Complete-active-space second-order perturbation theory (CASPT2//CASSCF) study of the dissociative electron attachment in canonical DNA nucleobases caused by low-energy electrons (0-3 eV)

Energy Technology Data Exchange (ETDEWEB)

Francés-Monerris, Antonio; Segarra-Martí, Javier; Merchán, Manuela; Roca-Sanjuán, Daniel, E-mail: Daniel.Roca@uv.es [Instituto de Ciencia Molecular, Universitat de València, P.O. Box 22085, 46071 València (Spain)

2015-12-07

Low-energy (0-3 eV) ballistic electrons originated during the irradiation of biological material can interact with DNA/RNA nucleobases yielding transient-anion species which undergo decompositions. Since the discovery that these reactions can eventually lead to strand breaking of the DNA chains, great efforts have been dedicated to their study. The main fragmentation at the 0-3 eV energy range is the ejection of a hydrogen atom from the specific nitrogen positions. In the present study, the methodological approach introduced in a previous work on uracil [I. González-Ramírez et al., J. Chem. Theory Comput. 8, 2769-2776 (2012)] is employed to study the DNA canonical nucleobases fragmentations of N–H bonds induced by low-energy electrons. The approach is based on minimum energy path and linear interpolation of internal coordinates computations along the N–H dissociation channels carried out at the complete-active-space self-consistent field//complete-active-space second-order perturbation theory level. On the basis of the calculated theoretical quantities, new assignations for the adenine and cytosine anion yield curves are provided. In addition, the π{sub 1}{sup −} and π{sub 2}{sup −} states of the pyrimidine nucleobases are expected to produce the temporary anions at electron energies close to 1 and 2 eV, respectively. Finally, the present theoretical results do not allow to discard neither the dipole-bound nor the valence-bound mechanisms in the range of energies explored, suggesting that both possibilities may coexist in the experiments carried out with the isolated nucleobases.

Interactions between Al₁₂X (X = Al, C, N and P) nanoparticles and DNA nucleobases/base pairs: implications for nanotoxicity.

Science.gov (United States)

Jin, Peng; Chen, Yongsheng; Zhang, Shengbai B; Chen, Zhongfang

2012-02-01

The interactions between neutral Al(12)X(I ( h )) (X = Al, C, N and P) nanoparticles and DNA nucleobases, namely adenine (A), thymine (T), guanine (G) and cytosine (C), as well as the Watson-Crick base pairs (BPs) AT and GC, were investigated by means of density functional theory computations. The Al(12)X clusters can tightly bind to DNA bases and BPs to form stable complexes with negative binding Gibbs free energies at room temperature, and considerable charge transfers occur between the bases/BPs and the Al(12)X clusters. These strong interactions, which are also expected for larger Al nanoparticles, may have potentially adverse impacts on the structure and stability of DNA and thus cause its dysfunction.

Pyrene–nucleobase conjugates: synthesis, oligonucleotide binding and confocal bioimaging studies

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Artur Jabłoński

2017-11-01

Full Text Available Fluorescent pyrene–linker–nucleobase (nucleobase = thymine, adenine conjugates with carbonyl and hydroxy functionalities in the linker were synthesized and characterized. X-ray single-crystal structure analysis performed for the pyrene–C(OCH2CH2–thymine (2 conjugate reveals dimers of molecules 2 stabilized by hydrogen bonds between the thymine moieties. The photochemical characterization showed structure-dependent fluorescence properties of the investigated compounds. The conjugates bearing a carbonyl function represent weak emitters as compared to compounds with a hydroxy function in the linker. The self-assembly properties of pyrene nucleobases were investigated in respect to their binding to single and double strand oligonucleotides in water and in buffer solution. In respect to the complementary oligothymidine T10 template in water, compounds 3 and 5 both show a self-assembling behavior according to canonical base–base pairing. However, in buffer solution, derivative 5 was much more effective than 3 in binding to the T10 template. Furthermore the adenine derivative 5 binds to the double-stranded (dA10–T10 template with a self-assembly ratio of 112%. Such a high value of a self-assembly ratio can be rationalized by a triple-helix-like binding, intercalation, or a mixture of both. Remarkably, compound 5 also shows dual staining pattern in living HeLa cells. Confocal microscopy confirmed that 5 predominantly stains mitochondria but it also accumulates in the nucleoli of the cells.

The Formation of Nucleobases from the Ultraviolet Photoirradiation of Purine in Simple Astrophysical Ice Analogues.

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Materese, Christopher K; Nuevo, Michel; Sandford, Scott A

2017-08-01

Nucleobases are the informational subunits of RNA and DNA and are essential to all known forms of life. The nucleobases can be divided into two groups of molecules: the pyrimidine-based compounds that include uracil, cytosine, and thymine, and the purine-based compounds that include adenine and guanine. Previous work in our laboratory has demonstrated that uracil, cytosine, thymine, and other nonbiological, less common nucleobases can form abiotically from the UV photoirradiation of pyrimidine in simple astrophysical ice analogues containing combinations of H 2 O, NH 3 , and CH 4 . In this work, we focused on the UV photoirradiation of purine mixed with combinations of H 2 O and NH 3 ices to determine whether or not the full complement of biological nucleobases can be formed abiotically under astrophysical conditions. Room-temperature analyses of the resulting photoproducts resulted in the detection of adenine, guanine, and numerous other functionalized purine derivatives. Key Words: Pyrimidine-Nucleobases-Interstellar; Ices-Cometary; Ices-Molecular processes-Prebiotic chemistry. Astrobiology 17, 761-770.

Extraterrestrial Nucleobases in Carbonaceous Chondrites

Science.gov (United States)

Martins, Z.; Botta, O.; Fogel, M.; Sephton, M.; Glavin, D.; Watson, J.; Dworkin, J.; Schwartz, A.; Ehrenfreund, P.

Nucleobases in Carbonaceous Chondrites Z. Martins (1), O. Botta (2), M. L. Fogel (3), M. A. Sephton (4), D. P. Glavin (2), J. S. Watson (5), J. P. Dworkin (2), A. W. Schwartz (6) and P. Ehrenfreund (1,6). (1) Astrobiology Laboratory, Leiden Institute of Chemistry, Leiden, The Netherlands, (2) NASA Goddard Space Flight Center, Goddard Center for Astrobiology, Greenbelt, MD, USA, (3) GL, Carnegie Institution of Washington, Washington DC, USA, (4) Impacts and Astromaterials Research Centre, Department of Earth Science and Engineering, South Kensington Campus, Imperial College, London, UK, (5) Planetary and Space Sciences Research Institute, The Open University, Walton Hall, Milton Keynes, UK, (6) Radboud University Nijmegen, Nijmegen, The Netherlands. E-mail: z.martins@chem.leidenuniv.nl/Phone:+31715274440 Nucleobases are crucial compounds in terrestrial biochemistry, because they are key components of DNA and RNA. Carbonaceous meteorites have been analyzed for nucleobases by different research groups [1-5]. However, significant quantitative and qualitative differences were observed, leading to the controversial about the origin of these nucleobases. In order to establish the origin of these compounds in carbonaceous chondrites and to assess the plausibility of their exogenous delivery to the early Earth, we have performed formic acid extraction of samples of the Murchison meteorite [6], followed by an extensive purification procedure, analysis and quantification by high-performance liquid chromatography with UV absorption detection and gas chromatography-mass spectrometry. Our results were qualitatively consistent with previous results [3, 4], but showed significant quantitative differences. Compound specific carbon isotope values were obtained, using gas chromatography-combustion- isotope ratio mass spectrometry. A soil sample collected in the proximity of the Murchison meteorite fall site was subjected to the same extraction, purification and analysis procedure

Nucleobase-Based Barbiturates: Their Protective Effect against DNA Damage Induced by Bleomycin-Iron, Antioxidant, and Lymphocyte Transformation Assay

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Bhaveshkumar D. Dhorajiya

2014-01-01

Full Text Available A number of nucleobase-based barbiturates have been synthesized by combination of nucleic acid bases and heterocyclic amines and barbituric acid derivatives through green and efficient multicomponent route and one pot reaction. This approach was accomplished efficiently using aqueous medium to give the corresponding products in high yield. The newly synthesized compounds were characterized by spectral analysis (FT-IR, 1H NMR, 13C NMR, HMBC, and UV spectroscopy and elemental analysis. Representative of all synthesized compounds was tested and evaluated for antioxidant, bleomycin-dependent DNA damage, and Lymphocyte Transformation studies. Compounds TBC > TBA > TBG showed highest lymphocyte transformation assay, TBC > TBA > BG showed inhibitory antioxidant activity using ABTS methods, and TBC > BPA > BAMT > TBA > 1, 3-TBA manifested the best protective effect against DNA damage induced by bleomycin.

The origin of efficient triplet state population in sulfur-substituted nucleobases

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Mai, Sebastian; Pollum, Marvin; Martínez-Fernández, Lara; Dunn, Nicholas; Marquetand, Philipp; Corral, Inés; Crespo-Hernández, Carlos E.; González, Leticia

2016-10-01

Elucidating the photophysical mechanisms in sulfur-substituted nucleobases (thiobases) is essential for designing prospective drugs for photo- and chemotherapeutic applications. Although it has long been established that the phototherapeutic activity of thiobases is intimately linked to efficient intersystem crossing into reactive triplet states, the molecular factors underlying this efficiency are poorly understood. Herein we combine femtosecond transient absorption experiments with quantum chemistry and nonadiabatic dynamics simulations to investigate 2-thiocytosine as a necessary step to unravel the electronic and structural elements that lead to ultrafast and near-unity triplet-state population in thiobases in general. We show that different parts of the potential energy surfaces are stabilized to different extents via thionation, quenching the intrinsic photostability of canonical DNA and RNA nucleobases. These findings satisfactorily explain why thiobases exhibit the fastest intersystem crossing lifetimes measured to date among bio-organic molecules and have near-unity triplet yields, whereas the triplet yields of canonical nucleobases are nearly zero.

Origin of the RNA world: The fate of nucleobases in warm little ponds

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Pearce, Ben K. D.; Pudritz, Ralph E.; Semenov, Dmitry A.; Henning, Thomas K.

2017-10-01

Before the origin of simple cellular life, the building blocks of RNA (nucleotides) had to form and polymerize in favorable environments on early Earth. At this time, meteorites and interplanetary dust particles delivered organics such as nucleobases (the characteristic molecules of nucleotides) to warm little ponds whose wet–dry cycles promoted rapid polymerization. We build a comprehensive numerical model for the evolution of nucleobases in warm little ponds leading to the emergence of the first nucleotides and RNA. We couple Earth's early evolution with complex prebiotic chemistry in these environments. We find that RNA polymers must have emerged very quickly after the deposition of meteorites (less than a few years). Their constituent nucleobases were primarily meteoritic in origin and not from interplanetary dust particles. Ponds appeared as continents rose out of the early global ocean, but this increasing availability of “targets” for meteorites was offset by declining meteorite bombardment rates. Moreover, the rapid losses of nucleobases to pond seepage during wet periods, and to UV photodissociation during dry periods, mean that the synthesis of nucleotides and their polymerization into RNA occurred in just one to a few wet–dry cycles. Under these conditions, RNA polymers likely appeared before 4.17 billion years ago.

Origin of the RNA world: The fate of nucleobases in warm little ponds.

Science.gov (United States)

Pearce, Ben K D; Pudritz, Ralph E; Semenov, Dmitry A; Henning, Thomas K

2017-10-24

Before the origin of simple cellular life, the building blocks of RNA (nucleotides) had to form and polymerize in favorable environments on early Earth. At this time, meteorites and interplanetary dust particles delivered organics such as nucleobases (the characteristic molecules of nucleotides) to warm little ponds whose wet-dry cycles promoted rapid polymerization. We build a comprehensive numerical model for the evolution of nucleobases in warm little ponds leading to the emergence of the first nucleotides and RNA. We couple Earth's early evolution with complex prebiotic chemistry in these environments. We find that RNA polymers must have emerged very quickly after the deposition of meteorites (less than a few years). Their constituent nucleobases were primarily meteoritic in origin and not from interplanetary dust particles. Ponds appeared as continents rose out of the early global ocean, but this increasing availability of "targets" for meteorites was offset by declining meteorite bombardment rates. Moreover, the rapid losses of nucleobases to pond seepage during wet periods, and to UV photodissociation during dry periods, mean that the synthesis of nucleotides and their polymerization into RNA occurred in just one to a few wet-dry cycles. Under these conditions, RNA polymers likely appeared before 4.17 billion years ago.

DNA Trojan Horses: Self-Assembled Floxuridine-Containing DNA Polyhedra for Cancer Therapy.

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Mou, Quanbing; Ma, Yuan; Pan, Gaifang; Xue, Bai; Yan, Deyue; Zhang, Chuan; Zhu, Xinyuan

2017-10-02

Based on their structural similarity to natural nucleobases, nucleoside analogue therapeutics were integrated into DNA strands through conventional solid-phase synthesis. By elaborately designing their sequences, floxuridine-integrated DNA strands were synthesized and self-assembled into well-defined DNA polyhedra with definite drug-loading ratios as well as tunable size and morphology. As a novel drug delivery system, these drug-containing DNA polyhedra could ideally mimic the Trojan Horse to deliver chemotherapeutics into tumor cells and fight against cancer. Both in vitro and in vivo results demonstrate that the DNA Trojan horse with buckyball architecture exhibits superior anticancer capability over the free drug and other formulations. With precise control over the drug-loading ratio and structure of the nanocarriers, the DNA Trojan horse may play an important role in anticancer treatment and exhibit great potential in translational nanomedicine. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nucleobases, nucleosides, and nucleotides: versatile biomolecules for generating functional nanomaterials.

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Pu, Fang; Ren, Jinsong; Qu, Xiaogang

2018-02-21

The incorporation of biomolecules into nanomaterials generates functional nanosystems with novel and advanced properties, presenting great potential for applications in various fields. Nucleobases, nucleosides and nucleotides, as building blocks of nucleic acids and biological coenzymes, constitute necessary components of the foundation of life. In recent years, as versatile biomolecules for the construction or regulation of functional nanomaterials, they have stimulated interest in researchers, due to their unique properties such as structural diversity, multiplex binding sites, self-assembly ability, stability, biocompatibility, and chirality. In this review, strategies for the synthesis of nanomaterials and the regulation of their morphologies and functions using nucleobases, nucleosides, and nucleotides as building blocks, templates or modulators are summarized alongside selected applications. The diverse applications range from sensing, bioimaging, and drug delivery to mimicking light-harvesting antenna, the construction of logic gates, and beyond. Furthermore, some perspectives and challenges in this emerging field are proposed. This review is directed toward the broader scientific community interested in biomolecule-based functional nanomaterials.

Solid-Phase Synthesis of Oligodeoxynucleotides Containing N4-[2-(t-butyldisulfanylethyl]-5-methylcytosine Moieties

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Sónia Pérez-Rentero

2010-08-01

Full Text Available An efficient route for the synthesis of the phosphoramidite derivative of 5-methylcytosine bearing a tert-butylsulfanyl group protected thiol is described. This building block is used for the preparation of oligonucleotides carrying a thiol group at the nucleobase at the internal position of a DNA sequence. The resulting thiolated oligonucleotides are useful intermediates to generate oligonucleotide conjugates carrying molecules of interest at internal positions of a DNA sequence.

Triazole nucleoside derivatives bearing aryl functionalities on the nucleobases show antiviral and anticancer activity.

Science.gov (United States)

Xia, Yi; Qu, Fanqi; Peng, Ling

2010-08-01

Synthetic nucleoside mimics are important candidates in the searing for antiviral and anticancer drugs. Ribavirin, the first antiviral nucleoside drug, is unique in its antiviral activity with mutilple modes of action, which are mainly due to its special triazole heterocycle as nucleobase. Additionally, introducing aromatic functionalities to the nucleobase is able to confer novel mechanisms of action for nucleoside mimics. With the aim to combine the special characteristics of unnatural triazole heterocycles with those of the appended aromatic groups on the nucleobases, novel 1,2,4-triazole nucleoside analogs bearing aromatic moieties were designed and developed. The present short review summarizes the molecular design, chemical synthesis and biological activity of these triazole nucleoside analogs. Indeed, the discovery of antiviral and anticancer activities shown by these triazole nucleosides as well as the new mechanism underlying the biological activity by one of the anticancer leads has validated the rationale for molecular design and impacted us to further explore the concept with the aim of developing structurally novel nucleoside drug candidates with new modes of action.

Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance

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Kristen K. Merritt

2014-10-01

Full Text Available 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

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

International Nuclear Information System (INIS)

Nishida, C.; Reinhard, P.; Linn, S.

1988-01-01

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

Charge transfer in pi-stacked systems including DNA

International Nuclear Information System (INIS)

Siebbeles, L.D.A.

2003-01-01

Charge migration in DNA is a subject of intense current study motivated by long-range detection of DNA damage and the potential application of DNA as a molecular wire in nanoscale electronic devices. A key structural element, which makes DNA a medium for long-range charge transfer, is the array of stacked base pairs in the interior of the double helix. The overlapping pi-orbitals of the nucleobases provide a pathway for motion of charge carriers generated on the stack. This 'pi-pathway' resembles the columnarly stacked macrocyclic cores in discotic materials such as triphenylenes. The structure of these pi-stacked systems is highly disordered with dynamic fluctuations occurring on picosecond to nanosecond time scales. Theoretical calculations, concerning the effects of structural disorder and nucleobase sequence in DNA, on the dynamics of charge carriers are presented. Electronic couplings and localization energies of charge carriers were calculated using density functional theory (DFT). Results for columnarly stacked triphenylenes and DNA nucleobases are compared. The results are used to provide insight into the factors that control the mobility of charge carriers. Further, experimental results on the site-selective oxidation of guanine nucleobases in DNA (hot spots for DNA damage) are analyzed on basis of the theoretical results

The photochemistry of pyrimidine in realistic astrophysical ices and the production of nucleobases

International Nuclear Information System (INIS)

Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.

2014-01-01

Nucleobases, together with deoxyribose/ribose and phosphoric acid, are the building blocks of DNA and RNA for all known life. The presence of nucleobase-like compounds in carbonaceous chondrites delivered to the Earth raises the question of an extraterrestrial origin for the molecules that triggered life on our planet. Whether these molecules are formed in interstellar/protostellar environments, in small parent bodies in the solar system, or both, is currently unclear. Recent experiments show that the UV irradiation of pyrimidine (C 4 H 4 N 2 ) in H 2 O-rich ice mixtures that contain NH 3 , CH 3 OH, or CH 4 leads to the formation of the pyrimidine-based nucleobases uracil, cytosine, and thymine. In this work, we discuss the low-temperature UV irradiation of pyrimidine in realistic astrophysical ice mixtures containing H 2 O, CH 3 OH, and NH 3 , with or without CH 4 , to search for the production of nucleobases and other prebiotic compounds. These experiments show the presence of uracil, urea, glycerol, hexamethylenetetramine, small amino acids, and small carboxylic acids in all samples. Cytosine was only found in one sample produced from ices irradiated with a higher UV dose, while thymine was not found in any sample, even after irradiation with a higher UV dose. Results are discussed to evaluate the role of the photochemistry of pyrimidine in the inventory of organic molecules detected in meteorites and their astrophysical/astrobiological implications.

Effects of inhibitors of DNA synthesis and protein synthesis on the rate of DNA synthesis after exposure of mammalian cells to ultraviolet light

International Nuclear Information System (INIS)

Griffiths, T.D.; Dahle, D.B.; Meechan, P.J.; Carpenter, J.G.

1981-01-01

Chinese hamster V-79 cells were treated with metabolic inhibitors of DNA or protein synthesis for various intervals of time after exposure of 3.0 or 5.0 J m -2 . After removal of the metabolic block(s) the rate of DNA synthesis was followed by measuring the incorporation of [ 14 C]thymidine into acid-insoluble material. A 2.5 or 5.0h incubation with cycloheximide or hydroxyurea was effective in delaying the onset of the recovery in the rate of DNA synthesis that normally becomes evident several hours after exposure to ultraviolet light. By using concentrations of cycloheximide or hydroxyurea that inhibit DNA synthesis by a similar amount (70%), but protein synthesis by vastly different amounts (95% for cycloheximide; 0% for hydroxyurea), it was apparent that the delay in recovery caused by the treatment of the cells with cycloheximide could be accounted for entirely by its inhibitory effect on DNA synthesis. This suggests that the recovery in DNA synthetic rates following exposure of V-79 cells to ultraviolet light does not appear to require de novo protein synthesis, and therefore does not appear to require the involvement of an inducible DNA repair process. (Auth.)

Steric restrictions of RISC in RNA interference identified with size-expanded RNA nucleobases.

Science.gov (United States)

Hernández, Armando R; Peterson, Larryn W; Kool, Eric T

2012-08-17

Understanding the interactions between small interfering RNAs (siRNAs) and the RNA-induced silencing complex (RISC), the key protein complex of RNA interference (RNAi), is of great importance to the development of siRNAs with improved biological and potentially therapeutic function. Although various chemically modified siRNAs have been reported, relatively few studies with modified nucleobases exist. Here we describe the synthesis and hybridization properties of siRNAs bearing size-expanded RNA (xRNA) nucleobases and their use as a novel and systematic set of steric probes in RNAi. xRNA nucleobases are expanded by 2.4 Å using benzo-homologation and retain canonical Watson-Crick base-pairing groups. Our data show that the modified siRNA duplexes display small changes in melting temperature (+1.4 to -5.0 °C); substitutions near the center are somewhat destabilizing to the RNA duplex, while substitutions near the ends are stabilizing. RNAi studies in a dual-reporter luciferase assay in HeLa cells revealed that xRNA nucleobases in the antisense strand reduce activity at some central positions near the seed region but are generally well tolerated near the ends. Most importantly, we observed that xRNA substitutions near the 3'-end increased activity over that of wild-type siRNAs. The data are analyzed in terms of site-dependent steric effects in RISC. Circular dichroism experiments show that single xRNA substitutions do not significantly distort the native A-form helical structure of the siRNA duplex, and serum stability studies demonstrated that xRNA substitutions protect siRNAs against nuclease degradation.

Differential sensitivity to aphidicolin of replicative DNA synthesis and ultraviolet-induced unscheduled DNA synthesis in vivo in mammalian cells

International Nuclear Information System (INIS)

Seki, Shuji; Hosogi, Nobuo; Oda, Takuzo

1984-01-01

In vivo in mammalian cells, ultraviolet-induced unscheduled DNA synthesis was less sensitive to aphidicolin than was replicative DNA synthesis. Replicative DNA synthesis in HeLa, HEp-2, WI-38 VA-13 and CV-1 cells was inhibited more than 97 % by aphidicolin at 10 μg/ml, whereas aphidicolin inhibition of DNA synthesis in ultraviolet-irradiated cells varied between 30 % and 90 % depending on cell types and assay conditions. Aphidicolin inhibition of unscheduled DNA synthesis (UDS) in HeLa cells increased gradually with increasing aphidicolin concentration and reached approximately 90 % at 100 μg/ml aphidicolin. A significant fraction of UDS in ultraviolet-irradiated HEp-2 cells was resistant to aphidicolin even at 300 μg/ml. Considered along with related information reported previously, the present results suggest that both aphidicolin-sensitive and insensitive DNA polymerases, DNA polymerase α and a non-α DNA polymerase (possibly DNA polymerase β), are involved in in situ UDS in these ultraviolet-irradiated cells. Comparison of staphylococcal nuclease sensitivity between DNAs repaired in the presence and in the absence of aphidicolin in HEp-2 cells suggested that the involvement of DNA polymerase α in UDS favored DNA synthesis in the intranucleosomal region. (author)

Spontaneous unscheduled DNA synthesis in human lymphocytes

International Nuclear Information System (INIS)

Forell, B.; Myers, L.S. Jr.; Norman, A.

1979-01-01

The rate of spontaneous unscheduled DNA synthesis in human lymphocytes was estimated from measurements of tritiated thymidine incorporation into double-stranded DNA (ds-DNA) during incubation of cells in vitro. The contribution of scheduled DNA synthesis to the observed incorporation was reduced by inhibiting replication with hydroxyurea and by separating freshly replicated single-stranded DNA (ss-DNA) from repaired ds-DNA by column chromatography. The residual contribution of scheduled DNA synthesis was estimated by observing effects on thymidine incorporation of: (a) increasing the rate of production of apurinic sites, and alternatively, (b) increasing the number of cells in S-phase. Corrections based on estimates of endogenous pool size were also made. The rate of spontaneous unscheduled DNA synthesis is estimated to be 490 +- 120 thymidine molecules incorporated per cell per hour. These results compare favorably with estimates made from rates of depurination and depyrimidination of DNA, measured in molecular systems if we assume thymidine is incorporated by a short patch mechanism which incorporates an average of four bases per lesion

NEXAFS characterization of DNA components and molecular-orientation of surface-bound DNA oligomers

International Nuclear Information System (INIS)

Samuel, Newton T.; Lee, C.-Y.; Gamble, Lara J.; Fischer, Daniel A.; Castner, David G.

2006-01-01

Single stranded DNA oligomers (ssDNA) immobilized onto solid surfaces forms the basis for several biotechnological applications such as DNA microarrays, affinity separations, and biosensors. Surface structure of Surface-bound oligomers is expected to significantly influence their biological activity and interactions with the environment. In this study near-edge X-ray absorption fine structure spectroscopy (NEXAFS) is used to characterize the components of DNA (nucleobases, nucleotides and nucleosides) and the orientation information of surface-bound ssDNA. The K-edges of carbon, nitrogen and oxygen have spectra with features that are characteristic of the different chemical species present in the nucleobases of DNA. The effect of addition of the DNA sugar and phosphate components on the NEXAFS K-edge spectra was also investigated. The polarization-dependent nitrogen K-edge NEXAFS data show significant changes for different orientations of surface bound ssDNA. These results establish NEXAFS as a powerful technique for chemical and structural characterization of surface-bound DNA oligomers

Nucleotides, Nucleosides, and Nucleobases

DEFF Research Database (Denmark)

Jensen, Kaj Frank; Dandanell, Gert; Hove-Jensen, Bjarne

2008-01-01

We review literature on the metabolism of ribo- and deoxyribonucleotides, nucleosides, and nucleobases in Escherichia coli and Salmonella,including biosynthesis, degradation, interconversion, and transport. Emphasis is placed on enzymology and regulation of the pathways, at both the level of gene...

A comprehensive theoretical investigation about the bio-functionalization capability of single walled CNT, BNNT and SiCNT using DNA/RNA nucleobases

Science.gov (United States)

Alinezhad, Heshmatollah; Ganji, Masoud Darvish; Soleymani, Elham; Tajbakhsh, Mahmood

2017-11-01

By means of Density Functional Theory (DFT) based calculations, we have elucidated the interactions between five nucleobases and three nanotubes, namely: CNT, BNNT and SiCNT. The energetics and equilibrium geometries have been calculated within the framework of revPBE method in combination with third version of Grimme's atom pair-wise dispersion corrections with Becke-Johnson damping (D3BJ). The obtained results in terms of adsorption energy values and geometrical parameters suggest that the overall interactions are divided into two parts: non-covalently and covalently bonded systems as the nucleobases are physisorbed onto the surface of CNT and BNNT (Eads ranges from -0.57 to -0.76 eV and -0.54 to -0.78 eV for CNT and BNNT complexes, respectively) while the type of interactions between nucleobase molecules and SiCNT has been found to be of covalent type with the Eads ranging from -0.61 to -1.8 eV. Moreover, the empirical dispersion corrections have been found to play crucial roles in obtaining reliable geometries and adsorption energy values for the non-covalently bonded systems. The role of solvation on the overall interactions has also been explored using the COSMO model within a media with dielectric constant of 78.39 which resembles the water environment and the results revealed that the interaction strength showed a decreasing trend with increasing the polarity of the system. Considering the adsorption energy differences between each nucleobase and the nanotubes, the SiCNT showed promising performance in differentiating between the nucleobase molecules and exhibited the highest affinity to be biofunctionalized in comparison to other nanotubes. The findings of the present work would be very useful for understanding the underlying phenomena behind the interface interactions and would aid future experimental investigations in the fields of biotechnology and materials science.

Electron transfer from nucleobase electron adducts to 5-bromouracil. Is guanine an ultimate sink for the electron in irradiated DNA?

International Nuclear Information System (INIS)

Nese, C.; Yuan, Z.; Schuchmann, M.N.; Sonntag, C. von

1992-01-01

Electron transfer to 5-bromouracil (5-BrU) from nucleobase (N) electron adducts (and their protonated forms) has been studied by product analysis and pulse radiolysis. When an electron is transferred to 5-BrU, the ensuing 5-BrU radical anion rapidly loses a bromide ion; the uracilyl radical thus formed reacts with added t-butanol, yielding uracil. From the uracil yields measured as the function of [N]/[5-BrU] after γ-radiolysis of Ar-saturated solutions it is concluded that thymine and adenine electron adducts and their heteroatom-protonated forms transfer electrons quantitatively to 5-BrU. The data raise the question whether in DNA the guanine moiety may act as the ultimate sink of the electron in competition with other processes such as protonation at C(6) of the thymine electron adduct. (Author)

Control of DNA synthesis in inhibited and activated Agrostemma githago seeds

Energy Technology Data Exchange (ETDEWEB)

Hecker, M [Sektion Biologie, FG Algemeine Botanik und Pflanzenphysiologie, Universitaet Greifswald (German Democratic Republic)

1975-01-01

The relationships between DNA synthesis and germination capacity of Agrostemma seeds had been studied. Protein synthesis and RNA synthesis were activated at the very beginning of imbibition, whereas DNA synthesis started in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30 degC), or aged seeds with a low germination capacity were characterized by a significantly reduced protein synthesis. DNA synthesis was also reduced. The inhibition of the protein synthesis of Agrostemma embryos fed with cycloheximide or actinomycin D caused a depression of DNA synthesis. The results indicated that the initiation of DNA synthesis of imbibing Agrostemma seeds depended on the synthesis of special proteins. Abscisic acid inhibited the growth as well as DNA synthesis of isolated Agrostemma embryos. Nitomycin inhibited germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also showed a reduced incorporation of /sup 3/H-thymidine by DNA. It is suggested that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, was involved in the mechanism of ripening of the Agrostemma seeds.

Control of DNA synthesis in inhibited and activated Agrostemma githago seeds

International Nuclear Information System (INIS)

Hecker, M.

1975-01-01

The relationships between DNA synthesis and germination capacity of Agrostemma seeds had been studied. Protein synthesis and RNA synthesis were activated at the very beginning of imbibition, whereas DNA synthesis started in the second part of the imbibition phase. Agrostemma seeds inhibited by higher temperature (30 degC), or aged seeds with a low germination capacity were characterized by a significantly reduced protein synthesis. DNA synthesis was also reduced. The inhibition of the protein synthesis of Agrostemma embryos fed with cycloheximide or actinomycin D caused a depression of DNA synthesis. The results indicated that the initiation of DNA synthesis of imbibing Agrostemma seeds depended on the synthesis of special proteins. Abscisic acid inhibited the growth as well as DNA synthesis of isolated Agrostemma embryos. Nitomycin inhibited germination and DNA synthesis to the same extent. Dormant seeds with an undiminished intensity of protein synthesis also showed a reduced incorporation of 3 H-thymidine by DNA. It is suggested that DNA synthesis of imbibed seeds, which is a necessary prerequisite for the radicle protrusion, was involved in the mechanism of ripening of the Agrostemma seeds. (author)

Enzymatic primer-extension with glycerol-nucleoside triphosphates on DNA templates.

Directory of Open Access Journals (Sweden)

Jesse J Chen

Full Text Available BACKGROUND: Glycerol nucleic acid (GNA has an acyclic phosphoglycerol backbone repeat-unit, but forms stable duplexes based on Watson-Crick base-pairing. Because of its structural simplicity, GNA is of particular interest with respect to the possibility of evolving functional polymers by in vitro selection. Template-dependent GNA synthesis is essential to any GNA-based selection system. PRINCIPAL FINDINGS: In this study, we investigated the ability of various DNA polymerases to use glycerol-nucleoside triphosphates (gNTPs as substrates for GNA synthesis on DNA templates. Therminator DNA polymerase catalyzes quantitative primer-extension by the incorporation of two glyceronucleotides, with much less efficient extension up to five glyceronucleotides. Steady-state kinetic experiments suggested that GNA synthesis by Therminator was affected by both decreased catalytic rates and weakened substrate binding, especially for pyrimidines. In an attempt to improve pyrimidine incorporation by providing additional stacking interactions, we synthesized two new gNTP analogs with 5-propynyl substituted pyrimidine nucleobases. This led to more efficient incorporation of gC, but not gT. CONCLUSIONS: We suggest that directed evolution of Therminator might lead to mutants with improved substrate binding and catalytic efficiency.

Radiation metagenesis and inhibition of DNA synthesis

International Nuclear Information System (INIS)

Dubinina, L.G.; Sergievskaya, S.P.; Kurashova, Z.I.; Dubinin, N.P.

1983-01-01

The study of modification of radiation mutagenesis and inhibition of the DNA synthesis by means of 1-β-D arabinofuranosylcytosine (ara-C) is carried out. It is shown that ara-C-acting on chromosomes in the G 1 phase and G 2 phase does not cause mutations in the C capillaris cells. The modification by means of ara-C radiation effect in the G 1 phase and G 2 phase correlates with duration and time of administering ara-C before and after irradiation. A new form of ara-C DNA synthesis inhibitor interaction with mutation processes has been found out. Protective effect of the DNA synthesis inhibitor (ara-C) from mutageneous radiation effect is stressed. Sensibilization of the radiation mutagenesis during cell treafment by the DNA synthesis inhibitor (ara-C) is shown. It is pointed out that emergence of sensibilization or protective effect, i. e. antimutagenesis phenomenon depends on conditions under which the synthesis inhibitor acted in G 1 and G 2 phases

2-Methoxypyridine as a Thymidine Mimic in Watson-Crick Base Pairs of DNA and PNA: Synthesis, Thermal Stability, and NMR Structural Studies.

Science.gov (United States)

Novosjolova, Irina; Kennedy, Scott D; Rozners, Eriks

2017-11-02

The development of nucleic acid base-pair analogues that use new modes of molecular recognition is important both for fundamental research and practical applications. The goal of this study was to evaluate 2-methoxypyridine as a cationic thymidine mimic in the A-T base pair. The hypothesis was that including protonation in the Watson-Crick base pairing scheme would enhance the thermal stability of the DNA double helix without compromising the sequence selectivity. DNA and peptide nucleic acid (PNA) sequences containing the new 2-methoxypyridine nucleobase (P) were synthesized and studied by using UV thermal melting and NMR spectroscopy. Introduction of P nucleobase caused a loss of thermal stability of ≈10 °C in DNA-DNA duplexes and ≈20 °C in PNA-DNA duplexes over a range of mildly acidic to neutral pH. Despite the decrease in thermal stability, the NMR structural studies showed that P-A formed the expected protonated base pair at pH 4.3. Our study demonstrates the feasibility of cationic unnatural base pairs; however, future optimization of such analogues will be required. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

UV laser photoactivation of hexachloroplatinate bound to individual nucleobases in vacuo as molecular level probes of a model photopharmaceutical.

Science.gov (United States)

Matthews, Edward; Sen, Ananya; Yoshikawa, Naruo; Bergström, Ed; Dessent, Caroline E H

2016-06-01

Isolated molecular clusters of adenine, cytosine, thymine and uracil bound to hexachloroplatinate, PtCl6(2-), have been studied using laser electronic photodissociation spectroscopy to investigate photoactivation of a platinum complex in the vicinity of a nucleobase. These metal complex-nucleobase clusters represent model systems for identifying the fundamental photochemical processes occurring in photodynamic platinum drug therapies that target DNA. This is the first study to explore the specific role of a strongly photoactive platinum compound in the aggregate complex. Each of the clusters studied displays a broadly similar absorption spectra, with a strong λmax ∼ 4.6 eV absorption band and a subsequent increase in the absorption intensity towards higher spectral-energy. The absorption bands are traced to ligand-to-metal-charge-transfer excitations on the PtCl6(2-) moiety within the cluster, and result in Cl(-)·nucleobase and PtCl5(-) as primary photofragments. These results demonstrate how selective photoexcitation can drive distinctive photodecay channels for a model photo-pharmaceutical. In addition, cluster absorption due to excitation of nucleobase-centred chromophores is observed in the region around 5 eV. For the uracil cluster, photofragments consistent with ultrafast decay of the excited state and vibrational predissociation on the ground-state surface are observed. However, this decay channel becomes successively weaker on going from thymine to cytosine to adenine, due to differential coupling of the excited states to the electron detachment continuum. These effects demonstrate the distinctive photophysical characteristics of the different nucleobases, and are discussed in the context of the recently recorded photoelectron spectra of theses clusters.

DNA synthesis in vitro in human fibroblast preparations

Energy Technology Data Exchange (ETDEWEB)

Kaufmann, W.K.

1983-01-01

When confluent cultures of human fibroblasts were ultraviolet irradiated and either permeabilized or lysed, three types of DNA synthesis were subsequently observed during incubation in vitro: (A) a low level of DNA replication, which ceased after 15-30 min incubation at 37/sup 0/C; (B) radiation-dependent reparative gap-filling, which also ceased after 15 min at 37/sup 0/C; and (C) radiation-independent DNA synthesis, which was not semiconservative and proceeded at a linear rate for 1 hr at 37/sup 0/C. Normal and xeroderma pigmentosum fibroblasts displayed different rates of radiation-dependent reparative gap-filling after lysis but similar rates of radiation-independent DNA synthesis. The rates of DNA replication and radiation-independent DNA synthesis were less in the permeable cell system than in the lysed cell system, whereas radiation-dependent reparative gap-filling was the same in both. Preparations of permeable and lysed cells activated radiation-dependent reparative gap-filling at about 15% of the rate estimated for intact cells. No radiation-dependent DNA strand breaks, as assayed by alkaline elution, were observed in the lysed cell preparation. Some radiation-dependent breaks were observed in the permeable cell preparation, but radiation-dependent DNA breakage was less than that seen in intact cells. This inability to incise DNA at damaged sites could account for the low rate of activation of reparative gap-filling in vitro. DNA strand breaks were produced in fibroblast preparations nonspecifically during lysis or permeabilization and incubation in vitro, and this breakage of DNA probably was responsible for the radiation-independent DNA synthesis.

DnaB gene product-independence of DNA polymerase III-directed repair synthesis in Escherichia coli K-12

International Nuclear Information System (INIS)

Billen, D.; Hellermann, G.R.

1977-01-01

An investigation has been carried out into the role of dnaB gene product in X-ray-induced repair synthesis carried out by DNA polymerase III in toluene-treated Escherichia coli K-12. A polAl polBlOO dnaB mutant deficient in both DNA polymerase I and II activities was used, and it was shown that the level of X-ray-induced, ATP-dependent, non-conservative DNA synthesis was, unlike semi-conservative DNA synthesis, unaffected by a temperature shift from 30 0 to 42 0 C. The dnaB gene product was not therefore necessary for DNA polymerase III-directed repair synthesis, which occurred in the absence of replicative synthesis. (U.K.)

Biomarkers of cigarette smoking and DNA methylating agents: Raman, SERS and DFT study of 3-methyladenine and 7-methyladenine

Science.gov (United States)

Harroun, Scott G.; Zhang, Yaoting; Chen, Tzu-Heng; Ku, Ching-Rong; Chang, Huan-Tsung

2017-04-01

3-Methyladenine and 7-methyladenine are biomarkers of DNA damage from exposure to methylating agents. For example, the concentration of 3-methyladenine increases significantly in the urine of cigarette smokers. Surface-enhanced Raman spectroscopy (SERS) has shown much potential for detection of biomolecules, including DNA. Much work has been dedicated to the canonical nucleobases, with comparatively fewer investigations of modified DNA and modified DNA nucleobases. Herein, Raman spectroscopy and SERS are used to examine the adsorption orientations of 3-methyladenine and 7-methyladenine on Ag nanoparticles. Density functional theory (DFT) calculations at the B3LYP level are used to support the conclusions via simulated spectra of the nucleobases and of Ag+/nucleobase complexes. The results herein show that 7-methyladenine adsorbs upright via its N3 and N9 atoms side, similarly to adenine. 3-Methyladenine adsorbs in a very tilted or flat orientation on the Ag nanoparticles. These findings will be useful for future SERS or other nanoparticle-based bioanalytical assays for detection of these methyladenines or other modified nucleobases.

