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Sample records for dna interaction studies

  1. 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.

  2. Intersegmental interactions in supercoiled DNA: atomic force microscope study

    Energy Technology Data Exchange (ETDEWEB)

    Shlyakhtenko, Luda S.; Miloseska, Lela; Potaman, Vladimir N.; Sinden, Richard R.; Lyubchenko, Yuri L

    2003-10-15

    Intersegmental interactions in DNA facilitated by the neutralization of electrostatic repulsion was studied as a function of salt concentration and DNA supercoiling. DNA samples with defined superhelical densities were deposited onto aminopropyl mica at different ionic conditions and imaged in air after drying of the samples. Similar to hydrodynamic data, we did not observe a collapse of supercoiled DNA, as proposed earlier by cryo-EM studies. Instead, the formation of the contacts between DNA helices within supercoiled loops with no visible space between the duplexes was observed. The length of such close contacts increased upon increasing NaCl concentration. DNA supercoiling was a critical factor for the stabilization of intersegmental contacts. Implications of the observed effect for understanding DNA compaction in the cell and for regulation DNA transactions via interaction of distantly separated DNA regions are discussed.

  3. Studies of interaction between two alkaloids and double helix DNA

    International Nuclear Information System (INIS)

    Sun, Yantao; Peng, Tingting; Zhao, Lei; Jiang, Dayu; Cui, Yuncheng

    2014-01-01

    This article presents the study on the interaction of two alkaloids (matrine and evodiamine) and hs-DNA by absorption, fluorescence, circular dichroism (CD), DNA melting and viscosity experiments. The spectroscopic studies suggested that two alkaloids can bind to DNA through an intercalative mode. The viscosity measurement and thermal denaturation also indicated that two alkaloids can intercalate to DNA. The binding constants (K A ) and the number of binding sites (n) were determined. At the same time, some significant thermodynamic parameters of the binding of the alkaloids to DNA were obtained. Competitive binding studies revealed that alkaloids had an effect on ethidium bromide (EB) bound DNA. In addition, it was also proved that the fluorescence quenching was influenced by ionic strength. - Highlights: • Interaction between two alkaloids and DNA is studied by spectral methods. • The binding constant and the binding sites between two alkaloids and DNA are obtained. • There are a classical intercalative mode between alkaloids and DNA. • The binding of matrine with DNA is weaker than that of evodiamine. • It is important for us to understand the alkaloids–DNA interactions at a molecular level

  4. An electrochemical study of neutral red-DNA interaction

    International Nuclear Information System (INIS)

    Heli, H.; Bathaie, S.Z.; Mousavi, M.F.

    2005-01-01

    Electrochemical methods were used to investigate the interaction of neutral red (NR) with double-stranded calf thymus DNA, in solution as well as using a DNA-modified glassy carbon (GC-DNA) electrode. The results were compared with those obtained from bare glassy carbon (GC) electrode. The formal potential of NR was more positive when GC-DNA electrode was used although the rate of heterogeneous electron transfer is as high as that of using GC electrode. GC-DNA electrode enables preconcentration of NR for chosen times on the electrode surface, despite the fact that the mass transfer effects in the thin DNA layer adsorbed on the surface was still observed using cyclic voltammetry and electrochemical impedance spectroscopy techniques. Parameters, such as the diffusion coefficient of NR, binding site size in base pairs and the ratio of the binding constants for the oxidized and reduced forms of the bound species were obtained. A binding isotherm for NR at GC-DNA electrode was obtained from coulometric titrations and gave an affinity constant equal to 2.76 x 10 4 L mol -1 . From the studies of the interaction in solution, the diffusion coefficient of free and DNA-bound NR, binding constant and binding site size of the DNA-NR complex was also obtained simultaneously by non-linear fitting analysis of voltammetric data

  5. Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

    Science.gov (United States)

    Rauf, Sakandar; Nawaz, Haq; Akhtar, Kalsoom; Ghauri, Muhammad A; Khalid, Ahmad M

    2007-05-15

    The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

  6. Electrochemical behavior of antioxidants: Part 3. Electrochemical studies of caffeic Acid–DNA interaction and DNA/carbon nanotube biosensor for DNA damage and protection

    Directory of Open Access Journals (Sweden)

    Refat Abdel-Hamid

    2016-05-01

    Full Text Available Multi-walled carbon nanotubes-modified glassy carbon electrode biosensor was used for electrochemical studies of caffeic acid–dsDNA interaction in phosphate buffer solution at pH 2.12. Caffeic acid, CAF, shows a well-defined cyclic voltammetric wave. Its anodic peak current decreases and the peak potential shifts positively on the addition of dsDNA. This behavior was ascribed to an interaction of CAF with dsDNA giving CAF–dsDNA complex by intercalative binding mode. The apparent binding constant of CAF–dsDNA complex was determined using amperometric titrations. The oxidative damage caused to DNA was detected using the biosensor. The damage caused by the reactive oxygen species, hydroxyl radical (·−OH generated by the Fenton system on the DNA-biosensor was detected. It was found that CAF has the capability of scavenging the hydroxide radical and protecting the DNA immobilized on the GCE surface.

