Influence of phytohemagglutinin concentration

    SciTech Connect

    Segel, G.B.; Lukacher, A.; Gordon, B.R.; Lichtman, M.A.

    1980-04-01

    Glucocorticoids have been shown to suppress lectin-stimulated lymphocyte DNA synthesis in some studies, whereas in other studies, the hormones have had little effect. We have found that the position on the PHA dose-response curve that is studied is the most important determinant of whether cortisol inhibits /sup 3/H-thymidine incorporation into lymphocyte DNA. The proportion of monocytes in culture also influenced the cortisol effect, but it was quantitatively less important than PHA concentration. Cortisol (5 nM to 100 ..mu..M) had little effect on blastogenesis or thymidine incorporation into DNA in cultures that contained both a high concentration (14% +- 2 (S.E.)) of monocytes and a concentration of PHA (0.6 to 1.2 ..mu..g/ml) that produced maximal stimulation of mitogenesis. When monocytes were reduced from 14 to 1.4%, cortisol (5 ..mu..M) caused a 30% reduction in thymidine incorporation in cultures stimulated by 0.6 to 1.2 ..mu..g/ml PHA. Much greater cortisol suppression of thymidine incorporation occurred if the concentration of PHA was reduced. For example, reduction of the PHA concentration from 1.2 to 0.075 ..mu..g/ml resulted in an increase in suppression by 5 ..mu..M cortisol from 5 to 90% even in the presence of 14% monocytes. These data indicate that the suppressive effects of glucocorticoids on blastogenesis and thymidine incorporation in vitro depend principally on the concentration of PHA used to stimulate blastogenesis and secondarily on the proportion of monocytes in the culture system.

  8. REV1 restrains DNA polymerase zeta to ensure frame fidelity during translesion synthesis of UV photoproducts in vivo.

    PubMed

    Szüts, Dávid; Marcus, Adam P; Himoto, Masayuki; Iwai, Shigenori; Sale, Julian E

    2008-12-01

    Exposure to ultraviolet light induces a number of forms of damage in DNA, of which (6-4) photoproducts present the most formidable challenge to DNA replication. No single DNA polymerase has been shown to bypass these lesions efficiently in vitro suggesting that the coordinate use of a number of different enzymes is required in vivo. To further understand the mechanisms and control of lesion bypass in vivo, we have devised a plasmid-based system to study the replication of site-specific T-T(6-4) photoproducts in chicken DT40 cells. We show that DNA polymerase zeta is absolutely required for translesion synthesis (TLS) of this lesion, while loss of DNA polymerase eta has no detectable effect. We also show that either the polymerase-binding domain of REV1 or ubiquitinated PCNA is required for the recruitment of Polzeta as the catalytic TLS polymerase. Finally, we demonstrate a previously unappreciated role for REV1 in ensuring bypass synthesis remains in frame with the template. Our data therefore suggest that REV1 not only helps to coordinate the delivery of DNA polymerase zeta to a stalled primer terminus but also restrains its activity to ensure that nucleotides are incorporated in register with the template strand.

  9. Synthesis and characterization of DNA-quantum dot conjugates for the fluorescence ratiometric detection of unlabelled DNA.

    PubMed

    Page, Leah Elizabeth; Zhang, Xi; Tyrakowski, Christina Marie; Ho, Chiun-Teh; Snee, Preston Todd

    2016-11-21

    A quantum dot-based ratiometrically responsive fluorescent sensor for unlabeled single-stranded DNA (ssDNA) is reported. Several technical issues concerning the development of high yield ssDNA-QD conjugation chemistry were addressed. The DNA sensor was synthesized by conjugating methacrylic phosphoramidite-functional oligonucleotides to water-soluble cadmium zinc sulfide core/zinc sulfide shell quantum dots (CdZnS/ZnS QDs). Duplex DNA was formed when the QD-bound ssDNA was incubated with its complement. Next, titration with PicoGreen resulted in FRET energy transfer from the dot to the dsDNA intercalating dye. The resulting ratio of the dye to QD integrated emissions is a calibratable metric for label-free DNA detection with a LOD of 3.8 nmol.

  10. DNA polymerase delta, RFC and PCNA are required for repair synthesis of large looped heteroduplexes in Saccharomyces cerevisiae.

    PubMed

    Corrette-Bennett, Stephanie E; Borgeson, Claudia; Sommer, Debbie; Burgers, Peter M J; Lahue, Robert S

    2004-01-01

    Small looped mispairs are corrected by DNA mismatch repair (MMR). In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells. Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components. This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR. Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase delta efficiently inhibited LLR in extracts by blocking repair just prior to gap filling. Gap filling was inhibited regardless of whether the loop was retained or removed. These experiments suggest polymerase delta is uniquely required in yeast extracts for LLR-associated synthesis. Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e. LLR was inhibited just prior to gap filling for both loop removal and loop retention. Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR. These biochemical experiments support the idea that yeast polymerase delta, RFC and PCNA are required for large loop DNA repair synthesis.

  11. Synthesis, crystal structures, DNA binding and photoluminescence properties of [Cu(pzta)2Cl]Cl⋅H2O for DNA detection.

    PubMed

    Duan, Ran-ran; Wang, Lu; Huo, Wei-qiang; Chen, Shi; Zhou, Xiao-hua

    2014-07-15

    We report here the synthesis of a new copper(II) complex of 2,4-diamino-6-(2'-pyrazin)-1,3,5-triazine [Cu(pzta)2Cl]Cl·H2O and its characterization using UV and IR spectroscopy, elemental analysis, and X-ray diffraction. Fluorescence spectroscopy revealed that the complex was sensitive to oxygen and to the polarity of nonaqueous solvents. Binding of the complex to DNA was investigated using UV spectroscopy, ethidium bromide displacement from DNA, cyclic voltammetry, and viscometry. The results revealed the DNA binding mode was intercalation together with external static-electricity. However, the complex can be also used to DNA detection as DNA fluorescence probe with a LOD of 4.21 ng mL(-1) for the relative wide linear range between 0.2 and 17 μg mL(-1). In conclusion, that synthetic method of the complex was easy with low expense and was relatively rapid and sensitive compared to most toxic fluorescence dyes. This finding would indicate the complex may be a potential DNA-targeted probes and optical probes for oxygen-free environments in nonaqueous form. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. M-phase-specific protein kinase from mitotic sea urchin eggs: cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis.

    PubMed

    Arion, D; Meijer, L

    1989-08-01

    Histone H1 kinase (H1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles. The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs. Translocation of active H1K from particulate to soluble fraction does not seem to be responsible for this activation. H1K activation cannot be accounted for by the transient disappearance of a putative H1K inhibitor present in soluble fractions of homogenates. Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H1K activity. H1K activation therefore does not require DNA or RNA synthesis. Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis. H1K activation is highly dependent on the intracellular pH. Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H1K activation. Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H1K activation. Protein synthesis is an absolute requirement for H1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition. About 60 min after fertilization, H1K activation and cleavage become independent of protein synthesis. The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H1K, does not vary during the cell cycle and remains constant in emetine-treated cells. H1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit. Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H1K. These results suggest that a subunit or an activator of H1K is stored as an mRNA in the egg before mitosis and that full activation of H1K requires a phosphorylation.

  13. Highly sensitive and selective chemiluminescent imaging for DNA detection by ligation-mediated rolling circle amplified synthesis of DNAzyme.

    PubMed

    Dong, Haifeng; Wang, Chen; Xiong, Yi; Lu, Huiting; Ju, Huangxian; Zhang, Xueji

    2013-03-15

    A highly sensitive DNA biosensing method down to sub-femtomolar level with excellent selectivity was proposed by designing an amplified synthesis of horseradish peroxidase mimicking DNAzyme and introducing the amplified DNAzyme to chemiluminescent (CL) imaging. The amplified synthesis was achieved by combining a target DNA related ligase reaction with rolling circle amplification (RCA), which produced thousands of repeated sequences to bind hemin and form a mass of horseradish peroxidase-mimicing DNAzyme units. The amplification strategy greatly enhanced the CL emission of the luminol-H(2)O(2) system. The genotyping method displayed highly specific biochemistry in allele discrimination. The novel CL imaging strategy based on ligation-mediated RCA synthesis of DNAzyme showed high fidelity in discriminating single-base mismatch and efficiently facilitated signal amplification for sensitive target DNA detection. It could detect DNA ranging from 1×10(-15) M to 1×10(-11) M with a detection limit of 0.26 fM. The proposed approach provided a robust, cost-efficient, highly sensitive and specific platform for genetic target analysis in bioanalysis and clinic biomedical application. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

    PubMed

    Mounce, Bryan C; Tsan, Fei Chin; Droit, Lindsay; Kohler, Sarah; Reitsma, Justin M; Cirillo, Lisa A; Tarakanova, Vera L

    2011-11-25

    Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

  15. Inhibition of semiconservative DNA synthesis in ICR 2A frog cells by pyrimidine dimers and nondimer photoproducts induced by ultraviolet radiation

    SciTech Connect

    Rosenstein, B.S.

    1984-11-01

    DNA synthesis was examined in ultraviolet (uv)-irradiated ICR 2A frog cells in which either pyrimidine dimers or nondimer photoproducts represented the major class of DNA lesions. In addition, cells were exposed to /sup 60/Co ..gamma.. rays. The cultures were pulse-labeled and the size distribution of the DNA synthesized was estimated using both sucrose gradient sedimentation and alkaline step elution. Using either of these techniques, it was found that the presence of dimers resulted in a reduction principally in the synthesis of high molecular weight (MW) DNA. In contrast, nondimer photoproducts caused a strong inhibition in the synthesis of low MW DNA, as was also observed in ..gamma..-irradiated cells. Hence the induction of pyrimidine dimers in DNA mainly affected the elongation of replicons, whereas nondimer lesions primarily caused an inhibition of replicon initiation.

  16. Identification of the active region of the DNA synthesis inhibitory gene p21Sdi1/CIP1/WAF1.

    PubMed

    Nakanishi, M; Robetorye, R S; Adami, G R; Pereira-Smith, O M; Smith, J R

    1995-02-01

    The cloning of the negative growth regulatory gene, p21Sdi1, has led to the convergence of the fields of cellular senescence, cell cycle regulation and tumor suppression. This gene was first cloned as an inhibitor of DNA synthesis that was overexpressed in terminally non-dividing senescent human fibroblasts (SD11) and later as a p53 transactivated gene (WAF1) and a Cdk-interacting protein (CIP1, p21) that inhibited cyclin-dependent kinase activity. To identify the active region(s) of p21Sdi1, cDNA constructs encoding various deleted forms of the protein were analyzed. Amino acids 22-71 were found to be the minimal region required for DNA synthesis inhibition. Amino acids 49-71 were involved in binding to Cdk2, and constructs deleted in this region expressed proteins that were unable to inhibit Cdk2 kinase activity in vitro. The latter stretch of amino acids shared sequence similarity with amino acids 60-76 of the p27Kip1 protein, another Cdk inhibitor. Point mutations made in p21Sdi1 in this region confirmed that amino acids common to both proteins were involved in DNA synthesis inhibition. Additionally, a chimeric protein, in which amino acids 49-65 of p21Sdi1 were substituted with amino acids 60-76 of p27Kip1, had almost the same DNA synthesis inhibitory activity as the wild-type protein. The results indicate that the region of sequence similarity between p21Sdi1 and p27Kip1 encodes an inhibitory motif characteristic of this family of Cdk inhibitors.

  17. Design, Synthesis and DNA Interaction Study of New Potential DNA Bis-Intercalators Based on Glucuronic Acid

    PubMed Central

    Zhao, Jiuyang; Li, Wei; Ma, Rui; Chen, Shaopeng; Ren, Sumei; Jiang, Tao

    2013-01-01

    A series of novel potential DNA bis-intercalators were designed and synthesized, in which two glucuronic acids were linked by ethylenediamine, and the glucuronic acid was coupled with various chromophores, including quinoline, acridine, indole and purine, at the C-1 position. The preliminary binding properties of these compounds to calf thymus DNA (CT-DNA) have been investigated by UV-absorption and fluorescence spectroscopy. The results indicated that all the target compounds can interact with CT-DNA, and the acridine derivative, 3b, showed the highest key selection vector (KSV) value, which suggested that compound 3b binds most strongly to CT-DNA. PMID:23955268

  18. Synthesis and DNA-binding properties of novel DNA cyclo-intercalators containing purine-glucuronic acid hybrids.

    PubMed

    Zhang, Renshuai; Chen, Shaopeng; Wang, Xueting; Yu, Rilei; Li, Mingjing; Ren, Sumei; Jiang, Tao

    2016-06-24

    Novel DNA cyclo-intercalators, which incorporated two intercalator subunits linked by two bridges, were synthesized. Binding of the compounds to calf-thymus DNA was studied by fluorescence spectroscopy, and docking simulations were used to predict the binding modes of these cyclic compounds. The spectral data demonstrated that all of these compounds can interact with CT-DNA. The sugar moiety played an important role in the process of binding between the intercalators containing glucuronic acid and DNA. The length and flexibility of the connecting bridges affected the binding affinity of the resultant cyclo-intercalators. Docking simulations showed that compounds 7 and 8 interact with DNA as mono-intercalators.