DNA-repair synthesis in ataxia telangiectasia lymphoblastoid cells

Energy Technology Data Exchange (ETDEWEB)

Ford, M.D.; Houldsworth, J.; Lavin, M.F. (Queensland Univ., Brisbane (Australia). Dept. of Biochemistry)

1981-12-01

The ability of a number of Epstein-Barr virus-transformed lymphoblastoid cells from ataxia telangiectasia (AT) patients to repair ..gamma..-radiation damage to DNA was determined. All of these AT cells were previously shown to be hypersensitive to ..gamma..-radiation. Two methods were used to determine DNA-repair synthesis: isopycnic gradient analysis and a method employing hydroxyurea to inhibit semiconservative DNA synthesis. Control, AT heterozygote and AT homozygote cells were demonstrated to have similar capacities for repair of radiation damage to DNA. In addition at high radiation doses (10-40 krad) the extent of inhibition of DNA synthesis was similar in the different cell types.

Inhibition of DNA replication, DNA repair synthesis, and DNA polymerases α and δ by butylphenyl deoxyguanosine triphosphate

International Nuclear Information System (INIS)

Dreslor, S.L.; Frattini, M.G.

1987-01-01

Semiconservative DNA replication in growing mammalian cells and ultraviolet (UV)-induced DNA repair synthesis in nongrowing mammalian cells are mediated by one or both of the aphidicolin-sensitive DNA polymerases, α and/or δ. They have studied the inhibition of replication and repair synthesis in permeable human cells by N 2 (p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh dGTP), an agent which inhibits polymerase α strongly and polymerase δ weakly. Both processes are inhibited by BuPh-dGTP in competition with dGTP. The K/sub i/'s are, for replication, 2-3 μM and, for repair synthesis, 3-4 μM, consistent with the involvement of the same DNA polymerase in both processes. Inhibition of isolated human polymerase α by BuPh-dGTP is also competitive with dGTP, but the K/sub i/ is approximately 10 nM, several hundred-fold lower than the K/sub i/'s of replication and repair synthesis. Isolated polymerase δ is inhibited by BuPh-dGTP at doses similar to those which inhibit replication and repair synthesis, however, attempts to determine the K/sub i/ of polymerase δ were hampered by the finding that the dependence of δ activity on deoxyribunucleotide concentration is parabolic at low doses. This behavior differs from the behavior of polymerase α and of cellular DNA replication and repair synthesis, all of which show a simple, hyperbolic relationship between activity and deoxyribonucleotide concentration. Thus, inhibition of DNA replication and UV induced DNA repair synthesis by BuPh dGTP is quantitatively similar to DNA polymerase δ, but some other characteristics of the cellular processes are more similar to those of polymerase α

Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

International Nuclear Information System (INIS)

Zurlo, J.; Mignano, J.E.; Eustice, D.C.; Poirier, M.C.; Yager, J.D.

1986-01-01

One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. It was found that N-OH-AAF caused a dose-dependent inhibition of [ 3 H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF were cultured. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. The results show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with previous studies support the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis. (Auth.)

Binding mechanisms of DNA/RNA nucleobases adsorbed on graphene under charging: first-principles van der Waals study

Science.gov (United States)

Gürel, Hikmet Hakan; Salmankurt, Bahadır

2017-06-01

Graphene is a 2D material that has attracted much attention due to its outstanding properties. Because of its high surface area and unique chemical and physical properties, graphene is a good candidate for biological applications. For this reason, a deep understanding of the mechanism of interaction of graphene with biomolecules is required. In this study, theoretical investigation of van der Waals effects has been conducted using density functional theory. Here we show that the order of the binding energies of five nucleobases with graphene is G  >  A  >  T  >  C  >   U. This trend is in good agreement with most of the theoretical and experimental data. Also, the effects of charging on the electronic and structural properties of the graphene-nucleubase systems are studied for the first time. We show that the binding energy can be changed by adding or removing an electron from the system. The results presented in this work provide fundamental insights into the quantum interactions of DNA with carbon-based nanostructures and will be useful for developments in biotechnology and nanotechnology.

Hypochlorite-induced damage to DNA, RNA, and polynucleotides

DEFF Research Database (Denmark)

Hawkins, Clare Louise; Davies, Michael Jonathan

2002-01-01

favored exocyclic amines. EPR experiments have also provided evidence for the rapid addition of pyrimidine-derived nitrogen-centered radicals to other nucleobases to give dimers and the oxidation of DNA by radicals derived from preformed nucleoside chloramines. Direct reaction of HOCl with plasmid DNA...... on the nature of the nucleobase on which they are formed, with chloramines formed from ring heterocyclic amine groups being less stable than those formed on exocyclic amines (RNH2 groups). Evidence is presented for chlorine transfer from the former, kinetically favored, sites to the more thermodynamically...

DNA-synthesis inhibition and repair DNA-synthesis in CHO Ade- C cells: An alternative approach to genotoxicity testing

International Nuclear Information System (INIS)

Slamenova, D.; Papsova, E.; Gabelova, A.; Dusinska, M.; Collins, A.; Wsolova, L.

1997-01-01

We describe an alternative assay to determine genotoxicity. Its main feature is that it combines two measures in a single experiment; the inhibition of replicative DNA synthesis together with the stimulation of DNA repair. We show that, in tests of four different genotoxic agents, the assay gives results that are entirely consistent with what is known about the mode of action of these agents. In addition, we have demonstrated that chemical carcinogens requiring metabolic activation can be examined using a standard procedure of incubation with a microsomal activating fraction. We consider the combined assay for DNA synthesis inhibition and repair synthesis to be a useful way for the rapid pre-screening of chemicals suspected of genotoxic activity on the level of mammalian cells. (author)

Effects of DNA polymerase inhibitors on replicative and repair DNA synthesis in ultraviolet-irradiated HeLa cells

International Nuclear Information System (INIS)

Morita, T.; Nakamura, H.; Tsutsui, Y.; Nishiyama, Y.; Yoshida, S.

1982-01-01

Aphidicolin specifically inhibits eukaryotic DNA polymerase α, while 2',3'-dideoxythymidine 5'-triphosphate (d 2 TTP) inhibits DNA polymerase ν and ν but not α. 1-ν-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) inhibits both DNA polymerase α and ν although to a different extent. Here we measured the effects of these inhibitors on repair DNA synthesis of U.V.-irradiated HeLa cells by two different methods. Firstly, aphidicolin, 1-ν-D-arabinofuranosylcytosine (araC, a precursor of araCTP) and 2',3'-dideoxythimidine (d 2 Thd, a precursor of d 2 TTP) were added directly to the culture medium. In this case, aphidicolin and araC strongly inhibited replicative DNA synthesis of HeLa cells, and they also inhibited repair synthesis after U.V.-irradiation but to a much lesser extent. In contrast, high concentrations of d 2 Thd inhibited repair DNA synthesis to a higher extent than replicative DNA synthesis. Secondly, the active form of inhibitor, d 2 TTP, was microinjected directly into cytoplasm or nuclei or U.V.-irradiated HeLa cells. Microinjection of d 2 TTP effectively inhibited repair synthesis. The microinjection of d 2 TTP, into either cytoplasm or nucleus, strongly inhibited replicative synthesis. These results might indicate that multiple DNA polymerases are involved in repair synthesis as well as in replicative synthesis

DNA repair synthesis dependent on the uvrA,B gene products

International Nuclear Information System (INIS)

Moses, R.E.; Moody, E.E.M.

1975-01-01

Ultraviolet irradiation of toluene-treated Escherichia coli causes an inhibition of replicative DNA synthesis. This is followed by the appearance of nonconservative DNA repair synthesis which does not require either the polymerase or 5' → 3' exonucleolytic activities of DNA polymerase I. The repair synthesis may be catalyzed by DNA polymerase III activity but does not require a functional DNA polymerase II. The ultraviolet-induced synthesis requires ATP and is dependent on a functional uvrA and uvrB gene product. However, other uvr gene products are not required for the synthesis. The recB function is also not required

DNA functionalization by dynamic chemistry

Directory of Open Access Journals (Sweden)

Zeynep Kanlidere

2016-10-01

Full Text Available Dynamic combinatorial chemistry (DCC is an attractive method to efficiently generate libraries of molecules from simpler building blocks by reversible reactions under thermodynamic control. Here we focus on the chemical modification of DNA oligonucleotides with acyclic diol linkers and demonstrate their potential for the deoxyribonucleic acid functionalization and generation of libraries of reversibly interconverting building blocks. The syntheses of phosphoramidite building blocks derived from D-threoninol are presented in two variants with protected amino or thiol groups. The threoninol building blocks were successfully incorporated via automated solid-phase synthesis into 13mer oligonucleotides. The amino group containing phosphoramidite was used together with complementary single-strand DNA templates that influenced the Watson–Crick base-pairing equilibrium in the mixture with a set of aldehyde modified nucleobases. A significant fraction of all possible base-pair mismatches was obtained, whereas, the highest selectivity (over 80% was found for the guanine aldehyde templated by the complementary cytosine containing DNA. The elevated occurrence of mismatches can be explained by increased backbone plasticity derived from the linear threoninol building block as a cyclic deoxyribose analogue.

Involvement of DNA polymerase δ in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

International Nuclear Information System (INIS)

Dresler, S.L.; Gowans, B.J.; Robinson-Hill, R.M.; Hunting, D.J.

1988-01-01

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase δ as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, the authors describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, α or δ. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase α several hundred times more strongly than it inhibits DNA polymerase δ. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase δ. It appears that repair synthesis at late time after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase δ

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

International Nuclear Information System (INIS)

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

A Search for Amino Acids and Nucleobases in the Martian Meteorite Roberts Massif 04262 Using Liquid Chromatography-Mass Spectrometry

Science.gov (United States)

Callahan, Michael P.; Burton, Aaron S.; Elsila, Jamie E.; Baker, Eleni M.; Smith, Karen E.; Glavin, Daniel P.; Dworkin, Jason P.

2013-01-01

The investigation into whether Mars contains signatures of past or present life is of great interest to science and society. Amino acids and nucleobases are compounds that are essential for all known life on Earth and are excellent target molecules in the search for potential Martian biomarkers or prebiotic chemistry. Martian meteorites represent the only samples from Mars that can be studied directly in the laboratory on Earth. Here, we analyzed the amino acid and nucleobase content of the shergottite Roberts Massif (RBT) 04262 using liquid chromatography-mass spectrometry. We did not detect any nucleobases above our detection limit in formic acid extracts; however, we did measure a suite of protein and nonprotein amino acids in hot-water extracts with high relative abundances of beta-alanine and gamma-amino-eta-butyric acid. The presence of only low (to absent) levels of several proteinogenic amino acids and a lack of nucleobases suggest that this meteorite fragment is fairly uncontaminated with respect to these common biological compounds. The distribution of straight-chained amine-terminal eta-omega-amino acids in RBT 04262 resembled those previously measured in thermally altered carbonaceous meteorites. A carbon isotope ratio of -24(0/00) +/- 6(0/00) for beta-alanine in RBT 04262 is in the range of reduced organic carbon previously measured in Martian meteorites (Steele et al. 2012). The presence of eta-omega-amino acids may be due to a high temperature Fischer-Tropschtype synthesis during igneous processing on Mars or impact ejection of the meteorites from Mars, but more experimental data are needed to support these hypotheses.

One-electron oxidation of DNA: mechanism and consequences.

Science.gov (United States)

Schuster, Gary B

2009-01-01

All living organisms store the information necessary to maintain life in their DNA. Any process that damages DNA and causes loss or corruption of that information threatens the viability of the organism. One-electron oxidation is such a process. Loss of an electron from DNA generates a radical cation that is located primarily on its nucleobases. The radical cation migrates reversibly through duplex DNA by hopping until it is eventually trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In its normal aqueous solutions, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counter ions (typically Na(+)) to the phosphate groups play an important role in facilitating both the migration of the radical cation and in its eventual reaction with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA that is comprised of the four common DNA nucleobases, reaction occurs most commonly at a guanine and results in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step primarily by a tandem process. The general outcomes revealed in the one-electron oxidation of DNA oligomers in solution appear to be generally valid also for more complex DNA structures and for the cellular DNA of living organisms.

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

Science.gov (United States)

Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

2018-01-01

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

Stimulation of DNA synthesis in bacterial DNA-membrane complexes after low doses of ionizing radiation

Energy Technology Data Exchange (ETDEWEB)

Watkins, D K [Hammersmith Hospital, London (UK). M.R.C. Experimental Radiopathology Unit

1980-09-01

DNA-membrane complexes from three strains of E. coli were irradiated and changes in the rates of DNA synthesis were observed. Doses from 1-10 krad to complexes from W3110 and pol A1 strains gave up to a 100 per cent increase in DNA synthesis; under the same conditions, no change was observed in Bsub(s-1). The degree of stimulation did not depend on the presence of oxygen during irradiation, and a post-irradiation incubation was necessary to achieve activation. The properties of all three complexes were similar when unirradiated. Irradiation of intact organisms under conditions which produced marked, oxygen-dependent inhibition of the Bsub(s-1) complex had no significant effect on those from W3110 and pol A1. Enhanced DNA synthesis is concluded to be due wholly to repair of pre-existing DNA. It is further postulated that DNA synthesis in untreated complexes (E.coli B's,W3110 and pol A1) is mainly of the repair-type and does not necessarily take place at the site of DNA-membrane attachment.

Theoretical investigation of nuclear quadrupole interactions in DNA at first-principles level

Energy Technology Data Exchange (ETDEWEB)

Mahato, Dip N. [State University of New York at Albany, Department of Physics (United States); Dubey, Archana [University of Central Florida, Department of Physics (United States); Pink, R. H. [State University of New York at Albany, Department of Physics (United States); Scheicher, R. H. [Uppsala University, Condensed Matter Theory Group, Department of Physics and Materials Science (Sweden); Badu, S. R. [State University of New York at Albany, Department of Physics (United States); Nagamine, K. [University of California at Riverside, Department of Physics (United States); Torikai, E. [Yamanashi University, Department of Electrical Engineering (Japan); Saha, H. P.; Chow, Lee [University of Central Florida, Department of Physics (United States); Huang, M. B. [State University of New York at Albany, College of Nanoscale Science and Engineering (United States); Das, T. P., E-mail: tpd56@albany.edu [State University of New York at Albany, Department of Physics (United States)

2008-01-15

We have studied the nuclear quadrupole interactions (NQI) of the {sup 14}N, {sup 17}O and {sup 2}H nuclei in the nucleobases cytosine, adenine, guanine and thymine in the free state as well as when they are bonded to the sugar ring in DNA, simulated through a CH{sub 3} group attached to the nucleobases. The nucleobase uracil, which replaces thymine in RNA, has also been studied. Our results show that there are substantial indirect effects of the bonding with the sugar group in the nucleic acids on the NQI parameters e{sup 2}qQ/h and {eta}. It is hoped that measurements of these NQI parameters in DNA will be available in the future to compare with our predictions. Our results provide the conclusion that for any property dependent on the electronic structures of the nucleic acids, the effects of the bonding between the nucleobases and the nucleic acid backbones have to be included.

γ-irradiation induces radioresistant DNA synthesis in HeLa cells

International Nuclear Information System (INIS)

Synzynys, B.I.; Aminev, A.G.; Konstantinova, S.A.; Saenko, A.S.

1987-01-01

Cells of suspension HeLa culture at the logarithmic phase of growth were exposed to 60 Co-γ-rays (5 Gy), incubated in the nutritious medium, and in 4 h subjected to repeated irradiation: the dose-response function and the dynamics of DNA synthesis inhibition were determined. It was shown that DNA synthesis was inhibited to a lesser extent after preirradiation, in other words, DNA synthesis was radioresistant. A correlation between this synthesis and reproductive cell death is discussed

Radicals derived from histone hydroperoxides damage nucleobases in RNA and DNA

DEFF Research Database (Denmark)

Luxford, C; Dean, R T; Davies, Michael Jonathan

2000-01-01

Exposure of individual histone proteins (H1, H2A, H2B, H3, or H4) and histone octamers (consisting of two molecules each of H2A, H2B, H3, and H4) to hydroxyl radicals, generated by gamma-irradiation, in the presence of O(2) generates protein-bound hydroperoxides in a dose-dependent fashion......; this is in accord with previous studies with other proteins. These histone hydroperoxides are stable in the absence of exogenous catalysts (e.g., heat, light, and transition metal ions), but in the presence of these agents decompose rapidly to give a variety of radicals which have been identified by EPR spin...... trapping. Histone hydroperoxide-derived radicals generated on decomposition of the hydroperoxides with Cu(+) react with both pyrimidine and purine nucleobases. Thus, with uridine the histone hydroperoxide-derived radicals undergo addition across the C(5)-C(6) double bond of the pyrimidine ring to give...

DNA-membrane complex restoration in Micrococcus radiodurans after X-irradiation: relation to repair, DNA synthesis and DNA degradation

Energy Technology Data Exchange (ETDEWEB)

Dardalhon-Samsonoff, M; Averbeck, D [Institut du Radium, 75 - Paris (France). Lab. Curie

1980-07-01

The DNA-membrane complex in Micrococcus radiodurans was shown to be essentially constituted of proteins, lipids and DNA. The complex was dissociated immediately after X-irradiation of cells and restored during post-incubation in complete medium. In X-irradiated protoplasts some DNA remained associated with the complex. Restoration of the complex during post-incubation was only seen in a medium favouring DNA polymerase and ligase activities. Under this condition no DNA synthesis occurred, suggesting that complex restoration may involve ligase activity. The complex restoration in the wild type and the X-ray sensitive mutant UV17 of M. radiodurans was strictly dependent on the X-ray dose. It was correlated with survival and DNA degradation but always preceded the onset of DNA synthesis after X-irradiation. At the same dose the complex restoration was about 2 fold lower in mutant than in wild type cells indicating that the restoration of the complex is related to repair capacity. The results are consistent with the idea that the complex protects X-irradiated DNA of M. radiodurans from further breakdown and, subsequently, permits DNA synthesis and repair to occur.

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

International Nuclear Information System (INIS)

Dresler, S.; Frattini, M.G.; Robinson-Hill, R.M.

1988-01-01

Using permeable diploid human fibroblasts, the authors have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent K m values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 μM. For UV-induced DNA repair synthesis, the apparent K m values were substantially lower, ranging from 0.11 to 0.44 μM for AG1518 cells and from 0.06 to 0.24 μM for IMR-90 cells. Recent data implicate DNA polymerase δ in UV-induced repair synthesis and suggest that DNA polymerases α and δ are both involved in semiconservative replication. They measured K m values for dGTP and dTTP for polymerases α and δ, for comparison with the values for replication and repair synthesis. The deoxyribonucleotide K m values for DNA polymerase δ are much greater than the K m values for UV-induced repair synthesis, suggesting that when polymerase δ functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases. The K m values for UV-induced repair synthesis are 5-80-fold lower than deoxyribonucleotide concentrations that have been reported for intact cultured diploid human fibroblasts. For replication, however, the K m for dGTP is only slightly lower than the average cellular dGTP concentration that has been reported for exponentially growing human fibroblasts. This finding is consistent with the concept that nucleotide compartmentation is required for the attainment of high rates of DNA replication in vivo

Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

Science.gov (United States)

Makarova, A V; Kulbachinskiy, A V

2012-06-01

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

Action of some drugs on enzymes involved in DNA-repair and semiconservative DNA-synthesis

International Nuclear Information System (INIS)

Wawra, E.; Klein, W.; Kocsis, F.; Weniger, P.

1975-07-01

Different antirheumatic and cytostatic drugs had been tested by measurement of the thymidine incorporation into DNA of spleen cells under conditions, under which either DNA-synthesis or repair after gamma- or UV-irradiation takes place. There are substances, which inhibit either only the semiconservative DNA-synthesis (vinblastine, isonicotinic acid hydracide) or only DNA-repair after gamma-irradiation (mixture of penicillin-G and procaine-penicillin-G) or both (cyclophosphamide, phenylbutazone, procarbazine, nalidixic acid). Vincristine shows no effect on the thymidine incorporation in DNA, but by density gradient centrifugation it has been found that it influences the ligase reaction. Two DNA polymerases had been isolated from spleen cells, one of the low molecular and one of the high molecular weight type. The influences of the described drugs on these enzymes and on a deoxyribonuclease I from beef pancreas have been tested in ''in vitro'' systems. In all cases, it has been found that there is no effect or only a very small one, compared with the action of well known inhibitors as e.g. ethidium bromide and p-chloromercuribenzoate, and this cannot be responsible for the suppressions found in DNA-repair and semiconservative DNA-synthesis. (author)

DNA polymerase I-mediated ultraviolet repair synthesis in toluene-treated Escherichia coli

International Nuclear Information System (INIS)

Dorson, J.W.; Moses, R.E.

1978-01-01

DNA synthesis after ultraviolet irradiation is low in wild type toluene-treated cells. The level of repair incorporation is greater in strains deficient in DNA polymerase I. The low level of repair synthesis is attributable to the concerted action of DNA polymerase I and polynucleotide ligase. Repair synthesis is stimulated by blocking ligase activity with the addition of nicotinamide mononucleotide (NMN) or the use of a ligase temperature-sensitive mutant. NMN stimulation is specific for DNA polymerase I-mediated repair synthesis, as it is absent in isogenic strains deficient in the polymerase function or the 5' yields 3' exonuclease function associated with DNA polymerase I. DNA synthesis that is stimulated by NMN is proportional to the ultraviolet exposure at low doses, nonconservative in nature, and is dependent on the uvrA gene product but is independent of the recA gene product. These criteria place this synthesis in the excision repair pathway. The NMN-stimulated repair synthesis requires ATP and is N-ethylmaleimide-resistant. The use of NMN provides a direct means for evaluating the involvement of DNA polymerase I in excision repair

ATP-independent DNA synthesis in Vaccinia-infected L cells

International Nuclear Information System (INIS)

Berger, N.A.; Kauff, R.A.; Sikorski, G.W.

1978-01-01

Mouse L cells can be made permeable to exogenous nucleotides by a cold shock in 0.01 M Tris . HCl pH 7.8, 0.25 M sucrose, 1 mM EDTA, 30 mM 2-mercaptoethanol and 4 mM MgCl 2 . DNA synthesis in permeabilized L cells requires ATP whereas DNA synthesis in permeabilized L cells that are infected with Vaccinia virus is ATP-independent. Permeabilized L cells that are infected with ultraviolet-irradiated virus show a marked suppression of DNA synthesis which is not corrected by an excess of deoxynucleoside triphosphates and ATP. The ATP-dependent and ATP-independent processes of DNA synthesis are inhibited to the same extent by Mal-Net, pHMB, ara CTP and phosphonoacetate. Concentrations of daunorubicin and cytembena, which cause marked inhibition of the ATP-dependent enzymes, only cause partial inhibition of the ATP-independent enzymes. (Auth.)

Damage of DNA by Low Energy Electrons (< 3 eV)

International Nuclear Information System (INIS)

Bald, Ilko; Illenberger, Eugen; Kopyra, Janina

2012-01-01

Recent experiments on low energy electron attachment to DNA and its components in the condensed phase and in the gas phase are reviewed and analysed. From different condensed phase experiments the sensitivity of DNA towards low energy electrons is well documented and strand breaks in DNA are observed at subexcitation energies (< 3 eV) and also in ultrafast electron transfer experiments involving electrons in presolvated states. Gas phase experiments indicate that all building blocks of DNA (the nucleobases, the sugar and the phosphate moiety) undergo resonant dissociative electron attachment (DEA) in the subexcitation regime which may ultimately lead to strand breaks. From very recent gas phase experiments on an entire nucleotide it can be concluded that most strand breaks result from direct electron attachment to the DNA backbone, but also initial electron capture by the nucleobase following electron transfer to the backbone contributes.

Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants of Ustilago maydis

Energy Technology Data Exchange (ETDEWEB)

Unrau, P.

1977-01-01

The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungus Ustilago maydis after incubation at the restrictive temperature (32/sup 0/C) for eight hours. Mutants ts-220, ts-207, ts-432 and ts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutants ts-20, tsd 1-1, ts-84 and pol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutant pol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutant ts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32/sup 0/C. tsd 1-1 and ts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis which correlates to increasing UV sensitivity of these strains on incubation at 32/sup 0/C. A pol 1-1 ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.

Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids.

Science.gov (United States)

Ordonez, Heather; Shuman, Stewart

2013-05-17

We are interested in the distinctive roster of helicases of Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis Lhr as the exemplar of a novel clade of superfamily II helicases, by virtue of its biochemical specificities and signature domain organization. Lhr is a 1507-amino acid monomeric nucleic acid-dependent ATPase that uses the energy of ATP hydrolysis to drive unidirectional 3'-to-5' translocation along single strand DNA and to unwind duplexes en route. The ATPase is more active in the presence of calcium than magnesium. ATP hydrolysis is triggered by either single strand DNA or single strand RNA, yet the apparent affinity for a DNA activator is 11-fold higher than for an RNA strand of identical size and nucleobase sequence. Lhr is 8-fold better at unwinding an RNA:DNA hybrid than it is at displacing a DNA:DNA duplex of identical nucleobase sequence. The truncated derivative Lhr-(1-856) is an autonomous ATPase, 3'-to-5' translocase, and RNA:DNA helicase. Lhr-(1-856) is 100-fold better RNA:DNA helicase than DNA:DNA helicase. Lhr homologs are found in bacteria representing eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria (including Escherichia coli).

Second-strand cDNA synthesis: classical method

International Nuclear Information System (INIS)

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 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

Radioresistant DNA synthesis in fibroblasts of a patient with Down's syndrome

International Nuclear Information System (INIS)

Barenfel'd, L.S.; Bil'din, V.N.; Pleskach, N.M.; Prokof'eva, V.V.

1985-01-01

Ionizing radiation effect on DNA replication on fibroblasts of a healthy donor and a patient with Down's syndrome either by direct 3 H-thymidine inclusion into DNA, or by analysis of the sizes of daughter DNA moleculs at the state of stable distribution in acid saccharose, gradients was studied. Gamma-radiation doses (5-10 Gy) suppressing DNA synthesis in normal fibroblasts practically had no effect on DNA synthesisin fibroblasts of a patient with Down's syndrome. Radioresistant DNA synthesis in Down's syndrome is conditioned by a far less supression of replicon initiation as compared with the one in normal cells. So, it is stated that in Down's disease there is no delay in DNA synthesis by ionizing radiation that enables the normal cells to repair DNA damages before replication renewal

Radiation-induced depression of DNA synthesis in cultured mammalian cells

International Nuclear Information System (INIS)

Povirk, L.F.

1977-01-01

A 313-nm light source was constructed in order to study the mechanisms by which ultraviolet and ionizing radiations inhibit DNA synthesis. It was found that in CHO, MDBK and HeLa cells, grown for one generation in the DNA sensitizer bromodeoxyuridine (BrdUrd), 313-nm light inhibited DNA synthesis with a pattern similar to that of the effect of x-rays on normal cells. A biphasic dose response curve for inhibition of total synthesis was observed, with a sensitive component representing depression of initiation of new replicons and a resistant component representing interference with elongation of replicons already growing at the time of irradiation. Since the BrdUrd plus 313-nm light treatment produces DNA lesions similar to those produced by x-rays (base damage, strand breaks, crosslinks) these results suggest that the effect of x-rays on DNA synthesis is mediated by DNA damage. In experiments with synchronized cells, it was found that in cells in which about half the chromosomes had incorporated BrdUrd, 313-nm light inhibited replication of the BrdUrd-containing DNA, but had no effect on the replication of the unsubstituted DNA in the same cell. Thus the information that DNA is damaged appears to be propagated along the DNA molecule from the sites of damage to the replication initiation sites as some kind of conformational change, possibly a relaxation of superhelical tension. Target theory calculations suggest that a single DNA lesion prevents the initiation of several adjacent replicons

Unlocked nucleic acids with a pyrene-modified uracil: Synthesis, hybridization studies, fluorescent properties and i-motif stability

DEFF Research Database (Denmark)

Perlíková, P.; Karlsen, K.K.; Pedersen, E.B.

2014-01-01

The synthesis of two new phosphoramidite building blocks for the incorporation of 5-(pyren-1-yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene-modified nucleobase component were found to destabilize an i-motif structure at pH 5...... intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene-modified UNA monomers was observed after formation of i-motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene-modified nucleic acids might be useful for designing antisense oligonucleotides...

Sickle erythrocytes inhibit human endothelial cell DNA synthesis

International Nuclear Information System (INIS)

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

1990-01-01

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

Nucleobase assemblies supported by uranyl cation coordination ...

Indian Academy of Sciences (India)

organic hybrid materials using smaller building blocks, which can be ..... nucleobase analogs, in addition to the presence of car- boxylate ... R 2004 Cryst. Eng. Comm. .... Sheldrick G M 2000 SADABS 2.0 (Germany: University of Göttingen). 76.

Probing nucleobase photoprotection with soft x-rays

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Osipov T.

2013-03-01

Full Text Available Nucleobases absorb strongly in the ultraviolet region, leading to molecular excitation into reactive states. The molecules avoid the photoreactions by funnelling the electronic energy into less reactive states on an ultrafast timescale via non-Born-Oppenheimer dynamics. Current theory on the nucleobase thymine discusses two conflicting pathways for the photoprotective dynamics. We present our first results of our free electron laser based UV-pump soft x-ray-probe study of the photoprotection mechanism of thymine. We use the high spatial sensitivity of the Auger electrons emitted after the soft x-ray pulse induced core ionization. Our transient spetra show two timescales on the order of 200 fs and 5 ps, in agreement with previous (all UV ultrafast experiments. The timescales appear at different Auger kinetic energies which will help us to decipher the molecular dynamics.

67Ga-citrate incorporation and DNA synthesis in tumors

International Nuclear Information System (INIS)

Hammersley, P.A.G.; Taylor, D.M.

1975-01-01

The results obtained in these studies suggest that in the tumors studied there is some form of relationship between 67 Ga uptake and the rate of DNA synthesis. However, the observations in the HP melanoma, in which small tumors showed a negative correlation between 67 Ga uptake and rate of DNA synthesis and larger tumors showed a positive correlation, coupled with the virtually constant uptake of 67 Ga over a wide range of rates of DNA synthesis in the drug- and radiation-treated tumors, suggest that the uptake of the radionuclide is not simply related to the rate of DNA synthesis per se. Studies in embryonic mouse tissues suggested that 67 Ga uptake was not related to the rate of DNA synthesis and regenerating liver does not show a greater 67 Ga uptake than normal liver. Phytohemagglutinin-treated human lymphocytes show increased 67 Ga uptake compared to unstimulated lymphocytes, and it has been suggested that this is related to the stimulus to divide rather than to events occurring in a specific phase of the cell cycle. This suggests that proliferating cells may exhibit membrane changes which either result in increased transport of 67 Ga into the cell or permit a greater degree of binding of the radionuclide to the cell membrane than can occur in resting cells. The membrane-binding hypothesis is supported by the observations on phytohemagglutinin-stimulated lymphocytes but not by observations of the subcellular distribution of 67 Ga in these tumors which confirm the suggestion that the radionuclide is concentrated in lysosomes. Thus it appears that although in tumor cells, at least, there is some correlation between 67 Ga uptake and the rate of DNA synthesis and hence by implication of cell proliferation, the nature of this link remains obscure, and more detailed studies are needed to increase our understanding of the relationship

Correspondence: chromosomal localization of uv-induced unscheduled DNA synthesis

International Nuclear Information System (INIS)

Berliner, J.; Mello, R.S.; Norman, A.

1976-01-01

We have measured the grain density - the number of grains per unit length - over the centromere and noncentromere regions of metaphase chromosomes in autoradiographs of human lymphocytes. When the chromosomes were labeled in G 0 by uv-induced unscheduled DNA synthesis, the grain density was two to four times larger over the centromere than over the noncentromere regions. When the labeling was done by scheduled DNA synthesis in S or unscheduled synthesis in M, the grain densities were approximately equal over both regions

Spectroscopic study of site selective DNA damage induced by intense soft X-rays

CERN Document Server

Fujii, K

2003-01-01

To investigate the mechanisms of DNA damage induced by direct photon impact, we observed the near edge X-ray absorption fine structures (NEXAFS) of DNA nucleobases using monochromatic synchrotron soft X-rays around nitrogen and oxygen K-shell excitation regions. Each spectrum obtained has unique structure corresponding to pi* excitation of oxygen or nitrogen 1s electron. These aspects open a way of nucleobase-selective photo-excitation in a DNA molecule using high resolution monochromatized soft X-rays. From the analysis of polarization-dependent intensities of the pi* resonance peak, it is clarified that adenine, guanine an uracil form orientated surface structure. Furthermore from the direct measurement of positive ions desorbed from photon irradiated DNA components, it is revealed that the sugar moiety is a fragile site in a DNA molecule. (author)

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

Science.gov (United States)

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

Proton Transfer in Nucleobases is Mediated by Water

Energy Technology Data Exchange (ETDEWEB)

Khistyaev, Kirill; Golan, Amir; Bravaya, Ksenia B.; Orms, Natalie; Krylov, Anna I.; Ahmed, Musahid

2013-08-08

Water plays a central role in chemistry and biology by mediating the interactions between molecules, altering energy levels of solvated species, modifying potential energy proles along reaction coordinates, and facilitating ecient proton transport through ion channels and interfaces. This study investigates proton transfer in a model system comprising dry and microhydrated clusters of nucleobases. With mass spectrometry and tunable vacuum ultraviolet synchrotron radiation, we show that water shuts down ionization-induced proton transfer between nucleobases, which is very ecient in dry clusters. Instead, a new pathway opens up in which protonated nucleo bases are generated by proton transfer from the ionized water molecule and elimination of a hydroxyl radical. Electronic structure calculations reveal that the shape of the potential energy prole along the proton transfer coordinate depends strongly on the character of the molecular orbital from which the electron is removed, i.e., the proton transfer from water to nucleobases is barrierless when an ionized state localized on water is accessed. The computed energetics of proton transfer is in excellent agreement with the experimental appearance energies. Possible adiabatic passage on the ground electronic state of the ionized system, while energetically accessible at lower energies, is not ecient. Thus, proton transfer is controlled electronically, by the character of the ionized state, rather than statistically, by simple energy considerations.

Continuous induction of unscheduled DNA synthesis by gamma irradiation

International Nuclear Information System (INIS)

Weniger, P.; Klein, W.; Ott, E.; Kocsis, F.; Altmann, H.

1990-01-01

The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of DNA. Usually, in an in vivo - in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation was observed over a long period of time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten time intervals during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process - with centrifugation in alkaline sucrose a half-life of some minutes is found - an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation. (author) 4 figs

Continuous induction of unscheduled DNA synthesis by gamma irradiation

International Nuclear Information System (INIS)

Weniger, P.; Klein, W.; Ott, E.; Kocsis, F.; Altmann, H.

1988-08-01

The induction of DNA-synthesis in non-S-phase cells is a very sensitive measure of a preceding damage of the DNA. Usually, in an in vivo -in vitro test (treatment of an animal, incorporation of H3-thymidine in a cell suspension) the damaging of DNA takes place hours to days before the evaluation. In this case, the time course of the UDS-induction after a single dose of 1 Gy gamma irradiation should be observed for a long time (21 months). C57 black mice served as test animals. In an age of about 80 days they were irradiated and the induction of unscheduled DNA synthesis was measured at ten points of time during the whole life-span of the animals. Although the repair in this gamma radiation damage in DNA is a very quick process - with centrifugation in alkaline sucrose you find a half time of some minutes - an induction of unscheduled DNA synthesis could be seen at the irradiated animals until the end of their life (640 days). The reason for this could be permanent disorders in cellular regulation caused by the gamma irradiation. 4 figs. (Author)

Replication stress activates DNA repair synthesis in mitosis

DEFF Research Database (Denmark)

Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A

2015-01-01

Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps...... into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early...... mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest...

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

Science.gov (United States)

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.

Using CdTe/ZnSe core/shell quantum dots to detect DNA and damage to DNA

Directory of Open Access Journals (Sweden)

Moulick A

2017-02-01

Full Text Available Amitava Moulick,1,2 Vedran Milosavljevic,1,2 Jana Vlachova,1,2 Robert Podgajny,3 David Hynek,1,2 Pavel Kopel,1,2 Vojtech Adam1,2 1Department of Chemistry and Biochemistry, Mendel University, 2Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic; 3Faculty of Chemistry, Jagiellonian University, Krakow, Poland Abstract: CdTe/ZnSe core/shell quantum dot (QD, one of the strongest and most highly luminescent nanoparticles, was directly synthesized in an aqueous medium to study its individual interactions with important nucleobases (adenine, guanine, cytosine, and thymine in detail. The results obtained from the optical analyses indicated that the interactions of the QDs with different nucleobases were different, which reflected in different fluorescent emission maxima and intensities. The difference in the interaction was found due to the different chemical behavior and different sizes of the formed nanoconjugates. An electrochemical study also confirmed that the purines and pyrimidines show different interactions with the core/shell QDs. Based on these phenomena, a novel QD-based method is developed to detect the presence of the DNA, damage to DNA, and mutation. The QDs were successfully applied very easily to detect any change in the sequence (mutation of DNA. The QDs also showed their ability to detect DNAs directly from the extracts of human cancer (PC3 and normal (PNT1A cells (detection limit of 500 pM of DNA, which indicates the possibilities to use this easy assay technique to confirm the presence of living organisms in extreme environments. Keywords: nanoparticles, nucleobases, biosensor, fluorescence, mutation

Mechanism Underlying the Nucleobase-Distinguishing Ability of Benzopyridopyrimidine (BPP).

Science.gov (United States)

Kochman, Michał A; Bil, Andrzej; Miller, R J Dwayne

2017-11-02

Benzopyridopyrimidine (BPP) is a fluorescent nucleobase analogue capable of forming base pairs with adenine (A) and guanine (G) at different sites. When incorporated into oligodeoxynucleotides, it is capable of differentiating between the two purine nucleobases by virtue of the fact that its fluorescence is largely quenched when it is base-paired to guanine, whereas base-pairing to adenine causes only a slight reduction of the fluorescence quantum yield. In the present article, the photophysics of BPP is investigated through computer simulations. BPP is found to be a good charge acceptor, as demonstrated by its positive and appreciably large electron affinity. The selective quenching process is attributed to charge transfer (CT) from the purine nucleobase, which is predicted to be efficient in the BPP-G base pair, but essentially inoperative in the BPP-A base pair. The CT process owes its high selectivity to a combination of two factors: the ionization potential of guanine is lower than that of adenine, and less obviously, the site occupied by guanine enables a greater stabilization of the CT state through electrostatic interactions than the one occupied by adenine. The case of BPP illustrates that molecular recognition via hydrogen bonding can enhance the selectivity of photoinduced CT processes.

Dissociation of histone and DNA synthesis in x-irradiated HeLa cells

International Nuclear Information System (INIS)

Bases, R.; Mendez, F.

1971-01-01

Although histone synthesis and DNA synthesis are normally very well coordinated in HeLa cells, their histone synthesis proved relatively resistant to inhibition by ionizing radiation. During the first 24 h after 1,000 R the rate of cellular DNA synthesis progressively fell to small fractions of control values while histone synthesis with much less relative reduction. Acrylamide gel electropherograms of the acid soluble nuclear histones synthesized by irradiated HeLa cells were qualitatively normal

Induction of unscheduled DNA synthesis on the nuclear matrix of rat hepatocytes after whole-body γ-irradiation

International Nuclear Information System (INIS)

Bezlepkin, V.G.; Malinovskij, Yu.Yu.; Kuznetsova, E.A.; Namvar, R.A.; Gaziev, A.I.

1986-01-01

DNA synthesis in hepatocytes was studied by incorporation of [ 3 H]thymidine administered of portal vein of γ-irradiated (80 Gy) rats. It was shown that the rate of replicative DNA synthesis decreased in hepatocytes of the regenerating liver and unscheduled DNA synthesis was induced at the nuclear matrix of resting cells of the intact liver. In addition to repair synthesis, DNA synthesis resembling replicative one (''aberrant'' DNA synthesis) accounts for a considerable fraction of γ-radiation-induced synthesis of DNA at the nuclear matrix

Neurotensin enhances estradiol induced DNA synthesis in immature rat uterus

Energy Technology Data Exchange (ETDEWEB)

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.

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

International Nuclear Information System (INIS)

Hammond, R.A.; Miller, M.R.; McClung, J.K.

1990-01-01

The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

Synthesis and biological evaluation of conformationally restricted and nucleobase-modified analogs of the anticancer compound 3'-C-ethynylcytidine (ECyd)

DEFF Research Database (Denmark)

Hrdlicka, Patrick J; Jepsen, Jan S; Wengel, Jesper

2005-01-01

A series of conformationally restricted and nucleobase-modified analogs of the anticancer compound 3'-C-ethynylcytidine (ECyd) and its uracil analog (EUrd) have been synthesized. While none of the conformationally restricted analogs displayed anticancer activity, 5-iodo-EUrd and 5-bromo-EUrd disp......-EUrd displayed potent anticancer activity with IC50 values of 35 nM and 0. 73 microM....

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

DEFF Research Database (Denmark)

Skov, Joan; Bryld, Torsten; Lindegaard, Dorthe

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 stabilizati...