  7. [Study of the interaction mechanism between brodifacoum and DNA by spectroscopy].

    Science.gov (United States)

    Duan, Yun-qing; Min, Shun-geng

    2009-04-01

    The interaction between brodifacoum (3-[3-(4'-bromophenyl-4) 1,2,3,4-tetralin-10]-4-hydroxyl-coumarin) (BDF), an anticoagulant rodenticide, and calf thymus DNA (ct-DNA) was studied by UV spectrum and fluorescence spectrum. The results were summarized as follows: There was a hypochromic effect of low concentration ct-DNA on the UV spectra. The fluorescence quenching studies showed a regular decrease in the fluorescence intensity after addition of ct-DNA by the static quenching mode with a quenching constant (Ksv) of 1.21 x 10(4) L x mol(-1) at 27 degrees C. The BDF possibly bonded to ct-DNA mainly via Van der Waals forces by the corresponding thermodynamics parameter. KI quenching experiment found that there was not obvious protection of ct-DNA to BDF. The fluorescence intensity of BDF/ct-DNA system changed with the variation in ionic strength Quenching of ct-DNA on the fluorescence of BDF/beta-CD inclusion complex was reduced in contrast with the free BDF, which showed that beta-CD could provide BDF with protection. So the comprehensive interaction mode of BDF with ct-DNA may be the groove binding by the above results. It was indicated that there had been static-electro interaction between BDF and ct-DNA at the same time. The conjunct action of Van der Waals forces and electrostatic attraction favorably provide BDF bonding interaction in the groove of ct-DNA.

  8. The study of genomic DNA adsorption and subsequent interactions using total internal reflection ellipsometry.

    Science.gov (United States)

    Nabok, Alexei; Tsargorodskaya, Anna; Davis, Frank; Higson, Séamus P J

    2007-10-31

    The adsorption of genomic DNA and subsequent interactions between adsorbed and solvated DNA was studied using a novel sensitive optical method of total internal reflection ellipsometry (TIRE), which combines spectroscopic ellipsometry with surface plasmon resonance (SPR). Single strands of DNA of two species of fish (herring and salmon) were electrostatically adsorbed on top of polyethylenimine films deposited upon gold coated glass slides. The ellipsometric spectra were recorded and data fitting utilized to extract optical parameters (thickness and refractive index) of adsorbed DNA layers. The further adsorption of single stranded DNA from an identical source, i.e. herring ss-DNA on herring ss-DNA or salmon ss-DNA on salmon ss-DNA, on the surface was observed to give rise to substantial film thickness increases at the surface of about 20-21 nm. Conversely adsorption of DNA from alternate species, i.e. salmon ss-DNA on herring ss-DNA or herring ss-DNA on salmon ss-DNA, yielded much smaller changes in thickness of 3-5 nm. AFM studies of the surface roughness of adsorbed layers were in line with the TIRE data.

  9. Structural Analysis of DNA Interactions with Magnesium Ion Studied by Raman Spectroscopy

    OpenAIRE

    S. Ponkumar; P. Duraisamy; N. Iyandurai

    2011-01-01

    Problem statement: In the present study, FT Raman spectroscopy had been used to extend our knowledge about Magnesium ion - DNA interactions at various volume ratios (1:50, 1:20, 1:10 and 1:5). Approach: The analysis of FT Raman data supported the existence of structural specificities in the interaction and also the stability of DNA secondary structure. Results: Results from the Raman spectra clearly indicate that the interaction of Magnesium ion with DNA is mainly through the phosphate groups...

  10. Studies on the Interaction between Zinc-Hydroxybenzoite Complex and Genomic DNA

    Directory of Open Access Journals (Sweden)

    Hacali Necefoglu

    2006-04-01

    Full Text Available Zinc-Hydroxybenzoite ([Zn (H206] (p-HO-C6H4COO22H20 complex which wassynthesized and characterized by instrumental methods and the DNA samples which hadbeen isolated from cattle were allowed to interact at 37 oC for different time periods. Theinteraction of genomic DNA with this complex has been followed by agarose gelelectrophoresis at 50 V for 2 h. When DNA samples were allowed to interact with this metalcomplex, it was found that band intensities changed with the concentrations of the complex.In the result of interaction between this complex and genomic DNA samples, it wasdetermined that the intensities of bands were changed at the different concentrations of thecomplex. The brightness of the bands was increased and mobility of the bands wasdecreased, indicating the occurrence of increased covalent binding of the metal complexwith DNA. In this study it was concluded that the damage effect of ascorbate was reducedby Zinc-Hydroxybenzoite.