  19. [Activation of deoxyribonucleotide synthesis by radioprotectants and antioxidants as a key stage in formation of body resistance to DNA-damaging factors].

    PubMed

    Sharygin, V L; Pulatova, M K; Shliakova, T G; Mitrokhin, Iu I; Todorov, I N

    2005-01-01

    The responses of the systems of synthesis of deoxyribonucleotides (dNTPs), DNA, and proteins in hematopoietic organs and liver of animals to gamma-radiation, administration of radioprotectants and antioxidants as well as the dependence of these responses on the doses of radiation and drugs were studied. Radioprotectants of acute (indralin) and durable effects (indomethaphen) as well as natural (alpha2-tocopherol) and synthetic anti-oxidants (ionol or 2,6-di-tert-butyl-4-methylphenol) efficient in survival test were used. Three stages could be recognized in the standard unspecific response of the studied systems to radiation: (1) immediate increase in ribonucleotide reductase activity in the tissues within the first 30 min as a part of the integrated SOS response to DNA damage, which activates dNTP synthesis; (2) inhibition of the synthesis of dNTPs, DNA, and and (3) restoring ribonucleotide reductase activity and integral increase in the production of dNTPs, DNA, and total protein, which is essential for the development of compensatory and restorative responses of the organism. The radioprotectants significantly increased ribonucleotide reductase activity, which increased intracellular concentrations of the four dNTP types in organs during radiation exposure and three following days. Within this period, ribonucleotide reductase activity was inhibited by 40-50% in animals not treated with radioprotectants as compared to control. Balanced high pools of dNTPs in the organs of radioprotectant-treated animals provided for high-performance repair of DNA damage. The radioprotectant-induced activation of dNTP synthesis during the development of compensatory and restorative responses provides for an earlier restoration of the cellular composition and functioning of the organs. Antioxidants stimulated the synthesis of dNTPs, DNA, and proteins in animal tissues in a strict dose interval. Their effect on the studied syntheses was dose-dependent: single or multiple long

  20. DNA synthesis, cell proliferation index in normal and abnormal gallbladder epithelium.

    PubMed

    Lamote, J; Willems, G

    1997-09-15

    The observation of mitotic figures in the epithelium of the normal gallbladder is exceptional because cell renewal is occurring at a very slow rate. It is only after using 3H-thymidine and autoradiography to observe the cells in DNA synthesis that evidence of a significant epithelial cell replication has been provided. Because numerous mitotic figures and increased 3H-thymidine uptake have been observed after intraluminal introduction of foreign bodies or after ligation of the common bile duct in animals, mechanical distension has been supposed to represent an important trigger factor of cell proliferation in this hollow organ. An increased epithelial cell renewal was also observed in human gallbladders of patients with a complete obstruction of the common bile duct causing the distension. However, the absence of correlation between the degree of gallbladder distension and the proliferative response was suggesting that factors other than distension could be involved. In studies on experimental lithiasis cell proliferation appeared to be enhanced in the gallbladder epithelium of mice fed on a cholesterol-cholic acid-rich lithogenic diet. The fact that the increase in proliferative activity was preceding the formation of gallstones was another indication that factors other than mechanical stimulation by stretching or by the stones may stimulate cell renewal in this organ. Factors in the bile of animals receiving a lithogenic diet could be involved which might cause cellular death and, hence, a regenerative reaction. Direct mitogenic effect of an unknown factor in the bile of these animals is an alternative possibility. On the other hand the stimulating effect of postprandial hormones on gallbladder cell renewal suggested by the observation of a DNA synthesis peak after feeding has been established. Synthetic cholecystokinin analogues have been shown to increase the proliferative activity and to induce epithelial hyperplasia in this organ. In one recent study using

  1. Rational design, synthesis, pharmacophore modeling, and docking studies for identification of novel potent DNA-PK inhibitors.

    PubMed

    Ihmaid, Saleh; Ahmed, Hany E A; Al-Sheikh Ali, Adeeb; Sherif, Yousery E; Tarazi, Hamadeh M; Riyadh, Sayed M; Zayed, Mohamed F; Abulkhair, Hamada S; Rateb, Heba S

    2017-06-01

    Drugs of cancer based upon ionizing radiation or chemotherapeutic treatment may affect breaking of DNA double strand in cell. DNA-PK enzyme has emerged as an attractive target for drug discovery efforts toward DNA repair pathways. Hence, the search for potent and selective DNA-PK inhibitors has particularly considered state-of-the art and several series of inhibitors have been designed. In this article, a novel benchmark DNA-PK database of 43 compounds was built and described. Ligand-based approaches including pharmacophore and QSAR modeling were applied and novel models were introduced and analyzed for predicting activity test for DNA-PK drug candidates. Based upon the modeling results, we gave a report of synthesis of fifteen novel 2-((8-methyl-2-morpholino-4-oxo-4H-benzo[e][1,3]oxazin-7-yl)oxy)acetamide derivatives and in vitro evaluation for DNA-PK inhibitory and antiproliferative activities. These fifteen compounds overall are satisfied with Lipinski's rule of five. The biological testing of target compounds showed five promising active compounds 7c, 7d, 7f, 9e and 9f with micromolar DNA-PK activity range from 0.25 to 5µM. In addition, SAR of the compounds activity was investigated and confirmed that the terminal aryl moiety was found to be quite crucial for DNA-PK activity. Moreover flexible docking simulation was done for the potent compounds into the putative binding site of the 3D homology model of DNA-PK enzyme and the probable interaction model between DNA-PK and the ligands was investigated and interpreted. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. A new class of cleavable fluorescent nucleotides: synthesis and optimization as reversible terminators for DNA sequencing by synthesis†

    PubMed Central

    Turcatti, Gerardo; Romieu, Anthony; Fedurco, Milan; Tairi, Ana-Paula

    2008-01-01

    Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3′-OH unprotected cleavable fluorescent 2′-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor® 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal–no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates. PMID:18263613

  3. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers

    PubMed Central

    Briggs, Adrian W.; Rios, Xavier; Chari, Raj; Yang, Luhan; Zhang, Feng; Mali, Prashant; Church, George M.

    2012-01-01

    DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin ‘capping’ oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical. PMID:22740649

  4. Effects of inhibitors of DNA, RNA and protein synthesis on frequencies and types of premature chromosome condensation from X-ray induced micronuclei.

    PubMed

    Madle, S; Nowak, J; Obe, G

    1976-10-28

    Cells containing X-ray induced micronuclei were treated for a few hours before fixation with inhibitors of DNA synthesis (cytosine arabinoside; azathioprine; thymidine; trenimon), of RNA synthesis (actinomycin D; ethidium bromide), and of protein synthesis (puromycin). Only the inhibitors of DNA synthesis lead to a significant suppression of the frequencies of mitoses with micronucleus derived premature chromosome condensation (PCC). We tend to interprete the result as follows: Micronuclei that are in the G1 phase of their cell cycles are accumulated at the G1/S border or in the early S phase of their cell cycles under the influence of the inhibitors of the DNA synthesis. Micronuclei blocked in this way cannot be induced to undergo PCC and seem to disappear from the cells.

  5. Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent.

    PubMed Central

    Kuijpers, W H; Huskens, J; Koole, L H; van Boeckel, C A

    1990-01-01

    A new deprotection procedure in the synthesis of (partially) phosphate-methylated oligodeoxynucleotides has been developed, involving treatment of fully protected DNA fragments with methanolic potassium carbonate. It is shown that base deprotection can be accomplished in potassium carbonate/methanol without affecting the methyl phosphotriesters. This methodology enables us to synthesize, both in solution and on a solid support, DNA fragments which are phosphate-methylated at defined positions. The solid phase synthesis, however, turns out to be accompanied by considerable demethylation of the phosphotriesters. It is demonstrated that this demethylation does not occur during the deprotection or work-up procedure. Furthermore, it was found that the latter side-reaction is suppressed when the standard capping procedure with acetic anhydride is included. PMID:2402444

  6. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  7. Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia.

    PubMed

    Pearce, Kaycey; Cai, Diancai; Roberts, Adam C; Glanzman, David L

    2017-01-09

    Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (partial) training following its disruption by reconsolidation blockade and inhibition of PKM (Chen et al., 2014). Here, we report that LTM can be induced by partial training after disruption of original consolidation by protein synthesis inhibition (PSI) begun shortly after training. But when PSI occurs during training, partial training cannot subsequently establish LTM. Furthermore, we find that inhibition of DNA methyltransferase (DNMT), whether during training or shortly afterwards, blocks consolidation of LTM and prevents its subsequent induction by truncated training; moreover, later inhibition of DNMT eliminates consolidated LTM. Thus, the consolidation of LTM depends on two functionally distinct phases of protein synthesis: an early phase that appears to prime LTM; and a later phase whose successful completion is necessary for the normal expression of LTM. Both the consolidation and maintenance of LTM depend on DNA methylation.

  8. Educational Price Differentials: A Clarification of the Issues.

    ERIC Educational Resources Information Center

    Chambers, Jay G.

    This paper provides a clarification of two factors--the differences in pupil need (as reflected by the proportion of disadvantaged or handicapped pupils in a district) and the differences in the prices of school inputs across districts--that affects the cost of educational services and a conceptual framework that may be useful in making welfare…

  9. The Impact of a Classroom Values Clarification Program

    ERIC Educational Resources Information Center

    Logan, Donald; And Others

    1977-01-01

    The Benton High School values clarification program is an orderly sequence of structured group activities directed toward clarifying students' values and decisions as part of a one-semester elective sociology class open to all juniors and seniors. Evaluation indicates program participants moved toward a stronger belief in internal control in…

  10. 28 CFR 513.12 - Inmate request for record clarification.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Inmate request for record clarification. 513.12 Section 513.12 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE GENERAL MANAGEMENT AND ADMINISTRATION ACCESS TO RECORDS Production or Disclosure of FBI/NCIC Information § 513.12...

  11. 76 FR 56304 - Hazardous Materials: Minor Editorial Corrections and Clarifications

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-13

    ... Pipeline and Hazardous Materials Safety Administration 49 CFR Parts 105, 106, 107, 130, 171, 172, 173, 174, 176, and 177 RIN 2137-AE77 Hazardous Materials: Minor Editorial Corrections and Clarifications AGENCY: Pipeline and Hazardous Materials Safety Administration (PHMSA), DOT. ACTION: Final rule. SUMMARY: This...

  12. Values Clarification and International Education: The Yonsei Experience.

    ERIC Educational Resources Information Center

    Bond, Douglas G.

    This paper describes selected experiences of Korean-American students and students with a Korean heritage from other countries who live and study at the undergraduate level in Korea. Korean-American students' search for their roots and the process of cultural values clarification are examined, with particular emphasis on identifying cultural…

  13. 76 FR 80831 - Clarification of Policy Regarding Approved Training Programs

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-27

    ... Federal Aviation Administration 14 CFR Part 135 Clarification of Policy Regarding Approved Training... Notice that would require FAA inspectors to review 14 CFR part 135 approved training programs to identify and correct those programs which erroneously issued credit for previous training or checking....

  14. An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family.

    PubMed

    Jozwiakowski, Stanislaw K; Keith, Brian J; Gilroy, Louise; Doherty, Aidan J; Connolly, Bernard A

    2014-09-01

    A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol ζ (a family-B translesion synthesis polymerase). Inactivation of the 3'-5' proof-reading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with a marked tendency to make changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an error-prone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family

    PubMed Central

    Jozwiakowski, Stanislaw K.; Keith, Brian J.; Gilroy, Louise; Doherty, Aidan J.; Connolly, Bernard A.