Stimulatory effect of low dose radionuclide on DNA synthesis and UDS in splenic lymphocytes

International Nuclear Information System (INIS)

Zhu Shoupeng; Yang Zhanshan

1999-12-01

To study the stimulatory effect on DNA synthesis and unscheduled DNA synthesis (UDS) in splenic lymphocytes induced by low dose enriched uranium 235 U. By using 3 H-TdR incorporation assay technique, the DNA replicative synthesis in PHA and LPS stimulated splenic lymphocytes was observed. By using DNA synthesis inhibitor such as hydroxyurea, the UV-induced unscheduled DNA synthesis in splenic lymphocytes occurred. When the injected low dose of enriched uranium 235 u was 0.1 μg/kg body weight, the transformation capacity was elevated for splenic T lymphocytes, simultaneously the stimulative index increased. The UDS of splenic lymphocytes induced by ultra-violate revealed a statistically significant increase by low dose of enriched uranium 235 U at the range of 0.1-20 μg/kg body weight. A stimulatory action of low dose enriched uranium 235 U on DNA replicative synthesis as well as on UV-induced UDS in splenic lymphocytes was detected

Multiple nucleobase transporters contribute to boscalid sensitivity in Aspergillus nidulans.

Science.gov (United States)

Kalampokis, Ioannis F; Kapetanakis, George C; Aliferis, Konstantinos A; Diallinas, George

2018-03-01

The development of fungicide-resistant fungal populations represents a major challenge for the agrochemical and agri-food sectors, which threatens food supply and security. The issue becomes complex for fungi that cause quantitative and qualitative losses due to mycotoxin biosynthesis. Nonetheless, currently, the molecular details underlying fungicide action and fungal resistance mechanisms are partially known. Here, we have investigated whether plasma membrane transporters contribute to specific fungicide uptake in the model fungus Aspergillus nidulans. Independent physiological tests and toxicity screening of selected fungicides provided evidence that the antifungal activity of Succinate Dehydrogenase Inhibitors (SDHIs) is associated with the expression of several nucleobase-related transporters. In particular, it was shown that a strain genetically inactivated in all seven nucleobase-related transporters is resistant to the fungicide boscalid, whereas none of the single null mutants exhibited significant resistance level. By constructing and testing isogenic strains that over-express each one of the seven transporters, we confirmed that five of them, namely, UapC, AzgA, FycB, CntA, and FurA, contribute to boscalid uptake. Additionally, by employing metabolomics we have examined the effect of boscalid on the metabolism of isogenic strains expressing or genetically lacking boscalid-related nucleobase transporters. The results confirmed the involvement of specific nucleobase transporters in fungicide uptake, leading to the discovery of corresponding metabolites-biomarkers. This work is the first report on the involvement of specific transporters in fungicide uptake and toxicity and their impact on fungal metabolism regulation and results might be further exploited towards the deeper understanding of fungal resistance to fungicides. Copyright © 2018 Elsevier Inc. All rights reserved.

Synthesis and biological evaluation of nucleobase-modified analogs of the anticancer compounds 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd)

DEFF Research Database (Denmark)

Hrdlicka, Patrick J; Jepsen, Jan S; Nielsen, Claus

2005-01-01

convergent synthetic route utilizing diacetone-alpha-D-glucose as starting material. 5-Iodo-substituted EUrd was the most potent inhibitor among the novel nucleobase-modified analogs in in vitro assays against human adenocarcinoma breast and prostate cancer cells with IC50 values down to 35 nM....

Analytical Devices Based on Direct Synthesis of DNA on Paper.

Science.gov (United States)

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.

Unscheduled DNA synthesis and elimination of DNA damage in liver cells of. gamma. -irradiated senescent mice

Energy Technology Data Exchange (ETDEWEB)

Gaziev, A.I.; Malakhova, L.V. (AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki)

1982-10-01

The level of 'spontaneous' and ..gamma..-radiation-induced DNA synthesis which is not inhibited with hydroxyurea (unscheduled synthesis) is considerably lower in hepatocytes of 18-22-month-old mice than that of 1.5-2-month-old mice. The dose-dependent increase (10-300 Gy) of unscheduled DNA synthesis (UDS) in hepatocytes of senescent mice is higher than in young animals. The elimination of damage in DNA of ..gamma..-irradiated hepatocytes (100 Gy) was examined by using an enzyme system (M. luteus extract and DNA-polymerase I of E. coli). It was found that the rate of elimination of the DNA damage in hepatocytes of 20-month-old mice is lower than that of 2-month-old mice although the activities of DNA-polymerase ..beta.. and apurinic endonuclease remain equal in the liver of both senescent and young mice. However, the nucleoids from ..gamma..-irradiated liver nuclei of 2-month-old mice are relaxed to a greater extent (as judged by the criterion of ethidium-binding capacity) than those of 20-month-old mice. The results suggest that there are limitations in the functioning of repair enzymes and in their access to damaged DNA sites in the chromatin of senescent mouse liver cells.

Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

International Nuclear Information System (INIS)

Barenfeld, L.S.; Pleskach, N.M.; Bildin, V.N.; Prokofjeva, V.V.; Mikhelson, V.M.

1986-01-01

The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [ 3 H]DNA in alkaline sucrose gradients or by direct assay of the amount of [ 3 H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations. (orig.)

Cell-free assay measuring repair DNA synthesis in human fibroblasts

International Nuclear Information System (INIS)

Ciarrocchi, G.; Linn, S.

1978-01-01

Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

Role of Achiral Nucleobases in Multicomponent Chiral Self-Assembly: Purine-Triggered Helix and Chirality Transfer.

Science.gov (United States)

Deng, Ming; Zhang, Li; Jiang, Yuqian; Liu, Minghua

2016-11-21

Chiral self-assembly is a basic process in biological systems, where many chiral biomolecules such as amino acids and sugars play important roles. Achiral nucleobases usually covalently bond to saccharides and play a significant role in the formation of the double helix structure. However, it remains unclear how the achiral nucleobases can function in chiral self-assembly without the sugar modification. Herein, we have clarified that purine nucleobases could trigger N-(9-fluorenylmethox-ycarbonyl) (Fmoc)-protected glutamic acid to self-assemble into helical nanostructures. Moreover, the helical nanostructure could serve as a matrix and transfer the chirality to an achiral fluorescence probe, thioflavin T (ThT). Upon chirality transfer, the ThT showed not only supramolecular chirality but also circular polarized fluorescence (CPL). Without the nucleobase, the self-assembly processes cannot happen, thus providing an example where achiral molecules played an essential role in the expression and transfer of the chirality. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Radiosensitive Down syndrome lymphoblastoid lines have normal ionizing-radiation-induced inhibition of DNA synthesis

International Nuclear Information System (INIS)

Ganges, M.B.; Robbins, J.H.; Jiang, H.; Hauser, C.; Tarone, R.E.

1988-01-01

The extent of X-ray-induced inhibition of DNA synthesis was determined in radiosensitive lymphoblastoid lines from 3 patients with Down syndrome and 3 patients with ataxia telangiectasia (AT). Compared to 6 normal control lines, the 3 AT lines were abnormally resistant to X-ray-induced inhibition of DNA synthesis, while the 3 Down syndrome lines had normal inhibition. These results demonstrate that radiosensitive human cells can have normal X-ray-induced inhibition of DNA synthesis and provide new evidence for the dissociation of radioresistant DNA synthesis. (author). 27 refs.; 1 fig.; 1 tab

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress.

Science.gov (United States)

Gan, Haiyun; Yu, Chuanhe; Devbhandari, Sujan; Sharma, Sushma; Han, Junhong; Chabes, Andrei; Remus, Dirk; Zhang, Zhiguo

2017-10-19

The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA synthesis in the imaginal wing discs of the American bollworm ...

Indian Academy of Sciences (India)

Unknown

The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids. 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instar Helicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of.

Impairment of DNA synthesis in Gunn rat cerebellum.

Science.gov (United States)

Yamada, N; Sawasaki, Y; Nakajima, H

1977-05-06

Brain DNA synthesis was developmentally investigated in Gunn rat with marked cerebellar hypoplasia due to hereditary hyperbilirubinemia. In this mutant rat, the Purkinje cell was nearly selectively affected in the cerebellar cortex by bilirubin. The impaired DNA synthesis was observed in homozygous (jj) Gunn rat cerebellum, in which the DNA content and [3H]thymidine incorporation rate into DNA decreased after 10 days of age compared to those in the heterozygous (Jj)littermate. In contrast, these impairments were not found in the non-cerebellar parts of the brain and liver of jj Gunn rat. The activity of cerebellar thymidine kinase in jj Gunn rat decreased from a very early stae, being 80% of Jj rat at 6 days, and 50% at 10 days of age. The enzyme activity was not affected in the non-cerebellar parts of the brain. Although bilirubin competitively inhibited cerebellar thymidine kinase activity in vitro (15% at 10(-5) M), such bilirubin level was found to be about 1000-fold that in vivo. Moreover, photo-degradation of bilirubin in jj cerebellum exhibited no improvement in thymidine kinase activity, and the presence of an enzyme inactivator was not suggested in jj cerebellum. These results seem to indicate that the induction of thymidine kinase might be affected in jj Gunn rat cerebellum. The possibility that the impaired DNA synthesis in the external granular cells in jj cerebellum may be due to Purkinje cell damage is discussed.

Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C.elegans embryos?

International Nuclear Information System (INIS)

Hartman, Phil; Reddy, Jennifer; Svendsen, Betty-Ann

1991-01-01

Over 10-fold larger fluences were required to inhibit both DNA synthesis and cell division in wild-type C.elegans embryos as compared with other model systems or C.elegans rad mutants. In addition, unlike in other organisms, the molecular weight of daughter DNA strands was reduced only after large, superlethal fluences. The molecular weight of nascent DNA fragments exceeded the interdimer distance by up to 19-fold, indicating that C.elegans embryos can replicate through non-instructional lesions. This putative trans-lesion synthetic capability may explain the refractory nature of UV-radiation on embryonic DNA synthesis and nuclear division in C.elegans. (author). 42 refs.; 7 figs

Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C. elegans embryos

Energy Technology Data Exchange (ETDEWEB)

Hartman, Phil; Reddy, Jennifer; Svendsen, Betty-Ann [Texas Christian Univ., Fort Worth, TX (United States). Dept. of Biology

1991-09-01

Over 10-fold larger fluences were required to inhibit both DNA synthesis and cell division in wild-type C.elegans embryos as compared with other model systems or C.elegans rad mutants. In addition, unlike in other organisms, the molecular weight of daughter DNA strands was reduced only after large, superlethal fluences. The molecular weight of nascent DNA fragments exceeded the interdimer distance by up to 19-fold, indicating that C.elegans embryos can replicate through non-instructional lesions. This putative trans-lesion synthetic capability may explain the refractory nature of UV-radiation on embryonic DNA synthesis and nuclear division in C.elegans. (author). 42 refs.; 7 figs.

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

International Nuclear Information System (INIS)

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

Anhydrous crystals of DNA bases are wide gap semiconductors.

Science.gov (United States)

Maia, F F; Freire, V N; Caetano, E W S; Azevedo, D L; Sales, F A M; Albuquerque, E L

2011-05-07

We present the structural, electronic, and optical properties of anhydrous crystals of DNA nucleobases (guanine, adenine, cytosine, and thymine) found after DFT (Density Functional Theory) calculations within the local density approximation, as well as experimental measurements of optical absorption for powders of these crystals. Guanine and cytosine (adenine and thymine) anhydrous crystals are predicted from the DFT simulations to be direct (indirect) band gap semiconductors, with values 2.68 eV and 3.30 eV (2.83 eV and 3.22 eV), respectively, while the experimentally estimated band gaps we have measured are 3.83 eV and 3.84 eV (3.89 eV and 4.07 eV), in the same order. The electronic effective masses we have obtained at band extremes show that, at low temperatures, these crystals behave like wide gap semiconductors for electrons moving along the nucleobases stacking direction, while the hole transport are somewhat limited. Lastly, the calculated electronic dielectric functions of DNA nucleobases crystals in the parallel and perpendicular directions to the stacking planes exhibit a high degree of anisotropy (except cytosine), in agreement with published experimental results.

Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

Science.gov (United States)

Makarova, Alena V; Grabow, Corinn; Gening, Leonid V; Tarantul, Vyacheslav Z; Tahirov, Tahir H; Bessho, Tadayoshi; Pavlov, Youri I

2011-01-31

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+) ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA"). We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

Directory of Open Access Journals (Sweden)

Alena V Makarova

2011-01-01

Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC

Energy Technology Data Exchange (ETDEWEB)

Shi, Rongxin; Mullins, Elwood A.; Shen, Xing; #8208; Xing; Lay, Kori T.; Yuen, Philip K.; David, Sheila S.; Rokas, Antonis; Eichman, Brandt F. (UCD); (Vanderbilt)

2017-10-20

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

The extension of a DNA double helix by an additional Watson-Crick base pair on the same backbone

DEFF Research Database (Denmark)

Kumar, P.; Sharma, P. K.; Madsen, Charlotte S.

2013-01-01

Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand.......Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand....

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

KAUST Repository

Fornander, Louise H

2013-12-03

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

Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

International Nuclear Information System (INIS)

Sawada, N.; Tomomura, A.; Sattler, C.A.; Sattler, G.L.; Kleinman, H.K.; Pitot, H.C.

1986-01-01

The effects of several extracellular matrix components (EMCs) - fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen - on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [ 3 H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [ 3 H]thymidine uptake exhibited in the cell cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density

Improved DNA clamps by stacking to adjacent nucleobases

DEFF Research Database (Denmark)

Fatthalla, M.I.; Pedersen, Erik Bjerregaard

2012-01-01

Three or four aromatic rings interconnected by acetylene bridges form a stiff conjugated system with sufficient conformational freedom to make it useful to link together the two strands of a DNA clamp. Upon targeting a ssDNA, the conformational flexibility allows better stacking of the linker...... to the underlying non-planar base triplet in the formed triplex. This type of triplexes has a substantially higher thermal melting temperature which can be further improved by inserting locked nucleic acids (LNAs) in the Hoogsteen part of the clamp. An extremely high sensitivity to mismatches is observed...

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements

Directory of Open Access Journals (Sweden)

Modesto Redrejo-Rodríguez

2017-11-01

Full Text Available Family B DNA polymerases (PolBs play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB, that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance.

Efficiency and Fidelity of Human DNA Polymerases λ and β during Gap-Filling DNA Synthesis

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Brown, Jessica A.; Pack, Lindsey R.; Sanman, Laura E.; Suo, Zucai

2010-01-01

The base excision repair (BER) pathway coordinates the replacement of 1 to 10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1- to 10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5′-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER. PMID:20961817

High-Throughput Block Optical DNA Sequence Identification.

Science.gov (United States)

Sagar, Dodderi Manjunatha; Korshoj, Lee Erik; Hanson, Katrina Bethany; Chowdhury, Partha Pratim; Otoupal, Peter Britton; Chatterjee, Anushree; Nagpal, Prashant

2018-01-01

Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label-free optical DNA sequencing technique will require nanoscale focusing of light, a high-throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the "fingerprinting region" from ≈400-1400 cm -1 . Here, surface-enhanced Raman spectroscopy is used to demonstrate label-free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer-scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k-mers. The content of each nucleotide in a DNA block can be a unique and high-throughput method for identifying sequences, genes, and other biomarkers as an alternative to single-letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high-throughput block optical sequencing method with lossy genomic data compression using k-mer identification from multiplexed optical data acquisition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA repair and DNA synthesis in leukemic and virus infected cells

International Nuclear Information System (INIS)

Tuschl, H.; Altmann, H.; Kovac, R.; Topaloglou, A.; Stacher, A.; Fanta, D.

1978-09-01

Autoradiographic determinations of unscheduled DNA synthesis in peripheral lymphocytes of leukemic patients showed strongly different results according to various types of disease of different forms of therapy, respectively. Similar investigations performed with lymphocytes of Herpes simplex infected persons during symptom-free intervals revealed imbalances of the repair system caused by virus infection. BND cellulose chromatography and measurement of 3 H-thymidine incorporation into single- and double stranded DNA fractions showed an increase in velocity of the rejoining process, but a decrease in total incorporation. Because of these results and the demonstration of the supercoiled structure of DNA it is suggested that virusinfections cause a faster rejoining of gaps, but at the same time leave a number of failures within DNA unrecognized. (author)

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

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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.

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

Science.gov (United States)

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

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes.

Science.gov (United States)

van Erp, Y H; Koopmans, M J; Heirbaut, P R; van der Hoeven, J C; Weterings, P J

1992-06-01

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.

Functionalized Solid Electrodes for Electrochemical Biosensing of Purine Nucleobases and Their Analogues: A Review

Science.gov (United States)

Sharma, Vimal Kumar; Jelen, Frantisek; Trnkova, Libuse

2015-01-01

Interest in electrochemical analysis of purine nucleobases and few other important purine derivatives has been growing rapidly. Over the period of the past decade, the design of electrochemical biosensors has been focused on achieving high sensitivity and efficiency. The range of existing electrochemical methods with carbon electrode displays the highest rate in the development of biosensors. Moreover, modification of electrode surfaces based on nanomaterials is frequently used due to their extraordinary conductivity and surface to volume ratio. Different strategies for modifying electrode surfaces facilitate electron transport between the electrode surface and biomolecules, including DNA, oligonucleotides and their components. This review aims to summarize recent developments in the electrochemical analysis of purine derivatives, as well as discuss different applications. PMID:25594595

Functionalized Solid Electrodes for Electrochemical Biosensing of Purine Nucleobases and Their Analogues: A Review

Directory of Open Access Journals (Sweden)

Vimal Kumar Sharma

2015-01-01

Full Text Available Interest in electrochemical analysis of purine nucleobases and few other important purine derivatives has been growing rapidly. Over the period of the past decade, the design of electrochemical biosensors has been focused on achieving high sensitivity and efficiency. The range of existing electrochemical methods with carbon electrode displays the highest rate in the development of biosensors. Moreover, modification of electrode surfaces based on nanomaterials is frequently used due to their extraordinary conductivity and surface to volume ratio. Different strategies for modifying electrode surfaces facilitate electron transport between the electrode surface and biomolecules, including DNA, oligonucleotides and their components. This review aims to summarize recent developments in the electrochemical analysis of purine derivatives, as well as discuss different applications.

Quantum Point Contact Single-Nucleotide Conductance for DNA and RNA Sequence Identification.

Science.gov (United States)

Afsari, Sepideh; Korshoj, Lee E; Abel, Gary R; Khan, Sajida; Chatterjee, Anushree; Nagpal, Prashant

2017-11-28

Several nanoscale electronic methods have been proposed for high-throughput single-molecule nucleic acid sequence identification. While many studies display a large ensemble of measurements as "electronic fingerprints" with some promise for distinguishing the DNA and RNA nucleobases (adenine, guanine, cytosine, thymine, and uracil), important metrics such as accuracy and confidence of base calling fall well below the current genomic methods. Issues such as unreliable metal-molecule junction formation, variation of nucleotide conformations, insufficient differences between the molecular orbitals responsible for single-nucleotide conduction, and lack of rigorous base calling algorithms lead to overlapping nanoelectronic measurements and poor nucleotide discrimination, especially at low coverage on single molecules. Here, we demonstrate a technique for reproducible conductance measurements on conformation-constrained single nucleotides and an advanced algorithmic approach for distinguishing the nucleobases. Our quantum point contact single-nucleotide conductance sequencing (QPICS) method uses combed and electrostatically bound single DNA and RNA nucleotides on a self-assembled monolayer of cysteamine molecules. We demonstrate that by varying the applied bias and pH conditions, molecular conductance can be switched ON and OFF, leading to reversible nucleotide perturbation for electronic recognition (NPER). We utilize NPER as a method to achieve >99.7% accuracy for DNA and RNA base calling at low molecular coverage (∼12×) using unbiased single measurements on DNA/RNA nucleotides, which represents a significant advance compared to existing sequencing methods. These results demonstrate the potential for utilizing simple surface modifications and existing biochemical moieties in individual nucleobases for a reliable, direct, single-molecule, nanoelectronic DNA and RNA nucleotide identification method for sequencing.

Survey of current trends in DNA synthesis core facilities.

Science.gov (United States)

Hager, K M; Fox, J W; Gunthorpe, M; Lilley, K S; Yeung, A

1999-12-01

The Nucleic Acids Research Group of the Association of Biomolecular Resource Facilities (ABRF) last surveyed DNA synthesis core facilities in April 1995. Because of the introduction of new technologies and dramatic changes in the market, we sought to update survey information and to determine how academic facilities responded to the challenge presented by commercial counterparts. The online survey was opened in January 1999 by notifying members and subscribers to the ABRF electronic discussion group. The survey consisted of five parts: general facility information, oligonucleotide production profile, oligonucleotide charges, synthesis protocols, and trends in DNA synthesis (including individual comments). All submitted data were anonymously coded. Respondents from DNA synthesis facilities were primarily from the academic category and were established between 1984 and 1991. Typically, a facility provides additional services such as DNA sequencing and has upgraded to electronic ordering. There is stability in staffing profiles for these facilities in that the total number of employees is relatively unchanged, the tenure for staff averages 5.9 years, and experience is extensive. On average, academic facilities annually produce approximately 1/16 the number of oligonucleotides produced by the average commercial facilities, but all facilities report an increase in demand. Charges for standard oligonucleotides from academic facilities are relatively higher than from commercial companies; however, the opposite is true for modified phosphoramidites. Subsidized facilities charge less than nonsubsidized facilities. Synthesis protocols and reagents are standard across the categories. Most facilities offer typical modifications such as biotinylation. Despite the competition by large commercial facilities that have reduced costs dramatically, academic facilities remain a stable entity. Academic facilities enhance the quality of service by focusing on nonstandard

Multi-line split DNA synthesis: a novel combinatorial method to make high quality peptide libraries

Directory of Open Access Journals (Sweden)

Ueno Shingo

2004-09-01

Full Text Available Abstract Background We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis. Results A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 1016 diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons. Conclusions Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases.

Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

International Nuclear Information System (INIS)

Vikhanskaya, F.L.; Khrebtukova, I.A.; Manuilova, E.S.

1985-01-01

A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m 2 ) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m 2 ) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair. (orig.)

Histone gene expression remains coupled to DNA synthesis during in vitro cellular senescence

International Nuclear Information System (INIS)

Zambetti, G.; Stein, G.; Stein, J.; Dell'Orco, R.

1987-01-01

Despite a decrease in the extent to which confluent monolayers of late compared to early passage CF3 human diploid fibroblasts can be stimulated to proliferate, the time course of DNA synthesis onset is similar regardless of the in vitro age of the cells. A parallel and stoichiometric relationship is maintained between the rate of DNA synthesis and the cellular levels of histone mRNA independent of the age of the cell cultures. Furthermore, DNA synthesis and cellular histone mRNA levels decline in a coordinate manner after inhibition of DNA replication by hydroxyurea treatment. These results indicate that while the proliferative activity of human diploid fibroblasts decreases with passage in culture, those cells that retain the ability to proliferate continue to exhibit a tight coupling of DNA replication and histone gene expression

Stimulation of DNA synthesis in cultured rat alveolar type II cells

International Nuclear Information System (INIS)

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 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 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

Loss of DNA-membrane interactions and cessation of DNA synthesis in myeloperoxidase-treated Escherichia coli

International Nuclear Information System (INIS)

Rosen, H.; Orman, J.; Rakita, R.M.; Michel, B.R.; VanDevanter, D.R.

1990-01-01

Neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host defense functions. The mechanisms of action of most of these systems are incompletely understood. The present report indicates that microbicidal activity by a neutrophil-derived antimicrobial system, consisting of myeloperoxidase, enzymatically generated hydrogen peroxide, and chloride ion, is accompanied by prompt cessation of DNA synthesis in Escherichia coli, as determined by markedly reduced incorporation of [ 3 H]thymidine into trichloracetic acid-precipitable material. Simultaneously, the myeloperoxidase system mediates a decline in the ability of E. coli membranes to bind hemimethylated DNA sequences containing the E. coli chromosomal origin of replication (oriC). Binding of oriC to the E. coli membrane is an essential element of orderly chromosomal DNA replication. Comparable early changes in DNA synthesis and DNA-membrane interactions were not observed with alternative oxidant or antibiotic-mediated microbicidal systems. It is proposed that oxidants generated by the myeloperoxidase system modify the E. coli membrane in such a fashion that oriC binding is markedly impaired. As a consequence chromosomal DNA replication is impaired and organisms can no longer replicate

Synthesis of CdS nanoparticles based on DNA network templates

International Nuclear Information System (INIS)

Yao Yong; Song Yonghai; Wang Li

2008-01-01

CdS nanoparticles have been successfully synthesized by using DNA networks as templates. The synthesis was carried out by first dropping a mixture of cadmium acetate and DNA on a mica surface for the formation of the DNA network template and then transferring the sample into a heated thiourea solution. The Cd 2+ reacted with thiourea at high temperature and formed CdS nanoparticles on the DNA network template. UV-vis spectroscopy, photoluminescence, x-ray diffraction and atomic force microscopy (AFM) were used to characterize the CdS nanoparticles in detail. AFM results showed that the resulted CdS nanoparticles were directly aligned on the DNA network templates and that the synthesis and assembly of CdS nanoparticles was realized in one step. CdS nanoparticles fabricated with this method were smaller than those directly synthesized in a thiourea solution and were uniformly aligned on the DNA networks. By adjusting the density of the DNA networks and the concentration of Cd 2+ , the size and density of the CdS nanoparticles could be effectively controlled and CdS nanoparticles could grow along the DNA chains into nanowires. The possible growth mechanism has also been discussed in detail

Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents

International Nuclear Information System (INIS)

Hsia, M.T.

1987-01-01

The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs

Relative ultraviolet radiation sensitivity of certain functions of polyoma virus. Stimulation of cell DNA synthesis

International Nuclear Information System (INIS)

Barra, Yves; Imbert, Jean; Planche, Jacqueline; Meyer, Georges.

1977-01-01

Peritoneal Mouse macrophages were used to study the stimulation of cell DNA synthesis by polyoma virus. Using ultraviolet-irradiated polyoma virus, it was possible to show a difference between the inactivation of infectivity and of induction of DNA synthesis. By statistical analysis of these two phenomena it was found that 39% of the viral genome is necessary for the induction of cell DNA synthesis [fr

Irreversible electron attachment--a key to DNA damage by solvated electrons in aqueous solution.

Science.gov (United States)

Westphal, K; Wiczk, J; Miloch, J; Kciuk, G; Bobrowski, K; Rak, J

2015-11-07

The TYT and TXT trimeric oligonucleotides, where X stands for a native nucleobase, T (thymine), C (cytosine), A (adenine), or G (guanine), and Y indicates a brominated analogue of the former, were irradiated with ionizing radiation generated by a (60)Co source in aqueous solutions containing Tris as a hydroxyl radical scavenger. In the past, these oligomers were bombarded with low energy electrons under an ultra-high vacuum and significant damage to TXT trimers was observed. However, in aqueous solution, hydrated electrons do not produce serious damage to TXT trimers although the employed radiation dose exceeded many times the doses used in radiotherapy. Thus, our studies demonstrate unequivocally that hydrated electrons, which are the major form of electrons generated during radiotherapy, are a negligible factor in damage to native DNA. It was also demonstrated that all the studied brominated nucleobases have a potential to sensitize DNA under hypoxic conditions. Strand breaks, abasic sites and the products of hydroxyl radical attachment to nucleobases have been identified by HPLC and LC-MS methods. Although all the bromonucleobases lead to DNA damage under the experimental conditions of the present work, bromopyrimidines seem to be the radiosensitizers of choice since they lead to more strand breaks than bromopurines.

Translesion synthesis DNA polymerases promote error-free replication through the minor-groove DNA adduct 3-deaza-3-methyladenine.

Science.gov (United States)

Yoon, Jung-Hoon; Roy Choudhury, Jayati; Park, Jeseong; Prakash, Satya; Prakash, Louise

2017-11-10

N3-Methyladenine (3-MeA) is formed in DNA by reaction with S -adenosylmethionine, the reactive methyl donor, and by reaction with alkylating agents. 3-MeA protrudes into the DNA minor groove and strongly blocks synthesis by replicative DNA polymerases (Pols). However, the mechanisms for replicating through this lesion in human cells remain unidentified. Here we analyzed the roles of translesion synthesis (TLS) Pols in the replication of 3-MeA-damaged DNA in human cells. Because 3-MeA has a short half-life in vitro , we used the stable 3-deaza analog, 3-deaza-3-methyladenine (3-dMeA), which blocks the DNA minor groove similarly to 3-MeA. We found that replication through the 3-dMeA adduct is mediated via three different pathways, dependent upon Polι/Polκ, Polθ, and Polζ. As inferred from biochemical studies, in the Polι/Polκ pathway, Polι inserts a nucleotide (nt) opposite 3-dMeA and Polκ extends synthesis from the inserted nt. In the Polθ pathway, Polθ carries out both the insertion and extension steps of TLS opposite 3-dMeA, and in the Polζ pathway, Polζ extends synthesis following nt insertion by an as yet unidentified Pol. Steady-state kinetic analyses indicated that Polι and Polθ insert the correct nt T opposite 3-dMeA with a much reduced catalytic efficiency and that both Pols exhibit a high propensity for inserting a wrong nt opposite this adduct. However, despite their low fidelity of synthesis opposite 3-dMeA, TLS opposite this lesion replicates DNA in a highly error-free manner in human cells. We discuss the implications of these observations for TLS mechanisms in human cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA-Compatible Nitro Reduction and Synthesis of Benzimidazoles.

Science.gov (United States)

Du, Huang-Chi; Huang, Hongbing

2017-10-18

DNA-encoded chemical libraries have emerged as a cost-effective alternative to high-throughput screening (HTS) for hit identification in drug discovery. A key factor for productive DNA-encoded libraries is the chemical diversity of the small molecule moiety attached to an encoding DNA oligomer. The library structure diversity is often limited to DNA-compatible chemical reactions in aqueous media. Herein, we describe a facile process for reducing aryl nitro groups to aryl amines. The new protocol offers simple operation and circumvents the pyrophoric potential of the conventional method (Raney nickel). The reaction is performed in aqueous solution and does not compromise DNA structural integrity. The utility of this method is demonstrated by the versatile synthesis of benzimidazoles on DNA.

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

Energy Technology Data Exchange (ETDEWEB)

Jones, R.A. [Rutgers, The State Univ. of New Jersey, Piscataway, NJ (United States)

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.

Semiconservative and unscheduled DNA-synthesis of rat thymocytes under the influence of some radioprotecting and radiosensitizing agents

Energy Technology Data Exchange (ETDEWEB)

Tempel, K.; Wulffius-Kock, M.; Winkle, J.; Schmerold, I.

1982-02-01

The effects of aminoethylisothiuroniumbromide (AET), cysteamine (CY-A), cysteine (CY-E), glutathione (GLU), mercaptoethanol (MA), mercaptopropionylglycine (MPG), N-ethylmaleimide (NEM), metronidazole (MNA), nitroacetophenone (NAP), nitrofurazone (NFA), arabinofuranosylcytosine (araC), fluorouracil (FU), adriamycin (AM), ethidiumbromide (E), bleomycin (BM), and diethyldithiocarbamate (DDC) on the semiconservative and unscheduled incorporation of /sup 3/H-thymidine into the DNA were tested on rat thymocytes in vitro. DNA damage has been measured using the hydroxylapatite system. Unscheduled DNA synthesis was induced by UV-light and/or X-irradiation. The semiconservative DNA synthesis was inhibited by the above substances-with exception of MA and MPG. Aminothioles, NAP, NFA, and BM enhanced, araC, FU, AM, E, and DDC diminished unscheduled DNA synthesis. After alkaline unwinding, the duplex form of DNA decreased under the influence of CY-A, CY-E, GLU, MPG, NEM, NAP, NFA, araC, FU, AM, E, and BM. It is suggested that stimulation of unscheduled DNA synthesis combined with a transient decrease of semiconservative DNA synthesis will amplify the DNA repair capacity of thymocytes, whereas radiation damage may be intensified by araC, FU, AM,E, and DDC - at least partly, through inhibition of unscheduled DNA synthesis. With respect to the action of NAP, NFA, and BM, DNA repair may be concerned in a more indirect manner.

Semiconservative and unscheduled DNA-synthesis of rat thymocytes under the influence of some radioprotecting and radiosensitizing agents

International Nuclear Information System (INIS)

Tempel, K.; Wulffius-Kock, M.; Winkle, J.; Schmerold, I.

1982-01-01

The effects of aminoethylisothiuroniumbromide (AET), cysteamine (CY-A), cysteine (CY-E), glutathione (GLU), mercaptoethanol (MA), mercaptopropionylglycine (MPG), N-ethylmaleimide (NEM), metronidazole (MNA), nitroacetophenone (NAP), nitrofurazone (NFA), arabinofuranosylcytosine (araC), fluorouracil (FU), adriamycin (AM), ethidiumbromide (E), bleomycin (BM), and diethyldithiocarbamate (DDC) on the semiconservative and unscheduled incorporation of 3 H-thymidine into the DNA were tested on rat thymocytes in vitro. DNA damage has been measured using the hydroxylapatite system. Unscheduled DNA synthesis was induced by UV-light and/or X-irradiation. The semiconservative DNA synthesis was inhibited by the above subtrances-with exception of MA and MPG. Aminothioles, NAP, NFA, and BM enhanced, araC, FU, AM, E, and DDC diminished unscheduled DNA synthesis. After alkaline unwinding, the duplex form of DNA decreased under the influence of CY-A, CY-E, GLU, MPG, NEM, NAP, NFA, araC, FU, AM, E, and BM. It is suggested that stimulation of unscheduled DNA synthesis combined with a transient decrease of semiconservative DNA synthesis will amplify the DNA repair capacity of thymocytes, whereas radiation damage may be intensified by araC, FU, AM,E, and DDC - at least partly, through inhibition of unscheduled DNA synthesis. With respect to the action of NAP, NFA, and BM, DNA repair may be concerned in a more indirect manner. (orig.) [de

Inhibitory effect of benzene metabolites on nuclear DNA synthesis in bone marrow cells

International Nuclear Information System (INIS)

Lee, E.W.; Johnson, J.T.; Garner, C.D.

1989-01-01

Effects of endogenously produced and exogenously added benzene metabolites on the nuclear DNA synthetic activity were investigated using a culture system of mouse bone marrow cells. Effects of the metabolites were evaluated by a 30-min incorporation of [ 3 H]thymidine into DNA following a 30-min interaction with the cells in McCoy's 5a medium with 10% fetal calf serum. Phenol and muconic acid did not inhibit nuclear DNA synthesis. However, catechol, 1,2,4-benzenetriol, hydroquinone, and p-benzoquinone were able to inhibit 52, 64, 79, and 98% of the nuclear DNA synthetic activity, respectively, at 24 μM. In a cell-free DNA synthetic system, catechol and hydroquinone did not inhibit the incorporation of [ 3 H]thymidine triphosphate into DNA up to 24 μM but 1,2,4-benzenetriol and p-benzoquinone did. The effect of the latter two benzene metabolites was completely blocked in the presence of 1,4-dithiothreitol (1 mM) in the cell-free assay system. Furthermore, when DNA polymerase α, which requires a sulfhydryl (SH) group as an active site, was replaced by DNA polymerase 1, which does not require an SH group for its catalytic activity, p-benzoquinone and 1,2,4-benzenetriol were unable to inhibit DNA synthesis. Thus, the data imply the p-benzoquinone and 1,2,4-benzenetriol inhibited DNA polymerase α, consequently resulting in inhibition of DNA synthesis in both cellular and cell-free DNA synthetic systems. The present study identifies catechol, hydroquinone, p-benzoquinone, and 1,2,4-benzenetriol as toxic benzene metabolites in bone marrow cells and also suggests that their inhibitory action on DNA synthesis is mediated by mechanism(s) other than that involving DNA damage as a primary cause

Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA

International Nuclear Information System (INIS)

Setlow, B.; Hand, A.R.; Setlow, P.

1991-01-01

Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores

Dissociation between insulin secretion and DNA synthesis in cultured pancreatic islets

DEFF Research Database (Denmark)

Nielsen, Jens Høiriis

1985-01-01

-Tdr incorporation. However, long-term exposure to IBMX did not result in increased DNA content of the islets. Inhibition of the DNA synthesis by 5 mM hydroxyurea resulted in a marked reduction in DNA content of the islets but no decrease in either insulin release or insulin content when expressed per ng DNA...

DNA synthesis during development and proliferation of glial cells in organotypic rat cerebellar culture

International Nuclear Information System (INIS)

Renkawek, K.

1977-01-01

DNA synthesis was investigated in glial cells in vitro with 3 H thymidine in concentration 1 μCi/ml medium. Incorporation of isotope into the glial nuclei has been found both in the explant (7-21%) and in the outgrowth (22-56%). DNA synthesis was dependent on the age of culture and due to the contact inhibition in the outgrowth. Results point out that marked DNA synthesis is a characteristic feature of glia differentiation and of reactive character of glial cells in vitro. (author)

The proofreading 3'→5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis

International Nuclear Information System (INIS)

Khare, Vineeta; Eckert, Kristin A.

2002-01-01

The 3'→5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase γ (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'→5' exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity

Lesion Orientation of O4-Alkylthymidine Influences Replication by Human DNA Polymerase η.

Science.gov (United States)

O'Flaherty, D K; Patra, A; Su, Y; Guengerich, F P; Egli, M; Wilds, C J

2016-08-01

DNA lesions that elude repair may undergo translesion synthesis catalyzed by Y-family DNA polymerases. O 4 -Alkylthymidines, persistent adducts that can result from carcinogenic agents, may be encountered by DNA polymerases. The influence of lesion orientation around the C4- O 4 bond on processing by human DNA polymerase η (hPol η ) was studied for oligonucleotides containing O 4 -methylthymidine, O 4 -ethylthymidine, and analogs restricting the O 4 -methylene group in an anti -orientation. Primer extension assays revealed that the O 4 -alkyl orientation influences hPol η bypass. Crystal structures of hPol η •DNA•dNTP ternary complexes with O 4 -methyl- or O 4 -ethylthymidine in the template strand showed the nucleobase of the former lodged near the ceiling of the active site, with the syn - O 4 -methyl group engaged in extensive hydrophobic interactions. This unique arrangement for O 4 -methylthymidine with hPol η , inaccessible for the other analogs due to steric/conformational restriction, is consistent with differences observed for nucleotide incorporation and supports the concept that lesion conformation influences extension across DNA damage. Together, these results provide mechanistic insights on the mutagenicity of O 4 MedT and O 4 EtdT when acted upon by hPol η .

DNA synthesis and uv resistance in Escherichia coli K12 cells

Energy Technology Data Exchange (ETDEWEB)

Slezarikova, V [Slovenska Akademia Vied, Bratislava (Czechoslovakia). Vyskumny Ustav Onkologicky

1976-01-01

The influence was studied of preirradiation inhibition of proteosynthesis by amino acids starvation on survival and DNA synthesis in E. coli K 12 cells, which differ by their genetic features with regard to a certain type of repair. The surviving fraction was studied by appropriate dilution of cell suspension and spreading on agar plates. DNA synthesis was investigated by the incorporation of thymine-2-/sup 14/C. In our conditions a correlation was found between cell survival and the resistance of DNA replication to UV radiation in cells proficient in excision and post-replication repair. This correlation was not found in the excision deficient strain. It is concluded that enhanced resistance of DNA replication is not a sufficient condition for enhanced cell resistance.

Characterization of nucleosides and nucleobases in fruits of Ziziphus jujuba by UPLC-DAD-MS.

Science.gov (United States)

Guo, Sheng; Duan, Jin-Ao; Tang, Yu-Ping; Zhu, Zhen-Hua; Qian, Ye-Fei; Yang, Nian-Yun; Shang, Er-Xin; Qian, Da-Wei

2010-10-13

The fruit of Ziziphus jujuba , named dazao in Chinese, has been utilized as food as well as crude drugs in China for thousands of years. To explore the profiles of the nucleosides and nucleobases in this fruit, an ultraperformance liquid chromatograph coupled with a photodiode array detector and electrospray ionization-mass spectrometer method (UPLC-DAD-MS) has been established and validated in this paper. The validated method was successfully applied for the simultaneous characterization and quantitation of 9 nucleosides and nucleobases in 49 dazao samples, which comprised 43 cultivars from 26 cultivation regions. Furthermore, principal component analysis (PCA) was performed to classify the samples on the basis of the contents of the nine analyzed compounds. The results showed that almost all of these dazao samples were rich in nucleosides and nucleobases, although their contents were obviously various, and the proposed method could serve as a prerequisite for quality control of jujube products.