    2014-01-01

    A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol ζ (a family-B translesion synthesis polymerase). Inactivation of the 3′–5′ proof-reading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with a marked tendency to make changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an error-prone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA. PMID:25063297

  16. The Rec1 Gene of Ustilago Maydis, Which Encodes a 3' -> 5' Exonuclease, Couples DNA Repair and Completion of DNA Synthesis to a Mitotic Checkpoint

    PubMed Central

    Onel, K.; Koff, A.; Bennett, R. L.; Unrau, P.; Holloman, N. K.

    1996-01-01

    Mutation in the REC1 gene of Ustilago maydis results in extreme sensitivity to killing by ultraviolet light. The lethality of the rec1-1 mutant was found to be partially suppressed if irradiated cells were held artificially in G2-phase by addition of a microtubule inhibitor. This mutant was also found to be sensitive to killing when DNA synthesis was inhibited by external means through addition of hydroxyurea or by genetic control in a temperature-sensitive mutant strain defective in DNA synthesis. Flow cytometric analysis of exponentially growing cultures indicated that wild-type cells accumulated in G2 after UV irradiation, while rec1-1 cells appeared to exit from G2 and accumulate in G1/S. Analysis of mRNA levels in synchronized cells indicated that the REC1 gene is periodically expressed with the cell cycle and reaches maximal levels at G1/S. The results are interpreted to mean that a G2-M checkpoint is disabled in the rec1-1 mutant. It is proposed that the REC1 gene product functions in a surveillance system operating during S-phase and G2 to find and repair stretches of DNA with compromised integrity and to communicate with the cell cycle apparatus. PMID:8722772

  17. Scorpion (Odontobuthus doriae) venom induces apoptosis and inhibits DNA synthesis in human neuroblastoma cells.

    PubMed

    Zargan, Jamil; Sajad, Mir; Umar, Sadiq; Naime, M; Ali, Shakir; Khan, Haider A

    2011-02-01

    Scorpion and its organs have been used to cure epilepsy, rheumatism, and male impotency since medieval times. Scorpion venom which contains different compounds like enzyme and non-enzyme proteins, ions, free amino acids, and other organic inorganic substances have been reported to posses antiproliferative, cytotoxic, apoptogenic, and immunosuppressive properties. We for the first time report the apoptotic and antiproliferative effects of scorpion venom (Odontobuthus doriae) in human neuroblastoma cells. After exposure of cells to medium containing varying concentrations of venom (10, 25, 50, 100, and 200 μg/ml), cell viability decreased to 90.75, 75.53, 55.52, 37.85, and 14.30%, respectively, after 24 h. Cells expressed morphological changes like swelling, inhibition of neurite outgrowth, irregular shape, aggregation, rupture of membrane, and release of cytosolic contents after treatment with venom. Lactate dehydrogenase (LDH) level increased in 50 and 100 μg/ml as compared to control, but there was no significant increase in LDH level at a dose of 10 and 20 μg/ml. Two concentrations viz. 50 and 100 μ/ml were selected because of the profound effect of these concentrations on the cellular health and population. Treatment with these two concentrations induced reactive nitrogen intermediates and depolarization in mitochondria. While caspase-3 activity increased in a concentration-dependent manner, only 50 μg/ml was able to fragment DNA. It was interesting to note that at higher dose, i.e., 100 μg/ml, the cells were killed, supposedly by acute necrosis. DNA synthesis evidenced by bromodeoxyuridine (BrdU) incorporation was inhibited in a concentration-dependent manner. The cells without treatment incorporated BrdU with high affinity confirming their cancerous nature whereas very less incorporation was noticed in treated cells. Our results show apoptotic and antiproliferative potential of scorpion venom (O. doriae) in human neuroblastoma cells. These properties

  18. mTOR signaling regulates myotube hypertrophy by modulating protein synthesis, rDNA transcription, and chromatin remodeling.

    PubMed

    von Walden, Ferdinand; Liu, Chang; Aurigemma, Nicole; Nader, Gustavo A

    2016-10-01

    Ribosome production is an early event during skeletal muscle hypertrophy and precedes muscle protein accretion. Signaling via mTOR is crucial for ribosome production and hypertrophy; however, the mechanisms by which it regulates these processes remain to be identified. Herein, we investigated the activation of mTOR signaling in hypertrophying myotubes and determined that mTOR coordinates various aspects of gene expression important for ribosome production. First, inhibition of translation with cycloheximide had a more potent effect on protein synthesis than rapamycin indicating that mTOR function during hypertrophy is not on general, but rather on specific protein synthesis. Second, blocking Pol II transcription had a similar effect as Rapamycin and, unexpectedly, revealed the necessity of Pol II transcription for Pol I transcription, suggesting that mTOR may regulate ribosome production also by controlling Class II genes at the transcriptional level. Third, Pol I activity is essential for rDNA transcription and, surprisingly, for protein synthesis as selective Pol I inhibition blunted rDNA transcription, protein synthesis, and the hypertrophic response of myotubes. Finally, mTOR has nuclear localization in muscle, which is not sensitive to rapamycin. Inhibition of mTOR signaling by rapamycin disrupted mTOR-rDNA promoter interaction and resulted in altered histone marks indicative of repressed transcription and formation of higher-order chromatin structure. Thus mTOR signaling appears to regulate muscle hypertrophy by affecting protein synthesis, Class I and II gene expression, and chromatin remodeling. Copyright © 2016 the American Physiological Society.

  19. Involvement of cysteinyl leukotrienes in airway smooth muscle cell DNA synthesis after repeated allergen exposure in sensitized Brown Norway rats

    PubMed Central

    Salmon, Michael; Walsh, David A; Huang, Tung-Jung; Barnes, Peter J; Leonard, Thomas B; Hay, Douglas W P; Chung, K Fan

    1999-01-01

    Airway smooth muscle thickening is a characteristic feature of airway wall remodelling in chronic asthma. We have investigated the role of the leukotrienes in airway smooth muscle (ASM) and epithelial cell DNA synthesis and ASM thickening following repeated allergen exposure in Brown Norway rats sensitized to ovalbumin. There was a 3 fold increase in ASM cell DNA synthesis, as measured by percentage bromodeoxyuridine (BrdU) incorporation, in repeatedly ovalbumin-exposed (4.1%, 3.6–4.6; mean, 95% c.i.) compared to chronically saline-exposed rats (1.3%, 0.6–2.1; P<0.001). Treatment with a 5-lipoxygenase enzyme inhibitor (SB 210661, 10 mg kg−1, p.o.) and a specific cysteinyl leukotriene (CysLT1) receptor antagonist, pranlukast (SB 205312, 30 mg kg−1, p.o.), both attenuated ASM cell DNA synthesis. Treatment with a specific leukotriene B4 (BLT) receptor antagonist (SB 201146, 15 mg kg−1, p.o.) had no effect. There was also a significant, 2 fold increase in the number of epithelial cells incorporating BrdU per unit length of basement membrane after repeated allergen exposure. This response was not inhibited by treatment with SB 210661, pranlukast or SB 201146. A significant increase in ASM thickness was identified following repeated allergen exposure and this response was attenuated significantly by SB 210661, pranlukast and SB 201146. Rats exposed to chronic allergen exhibited bronchial hyperresponsiveness to acetylcholine and had significant eosinophil recruitment into the lungs. Treatment with SB 210661, pranlukast or SB 201146 significantly attenuated eosinophil recruitment into the lungs, whilst having no significant effect on airway hyperresponsiveness. These data indicate that the cysteinyl leukotrienes are important mediators in allergen-induced ASM cell DNA synthesis in rats, while both LTB4 and cysteinyl leukotrienes contribute to ASM thickening and eosinophil recruitment following repeated allergen exposure. PMID:10455261

  20. Interleukin-8 induces DNA synthesis, migration and down-regulation of cleaved caspase-3 in cultured human gingival epithelial cells.

    PubMed

    Fujita, T; Yoshimoto, T; Matsuda, S; Kajiya, M; Kittaka, M; Imai, H; Iwata, T; Uchida, Y; Shiba, H; Kurihara, H

    2015-08-01

    Migration of the junctional epithelium occurs in association with the formation of a periodontal pocket. Although the migration of junctional epithelium is known to be related to the proliferation and migration of gingival junctional epithelial cells, the mechanism has not been clarified. In patients with periodontitis, the levels of interleukin-8 (IL-8) in both gingival tissue and gingival crevicular fluid are dramatically increased. IL-8 has broad bioactive functions. In this study, we examined the role of IL-8 in DNA synthesis, migration and protection against apoptosis in cultured human gingival epithelial cells (HGEC). DNA synthesis was estimated by measuring the incorporation of bromodeoxyuridine. The migration of gingival epithelial cells was assessed in a wound-healing assay. The expression of integrin beta-1 was analyzed using immunofluorescence confocal microscopy and western blotting. Cleaved caspase-3 was detected using western blotting and a Caspase-Glo assay kit. IL-8 increased the synthesis of DNA in HGEC, and the maximal effect was seen at 25 or 50 ng/mL of IL-8. In addition, 50 ng/mL of IL-8 induced cell migration, and a neutralizing antibody of integrin beta-1 inhibited the migration. IL-8 also activated expression of integrin beta-1. Furthermore, IL-8 reduced the Aggregatibacter actinomycetemcomitans-induced increase in caspase-3 expression in HGEC. IL-8 may facilitate the migration of gingival junctional epithelium by enhancing DNA synthesis, migration and preventing apoptosis of gingival epithelial cells. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Extracellular calcium stimulates DNA synthesis in synergism with zinc, insulin and insulin-like growth factor I in fibroblasts.

    PubMed

    Huang, J S; Mukherjee, J J; Chung, T; Crilly, K S; Kiss, Z

    1999-12-01

    In serum-starved mouse NIH 3T3 fibroblasts cultured in 1.8 mM Ca2+-containing medium, addition of 0.75-2 mM extra Ca2+ stimulated DNA synthesis in synergism with zinc (15-60 microM), insulin and insulin-like growth factor I. Extra Ca2+ stimulated phosphorylation/activation of p42/p44 mitogen-activated protein kinases by an initially (10 min) zinc-independent mechanism; however, insulin, and particularly zinc, significantly prolonged Ca2+-induced mitogen-activated protein kinase phosphorylation. In addition, extra Ca2+ activated p70 S6 kinase by a zinc-dependent mechanism and enhanced the stimulatory effect of zinc on choline kinase activity. Insulin and insulin-like growth factor I also commonly increased both p70 S6 kinase and choline kinase activities. In support of the role of the choline kinase product phosphocholine in the mediation of mitogenic Ca2+ effects, cotreatments with the choline kinase substrate choline (250 microM) and the choline kinase inhibitor hemicholinium-3 (2 mM) enhanced and inhibited, respectively, the combined stimulatory effect of extra Ca2+ (3.8 mM total) and zinc on DNA synthesis. In various human skin fibroblast lines, 1-2 mM extra Ca2+ also stimulated DNA synthesis in synergism with zinc and insulin. The results show that in various fibroblast cultures, high concentrations of extracellular Ca2+ can collaborate with zinc and certain growth factors to stimulate DNA synthesis. Considering the high concentration of extracellular Ca2+ in the dermal layer, Ca2+ may promote fibroblast growth during wound healing in concert with zinc, insulin growth factor-I insulin, and perhaps other growth factors.

  2. Initiation of DNA synthesis by human thrombin: relationships between receptor binding, enzymic activity, and stimulation of 86Rb+ influx.

    PubMed

    Stiernberg, J; Carney, D H; Fenton, J W; LaBelle, E F

    1984-09-01

    Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human alpha-thrombin was converted to gamma-thrombin, nitro-alpha-thrombin, and diisopropylphospho (DIP)-alpha-thrombin. These derivatives retain either the capacity to bind cell surface alpha-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of alpha-thrombin or the various thrombin derivatives, only alpha-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-alpha-thrombin that saturate the alpha-thrombin receptors (up to 2 micrograms/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-alpha-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either gamma-thrombin or nitro-alpha-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.

  3. Effect of pH on the base-mispairing properties of 5-bromouracil during DNA synthesis

    SciTech Connect

    Driggers, P.H.; Beattie, K.L.

    1988-03-08

    The authors have utilized an electrophoretic assay of misincorporation and autoradiography to investigate the possibility that ionization of 5-bromouracil (BU) may play a role in its mispairing during DNA synthesis in vitro. They examined the effects of increasing pH on the relative rates of formation of BU x G and T x G mispairs during chain elongation catalyzed by various DNA polymerases. For the Klenow fragment of Escherichia coli DNA polymerase I, increasing pH facilitated BU x G mispair formation (relative to T x G mispairing) when BU was present in the template strand. This effect showed a strong dependence on sequence context. Increasing pH had little effect on the relative rate of misincorporation of BrdUMP versus dTMP (at template G) by the Klenow polymerase. Misincorporation opposite template BU residues catalyzed by Maloney murine leukemia virus DNA polymerase and DNA polymerase ..beta.. (Novikoff hepatoma) also increased with pH, but for these two enzymes, there was no apparent dependence on sequence context. With T4 DNA polymerase and E. coli DNA polymerase III holoenzyme, a similar occurrence of BU x G and T x G mispairing during polymerization was observed, whether BU was present in the template or in the incoming nucleotide, and there was little effect of pH. The results reported here are consistent with a mispairing mechanism for template BU wherein the anionic form of the base mispairs with G.

  4. Synthesis of 4-aminophthalimide and 2,4-diaminopyrimidine C-nucleosides as isosteric fluorescent DNA base substitutes.

    PubMed

    Weinberger, Michael; Berndt, Falko; Mahrwald, Rainer; Ernsting, Nikolaus P; Wagenknecht, Hans-Achim

    2013-03-15

    The 4-aminophthalimide C-nucleoside 1 was designed as an isosteric DNA base surrogate, and a synthetic route to nucleoside 1 together with the 2,4-diaminopyrimidine-C-nucleoside 2 as a potential counterbase was worked out. The key steps in both synthetic routes represent a stereoselective Heck-type palladium-catalyzed cross-coupling with 2'-deoxyribofuranoside glycal followed by stereoselective reduction with NaBH(OAc)3. The nucleoside 1 shows a solvatofluorochromic behavior and significantly red-shifted fluorescence in solvents of high polarity and with hydrogen bonding capabilities. Both nucleosides 1 and 2 can be further processed to the corresponding phosphoramidite as DNA building blocks that allow incorporation of these chromophores as artificial DNA bases by automated DNA synthesis. The combination of the poor stacking properties of 1 and the hydrogen bonding interface at the phthalimide functionality that does not fit to any of natural DNA bases in the counterstrand yields destabilization of the duplex by 4-11 °C. The fluorescence of 1 in a representative double stranded DNA is characterized by a large Stokes' shift and a quantum yield of approximately 12%. These are remarkable optical properties considering the very small size of the chromophore and indicate a high potential of these nucleoside analogues for fluorescent DNA analytics and imaging.

  5. Interacting RNA polymerase motors on a DNA track: Effects of traffic congestion and intrinsic noise on RNA synthesis

    NASA Astrophysics Data System (ADS)

    Tripathi, Tripti; Chowdhury, Debashish

    2008-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechanochemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two different measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions.

  6. Interacting RNA polymerase motors on a DNA track: effects of traffic congestion and intrinsic noise on RNA synthesis.

    PubMed

    Tripathi, Tripti; Chowdhury, Debashish

    2008-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechanochemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two different measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions.

  7. Days weaving the lagging strand synthesis of DNA - A personal recollection of the discovery of Okazaki fragments and studies on discontinuous replication mechanism.

    PubMed

    Okazaki, Tsuneko

    2017-01-01

    At DNA replication forks, the overall growth of the antiparallel two daughter DNA chains appears to occur 5'-to-3' direction in the leading-strand and 3'-to-5' direction in the lagging-strand using enzyme system only able to elongate 5'-to-3' direction, and I describe in this review how we have analyzed and proved the lagging strand multistep synthesis reactions, called Discontinuous Replication Mechanism, which involve short RNA primer synthesis, primer-dependent short DNA chains (Okazaki fragments) synthesis, primer removal from the Okazaki fragments and gap filling between Okazaki fragments by RNase H and DNA polymerase I, and long lagging strand formation by joining between Okazaki fragments with DNA ligase.

  8. Days weaving the lagging strand synthesis of DNA — A personal recollection of the discovery of Okazaki fragments and studies on discontinuous replication mechanism —

    PubMed Central

    OKAZAKI, Tsuneko

    2017-01-01

    At DNA replication forks, the overall growth of the antiparallel two daughter DNA chains appears to occur 5′-to-3′ direction in the leading-strand and 3′-to-5′ direction in the lagging-strand using enzyme system only able to elongate 5′-to-3′ direction, and I describe in this review how we have analyzed and proved the lagging strand multistep synthesis reactions, called Discontinuous Replication Mechanism, which involve short RNA primer synthesis, primer-dependent short DNA chains (Okazaki fragments) synthesis, primer removal from the Okazaki fragments and gap filling between Okazaki fragments by RNase H and DNA polymerase I, and long lagging strand formation by joining between Okazaki fragments with DNA ligase. PMID:28496054

  9. Synthesis of core-shell gold coated magnetic nanoparticles and their interaction with thiolated DNA.

    PubMed

    Robinson, Ian; Tung, Le D; Maenosono, Shinya; Wälti, Christoph; Thanh, Nguyen T K

    2010-12-01

    Core-shell magnetic nanoparticles have received significant attention recently and are actively investigated owing to their large potential for a variety of applications. Here, the synthesis and characterization of bimetallic nanoparticles containing a magnetic core and a gold shell are discussed. The gold shell facilitates, for example, the conjugation of thiolated biological molecules to the surface of the nanoparticles. The composite nanoparticles were produced by the reduction of a gold salt on the surface of pre-formed cobalt or magnetite nanoparticles. The synthesized nanoparticles were characterized using ultraviolet-visible absorption spectroscopy, transmission electron microscopy, energy dispersion X-ray spectroscopy, X-ray diffraction and super-conducting quantum interference device magnetometry. The spectrographic data revealed the simultaneous presence of cobalt and gold in 5.6±0.8 nm alloy nanoparticles, and demonstrated the presence of distinct magnetite and gold phases in 9.2±1.3 nm core-shell magnetic nanoparticles. The cobalt-gold nanoparticles were of similar size to the cobalt seed, while the magnetite-gold nanoparticles were significantly larger than the magnetic seeds, indicating that different processes are responsible for the addition of the gold shell. The effect on the magnetic properties by adding a layer of gold to the cobalt and magnetite nanoparticles was studied. The functionalization of the magnetic nanoparticles is demonstrated through the conjugation of thiolated DNA to the gold shell.

  10. Synthesis and investigation of the specific activity of the DNA-doxorubicin conjugates

    NASA Astrophysics Data System (ADS)

    Kokorev, A. V.; Zaborovskiy, A. V.; Kotlyarov, A. A.; Balykova, L. A.; Malkina, M. A.; Kargina, I. V.; Gromova, E. V.; Medvezhonkov, V. Yu; Gurevich, K. G.; Shchukin, S. A.; Pyataev, N. A.

    2017-01-01

    In the present work, the method of obtaining the conjugate of the anticancer chemotherapeutic agent doxorubicin to the exogenous double-stranded DNA of the sturgeons is proposed (the source: commercial drug “Derinat”). The optimal conditions for synthesis of conjugate (pH, temperature and the mass ratio of the components), ensuring the highest degree of binding the chemotherapeutic agent to a carrier, were picked out. Clearing the conjugate from the non-encapsulated chemotherapeutic agent was being made by ultrafiltration method. The investigation of the toxicity and specific antineoplastic activity of the synthesized complex was conducted. The performance of the drug toxicity were established on the intact mice in compliance with the accepted standards. The antineoplastic activity was evaluated upon the Tumor Growth Inhibition Index and Metastasis Inhibition Index on mice with the transplanted Lewis lung carcinoma (LLC). It was demonstrated that the conjugate toxicity is approximately lower that the one of the unconjugated doxorubicin (LD 50 was equal 14.6 mg/kg and 9.9 mg/kg for the conjugate and doxorubicin, respectively). The specific antineoplastic activity was investigated in equitoxic doses of the drug. It was established that the conjugate being administered in equitoxic doses possesses a stronger antineoplastic activity, than the water-soluble drug (maximum 35% more as to the tumor volume and 51% more as to the Tumor Growth Inhibition index).

  11. Stimulation of DNA synthesis by ascorbate in cultures of articular chondrocytes

    SciTech Connect

    Krystal, G.; Morris, G.M.; Sokoloff, L.

    1982-03-01

    The addition of 0.2 mM Na L-ascorbate increased the incorporation of 3H-thymidine by rabbit articular chondrocytes in cell and organ culture. The stimulatory response of explants to ascorbate was potentiated by pretreatment of the cartilage with 0.2% clostridial collagenase (type 1) or trypsin for 15-30 minutes. In explants there was a latent period of 3 to 4 days before increased labeling of the nuclei could be detected. The effect was transient and declined after 8 days of culture. It was more evident in organ cultures of immature (3-month-old) than 2- to 3-year-old rabbits. Age differences were not detected in cell cultures. Explants of adult human articular cartilage were stimulated by ascorbate when the medium was supplemented with 10% fresh human serum but not by fetal bovine serum. The findings indicated that synthesis of DNA by articular chondrocytes in situ is regulated by responsiveness of the cells proper to compounds such as vitamin C, by properties of the extracellular matrix, and by factors in the serum. Ascorbate was cytotoxic at concentrations greater than 0.2 mM in the presence of certain batches of serum.

  12. Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins.

    PubMed

    McIntyre, Justyna; Woodgate, Roger

    2015-05-01

    Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo.

  13. Dihydrochelerythrine and its derivatives: Synthesis and their application as potential G-quadruplex DNA stabilizing agents.

    PubMed

    Malhotra, Rajesh; Rarhi, Chhanda; Diveshkumar, K V; Barik, Rajib; D'cunha, Ruhee; Dhar, Pranab; Kundu, Mrinalkanti; Chattopadhyay, Subrata; Roy, Subho; Basu, Sourav; Pradeepkumar, P I; Hajra, Saumen

    2016-07-01

    A convenient route was envisaged toward the synthesis of dihydrochelerythrine (DHCHL), 4 by intramolecular Suzuki coupling of 2-bromo-N-(2-bromobenzyl)-naphthalen-1-amine derivative 5 via in situ generated arylborane. This compound was converted to (±)-6-acetonyldihydrochelerythrine (ADC), 3 which was then resolved by chiral prep-HPLC. Efficiency of DHCHL for the stabilization of promoter quadruplex DNA structures and a comparison study with the parent natural alkaloid chelerythrine (CHL), 1 was performed. A thorough investigation was carried out to assess the quadruplex binding affinity by using various biophysical and biochemical studies and the binding mode was explained by using molecular modeling and dynamics studies. Results clearly indicate that DHCHL is a strong G-quadruplex stabilizer with affinity similar to that of the parent alkaloid CHL. Compounds ADC and DHCHL were also screened against different human cancer cell lines. Among the cancer cells, (±)-ADC and its enantiomers showed varied (15-48%) inhibition against human colorectal cell line HCT116 and breast cancer cell line MDA-MB-231 albeit low enantio-specificity in the inhibitory effect; whereas DHCHL showed 30% inhibition against A431 cell line only, suggesting the compounds are indeed cancer tissue specific.

  14. EGF stimulates rat spermatogonial DNA synthesis in seminiferous tubule segments in vitro.

    PubMed

    Wahab-Wahlgren, Aida; Martinelle, Nina; Holst, Mikael; Jahnukainen, Kirsi; Parvinen, Martti; Söder, Olle

    2003-03-28

    Epidermal growth factor (EGF) superfamily of peptide growth factors (EGF-GFs) plays a role in male germ cell development, but the precise function is yet to be defined. The present study shows that EGF-GFs stimulate spermatogonial proliferation in vitro. The EGF-GF ligands, EGF, transforming growth factor-alpha and betacellulin all stimulated DNA synthesis in microdissected stage I segments of rat testis seminiferous tubules in vitro, as revealed by 3H-thymidine incorporation and 5-bromo-2'-deoxyuridine (BrdU) labeling. A fourfold increase over control of BrdU labeled cells, identified as spermatogonia, was seen after treatment with EGF. RT-PCR analysis revealed that the EGF receptors erbB1, erbB2, erbB3 and erbB4 were expressed at all stages of the spermatogenic wave, whereas differential expression was found in isolated Leydig, Sertoli and peritubular cells. The results show that EGF-GFs is spermatogonial growth factor(s) in vitro, although we have not discriminated between a direct action and an indirect effect via somatic cells. We suggest that EGF-GFs is involved in the paracrine control of spermatogenesis in vivo.