The rate of DNA synthesis in normal human and ataxia telangiectasia cells after exposure to X-irradiation

International Nuclear Information System (INIS)

Wit, J. de; Bootsma, D.; Jaspers, N.G.J.; Rijksverdedigingsorganisatie TNO, Rijswijk

1981-01-01

The rate of DNA synthesis was studied in normal cell strains and in strains from patients suffering from the inherited disorder ataxia telangiectasia (AT). After exposure to relatively low doses of oxic X-rays (0- 4 krad) DNA synthesis was depressed in AT cell strains to a significantly lesser extent than in normal cells. This response was observed in both an excision-deficient and an excision-proficient strain. In contrast, there was no difference in DNA-synthesis inhibition between AT and normal cells after UV exposure. After X-irradiation of cells from patients with xeroderma pigmentosum, both complementation group A and XP variants, the observed rate of DNA synthesis was equal to that in normal cells. An exception was the strain XP3BR which has been shown to be X-ray-sensitive. This strain exhibited diminished DNA synthesis inhibition after X-ray doses below 1 krad. These data suggest a relationship between hypersensitivity to X-rays and diminished depression of DNA synthesis. (orig.)

Ultraviolet Irradiation of Pyrimidine in Interstellar Ice Analogs: Formation and Photo-Stability of Nucleobases

Science.gov (United States)

Nuevo, Michel; Milam, Stefanie N.; Sandford, Scott A.; Elsila, Jamie E.; Dworkin, Jason P.

2010-01-01

Astrochemistry laboratory experiments recently showed that molecules of prebiotic interest can potentially form in space, as supported by the detection of amino acids in organic residues formed by the UV photolysis of ices simulating interstellar and cometary environments (H2O, CO, CO2, CH3OH, NH3, etc.). Although the presence of amino acids in the interstellar medium (ISM) is still under debate, experiments and the detection of amino acids in meteorites both support a scenario in which prebiotic molecules could be of extraterrestrial origin, before they are delivered to planets by comets, asteroids, and interplanetary dust particles. Nucleobases, the informational subunits of DNA and RNA, have also been detected in meteorites, although they have not yet been observed in the ISM. Thus, these molecules constitute another family of prebiotic compounds that can possibly form via abiotical processes in astrophysical environments. Nucleobases are nitrogen-bearing cyclic aromatic species with various functional groups attached, which are divided into two classes: pyrimidines (uracil, cytosine, and thymine) and purines (adenine and guanine). In this work, we study how UV irradiation affects pyrimidine mixed in interstellar ice analogs (H2O, NH3, CH3OH). In particular, we show that the UV irradiation of H2O:pyrimidine mixtures leads to the production of oxidized compounds including uracil, and show that both uracil and cytosine are formed upon irradiation of H2O:NH3:pyrimidine mixtures. We also study the photostability of pyrimidine and its photoproducts formed during these experiments.

DNA synthesis in the pituitary gland of the rat: effect of sulpiride and clomiphene.

Science.gov (United States)

Burdman, J A; Szijan, I; Jahn, G A; Machiavelli, G; Kalbermann, L E

1979-09-15

Sulpiride administration to rats releases prolactin and increases DNA replication in the anterior pituitary gland. Clomiphene prevents the stimulation of DNA synthesis produced by sulpiride, but does not affect prolactin release from the gland. These findings suggest that the intracellular prolactin content of the anterior pituitary gland plays a role in the regulation of DNA synthesis through a mechanism mediated by oestrogens.

Age-related variation in the DNA-repair synthesis after UV-C irradiation in unstimulated lymphocytes of healthy blood donors

International Nuclear Information System (INIS)

Kovacs, E.; Weber, W.; Mueller, H.

1984-01-01

UV-C light-induced DNA-repair synthesis was studied in unstimulated lymphocytes of 51 healthy blood donors aged between 17 and 74 years. The evaluation included (1) the spontaneous DNA-synthesis in unirradiated lymphocytes with and without hydroxyurea, (2) the DNA-repair synthesis in lymphocytes irradiated with UV-light. The interindividual variation was significantly higher than the methodological variation ascertained in 24 persons in whom 2 determinations were carried out. In blood donors aged between 17 and 39 years, the spontaneous DNA synthesis, both with and without hydroxyurea, was significantly lower than in older individuals. The DNA-repair synthesis was dependent on the dose of UV-C light between 2 and 16 J/m 2 . There were no significant differences in DNA-repair synthesis in the age range 17-74 years. The variations in rate of DNA-repair synthesis were wider in older (44-74 years), than in younger individuals. (orig.)

Herpes virus and viral DNA synthesis in ultraviolet light-irradiated cells

Energy Technology Data Exchange (ETDEWEB)

Coppey, J; Nocentini, S [Institut du Radium, 75 - Paris (France). Lab. Curie

1976-07-01

The rate of virus DNA synthesis and the production of infectious virus are impaired in stationary monkey kidney CV-I cells irradiated with u.v. before infection with herpes simplex virus (HSV). The inhibition of HSV multiplication is due to u.v.-induced damage in cell DNA. CV-I cells recover their capacity to support HSV growth during the 40 to 48 h after irradiation, and the final virus yield is enhanced by factor of 10. The time course of the recovery is similar to that of the excision repair process occurring in u.v.-irradiated mammalian cells. Caffeine, hydroxyurea and cycloheximide inhibit the recovery. Fluorodeoxyuridine is without effect. A small but significant amount of labelled dThd coming from irradiated cell DNA is incorporated into virus DNA. HSV specified thymidine kinase seems to be more effective for virus DNA synthesis in irradiated than in control cells.

Direct on-chip DNA synthesis using electrochemically modified gold electrodes as solid support

Science.gov (United States)

Levrie, Karen; Jans, Karolien; Schepers, Guy; Vos, Rita; Van Dorpe, Pol; Lagae, Liesbet; Van Hoof, Chris; Van Aerschot, Arthur; Stakenborg, Tim

2018-04-01

DNA microarrays have propelled important advancements in the field of genomic research by enabling the monitoring of thousands of genes in parallel. The throughput can be increased even further by scaling down the microarray feature size. In this respect, microelectronics-based DNA arrays are promising as they can leverage semiconductor processing techniques with lithographic resolutions. We propose a method that enables the use of metal electrodes for de novo DNA synthesis without the need for an insulating support. By electrochemically functionalizing gold electrodes, these electrodes can act as solid support for phosphoramidite-based synthesis. The proposed method relies on the electrochemical reduction of diazonium salts, enabling site-specific incorporation of hydroxyl groups onto the metal electrodes. An automated DNA synthesizer was used to couple phosphoramidite moieties directly onto the OH-modified electrodes to obtain the desired oligonucleotide sequence. Characterization was done via cyclic voltammetry and fluorescence microscopy. Our results present a valuable proof-of-concept for the integration of solid-phase DNA synthesis with microelectronics.

DNA-like double helix formed by peptide nucleic acid

DEFF Research Database (Denmark)

Wittung, P; Nielsen, Peter E.; Buchardt, O

1994-01-01

Although the importance of the nucleobases in the DNA double helix is well understood, the evolutionary significance of the deoxyribose phosphate backbone and the contribution of this chemical entity to the overall helical structure and stability of the double helix is not so clear. Peptide nucleic...

Desoxyribonucleic acid (DNA) synthesis in vitro by thymus and spleen cells of the rat after hyperthermia

Energy Technology Data Exchange (ETDEWEB)

Tempel, K.; Spath, A.

1988-03-01

The inhibition of the semiconservative and restorative DNA synthesis caused by hyperthermia (30 to 60 min, 43/sup 0/C) was significantly higher in spleen cells than in thymus cells. The DNA repair synthesis of thymus cells measured at 37/sup 0/C was increased by about two times the initial value after a pre-incubation of 30 to 90 min and 30 to 60 min, respectively, with 37 and 43/sup 0/C, respectively. Under the same conditions, the /sup 3/H-thymidine incorporation into the DNA of spleen cells diminished proportionally to the pre-incubation time after a pre-incubation of 30 and 45 min, respectively, with 43 and 37/sup 0/C, respectively. When hyperthermia and inhibitors of DNA synthesis or DNA repair (hydroxyurea, 1-..beta..-D-arabinofuranosylcytosine, 3', 5'-didesoxythymidine, and 3-aminobenzamide) were combined, overadditive effects - without cellspecific particularities - were seen only in the case of 3-aminobenzamide. Only in thymus cells, the inhibitor of DNA topoisomerase II novobiocin caused an overadditive reinforcement of the inhibition induced by hyperthermia of the semiconservative DNA synthesis. The stimulation of DNA repair synthesis in thymus cells caused by novobiocin with the aid of DNA polymerase ..beta.. could be compensated by hyperthermia. The sedimentation of thymus and spleen cell nucleoids was increased after hyperthermia. The results suggest a special importance of DNA topology and of the DNA polymerase ..beta.. activity for the cellular effect of hyperthermia.

DNA synthesis in permeabilized WI38 and MRC5 cells

International Nuclear Information System (INIS)

Griffiths, T.D.; Carpenter, J.G.

1980-01-01

DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident

Postirradiation DNA synthesis is inversely related to cell survival

International Nuclear Information System (INIS)

Kapiszewska, M.; Lange, C.S.

1987-01-01

Postirradiation (PI) events which might lead to cellular reproductive death or survival were studied in L5178Y-S (LY-S) cells. PI incubation at 25 0 C protects LY-S cells against the PLD fixation which takes place at 37 0 C. An optimal condition for the repair of PLD is 1h at 37 0 C followed by 4h holding at 25 0 C prior to the second half of a split dose, or 5L holding at 25 0 C without a 37 0 C incubation. Longer incubations at 37 0 C resulted in progressively decreased survivals. Postirradiation inhibition of DNA synthesis at 37 0 C was observed only during the first 30 min; thereafter, /sup 3/H-dThd incorporation was higher than in unirradiated controls. This excess synthesis effect was removed by shifting irradiated cells to 25 0 C holding. The inhibition observed at 25 0 C was reversed by shifting to 37 0 C. Thus the degree of postirradiation DNA synthesis is inversely related to PLD/SLD repair. DNA filter elution shows complete SSB repair by 3h at both temperatures (with faster kinetics at 37 0 C), and DSB repair plateaus at 80% (37 0 C) and 60% (25 0 C) after 90 min

The influence of some prostaglandins on DNA synthesis and DNA excision repair in mouse spleen cells ''in vitro''

International Nuclear Information System (INIS)

Klein, W.; Altmann, H.; Kocsis, F.; Egg, D.; Guenther, R.

1978-03-01

''In vitro'' experiments were performed on mouse spleen cells to establish possible influences of some naturally occurring prostaglandins on DNA synthesis and DNA excision repair. The prostaglandins A 1 , B 1 , E 1 , E 2 and Fsub(2α) were tested in concentrations of 10 pg, 5 ng and 2,5μg per ml cell suspension. DNA synthesis was significantly increased by PgFsub(2α) in all the three concentrations tested, while the other tested prostaglandins were essentially ineffective. DNA excision repair was significantly inhibited by PgE 1 and PgE 2 at 5 ng/ml and at 2,5 μg/ml but increased by PgFsub(2α) in the two lower concentrations. The rejoining of DNA-strand breaks after gamma-irradiation was slightly reduced by PgE 1 , PgE 2 and PgF 2 at 2,5 μg/ml. (author)

Yield of DNA strand breaks and their relationship to DNA polymerase I-dependent repair synthesis and ligation following x-ray exposure of toluene-treated Escherichia coli

International Nuclear Information System (INIS)

Billen, D.

1981-01-01

In Escherichia coli made permeable to nucleotides by toluene treatment, a DNA polymerase I-directed repair synthesis is observed. This is an exaggerated repair synthesis which can be abruptly terminated by the addition of the DNA ligase cofactor, nicotinamide adenine dinucleotide. This communication describes experiments which bear on the relationship between measurable strand breaks, DNA polymerase I-directed, exaggerated repair synthesis, and strand-break repair

Resonance Enhanced Multi-photon Spectroscopy of DNA

Science.gov (United States)

Ligare, Marshall Robert

For over 50 years DNA has been studied to better understand its connection to life and evolution. These past experiments have led to our understanding of its structure and function in the biological environment but the interaction of DNA with UV radiation at the molecular level is still not very well understood. Unique mechanisms in nucleobase chromaphores protect us from adverse chemical reactions after UV absorption. Studying these processes can help develop theories for prebiotic chemistry and the possibility of alternative forms of DNA. Using resonance enhanced multi-photon spectroscopic techniques in the gas phase allow for the structure and dynamics of individual nucleobases to be studied in detail. Experiments studying different levels of structure/complexity with relation to their biological function are presented. Resonant IR multiphoton dissociation spectroscopy in conjunction with molecular mechanics and DFT calculations are used to determine gas phase structures of anionic nucleotide clusters. A comparison of the identified structures with known biological function shows how the hydrogen bonding of the nucleotides and their clusters free of solvent create favorable structures for quick incorporation into enzymes such as DNA polymerase. Resonance enhanced multi-photon ionization (REMPI) spectroscopy techniques such as resonant two photon ionization (R2PI) and IR-UV double resonance are used to further elucidate the structure and excited state dynamics of the bare nucleobases thymine and uracil. Both exhibit long lived excited electronic states that have been implicated in DNA photolesions which can ultimately lead to melanoma and carcinoma. Our experimental data in comparison with many quantum chemical calculations suggest a new picture for the dynamics of thymine and uracil in the gas phase. A high probability of UV absorption from a vibrationally hot ground state to the excited electronic state shows that the stability of thymine and uracil comes from

Inhibition by hyperthermia of repair synthesis and chromatin reassembly of ultraviolet-induced damage to DNA

International Nuclear Information System (INIS)

Bodell, W.J.; Cleaver, J.E.; Roti Roti, J.L.

1984-01-01

The authors have investigated the effects of hyperthermia treatment on sequential steps of the repair of UV-induced DNA damage in HeLa cells. DNA repair synthesis was inhibited by 40% after 15 min of hyperthermia treatment at 45 0 C; greater inhibition of repair synthesis occurred with prolonged incubation at 45 0 C. Enzymatic digestion of repair-labeled DNA with Exonuclease III indicated that once DNA repair was initiated, the DNA repair patch was synthesized to completion and that ligation of the DNA repair patch occurred. Thus, the observed inhibition of UV-induced DNA repair synthesis by hyperthermia treatment may be the result of inhibition of enzymes involved in the initiating steps(s) of DNA repair. DNA repair patches synthesized in UV-irradiated cells labeled at 37 0 C with[ 3 H]Thd were 2.2-fold more sensitive to micrococcal nuclease digestion than was parental DNA; if the length of the labeling period was prolonged, the nuclease sensitivity of the repair patch synthesized approached that of the parental DNA. DNA repair patches synthesized at 45 0 C, however, remained sensitive to micrococcal nuclease digestion even after long labeling periods, indicating that heat treatment inhibits the reassembly of the DNA repair patch into nucleosomal structures. 23 references, 3 figures, 2 tables

Overproduction of the poly(ADP-ribose)polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

NARCIS (Netherlands)

M. Molinete; W. Vermeulen (Wim); A. Bürkle; J. Mé nissier-de Murcia; J.H. Küpper; J.H.J. Hoeijmakers (Jan); G. de Murcia

1993-01-01

textabstractThe zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during

Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

International Nuclear Information System (INIS)

Burkovics, Peter; Sebesta, Marek; Kolesar, Peter; Sisakova, Alexandra; Marini, Victoria; Plault, Nicolas; Szukacsov, Valeria; Pinter, Lajos; Haracska, Lajos; Robert, Thomas; Kolesar, Peter; Gangloff, Serge; Krejci, Lumir

2013-01-01

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability. (authors)

On DNA synthesis during C14O2 assimilation by peas seedlings

International Nuclear Information System (INIS)

Karimov, Kh.Kh.; Nikolaeva, M.I.

1976-01-01

In this article authors try to determine how much p articipate t hephotosynthesis in the new formation of DNA seedlings, depends this processfrom the light and realize at this the synthesis DNA in chloroplasts

Hole transfer in DNA studied by pulse radiolysis

International Nuclear Information System (INIS)

Majima, T.; Kawai, K.; Takada, T.

2003-01-01

Attention has been paid to charge transfer in DNA with respect to oxidation damage of DNA and nano bio-devices such as DNA molecular wire. We report hole transfer in DNA during pulse radiolysis of molecule-conjugated DNA (M-DNA). Direct measurement of the charge transfer in DNA has never been reported due to the low extinction coefficient of nucleobase radical ions and to difficult definition of nucleobases. We have used M-DNA in which M radical cation has enough extinction coefficient and distinct absorption. Radical cation was generated in M-DNA during pulse radiolysis in water, and hole transfer through DNA was monitored by time-resolved transient absorption spectra of the radical cations. Hole was generated in Py-DNA by pulse radiolysis in water. Hole transfer to Py through DNA was monitored by transient absorption of Py'+ at 465 nm. The hole hopping rate (k) from G-region to Py was determined to be 104 s-1 which decreased with intervening A-T base-pairs between Py and G-region. We suppose that G(-H+)-radical and C(+H+) basepair can alive in DNA more than 100 us and that this long lifetime is responsible to the long-distance hole transfer. The dependence of k against the distance between the G-region and Py led to the slope of 0.3 Angstroms-1 which is due to multi-step k with the smaller distance dependence. On the other hand, beta = 0.6 Angstroms-1 was found for the single-step k in DNA. On the basis of pulse radiolytic studied on various molecule-conjugated DNA, we found that hole transfer between two chromophores (A and B)-conjugated DNA increased with decreasing the distance between A and B and was accelerated slightly with increasing the number of Gs of the bridge between A and B, and that k was modulated by the bridged base sequences. We also found that weak distance dependent hole transfer in DNA by adenine hopping mechanism

Gauging the Nanotoxicity of h2D-C2N toward Single-Stranded DNA: An in Silico Molecular Simulation Approach.

Science.gov (United States)

Mukhopadhyay, Titas Kumar; Bhattacharyya, Kalishankar; Datta, Ayan

2018-04-12

Recent toxicological assessments of graphene, graphene oxides, and some other two-dimensional (2D) materials have shown them to be substantially toxic at the nanoscale, where they inhibit and eventually disrupt biological processes. These shortfalls of graphene and analogs have resulted in a quest for novel biocompatible 2D materials with minimum cytotoxicity. In this article, we demonstrate C 2 N (h2D-C 2 N), a newly synthesized 2D porous graphene analog, to be non-nanotoxic toward genetic materials from an "in-silico" point of view through sequence-dependent binding of different polynucleotide single-stranded DNA (ssDNA) onto it. The calculated binding energy of nucleobases and the free energy of binding of polynucleotides follow the common trait, cytosine > guanine > adenine > thymine, and are well within the limits of physisorption. Ab-initio simulations completely exclude the possibility of any chemical reaction, demonstrating purely noncovalent binding of nucleobases with C 2 N through a crucial interplay between hydrogen bonding and π-stacking interactions with the surface. Further, we show that the extent of distortion inflicted upon ssDNA by C 2 N is negligible. Analysis of the density of states of the nucleobase-C 2 N hybrids confirms minimum electronic perturbation of the bases after adsorption. Most importantly, we demonstrate the potency of C 2 N in nucleic acid transportation via reversible binding of ssDNA. The plausible use of C 2 N as a template for DNA repair is illustrated through an example of C 2 N-assisted complementary ssDNA winding.

A fluorescence study of the molecular interactions of harmane with the nucleobases, their nucleosides and mononucleotides.

Science.gov (United States)

Balón, M; Muñoz, M A; Carmona, C; Guardado, P; Galán, M

1999-07-19

Fluorescence binding studies of harmane to the elemental components of the nucleic acids were undertaken to investigate the origin of the interaction between the drug and DNA. Most of the tested substrates have been found to induce hypochromism in the absorption spectrum of harmane and to quench its fluorescence. The quenching process induced by the nucleobases and their nucleosides is mainly due to the formation of ground state 1:1 complexes. However, in the case of the mononucleotides a dynamic quenching component is also observed. This quenching component is likely due to the excited state interaction of harmane with the phosphate group of the nucleotides. UV-vis spectral changes and quenching measurements have been used to quantify the ground state association constants of the complexes and the quenching rate constants.

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

Science.gov (United States)

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.

Spectroscopic approaches to study DNA damage induced in genome exposed to ionizing radiation and its enzymatic repair

International Nuclear Information System (INIS)

Yokoya, Akinari; Fujii, Kentaro; Oka, Toshitaka; Watanabe, Ritsuko

2012-01-01

Recent progress on spectroscopic study on physicochemical process of DNA damage induction will be reported. It has been predicted by computer track simulation studies that complex DNA damage, so called clustered DNA damage sites, is produced along the tack particularly of high Linear Energy Transfer (LET) ions. The clustered DNA damage, consisting of two or more isolated lesions such as single strand breaks or nucleobase lesions, is thought to compromise DNA repair enzymes. We have revealed that the nucleobase lesions produced by He 2+ ion impact to simple model DNA (plasmid) are hardly processed by base excision repair enzymes (E. coli DNA glycosylases). Using the third generation synchrotron radiation facility (SPring-8), we have studied unpaired electron species or desorbed ions as intermediates of DNA damage using an EPR apparatus or mass spectrometer installed in the soft X-ray beamline in SPring-8. These aspects are compared with the yields of final products of single- and double-strand breaks and base lesions revealed biochemical techniques. Models of complex DNA damage induction will be proposed considering various modification factors of the damage induction, ionization of valence and inner-shell electrons, OH radicals, hydration layer and the impact of secondary electrons. (author)

DNA hydrogel as a template for synthesis of ultrasmall gold nanoparticles for catalytic applications.

Science.gov (United States)

Zinchenko, Anatoly; Miwa, Yasuyuki; Lopatina, Larisa I; Sergeyev, Vladimir G; Murata, Shizuaki

2014-03-12

DNA cross-linked hydrogel was used as a matrix for synthesis of gold nanoparticles. DNA possesses a strong affinity to transition metals such as gold, which allows for the concentration of Au precursor inside a hydrogel. Further reduction of HAuCl4 inside DNA hydrogel yields well dispersed, non-aggregated spherical Au nanoparticles of 2-3 nm size. The average size of these Au nanoparticles synthesized in DNA hydrogel is the smallest reported so far for in-gel metal nanoparticles synthesis. DNA hybrid hydrogel containing gold nanoparticles showed high catalytic activity in the hydrogenation reaction of nitrophenol to aminophenol. The proposed soft hybrid material is promising as environmentally friendly and sustainable material for catalytic applications.

Two dimensional molecular electronics spectroscopy for molecular fingerprinting, DNA sequencing, and cancerous DNA recognition.

Science.gov (United States)

Rajan, Arunkumar Chitteth; Rezapour, Mohammad Reza; Yun, Jeonghun; Cho, Yeonchoo; Cho, Woo Jong; Min, Seung Kyu; Lee, Geunsik; Kim, Kwang S

2014-02-25

Laser-driven molecular spectroscopy of low spatial resolution is widely used, while electronic current-driven molecular spectroscopy of atomic scale resolution has been limited because currents provide only minimal information. However, electron transmission of a graphene nanoribbon on which a molecule is adsorbed shows molecular fingerprints of Fano resonances, i.e., characteristic features of frontier orbitals and conformations of physisorbed molecules. Utilizing these resonance profiles, here we demonstrate two-dimensional molecular electronics spectroscopy (2D MES). The differential conductance with respect to bias and gate voltages not only distinguishes different types of nucleobases for DNA sequencing but also recognizes methylated nucleobases which could be related to cancerous cell growth. This 2D MES could open an exciting field to recognize single molecule signatures at atomic resolution. The advantages of the 2D MES over the one-dimensional (1D) current analysis can be comparable to those of 2D NMR over 1D NMR analysis.

RBE comparison between rapid electrons of 20 MeV and 45 MeV with survival rate, DNA synthesis, DNA reparation and nucleoid sedimentation

International Nuclear Information System (INIS)

Alth, G.; Weniger, P.; Turtzer, K.; Klein, W.; Kocsis, F.; Krankenhaus der Stadt Wien-Lainz; Oesterreichisches Forschungszentrum Seibersdorf G.m.b.H. Inst. fuer Biologie)

1982-01-01

In order to find out possible differences of the biologic efficacy of rapid electrons of different energies, the authors examined the influence of rapid electrons of 20 MeV and 45 MeV upon the survival rate of Hela cells S3, their cell growth, DNA synthesis, DNA reparation, and sedimentation of nucleoids. The results show a difference only for the nucleoid sedimentation, i.e. there are more fractured DNA cords after 45 MeV irradiation. No significant differences could be demonstrated for the parameters of the survival curve, DNA synthesis and DNA reparation. Four double tests were carried out corresponding to the indicated types of examination. (orig.) [de

DNA synthesis in HeLa cells and isolated nuclei after treatment with an inhibitor of spermidine synthesis, methyl glyoxal bis(guanylhydrazone).

Science.gov (United States)

Krokan, H; Eriksen, A

1977-02-01

Addition of methyl glyoxal bis(guanylhydrazone) to HeLa S3 suspension cultures resulted in increased putrescine levels and decreased spermidine and spermine levels preceding a drop in incorporation of [3H]thymidine, [3H]uridine and [14C]leucine into macromolecules. When putrescine, spermidine, spermine or cadaverine was added simultaneously with methyl glyoxal bis(guanylhydrazone), the drug had no detectable effect on the synthesis of macromolecules. In nuclei isolated from cells treated with methyl glyoxal bis(guanylhydrazone) the reduction in the rate of DNA synthesis was equal to the reduction of [3H]thymidine incorporation in the corresponding whole cells. The capability of the nuclei to synthesize DNA could not be restored by adding spermidine or spermine to the system in vitro. The rate of DNA chain elongation was only reduced slightly by methyl glyoxal bis(guanylhydrazone) indicating that decreased levels of spermidine and spermine lead to a decrease in the number of replication units active in DNA synthesis within each cell.

Bronchoalveolar lavage fluid from normal rats stimulates DNA synthesis in rat alveolar type II cells

International Nuclear Information System (INIS)

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

1989-01-01

Proliferation of alveolar type II cells after lung injury is important for the restoration of the alveolar epithelium. Bronchoalveolar lavage fluid (BALF) may represent an important source of growth factors for alveolar type II cells. To test this possibility, BALF fluid was collected from normal rats, concentrated 10-fold by Amicon filtration, and tested for its ability to stimulate DNA synthesis in rat alveolar type II cells in primary culture. BALF induced a dose-dependent increase in type II cell DNA synthesis resulting in a 6-fold increase in [3H]thymidine incorporation. Similar doses also stimulated [3H]thymidine incorporation into rat lung fibroblasts by 6- to 8-fold. Removal of pulmonary surface active material by centrifugation did not significantly reduce the stimulatory activity of BALF for type II cells. The stimulation of type II cell DNA synthesis by BALF was reduced by 100% after heating at 100 degrees C for 10 min, and by approximately 80% after reduction with dithiothreitol, and after trypsin treatment. Dialysis of BALF against 1 N acetic acid resulted in a 27% reduction in stimulatory activity. The effect of BALF in promoting type II cell DNA synthesis was more pronounced when tested in the presence of serum, although serum itself has very little effect on type II cell DNA synthesis. When BALF was tested in combination with other substances that stimulate type II cell DNA synthesis (cholera toxin, insulin, epidermal growth factor, and acidic fibroblast growth factor), additive effects or greater were observed. When BALF was chromatographed over Sephadex G150, the activity eluted with an apparent molecular weight of 100 kDa

CdS nanowires formed by chemical synthesis using conjugated single-stranded DNA molecules

Science.gov (United States)

Sarangi, S. N.; Sahu, S. N.; Nozaki, S.

2018-03-01

CdS nanowires were successfully grown by chemical synthesis using two conjugated single-stranded (ss) DNA molecules, poly G (30) and poly C (30), as templates. During the early stage of the synthesis with the DNA molecules, the Cd 2+ interacts with Poly G and Poly C and produces the (Cd 2+)-Poly GC complex. As the growth proceeds, it results in nanowires. The structural analysis by grazing angle x-ray diffraction and transmission electron microscopy confirmed the zinc-blende CdS nanowires with the growth direction of . Although the nanowires are well surface-passivated with the DNA molecules, the photoluminescence quenching was caused by the electron transfer from the nanowires to the DNA molecules. The quenching can be used to detect and label the DNAs.

Synthesis of 2,6-Substituted 7-(Het)aryl-7-deazapurine Nucleobases (2,4-Disubstituted 5-(Het)aryl-pyrrolo[2,3-d]pyrimidines)

Czech Academy of Sciences Publication Activity Database

Sabat, Nazarii; Smolen, Sabina; Nauš, Petr; Perlíková, Pavla; Cebová, M.; Poštová Slavětínská, Lenka; Hocek, Michal

2017-01-01

Roč. 49, č. 20 (2017), s. 4623-4650 ISSN 0039-7881 R&D Projects: GA ČR(CZ) GA16-00178S; GA MZd(CZ) NV15-31984A Grant - others:AV ČR(CZ) AP1501 Program:Akademická prémie - Praemium Academiae Institutional support: RVO:61388963 Keywords : deazapurines * pyrrolo[2,3-d]pyrimidines * nucleobases * Suzuki-Miyaura cross-coupling * deprotection * demethylation Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 2.650, year: 2016

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

International Nuclear Information System (INIS)

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

The effect of heat and radiation on the initiation and elongation processes of DNA synthesis

International Nuclear Information System (INIS)

Davies, R.C.; Bowden, G.T.; Cress, A.E.

1983-01-01

The pH step alkaline elution and alkaline sucrose gradient techniques were utilized to evaluate alterations in DNA replication (initiation and elongation) induced by heat and low dose X-irradiation in synchronized Chinese hamster ovary cells. The initiation and elongation processes of DNA synthesis were radioresistant at the G 1 /S boundary (4 hours after mitosis) while in mid S phase (9 hours after mitosis) DNA initiation and elongation were sensitive to X-irradiation. The initiation and elongation processes of DNA synthesis which were radiation resistant at the G 1 /S boundary could be inhibited by a hyperthermia treatment (43 0 C for 1 hour beginning at 4 hours after mitosis). The impairment of initiation in the heated cells was maintained through late S phase while that of elongation was reversible as judged by full recovery at 15 hours after mitosis. These data suggest that the known synergistic lethality of heat and radiation may be mediated by an impairment of initiation of DNA synthesis. (author)

Influence of vinyl chloride monomer and vinyl chloride monomer derivatives on hepatic DNA synthesis

International Nuclear Information System (INIS)

Brenner, E.A.

1982-01-01

Vinyl chloride monomer (VCM) is used extensively in the chemical industry, mainly in the production of polyvinyl chloride. It has recently been found to cause hepatic angiosarcoma. As VCM has also been shown to be mutagenic after metabolic activation the effect of VCM on DNA synthesis was investigated. [ 3 H]Thymidine incorporation into DNA was used to measure the rate of DNA synthesis in regenerating rat liver. A possible direct toxic effect of VCM or its metabolites on liver cell metabolism was examined by two unrelated techniques, viz. the measurement of adenine nucleotide concentrations in regenerating livers and the influence on transmembrane potentials in hepatocytes. The distribution of radioactivity in subcellular fractions following [ 14 C]VCM administration suggested microsomal conversion of VCM to an active form which was selectively retained in the nuclear fraction. Measurement of the activities of thymidine kinase and DNA polymerase in regenerating liver indicated that the induction of these enzymes which normally occurs after partial hepatectomy was not prevented by VCM treatment. Three techniques were used to test the hypothesis that the retardation in DNA synthesis was due to DNA damage: the prophage lambda induction test for DNA damage, autoradiographic detection of unscheduled thymidine incorporation into DNA, and detection of DNA strand breaks in alkaline sucrose gradients. All three provided evidence of DNA damage and led to the development of a novel technique to confirm these findings. This involved centrifugation in neutral sucrose gradients on intact double-stranded DNA contained in hepatocyte nucleoids and showed conclusively that VCM administration causes DNA strand breaks. Subsequent repair of DNA was also assessed by this technique. The site of the VCM/metabolite: DNA reaction was characterized by DNA thermal denaturation and renaturation studies

Typical xeroderma pigmentosum complementation group A fibroblasts have detectable ultraviolet light-induced unscheduled DNA synthesis

International Nuclear Information System (INIS)

Petinga, R.A.; Andrews, A.D.; Robbins, J.H.; Tarone, R.E.

1977-01-01

Ultraviolet-induced nuclear uptake of tritiated thymidine [ 3 H]dThd demonstrable by autoradiography in non-synthesis phases of the cell cycle is known as unscheduled DNA synthesis and reflects repair replication of ultraviolet-damaged DNA. We have reported that the rate of any such unscheduled DNA synthesis in typical group A xeroderma pigmentosum fibroblasts, if present, is less than 2% of the normal rate. We have now performed experiments to determine whether these fibroblasts have any unscheduled DNA synthesis. Fibroblast coverslip cultures of four xeroderma pigmentosum group A strains were prepared. Irradiated (254 nm ultraviolet light) and unirradiated cultures from each strain were incubated with [ 3 H]dThd at 37degC, and autoradiograms were prepared using NTB-3 emulsion. A nuclear grain count was made of 100 consecutive nuclei of non-S-phase irradiated and unirradiated cells. A slide background grain count was simultaneously made from an acellular area adjacent to each cell analyzed. When a strain's irradiated and unirradiated autoradiograms having similar slide background grain count averages were compared, the nuclear grain count average of the irradiated cells was always higher than that of the unirradiated cells. This ultraviolet-induced increase in the mean nuclear grain count ranged from 0.4 to 1.3% of that given by normal non-xeroderma pigmentosum fibroblasts and was not reduced by 10 -2 M hydroxyurea. Planimetric studies showed that the ultraviolet-induced increase in nuclear grain count is not due to an increased nuclear area in irradiated cells. We conclude that these typical group A xeroderma pigmentosum strains perform very low, but detectable, ultraviolet-induced unscheduled DNA synthesis which probably reflects repair replication. We cannot, however, determine if there are significantly different rates of ultraviolet-induced unscheduled DNA synthesis among these ultraviolet strains

Effect of Vaccinia virus infection on poly(ADP-ribose)synthesis and DNA metabolism in different cells

Energy Technology Data Exchange (ETDEWEB)

Topaloglou, A.; Ott, E.; Altmann, H. (Oesterreichisches Forschungszentrum Seibersdorf G.m.b.H. Inst. fuer Biologie); Zashukhina, G.D.; Sinelschikova, T.A. (AN SSSR, Moscow. Inst. Obshchej Genetiki)

1983-07-14

In Chang liver cells and rat spleen cells infected with Vaccinia virus, DNA synthesis, repair replication after UV irradiation and poly(ADP-ribose)(PAR) synthesis were determined. In the time post infection semiconservative DNA synthesis showed only a slight reduction. DNA repair replication was not very different from controls 4 hours p.i. but was enhanced 24 hours after infection compared to noninfected cells. PAR synthesis was also not changed very much 4 hours p.i. but was decreased significantly after 24 hours. The determination of radioactivity resulting from /sup 3/H-NAD, showed a marked reduction of PAR in the spacer region of chromatin 24 hours p.i., but in addition, PAR located in the core region, was reduced, too.

Histoautoradiographic and liquid scintillometric studies on DNA synthesis in the liver, kidneys, spleen and tongue after bilateral adrenalectomy in rats

International Nuclear Information System (INIS)

Schneider, A.

1981-01-01

Historadiographies and liquid scintillometries were carried out in 163 male Wistar rats in order to determine the effects of bilateral adrenalectomy on DNA synthesis in the liver, kidneys, spleen, and tongue. Both DNA synthesis and mitotic index are significantly increased from the 1st day p.o. onwards, with broad synthesis peaks between the 2nd and the 4th day. The intensity of DNA synthesis shows a gradual decrease with increasing duration of the experiment. In contrast to the adrenalectonized animals, the synthesis rate and mitotic index in the organs of sham-operated animals were significantly lower, although enhanced proliferation was observed after surgery. The enhanced DNA synthesis after bilateral adrenalectomy is interpreted in terms of a disinhibition; corticosteroids are assumed to play a key role. The effects of bilateral adrenalectromy on untreated organs are not organ-specific. The highest synthesis rate was observed in the tubular epithelia of the convoluted main parts, while the DNA synthesis in the tongue. The findings of autoradiography and liquid scintillometry are well correlated. (orig./MG) [de

Design and synthesis of DNA four-helix bundles

Energy Technology Data Exchange (ETDEWEB)

Rangnekar, Abhijit; Gothelf, Kurt V [Department of Chemistry, Centre for DNA Nanotechnology (CDNA) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C (Denmark); LaBean, Thomas H, E-mail: kvg@chem.au.dk, E-mail: thl@cs.duke.edu [Department of Chemistry, Duke University, Durham, NC 27708 (United States)

2011-06-10

The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

Design and synthesis of DNA four-helix bundles

International Nuclear Information System (INIS)

Rangnekar, Abhijit; Gothelf, Kurt V; LaBean, Thomas H

2011-01-01

The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

Synthesis, X-ray crystal structure, DNA binding and Nuclease activity ...

Indian Academy of Sciences (India)

s12039-016-1125-x. Synthesis, X-ray crystal structure, DNA binding and Nuclease activity of lanthanide(III) complexes of 2-benzoylpyridine acetylhydrazone. KARREDDULA RAJA, AKKILI SUSEELAMMA and KATREDDI HUSSAIN REDDY. ∗.

Radiation effect and response of DNA synthesis in lymphocytes induced by low dose irradiation

International Nuclear Information System (INIS)

Zhao Yujie; Su Liaoyuan; Zou Huawei; Kong Xiangrong

1999-01-01

The ability of DNA synthesis in lymphocytes were measured by using 3 H-TdR incorporation method. This method was used to observe the damage of lymphocytes irradiated by several challenge doses (0.5-0.8 Gy) and adaptive response induced by previous low dose irradiation. The results show that DNA synthesis was inhibited by challenge dose of radiation and was adapted by previous 0.048 Gy irradiation

On the recovery of the DNA-synthesis after X-irradiation in the spleen of mice and its modification by the NAD-metabolism

International Nuclear Information System (INIS)

Streffer, C.

1974-01-01

The incorporation of tritium-labelled thymidine into the DNA of mice spleen cells after whole body irradiation with X-rays was measured in order to study the decrease of DNA synthesis is decreased for several hours after irradiation with low doses. Recovery effects become operative after six hours. The radiation effect on the NAD metabolism, known to be related to DNA synthesis, was also investigated. The rate of NAD synthesis is influenced via the extremely radiosensitive metabolic process in the nucleus. Conversely, inhibition of DNA synthesis by injection of NAD enhances the recovery of DNA synthesis after irradiaton. (G.G.)

DNA synthesis in toluene-treated bacteriophage-infected minicells of Bacillus subtilis

International Nuclear Information System (INIS)

Amann, E.; Reeve, J.N.

1978-01-01

Bateriophage (phi29, SPP1, or SP01)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthesis as measured by incorporation of [ 3 H]dTTP into a trichloroacetic acid-precipitable form. The [ 3 H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2.10 5 ). Short exposure of the DNA molecules containing the incorporated [ 3 H]dTMP to Escherichia coli exonuclease III results in over 90% of the [ 3 H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, nalidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p-hydroxyphenylazo)-uracil. (Auth.)

DNA synthesis and degradation in UV-irradiated toluene treated cells of E. coli K12: the role of polynucleotide ligase

International Nuclear Information System (INIS)

Strike, P.

1977-01-01

Toluene treated cells have been used to study the processes of DNA synthesis and DNA degradation in ultra-violet irradiated Escherichia coli K12. Synthesis and degradation are both shown to occur extensively if polynucleotide ligase is inhibited, and to occur to a much lesser extent if ligase activity is optimal. Extensive UV-induced DNA synthesis in toluene-treated cells requires ATP for the initial incision step, and DNA polymerase I. Extensive degradation also depends on the early ATP-dependent incision step, and the subsequent degradation shows a partial requirement for ATP. Curtailment of degradation by ligase requires DNA polymerase activity, but is not dependent upon DNA polymerase I. Apparently this process can be carried out with equal facility by either DNA polymerase II or polymerase III. These observations suggest that extensive DNA polymerase I-dependent repair synthesis and extensive DNA degradation are facets of two divergent pathways of excision repair, both of which depend upon the early uvrABC determined ATP-dependent incision step. (orig.) [de

Correlation between survival, ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in a rec- mutant of Escherichia coli K12

International Nuclear Information System (INIS)

Pirsel, M.; Slezarikova, V.

1977-01-01

A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA - ) or by chloramphenicol (CAP) treatment prior to UV irradiation (2.5 J m -2 ) increased more than tenfold the resistance of the strain Escherichia coli K12 SR19 to UV radiation. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. In Escherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation, and a higher stability of both parental and newly synthesized DNAs could be demonstrated. (author)

Decreased UV-induced DNA repair synthesis in peripheral leukocytes from patients with the nevoid basal cell carcinoma syndrome

International Nuclear Information System (INIS)

Ringborg, U.; Lambert, B.; Landergen, J.; Lewensohn, R.