  15. Stimulators and inhibitors of lymphocyte DNA synthesis in supernatants from human lymphoid cell lines.

    PubMed

    Vesole, D H; Goust, J M; Fett, J W; Fudenberg, H H

    1979-09-01

    Some T and B lymphoid cell lines (LCL) were found to secrete into their supernatants a substance able to stimulate lymphocyte proliferation. This substance produced an increase in [3H]thymidine uptake by mononuclear cells when added to unstimulated cultures (mitogenic effect) or when added to cultures stimulated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) (potentiating effect). When complete supernatants were used, the potentiating effect was sometimes masked by an inhibitor of DNA synthesis. Fractionation on Sephadex G-100 separated these two activities. The stimulatory substance eluted at a m.w. range of 15,000 to 30,000, and the inhibitor eluted with the albumin peak. B cells with or without monocytes were the most sensitive to the mitogenic effect, whereas T cells were unaffected. Responses to PHA and PWM were potentiated when T cells were present, but the maximum effect was observed when the proportion of T cells was less than 50%. The stimulatory material may be similar to lymphocyte mitogenic factor and may function as a T cell-replacing factor in B cell stimulation.

  16. Synthesis, spectroscopic characterization, biological screenings, DNA binding study and POM analyses of transition metal carboxylates

    NASA Astrophysics Data System (ADS)

    Uddin, Noor; Sirajuddin, Muhammad; Uddin, Nizam; Tariq, Muhammad; Ullah, Hameed; Ali, Saqib; Tirmizi, Syed Ahmed; Khan, Abdur Rehman

    2015-04-01

    This article contains the synthesis of a novel carboxylic acid derivative, its transition metal complexes and evaluation of biological applications. Six carboxylate complexes of transition metals, Zn(II) and Hg(II), have been successfully synthesized and characterized by FT-IR and NMR (1H, 13C). The ligand, HL, (4-[(2,6-Diethylphenyl)amino]-4-oxobutanoic acid) was also characterized by single crystal X-ray analysis. The complexation occurs via oxygen atoms of the carboxylate moiety. FT-IR date show the bidentate nature of the carboxylate moiety of the ligand as the Δν value in all complexes is less than that of the free ligand. The ligand and its complexes were screened for antifungal and antileishmanial activities. The results showed that the ligand and its complexes are active with few exceptions. UV-visible spectroscopy and viscometry results reveal that the ligand and its complexes interact with the DNA via intercalative mode of interaction. A new and efficient strategy to identify the pharmacophores and anti-pharmacophores sites in carboxylate derivatives for the antibacterial/antifungal activity using Petra, Osiris and Molinspiration (POM) analyses was also carried out.

  17. Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, β, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

    PubMed

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

    2016-03-01

    Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo(-) (KF exo(-)) and DNA polymerases (Pols) α, β, ζ, η, ι, κ and REV1. We found that KF exo(-) and Pols α, β, ι and REV1 inserted one nucleotide opposite the 3' Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3' or 5' Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society.

  18. Replication of phage phi 29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length phi 29 DNA.

    PubMed Central

    Blanco, L; Salas, M

    1985-01-01

    A system that replicates bacteriophage phi 29 DNA with protein p3 covalently attached to the two 5' ends, using as the only proteins the phi 29 DNA polymerase and the terminal protein, is described. Restriction analysis of the 32P-labeled DNA synthesized in vitro showed that all phi 29 DNA fragments were labeled. Analysis by alkaline sucrose gradient centrifugation of the DNA labeled during a 10-min pulse showed that, after a 20-min chase, about half of the DNA molecules had reached apparently full-length phi 29 DNA (approximately equal to 18,000 nucleotides). Ammonium ions strongly stimulated phi 29 DNA-protein p3 replication, the effect being due to stimulation of the initiation reaction. ATP was not required for phi 29 DNA-protein p3 replication, either in the initiation or elongation steps. The results show that the phi 29 DNA polymerase functions, not only in the formation of the p3-dAMP covalent initiation complex but also in the elongation of the latter, as the only DNA polymerase to produce full-length phi 29 DNA. Images PMID:3863101

  19. Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance

    PubMed Central

    Merritt, Kristen K; Bradley, Kevin M; Hutter, Daniel; Matsuura, Mariko F; Rowold, Diane J

    2014-01-01

    Summary Background: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide “letters”, the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson–Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. Results: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson–Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2’-deoxyisocytidine and 2’-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with

  20. Effects of N(2)-alkylguanine, O(6)-alkylguanine, and abasic lesions on DNA binding and bypass synthesis by the euryarchaeal B-family DNA polymerase vent (exo(-)).

    PubMed

    Lim, Seonhee; Song, Insil; Guengerich, F Peter; Choi, Jeong-Yun

    2012-08-20

    Archaeal and eukaryotic B-family DNA polymerases (pols) mainly replicate chromosomal DNA but stall at lesions, which are often bypassed with Y-family pols. In this study, a B-family pol Vent (exo(-)) from the euryarchaeon Thermococcus litoralis was studied with three types of DNA lesions-N(2)-alkylG, O(6)-alkylG, and an abasic (AP) site-in comparison with a model Y-family pol Dpo4 from Sulfolobus solfataricus, to better understand the effects of various DNA modifications on binding, bypass efficiency, and fidelity of pols. Vent (exo(-)) readily bypassed N(2)-methyl(Me)G and O(6)-MeG, but was strongly blocked at O(6)-benzyl(Bz)G and N(2)-BzG, whereas Dpo4 efficiently bypassed N(2)-MeG and N(2)-BzG and partially bypassed O(6)-MeG and O(6)-BzG. Vent (exo(-)) bypassed an AP site to an extent greater than Dpo4, corresponding with steady-state kinetic data. Vent (exo(-)) showed ~110-, 180-, and 300-fold decreases in catalytic efficiency (k(cat)/K(m)) for nucleotide insertion opposite an AP site, N(2)-MeG, and O(6)-MeG but ~1800- and 5000-fold decreases opposite O(6)-BzG and N(2)-BzG, respectively, as compared to G, whereas Dpo4 showed little or only ~13-fold decreases opposite N(2)-MeG and N(2)-BzG but ~260-370-fold decreases opposite O(6)-MeG, O(6)-BzG, and the AP site. Vent (exo(-)) preferentially misinserted G opposite N(2)-MeG, T opposite O(6)-MeG, and A opposite an AP site and N(2)-BzG, while Dpo4 favored correct C insertion opposite those lesions. Vent (exo(-)) and Dpo4 both bound modified DNAs with affinities similar to unmodified DNA. Our results indicate that Vent (exo(-)) is as or more efficient as Dpo4 in synthesis opposite O(6)-MeG and AP lesions, whereas Dpo4 is much or more efficient opposite (only) N(2)-alkylGs than Vent (exo(-)), irrespective of DNA-binding affinity. Our data also suggest that Vent (exo(-)) accepts nonbulky DNA lesions (e.g., N(2)- or O(6)-MeG and an AP site) as manageable substrates despite causing error-prone synthesis, whereas Dpo4

  1. Decay of ccc-DNA marks persistence of intrahepatic viral DNA synthesis under tenofovir in HIV-HBV co-infected patients.

    PubMed

    Boyd, Anders; Lacombe, Karine; Lavocat, Fabien; Maylin, Sarah; Miailhes, Patrick; Lascoux-Combe, Caroline; Delaugerre, Constance; Girard, Pierre-Marie; Zoulim, Fabien

    2016-10-01

    In the presence of highly-potent antivirals, persistence of hepatitis B virus (HBV) is most well-characterized by covalently-closed circular DNA (cccDNA) and total intrahepatic DNA (IH-DNA). We sought to determine how antiviral therapy could affect their levels during human immunodeficiency virus (HIV)-HBV co-infection. Sixty co-infected patients from a well-defined cohort with ⩾1 liver biopsy were studied. HBV cccDNA and total IH-DNA were extracted from biopsies and quantified by real-time PCR. Factors associated with intrahepatic viral load were determined using mixed-effect linear regression and half-life viral kinetics during reconstructed follow-up using non-linear exponential decay models. At biopsy, 35 (58.3%) patients were hepatitis B "e" antigen (HBeAg)-positive and 33 (55.0%) had detectable plasma HBV-DNA (median=4.58log10IU/ml, IQR=2.95-7.43). Overall, median cccDNA was -0.95log10copies/cell (IQR=-1.70, -0.17) and total IH-DNA was 0.27log10copies/cell (IQR=-0.39, 2.00). In multivariable analysis, significantly lower levels of cccDNA and total IH-DNA were observed in patients with HBeAg-negative serology, nadir CD4(+) cell counts >250/mm(3), and longer cumulative TDF-duration, but not lamivudine- or adefovir-duration. In post-hoc analysis using reconstructed TDF-duration (median 29.6months, IQR=15.0-36.1, n=31), average half-life of cccDNA was estimated at 9.2months (HBeAg-positive=8.6, HBeAg-negative=26.2) and total IH DNA at 5.8months (HBeAg-positive=1.3, HBeAg-negative=13.6). Intrahepatic viral loads remained detectable for all patients, even with prolonged TDF-exposure. In co-infection, TDF-use is associated with lower levels of HBV replication intermediates and cccDNA. Slow decay of intrahepatic viral loads underscores that TDF is unable to completely block intracellular viral DNA synthesis, which possibly accounts for continuous replenishment of the cccDNA pool. Chronic hepatitis B virus (HBV) is a persistent infection, while the only real way of

  2. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis.

    PubMed

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia; Altmannova, Veronika; Sebesta, Marek; Pacesa, Martin; Fugger, Kasper; Sorensen, Claus Storgaard; Lee, Marietta Y W T; Haracska, Lajos; Krejci, Lumir

    2016-04-20

    Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.

  3. Compaction agent clarification of microbial lysates

    NASA Technical Reports Server (NTRS)

    DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

  4. Compaction agent clarification of microbial lysates

    NASA Technical Reports Server (NTRS)

    DeWalt, Brad W.; Murphy, Jason C.; Fox, George E.; Willson, Richard C.

    2003-01-01

    Recombinant proteins are often purified from microbial lysates containing high concentrations of nucleic acids. Pre-purification steps such as nuclease addition or precipitation with polyethyleneimine or ammonium sulfate are normally required to reduce viscosity and to eliminate competing polyanions before anion exchange chromatography. We report that small polycationic compaction agents such as spermine selectively precipitate nucleic acids during or after Escherichia coli lysis, allowing DNA and RNA to be pelleted with the insoluble cell debris. Analysis by spectrophotometry and protein assay confirmed a significant reduction in the concentration of nucleic acids present, with preservation of protein. Lysate viscosity is greatly reduced, facilitating subsequent processing. We have used 5mM spermine to remove nucleic acids from E. coli lysate in the purification of a hexahistidine-tagged HIV reverse transcriptase.

  5. Synthesis of cobalt ferrite core/metallic shell nanoparticles for the development of a specific PNA/DNA biosensor.

    PubMed

    Pita, Marcos; Abad, José María; Vaz-Dominguez, Cristina; Briones, Carlos; Mateo-Martí, Eva; Martín-Gago, José Angel; Morales, Maria del Puerto; Fernández, Víctor M

    2008-05-15

    Controlled synthesis of cobalt ferrite superparamagnetic nanoparticles covered with a gold shell has been achieved by an affinity and trap strategy. Magnetic nanoparticles are functionalized with a mixture of amino and thiol groups that facilitate the electrostatic attraction and further chemisorption of gold nanoparticles, respectively. Using these nanoparticles as seeds, a complete coating shell is achieved by gold salt-iterative reduction leading to monodisperse water-soluble gold-covered magnetic nanoparticles, with an average diameter ranging from 21 to 29 nm. These constitute a versatile platform for immobilization of biomolecules via thiol chemistry, which is exemplified by the immobilization of peptide nucleic acid (PNA) oligomers that specifically hybridize with complementary DNA molecules in solution. Hybridation with DNA probes has been measured using Rhodamine 6G fluorescence marker and the detection of a single nucleotide mutation has been achieved. These results suggest the PNA-nanoparticles application as a biosensor for DNA genotyping avoiding commonly time-consuming procedures employed.

  6. Synthesis of trifunctional PNA-benzophenone derivatives for mitochondrial targeting, selective DNA binding, and photo-cross-linking.