1981-01-01

The uv-induced DNA repair synthesis in peripheral leukocytes from 7 patients with the nevoid basal cell carcinoma syndrome was compared to that in peripheral leukocytes from 5 patients with basal cell carcinomas and 39 healthy subjects. A dose response curve was established for each individual, and maximum DNA repair synthesis was used as a measure of the capacity for DNA repair. The patients with the nevoid basal cell carcinoma syndrome had about 25% lower level of maximum DNA repair synthesis as compared to the patients with basal cell carcinomas and control individuals. The possibility that DNA repair mechanisms may be involved in the etiology to the nevoid basal cell carcinoma syndrome is discussed

Effects of gamma- and UV-radiation on DNA synthesis in permeable cells of Bacillus stearothermophilus

International Nuclear Information System (INIS)

Trofimenko, A.F.; Vorob'eva, A.M.; Gaziev, A.I.

1981-01-01

It was shown that the most of the DNA synthesis is repaired in permeable cells of Bacillus stearothermophilus not affected by injurious agents. γ-irradiation stimulates the reparative synthesis and degradation of DNA whereas UV-radiation decreases the activity of these processes. The reason for such an unusual response of thermophiles to irradiation lies perhaps in high temperatures at which the cells exist

Programmable autonomous synthesis of single-stranded DNA

Science.gov (United States)

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

2018-02-01

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

GAS PHASE SYNTHESIS OF (ISO)QUINOLINE AND ITS ROLE IN THE FORMATION OF NUCLEOBASES IN THE INTERSTELLAR MEDIUM

International Nuclear Information System (INIS)

Parker, Dorian S. N.; Kaiser, Ralf I.; Kostko, Oleg; Troy, Tyler P.; Ahmed, Musahid; Mebel, Alexander M.; Tielens, Alexander G. G. M.

2015-01-01

Nitrogen-substituted polycyclic aromatic hydrocarbons (NPAHs) have been proposed to play a key role in the astrochemical evolution of the interstellar medium, yet the formation mechanisms of even their simplest prototypes—quinoline and isoquinoline—remain elusive. Here, we reveal a novel concept that under high temperature conditions representing circumstellar envelopes of carbon stars, (iso)quinoline can be synthesized via the reaction of pyridyl radicals with two acetylene molecules. The facile gas phase formation of (iso)quinoline in circumstellar envelopes defines a hitherto elusive reaction class synthesizing aromatic structures with embedded nitrogen atoms that are essential building blocks in contemporary biological-structural motifs. Once ejected from circumstellar shells and incorporated into icy interstellar grains in cold molecular clouds, these NPAHs can be functionalized by photo processing forming nucleobase-type structures as sampled in the Murchison meteorite

On modeling biomolecular–surface nonbonded interactions: application to nucleobase adsorption on single-wall carbon nanotube surfaces

International Nuclear Information System (INIS)

Akdim, B; Pachter, R; Day, P N; Kim, S S; Naik, R R

2012-01-01

In this work we explored the selectivity of single nucleobases towards adsorption on chiral single-wall carbon nanotubes (SWCNTs) by density functional theory calculations. Specifically, the adsorption of molecular models of guanine (G), adenine (A), thymine (T), and cytosine (C), as well as of AT and GC Watson–Crick (WC) base pairs on chiral SWCNT C(6, 5), C(9, 1) and C(8, 3) model structures, was analyzed in detail. The importance of correcting the exchange–correlation functional for London dispersion was clearly demonstrated, yet limitations in modeling such interactions by considering the SWCNT as a molecular model may mask subtle effects in a molecular–macroscopic material system. The trend in the calculated adsorption energies of the nucleobases on same diameter C(6, 5) and C(9, 1) SWCNT surfaces, i.e. G > A > T > C, was consistent with related computations and experimental work on graphitic surfaces, however contradicting experimental data on the adsorption of single-strand short homo-oligonucleotides on SWCNTs that demonstrated a trend of G > C > A > T (Albertorio et al 2009 Nanotechnology 20 395101). A possible role of electrostatic interactions in this case was partially captured by applying the effective fragment potential method, emphasizing that the interplay of the various contributions in modeling nonbonded interactions is complicated by theoretical limitations. Finally, because the calculated adsorption energies for Watson–Crick base pairs have shown little effect upon adsorption of the base pair farther from the surface, the results on SWCNT sorting by salmon genomic DNA could be indicative of partial unfolding of the double helix upon adsorption on the SWCNT surface. (paper)

Aryl sulfonate based anticancer alkylating agents.

Science.gov (United States)

Sheikh, Hamdullah Khadim; Arshad, Tanzila; Kanwal, Ghazala

2018-05-01

This research work revolves around synthesis of antineoplastic alkylating sulfonate esters with dual alkylating sites for crosslinking of the DNA strands. These molecules were evaluated as potential antineoplastic cross linking alkylating agents by reaction with the nucleoside of Guanine DNA nucleobase at both ends of the synthesized molecule. Synthesis of the alkylating molecules and the crosslinking with the guanosine nucleoside was monitored by MALDITOF mass spectroscopy. The synthesized molecule's crosslinking or adduct forming rate with the nucleoside was compared with that of 1,4 butane disulfonate (busulfan), in form of time taken for the appearance of [M+H] + . It was found that aryl sulfonate leaving group was causing higher rate of nucleophilic attack by the Lewis basic site of the nucleobase. Furthermore, the rate was also found to be a function of electron withdrawing or donating nature of the substituent on the aryl ring. Compound with strong electron withdrawing substituent on the para position of the ring reacted fastest. Hence, new alkylating agents were synthesized with optimized or desired reactivity.

Self-assembled catalytic DNA nanostructures for synthesis of para-directed polyaniline.

Science.gov (United States)

Wang, Zhen-Gang; Zhan, Pengfei; Ding, Baoquan

2013-02-26

Templated synthesis has been considered as an efficient approach to produce polyaniline (PANI) nanostructures. The features of DNA molecules enable a DNA template to be an intriguing template for fabrication of emeraldine PANI. In this work, we assembled HRP-mimicking DNAzyme with different artificial DNA nanostructures, aiming to manipulate the molecular structures and morphologies of PANI nanostructures through the controlled DNA self-assembly. UV-vis absorption spectra were used to investigate the molecular structures of PANI and monitor kinetic growth of PANI. It was found that PANI was well-doped at neutral pH and the redox behaviors of the resultant PANI were dependent on the charge density of the template, which was controlled by the template configurations. CD spectra indicated that the PANI threaded tightly around the helical DNA backbone, resulting in the right handedness of PANI. These reveal the formation of the emeraldine form of PANI that was doped by the DNA. The morphologies of the resultant PANI were studied by AFM and SEM. It was concluded from the imaging and spectroscopic kinetic results that PANI grew preferably from the DNAzyme sites and then expanded over the template to form 1D PANI nanostructures. The strategy of the DNAzyme-DNA template assembly brings several advantages in the synthesis of para-coupling PANI, including the region-selective growth of PANI, facilitating the formation of a para-coupling structure and facile regulation. We believe this study contributes significantly to the fabrication of doped PANI nanopatterns with controlled complexity, and the development of DNA nanotechnology.

Formation of a Thymine-Hg-II-Thymine Metal-Mediated DNA Base Pair: Proposal and Theoretical Calculation of the Reaction Pathway

Czech Academy of Sciences Publication Activity Database

Šebera, Jakub; Burda, J.; Straka, Michal; Ono, A.; Kojima, C.; Tanaka, Y.; Sychrovský, Vladimír

2013-01-01

Roč. 19, č. 30 (2013), s. 9884-9894 ISSN 0947-6539 R&D Projects: GA ČR GAP205/10/0228; GA MŠk(CZ) LH11033 Institutional support: RVO:61388963 Keywords : DNA structures * mercury * metalation * metal-DNA binding * nucleobases Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 5.696, year: 2013

Action of cytochalasin D on DNA synthesis in cells in culture

International Nuclear Information System (INIS)

Glushankova, N.A.

1986-01-01

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 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

Lethality and the depression on DNA synthesis in UV-irradiated normal human and xeroderma pigmentosum cells

Energy Technology Data Exchange (ETDEWEB)

Shinohara, K. (Kobe Univ. (Japan). School of Medicine)

1983-12-01

Ultraviolet radiation suppresses the semiconservative DNA replication in mammalian cells. The rate of DNA synthesis is initially depressed and later recovers after low doses of UV radiation in human cells. Such a response is more sensitive to UV radiation in cells derived from patients with xeroderma pigmentosum (XP) than that in normal human cells. The relative rate of DNA synthesis is not always correlated with cell survival because, unlike cell survival, the dose-response curve of the relative rate of DNA synthesis shows the biphasic nature of the sensitivity. In the experiments reported herein, the total amount (not the rate) of DNA synthesized during a long interval of incubation which covers the period of inhibition and recovery (but not longer than one generation time) after irradiation with various doses of UV radiation was examined in normal human and XP cells, and was found to be well correlated with cell survival in all the cells tested.

Synthesis of base-modified 2'-deoxyribonucleoside triphosphates and their use in enzymatic synthesis of modified DNA for applications in bioanalysis and chemical biology.

Science.gov (United States)

Hocek, Michal

2014-11-07

The synthesis of 2'-deoxyribonucleoside triphosphates (dNTPs) either by classical triphosphorylation of nucleosides or by aqueous cross-coupling reactions of halogenated dNTPs is discussed. Different enzymatic methods for synthesis of modified oligonucleotides and DNA by polymerase incorporation of modified nucleotides are summarized, and the applications in redox or fluorescent labeling, as well as in bioconjugations and modulation of interactions of DNA with proteins, are outlined.

ATM Protein Physically and Functionally Interacts with Proliferating Cell Nuclear Antigen to Regulate DNA Synthesis*

Science.gov (United States)

Gamper, Armin M.; Choi, Serah; Matsumoto, Yoshihiro; Banerjee, Dibyendu; Tomkinson, Alan E.; Bakkenist, Christopher J.

2012-01-01

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner. PMID:22362778

Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light

International Nuclear Information System (INIS)

Painter, R.B.

1985-01-01

The inhibition of DNA synthesis in normal human cells by UV is a complex function of fluence because it has several causes. At low fluences, inhibition of replicon initiation is most important. This is made clear by the fact that it occurs to a lesser degree in cells from patients with ataxia telangiectasia (AT). Assuming that only leading strand synthesis is blocked by UV-induced lesions, single lesions between replicons in parental strands for leading strand synthesis inhibit DNA synthesis by acting as temporary blocks until they are replicated by extension of the lagging strand of the adjacent replicon. A more severe inhibition occurs when two lesions are induced between adjacent growing replicons, because one in four possible configurations may result in a long-lived unreplicated region (LLUR). In the absence of excision repair, these may eventually be replicated by activation of an otherwise unused origin within the LLUR. The frequency of LLURs increases steeply with fluence. Activation of normally unused origins to replicate LLURs may facilitate recovery from inhibition of DNA synthesis, but repair of lesions is probably more important. In excision-repair-defective cells, an LLUR without an origin to initiate its replication may be a lethal lesion. (orig.)

Unexpected Hydration of a Triple Bond During DNA Synthesis

DEFF Research Database (Denmark)

Fatthalla, Maha I.; Pedersen, Erik B.

2016-01-01

acidic conditions, polarizes the triple bond in the intercalator and this makes hydration of the triple bond possible during the DNA synthesis and an oligonucleotide with 1-(indol-3-yl)-2-(pyren-1-yl)ethanone as the intercalator is formed. Insertion of the unhydrated and hydrated linker systems gave...

RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress

DEFF Research Database (Denmark)

Bhowmick, Rahul; Minocherhomji, Sheroy; Hickson, Ian D

2016-01-01

Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis...... replication stress at CFS loci during S-phase. In contrast, MiDAS is RAD52 dependent, and RAD52 is required for the timely recruitment of MUS81 and POLD3 to CFSs in early mitosis. Our results provide further mechanistic insight into MiDAS and define a specific function for human RAD52. Furthermore, selective...

Escape from X-ray-induced arrest for lens cells stimulated from quiescence: time relationship to RNA, protein, and DNA synthesis

International Nuclear Information System (INIS)

Lindgren, A.L.; Miller, R.C.; Guernsey, D.L.; Riley, E.F.

1988-01-01

Quiescent cells of the central zone region of the rat lens epithelium were stimulated to enter the proliferation cycle by wounding. RNA synthesis and a corresponding increase in poly(A)+/total RNA reached a peak by Hour 4. Cells progressed into the G1B compartment by Hour 10. A rise in protein synthesis began at Hour 8, and onset of DNA synthesis occurred by Hour 14. The timing of cell cycle progression that allowed escape from a dose of X irradiation that completely inhibited DNA synthesis was investigated. A growth-arrest point was identified at Hour 9 where 10 GY of X irradiation given before, but not after, completely inhibited earliest responding cells from entering DNA synthesis on schedule. Increased quantities of cells entered DNA synthesis on schedule as timing of the X irradiation was moved closer to the end of G1. Based on time relationships, the rise in protein synthesis is correlated with the sufficient event for the escape

Synthesis of 4'-Selenoribonucleosides, the Building Blocks of 4'-SelenoRNA, Using a Hypervalent Iodine.

Science.gov (United States)

Saito-Tarashima, Noriko; Ota, Masashi; Minakawa, Noriaki

2017-09-18

Herein is described a detailed protocol for the synthesis of 4'-selenoribonucleoside derivatives that involves the use of a hypervalent iodine species. These derivatives are versatile units for the preparation of 4'-selenoRNA. Large-scale synthesis of a 4-selenosugar starting from D-ribose is achieved in eight steps, including a final chromatographic purification. The resulting 4-selenosugar is then subjected to the one-pot Pummerer-like reaction using hypervalent iodine in the presence of silylated nucleobases. The reaction with silylated uracil affords the desired 4'-selenouridine derivatives with excellent β-selectivity and in good yield. Conversely, when purine nucleobases are used in the Pummerer-like reaction, N7 4'-selenoribonucleoside isomers are obtained alongside the desired N9 isomers. However, the undesired N7 isomers can be converted to the desired N9 ones under acidic conditions. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Distribution of ultraviolet-induced DNA repair synthesis in nuclease sensitive and resistant regions of human chromatin

International Nuclear Information System (INIS)

Smerdon, M.J.; Tlsty, T.D.; Lieberman, M.W.

1978-01-01

The distribution of ultraviolet radiation (uv) induced DNA repair synthesis within chromatin was examined in cultured human diploid fibroblasts (IMR-90). Measurement of the time course of repair synthesis yielded two distinct phases: An initial rapid phase (fast repair) which occurs during the first 2 to 3 h after damage and a slower phase (slow repair) associated with a tenfold decrease in the rate of nucleotide incorporation, which persists for at least 35 h after damage. Staphylococcal nuclease digests of nuclei from cells damaged with uv and labeled during the fast-repair phase revealed a marked preference of fast-repair synthesis for the nuclease-sensitive regions. A new method was developed to analyze the digestion data and showed that approximately 50% of the nucleotides incorporated during the fast-repair phase are located in staphylococcal nuclease-sensitive regions, which comprise about 30% of the genome. Calculations from these data indicate that in the staphylococcal nuclease-sensitive regions the number of newly inserted nucleotides per unit DNA is about twice that of resistant regions. These results were supported by electrophoresis studies which demonstrated a decreased representation of fast-repair synthesis in core particle DNA. In contrast, the distribution within chromatin of nucleotides incorporated during the slow-repair phase was found to be much more homogeneous with about 30% of the repair sites located in 25% of the genome. Digestion studieswith DNase I indicated a slight preference of repair synthesis for regions sensitive to this enzyme; however, no marked difference between the distributions of fast- and slow-repair synthesis was observed. This study provides evidence that the structural constraints placed upon DNA in chromatin also place constraints upon uv-induced DNA repair synthesis in human cells

Real-time analysis of self-assembled nucleobases by Venturi easy ambient sonic-spray ionization mass spectrometry.

Science.gov (United States)

Na, Na; Shi, Ruixia; Long, Zi; Lu, Xin; Jiang, Fubin; Ouyang, Jin

2014-10-01

In this study, the real-time analysis of self-assembled nucleobases was employed by Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS). With the analysis of three nucleobases including 6-methyluracil (6MU), uracil (U) and thymine (T) as examples, different orders of clusters centered with different metal ions were recorded in both positive and negative modes. Compared with the results obtained by traditional electrospray ionization mass spectrometry (ESI-MS) under the same condition, more clusters with high orders, such as [6MU7+Na](+), [6MU15+2NH4](2+), [6MU10+Na](+), [T7+Na](+), and [T15+2NH4](2+) were detected by V-EASI-MS, which demonstrated the soft ionization ability of V-EASI for studying the non-covalent interaction in a self-assembly process. Furthermore, with the injection of K(+) to the system by a syringe pumping, the real-time monitoring of the formation of nucleobases clusters was achieved by the direct extraction of samples from the system under the Venturi effect. Therefore, the effect of cations on the formation of clusters during self-assembly of nucleobases was demonstrated, which was in accordance with the reports. Free of high voltage, heating or radiation during the ionization, this technique is much soft and suitable for obtaining the real-time information of the self-assembly system, which also makes it quite convenient for extraction samples from the reaction system. This "easy and soft" ionization technique has provided a potential pathway for monitoring and controlling the self-assembly processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Intestinal DNA concentration and protein synthesis in response to ...

African Journals Online (AJOL)

Performance, protein synthesis and mucosal DNA in small intestine of Leghorn hens may be affected by low quality feedstuff. An experiment was conducted in completely randomized design (CRD) in 2 × 2 factorial arrangement. Main factors included diets containing 20 and 40 % barley and black and blue strains of leghorn ...

UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions

Science.gov (United States)

Brem, Reto; Zhang, Xiaohui; Xu, Yao-Zhong; Karran, Peter

2015-01-01

Photochemotherapy, the combination of a photosensitiser and ultraviolet (UV) or visible light, is an effective treatment for skin conditions including cancer. The high mutagenicity and non-selectivity of photochemotherapy regimes warrants the development of alternative approaches. We demonstrate that the thiopyrimidine nucleosides 5-bromo-4-thiodeoxyuridine (SBrdU) and 5-iodo-4-thiodeoxyuridine (SIdU) are incorporated into the DNA of cultured human and mouse cells where they synergistically sensitise killing by low doses of UVA radiation. The DNA halothiopyrimidine/UVA combinations induce DNA interstrand crosslinks, DNA-protein crosslinks, DNA strand breaks, nucleobase damage and lesions that resemble UV-induced pyrimidine(6-4)pyrimidone photoproducts. These are potentially lethal DNA lesions and cells defective in their repair are hypersensitive to killing by SBrdU/UVA and SIdU/UVA. DNA SIdU and SBrdU generate lethal DNA photodamage by partially distinct mechanisms that reflect the different photolabilities of their C–I and C–Br bonds. Although singlet oxygen is involved in photolesion formation, DNA SBrdU and SIdU photoactivation does not detectably increase DNA 8-oxoguanine levels. The absence of significant collateral damage to normal guanine suggests that UVA activation of DNA SIdU or SBrdU might offer a strategy to target hyperproliferative skin conditions that avoids the extensive formation of a known mutagenic DNA lesion. PMID:25747491

Hypothesis of Lithocoding: Origin of the Genetic Code as a "Double Jigsaw Puzzle" of Nucleobase-Containing Molecules and Amino Acids Assembled by Sequential Filling of Apatite Mineral Cellules.

Science.gov (United States)

Skoblikow, Nikolai E; Zimin, Andrei A

2016-05-01

The hypothesis of direct coding, assuming the direct contact of pairs of coding molecules with amino acid side chains in hollow unit cells (cellules) of a regular crystal-structure mineral is proposed. The coding nucleobase-containing molecules in each cellule (named "lithocodon") partially shield each other; the remaining free space determines the stereochemical character of the filling side chain. Apatite-group minerals are considered as the most preferable for this type of coding (named "lithocoding"). A scheme of the cellule with certain stereometric parameters, providing for the isomeric selection of contacting molecules is proposed. We modelled the filling of cellules with molecules involved in direct coding, with the possibility of coding by their single combination for a group of stereochemically similar amino acids. The regular ordered arrangement of cellules enables the polymerization of amino acids and nucleobase-containing molecules in the same direction (named "lithotranslation") preventing the shift of coding. A table of the presumed "LithoCode" (possible and optimal lithocodon assignments for abiogenically synthesized α-amino acids involved in lithocoding and lithotranslation) is proposed. The magmatic nature of the mineral, abiogenic synthesis of organic molecules and polymerization events are considered within the framework of the proposed "volcanic scenario".

Excited state dynamics of DNA bases

Czech Academy of Sciences Publication Activity Database

Kleinermanns, K.; Nachtigallová, Dana; de Vries, M. S.

2013-01-01

Roč. 32, č. 2 (2013), s. 308-342 ISSN 0144-235X R&D Projects: GA ČR GAP208/12/1318 Grant - others:National Science Foundation(US) CHE-0911564; NASA (US) NNX12AG77G; Deutsche Forschungsgemeinschaft(DE) SFB 663; Deutsche Forschungsgemeinschaft(DE) KI 531-29 Institutional support: RVO:61388963 Keywords : DNA bases * nucleobases * excited state * dynamics * computations * gas phase * conical intersections Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.920, year: 2013

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Termination of DNA synthesis in vitro at apurinic sites but not at ethyl adducts of the template

Energy Technology Data Exchange (ETDEWEB)

Lockhart, M.L.; Deutsch, J.F.; Yamaura, I.; Cavalieri, L.F.; Rosenberg, B.H.

1982-01-01

The effects of DNA lesions produced by the carcinogenic alkylating agents ethylnitrosourea and diethylsulfate on the extent of DNA synthesis have been studied in a system utilizing circular single-stranded phi X174 DNA as template and a 392-base restriction fragment as primer with E. coli polymerase I (Klenow fragment). Apurinic sites produced by loss of unstable ethylated bases from the template terminate DNA synthesis at the first such site encountered, but ethyl adducts at most, if not all, locations permit readthrough. 22 references, 3 figures, 1 table.

Action of caffeine on x-irradiated HeLa cells. I. Delayed inhibition of DNA synthesis

International Nuclear Information System (INIS)

Tolmach, L.J.; Jones, R.W.; Busse, P.M.

1977-01-01

Treatment of HeLa S3 cells with 1 mM caffeine delays progression through G1 by 1.5 hours but causes no other detectable inhibition of cell progression; it sometimes results in a large stimulation of thymidine incorporation. When this concentration is applied to cells that have been irradiated with 1-krad doses of 220-kV x rays, there is a marked suppression of both the inhibition of DNA synthesis and G2 arrest induced by the radiation. Larger doses require higher concentrations of caffeine to suppress the inhibition of DNA synthesis. Delaying addition until the rate of synthesis is at its minimum (1.5 hours after irradiation with 1 krad) results in a slightly accelerated recovery of the rate. Treatment before or during irradiation is without effect on the inhibition. Removal of the caffeine as late as 6 hours after its addition at the time of irradiation results in a prompt inhibition in DNA synthesis that mimics that observed immediately after irradiation in the absence of caffeine. These findings raise the possibility that the depression in rate of DNA systhesis might not result from radiation damage introduced into the replicon initiation system, but rather may be an indirect consequence of damage residing elsewhere in the irradiated cell

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

Science.gov (United States)

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.

Recovery of DNA synthesis from inhibition by ultraviolet light in mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Ventura, A M; Ortega, J M; Schumacher, R I; Meneghini, R

1987-01-01

In general mammalian cells recover from DNA synthesis inhibition by ultraviolet light (u.v.) before most of the pyrimidine dimers have been removed from the genome. Using metabolic inhibitors, it has been shown that (1) even the low repair rate exhibited by V79 cells is important for recovery; although most of the dimers remain in the V79 genome after recovery of DNA synthesis, either the removal of lesions from some important region of chromatin or the activity of the repair process itself is important for the recovery; (2) the recovery mechanism is induced and depends on RNA synthesis and the production of specific factors. Finally, we have observed that cells previously treated with fluorodeoxyuridine become more resistant to inhibition by u.v. Since it has been shown that this drug activates unused origins of replication in Chinese hamster cells, reducing the average replicon size, we assume that the acquired resistance has to do with the operation of a larger number of small replicons.

Sister chromatid exchanges in X-ray irradiated blood lymphocytes from patients with hereditary diseases with radioresistant DNA synthesis

International Nuclear Information System (INIS)

Pleskach, N.M.; Andriadze, M.I.; Mikhel'son, V.M.; Zhestyanikov, V.D.

1988-01-01

X-ray irradiation induced sister chromatid exchanges (SCE) in blood lymphocytes from patient with Down's syndrome and adult progeria (in both the cases radioresistant DNA synthesis takes place). In normal lymphocytes (in which ionizing radiation inhibits the replicative synthesis of DNA) the rate of SCE rises with the rise of radiation dose. Thus, the rate of SCE in X-ray irradiated lymphocytes is in reverse dependence with radioresistance of replicative synthesis of DNA. The data obtained are explained in accordance with the replicative hypothesis of the SCE nature (Painter, 1980a): in cells of patients with Down's syndrome, xeroderma pigmentosum from 2 and progeria of adults the time of existence of partly replicated clusters of replicons is decreased due to radioresistant replicative synthesis of DNA, but the presence of partly replicated clusters of replicons in necessary for SCE formation. Therefore the rate of SCF in X-irradiated cells of these patients decreases

Synthesis, Improved Antisense Activity and Structural Rationale for the Divergent RNA Affinities of 3;#8242;-Fluoro Hexitol Nucleic Acid (FHNA and Ara-FHNA) Modified Oligonucleotides

Energy Technology Data Exchange (ETDEWEB)

Egli, Martin; Pallan, Pradeep S.; Allerson, Charles R.; Prakash, Thazha P.; Berdeja, Andres; Yu, Jinghua; Lee, Sam; Watt, Andrew; Gaus, Hans; Bhat, Balkrishen; Swayze, Eric E.; Seth, Punit P. (Isis Pharm.); (Vanderbilt)

2012-03-16

The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F {hor_ellipsis} H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py) {hor_ellipsis} H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.

Nucleobase modification as redox DNA labelling for electrochemical detection

Czech Academy of Sciences Publication Activity Database

Hocek, Michal; Fojta, Miroslav

2011-01-01

Roč. 40, č. 12 (2011), s. 5802-5814 ISSN 0306-0012 R&D Projects: GA MŠk(CZ) LC06035; GA MŠk LC512; GA AV ČR(CZ) IAA400040901; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : nucleotides * oligonucleotides * DNA * electrochemistry * redox labeling Subject RIV: CC - Organic Chemistry Impact factor: 28.760, year: 2011

Influence of metronidazole on the survival rate of whole-body irradiated mice and on the DNA repair synthesis of lymphocytes

International Nuclear Information System (INIS)

Magdon, E.; Schroeder, E.

1978-01-01

With reference to literature reports the effect of Metronidazole [1-(hydroxyethyl)-5-nitro-2-methyl-imidazole] on the survival rate of C 3 H inbred mice following whole-body doses ranging from 5 to 15 Gy was determined under oxic and hypoxic conditions. Ehrlich ascites tumor cells were used to study the influence of Metronidazole on radiation-induced alterations of the DNA sedimentation behavior in the alkaline sucrose gradient under oxic conditions in vitro. The effect of Metronidazole on the semiconservative DNA synthesis was investigated under oxic and hypoxic conditions in Ehrlich ascites carcinoma cells and L5178Y lymphoma cells. Furthermore, it was examined whether the radiation-induced inhibition of semiconservative DNA synthesis in L5178Y lymphoma cells and the radiation-induced repair synthesis in lymphocytes is influenced by Metronidazole. From the values of the LDsub(50/30) after whole-body irradiation a sensitilization factor of 1.3 was derived for Metronidazole under hypoxic conditions. Under atmospheric conditions an increase of the radiation effect by a factor of 1.1 was obtained. The protective factor of hypoxia was 1.6 and thus greater than the radiosensibilization caused by Metronidazole. The DNA synthesis was slightly inhibited by Metronidazole under both hypoxic and euoxic conditions. The studies revealed no significant influence of Metronidazole on radiation-induced changes of the DNA sedimentation behavior and of the DNA repair synthesis as well as on the radiation induced inhibition of semiconservative DNA synthesis. (author)

Recovery from DNA synthesis in V 79 chinese hamster cells irradiated with UV light

International Nuclear Information System (INIS)

Ventura, A.M.

1987-01-01

Mammalian cells recover from DNA synthesis inhibition by UV light before most of the pyrimidine dimers have been removed from the genome. Most of the rodent cells show a deficient dimer excision repair compared with normal human fibroblasts. Despite this fact they recover efficiently from DNA synthesis inhibition after UV. In Chinese hamster V 79 cells was found that this recovery takes place in the absence of a significant excision repair, and it seems to be directly coupled to a recovery in the rate of movement of the replication fork. 120 refs, 31 figs. (author)

Inhibition and recovery of the rate of DNA synthesis in V79 Chinese hamster cells following ultraviolet light irradiation

International Nuclear Information System (INIS)

Ventura, A.M.; Meneghini, R.

1984-01-01

Chinese hamster fibroblasts (V79 cell line) exhibit the phenomenon of recovery of DNA synthesis from the initial inhibition observed after ultraviolet light irradiation, in the absence of significant excision of pyrimidine dimers. In an attempt to determine whether the initial inhibition and subsequent recovery can be accounted for by parallel variations in the rate of movement of the replication fork, the cells were pulse-labeled with radioactive bromodeoxyuridine at different times following irradiation and their DNA centrifuged in neutral CsCl density gradients. When DNA synthesis inhibition was at a maximum, an accumulation of DNA, of density intermediate between hybrid and nonsubstituted DNA, was noticed in the density-distribution profiles. The density distribution of DNA along the gradient can provide an estimate of the rate of movement of the replication fork, and the results indicate that most of the variation in the overall rate of DNA synthesis can be accounted for by a parallel variation in the rate of fork movement. (Auth.)

Complexes of DNA bases and Watson-Crick base pairs with small neutral gold clusters.

Science.gov (United States)

Kryachko, E S; Remacle, F

2005-12-08

The nature of the DNA-gold interaction determines and differentiates the affinity of the nucleobases (adenine, thymine, guanine, and cytosine) to gold. Our preliminary computational study [Kryachko, E. S.; Remacle, F. Nano Lett. 2005, 5, 735] demonstrates that two major bonding factors govern this interaction: the anchoring, either of the Au-N or Au-O type, and the nonconventional N-H...Au hydrogen bonding. In this paper, we offer insight into the nature of nucleobase-gold interactions and provide a detailed characterization of their different facets, i.e., geometrical, energetic, and spectroscopic aspects; the gold cluster size and gold coordination effects; proton affinity; and deprotonation energy. We then investigate how the Watson-Crick DNA pairing patterns are modulated by the nucleobase-gold interaction. We do so in terms of the proton affinities and deprotonation energies of those proton acceptors and proton donors which are involved in the interbase hydrogen bondings. A variety of properties of the most stable Watson-Crick [A x T]-Au3 and [G x C]-Au3 hybridized complexes are described and compared with the isolated Watson-Crick A x T and G x C ones. It is shown that enlarging the gold cluster size to Au6 results in a rather short gold-gold bond in the Watson-Crick interbase region of the [G x C]-Au6 complex that bridges the G x C pair and thus leads to a significant strengthening of G x C pairing.

Occurrence and stability of lone pair–π stacking interactions between ribose and nucleobases in functional RNAs

KAUST Repository

Chawla, Mohit; Chermak, Edrisse; Zhang, Qingyun; Bujnicki, Janusz M.; Oliva, Romina; Cavallo, Luigi

2017-01-01

The specific folding pattern and function of RNA molecules lies in various weak interactions, in addition to the strong base-base pairing and stacking. One of these relatively weak interactions, characterized by the stacking of the O4′ atom of a ribose on top of the heterocycle ring of a nucleobase, has been known to occur but has largely been ignored in the description of RNA structures. We identified 2015 ribose–base stacking interactions in a high-resolution set of non-redundant RNA crystal structures. They are widespread in structured RNA molecules and are located in structural motifs other than regular stems. Over 50% of them involve an adenine, as we found ribose-adenine contacts to be recurring elements in A-minor motifs. Fewer than 50% of the interactions involve a ribose and a base of neighboring residues, while approximately 30% of them involve a ribose and a nucleobase at least four residues apart. Some of them establish inter-domain or inter-molecular contacts and often implicate functionally relevant nucleotides. In vacuo ribose-nucleobase stacking interaction energies were calculated by quantum mechanics methods. Finally, we found that lone pair–π stacking interactions also occur between ribose and aromatic amino acids in RNA–protein complexes.

Occurrence and stability of lone pair–π stacking interactions between ribose and nucleobases in functional RNAs

KAUST Repository

Chawla, Mohit

2017-08-18

The specific folding pattern and function of RNA molecules lies in various weak interactions, in addition to the strong base-base pairing and stacking. One of these relatively weak interactions, characterized by the stacking of the O4′ atom of a ribose on top of the heterocycle ring of a nucleobase, has been known to occur but has largely been ignored in the description of RNA structures. We identified 2015 ribose–base stacking interactions in a high-resolution set of non-redundant RNA crystal structures. They are widespread in structured RNA molecules and are located in structural motifs other than regular stems. Over 50% of them involve an adenine, as we found ribose-adenine contacts to be recurring elements in A-minor motifs. Fewer than 50% of the interactions involve a ribose and a base of neighboring residues, while approximately 30% of them involve a ribose and a nucleobase at least four residues apart. Some of them establish inter-domain or inter-molecular contacts and often implicate functionally relevant nucleotides. In vacuo ribose-nucleobase stacking interaction energies were calculated by quantum mechanics methods. Finally, we found that lone pair–π stacking interactions also occur between ribose and aromatic amino acids in RNA–protein complexes.

Influence of the nucleobase on the physicochemical characteristics and biological activities of Sb{sup V}-ribonucleoside complexes

Energy Technology Data Exchange (ETDEWEB)

Ferreira, Claudio S.; Demicheli, Cynthia, E-mail: demichel@netuno.lcc.ufmg.b [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Quimica; Rocha, Iara C.M. da; Melo, Maria N. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia; Monte Neto, Rubens L.; Frezard, Frederic [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Fisiologia e Biofisica

2010-07-01

The influence of the nucleobase (uracyl, U; cytosine, C; adenine, A; guanine, G) on the physicochemical characteristics and in vitro biological activities of Sb{sup V}-ribonucleoside complexes has been investigated. The 1:1 Sb-U and Sb-C complexes were characterized by NMR and ESI-MS spectroscopies and elemental analysis. The stability constant and the apparent association and dissociation rate constants of 1:1 Sb{sup V}-U, Sb{sup V}-C and Sb{sup V}-A complexes were determined. Although Sb{sup V} most probably binds via oxygen atoms to the same 2' and 3' positions in the different nucleosides, the ribose conformational changes and the physicochemical characteristics of the complex depend on the nucleobase. The nucleobase had a strong influence on the cytotoxicity against macrophages and the antileishmanial activity of the Sb{sup V}-ribonucleoside complexes. The Sb{sup V}-purine complexes were more cytotoxic and more effective against Leishmania chagasi than the Sb{sup V}-pyrimidine complexes, supporting the model that the interaction of Sb{sup V} with purine nucleosides may mediate the antileishmanial activity of pentavalent antimonial drugs. (author)

Dynamic changes of peripheral blood T-lymphocyte DNA-Synthesis in rabbits after fractionated and single exposure to 60Co-γ rays

International Nuclear Information System (INIS)

Wang Zongwu; Chen Tiehe; Yu Zhijie; Han Ling; Pan Yusha; Su Fuqiang

1988-01-01

The experiments in 59 rabbits γ-irradiated with doses of 0, 0.5, 1.0, 2.0, and 3.0 Gy in fractional and single exposure to 60 Co-γ rays were reported, respectively · Dynamics of the changes of DNA-Synthesis in T-lymphocytes of peripheral blood was obserced during 29 days after γ-irradiation. Marked inhibition in DNA-synthesis was found on 1st day after irradiation. Recovery was observed in 3rd day after irradiation. The levels of DNA-synthesis before irradiation was recovered on 7th day after exposure for all groups. For fractionated irradiation, however, an increase, rather than a decrese, of DNA-synthesis was in the group of 1.0 Gy

Immediate effects of grenz rays on epidermal DNA synthesis in the flanks of guinea pigs

International Nuclear Information System (INIS)

Daikeler, G.

1976-01-01

The following findings were obtained by autoradiography: 1) Labelling index (number of labelled cell nuclei per 1,000 based cells): Significant decrease immediately after exposure to grenz rays. 2) Silver grain index (number of silver cells as a function of the labelled basal cells): Significant decrease after irradiation. 3) DNA synthesis index (product of labelling index and silver grain index): Sifnificant decrease of the actual DNA synthesis rate of the reproductive cell cluster after exposure to grenz rays. (orig./AJ) [de

Platinated DNA oligonucleotides: new probes forming ultrastable conjugates with graphene oxide

Science.gov (United States)

Wang, Feng; Liu, Juewen

2014-05-01

Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials.Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate

NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

Science.gov (United States)

Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

2015-01-01

Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues.

The impact of cofactors and inhibitors on DNA repair synthesis after γ-irradiation in semi-permeable Escherichia coli cells

International Nuclear Information System (INIS)

Gaertner, C.

1981-01-01

The DNA-repair synthesis in tuluol-permeable E. coli cells after γ-irradiation has been investigated in dependence on the co-facotrs. ATB and NAD by means of enzyme kinetics. A partly repair-deficient mutants were taken into consideration which are well characterized in view of molecular biology; they showed which enzyme functions participate in the γ-induced DNA repair synthesis. The inhibition of the DNA-repair synthesis by the intercalary substances Adriamycin and Proflavin has been described and compared with the survival rates after irradiation and after combined treatment by irradiation and intercalary agents. (orig./AJ) [de

Scheduled and unscheduled DNA synthesis in chick embryo liver following X-irradiation and treatment with DNA repair inhibitors in vivo

Energy Technology Data Exchange (ETDEWEB)

Stammberger, I.; Tempel, K. (Muenchen Univ. (Germany, F.R.). Inst. fuer Pharmakologie und Toxikologie); Schmahl, W. (Gesellschaft fuer Strahlen- und Umweltforschung m.b.H. Muenchen, Neuherberg (Germany, F.R.). Inst. fuer Pathologie)

1989-09-01

Three hours following X-irradiation of chick embryos with doses of 4 and 8 Gy the in vitro incorporation of tritiated thymidine (({sup 3}H)dT) into DNA (scheduled DNA synthesis, ss) of hepatocytes was reduced to about one-third. Within 24 h after the exposure, ss returned to control values. The return of ss to a normal rate could be strongly inhibited by 2', 3'-dideoxythymidine (ddT), and to a lesser extent by 1-beta-D-arabinofuranosylcytosine (araC). In strong contrast to ss, the hydroxyurea (hu)-resistant ({sup 3)H}dT incorporation (unscheduled DNA synthesis, us) showed a highly significant increase 24 h after treatment of the embryos with araC and/or X-irradiation. Autoradiographic studies revealed no change of total ({sup 3}H)dT labelling frequency in the whole chick embryo liver 24 h after treatment with araC and/or X-irradiation, but a persistent depression of ss and a simultaneous increase of us. (author).

Sensitization of human cells by inhibitors of DNA synthesis following the action of DNA-damaging agents

Energy Technology Data Exchange (ETDEWEB)

Filatov, M.V.; Noskin, L.A. (Leningrad Inst. of Nuclear Physics, Gatchina (USSR))

1983-08-01

Inhibitors of DNA synthesis 1-..beta..-arabinofuranosylcytosine (Ac) and hydroxyurea (Hu) taken together drastically sensitized human cells to the killing effect of DNA-damaging agents. For UV-irradiation this sensitization depended on the cells' ability for excision repair. By using viscoelastometric methods of measurement of double-strand breaks (DSB) in the genome, it was established that the first DSB were generated after incubation of the damaged cells in the mixture of inhibitors at about the same dose when sensitization appeared. A scheme is proposed to describe molecular events associated with the phenomenon studied. 35 refs.

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA.

Science.gov (United States)

Cadet, Jean; Wagner, J Richard; Shafirovich, Vladimir; Geacintov, Nicholas E

2014-06-01

The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation.

Characterisation of nucleosides and nucleobases in Mactra veneriformis by high performance liquid chromatography coupled with diode array detector-mass spectrometry (HPLC-DAD-MS).