    PubMed

    Ross, Günther F; Smith, Paul M; McGregor, Alistair; Turnbull, Douglass M; Lightowlers, Robert N

    2003-01-01

    Mutations in mitochondrial DNA (mtDNA) cause a variety of human pathologies. In many patients, mutated and wild-type mtDNAs coexist in the same cell, a situation termed mtDNA heteroplasmy. In the absence of standard therapies for these disorders, a genetic strategy for treatment has been proposed whereby replication of mutated mtDNA is inhibited by the selective hybridization of a nucleic acid derivative, allowing propagation of the wild-type genome and correction of the associated defects. To allow for selective binding under physiological conditions, peptide nucleic acids (PNA) are being used. Two other problems, however, have to be resolved: mitochondrial import and attachment of the PNA to the target DNA to inhibit replication. Mitochondrial localization can be achieved by the addition of a caged lipophilic cation and addition of a photo-cross-linking reagent should facilitate covalent attachment. We therefore report the synthesis of benzophenone-PNA derivatives carrying a triphenylphosphonium moiety and demonstrate irreversible binding selectivity between two DNA molecules that differ by a single nucleotide.

  7. Exploration of cellular DNA lesion, DNA-binding and biocidal ordeal of novel curcumin based Knoevenagel Schiff base complexes incorporating tryptophan: Synthesis and structural validation

    NASA Astrophysics Data System (ADS)

    Chandrasekar, Thiravidamani; Raman, Natarajan

    2016-07-01

    A few novel Schiff base transition metal complexes of general formula [MLCl] (where, L = Schiff base, obtained by the condensation reaction of Knoevenagel condensate of curcumin, L-tryptophan and M = Cu(II), Ni(II), Co(II), and Zn(II)), were prepared by stencil synthesis. They were typified using UV-vis, IR, EPR spectral techniques, micro analytical techniques, magnetic susceptibility and molar conductivity. Geometry of the metal complexes was examined and recognized as square planar. DNA binding and viscosity studies revealed that the metal(II) complexes powerfully bound via an intercalation mechanism with the calf thymus DNA. Gel-electrophoresis technique was used to investigate the DNA cleavage competence of the complexes and they establish to approve the cleavage of pBR322 DNA in presence of oxidant H2O2. This outcome inferred that the synthesized complexes showed better nuclease activity. Moreover, the complexes were monitored for antimicrobial activities. The results exposed that the synthesized compounds were forceful against all the microbes under exploration.

  8. Boron Clusters as a Platform for New Materials: Synthesis of Functionalized o-Carborane (C2 B10 H12 ) Derivatives Incorporating DNA Fragments.

    PubMed

    Janczak, Slawomir; Olejniczak, Agnieszka; Balabańska, Sandra; Chmielewski, Marcin K; Lupu, Marius; Viñas, Clara; Lesnikowski, Zbigniew J

    2015-10-19

    A synthetic strategy for functionalization of the three vertices of o-carborane and the attachment of the obtained triped to the solid support was developed. Further functionalization of the triped with short DNA sequences by automated DNA synthesis was achieved. The proposed methodology is a first example of boron cluster chemistry on a solid support opening new perspectives in boron cluster functionalization.

  9. Clickable Cγ-azido(methylene/butylene) peptide nucleic acids and their clicked fluorescent derivatives: synthesis, DNA hybridization properties, and cell penetration studies.

    PubMed

    Jain, Deepak R; Ganesh, Krishna N

    2014-07-18

    Synthesis, characterization, and DNA complementation studies of clickable C(γ)-substituted methylene (azm)/butylene (azb) azido PNAs show that these analogues enhance the stability of the derived PNA:DNA duplexes. The fluorescent PNA oligomers synthesized by their click reaction with propyne carboxyfluorescein are seen to accumulate around the nuclear membrane in 3T3 cells.

  10. Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword.

    PubMed

    Jaszczur, Malgorzata; Bertram, Jeffrey G; Robinson, Andrew; van Oijen, Antoine M; Woodgate, Roger; Cox, Michael M; Goodman, Myron F

    2016-04-26

    1953, the year of Watson and Crick, bore witness to a less acclaimed yet highly influential discovery. Jean Weigle demonstrated that upon infection of Escherichia coli, λ phage deactivated by UV radiation, and thus unable to form progeny, could be reactivated by irradiation of the bacterial host. Evelyn Witkin and Miroslav Radman later revealed the presence of the SOS regulon. The more than 40 regulon genes are repressed by LexA protein and induced by the coproteolytic cleavage of LexA, catalyzed by RecA protein bound to single-stranded DNA, the RecA* nucleoprotein filament. Several SOS-induced proteins are engaged in repairing both cellular and extracellular damaged DNA. There's no "free lunch", however, because error-free repair is accompanied by error-prone translesion DNA synthesis (TLS), involving E. coli DNA polymerase V (UmuD'2C) and RecA*. This review describes the biochemical mechanisms of pol V-mediated TLS. pol V is active only as a mutasomal complex, pol V Mut = UmuD'2C-RecA-ATP. RecA* donates a single RecA subunit to pol V. We highlight three recent insights. (1) pol V Mut has an intrinsic DNA-dependent ATPase activity that governs polymerase binding and dissociation from DNA. (2) Active and inactive states of pol V Mut are determined at least in part by the distinct interactions between RecA and UmuC. (3) pol V is activated by RecA*, not at a blocked replisome, but at the inner cell membrane.

  11. Bloodstream form-specific up-regulation of silent vsg expression sites and procyclin in Trypanosoma brucei after inhibition of DNA synthesis or DNA damage.

    PubMed

    Sheader, Karen; te Vruchte, Daniëlle; Rudenko, Gloria

    2004-04-02

    The African trypanosome Trypanosoma brucei transcribes the active variant surface glycoprotein (VSG) gene from one of about 20 VSG expression sites (ESs). In order to study ES control, we made reporter lines with a green fluorescent protein gene inserted behind the promoter of different ESs. We attempted to disrupt the silencing machinery, and we used fluorescence-activated cell sorter analysis for the rapid and sensitive detection of ES up-regulation. We find that a range of treatments that either block nuclear DNA synthesis, like aphidicolin, or modify DNA-like cisplatin and 1-methyl-3-nitro-1-nitrosoguanidine results in up-regulation of silent ESs. Aphidicolin treatment was the most effective, with almost 80% of the cells expressing green fluorescent protein from a silent ES. All of these treatments blocked the cells in S phase. In contrast, a range of toxic chemicals had little or no effect on expression. These included berenil and pentamidine, which selectively cleave the mitochondrial kinetoplast DNA, the metabolic inhibitors suramin and difluoromethylornithine, and the mitotic inhibitor rhizoxin. Up-regulation also affected other RNA polymerase I (pol I) transcription units, as procyclin genes were also up-regulated after cells were treated with either aphidicolin or DNA-modifying agents. Strikingly, this up-regulation of silent pol I transcription units was bloodstream form-specific and was not observed in insect form T. brucei. We postulate that the redistribution of a limiting bloodstream form-specific factor involved in both silencing and DNA repair results in the derepression of normally silenced pol I transcription units after DNA damage.

  12. Synthesis and study of antiproliferative, antitopoisomerase II, DNA-intercalating and DNA-damaging activities of arylnaphthalimides.

    PubMed

    Quintana-Espinoza, Patricia; García-Luis, Jonay; Amesty, Angel; Martín-Rodríguez, Patricia; Lorenzo-Castrillejo, Isabel; Ravelo, Angel G; Fernández-Pérez, Leandro; Machín, Félix; Estévez-Braun, Ana

    2013-11-01

    A series of arylnaphthalimides were designed and synthesized to overcome the dose-limiting cytotoxicity of N-acetylated metabolites arising from amonafide, the prototypical antitumour naphthalimide whose biomedical properties have been related to its ability to intercalate the DNA and poison the enzyme Topoisomerase II. Thus, these arylnaphthalimides were first evaluated for their antiproliferative activity against two tumour cell lines and for their antitopoisomerase II in vitro activities, together with their ability to intercalate the DNA in vitro and also through docking modelization. Then, the well-known DNA damage response in Saccharomyces cerevisiae was employed to critically evaluate whether these novel compounds can damage the DNA in vivo. By performing all these assays we conclude that the 5-arylsubstituted naphthalimides not only keep but also improve amonafide's biological activities. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. The Affinity of EBNA1 for Its Origin of DNA Synthesis Is a Determinant of the Origin's Replicative Efficiency▿ †

    PubMed Central

    Lindner, Scott E.; Zeller, Krisztina; Schepers, Aloys; Sugden, Bill

    2008-01-01

    Epstein-Barr virus (EBV) replicates its genome as a licensed plasmid in latently infected cells. Although replication of this plasmid is essential for EBV latent infection, its synthesis still fails for 16% of the templates in S phase. In order to understand these failures, we sought to determine whether the affinity of the initiator protein (EBNA1) for its binding sites in the origin affects the efficiency of plasmid replication. We have answered this question by using several engineered origins modeled upon the arrangement of EBNA1-binding sites found in DS, the major plasmid origin of EBV. The human TRF2 protein also binds to half-sites in DS and increases EBNA1's affinity for its own sites; we therefore also tested origin efficiency in the presence or absence of these sites. We have found that if TRF2-half-binding sites are present, the efficiency of supporting the initiation of DNA synthesis and of establishing a plasmid bearing that origin directly correlates with the affinity of EBNA1 for that origin. Moreover, the presence of TRF2-half-binding sites also increases the average level of EBNA1 and ORC2 bound to those origins in vivo, as measured by chromatin immunoprecipitation. Lastly, we have created an origin of DNA synthesis from high-affinity EBNA1-binding sites and TRF2-half-binding sites that functions severalfold more efficiently than does DS. This finding indicates that EBV has selected a submaximally efficient origin of DNA synthesis for the latent phase of its life cycle. This enhanced origin could be used practically in human gene vectors to improve their efficiency in therapy and basic research. PMID:18385243

  14. The affinity of EBNA1 for its origin of DNA synthesis is a determinant of the origin's replicative efficiency.

    PubMed

    Lindner, Scott E; Zeller, Krisztina; Schepers, Aloys; Sugden, Bill

    2008-06-01

    Epstein-Barr virus (EBV) replicates its genome as a licensed plasmid in latently infected cells. Although replication of this plasmid is essential for EBV latent infection, its synthesis still fails for 16% of the templates in S phase. In order to understand these failures, we sought to determine whether the affinity of the initiator protein (EBNA1) for its binding sites in the origin affects the efficiency of plasmid replication. We have answered this question by using several engineered origins modeled upon the arrangement of EBNA1-binding sites found in DS, the major plasmid origin of EBV. The human TRF2 protein also binds to half-sites in DS and increases EBNA1's affinity for its own sites; we therefore also tested origin efficiency in the presence or absence of these sites. We have found that if TRF2-half-binding sites are present, the efficiency of supporting the initiation of DNA synthesis and of establishing a plasmid bearing that origin directly correlates with the affinity of EBNA1 for that origin. Moreover, the presence of TRF2-half-binding sites also increases the average level of EBNA1 and ORC2 bound to those origins in vivo, as measured by chromatin immunoprecipitation. Lastly, we have created an origin of DNA synthesis from high-affinity EBNA1-binding sites and TRF2-half-binding sites that functions severalfold more efficiently than does DS. This finding indicates that EBV has selected a submaximally efficient origin of DNA synthesis for the latent phase of its life cycle. This enhanced origin could be used practically in human gene vectors to improve their efficiency in therapy and basic research.

  15. Effects of 8-halo-7-deaza-2'-deoxyguanosine triphosphate on DNA synthesis by DNA polymerases and cell proliferation.

    PubMed

    Yin, Yizhen; Sasaki, Shigeki; Taniguchi, Yosuke

    2016-08-15

    8-OxodG (8-oxo-2'-deoxyguanosine) is representative of nucleoside damage and shows a genotoxicity. To significantly reveal the contributions of 7-NH and C8-oxygen to the mutagenic effect of 8-oxodG by DNA polymerases, we evaluated the effects of the 8-halo-7-deaza-dG (8-halogenated 7-deaza-2'-deoxyguanosine) derivatives by DNA polymerases. 8-Halo-7-deaza-dGTPs were poorly incorporated by both KF(exo(-)) and human DNA polymerase β opposite dC or dA into the template DNA. Furthermore, it was found that KF(exo(-)) was very sensitive to the introduction of the C8-halogen, while polymerase β can accommodate the C8-halogen resulting in an efficient dCTP insertion opposite the 8-halo-7-deaza-dG in the template DNA. These results indicate that strong hydrogen bonding between 7-NH in the 8-oxo-G nucleobase and 1-N in the adenine at the active site of the DNA polymerase is required for the mutagenic effects. Whereas, I-deaza-dGTP shows an antiproliferative effect for the HeLa cells, suggesting that it could become a candidate as a new antitumor agent. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. The telomerase essential N-terminal domain promotes DNA synthesis by stabilizing short RNA–DNA hybrids

    PubMed Central

    Akiyama, Benjamin M.; Parks, Joseph W.; Stone, Michael D.