Science.gov (United States)

Liu, Rui; Ji, Jing; Wang, Lingchong; Chen, Shiyong; Guo, Sheng; Wu, Hao

2012-11-15

Mactra veneriformis has been used as sea food and traditional Chinese medicine (TCM) for thousands of years in China. In the present study, a high performance liquid chromatograph coupled with photodiode array detector and electrospray ionisation-mass spectrometer (HPLC-DAD-ESI-MS) method was established for detection of the nucleosides and nucleobases in M. veneriformis from four aquaticultural area of Jiangsu during different harvest time of one year. The validated method was successfully applied to identifying 10 nucleosides and nucleobases in 48 M. veneriformis samples. Quantitative analysis showed that nucleosides and nucleobases are rich in all M. veneriformis samples. However, their contents vary in different areas and harvest times. Principal component analysis (PCA) was used to classify the 48 samples based on the contents of the nucleosides and nucleobases. As a result, the samples could be mainly clustered into four groups, which was similar as aquaticultural areas classification. Based on the results, present method might be applicable for the quality control of M. veneriformis, or even other marine shellfish aquiculture and their products, and the quality of M. veneriformis might be more related with aquaticultural areas. Copyright © 2012 Elsevier Ltd. All rights reserved.

Inhibition and recovery of semiconservative DNA synthesis in normal and solar UV sensitive ICR 2A frog cell lines following the induction of non-dimer DNA damage by sunlamp UV > 315 nm

Energy Technology Data Exchange (ETDEWEB)

Rosenstein, B.S. (Brown Univ., Providence, RI (USA). Dept. of Radiation Medicine)

1989-08-01

Cultures of solar UV-sensitive cell lines DRP 36 and DRP 153, and of the parental ICR 2A cell line, were exposed to 150 kJ/m{sup 2} of sunlamp UV>315nm plus photoreactivating light, resulting in the induction primarily of non-dimer DNA damage. Following either 0, 3, 6, 12 or 24 h incubation, cultures were pulse-labelled with ({sup 3}H) thymidine, and the synthesis of different size classes of replicon intermediates measured using alkaline step elution assay. For all three cell lines tested, an immediate depression of low molecular weight DNA synthesis was observed, followed by inhibition of all size classes of replicon intermediates. Within 12 h following irradiation, recovery of DNA synthesis was observed, generally most apparent for low molecular weight DNA. The ICR 2A cells exhibited a nearly full recovery in all size classes of DNA synthesized by 24 h. A much smaller recovery of continued inhibition was primarily in the synthesis of full replicon size DNA, and most pronounced for DRP 36 cells. (author).

Effects of low doses of gamma radiation on DNA synthesis in the developing rat brain

International Nuclear Information System (INIS)

Cerda, H.

1983-01-01

Rats of one or ten days of age were irradiated with low doses of gamma radiation, and synthesis of DNA was examined by the incorporation of 3 H-thymidine in the cerebellum and the rest of the brain in vivo. DNA synthesis was depressed in both parts of the brain but the effects were larger in cerebellum. A minimum was found about 10 hours after irradiation in the older rats and later (18 h) in the younger ones. The dose response in 10 day-old rats, was biphasic and showed that cerebellum was more affected. Autoradiographs showed that fewer cells entered the cycle and those synthesizing showed a depressed rate of synthesis. These findings are discussed in relation to induction of cell death. (Auth.)

Spectroscopic Tools for Quantitative Studies of DNA Structure and Dynamics

DEFF Research Database (Denmark)

Preus, Søren

The main objective of this thesis is to develop quantitative fluorescence-based, spectroscopic tools for probing the 3D structure and dynamics of DNA and RNA. The thesis is founded on six peer-reviewed papers covering mainly the development, characterization and use of fluorescent nucleobase...... analogues. In addition, four software packages is presented for the simulation and quantitative analysis of time-resolved and steady-state UV-Vis absorption and fluorescence experiments....

DNA-Conjugated Organic Chromophores in DNA Stacking Interactions

DEFF Research Database (Denmark)

Filichev, Vyacheslav V.; Pedersen, Erik Bjerregaard

2009-01-01

Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic ch...... review presents those efforts in the design of intercalators/organic chromophores as oligonucleotide conjugates that form a foundation for the generation of novel nucleic acid architectures......Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic...

Synthesis and DNA cleavage activity of Bis-3-chloropiperidines as alkylating agents.

Science.gov (United States)

Zuravka, Ivonne; Roesmann, Rolf; Sosic, Alice; Wende, Wolfgang; Pingoud, Alfred; Gatto, Barbara; Göttlich, Richard

2014-09-01

Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3-chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis-3-chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide-catalyzed double cyclization of unsaturated bis-N-chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen-bridged bis-3-chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

Science.gov (United States)

Stone, G. E.; Miller, O. L.; Prescott, D. M.

1965-01-01

The formation of a soluble H3-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H3-thymidine during G1 or G2. Uptake of H3-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H3-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H3-thymidine, the pool does not turn over during non-S periods. PMID:19866660

Inhibition of the synthesis of polyamines and DNA in activated lymphocytes by a combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone).

Science.gov (United States)

Morris, D R; Jorstad, C M; Seyfried, C E

1977-09-01

The cancer chemotherapeutic drug, methylglyoxal bis(guanylhydrazone), inhibits the synthesis of spermidine and spermine, but allows continued putrescine production in small lymphocytes stimulated by concanavalin A. DNA replication in these cells is inhibited 50% while the synthesis of protein and RNA continues normally. When excess putrescine accumulation in the presence of methylglyoxal bis(guanylhydrazone) was inhibited with alpha-methylornithine, a competitive inhibitor of ornithine decarboxylase, the inhibition of DNA replication was accentuated, with still no effect on protein or RNA synthesis. No inhibition of DNA synthesis by the combination of alpha-methylornithine and methylglyoxal bis(guanylhydrazone) was observed when the inhibitors were added after accumulation of cellular polyamines. In addition, inhibition was reversed by exogenous putrescine, spermidine, or spermine. We conclude that putrescine can fulfill in part the role normally played by spermidine and spermine in DNA replication, and that blocking putrescine synthesis in the presence of methylglyoxal bis(guanylhydrazone) amplifies the polyamine requirement. The implications of this with regard to polyamine synthesis as a site of chemotherapy are discussed.

Opportunities for measuring DNA synthesis time by quantitative autoradiography

International Nuclear Information System (INIS)

Vasileva, D.

1980-01-01

DNA sysntesis time (Tsub(s)) in cells of the canine erythropoiesis and myelopoiesis pools was determined by quantitative autoradiography according to Doermer. In contrast to mitosis labelling for Tsub(s) estimation as so far applied, this technique uses well-differentiated cells. After blocking endogeneous DNA synthesis with 5-fluorodeoxyuridine, its further course becomes dependent on exogeneous supply of thymidine, in the form of 14 C-thymidine. From incroporation of the latter into the individual cell within a definite time span (3-7 min) and taking into account its total amount, Tsub(s) may be calculated. The data thus obtained were found to agree with Tsub(s) values as estimated from the labelled mitosis curve

Unusually high dispersion of nitrogen-doped carbon nanotubes in DNA solution.

Science.gov (United States)

Kim, Jin Hee; Kataoka, Masakazu; Fujisawa, Kazunori; Tojo, Tomohiro; Muramatsu, Hiroyuki; Vega-Díaz, Sofía M; Tristán-López, F; Hayashi, Takuya; Kim, Yoong Ahm; Endo, Morinobu; Terrones, Mauricio; Dresselhaus, Mildred S

2011-12-08

The dispersibility in a DNA solution of bundled multiwalled carbon nanotubes (MWCNTs), having different chemical functional groups on the CNT sidewall, was investigated by optical spectroscopy. We observed that the dispersibility of nitrogen (N)-doped MWCNTs was significantly higher than that of pure MWCNTs and MWCNTs synthesized in the presence of ethanol. This result is supported by the larger amount of adsorbed DNA on N-doped MWCNTs, as well as by the higher binding energy established between nucleobases and the N-doped CNTs. Pure MWCNTs are dispersed in DNA solution via van der Waals and hydrophobic interactions; in contrast, the nitrogenated sites within N-doped MWCNTs provided additional sites for interactions that are important to disperse nanotubes in DNA solutions. © 2011 American Chemical Society

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

International Nuclear Information System (INIS)

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

1991-01-01

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

Effect of haloperidol on the synthesis of DNA in the pituitary gland of the rat.

Science.gov (United States)

Machiavelli, G A; Jahn, G A; Kalbermann, L E; Szijan, I; Alonso, G E; Burdman, J A

1982-03-01

The administration of haloperidol increased serum prolactin and decreased the pituitary concentration of prolactin 15 min after its administration. Concomitantly there was a stimulation in the synthesis of DNA and the activity of DNA polymerase alpha in the anterior pituitary gland that was greater in oestrogenized than in non-oestrogenized male rats. Both these effects were greatly reduced by clomiphene in the oestrogenized male rats, although it did not affect the release of prolactin produced by haloperidol. In non-oestrogenized animals clomiphene abolished the stimulatory effect of haloperidol on the synthesis of DNA. These results suggest that the reduction in the intracellular levels of prolactin are a primary event in the oestrogen mediated stimulation of cell proliferation by prolactin releasing agents.

Photoreactions of ruthenium(II) and osmium(II) complexes with deoxyribonucleic acid (DNA).

Science.gov (United States)

Moucheron, C; Kirsch-De Mesmaeker, A; Kelly, J M

1997-09-01

The design of Ru(II) and Os(II) complexes which are photoreactive with deoxyribonucleic acid (DNA) represents one of the main targets for the development of novel molecular tools for the study of DNA and, in the future, for the production of new, metal-based, anti-tumor drugs. In this review, we explain how it is possible to make a complex photoreactive with nucleobases and nucleic acids. According to the photophysical behaviour of the Ru(II) compounds, two types of photochemistry are expected: (1) photosubstitution of a ligand by a nucleobase and another monodentate ligand, which takes place from the triplet, metal-centred (3MC) state; this state is populated thermally from the lowest lying triplet metal to ligand charge transfer (3MLCT) state; (2) photoreaction from the 3MLCT state, corresponding to photoredox processes with DNA bases. The two photoreactivities are in competition. By modulating appropriately the redox properties of the 3MLCT state, an electron transfer process from the base to the excited complex takes place, and is directly correlated with DNA cleavage or the formation of an adduct of the complex to DNA. In this adduct, guanine is linked by N2 to the alpha-position of a non-chelating nitrogen of the polyazaaromatic ligand without destruction of the complex. Different strategies are explained which increase the affinity of the complexes for DNA and direct the complex photoreactivity to sites of special DNA topology or targeted sequences of bases. Moreover, the replacement of the Ru(II) ion by the Os(II) ion in the photoreactive complexes leads to an increased specificity of photoreaction. Indeed, only one type of photoreactivity (from the 3MLCT state) is present for the Os(II) complexes because the 3MC state is too high in energy to be populated at room temperature.

DNA repair genes RAD52 and SRS2, a cell wall synthesis regulator gene SMI1, and the membrane sterol synthesis scaffold gene ERG28 are important in efficient Agrobacterium-mediated yeast transformation with chromosomal T-DNA.

Science.gov (United States)

Ohmine, Yuta; Satoh, Yukari; Kiyokawa, Kazuya; Yamamoto, Shinji; Moriguchi, Kazuki; Suzuki, Katsunori

2016-04-02

Plant pathogenic Agrobacterium strains can transfer T-DNA regions of their Ti plasmids to a broad range of eukaryotic hosts, including fungi, in vitro. In the recent decade, the yeast Saccharomyces cerevisiae is used as a model host to reveal important host proteins for the Agrobacterium-mediated transformation (AMT). Further investigation is required to understand the fundamental mechanism of AMT, including interaction at the cell surface, to expand the host range, and to develop new tools. In this study, we screened a yeast mutant library for low AMT mutant strains by advantage of a chromosome type T-DNA, which transfer is efficient and independent on integration into host chromosome. By the mutant screening, we identified four mutant strains (srs2Δ, rad52Δ, smi1Δ and erg28Δ), which showed considerably low AMT efficiency. Structural analysis of T-DNA product replicons in AMT colonies of mutants lacking each of the two DNA repair genes, SRS2 and RAD52, suggested that the genes act soon after T-DNA entry for modification of the chromosomal T-DNA to stably maintain them as linear replicons and to circularize certain T-DNA simultaneously. The cell wall synthesis regulator SMI1 might have a role in the cell surface interaction between the donor and recipient cells, but the smi1Δ mutant exhibited pleiotropic effect, i.e. low effector protein transport as well as low AMT for the chromosomal T-DNA, but relatively high AMT for integrative T-DNAs. The ergosterol synthesis regulator/enzyme-scaffold gene ERG28 probably contributes by sensing a congested environment, because growth of erg28Δ strain was unaffected by the presence of donor bacterial cells, while the growth of the wild-type and other mutant yeast strains was suppressed by their presence. RAD52 and the DNA helicase/anti-recombinase gene SRS2 are necessary to form and maintain artificial chromosomes through the AMT of chromosomal T-DNA. A sterol synthesis scaffold gene ERG28 is important in the high

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

Science.gov (United States)

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

2017-10-17

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

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

International Nuclear Information System (INIS)

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

1990-01-01

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

Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression

International Nuclear Information System (INIS)

Kim, Sunyoung; Baltimore, D.; Byrn, R.; Groopman, J.

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. The authors 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

Unscheduled DNA synthesis in human skin after in vitro ultraviolet-excimer laser ablation

International Nuclear Information System (INIS)

Green, H.A.; Margolis, R.; Boll, J.; Kochevar, I.E.; Parrish, J.A.; Oseroff, A.R.

1987-01-01

DNA damage repaired by the excision repair system and measured as unscheduled DNA synthesis (UDS) was assessed in freshly excised human skin after 193 and 248 nm ultraviolet (UV)-excimer laser ablative incisions. Laser irradiation at 248 nm induced DNA damage throughout a zone of cells surrounding the ablated and heat-damaged area. In contrast, with 193 nm irradiation UDS was not detected in cells adjacent to the ablated area, even though DNA strongly absorbs this wavelength. Our results suggest that the lack of UDS after 193 nm irradiation is due to: ''shielding'' of DNA by the cellular interstitium, membrane, and cytoplasm, DNA damage that is not repaired by excision repair, or thermal effects that either temporarily or permanently inhibit the excision repair processes

The acute effects of ionizing radiation on DNA synthesis and the development of antibody-producing cells

International Nuclear Information System (INIS)

Harris, G.; Olsen, I.; Cramp, W.A.

1981-01-01

Ionizing radiation inhibited the development of specific haemolysin-producing cells (PFC) and depressed the incorporation of ( 3 H) thymidine by rabbit spleen explants responding to SRC in the culture medium. In contrast to these effects, the rates of incorporation of precursors for protein and RNA synthesis were much less affected. The depression of ( 3 H) thymidine incorporation was found to result from a quantitative reduction of new DNA synthesis, without any change in the proportion of labelled cells, at any time after irradiation. The DNA synthesis occurring in these cells preparing to develop antibody-producing capacity was thus radio-sensitive, but the exact nature of the defect resulting from exposure to radiation requires further study. (orig.)

Novel pattern of post-γ ray de novo DNA synthesis in a radioresistant human strain

International Nuclear Information System (INIS)

Mirzayans, R.; Gentner, N.E.; Paterson, M.C.

1985-01-01

Enhanced resistance to radiation cytotoxicity in a fibroblast strain from an afflicted member of a Li-Fraumeni syndrome family may be largely ascribable to a change in the pattern of DNA replicative synthesis following γ ray exposure. That is, the extent of the initial radiogenic inhibition of replicative synthesis and the time interval before its subsequent recovery were both found to be greater in radioresistant (RR) compared to normal cells. In addition, the post-recovery replication rates in the RR cells were both higher and longer lasting than those in the control cells. A similar differential pattern was also seen following treatment with 4NQO, another DNA-damaging agent to which this RR strain displays enhanced resistance. Moreover, several conventional DNA repair assays indicated that the RR cells repair radiogenic damage at normal rates. The authors therefore suggest that the increased inhibition and prolonged lag in resumption of replicative synthesis seen in the RR strain upon exposure to certain genotoxic agents may enhance cellular recovery by ''buying additional time'' for processing of potentially lethal lesions

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

Science.gov (United States)

Seco, Elena M.

2017-01-01

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

Effects of the Nd:YAG laser on DNA synthesis and collagen production in human skin fibroblast cultures

Energy Technology Data Exchange (ETDEWEB)

Castro, D.J.; Abergel, R.P.; Meeker, C.; Dwyer, R.M.; Lesavoy, M.A.; Uitto, J.

1983-09-01

Human skin fibroblasts were subjected to treatment with a Neodymium:YAG laser at 1060 nm with varying levels of energy determined by a reproducible method of dosimetry. DNA synthesis in the cells was measured by the incorporation of (3H)thymidine, and collagen production was monitored by the synthesis of nondialyzable (3H)hydroxyproline after incubation of cells with (3H)proline. Using energy levels equal to 1.7 X 10(3) J/cm2, a significant reduction in DNA synthesis was noted, while the cells remained viable as tested by the trypan blue exclusion test. With energy levels higher or equal to 2.3 X 10(3) J/cm2, the suppression of DNA synthesis was accompanied by cell nonviability. The collagen production, when measured immediately following the treatment with 1.7 X 10(3) J/cm2, was markedly reduced, and similar effects were observed with higher energy levels. However, when the cells were tested for collagen production at 20 hours following laser treatment, there was a significant decrease in collagen production at energy levels as low as 1.1 X 10(3) J/cm2, a dose that did not affect DNA synthesis or cell viability. Thus, the results indicate that the Nd:YAG laser can selectively suppress collagen production without affecting cell proliferation. These observations suggest that laser treatment could potentially be used to reduce collagen deposition in conditions such as keloids and hypertrophic scars.

Synthesis of DNA

Science.gov (United States)

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.

Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function

International Nuclear Information System (INIS)

Minor, P.D.; Dimmock, N.J.

1977-01-01

Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and α-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at high concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent

Simple Laboratory methods to measure cell proliferation using DNA synthesis property

Directory of Open Access Journals (Sweden)

Madhavan H N

2007-01-01

Full Text Available This is a mini-review on the techniques to measure proliferation of cells by estimation of DNA synthesis. This is not an exhaustive review of literature, but a bird’s eye view of a few selected articles which may provide the technical details to the readers.The nucleus of a cell occupies about 10-30% of the cells space, depends on the type of genetic material (DNA -DeoxyriboNucleic Acid. DNA is a long, double-stranded, helical molecule which carries the genetic information. Duplication of the DNA takes place by the phenomena of replication. One copy of double-stranded DNA molecule forms two double-stranded DNA molecules. DNA replication is the fundamental process used in all living organisms as it is the basis for biological inheritance. This process is known also as Mitosis in somatic cells. In Mitosis, the duplication process results in two genetically identical "daughter" cells from a single "parent" cell. The resulting double-stranded DNA molecules are identical; proof reading and error-checking mechanisms exist to ensure near perfect pair. Mitosis is divided into six phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis.

The effect of caffeine and adenine on radiation induced suppression of DNA synthesis, and cell survival

International Nuclear Information System (INIS)

Wilcoxson, L.T.; Griffiths, T.D.

1984-01-01

Exposure of cultured mammalian cells to ionizing radiation or UV light results in a transient decrease in the rate of DNA synthesis. This depression in synthetic rate may be attenuated or deferred via a post-irradiation treatment with caffeine or adenine. It has been suggested that this attenuation may increase the fixation of damage and, therefore, increase radiation sensitivity. However, it has been previously reported that, for V79 cells treated with caffeine or adenine, no correlation exists between the extent of depression and cell survival. The present investigation expands upon these findings by examining the effect of caffeine or adenine post-irradiation treatment on two cell lines with normal UV sensitivity, mouse 3T3 and CHO AA8 cells, and one UV sensitive cell line, CHO UV5 cells. Both caffeine and adenine have been found to reduce, or delay, the suppression in DNA synthesis in all three cell lines. Surprisingly, caffeine appeared to induced even the UV5 cells to recover DNA synthetic ability. The amount of reduction in suppression of DNA synthesis, however, varies between the different cell lines and no consistent relationship with cell survival has emerged

Origin of DNA-Induced Circular Dichroism in a Minor-Groove Binder.

Science.gov (United States)

Holmgaard List, Nanna; Knoops, Jérémie; Rubio-Magnieto, Jenifer; Idé, Julien; Beljonne, David; Norman, Patrick; Surin, Mathieu; Linares, Mathieu

2017-10-25

Induced circular dichroism (ICD) of DNA-binding ligands is well known to be strongly influenced by the specific mode of binding, but the relative importance of the possible mechanisms has remained undetermined. With a combination of molecular dynamics simulations, CD response calculations, and experiments on an AT-sequence, we show that the ICD of minor-groove-bound 4',6-diamidino-2-phenylindole (DAPI) originates from an intricate interplay between the chiral imprint of DNA, off-resonant excitonic coupling to nucleobases, charge-transfer, and resonant excitonic coupling between DAPIs. The significant contributions from charge-transfer and the chiral imprint to the ICD demonstrate the inadequacy of a standard Frenkel exciton theory of the DAPI-DNA interactions.

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

Science.gov (United States)

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.

TRX-LOGOS - a graphical tool to demonstrate DNA information content dependent upon backbone dynamics in addition to base sequence.

Science.gov (United States)

Fortin, Connor H; Schulze, Katharina V; Babbitt, Gregory A

2015-01-01

It is now widely-accepted that DNA sequences defining DNA-protein interactions functionally depend upon local biophysical features of DNA backbone that are important in defining sites of binding interaction in the genome (e.g. DNA shape, charge and intrinsic dynamics). However, these physical features of DNA polymer are not directly apparent when analyzing and viewing Shannon information content calculated at single nucleobases in a traditional sequence logo plot. Thus, sequence logos plots are severely limited in that they convey no explicit information regarding the structural dynamics of DNA backbone, a feature often critical to binding specificity. We present TRX-LOGOS, an R software package and Perl wrapper code that interfaces the JASPAR database for computational regulatory genomics. TRX-LOGOS extends the traditional sequence logo plot to include Shannon information content calculated with regard to the dinucleotide-based BI-BII conformation shifts in phosphate linkages on the DNA backbone, thereby adding a visual measure of intrinsic DNA flexibility that can be critical for many DNA-protein interactions. TRX-LOGOS is available as an R graphics module offered at both SourceForge and as a download supplement at this journal. To demonstrate the general utility of TRX logo plots, we first calculated the information content for 416 Saccharomyces cerevisiae transcription factor binding sites functionally confirmed in the Yeastract database and matched to previously published yeast genomic alignments. We discovered that flanking regions contain significantly elevated information content at phosphate linkages than can be observed at nucleobases. We also examined broader transcription factor classifications defined by the JASPAR database, and discovered that many general signatures of transcription factor binding are locally more information rich at the level of DNA backbone dynamics than nucleobase sequence. We used TRX-logos in combination with MEGA 6.0 software

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

International Nuclear Information System (INIS)

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

1986-01-01

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

Dual door entry to exciplex emission in a chimeric DNA duplex containing non-nucleoside-nucleoside pair.

Science.gov (United States)

Bag, Subhendu Sekhar; Talukdar, Sangita; Kundu, Rajen; Saito, Isao; Jana, Subhashis

2014-01-25

Dual door entry to exciplex formation was established in a chimeric DNA duplex wherein a fluorescent non-nucleosidic base surrogate () is paired against a fluorescent nucleosidic base surrogate (). Packing of the nucleobases via intercalative stacking interactions led to an exciplex emission either via FRET from the donor or direct excitation of the FRET acceptor .

Effect of different BNCT protocols on DNA synthesis in precancerous and normal tissues in an experimental model of oral cancer

International Nuclear Information System (INIS)

Heber, Elisa M.; Aromando, Romina; Trivillin, Veronica A.; Itoiz, Maria E.; Kreimann, Erica L.; Schwint, Amanda E.; Nigg, David W.

2006-01-01

We previously reported the therapeutic success of different BNCT protocols in the treatment of oral cancer, employing the hamster cheek pouch model. The aim of the present study was to evaluate the effect of these BNCT protocols on DNA synthesis in precancerous and normal tissue in this model and assess the potential lag in the development of second primary tumors in precancerous tissue. The data are relevant to potential control of field cancerized tissue and tolerance of normal tissue. We evaluated DNA synthesis in precancerous and normal pouch tissue 1-30 days post-BNCT mediated by BPA, GB-10 or BPA + GB-10 employing incorporation of bromo-deoxyuridine as an end-point. The BNCT-induced potential lag in the development of second primary tumors in precancerous tissue was monitored. A drastic, statistically significant reduction in DNA synthesis occurred in pacancerous tissue as early as 1 day post-BNCT and was sustained at virtually all time points until 30 days post-BNCT for all protocols. The histological categories evaluated individually within precancerous tissue (dysplasia, hyperplasia and NUMF [no unusual microscopic features]) responded similarly. DNA synthesis in normal tissue treated with BNCT oscillated around the very low pre-treatment values. A BNCT-induced lag in the development of second primary tumors was observed. BNCT induced a drastic fall in DNA synthesis in precancerous tissue that would be associated to the observed lag in the development of second primary tumors. The minimum variations in DNA synthesis in BNCT-treated normal tissue would correlate with the absence of normal tissue radiotoxicity. The present data would contribute to optimize therapeutic efficacy in the treatment of field-cancerized areas. (author)

Nuclear DNA synthesis rate and labelling index: effects of carcinogenic and non-carcinogenic chemicals on its behaviour in the organism of growing CBA mice

International Nuclear Information System (INIS)

Amlacher, E.; Rudolph, C.

1978-01-01

Well known bioassays have been compared with the author's thymidine incorporation-screening system and other assays based on biochemical quantification of DNA synthesis as a possibility of identification of carcinogens. The partial inhibition of the whole DNA synthesis in a proliferating cell population after treatment with toxic and carcinogenic chemicals is an early common response especially in hepatectomized animal, livers caused by the effects of those substances. However, by quantitative evaluation of the nuclear DNA synthesis rate as a basic parameter, using autoradiographs of kidney and liver of juvenile growing CBA mice, it is possible to differentiate carcinogenic from non-carcinogenic chemicals by means of silver grain counting after 3 H-TdR incorporation. On the contrary, the whole DNA synthesis, expressed by the 3 H-labelling index (in per cent) of kidney and liver, did not permit such a differentiation in the experimental arrangement used. It could be demonstrated that carcinogenic compounds of different chemical classes partially inhibit the nuclear DNA synthesis rate significantly over a period of more than 24 hours. The tested non-carcinogenic compounds did not show this suppressive effect on the nuclear DNA synthesis rate. (author)

DNA synthesis time in germinating rice and pattern of diethylsulphate induced mutations in pre-soaked seeds

International Nuclear Information System (INIS)

Narahari, P.

1978-01-01

DNA synthesis pattern in germinating rice seeds, pre-soaked in water for varying periods upto 48 hr, was determined by following the pulse incorporation of 3 H-thymidine into the TCA-insoluble nucleoprotein. Synthesis of DNA commenced at 24 hr, progressively increased to a first peak at about 38 hr, thereafter showed a 1/3rd drop and subsequently increased to a 2nd and still higher peak at 46 to 48 hr of pre-soaking. Treatments of diethylsulphate (dES) at a low concentration (0.2%-2hr) administered at various progressing stages of DNA synthesis resulted in decrease in seedling height and survival, and increase in mutation frequency at 45 hr. pre-soaking, maximum mutation frequencies of 20, 10 and 2% on M 1 plants, M 1 spikes and M 2 seedling bases, respectively were observed. Higher dES concentration (0.3%-2hr) given at later periods of pre-soaking showed near lethal effects and consequently decreased mutation frequencies. Treatments of sodium fluoride given singly or in combination with dES did not show any substantially different results as compared to those of the respective controls. Mutation spectra observed after dES treatments to germinating seeds, at different pre-soaking periods, were quite dissimilar. Specific mutations of economic importance like semi-dwarf mutants were isolated from the treatment of germinating seeds pre-soaked for 37.5 hr or more when shoot apex cells were undergoing DNA synthesis. (author)

Unscheduled DNA synthesis in human skin after in vitro ultraviolet-excimer laser ablation

Energy Technology Data Exchange (ETDEWEB)

Green, H.A.; Margolis, R.; Boll, J.; Kochevar, I.E.; Parrish, J.A.; Oseroff, A.R.

1987-08-01

DNA damage repaired by the excision repair system and measured as unscheduled DNA synthesis (UDS) was assessed in freshly excised human skin after 193 and 248 nm ultraviolet (UV)-excimer laser ablative incisions. Laser irradiation at 248 nm induced DNA damage throughout a zone of cells surrounding the ablated and heat-damaged area. In contrast, with 193 nm irradiation UDS was not detected in cells adjacent to the ablated area, even though DNA strongly absorbs this wavelength. Our results suggest that the lack of UDS after 193 nm irradiation is due to: ''shielding'' of DNA by the cellular interstitium, membrane, and cytoplasm, DNA damage that is not repaired by excision repair, or thermal effects that either temporarily or permanently inhibit the excision repair processes.

Murine scid cells complement ataxia-telangiectasia cells and show a normal port-irradiation response of DNA synthesis

International Nuclear Information System (INIS)

Komatsu, K.; Yoshida, M.; Okumura, Y.

1993-01-01

The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a deficit in lymphoid variable-(diversity)-joining (V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Significantly reduced D 0 and n values were demonstrated in scid cells and were similar to ataxia-telangiectasia (AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Results indicate that the radiobiological character of scid is similar to AT but is presumably caused by different mechanisms. (author)

Possible roles of HIV-1 nucleocapsid protein in the specificity of proviral DNA synthesis and in its variability.

Science.gov (United States)

Lapadat-Tapolsky, M; Gabus, C; Rau, M; Darlix, J L

1997-05-02

Retroviral nucleocapsid (NC) protein is an integral part of the virion nucleocapsid where it coats the dimeric RNA genome. Due to its nucleic acid binding and annealing activities, NC protein directs the annealing of the tRNA primer to the primer binding site and greatly facilitates minus strand DNA elongation and transfer while protecting the nucleic acids against nuclease degradation. To understand the role of NCp7 in viral DNA synthesis, we examined the influence of NCp7 on self-primed versus primer-specific reverse transcription. The results show that HIV-1 NCp7 can extensively inhibit self-primed reverse transcription of viral and cellular RNAs while promoting primer-specific synthesis of proviral DNA. The role of NCp7 vis-a-vis the presence of mutations in the viral DNA during minus strand elongation was examined. NCp7 maximized the annealing between a cDNA(-) primer containing one to five consecutive errors and an RNA representing the 3' end of the genome. The ability of reverse transcriptase (RT) in the presence of NCp7 to subsequently extend the mutated primers depended upon the position of the mismatch within the primer:template complex. When the mutations were at the polymerisation site, primer extension by RT in the presence of NCp7 was very high, about 40% for one mismatch and 3% for five consecutive mismatches. Mutations within the DNA primer or at its 5' end had little effect on the extension of viral DNA by RT. Taken together these results indicate that NCp7 plays major roles in proviral DNA synthesis within the virion core due to its ability to promote prime-specific proviral DNA synthesis while concurrently inhibiting non-specific reverse transcription of viral and cellular RNAs. Moreover, the observation that NCp7 enhances the incorporation of mutations during minus strand DNA elongation favours the notion that NCp7 is a factor contributing to the high mutation rate of HIV-1.

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

Science.gov (United States)

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 cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

Increased cellular levels of spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A.

Science.gov (United States)

Fillingame, R H; Jorstad, C M; Morris, D R

1975-01-01

There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) blocks synthesis of these polyamines by inhibiting S-adenosylmethionine decarboxylase. Previous results showed that when cells are activated in the presence of MGBG the synthesis and processing of RNA, as well as protein synthesis, proceed as in the absence of the drug. In contrast, the incorporation of [methyl-3H]thymidine into DNA and the rate of entry of the cells into mitosis are inhibited by 60% in the presence of MGBG. Several experiments suggest that MGBG inhibits cell proliferation by directly blocking polyamine synthesis and not by an unrelated pharmacological effect: (1) the inhibitory action of MGBG is reversed by exogenously added spermidine or spermine; (2) inhibition of DNA synthesis by MGBG shows the same dose-response curve as does inhibition of spermidine and spermine synthesis; and (3) if MGBG is added to cells which have been allowed to accumulate their maximum complement of polyamines, there is no inhibition of thymidine incorporation. MGBG-treated and control cultures initiate DNA synthesis at the same time and show the same percentage of labeled cells by autoradiography. Therefore, it appears that in the absence of increased cellular levels of polyamines, lymphocytes progress normally from G0 through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication. PMID:1060087

Excision of pyrimidine dimers from epidermal DNA and nonsemiconservative epidermal DNA synthesis following ultraviolet irradiation of mouse skin

International Nuclear Information System (INIS)

Bowden, G.T.; Trosko, J.E.; Shapas, B.G.; Boutwell, R.K.

1975-01-01

Pyrimidine dimer production and excision in epidermal DNA were studied at five different dose levels of ultraviolet light in the skin of intact mice. Dimer production increased with dose up to 50,400 ergs/sq mm. Approximately 30 percent of the thymine-containing dimers were excised by 24 hr after irradiation at three lower dose levels of ultraviolet light. Nonsemiconservative DNA replication in ultraviolet-irradiated mouse skin was shown to continue for at least 18 hr. The rate of nonsemiconservative replication decreased with time, but did so slowly. The initial rates of nonsemiconservative replication increased with ultraviolet light dose levels up to about 4200 ergs/sq mm, after which the initial rates were decreased. Semiconservative epidermal DNA synthesis was shown to be inhibited by hydroxyurea, but hydroxyurea had no effect on ultraviolet light-induced nonsemiconservative DNA replication. The observed pyrimidine dimer excision and nonsemiconservative DNA replication suggest that in the intact mouse the cells of the epidermis are capable of DNA excision repair after ultraviolet irradiation of mouse skin

Unscheduled DNA synthesis after β-irradiation of mouse skin in situ

International Nuclear Information System (INIS)

Ootsuyama, Akira; Tanooka, Hiroshi

1986-01-01

The skin of ICR mouse was irradiated with β-rays from 90 Sr- 90 Y with surface doses up to 30 krad. Unscheduled DNA synthesis (UDS) was measured by autoradiography after labeling the skin with radioactive thymidine using the forceps-clamping method. The level of UDS in epithelial cells of the skin was detected as an increasing function of radiation dose. Fibroblastic cells, compared with epithelial cells and hair follicle cells at the same depth of the skin, showed a lower level of UDS, indicating a lower DNA repair activity in fibroblasts. Cancer risk of the skin was discussed. (Auth.)

Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates

International Nuclear Information System (INIS)

Piette, J.; Hearst, J.

1985-01-01

Single-stranded DNA has been photochemically induced to react with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and used as substrate for DNA replication with E. coli DNA polymerase I large fragment. By using the dideoxy sequencing procedure, it is possible to map the termination sites on the template photoreacted with HMT. These sites occur at the nucleotides preceding each thymine residue (and a few cytosine residues), emphasizing the fact that in a single-stranded stretch of DNA, HMT reacts with each thymine residue without any specificity regarding the flanking base sequence of the thymine residues. In addition, termination of DNA synthesis due to psoralen-adducted thymine is not influenced by the efficiency of the 3'-5' exonuclease proof-reading activity of the DNA polymerase. (author)

Cell growth state determines susceptibility of repair DNA synthesis to inhibition by hydroxyurea and 1-beta-D-arabinofuranosylcytosine

International Nuclear Information System (INIS)

Mullinger, A.M.; Collins, A.R.; Johnson, R.T.

1983-01-01

The effects of inhibitors of replicative DNA synthesis on repair DNA synthesis have been examined by autoradiography in several different cell types and in cells in different growth states. Hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), administered together, influence unscheduled DNA synthesis (UDS) in a manner which is independent of the status of the cell culture (normal or transformed) and of the species, but which is strongly affected by whether the cells are proliferating or quiescent. In proliferating human, Chinese hamster and Microtus cell cultures, UDS is not inhibited by HU and ara C, and may even appear to be stimulated. In quiescent cultures of these cells UDS is reduced by HU and ara C. In cells reseeded from a confluent culture and followed during proliferation and back to quiescence the effect of inhibitors parallels the growth pattern. The results are interpreted in terms of changes in the sizes of endogenous DNA precursor pools; they underline the potential problems associated with quantitating UDS in the presence of inhibitors

DNA damage and genetic methylation changes caused by Cd in Arabidopsis thaliana seedlings.

Science.gov (United States)

Li, Zhaoling; Liu, Zhihong; Chen, Ruijuan; Li, Xiaojun; Tai, Peidong; Gong, Zongqiang; Jia, Chunyun; Liu, Wan

2015-09-01

Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MASP) techniques are sensitive to deoxyribonucleic acid (DNA) damage and genetic methylation, respectively. Using these 2 techniques, Arabidopsis thaliana cultured with 0 mg/L (control), 0.5 mg/L, 1.5 mg/L, and 5.0 mg/L Cd(2+) for 16 d was used to analyze the DNA damage and methylation changes as a result of cadmium (Cd). The DNA was amplified by 14 AFLP primer pairs and 13 MSAP primer combinations. In the AFLP experiment, 62 polymorphic sites were found in the patterns of 11 primer combinations and a total of 1116 fragments were obtained in these patterns. There were no polymorphic bands in the remaining 3 pairs. The proportions of polymorphic sites in the 0.5-mg/L Cd(2+) and 5.0-mg/L Cd(2+) treatments were significantly different. Seven polymorphic fragments were then separated and successfully sequenced, yielding 6 nucleobase substitutions and 1 nucleobase deletion. Similarly, in the MSAP experiment, the MSAP% and number of demethylated-type bands were unchanged after Cd treatment, but the number of methylated-type bands was increased significantly in the 5.0-mg/L Cd(2+) treatment group, a finding that may be associated with the AFLP results. The polymorphic bands were also sequenced and the functions of their homologous genes were determined. The DNA damage and methylation changes may be the primary cause of certain pathology changes as a result of Cd uptake in plants. © 2015 SETAC.

Influence of some radioprotective and radiosensitizing compounds on the replicative and repair induced DNA synthesis of rats spleen cells in vitro

International Nuclear Information System (INIS)

Goette, A.

1982-01-01

The effect of cysteine, dithiothreitol, N-ethylmaleimide, cytosinearabinoside, ethidiumbromide, bleomycine and diethyldithiocarbamate on the replicative and repair induced DNA synthesis in vitro was tested by using rats spleen cells. Besides the incorporation of a labeled DNA precursor (TdR- 3 H) the sedimentation of DNA in sucrose gradients was inquired. With respect to the DNA synthesis an uniform mechanism of action for the radioprotective substances can't be seen. Thymocytes and spleen cells seem to possess different systems of repair; this may be an explanation for their different sensibility against ionizing radiation. (orig./MG) [de

Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system.

Science.gov (United States)

Villalva, C; Touriol, C; Seurat, P; Trempat, P; Delsol, G; Brousset, P

2001-07-01

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

Autoradiographic study of gamma-ray induced unscheduled DNA synthesis in bean root meristem cells

International Nuclear Information System (INIS)

Liu Zhenshen; Qiu Quanfa; Chen Dongli

1989-01-01

The gamma-ray induced unscheduled DNA synthesis in root meristem cells of Vica faba was studied autoradiographically by calculating the number of cells with different 3H-thymidine labelling degree. It was found that the level of unscheduled synthesis in cells with intermediate dose (500 R) irradiation was higher than that in cells with lower dose (250 R) irradiation; however, higher dose (1000 R) irradiation would inhibit the reparative replication

Unscheduled DNA synthesis in xeroderma pigmentosum cells after microinjection of yeast photoreactivating enzyme.

NARCIS (Netherlands)

J.C.M. Zwetsloot; J.H.J. Hoeijmakers (Jan); W. Vermeulen (Wim); A.P.M. Eker (André); D. Bootsma (Dirk)

1986-01-01

textabstractPhotoreactivating enzyme (PRE) from yeast causes a light-dependent reduction of UV-induced unscheduled DNA synthesis (UDS) when injected into the cytoplasm of repair-proficieint human fibroblasts (Zwetsloot et al., 1985). This result indicates that the exogenous PRE monomerizers

DNA Photosensitization by an "Insider": Photophysics and Triplet Energy Transfer of 5-Methyl-2-pyrimidone Deoxyribonucleoside.

Science.gov (United States)

Bignon, Emmanuelle; Gattuso, Hugo; Morell, Christophe; Dumont, Elise; Monari, Antonio

2015-08-03

The main chromophore of (6-4) photoproducts, namely, 5-methyl-2-pyrimidone (Pyo), is an artificial noncanonical nucleobase. This chromophore has recently been reported as a potential photosensitizer that induces triplet damage in thymine DNA. In this study, we investigate the spectroscopic properties of the Pyo unit embedded in DNA by means of explicit solvent molecular-dynamics simulations coupled to time-dependent DFT and quantum-mechanics/molecular-mechanics techniques. Triplet-state transfer from the Pyo to the thymine unit was monitored in B-DNA by probing the propensity of this photoactive pyrimidine analogue to induce a Dexter-type triplet photosensitization and subsequent DNA damage. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The extension of a DNA double helix by an additional Watson-Crick base pair on the same backbone.