    2015-01-01

    Telomerase is an enzyme that adds repetitive DNA sequences to the ends of chromosomes and consists of two main subunits: the telomerase reverse transcriptase (TERT) protein and an associated telomerase RNA (TER). The telomerase essential N-terminal (TEN) domain is a conserved region of TERT proposed to mediate DNA substrate interactions. Here, we have employed single molecule telomerase binding assays to investigate the function of the TEN domain. Our results reveal telomeric DNA substrates bound to telomerase exhibit a dynamic equilibrium between two states: a docked conformation and an alternative conformation. The relative stabilities of the docked and alternative states correlate with the number of basepairs that can be formed between the DNA substrate and the RNA template, with more basepairing favoring the docked state. The docked state is further buttressed by the TEN domain and mutations within the TEN domain substantially alter the DNA substrate structural equilibrium. We propose a model in which the TEN domain stabilizes short RNA–DNA duplexes in the active site of the enzyme, promoting the docked state to augment telomerase processivity. PMID:25940626

  17. Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases.

    PubMed

    Günther Sillero, María A; de Diego, Anabel; Pérez-Zúñiga, Francisco J; Sillero, Antonio

    2008-05-15

    T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP beta,gamma-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH(3))p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K(m) values and the rate of synthesis k(cat) (s(-1)), determined for the following compounds were, respectively: etidronate, 0.73+/-0.09 mM and (70+/-10)x10(-3) s(-1); clodronate (pCCl(2)p), 0.08+/-0.01 mM and (4.1+/-0.3)x10(-3) s(-1); methylenebisphosphonate (pCH(2)p), 0.024+/-0.001 mM and (0.6+/-0.1)x10(-3) s(-1); tripolyphosphate (P(3)) (in the synthesis of adenosine 5'-tetraphosphate, p(4)A), 1.30+/-0.30 mM and (6.2+/-1.1)x10(-3) s(-1); (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap(4)G was observed with clodronate but not with pamidronate (pC(OH)(CH(2)-CH(2)-NH(3))p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative (K(m)=0.36+/-0.09 mM and k(cat)=0.15+/-0.02 s(-1)). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.

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

    PubMed Central

    Biocca, S; Cattaneo, A; Calissano, P

    1984-01-01

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

  19. DNA synthesis in pulmonary alveolar macrophages and type II cells: effects of ozone exposure and treatment with alpha-difluoromethylornithine

    SciTech Connect

    Wright, E.S.; White, D.M.; Brady, A.N.; Li, L.C.; D'Arcy, J.B.; Smiler, K.L.

    1987-01-01

    An increase in the number of pulmonary alveolar macrophages (AM) can be induced by a number of toxic insults to the lung, including ozone, an important photochemical oxidant air pollutant. This increase could arise from an influx of monocytes from the vascular or interstitial compartments, or from proliferation of AM in situ. While proliferation of alveolar type II cells after oxidant exposure has been well documented, it is not clear whether AM are also capable of this response. Rats were exposed to air or to 0.12, 0.25, or 0.50 ppm ozone for 1, 2, 3, 7, or 14 d, 20 h/d. The labeling index in both AM and type II cells increased about 10-fold after 2 d of exposure to 0.25 and 0.50 ppm of ozone, but returned to control levels by the end of 1 wk of exposure. These changes closely paralleled the temporal and dose-response characteristics of changes in total lung DNA synthesis. alpha-Difluoromethylornithine (DFMO) administered to rats during a 2-d exposure to 0.50 ppm ozone did not inhibit the ozone-induced increase in labeling index in AM or type II cells, although evidence of inhibition of lung ornithine decarboxylase activity was obtained, and the ozone-induced increase in total lung DNA synthesis was inhibited by 23%. These results suggest that, like type II cells, AM are capable of entering the cell cycle and synthesizing new DNA in situ in response to short-term exposure to environmentally relevant doses of ozone, and that the ozone-induced stimulation of DNA synthesis in these cell types was refractory to inhibition by DFMO.

  20. Unscheduled DNA synthesis in rat pleural mesothelial cells treated with mineral fibres.

    PubMed

    Renier, A; Lévy, F; Pillière, F; Jaurand, M C

    1990-08-01

    Unscheduled DNA synthesis (UDS) was studied in confluent rat pleural mesothelial cells (RPMCs) arrested in G0/G1 with hydroxyurea (HU) and treated with various fibre types, i.e., chrysotile, crocidolite or attapulgite. In addition, the effects of UV light and of benzo[a]pyrene were determined as references. Using autoradiography after [3H]thymidine incorporation ([3H]dThd), RPMCs treated with 4 micrograms/cm2 of chrysotile fibres exhibited a low but significant enhancement of net grains compared to untreated cells. Treatment with higher doses of chrysotile was not possible because of the impairment of microscopic observation due to the presence of the fibres. Using liquid scintillation counting, RPMCs treated with chrysotile or crocidolite showed a significant dose-dependent increase in [3H]dThd incorporation compared to untreated cells. In contrast, attapulgite did not enhance [3H]dThd incorporation compared to untreated cells. Treatment of RPMCs with 1, 2 or 4 micrograms/ml of benzo[a]pyrene resulted in a significant increase in [3H]dThd incorporation. In order to discount a possible role of S cells in the augmentation of [3H]dThd incorporation, despite the presence of 5 mM HU, S cells were counted by autoradiography. Results indicated that the percentage of S cells was similar in asbestos-treated and untreated cultures. Stimulation of the S phase also seems unlikely because treatment of RPMCs with asbestos fibres in the absence of HU resulted in a reduction of [3H]dThd incorporation attributed to an impairment of the S phase by the fibres. 1-4 micrograms/ml benzo[a]pyrene or 10-50 J/m2 UV light resulted in an approximate doubling of [3H]dThd incorporation. The effects of inhibitors of DNA repair were determined in chrysotile-treated RPMCs. [3H]dThd incorporation was inhibited by cytosine arabinoside and nalidixic acid. These results show that asbestos produces UDS in RPMCs.

  1. Breast cancer proteins PALB2 and BRCA2 stimulate polymerase η in recombination-associated DNA synthesis at blocked replication forks.

    PubMed

    Buisson, Rémi; Niraj, Joshi; Pauty, Joris; Maity, Ranjan; Zhao, Weixing; Coulombe, Yan; Sung, Patrick; Masson, Jean-Yves

    2014-02-13

    One envisioned function of homologous recombination (HR) is to find a template for DNA synthesis from the resected 3'-OH molecules that occur during double-strand break (DSB) repair at collapsed replication forks. However, the interplay between DNA synthesis and HR remains poorly understood in higher eukaryotic cells. Here, we reveal functions for the breast cancer proteins BRCA2 and PALB2 at blocked replication forks and show a role for these proteins in stimulating polymerase η (Polη) to initiate DNA synthesis. PALB2, BRCA2, and Polη colocalize at stalled or collapsed replication forks after hydroxyurea treatment. Moreover, PALB2 and BRCA2 interact with Polη and are required to sustain the recruitment of Polη at blocked replication forks. PALB2 and BRCA2 stimulate Polη-dependent DNA synthesis on D loop substrates. We conclude that PALB2 and BRCA2, in addition to their functions in D loop formation, play crucial roles in the initiation of recombination-associated DNA synthesis by Polη-mediated DNA repair. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Developing Inhibitors of Translesion DNA Synthesis as Therapeutic Agents Against Lung Cancer

    DTIC Science & Technology

    2014-10-01

    have demonstrated that high-fidelity DNA polymerases (eukaryotic pol delta and bacteriophage T4 DNA polymerase) display error-prone tendencies when...modified nucleotides opposite this lesion. Although high-fidelity DNA polymerases (eukaryotic pol  and bacteriophage T4 DNA polymerase) display error...Exonuclease-deficient bacteriophage T4 DNA polymerase (gp43exo− (Asp-219 to Ala mutation)) was purified and quantified as previously described (26

  3. Synthesis of core-shell gold coated magnetic nanoparticles and their interaction with thiolated DNA

    NASA Astrophysics Data System (ADS)

    Robinson, Ian; Tung, Le D.; Maenosono, Shinya; Wälti, Christoph; Thanh, Nguyen T. K.

    2010-12-01

    Core-shell magnetic nanoparticles have received significant attention recently and are actively investigated owing to their large potential for a variety of applications. Here, the synthesis and characterization of bimetallic nanoparticles containing a magnetic core and a gold shell are discussed. The gold shell facilitates, for example, the conjugation of thiolated biological molecules to the surface of the nanoparticles. The composite nanoparticles were produced by the reduction of a gold salt on the surface of pre-formed cobalt or magnetite nanoparticles. The synthesized nanoparticles were characterized using ultraviolet-visible absorption spectroscopy, transmission electron microscopy, energy dispersion X-ray spectroscopy, X-ray diffraction and super-conducting quantum interference device magnetometry. The spectrographic data revealed the simultaneous presence of cobalt and gold in 5.6 +/- 0.8 nm alloy nanoparticles, and demonstrated the presence of distinct magnetite and gold phases in 9.2 +/- 1.3 nm core-shell magnetic nanoparticles. The cobalt-gold nanoparticles were of similar size to the cobalt seed, while the magnetite-gold nanoparticles were significantly larger than the magnetic seeds, indicating that different processes are responsible for the addition of the gold shell. The effect on the magnetic properties by adding a layer of gold to the cobalt and magnetite nanoparticles was studied. The functionalization of the magnetic nanoparticles is demonstrated through the conjugation of thiolated DNA to the gold shell.Core-shell magnetic nanoparticles have received significant attention recently and are actively investigated owing to their large potential for a variety of applications. Here, the synthesis and characterization of bimetallic nanoparticles containing a magnetic core and a gold shell are discussed. The gold shell facilitates, for example, the conjugation of thiolated biological molecules to the surface of the nanoparticles. The composite

  4. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed Central

    DeWyngaert, M A; Hinkle, D C

    1980-01-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein. Images PMID:6997508

  5. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed

    DeWyngaert, M A; Hinkle, D C

    1980-02-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein.

  6. Models and strategies for value clarification in population education.

    PubMed

    Villanueva, C L

    1976-01-01

    Teachers, principals, supervisiors, superintendents, policymakers, and researchers all agree that value clarification is the most effective method for teaching value-laden population issues. It has been recognized that cultural attitudes and beliefs that are negative to family planning block acceptance of the population program. Education can become an effective vehicle in raising the level of awareness and acceptance of population practices. Teachers and students must 1st critically examine a set of alternative population values, for then they can weigh the pros and cons in order to arrive at value decisions regarding fertility behavior. Discussion and recommendations for practice and policy are presented for 9 problem areas: defining value clarification, affective versus cognitive process, teacher participation, open-ended versus closed-ended approach, use of resources and devices, target group, teacher personality, selecting appropriate issues, and dealing with controversial subjects.

  7. Phenomenology and the life sciences: Clarifications and complementarities.

    PubMed

    Sheets-Johnstone, Maxine

    2015-12-01

    This paper first clarifies phenomenology in ways essential to demonstrating its basic concern with Nature and its recognition of individual and cultural differences as well as commonalities. It furthermore clarifies phenomenological methodology in ways essential to understanding the methodology itself, its purpose, and its consequences. These clarifications show how phenomenology, by hewing to the dynamic realities of life itself and experiences of life itself, counters reductive thinking and "embodiments" of one kind and another. On the basis of these clarifications, the paper then turns to detailing conceptual complementarities between phenomenology and the life sciences, particularly highlighting studies in coordination dynamics. In doing so, it brings to light fundamental relationships such as those between mind and motion and between intrinsic dynamics and primal animation. It furthermore highlights the common concern with origins in both phenomenology and evolutionary biology: the history of how what is present is related to its inception in the past and to its transformations from past to present.

  8. A study on the unprimed poly (dA-dT) synthesis catalyzed by preparations of E. coli DNA polymerase I.

    PubMed Central

    Nazarenko, I A; Bobko, L E; Romashchenko, A G; Khripin, Y L; Salganik, R I

    1979-01-01

    Evidence was obtained indicating that the initiation of poly (dA-dT) de novo synthesis is provided by deoxynucleoside diphosphate: oligonucleotide deoxynucleotidyl transferase (dNDP-transferase present in preparations of E. coli DNA polymerase I and capable of catalyzing the unprimed polymerization of dNDP. dNDP-transferase synthesyzes short oligonucleotides which form template-primer complexes repeatedly replicated by DNA polymerase I. This conclusion was based on the following observations: the abolition of the lag period of poly (dA-dT) synthesis by preincubation of DNA-polymerase I preparations with dADP and dTDP; the presence of oligo (dA-dT) among the preincubation products; the suppressive effect of dithiothreitol and N-ethylmaleimide (inhibitors of dNDP-transferase) on the de novo, but not on the primed synthesis of poly (dA-dT), catalyzed by preparations of DNA-polymerase I. PMID:379822

  9. Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs

    PubMed Central

    Turner, Marie; Adhikari, Sajag; Subramanian, Senthil

    2013-01-01

    We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays. PMID:23673353

  10. Induction of Duplication Reversion in Human Fibroblasts, by Wild-Type and Mutated Sv40 T Antigen, Covaries with the Ability to Induce Host DNA Synthesis

    PubMed Central

    Shammas, M. A.; Xia, S. J.; Reis, RJS.

    1997-01-01

    Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis. PMID:9258684

  11. In vivo effects of endotoxin on DNA synthesis in rat nasal epithelium

    SciTech Connect

    Harkema, J.R.; Hotchkiss, J.A. )

    1993-12-01

    Airway inflammation in bacterial infections is characterized by the presence of neutrophils and often epithelial injury and repair. Release of endotoxin from bacteria may contribute to these processes. The purpose of this study was to determine the in vivo effects of repeated endotoxin exposure on DNA synthesis in rat nasal epithelium in the presence and absence of neutrophilic influx. Rats were intranasally instilled, once a day for 3 days, with endotoxin or saline (controls). Before the first and third instillations, half of the saline and endotoxin-instilled animals were depleted of circulating blood neutrophils by administering a rabbit anti-rat neutrophil antiserum. Rats were sacrificed 6 or 24 h after the last instillation. Two hours prior to sacrifice, rats were intraperitoneally injected with bromodeoxyuridine (BrdU), an analog of thymidine that is incorporated in the nucleus of cells in the S-phase of the cell cycle. Nasal tissues were processed for light microscopy and immunohistochemical detection of BrdU in nasal epithelial cells. The numbers of nasal epithelial cells, BrdU-labeled epithelial nuclei, and neutrophils per millimeter of basal lamina in the epithelium lining the nasal turbinates in the proximal nasal passages were determined by morphometric analysis. The authors did not observe a neutrophilic influx in the nasal tissues of neutrophil-depleted rats at 6 or 24 h after the last endotoxin instillation; however, the numbers of nasal epithelial cells and the BrdU-labeling index were significantly increased compared to saline-instilled controls. In contrast, non-neutrophil-depleted rats instilled with endotoxin had a marked neutrophilic influx, but no significant differences in the number of nasal epithelial cells at 6 or 24 h, compared to controls. In addition, the BrdU-labeling index in neutrophil-sufficient rats was increased only 6 h after the last instillation, compared to controls.

  12. Viral DNA Synthesis Defects in Assembly-Competent Rous Sarcoma Virus CA Mutants

    PubMed Central

    Cairns, Tina M.; Craven, Rebecca C.

    2001-01-01

    The major structural protein of the retroviral core (CA) contains a conserved sequence motif shared with the CA-like proteins of distantly related transposable elements. The function of this major region of homology (MHR) has not been defined, in part due to the baffling array of phenotypes in mutants of several viruses and the yeast TY3. This report describes new mutations in the CA protein of Rous sarcoma virus (RSV) that were designed to test whether these different phenotypes might indicate distinct functional subdomains in the MHR. A comparison of 25 substitutions at 10 positions in the RSV conserved motif argues against this possibility. Most of the replacements destroyed virus infectivity, although either of two lethal phenotypes was obtained depending on the residue introduced. At most of the positions, one or more replacements (generally the more conservative substitutions) caused a severe replication defect without having any obvious effects on virus assembly, budding, Gag-Pol and genome incorporation, or protein processing. The mutant particles exhibited a defect in endogenous viral DNA synthesis and showed increased sensitivity of the core proteins to detergent, indicating that the mutations interfere with the formation and/or activity of the virion core. The distribution of these mutations across the MHR, with no evidence of clustering, suggests that the entire region is important for a critical postbudding function. In contrast, a second class of lethal substitutions (those that destroyed virus assembly and release) consists of alterations that are expected to cause severe effects on protein structure by disruption either of the hydrophobic core of the CA carboxyl-terminal domain or of the hydrogen bond network that stabilizes the domain. We suggest that this duality of phenotypes is consistent with a role for the MHR in the maturation process that links the two parts of the life cycle. PMID:11119594

  13. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells.

    PubMed

    Maddocks, Oliver D K; Labuschagne, Christiaan F; Adams, Peter D; Vousden, Karen H

    2016-01-21

    Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Proteasome inhibition induces DNA damage and reorganizes nuclear architecture and protein synthesis machinery in sensory ganglion neurons.

    PubMed

    Palanca, Ana; Casafont, Iñigo; Berciano, María T; Lafarga, Miguel

    2014-05-01

    Bortezomib is a reversible proteasome inhibitor used as an anticancer drug. However, its clinical use is limited since it causes peripheral neurotoxicity. We have used Sprague-Dawley rats as an animal model to investigate the cellular mechanisms affected by both short-term and chronic bortezomib treatments in sensory ganglia neurons. Proteasome inhibition induces dose-dependent alterations in the architecture, positioning, shape and polarity of the neuronal nucleus. It also produces DNA damage without affecting neuronal survival, and severe disruption of the protein synthesis machinery at the central cytoplasm accompanied by decreased expression of the brain-derived neurotrophic factor. As a compensatory or adaptive survival response against proteotoxic stress caused by bortezomib treatment, sensory neurons preserve basal levels of transcriptional activity, up-regulate the expression of proteasome subunit genes, and generate a new cytoplasmic perinuclear domain for protein synthesis. We propose that proteasome activity is crucial for controlling nuclear architecture, DNA repair and the organization of the protein synthesis machinery in sensory neurons. These neurons are primary targets of bortezomib neurotoxicity, for which reason their dysfunction may contribute to the pathogenesis of the bortezomib-induced peripheral neuropathy in treated patients.

  15. Evaluation of predictive tools for cell culture clarification performance.

    PubMed

    Senczuk, Anna; Petty, Krista; Thomas, Anne; McNerney, Thomas; Moscariello, John; Yigzaw, Yinges

    2016-03-01

    Recent advances in the productivity of industrial mammalian cell culture processes have resulted in part in increased cell density. This increase and the associated increase in cellular debris are known to challenge harvest operations, however this understanding is limited and largely qualitative. Part of the issue arises from the heterogeneous size and composition of cellular debris, which makes harvest feed stream extremely difficult to characterize. Improved characterization methods would facilitate the development of clarification approaches that are consistent and scalable. This work describes how both particle size and cholesterol analysis can be used to characterize the feed stream. Particle size analysis by focused beam reflectance and dynamic light scattering are shown to be predictive of centrate filterability under certain harvest conditions. Because of the particle size range limitations of each detector, their applicability is limited to a particular stage or method of clarification. The measurement of cholesterol present in the cell culture supernatant or centrate was successfully used in providing relative amount of lysed cellular debris and enabled us to predict clarification performance of acid precipitated harvest regardless of particle size distribution profile. © 2015 Wiley Periodicals, Inc.

  16. Influence of prefermentary clarification on the composition of apple musts.

    PubMed

    Hubert, Bertrand; Baron, Alain; Le Quere, Jean-Michel; Renard, Catherine M G C

    2007-06-27

    The polyphenol contents and colors of cider apple juices were compared before (NCM, not clarified must) and after five clarification treatments: enzymatic depectinization by pectinases followed by (i) sedimentation (depectinized and decanted juice), (ii) tangential microfiltration (microfiltered juice) or (iii) fining using gelatin (gelatin-treated juice); (iv) enzymatic gelification of pectin by pectin methylesterase followed by natural keeving by a cider manufacturer (producer keeved juice), or (v) flotation (floated with nitrogen gas juice). The pressing of the apples led to the highly selective extraction of the flavan-3-ols with the lowest molecular weights: In the apples, the number average degree of polymerization of the flavanols was 14.7, and it dropped to 2.2 in the NCM. Keeving had the highest impact on the reduction of both flavanol content and number average degree of polymerization. The flavanol concentrations were decreased in the permeate by fining (30%) much more than by depectinization. The clarification step led to a further decrease of the number average degree of polymerization. Hydroxycinnamic acids were less affected by the extraction process (with extraction yields >50%) and not affected by clarification. The color evolved with all treatments: L*, a*, b*, and chromaticity distance index measures indicated a reduction of orange-yellow saturation except after sedimentation.

  17. High-rate stormwater clarification with polymeric flocculant addition.

    PubMed

    Wood, J; He, C; Rochfort, Q; Marsalek, J; Seto, P; Yang, M; Chessie, P; Kok, S

    2005-01-01

    Treatment of urban stormwater by clarification, with flocculant addition, was studied in Toronto, Canada using a pilot-scale clarifier with removable lamellar plates. Almost 90 stormwater runoff events were characterised at the study site and found fairly polluted. The previous research phase indicated good treatability of this stormwater by lamellar clarification with flocculant addition (total suspended solids, TSS, removal of 84%, at a surface load of 15 m/h), but there were concerns about cleaning plates after storm events. With the aid of numerical modelling, hydraulic improvements to the clarifier inlet zone were retrofitted in 2004 and permitted the removal of the lamellar pack without a loss in treatment efficiency. In the modified clarifier, a cationic polymeric flocculant dosage of 4 mg/L with conventional clarification provided a TSS removal of 77%, at surface loads up to 43 m/h. The use of the polymer did not increase the acute toxicity of the treated effluent. The clarifier sludge was severely polluted by several heavy metals and would require special disposal. The treatment process tested could be well applied in projects requiring intensive stormwater treatment at compact sites.

  18. Homologous recombination rescues ssDNA gaps generated by nucleotide excision repair and reduced translesion DNA synthesis in yeast G2 cells

    PubMed Central

    Ma, Wenjian; Westmoreland, James W.; Resnick, Michael A.

    2013-01-01

    Repair of DNA bulky lesions often involves multiple repair pathways such as nucleotide-excision repair, translesion DNA synthesis (TLS), and homologous recombination (HR). Although there is considerable information about individual pathways, little is known about the complex interactions or extent to which damage in single strands, such as the damage generated by UV, can result in double-strand breaks (DSBs) and/or generate HR. We investigated the consequences of UV-induced lesions in nonreplicating G2 cells of budding yeast. In contrast to WT cells, there was a dramatic increase in ssDNA gaps for cells deficient in the TLS polymerases η (Rad30) and ζ (Rev3). Surprisingly, repair in TLS-deficient G2 cells required HR repair genes RAD51 and RAD52, directly revealing a redundancy of TLS and HR functions in repair of ssDNAs. Using a physical assay that detects recombination between circular sister chromatids within a few hours after UV, we show an approximate three-fold increase in recombinants in the TLS mutants over that in WT cells. The recombination, which required RAD51 and RAD52, does not appear to be caused by DSBs, because a dose of ionizing radiation producing 20 times more DSBs was much less efficient than UV in producing recombinants. Thus, in addition to revealing TLS and HR functional redundancy, we establish that UV-induced recombination in TLS mutants is not attributable to DSBs. These findings suggest that ssDNA that might originate during the repair of closely opposed lesions or of ssDNA-containing lesions or from uncoupled replication may drive recombination directly in various species, including humans. PMID:23858457

  19. Base-modified thymidines capable of terminating DNA synthesis are novel bioactive compounds with activity in cancer cells

    PubMed Central

    Borland, Kayla M.; AbdulSalam, Safnas F.; Solivio, Morwena J.; Burke, Matthew P.; Wolfkiel, Patrick R.; Lawson, Sean M.; Stockman, Courtney A.; Andersen, Joel M.; Smith, Skyler; Tolstolutskaya, Julia N.; Gurjar, Purujit N.; Bercz, Aron P.; Merino, Edward J.; Litosh, Vladislav A.

    2015-01-01

    Current FDA-approved chemotherapeutic antimetabolites elicit severe side effects that warrant their improvement; therefore, we designed compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5′-triphosphates were exquisite DNA synthesis terminators; therefore, we synthesized a library of 35 thymidine analogs and evaluated their activity using an MTT cell viability assay of MCF7 breast cancer cells chosen for their vulnerability to these nucleoside derivatives. Compound 3a, having an α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy group, showed an IC50 of 9 ± 1 μM. T