Science.gov (United States)

Kumar, Pawan; Sharma, Pawan K; Madsen, Charlotte S; Petersen, Michael; Nielsen, Poul

2013-06-17

Additional base pair: The DNA duplex can be extended with an additional Watson-Crick base pair on the same backbone by the use of double-headed nucleotides. These also work as compressed dinucleotides and form two base pairs with cognate nucleobases on the opposite strand. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increased levels of unscheduled DNA synthesis in UV-irradiated human fibroblasts pretreated with sodium butyrate

International Nuclear Information System (INIS)

Williams, J.I.; Friedberg, E.C.

1982-01-01

Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 mM results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m 2 . The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine. (author)

Recovery of subchromosomal DNA synthesis in synchronous V-79 Chinese hamster cells after ultraviolet light exposure

International Nuclear Information System (INIS)

Meechan, P.J.; Carpenter, J.G.

1986-01-01

Previous work obtained from Chinese hamster V-79 cells indicated that, immediately following exposure, UV-induced lesions acted as blocks to elongation of nascent strands, but gradually lost that ability over a 10 h period after exposure to 10 J/m 2 . The work reported herein attempted to examine possible cell cycle mediated alterations in the recovery of DNA synthesis. Kinetic incorporation of radiolabeled thymidine studies indicated that there may have been a more rapid recover of DNA synthesis in cells irradiated in G 1 or G 2 vs cells irradiated in S phase. DNA fiber autoradiograms prepared from synchronous cells indicated that after irradiation in any phase of the cell cycle, the length of newly synthesized DNA was equal to control lengths 1 h after exposure to 5.0Jm 2 (or 1 h after entering S phase for cells irradiated in G 1 or G 2 ). This observed recovery was not solely due to an excision process. No cell cycle mediated difference in the number of dimers induced or removed as a function of cell cycle position was observed. These results appear to be consistent with a continuum of effects, with initiation effects dominating the response at low fluences, gapped synthesis at intermediate fluences and elongation inhibition at high fluences. The fluences at which each event dominates may be cell-line specific. (author)

Labelling of Cells Engaged in DNA Synthesis: Autoradiography and BrdU Staining

DEFF Research Database (Denmark)

Madsen, Peder Søndergaard

2010-01-01

The cell cycle is divided in four phases: G1 phase, S phase (DNA-synthesis), G2 phase (together termed interphase) and M phase (mitosis). Cells that have ceased proliferation enter a state of quiescence called G0. M phase is itself composed of two tightly coupled processes: mitosis, in which...

Inhibition of hydrogenase synthesis by DNA gyrase inhibitors in Bradyrhizobium japonicum

International Nuclear Information System (INIS)

Novak, P.D.; Maier, R.J.

1987-01-01

Derepression of an uptake hydrogenase in Bradyrhizobium japonicum is dependent on a microaerophilic environment. Addition of DNA gyrase inhibitors during derepression of hydrogenase specifically prevented expression of the hydrogenase enzyme. Antibodies to individual hydrogenase subunits failed to detect the protein after derepression in the presence of inhibitors, although there was no general inhibition of protein synthesis. The general pattern of proteins synthesized from 14 C-labeled amino acids during derepression was no significantly different whether proteins were labeled in the presence or in the absence of gyrase inhibitors. In contrast, if transcription or translation was inhibited by addition of inhibitors of those functions, virtually no proteins were labeled during derepression. This indicated that most of the 14 C-labeled proteins were synthesized de novo during derepression, synthesis of most proteins was unaffected by gyrase inhibitors, and the dependence of hydrogenase synthesis on gyrase activity was a specific one

Inhibition by 2-deoxy-D-ribose of DNA synthesis and growth in Raji cells

International Nuclear Information System (INIS)

Ulrich, F.

1988-01-01

When Raji cells were cultured for 3 days in serum-free medium, addition of 2-deoxy-D-ribose at the start of culture inhibited incorporation of [ 3 H]thymidine and cell division. At deoxyribose concentrations between 1 and 5 mM, viability was 80% or greater after 3 days of culture even though 5 mM deoxyribose inhibited thymidine incorporation 95-99%. Inhibition by deoxyribose could be completely reversed if the culture medium was replaced with fresh medium up to 8 hr after the start of culture. The inhibition was specific for deoxyribose since other monosaccharides had no effect. Inhibition of DNA synthesis did not appear to be due to depletion of essential nutrients in the medium since the percentage inhibition of thymidine incorporation by cells cultured either in suboptimal serum-free media or in media supplemented with 0.025-5% human AB serum was similar. When DNA repair synthesis was measured as hydroxyurea-resistant thymidine incorporation, addition of deoxyribose to Raji cultures caused increased thymidine incorporation. These results, together with data from others,suggest that deoxyribose damages DNA

Hydrophilic interaction ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry for determination of nucleotides, nucleosides and nucleobases in Ziziphus plants.

Science.gov (United States)

Guo, Sheng; Duan, Jin-ao; Qian, Dawei; Wang, Hanqing; Tang, Yuping; Qian, Yefei; Wu, Dawei; Su, Shulan; Shang, Erxin

2013-08-02

In this study, a rapid and sensitive analytical method was developed for the determination of 20 nucleobases, nucleosides and nucleotides in Ziziphus plants at trace levels by using hydrophilic interaction ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HILIC-UHPLC-TQ-MS/MS) in multiple-reaction monitoring (MRM) mode. Under the optimized chromatographic conditions, good separation for 20 target compounds were obtained on a UHPLC Amide column with sub-2μm particles within 10min. The overall LODs and LOQs were between 0.11-3.12ngmL(-1) and 0.29-12.48ngmL(-1) for the 20 analytes, respectively. It is the first report about simultaneous analysis of nucleobases, nucleosides and nucleotides in medicinal plants using HILIC-UHPLC-TQ-MS/MS method, which affords good linearity, precision, repeatability and accuracy. The developed method was successfully applied to Ziziphus plant (Z. jujuba, Z. jujuba var. spinosa and Z. mauritiana) samples. The analysis showed that the fruits and leaves of Ziziphus plants are rich in nucleosides and nucleobases as well as nucleotides, and could be selected as the healthy food resources. Our results in present study suggest that HILIC-UHPLC-TQ-MS/MS method could be employed as a useful tool for quality assessment of the samples from the Ziziphus plants as well as other medicinal plants or food samples using nucleotides, nucleosides and nucleobases as markers. Copyright © 2013 Elsevier B.V. All rights reserved.

Design, Synthesis, and Evaluation of Novel Tyrosine-Based DNA Gyrase B Inhibitors.

Science.gov (United States)

Cotman, Andrej E; Trampuž, Marko; Brvar, Matjaž; Kikelj, Danijel; Ilaš, Janez; Peterlin-Mašič, Lucija; Montalvão, Sofia; Tammela, Päivi; Frlan, Rok

2017-08-01

The discovery and synthesis of new tyrosine-based inhibitors of DNA gyrase B (GyrB), which target its ATPase subunit, is reported. Twenty-four compounds were synthesized and evaluated for activity against DNA gyrase and DNA topoisomerase IV. The antibacterial properties of selected GyrB inhibitors were demonstrated by their activity against Staphylococcus aureus and Enterococcus faecalis in the low micromolar range. The most promising compounds, 8a and 13e, inhibited Escherichia coli and S. aureus GyrB with IC 50 values of 40 and 30 µM. The same compound also inhibited the growth of S. aureus and E. faecalis with minimal inhibitory concentrations (MIC 90 ) of 14 and 28 µg/mL, respectively. © 2017 Deutsche Pharmazeutische Gesellschaft.

Extracellular calcium alters the effects of retinoic acid on DNA synthesis in cultured murine keratinocytes

International Nuclear Information System (INIS)

Tong, P.; Mayes, D.; Wheeler, L.

1986-01-01

The rate of proliferation of epidermal keratinocytes was manipulated by growing the cells in medium containing high or low concentrations of calcium. Keratinocytes cultured in high extracellular Ca ++ (1.4 mM and 2.8 mM) proliferated twice as fast as those grown in low Ca ++ medium (0.09 mM) as measured by incorporation of [ 3 H] thymidine into DNA. Exposure of high calcium keratinocytes to all-trans retinoic acid for 4 days caused a dose-related inhibition of DNA synthesis with an IC 50 of about 10 μM. In contrast, incubating low calcium keratinocytes with all-trans retinoic acid caused a dose-related stimulation of DNA synthesis with maximum increase of 278% over control at 10 μM. This increase was accompanied by increases in culture confluency with maximum increase of 109% in cell number of control at 10 μM. These results are of importance since they suggest Ca ++ may influence the effect of retinoids on keratinocytes

Assessment of DNA synthesis in Islet-1+ cells in the adult murine heart

International Nuclear Information System (INIS)

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

2015-01-01

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 + ) 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 + 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 ( 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 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 + cells. Whereas Islet − 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 + 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

Gamma-ray induced inhibition of DNA synthesis in ataxia telangiectasia fibroblasts is a function of excision repair capacity

International Nuclear Information System (INIS)

Smith, P.J.; Paterson, M.C.

1980-01-01

The extent of the deficiency in γ-ray induced DNA repair synthesis in an ataxia telangiectasia (AT) human fibroblast strain was found to show no oxygen enhancement, consistent with a defect in the repair of base damage. Repair deficiency, but not repair proficiency, in AT cells was accompanied by a lack of inhibition of DNA synthesis by either γ-rays or the radiomimetic drug bleomycin. Experiments with 4-nitroquinoline 1-oxide indicated that lack of inhibition was specific for radiogenic-type damage. Thus excision repair, perhaps by DNA strand incision or chromatin modification, appears to halt replicon initiation in irradiated repair proficient cells whereas in repair defective AT strains this putatively important biological function is inoperative

Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase.

Science.gov (United States)

Liu, Xinran; Musser, Derek M; Lee, Cheri A; Yang, Xiaorong; Arnold, Jamie J; Cameron, Craig E; Boehr, David D

2015-10-26

The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I) in the RNA-dependent RNA polymerase (RdRp). We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E) help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.

Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

International Nuclear Information System (INIS)

Sugaya, Shigeru; Nakanishi, Hiroshi; Tanzawa, Hideki; Sugita, Katsuo; Kita, Kazuko; Suzuki, Nobuo

2005-01-01

Fibroblast cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. Genes, whose expression is modulated in association with the DNA synthesis induction, were searched by using PCR-based mRNA differential display analysis in one of the NBCCS cell lines, NBCCS1 cells. Decreased levels of SMT3A gene expression were found in X-ray-irradiated NBCCS1 cells. This decrease was also shown by RT-PCR analysis in another cell line, NBCCS3 cells. In addition to NBCCS cells, normal fibroblast cells showed the DNA synthesis induction after X-ray irradiation when they were treated with antisense oligonucleotides (AO) for SMT3A. However, treatment of normal fibroblasts with the random oligonucleotides (RO) resulted in decreased levels of DNA synthesis after X-ray irradiation. Thus, down-regulation of SMT3A gene expression may be involved in the DNA synthesis induction after X-ray irradiation in the NBCCS cells at least tested

Down-regulation of SMT3A gene expression in association with DNA synthesis induction after X-ray irradiation in nevoid basal cell carcinoma syndrome (NBCCS) cells

Energy Technology Data Exchange (ETDEWEB)

Sugaya, Shigeru [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Nakanishi, Hiroshi [Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Tanzawa, Hideki [Department of Clinical Molecular Biology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Sugita, Katsuo [Department of Clinical Medicine, Faculty of Education, Chiba University, 1-33 Yayoi, Inage-ku, Chiba 263-8522 (Japan); Kita, Kazuko [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan); Suzuki, Nobuo [Department of Environmental Biochemistry, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670 (Japan)]. E-mail: nobuo@faculty.chiba-u.jp

2005-10-15

Fibroblast cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis after X-ray irradiation. Genes, whose expression is modulated in association with the DNA synthesis induction, were searched by using PCR-based mRNA differential display analysis in one of the NBCCS cell lines, NBCCS1 cells. Decreased levels of SMT3A gene expression were found in X-ray-irradiated NBCCS1 cells. This decrease was also shown by RT-PCR analysis in another cell line, NBCCS3 cells. In addition to NBCCS cells, normal fibroblast cells showed the DNA synthesis induction after X-ray irradiation when they were treated with antisense oligonucleotides (AO) for SMT3A. However, treatment of normal fibroblasts with the random oligonucleotides (RO) resulted in decreased levels of DNA synthesis after X-ray irradiation. Thus, down-regulation of SMT3A gene expression may be involved in the DNA synthesis induction after X-ray irradiation in the NBCCS cells at least tested.

Interactions of nucleobases with alkali earth metal cations--electrospray ionization mass spectrometric study.

Science.gov (United States)

Frańska, Magdalena

2007-01-01

Interactions of nucleobases with alkali earth metal cations have been studied by electrospray ionization mass spectrometry (ESI-MS). Nucleobases containing at least one oxygen atom form stable complexes with alkali earth metal cations. This phenomenon can be explained on the grounds of the well known theory of hard and soft acids and bases. Uracil and thymine make complexes only when in their deprotonoted forms. The cations of great radii (Sr(2+), Ba(2+)) are more prone to form complexes of stoichiometry 1:1 with uracil and thymine than the cations of small radii (Mg(2+), Ca(2+)). On the other hand, Mg(2+) forms complexes of stoichiometry 2:1 and 3:2 with uracil and thymine. Gas-phase stabilities of the 1:1 complexes are higher for the cations of small radii, in contrast to the solution stabilities. For cytosine and 9- methylhypoxantine the 1:1 complexes of their deprotonated forms are observed at higher cone voltage as a result of HCl molecule loss from the complexes containing the counter ion (Cl(-)). In solution, more stable complexes are formed with metal cations of low radii. Gas-phase stability of the complexes formed by deprotonated 9- methyl-hypoxantine increases with increasing metal cation radius.

The effect of purine phosphonomethoxyalkyl derivatives on DNA synthesis in Cho Chinese hamster cells

Energy Technology Data Exchange (ETDEWEB)

Stetina, R [Institute of Experimental Medicine, Laboratory of Developmental Toxicology, Academy of Sciences of Czech Republic, 51783 Olesnice v Orlickych horach (Czech Republic); Votruba, I; Holy, A; Merta, A [Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech Republic (Czech Republic)

1994-12-31

The inhibition of incorporation of {sup 3}H-thymidine and the changes of the rate of nascent DNA chain elongation were investigated in Cho Chinese hamster cells treated with (S)-(3-hydroxy-2-phosphonomethoxypropyl) (HPMP) and N-(2-phosphonomethoxyethyl) (PME) derivatives of adenine (A), guanine (G) and 2,6-diaminopurine (DAP). No direct correlation was observed in PME and HPMP derivatives between cytotoxicity, inhibition of {sup 3}H-thymidine incorporation and inhibition of nascent DNA chain elongation. The highest cytotoxicity and inhibition of DNA synthesis were caused by PMEG. The limited extent of inhibition of DNA elongation was encountered in the case of HPMPG and HPMPA. With PMEA, weak inhibition of elongation of DNA was observed only after a prolonged exposure (6 h). None of the investigated drugs induced DNA breaks. (author) 4 figs., 23 refs.

Effect of inhibition of DNA synthesis on recovery of X-irradiated L5178Y-S cells. I

International Nuclear Information System (INIS)

Kapiszewska, M.; Lange, C.S.

1989-01-01

Irradiated L5178Y-S cells (LY-S) were characterized by an exponential survival curve and the potentiation effect of split -dose irradiation. Previously it was found that in LY-S cells the reduction of DNA replicative synthesis rate affected the balance between the fixation and repair of sublethal damage (SLD) and of potentially lethal damage (PLD) in favor of repair. It was found now that a block of DNA synthesis by aphidicolin (APC), an inhibitor of DNA polymerase alpha, was sufficient to protect LY-S cells from fixation of PLD and SLD induced by X-rays. Treatment with APC 0.5 μg/ml for 2 h, efficiently inhibited DNA replication (95%) with minimal effect on survival. Inhibition of DNA synthesis by combined irradiation and APC was not significantly different from APC treatment alone. The level of protection by APC was dependent on the length of time between irradiation and APC application. An opposite effect was observed when the drug treatment had preceded irradiation: The killing effect of X-ray increased. The effect of aphidicolin treatment remained even after removal of APC and was dependent on the drug concentration and time between drug removal and irradiaton. These results are interpreted as indicating that X-ray damage was fixed in LY-S cells, because of their lack of ability to maintain the nucleotide pool balance, and that fixation took place during progression through the cell cycle. (author). 6 figs., 22 refs

Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

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Chenoufi, N.; Loreal, O.; Cariou, S.; Hubert, N.; Lescoat, G. [Univ. Hospital Pontchaillou, Unite de Recherches Hepatologiques, INSERM U 49, Rennes (France); Drenou, B. [Univ. Hospital Pontchaillou, Lab. d`Hematologie et d`Immunologie, Rennes (France); Leroyer, P.; Brissot, P. [Univ. Hospital Pontchaillou, Clinique des Maladies du Foie, Rennes (France)

1997-03-01

Background/Aims: Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. Methods: The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. Results: In EGF/pyruvate-stimulated cultures, the level of [{sup 3}H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [{sup 3}H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However,the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [{sup 3}H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. Conclusions: Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma. (au) 61 refs.

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

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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.

Automation of cDNA Synthesis and Labelling Improves Reproducibility

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Daniel Klevebring

2009-01-01

Full Text Available Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

Mechanisms by which herpes simplex virus DNA polymerase limits translesion synthesis through abasic sites.

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Zhu, Yali; Song, Liping; Stroud, Jason; Parris, Deborah S

2008-01-01

Results suggest a high probability that abasic (AP) sites occur at least once per herpes simplex virus type 1 (HSV-1) genome. The parameters that control the ability of HSV-1 DNA polymerase (pol) to engage in AP translesion synthesis (TLS) were examined because AP lesions could influence the completion and fidelity of viral DNA synthesis. Pre-steady-state kinetic experiments demonstrated that wildtype (WT) and exonuclease-deficient (exo-) pol could incorporate opposite an AP lesion, but full TLS required absence of exo function. Virtually all of the WT pol was bound at the exo site to AP-containing primer-templates (P/Ts) at equilibrium, and the pre-steady-state rate of excision by WT pol was higher on AP-containing than on matched DNA. However, several factors influencing polymerization work synergistically with exo activity to prevent HSV-1 pol from engaging in TLS. Although the pre-steady-state catalytic rate constant for insertion of dATP opposite a T or AP site was similar, ground-state-binding affinity of dATP for insertion opposite an AP site was reduced 3-9-fold. Single-turnover running-start experiments demonstrated a reduced proportion of P/Ts extended to the AP site compared to the preceding site during processive synthesis by WT or exo- pol. Only the exo- pol engaged in TLS, though inefficiently and without burst kinetics, suggesting a much slower rate-limiting step for extension beyond the AP site.

Protein, RNA, and DNA synthesis in cultures of skin fibroblasts from healthy subjects and patients with rheumatic diseases

International Nuclear Information System (INIS)

Abakumova, O.Y.; Kutsenko, N.G.; Panasyuk, A.F.

1985-01-01

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 14 C-protein hydrolysate, ( 14 C)uridine, and ( 14 C) thymidine. Stimulation was determined by measuring incorporation of ( 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

Neurotoxicity of cytarabine (Ara-C) in dorsal root ganglion neurons originates from impediment of mtDNA synthesis and compromise of mitochondrial function.

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Zhuo, Ming; Gorgun, Murat F; Englander, Ella W

2018-06-01

Peripheral Nervous System (PNS) neurotoxicity caused by cancer drugs hinders attainment of chemotherapy goals. Due to leakiness of the blood nerve barrier, circulating chemotherapeutic drugs reach PNS neurons and adversely affect their function. Chemotherapeutic drugs are designed to target dividing cancer cells and mechanisms underlying their toxicity in postmitotic neurons remain to be fully clarified. The objective of this work was to elucidate progression of events triggered by antimitotic drugs in postmitotic neurons. For proof of mechanism study, we chose cytarabine (ara-C), an antimetabolite used in treatment of hematological cancers. Ara-C is a cytosine analog that terminates DNA synthesis. To investigate how ara-C affects postmitotic neurons, which replicate mitochondrial but not genomic DNA, we adapted a model of Dorsal Root Ganglion (DRG) neurons. We showed that DNA polymerase γ, which is responsible for mtDNA synthesis, is inhibited by ara-C and that sublethal ara-C exposure of DRG neurons leads to reduction in mtDNA content, ROS generation, oxidative mtDNA damage formation, compromised mitochondrial respiration and diminution of NADPH and GSH stores, as well as, activation of the DNA damage response. Hence, it is plausible that in ara-C exposed DRG neurons, ROS amplified by the high mitochondrial content shifts from physiologic to pathologic levels signaling stress to the nucleus. Combined, the findings suggest that ara-C neurotoxicity in DRG neurons originates in mitochondria and that continuous mtDNA synthesis and reliance on oxidative phosphorylation for energy needs sensitize the highly metabolic neurons to injury by mtDNA synthesis terminating cancer drugs. Copyright © 2018 Elsevier Inc. All rights reserved.

Inhibition of DNA synthesis and radiosensitization effects of thalidomide on esophageal carcinoma TE1 cells

International Nuclear Information System (INIS)

Yu Jingping; Sun Suping; Sun Zhiqiang; Sun Meiling; Liu Fenju

2010-01-01

Objective: To explore the radiosensitization effect of thalidomide combined with X-ray on esophageal carcinoma TE1 cells. Methods: Cell scratch assay was used to detect the inhibition ability of different concentration of Thalidomide on cell invasion and metastasis. H 3 -TdR incorporation assay was used to investigate the inhibition of DNA synthesis in TE1 cells by treated with Thalidomide singly or combination with X-rays. The colony formation assay was used to analyze the radiosensitization of Thalidomide effect on TE1 cells. Results: Thalidomide had obvious inhibition effect on TE1 cell metastasis, DNA synthesis and colony formation, which were correlated with drug concentration. The values D 0 , D q and SF 2 in TE1 cells were gradually decreased with thalidomide concentration increased. When the concentration of thalidomide was 100μg/ml, the SER D 0 and SER D 0 and SER D q were (1.4±0.2) and (1.5±0.1), respectively, While the concentration of thalidomide was 150 μg/ml, the SER D 0 and SER D q were (1.5±0.2) and (1.8±0.2), respectively. Conclusions: Thalidomide could inhibit TE1 cell invasion, metastasis, DNA synthesis, and significantly enhance the radiosensitizing effect on esophageal carcinoma TE1 cells. (authors)

Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

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Konze-Thomas, B.; Hazard, R.M.; Maher, V.M.; McCormick, J.J. (Michigan State Univ., East Lansing (USA). Carcinogenesis Lab.)

1982-01-01

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G/sub 0/ state. Autoradiography studies of cells released from G/sub 0/ after 72 h and replated at lower densities (3-9 x 10/sup 3/ cells/cm/sup 2/) in fresh medium showed that semiconservative DNA synthesis (S phase) began approx. equal to 24 h after the replating. The task was to determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts.

Changes in the synthesis of DNA, RNA and protein during somatic embryogenesis in wheat (triticum aestivum L.)

International Nuclear Information System (INIS)

Cui Kairong; Wang Xiaozhe; Chen Xiong; Wang Yafu

1997-01-01

Embryogenic and non-embryogenic callus formed from immature embryo of wheat (Triticum aestivum L.) in N 6 B 5 MS medium I supplemented with 2,4-D 2 mg/L, KT 0.5 mg/L, LH300 mg/L, sucrose 3% were sub-cultured and transferred respectively to N 6 B 5 MS medium II (2,4-D was decreased to 0.5 mg/L and 4 mol/L proline was added). Somatic embryos obtained from embryogenic callus, and plantlet formed from non-embryogenic callus through organogenesis respectively. By incorporation of 3 H-thymidine, 3 H-uridine and 3 H-leucine into DNA, RNA and protein respectively, the rate of synthesis of DNA, RNA and protein during somatic embryogenesis were measured. A large amount of RNA and protein synthesized during the early somatic embryogenesis. The activities of RNA and protein synthesis reached the peak on the 4th and the 8th day respectively, then decreased a little, but kept a high level. The synthesis of DNA increased apparently during the early stage. No apparent change occurred when the embryogenic cell masses formed. The synthesis rate of RNA and protein in non-embryogenic callus were much less than that in embryogenic callus. Actinomycin and cycloheximide inhibited not only the synthesis of nucleic acid and protein, but also the growth of embryogenic callus and somatic embryogenesis. The earlier the inhibitors were added, the greater the influence was caused. The results indicate that the active expression of corresponding genes of wheat is the molecular base of somatic embryogenesis

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

Science.gov (United States)

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)

Theoretical studies on the electronic and optoelectronic properties of [A.2AP(w)/A*.2AP(WC)/C.2AP(w)/C*.2AP(WC)/C.A(w)/C*.A(WC)]-Au8 mismatch nucleobase complexes

Science.gov (United States)

Srivastava, Ruby

2018-01-01

The electronic and optoelectronic properties of [A.2AP(w)/A*.2AP(WC)/C.2AP(w)/C*.2AP(WC)/C.A(w)/ C*.A(WC)]-Au8 metal-mismatch nucleobase complexes are investigated by means of density functional theory and time-dependent methods. We selected these mispairs as 2-aminopurine (2AP) produces incorporation errors when binding with cytosine (C) into the wobble (w) C.2AP(w) mispair, and is tautomerised into Watson-Crick (WC)-like base mispair C*.2AP(WC) and less effectively produces A.2AP(w)/A*.2AP(WC) mispairs. The vertical ionisation potential, vertical electron affinity, hardness and electrophilicity index of these complexes have also been discussed. The modifications of energy levels and charge density distributions of the frontier orbitals are also analysed. The absorption spectra of these complexes lie in the visible region, which suggests their application in fluorescent-bio imaging. The mechanism of cooperativity effect is studied by molecular orbital potential (MEP), atoms-in-molecules (AIM) and natural bond orbital analyses. Most metalated pairs have smaller HOMO-LUMO band gaps than the isolated mismatch nucleobases which suggest interesting consequences for electron transfer through DNA duplexes.

UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein

International Nuclear Information System (INIS)

Carty, M.P.; Zernik-Kobak, M.; McGrath, S.; Dixon, K.

1994-01-01

We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus. (Author)

Automated quantum chemistry based molecular dynamics simulations of electron ionization induced fragmentations of the nucleobases Uracil, Thymine, Cytosine, and Guanine.

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Grimme, Stefan; Bauer, Christopher Alexander

2015-01-01

The gas-phase decomposition pathways of electron ionization (EI)-induced radical cations of the nucleobases uracil, thymine, cytosine, and guanine are investigated by means of mixed quantum-classical molecular dynamics. No preconceived fragmentation channels are used in the calculations. The results compare well to a plethora of experimental and theoretical data for these important biomolecules. With our combined stochastic and dynamic approach, one can access in an unbiased way the energetically available decomposition mechanisms. Additionally, we are able to separate the EI mass spectra of different tautomers of cytosine and guanine. Our method (previously termed quantum chemistry electron ionization mass spectra) reproduces free nucleobase experimental mass spectra well and provides detailed mechanistic in-sight into high-energy unimolecular decomposition processes.

Synthesis of acetylene linked double-nucleobase nucleos(t)ide building blocks and polymerase construction of DNA containing cytosines in the major groove

Czech Academy of Sciences Publication Activity Database

Kielkowski, Pavel; Pohl, Radek; Hocek, Michal

2011-01-01

Roč. 76, č. 9 (2011), s. 3457-3462 ISSN 0022-3263 R&D Projects: GA MŠk LC512; GA ČR GA203/09/0317 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleoside triphosphates * functionalized DNA * cross - coupling reactions * nucleic-acids Subject RIV: CC - Organic Chemistry Impact factor: 4.450, year: 2011

Hydrated Electron Transfer to Nucleobases in Aqueous Solutions Revealed by Ab Initio Molecular Dynamics Simulations.

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Zhao, Jing; Wang, Mei; Fu, Aiyun; Yang, Hongfang; Bu, Yuxiang

2015-08-03

We present an ab initio molecular dynamics (AIMD) simulation study into the transfer dynamics of an excess electron from its cavity-shaped hydrated electron state to a hydrated nucleobase (NB)-bound state. In contrast to the traditional view that electron localization at NBs (G/A/C/T), which is the first step for electron-induced DNA damage, is related only to dry or prehydrated electrons, and a fully hydrated electron no longer transfers to NBs, our AIMD simulations indicate that a fully hydrated electron can still transfer to NBs. We monitored the transfer dynamics of fully hydrated electrons towards hydrated NBs in aqueous solutions by using AIMD simulations and found that due to solution-structure fluctuation and attraction of NBs, a fully hydrated electron can transfer to a NB gradually over time. Concurrently, the hydrated electron cavity gradually reorganizes, distorts, and even breaks. The transfer could be completed in about 120-200 fs in four aqueous NB solutions, depending on the electron-binding ability of hydrated NBs and the structural fluctuation of the solution. The transferring electron resides in the π*-type lowest unoccupied molecular orbital of the NB, which leads to a hydrated NB anion. Clearly, the observed transfer of hydrated electrons can be attributed to the strong electron-binding ability of hydrated NBs over the hydrated electron cavity, which is the driving force, and the transfer dynamics is structure-fluctuation controlled. This work provides new insights into the evolution dynamics of hydrated electrons and provides some helpful information for understanding the DNA-damage mechanism in solution. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

Science.gov (United States)

Taggart, David J.; Camerlengo, Terry L.; Harrison, Jason K.; Sherrer, Shanen M.; Kshetry, Ajay K.; Taylor, John-Stephen; Huang, Kun; Suo, Zucai

2013-01-01

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases. PMID:23470999

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

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Weinberger, Florian, E-mail: f.weinberger@uke.de; Mehrkens, Dennis, E-mail: dennis.mehrkens@uk-koeln.de; Starbatty, Jutta, E-mail: starbatty@uke.uni-hamburg.de; Nicol, Philipp, E-mail: Philipp.Nicol@gmx.de; Eschenhagen, Thomas, E-mail: t.eschenhagen@uke.de

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.

DNA polymerase zeta cooperates with polymerases kappa and iota in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients.

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Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

2009-07-14

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase eta, the POLH gene product. A deficiency in DNA polymerase eta due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase zeta cooperates with DNA polymerases kappa and iota to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases zeta and kappa, but not iota, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.

DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Science.gov (United States)

Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

2009-01-01

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase η, the POLH gene product. A deficiency in DNA polymerase η due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase ζ cooperates with DNA polymerases κ and ι to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases ζ and κ, but not ι, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load. PMID:19564618

Nucleobase but not Sugar Fidelity is Maintained in the Sabin I RNA-Dependent RNA Polymerase

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Xinran Liu

2015-10-01

Full Text Available The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes (i.e., D53N, Y73H, K250E, and T362I in the RNA-dependent RNA polymerase (RdRp. We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes (i.e., D53N, Y73H, K250E help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.

Age-dependence of the x-ray-induced deficiency in DNA synthesis in HeLa S3 cells during generation 1

International Nuclear Information System (INIS)

Griffiths, T.D.; Tolmach, L.J.

1975-01-01

The radiation-induced deficiency in DNA synthesis in Generation 1 was studied as a function of the age of HeLa S3 cells at the time of exposure to 220 kV x rays in the previous generation (Generation 0). The amount of DNA synthesized is dependent on the stage in the generation cycle at which cells are irradiated. The smallest deficiency (20 to 35 percent after a dose of 500 rad) is observed in cells irradiated in early G1 or early G2, while the greatest deficiency (55 to 70 percent after 500 rad) is found in cells irradiated at mitosis or at the G1/S transition. The high sensitivity of cells at G1/S is also manifested by a steeper dose-response curve. Cells irradiated in late G2, past the point where their progression is temporarily blocked by x rays, synthesize a normal amount of DNA in Generation 1, while cells that are held up in the G2 block exhibit deficient synthesis in the next generation. The extent of the deficiency in early G1 cells can be enhanced by treatment with 1 mM hydroxyurea for several hours immediately following irradiation. The possibility that deficient DNA synthesis is related to cell killing, and the relation between the G2 block and deficient synthesis, are discussed

DNA-synthesis of lymphocytes in hyperthyroid and enthyroid subjects. Effect of 131I therapy on hyperthyroidism

International Nuclear Information System (INIS)

Lundell, G.; Wasserman, J.; Einhorn, N.; Granberg, P.-O.

1976-01-01

The DNA-synthesis of human lymphoid cells as estimated by the measurement of thymidine incorporation in vitro was investigated in healthy controls and in patients with various thyroid disorders before and after therapy. Hyperthyroid patients treated with 131 I and surgery (euthyroid at initial blood sampling before surgery), patients with atoxic nodular goitre treated by surgery and healthy untreated control individuals comprised the material. The synthesis of DNA in lymphocytes was higher in hyperthyroid patients in comparison with euthyroid individuals, and decreased subsequent to 131 I therapy in the hyperthyroid patients. No decrease was recorded in the other groups of patients. No evidence suggesting a change in the lymphocyte reactivity to thyroglobulin was found in any of the patient groups. (author)

Pharmacogenomic study using bio- and nanobioelectrochemistry: Drug-DNA interaction.

Science.gov (United States)

Hasanzadeh, Mohammad; Shadjou, Nasrin

2016-04-01

Small molecules that bind genomic DNA have proven that they can be effective anticancer, antibiotic and antiviral therapeutic agents that affect the well-being of millions of people worldwide. Drug-DNA interaction affects DNA replication and division; causes strand breaks, and mutations. Therefore, the investigation of drug-DNA interaction is needed to understand the mechanism of drug action as well as in designing DNA-targeted drugs. On the other hand, the interaction between DNA and drugs can cause chemical and conformational modifications and, thus, variation of the electrochemical properties of nucleobases. For this purpose, electrochemical methods/biosensors can be used toward detection of drug-DNA interactions. The present paper reviews the drug-DNA interactions, their types and applications of electrochemical techniques used to study interactions between DNA and drugs or small ligand molecules that are potentially of pharmaceutical interest. The results are used to determine drug binding sites and sequence preference, as well as conformational changes due to drug-DNA interactions. Also, the intention of this review is to give an overview of the present state of the drug-DNA interaction cognition. The applications of electrochemical techniques for investigation of drug-DNA interaction were reviewed and we have discussed the type of qualitative or quantitative information that can be obtained from the use of each technique. Copyright © 2015 Elsevier B.V. All rights reserved.

Electron collisions with biomolecules

International Nuclear Information System (INIS)

McKoy, V; Winstead, C

2008-01-01

We report on results of recent studies of collisions of low-energy electrons with nucleobases and other DNA constituents. A particular focus of these studies has been the identification and characterization of resonances that play a role in electron attachment leading to strand breaks in DNA. Comparison of the calculated resonance positions with results of electron transmission measurements is quite encouraging. However, the higher-lying π* resonances of the nucleobases appear to be of mixed elastic and core-excited character. Such resonant channel coupling raises the interesting possibility that the higher π*resonances in the nucleobases may promote dissociation of DNA by providing doorway states to triplet excited states.

Identification of proteins whose synthesis in Saccharomyces cerevisiae is induced by DNA damage and heat shock

International Nuclear Information System (INIS)

Gailit, James

1990-01-01

Protein synthesis in Saccharomyces cerevisiae after exposure to ultraviolet light (UV) was examined by two-dimensional gel electrophoresis of pulse-labelled proteins. The synthesis of 12 distinct proteins was induced by treatment with UV doses of 10-200 J/m 2 . The induced proteins differed in minimum dose necessary for induction, maximum dose at which induction still occurred and constitutive level present in unirradiated cells. A chemical mutagen, 4-nitroquinoline-1-oxide, induced synthesis of the same proteins. Induction after UV treatment was observed in seven different yeast strains, including three mutants deficient in DNA repair. Synthesis of five of the proteins was also induced by brief heat shock treatment. These five may be members of a family of proteins whose synthesis is regulated by two different pathways responding to different types of stress. (author)

Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

Directory of Open Access Journals (Sweden)

Stougaard Magnus

2007-11-01

Full Text Available Abstract Background In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature. Methods Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling. Results An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1 on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory. Conclusion Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as

Eukaryotic DNA Replication Fork.

Science.gov (United States)

Burgers, Peter M J; Kunkel, Thomas A

2017-06-20

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA

International Nuclear Information System (INIS)

Kastan, M.B.; Gowans, B.J.; Lieberman, M.W.

1982-01-01

Methylation of deoxycytidine incorporated by DNA excision-repair was studied in human diploid fibroblasts following damage with ultraviolet radiation, N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene. In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication in logarithmic-phase cultures a steady state level of 3.4% 5-methylcytosine is reached in less than 2 hr after cells are labeled with 6- 3H-deoxycytidine, following ultraviolet-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approximately 2.0% 5-methylcytosine in the repair patch. In cells from cultures in logarithmic-phase growth, 5-methylcytosine formation in ultraviolet-induced repair patches occurs faster and to a greater extent, reaching a level of approximately 2.7% in 10-20 hr. Preexisting hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites

DNA synthesis and cell survival after X-irradiation of mammalian cells treated with caffeine or adenine

International Nuclear Information System (INIS)

Griffiths, T.D.; Carpenter, J.G.; Dahle, D.B.

1978-01-01

The expression of the transient depression in the rate of DNA synthesis normally observed after exposure of randomly-dividing Chinese hamster V-79 or Chinese hamster CHO cells to ionizing radiation could be postponed by a post-irradiation treatment with 1.0 to 2.0 mM adenine or 1.5 mM caffeine. Caffeine may exert its effect by creating additional sites for replication in irradiated cells. Cells treated with caffeine or adenine for 2 or 4 hours after exposure to 3000 rad of 300 kVp X-rays exhibited depressed synthesis only after the removal of caffeine or adenine. These alterations in the timing of the X-ray-induced depression of the rate of DNA synthesis had no effect on X-ray-induced cell killing. Although a 4 hour post-irradiation treatment of randomly-dividing Chinese hamster V-79 cells with 1.0 or 2.0 mM caffeine potentiated X-ray-induced cell killing, this reduction in survival was due primarily to effects on cells not in S-phase. (author)

Threading DNA through nanopores for biosensing applications

International Nuclear Information System (INIS)

Fyta, Maria

2015-01-01

This review outlines the recent achievements in the field of nanopore research. Nanopores are typically used in single-molecule experiments and are believed to have a high potential to realize an ultra-fast and very cheap genome sequencer. Here, the various types of nanopore materials, ranging from biological to 2D nanopores are discussed together with their advantages and disadvantages. These nanopores can utilize different protocols to read out the DNA nucleobases. Although, the first nanopore devices have reached the market, many still have issues which do not allow a full realization of a nanopore sequencer able to sequence the human genome in about a day. Ways to control the DNA, its dynamics and speed as the biomolecule translocates the nanopore in order to increase the signal-to-noise ratio in the reading-out process are examined in this review. Finally, the advantages, as well as the drawbacks in distinguishing the DNA nucleotides, i.e., the genetic information, are presented in view of their importance in the field of nanopore sequencing. (topical review)

First-principles study of high-conductance DNA sequencing with carbon nanotube electrodes

KAUST Repository

Chen, X.

2012-03-26

Rapid and cost-effective DNA sequencing at the single nucleotide level might be achieved by measuring a transverse electronic current as single-stranded DNA is pulled through a nanometer-sized pore. In order to enhance the electronic coupling between the nucleotides and the electrodes and hence the current signals, we employ a pair of single-walled close-ended (6,6) carbon nanotubes (CNTs) as electrodes. We then investigate the electron transport properties of nucleotides sandwiched between such electrodes by using first-principles quantum transport theory. In particular, we consider the extreme case where the separation between the electrodes is the smallest possible that still allows the DNA translocation. The benzene-like ring at the end cap of the CNT can strongly couple with the nucleobases and therefore it can both reduce conformational fluctuations and significantly improve the conductance. As such, when the electrodes are closely spaced, the nucleobases can pass through only with their base plane parallel to the plane of CNT end caps. The optimal molecular configurations, at which the nucleotides strongly couple to the CNTs, and which yield the largest transmission, are first identified. These correspond approximately to the lowest energy configurations. Then the electronic structures and the electron transport of these optimal configurations are analyzed. The typical tunneling currents are of the order of 50 nA for voltages up to 1 V. At higher bias, where resonant transport through the molecular states is possible, the current is of the order of several μA. Below 1 V, the currents associated to the different nucleotides are consistently distinguishable, with adenine having the largest current, guanine the second largest, cytosine the third and, finally, thymine the smallest. We further calculate the transmission coefficient profiles as the nucleotides are dragged along the DNA translocation path and investigate the effects of configurational variations

Peripheral SLC6A4 DNA methylation is associated with in vivo measures of human brain serotonin synthesis and childhood physical aggression.

Directory of Open Access Journals (Sweden)

Dongsha Wang

Full Text Available The main challenge in addressing the role of DNA methylation in human behaviour is the fact that the brain is inaccessible to epigenetic analysis in living humans. Using positron emission tomography (PET measures of brain serotonin (5-HT synthesis, we found in a longitudinal sample that adult males with high childhood-limited aggression (C-LHPA had lower in vivo 5-HT synthesis in the orbitofrontal cortex (OBFC. Here we hypothesized that 5-HT alterations associated with childhood aggression were linked to differential DNA methylation of critical genes in the 5-HT pathway and these changes were also detectable in peripheral white blood cells. Using pyrosequencing, we determined the state of DNA methylation of SLC6A4 promoter in T cells and monocytes isolated from blood of cohort members (N = 25 who underwent a PET scan, and we examined whether methylation status in the blood is associated with in vivo brain 5-HT synthesis. Higher levels of methylation were observed in both T cells and monocytes at specific CpG sites in the C-LHPA group. DNA methylation of SLC6A4 in monocytes appears to be associated more reliably with group membership than T cells. In both cell types the methylation state of these CpGs was associated with lower in vivo measures of brain 5-HT synthesis in the left and right lateral OBFC (N = 20 where lower 5-HT synthesis in C-LHPA group was observed. Furthermore, in vitro methylation of the SLC6A4 promoter in a luciferase reporter construct suppresses its transcriptional activity supporting a functional role of DNA methylation in SLC6A4 promoter regulation. These findings indicate that state of SLC6A4 promoter methylation is altered in peripheral white blood cells of individuals with physical aggression during childhood. This supports the relevance of peripheral DNA methylation for brain function and suggests that peripheral SLC6A4 DNA methylation could be a marker of central 5-HT function.

Some new methyl-8-methoxypsoralens: synthesis, photobinding to DNA, photobiological properties and molecular modelling.

Science.gov (United States)

Gia, O; Anselmo, A; Pozzan, A; Antonello, C; Magno, S M; Uriarte, E

1997-01-01

The tricyclic structure of known natural photochemotherapeutic drugs such as 8-methoxypsoralen and 5-methoxypsoralen is often taken as a model in the search of new photosensitizer agents with less phototoxic and mutagenic effects. This paper describes the synthesis, characterization, photobinding to DNA, photobiological properties and computational chemistry of some 8-methoxypsoralen derivatives bearing two or three methyl groups at the key positions of the two photoactive double bonds. Results showed that photoreactivity and photobiological behaviour depend on the pattern of methyl substitutions. Antiproliferative activity in cell lines shows good correlation with DNA interaction data.

Oxidative stress and inflammation generated DNA damage by exposure to air pollution particles

DEFF Research Database (Denmark)

Møller, Peter; Danielsen, Pernille Høgh; Karottki, Dorina Gabriela

2014-01-01

at different locations (spatial variability), times (temporal variability) or particle size fraction across different experimental systems of acellular conditions, cultured cells, animals and humans. Nevertheless, there is substantial variation in the genotoxic, inflammation and oxidative stress potential......Generation of oxidatively damaged DNA by particulate matter (PM) is hypothesized to occur via production of reactive oxygen species (ROS) and inflammation. We investigated this hypothesis by comparing ROS production, inflammation and oxidatively damaged DNA in different experimental systems...... investigating air pollution particles. There is substantial evidence indicating that exposure to air pollution particles was associated with elevated levels of oxidatively damaged nucleobases in circulating blood cells and urine from humans, which is supported by observations of elevated levels of genotoxicity...

Charge transport properties of a twisted DNA molecule: A renormalization approach

Energy Technology Data Exchange (ETDEWEB)

Almeida, M.L. de; Ourique, G.S.; Fulco, U.L. [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Albuquerque, E.L., E-mail: eudenilson@gmail.com [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Moura, F.A.B.F. de; Lyra, M.L. [Instituto de Física, Universidade Federal de Alagoas, 57072-900 Maceió-AL (Brazil)

2016-10-20

In this work we study the charge transport properties of a nanodevice consisting of a finite segment of the DNA molecule sandwiched between two metallic electrodes. Our model takes into account a nearest-neighbor tight-binding Hamiltonian considering the nucleobases twist motion, whose solutions make use of a two-steps renormalization process to simplify the algebra, which can be otherwise quite involved. The resulting variations of the charge transport efficiency are analyzed by numerically computing the main features of the electron transmittance spectra as well as their I × V characteristic curves.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism.

Science.gov (United States)

Francis, Andrew J; Resendiz, Marino J E

2017-07-28

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5'-[CUA CGG AAU CAU]-3' containing zero, one, or two modifications located at the C2'-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2'-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in

Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis.

Science.gov (United States)

Pham, Phuong; Seitz, Erica M; Saveliev, Sergei; Shen, Xuan; Woodgate, Roger; Cox, Michael M; Goodman, Myron F

2002-08-20

SOS mutagenesis in Escherichia coli requires DNA polymerase V (pol V) and RecA protein to copy damaged DNA templates. Here we show that two distinct biochemical modes for RecA protein are necessary for pol V-catalyzed translesion synthesis. One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass. A separate RecA mode is necessary for translesion synthesis. The RecA1730 mutant protein, which was identified on the basis of its inability to promote pol V (UmuD'(2)C)-dependent UV-mutagenesis, appears proficient for the first mode of RecA action but is deficient in the second mode. Data are presented suggesting that the two RecA modes are "nonfilamentous". That is, contrary to current models for SOS mutagenesis, formation of a RecA nucleoprotein filament may not be required for copying damaged DNA templates. Instead, SOS mutagenesis occurs when pol V interacts with two RecA molecules, first at a 3' primer end, upstream of a template lesion, where RecA mode 1 stimulates pol V activity, and subsequently at a site immediately downstream of the lesion, where RecA mode 2 cocatalyzes lesion bypass. We posit that in vivo assembly of a RecA nucleoprotein filament may be required principally to target pol V to a site of DNA damage and to stabilize the pol V-RecA interaction at the lesion. However, it is only a RecA molecule located at the 3' filament tip, proximal to a damaged template base, that is directly responsible for translesion synthesis.

Comparative investigations about the DNA synthesis by thymus and spleen cells of rats in vitro under the influence of X-rays, UV radiation and radiomimetic substances

Energy Technology Data Exchange (ETDEWEB)

Tempel, K.; Schmerold, I.; Pfahler, W.; Goette, A.

1984-06-01

In order to further characterize the different repairing behavior of thymus and spleen cells of rats in vitro under the influence of X-rays, UV radiation and methylmethanesulfonate (MMS), the effect of bleomycine (BM), L-cysteine (CY-E), N-ethylmaleimide (NEM), I-..beta..-D-arabinofuranosylcytosine (araC), dideoxythymidine (ddT), and novobiocine (NB) on the semiconservative and restorative DNA synthesis as well as on the behavior of DNA under the alkaline elution was studied. The semiconservative DNA synthesis was inhibited by all examined agents except ddT, the restorative DNA synthesis only by NEM, araC, and NB. The stimulation of the restorative DNA synthesis was increased by UV radiation and MMS in spleen cells and by X-rays, BM and CY-E in thymus cells. Under the conditions of alkaline elution, there was a more sensitive reaction of spleen cells than of thymus cells to X-rays, BM and CY-E. The results show that thymus cells are especially qualified for the repair of short chains and spleen cells for the repair of long chains.

Dispersion Interactions between Urea and Nucleobases Contribute to the Destabilization of RNA by Urea in Aqueous Solution

Science.gov (United States)

Kasavajhala, Koushik; Bikkina, Swetha; Patil, Indrajit; MacKerell, Alexander D.; Priyakumar, U. Deva

2015-01-01

Urea has long been used to investigate protein folding and, more recently, RNA folding. Studies have proposed that urea denatures RNA by participating in stacking interactions and hydrogen bonds with nucleic acid bases. In this study, the ability of urea to form unconventional stacking interactions with RNA bases is investigated using ab initio calculations (RI-MP2 and CCSD(T) methods with the aug-cc-pVDZ basis set). A total of 29 stable nucleobase-urea stacked complexes are identified in which the intermolecular interaction energies (up to −14 kcal/mol) are dominated by dispersion effects. Natural bond orbital (NBO) and atoms in molecules (AIM) calculations further confirm strong interactions between urea and nucleobases. Calculations on model systems with multiple urea and water molecules interacting with a guanine base lead to a hypothesis that urea molecules along with water are able to form cage-like structures capable of trapping nucleic acid bases in extrahelical states by forming both hydrogen bonded and dispersion interactions, thereby contributing to the unfolding of RNA in the presence of urea in aqueous solution. PMID:25668757

α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.

Directory of Open Access Journals (Sweden)

Olivier Martínez

Full Text Available (S(C5', R(P α,β-D- Constrained Nucleic Acids (CNA are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

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

Science.gov (United States)

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

2009-10-30

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

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

Science.gov (United States)

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

2009-01-01

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

Differential responses of nascent DNA synthesis and chain elongation in V79 and V79/79 cells exposed to U.V. light and chemical mutagens

International Nuclear Information System (INIS)

Fox, M.; Bloomfield, M.E.; Hopkins, J.; Boyle, J.M.

1983-01-01

DNA repair after u.v., N-methyl-N-nitrosourea (MNU) and ethylmethane sulphonate (EMS) in Chinese hamster V79 cells and the mutagen sensitive derivative V79/79 was investigated by measurement of five parameters: production of strand breaks in template DNA, incorporation of [ 3 H]TdR, semi-conservative and repair synthesis, molecular weights of pulse labelled DNA after mutagen exposure (nascent synthesis) and molecular weights of DNA pulse labelled and chased after mutagen exposure (elongation and ligation). Equal template strand breakage was evident in both cell lines immediately after MNU and EMS exposure and by 4-5 h after MNU the extent of fragmentation was greater in V79/79 cells. After u.v. irradiation template fragmentation was evident in V79/79 but not in V79 cells, even though V79/79 cells failed to excise cyclobutane dimers and repair synthesis was demonstrable in V79 cells but not in V79/79 cells after exposure to all three mutagens. The rate of incorporation of [ 3 H]TdR during semi-conservative DNA synthesis was inhibited equally in a dose dependent manner after u.v. and MNU exposure; incorporation by V79/79 cells was inhibited to a greater extent than by V79 cells after EMS exposure. Nascent DNA synthesis was suppressed more in V79/79 cells than in V79 cells after u.v. but to similar extents in both cell lines after MNU and EMS treatment. Pulse chase experiments indicated a lower rate of elongation of nascent DNA in V79/79 cells after MNU and u.v. exposure but little difference was detectable after EMS

DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

International Nuclear Information System (INIS)

Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

1991-01-01

Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

Are dinucleoside monophosphates relevant models for the study of DNA intrastrand cross-link lesions? The example of g[8-5m]T.

Science.gov (United States)

Garrec, Julian; Dumont, Elise

2014-07-21

Oxidatively generated tandem lesions such as G[8-5m]T pose a potent threat to genome integrity. Direct experimental studies of the kinetics and thermodynamics of a specific lesion within DNA are very challenging, mostly due to the variety of products that can be formed in oxidative conditions. Dinucleoside monophosphates (DM) involving only the reactive nucleobases in water represent appealing alternative models on which most physical chemistry and structural techniques can be applied. However, it is not yet clear how relevant these models are. Here, we present QM/MM MD simulations of the cyclization step involved in the formation of G[8-5m]T from the guanine-thymine (GpT) DM in water, with the aim of comparing our results to our previous investigation of the same reaction in DNA ( Garrec , J. , Patel , C. , Rothlisberger , U. , and Dumont , E. ( 2012 ) J. Am. Chem. Soc. 134 , 2111 - 2119 ). We show that, despite the different levels of preorganization of the two systems, the corresponding reactions share many energetic and structural characteristics. The main difference lies in the angle between the G and T bases, which is slightly higher in the transition state (TS) and product of the reaction in water than in the reaction in DNA. This effect is due to the Watson-Crick H-bonds, which are absent in the {GpT+water} system and restrain the relative positioning of the reactive nucleobases in DNA. However, since the lesion is accommodated easily in the DNA macromolecule, the induced energetic penalty is relatively small. The high similarity between the two reactions strongly supports the use of GpT in water as a model of the corresponding reaction in DNA.

Aviram–Ratner rectifying mechanism for DNA base-pair sequencing through graphene nanogaps

International Nuclear Information System (INIS)

Agapito, Luis A; Gayles, Jacob; Wolowiec, Christian; Kioussis, Nicholas

2012-01-01

We demonstrate that biological molecules such as Watson–Crick DNA base pairs can behave as biological Aviram–Ratner electrical rectifiers because of the spatial separation and weak hydrogen bonding between the nucleobases. We have performed a parallel computational implementation of the ab initio non-equilibrium Green’s function (NEGF) theory to determine the electrical response of graphene—base-pair—graphene junctions. The results show an asymmetric (rectifying) current–voltage response for the cytosine–guanine base pair adsorbed on a graphene nanogap. In sharp contrast we find a symmetric response for the thymine–adenine case. We propose applying the asymmetry of the current–voltage response as a sensing criterion to the technological challenge of rapid DNA sequencing via graphene nanogaps. (paper)

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms

Directory of Open Access Journals (Sweden)

Olga A. Kladova

2018-03-01

Full Text Available Endonuclease III (Endo III or Nth is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tCO, a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5–37 °C. Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van’t Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU recognition and desolvation-accompanied entropy-driven adjustment of the enzyme–substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme–DNA complex formation.

Analysis of translesion DNA synthesis activity of the human REV1 protein, which is a key player in radiation-induced mutagenesis

International Nuclear Information System (INIS)

Masuda, Yuji; Kamiya, Kenji

2003-01-01

Ionizing radiation frequently causes oxidative DNA damage in cells. It has been suggested that functions of the REV1 gene are induction of mutations and prevention of cell death caused by ionizing radiation through the damage bypass DNA replication. The gene product possesses a deoxycytidyl transferase activity, which is required for translesion DNA synthesis of a variety of damaged bases and an abasic site. To elucidate molecular mechanisms of the mutagenesis and translesion DNA synthesis, it is important to characterize the enzymatic properties of the REV1 protein. Here, we describe a novel method for purifying the recombinant human REV1 protein and the anzymatic properties of the protein. We established an efficient system for induction of the recombinant human REV1 protein in Escherichia coli cells. The REV1 protein was purified to homogeneity using nickel-chelating sepharose, heparin sepharose and superdex 200 chromatography. When purified by this method, REV1 protein is free of endo-, exonuclease and DNA polymerase activities. The purified REV1 protein is suitable for enzymological studies, and we used this to biochemical characterization. The REV1 protein inserts dCMP opposite templates G, A, T, C and an abasic site and inserts dGMP and dTMP opposite template G. Kinetic analysis provided evidence for high efficiency for dCMP insertion opposite template G and an abasic site, suggesting that the REV1 protein play a role in translesion DNA synthesis of an abasic site. (author)

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

International Nuclear Information System (INIS)

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

1991-01-01

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 [ 3 H]proline and collagenase-digestible protein) at 100, 300, and 600 micrograms/ml. Release of [ 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 [ 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

Chemical Morphing of DNA Containing Four Noncanonical Bases.

Science.gov (United States)

Eremeeva, Elena; Abramov, Michail; Margamuljana, Lia; Rozenski, Jef; Pezo, Valerie; Marlière, Philippe; Herdewijn, Piet

2016-06-20

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular mechanisms of DNA photodamage

International Nuclear Information System (INIS)

Starrs, S.M.

2000-05-01

Photodamage in DNA, caused by ultraviolet (UV) light, can occur by direct excitation of the nucleobases or indirectly via the action of photosensitisers. Such, DNA photodamage can be potentially mutagenic or lethal. Among the methods available for detecting UV-induced DNA damage, gel sequencing protocols, utilising synthetic oligodeoxyribonucleotides as targets for UV radiation, allow photolesions to be mapped at nucleotide resolution. This approach has been applied to investigate both DNA damage mechanisms. Following a general overview of DNA photoreactivity, and a description of the main experimental procedures, Chapter 3 identifies the origin of an anomalous mobility shift observed in purine chemical sequence ladders that can confuse the interpretation of DNA cleavage results; measures to abolish this shift are also described. Chapters 4 and 5 examine the alkali-labile DNA damage photosensitised by representative nonsteroidal antiinflammatory drugs (NSAIDs) and the fluoroquinolone antibiotics. Suprofen was the most photoactive NSAID studied, producing different patterns of guanine-specific damage in single-stranded and duplex DNA. Uniform modification of guanine bases, typifying attack by singlet oxygen, was observed in single-stranded oligodeoxyribonucleotides. In duplex molecules, modification was limited to the 5'-G of GG doublets, which is indicative of an electron transfer. The effect of quenchers and photoproduct analysis substantiated these findings. The quinolone, nalidixic acid, behaves similarly. The random base cleavage photosensitised by the fluoroquinolones, has been attributed to free radicals produced during their photodecomposition. Chapter 6 addresses the photoreactivity of purines within unusual DNA structures formed by the repeat sequences (GGA) n and (GA) n , and a minihairpin. There was no definitive evidence for enhanced purine reactivity caused by direct excitation. Finally, Chapter 7 investigates the mutagenic potential of a dimeric

Different effects of inorganic and dimethylated arsenic compounds on cell morphology, cytoskeletal organization, and DNA synthesis in cultured Chinese hamster V79 cells

Energy Technology Data Exchange (ETDEWEB)

Ochi, Takafumi; Nakajima, Fumie [Department of Environmental Toxicology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa (Japan); Fukumori, Nobutaka [Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Hyakuninchou, Shinjyuku (Japan)

1998-09-01

Changes in cytoskeletal organization of cultured V79 cells exposed to arsenite and dimethylarsinic acid (DMAA), a methylated derivative of inorganic arsenics, and related changes, such as mitotic arrest and induction of multinucleated cells, were investigated in comparison with their effects on DNA synthesis. DMAA caused mitotic arrest and induction of multinucleated cells with a delay of 12 h relative to the mitotic arrest. By contrast, arsenite at equitoxic concentrations to DMAA was less effective than DMAA in causing mitotic arrest and in inducing multinucleated cells. Post-mitotic incubation of cells arrested in metaphase by 6 h incubation with 10 mM DMAA showed that the incidence of multinucleated cells increased conversely with a rapid decrease in metaphase cells. This suggests that metaphase-arrested cells can escape from metaphase, resulting in the appearance of multinucleated cells. The mitotic arrest caused by DMAA was accompanied by disruption of the microtubule network. By contrast, both arsenite and DMAA did not cause disorganization of actin stress fibers even when incubated at concentrations that caused a marked retardation of cell growth. Cells exposed to arsenite for 6 h showed marked inhibition of DNA synthesis, whereas inhibition by DMAA was not observed. When incubation was prolonged by 18 h, the arsenite-induced inhibition of DNA synthesis was mitigated. By contrast, inhibition of DNA synthesis by DMAA occurred in parallel with an increase in the population of mitotic cells. These results suggest that DMAA caused growth retardation and morphological changes via disruption of the microtubule network, and that arsenite-induced retardation of cell growth and inhibition of DNA synthesis were not attributable to the cytoskeletal changes. (orig.) (orig.) With 7 figs., 31 refs.

Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases.

Science.gov (United States)

Raindlová, Veronika; Janoušková, Martina; Slavíčková, Michaela; Perlíková, Pavla; Boháčová, Soňa; Milisavljevič, Nemanja; Šanderová, Hana; Benda, Martin; Barvík, Ivan; Krásný, Libor; Hocek, Michal

2016-04-20

DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Inhibition of DNA replication by ultraviolet light

International Nuclear Information System (INIS)

Edenberg, H.J.

1976-01-01

DNA replication in ultraviolet-irradiated HeLa cells was studied by two different techniques: measurements of the kinetics of semiconservative DNA synthesis, and DNA fiber autoradiography. In examining the kinetics of semiconservative DNA synthesis, density label was used to avoid measuring the incorporation due to repair replication. The extent of inhibition varied with time. After doses of less than 10 J/m 2 the rate was initially depressed but later showed some recovery. After higher doses, a constant, low rate of synthesis was seen for at least the initial 6 h. An analysis of these data indicated that the inhibition of DNA synthesis could be explained by replication forks halting at pyrimidine dimers. DNA fiber autoradiography was used to further characterize replication after ultraviolet irradiation. The average length of labeled segments in irradiated cells increased in the time immediately after irradiation, and then leveled off. This is the predicted pattern if DNA synthesis in each replicon continued at its previous rate until a lesion is reached, and then halted. The frequency of lesions that block synthesis is approximately the same as the frequency of pyrimidine dimers

Charge transport through exciton shelves in cadmium chalcogenide quantum dot-DNA nano-bioelectronic thin films

Science.gov (United States)

Goodman, Samuel M.; Noh, Hyunwoo; Singh, Vivek; Cha, Jennifer N.; Nagpal, Prashant

2015-02-01

Quantum dot (QD), or semiconductor nanocrystal, thin films are being explored for making solution-processable devices due to their size- and shape-tunable bandgap and discrete higher energy electronic states. While DNA has been extensively used for the self-assembly of nanocrystals, it has not been investigated for the simultaneous conduction of multiple energy charges or excitons via exciton shelves (ES) formed in QD-DNA nano-bioelectronic thin films. Here, we present studies on charge conduction through exciton shelves, which are formed via chemically coupled QDs and DNA, between electronic states of the QDs and the HOMO-LUMO levels in the complementary DNA nucleobases. While several challenges need to be addressed in optimizing the formation of devices using QD-DNA thin films, a higher charge collection efficiency for hot-carriers and our detailed investigations of charge transport mechanism in these thin films highlight their potential for applications in nano-bioelectronic devices and biological transducers.

Charge transport through exciton shelves in cadmium chalcogenide quantum dot-DNA nano-bioelectronic thin films

Energy Technology Data Exchange (ETDEWEB)

Goodman, Samuel M.; Singh, Vivek [Department of Chemical and Biological Engineering, University of Colorado Boulder, 3415 Colorado Avenue, Boulder, Colorado 80303 (United States); Noh, Hyunwoo [Department of Chemical and Biological Engineering, University of Colorado Boulder, 3415 Colorado Avenue, Boulder, Colorado 80303 (United States); Materials Science and Engineering Program and Department of Nanoengineering, University of California, 9500 Gilman Drive, La Jolla, San Diego, California 92093 (United States); Cha, Jennifer N. [Department of Chemical and Biological Engineering, University of Colorado Boulder, 3415 Colorado Avenue, Boulder, Colorado 80303 (United States); Materials Science and Engineering Program and Department of Nanoengineering, University of California, 9500 Gilman Drive, La Jolla, San Diego, California 92093 (United States); Materials Science and Engineering, University of Colorado Boulder, 3415 Colorado Avenue, Boulder, Colorado 80303 (United States); Nagpal, Prashant, E-mail: pnagpal@colorado.edu [Department of Chemical and Biological Engineering, University of Colorado Boulder, 3415 Colorado Avenue, Boulder, Colorado 80303 (United States); Materials Science and Engineering, University of Colorado Boulder, 3415 Colorado Avenue, Boulder, Colorado 80303 (United States); BioFrontiers Institute, University of Colorado Boulder, 3415 Colorado Avenue, Boulder, Colorado 80303 (United States); Renewable and Sustainable Energy Institute, University of Colorado Boulder, 2445 Kittredge Loop, Boulder, Colorado 80309 (United States)

2015-02-23

Quantum dot (QD), or semiconductor nanocrystal, thin films are being explored for making solution-processable devices due to their size- and shape-tunable bandgap and discrete higher energy electronic states. While DNA has been extensively used for the self-assembly of nanocrystals, it has not been investigated for the simultaneous conduction of multiple energy charges or excitons via exciton shelves (ES) formed in QD-DNA nano-bioelectronic thin films. Here, we present studies on charge conduction through exciton shelves, which are formed via chemically coupled QDs and DNA, between electronic states of the QDs and the HOMO-LUMO levels in the complementary DNA nucleobases. While several challenges need to be addressed in optimizing the formation of devices using QD-DNA thin films, a higher charge collection efficiency for hot-carriers and our detailed investigations of charge transport mechanism in these thin films highlight their potential for applications in nano-bioelectronic devices and biological transducers.

Charge transport through exciton shelves in cadmium chalcogenide quantum dot-DNA nano-bioelectronic thin films

International Nuclear Information System (INIS)

Goodman, Samuel M.; Singh, Vivek; Noh, Hyunwoo; Cha, Jennifer N.; Nagpal, Prashant

2015-01-01

Quantum dot (QD), or semiconductor nanocrystal, thin films are being explored for making solution-processable devices due to their size- and shape-tunable bandgap and discrete higher energy electronic states. While DNA has been extensively used for the self-assembly of nanocrystals, it has not been investigated for the simultaneous conduction of multiple energy charges or excitons via exciton shelves (ES) formed in QD-DNA nano-bioelectronic thin films. Here, we present studies on charge conduction through exciton shelves, which are formed via chemically coupled QDs and DNA, between electronic states of the QDs and the HOMO-LUMO levels in the complementary DNA nucleobases. While several challenges need to be addressed in optimizing the formation of devices using QD-DNA thin films, a higher charge collection efficiency for hot-carriers and our detailed investigations of charge transport mechanism in these thin films highlight their potential for applications in nano-bioelectronic devices and biological transducers

Gene assembly via one-pot chemical ligation of DNA promoted by DNA nanostructures

DEFF Research Database (Denmark)

Manuguerra, Ilenia; Croce, Stefano; El-Sagheer, Afaf H.

2018-01-01

Current gene synthesis methods are driven by enzymatic reactions. Here we report the one-pot synthesis of a chemically-ligated gene from 14 oligonucleotides. The chemical ligation benefits from the highly efficient click chemistry approach templated by DNA nanostructures, and produces modified DNA...

Design, synthesis and DNA-binding study of some novel morpholine linked thiazolidinone derivatives

Science.gov (United States)

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.

Host DNA synthesis-suppressing factor in culture fluid of tissue cultures infected with measles virus

International Nuclear Information System (INIS)

Minagawa, T.; Nakaya, C.; Iida, H.

1974-01-01

Host DNA synthesis is suppressed by the culture fluid of cell cultures infected with measles virus. This activity in the culture fluid is initiated somewhat later than the growth of infectious virus. Ninety percent of host DNA synthesis in HeLa cells is inhibited by culture fluid of 3-day-old cell cultures of Vero or HeLa cells infected with measles virus. This suppressing activity is not a property of the virion, but is due to nonvirion-associated componentnent which shows none of the activities of measles virus such as hemagglutination, hemolysis, or cell fusion nor does it have the antigenicity of measles virus as tested by complement-fixation or hemagglutination-inhibiting antibody blocking tests. Neutralization of the activity of this component is not attained with the pooled sera of convalescent measles patients. This component has molecular weights of about 45,000, 20,000, and 3,000 and appears to be a heat-stable protein. The production of host DNA suppressing factor (DSF) is blocked by cycloheximide. Neither uv-inactivated nor antiserum-neutralized measles virus produce DSF. Furthermore, such activity of nonvirion-associated component is not detected in the culture fluid of cultures infected with other RNA viruses such as poliovirus, vesicular stomatitis virus, or Sindbis virus. (auth)

One-by-one single-molecule detection of mutated nucleobases by monitoring tunneling current using a DNA tip.

Science.gov (United States)

Bui, Phuc Tan; Nishino, Tomoaki; Shiigi, Hiroshi; Nagaoka, Tsutomu

2015-01-31

A DNA molecule was utilized as a probe tip to achieve single-molecule genetic diagnoses. Hybridization of the probe and target DNAs resulted in electron tunneling along the emergent double-stranded DNA. Simple stationary monitoring of the tunneling current leads to single-molecule DNA detection and discovery of base mismatches and methylation.

Human DNA polymerase alpha uses a combination of positive and negative selectivity to polymerize purine dNTPs with high fidelity

Czech Academy of Sciences Publication Activity Database

Beckman, J.; Kincaid, K.; Hocek, Michal; Spratt, T.; Engels, J.; Cosstick, R.; Kuchta, R. D.

2007-01-01

Roč. 46, č. 2 (2007), s. 448-460 ISSN 0006-2960 R&D Projects: GA MŠk LC512 Grant - others:NIH(US) GM54194; NIH(US) TW007372-01; Army Research Office(US) W911NF-05-1-0172; DFG(DE) SFB 579 Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA polymerase * nucleoside triphosphates * modified nucleobases Subject RIV: CC - Organic Chemistry Impact factor: 3.368, year: 2007

The Importance of Short- and Long-Range Exchange on Various Excited State Properties of DNA Monomers, Stacked Complexes, and Watson-Crick Pairs.

Science.gov (United States)

Raeber, Alexandra E; Wong, Bryan M

2015-05-12

We present a detailed analysis of several time-dependent DFT (TD-DFT) methods, including conventional hybrid functionals and two types of nonempirically tuned range-separated functionals, for predicting a diverse set of electronic excitations in DNA nucleobase monomers and dimers. This large and extensive set of excitations comprises a total of 50 different transitions (for each tested DFT functional) that includes several n → π and π → π* valence excitations, long-range charge-transfer excitations, and extended Rydberg transitions (complete with benchmark calculations from high-level EOM-CCSD(T) methods). The presence of localized valence excitations as well as extreme long-range charge-transfer excitations in these systems poses a serious challenge for TD-DFT methods that allows us to assess the importance of both short- and long-range exchange contributions for simultaneously predicting all of these various transitions. In particular, we find that functionals that do not have both short- and full long-range exchange components are unable to predict the different types of nucleobase excitations with the same accuracy. Most importantly, the current study highlights the importance of both short-range exchange and a nonempirically tuned contribution of long-range exchange for accurately predicting the diverse excitations in these challenging nucleobase systems.

Involvement of DNA gyrase in replication and transcription of bacteriophage T7 DNA

International Nuclear Information System (INIS)

De Wyngaert, M.A.; Hinkle, D.C.

1979-01-01

Growth of bacteriophage T7 is inhibited by the antibiotic coumermycin A 1 , an inhibitor of the Escherichia coli DNA gyrase. Since growth of the phage is insensitive to the antibiotic in strains containing a coumermycin-resistent DNA gyrase, this enzyme appears to be required for phage growth. We have investigated the effect of coumermycin on the kinetics of DNA, RNA, and protein synthesis during T7 infection. DNA synthesis is completely inhibited by the antibiotic. In addition, coumermycin significantly inhibits transcription of late but not early genes. Thus, E. coli DNA gyrase may play an important role in transcription as well as in replication of T7 DNA

DNA-inspired hierarchical polymer design: electrostatics and hydrogen bonding in concert.

Science.gov (United States)

Hemp, Sean T; Long, Timothy E

2012-01-01

Nucleic acids and proteins, two of nature's biopolymers, assemble into complex structures to achieve desired biological functions and inspire the design of synthetic macromolecules containing a wide variety of noncovalent interactions including electrostatics and hydrogen bonding. Researchers have incorporated DNA nucleobases into a wide variety of synthetic monomers/polymers achieving stimuli-responsive materials, supramolecular assemblies, and well-controlled macromolecules. Recently, scientists utilized both electrostatics and complementary hydrogen bonding to orthogonally functionalize a polymer backbone through supramolecular assembly. Diverse macromolecules with noncovalent interactions will create materials with properties necessary for biomedical applications. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

TopBP1-mediated DNA processing during mitosis.

Science.gov (United States)

Gallina, Irene; Christiansen, Signe Korbo; Pedersen, Rune Troelsgaard; Lisby, Michael; Oestergaard, Vibe H

2016-01-01

Maintenance of genome integrity is crucial to avoid cancer and other genetic diseases. Thus faced with DNA damage, cells mount a DNA damage response to avoid genome instability. The DNA damage response is partially inhibited during mitosis presumably to avoid erroneous processing of the segregating chromosomes. Yet our recent study shows that TopBP1-mediated DNA processing during mitosis is highly important to reduce transmission of DNA damage to daughter cells. (1) Here we provide an overview of the DNA damage response and DNA repair during mitosis. One role of TopBP1 during mitosis is to stimulate unscheduled DNA synthesis at underreplicated regions. We speculated that such genomic regions are likely to hold stalled replication forks or post-replicative gaps, which become the substrate for DNA synthesis upon entry into mitosis. Thus, we addressed whether the translesion pathways for fork restart or post-replicative gap filling are required for unscheduled DNA synthesis in mitosis. Using genetics in the avian DT40 cell line, we provide evidence that unscheduled DNA synthesis in mitosis does not require the translesion synthesis scaffold factor Rev1 or PCNA ubiquitylation at K164, which serve to recruit translesion polymerases to stalled forks. In line with this finding, translesion polymerase η foci do not colocalize with TopBP1 or FANCD2 in mitosis. Taken together, we conclude that TopBP1 promotes unscheduled DNA synthesis in mitosis independently of the examined translesion polymerases.

Fast Simultaneous Determination of 13 Nucleosides and Nucleobases in Cordyceps sinensis by UHPLC–ESI–MS/MS

Directory of Open Access Journals (Sweden)

Shi-Yu Zong

2015-12-01

Full Text Available A reliable ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC–ESI–MS/MS method for the fast simultaneous determination of 13 nucleosides and nucleobases in Cordyceps sinensis (C. sinensis with 2-chloroadenosine as internal standard was developed and validated. Samples were ultrasonically extracted in an ice bath thrice, and the optimum analyte separation was performed on an ACQUITY UPLCTM HSS C18 column (100 mm × 2.1 mm, 1.8 μm with gradient elution. All targeted analytes were separated in 5.5 min. Furthermore, all calibration curves showed good linear regression (r > 0.9970 within the test ranges, and the limits of quantitation and detection of the 13 analytes were less than 150 and 75 ng/mL, respectively. The relative standard deviations (RSDs of intra- and inter-day precisions were <6.23%. Recoveries of the quantified analytes ranged within 85.3%–117.3%, with RSD < 6.18%. The developed UHPLC–ESI–MS/MS method was successfully applied to determine nucleosides and nucleobases in 11 batches of C. sinensis samples from different regions in China. The range for the total content in the analyzed samples was 1329–2057 µg/g.

Doxazosin blocks the angiotensin II-induced smooth muscle cell DNA synthesis in the media, but not in the neointima of the rat carotid artery after balloon injury

NARCIS (Netherlands)

van Kleef, E. M.; Smits, J. F.; Schwartz, S. M.; Daemen, M. J.

1996-01-01

Infusion of angiotensin II (AngII) during the third and fourth week after balloon injury of the left common carotid artery of the rat induces smooth muscle cell (SMC) DNA synthesis. In this study we wanted to investigate whether alpha 1-adrenoreceptors are involved in AngII-induced SMC DNA synthesis

Formic acid hydrolysis/liquid chromatography isotope dilution mass spectrometry: An accurate method for large DNA quantification.

Science.gov (United States)

Shibayama, Sachie; Fujii, Shin-Ichiro; Inagaki, Kazumi; Yamazaki, Taichi; Takatsu, Akiko

2016-10-14

Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) with formic acid hydrolysis was established for the accurate quantification of λDNA. The over-decomposition of nucleobases in formic acid hydrolysis was restricted by optimizing the reaction temperature and the reaction time, and accurately corrected by using deoxynucleotides (dNMPs) and isotope-labeled dNMPs as the calibrator and the internal standard, respectively. The present method could quantify λDNA with an expanded uncertainty of 4.6% using 10fmol of λDNA. The analytical results obtained with the present method were validated by comparing with the results of phosphate-base quantification by inductively coupled plasma-mass spectrometry (ICP-MS). The results showed good agreement with each other. We conclude that the formic acid hydrolysis/LC-IDMS method can quantify λDNA accurately and is promising as the primary method for the certification of DNA as reference material. Copyright © 2016 Elsevier B.V. All rights reserved.

pH dependent interaction of biofunctionalized CdS nanoparticles with nucleobases and nucleotides: A fluorimetric study

International Nuclear Information System (INIS)

Chatterjee, Anindita; Priyam, Amiya; Bhattacharya, Subhash C.; Saha, Abhijit

2007-01-01

The interaction of DNA bases and corresponding nucleotides with CdS nanoparticles (NPs), biofunctionalized by cysteine, has been investigated by absorption and fluorescence spectroscopy. Unique enhancement effect of adenine, in contrast to other nucleobases, on the luminescence of cysteine capped CdS (cys-CdS) NPs at both pH 7.5 and 10.5 was found, the extent of enhancement being much higher at pH 10.5. At the latter pH, the difference optical absorption spectra show development of new peak at 278 nm with corresponding decrease in the absorption of adenine at 260 nm, which is attributed to binding of adenine anion to the CdS surface through N7 of the purine ring. Appearance of a new band at 478 cm -1 and concomitant shift in the C 8 -N 7 vibrations to 1610 cm -1 in the FTIR spectra of cys-CdS NPs with adenine also suggest Cd-N7 binding on the particle surface. Amongst various nucleotides, ATP exhibited maximum luminescence enhancement on CdS NPs for a given change in concentration in the micro-molar range at physiological pH. A quantitative correlation between ATP concentration and PL enhancement of CdS NPs has been established, a step which in future might assist in developing new protocols for fluorescence sensing of adenine nucleotides under certain pathological conditions

Repair of Clustered Damage and DNA Polymerase Iota.

Science.gov (United States)

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

Highly Sensitive DNA Sensor Based on Upconversion Nanoparticles and Graphene Oxide.

Science.gov (United States)

Alonso-Cristobal, P; Vilela, P; El-Sagheer, A; Lopez-Cabarcos, E; Brown, T; Muskens, O L; Rubio-Retama, J; Kanaras, A G

2015-06-17

In this work we demonstrate a DNA biosensor based on fluorescence resonance energy transfer (FRET) between NaYF4:Yb,Er nanoparticles and graphene oxide (GO). Monodisperse NaYF4:Yb,Er nanoparticles with a mean diameter of 29.1 ± 2.2 nm were synthesized and coated with a SiO2 shell of 11 nm, which allowed the attachment of single strands of DNA. When these DNA-functionalized NaYF4:Yb,Er@SiO2 nanoparticles were in the proximity of the GO surface, the π-π stacking interaction between the nucleobases of the DNA and the sp(2) carbons of the GO induced a FRET fluorescence quenching due to the overlap of the fluorescence emission of the NaYF4:Yb,Er@SiO2 and the absorption spectrum of GO. By contrast, in the presence of the complementary DNA strands, the hybridization leads to double-stranded DNA that does not interact with the GO surface, and thus the NaYF4:Yb,Er@SiO2 nanoparticles remain unquenched and fluorescent. The high sensitivity and specificity of this sensor introduces a new method for the detection of DNA with a detection limit of 5 pM.

Recovery of DNA synthesis after ultraviolet irradiation of xeroderma pigmentosum cells depends on excision repair and is blocked by caffeine

International Nuclear Information System (INIS)

Park, S.D.; Cleaver, J.E.

1979-01-01

Normal human and xeroderma pigmentosum (XP, excision-defective group A) cells (both SV40-transformed) pulse-labeled with [ 3 H] thymidine at various times after irradiation with ultraviolet light showed a decline and recovery of both the molecular weights of newly synthesized DNA and the rated of synthesis per cell. At the same ultraviolet dose, both molecular weights and rates of synthesis were inhibited more in XP than in normal cells. This indicates that excision repair plays a role in minimizing the inhibition of chain growth, possibly by excision of dimers ahead of the growing point. The ability to synthesize normal-sized DNA recovered more rapidly than rates of synthesis in normal cells, but both parameters recovered in phase in XP cells. During recovery in normal cells there are therefore fewer actively replicating clusters of replicons because the single-strand breaks involved in the excision of dimers inhibit replicon initiation. XP cells have few excision repair events and therefore fewer breaks to interfere with initiation, but chain growth is blocked by unexcised dimers. In both cell types recovery of the ability to synthesize normal-sized DNA was prevented by growing cells in caffeine after irradiation, possibly because of competition between the DNA binding properties of caffeine and replication proteins. These observations imply that excision repair and semiconservative replication interact strongly in irradiated cells to produce a complex spectrum of changes in DNA replication which may be confused with parts of alternative systems such as post-replication repair. (author)

Protozoan ALKBH8 Oxygenases Display both DNA Repair and tRNA Modification Activities

DEFF Research Database (Denmark)

Zdżalik, Daria; Vågbø, Cathrine B; Kirpekar, Finn

2014-01-01

The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH......1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity...... was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but...

Structure of a DNA glycosylase that unhooks interstrand cross-links

Energy Technology Data Exchange (ETDEWEB)

Mullins, Elwood A.; Warren, Garrett M.; Bradley, Noah P.; Eichman, Brandt F. (Vanderbilt)

2017-04-10

DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

Non-transcriptional Function of FOXO1/DAF-16 Contributes to Translesion DNA Synthesis.

Science.gov (United States)

Daitoku, Hiroaki; Kaneko, Yuta; Yoshimochi, Kenji; Matsumoto, Kaori; Araoi, Sho; Sakamaki, Jun-Ichi; Takahashi, Yuta; Fukamizu, Akiyoshi

2016-08-22