  11. Theoretical study on the interaction of pregabalin and olanzapine with DNA

    Directory of Open Access Journals (Sweden)

    ZibaHooshiarSodagar

    2016-12-01

    Full Text Available This paper aims is to study the interaction of two drugs including pregabalin and olanzapine with DNA. For this purpose, density functional theory calculations and docking were used. The structure of pregabalin and olanzapine using B3Lyp theory level and the basis set 6-311 G(d,p was optimized. Highest occupied molecular orbital (HOMO and lowest unoccupied molecular orbital (LUMO calculated for each drugs. The obtained results showed that olanzapine is more reactive than pregabalin. Docking of drugs with DNA was performed and the results showed that binding affinity of olanzapine is higher than pregabalin. Also, the graphical results revealed that olanzapine interact with DNA via 5-terminal major groove of DNA, whereas pregabalin interact with DNA via 3- termind major groove.

  12. [Study on the aggregation behavior of cationic porphyrins and their interaction with ctDNA].

    Science.gov (United States)

    Ma, Hong-Min; Chen, Xin; Sun, Shu-Ting; Zhang, Li-Na; Wu, Dan; Zhu, Pei-Hua; Li, Yan; Du, Bin; Wei, Qin

    2009-02-01

    Interest in the interaction between cationic porphyrins, particularly derivatives of meso-tetra(N-methylpyridinium-4-yl) porphyrin(TMPyP), and DNA abounds because they are versatile DNA-binding agents that could find application in photodynamic therapy, cancer detection, artificial nucleases, virus inhibition and so on. The interaction of two water-soluble cationic porphyrins, meso-tetrakis(4-N, N, N-trimethylanilinium) porphyrin (TMAP) and 5-phenyl-10,15,20-tris[4-(N-methyl) pyridinium]porphyrin (TriMPyP), with calf thymus DNA (ctDNA) was studied by UV-Vis absorption spectroscopy, fluorescence spectroscopy and resonance light scattering technique. TriMPyP forms aggregate in water due to the molecular asymmetry while TMAP exists as monomers. At lower concentrations of ctDNA (R > 1, R = c(TMAP)/c(DNA) base pair), the interaction of TMAP with DNA leads to significant hypochromicity and bathochromic shift of absorption spectra. And the fluorescence of TMAP was quenched while it showed enhanced resonance light scattering signals. But the extent of enhancement of resonance light scattering signals is very small, so the aggregate of TMAP is not very high. These observations indicate the self-stacking of TMAP along the DNA surface. At higher concentrations of ctDNA (R TMAP association with DNA is via outside binding which is accompanied with hyperchromic effect and fluorescence enhancement while the resonance light scattering signals is reduced. DNA addition decreases the fluorescence intensity of TriMPyP and it shifts the peak to the higher wavelengths (red shift). The interaction with DNA promotes the aggregation of TriMPyP and no simple outside binding is observed even at higher concentrations of ctDNA. The steric effect of molecular distortion constrains the intercalation or further binding to DNA. The effect of ionic strength on the interaction was investigated at two DNA concentrations, 1.2 and 24.0 micromol x L(-1), for TMAP. The Interactions of both porphyrins

  13. Electrostatic study of Alanine mutational effects on transcription: application to GATA-3:DNA interaction complex.

    Science.gov (United States)

    El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges

    2015-01-01

    Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature.

  14. Interaction of anthraquinone anti-cancer drugs with DNA:Experimental and computational quantum chemical study

    Science.gov (United States)

    Al-Otaibi, Jamelah S.; Teesdale Spittle, Paul; El Gogary, Tarek M.

    2017-01-01

    Anthraquinones form the basis of several anticancer drugs. Anthraquinones anticancer drugs carry out their cytotoxic activities through their interaction with DNA, and inhibition of topoisomerase II activity. Anthraquinones (AQ4 and AQ4H) were synthesized and studied along with 1,4-DAAQ by computational and experimental tools. The purpose of this study is to shade more light on mechanism of interaction between anthraquinone DNA affinic agents and different types of DNA. This study will lead to gain of information useful for drug design and development. Molecular structures were optimized using DFT B3LYP/6-31 + G(d). Depending on intramolecular hydrogen bonding interactions two conformers of AQ4 were detected and computed as 25.667 kcal/mol apart. Molecular reactivity of the anthraquinone compounds was explored using global and condensed descriptors (electrophilicity and Fukui functions). Molecular docking studies for the inhibition of CDK2 and DNA binding were carried out to explore the anti cancer potency of these drugs. NMR and UV-VIS electronic absorption spectra of anthraquinones/DNA were investigated at the physiological pH. The interaction of the three anthraquinones (AQ4, AQ4H and 1,4-DAAQ) were studied with three DNA (calf thymus DNA, (Poly[dA].Poly[dT]) and (Poly[dG].Poly[dC]). NMR study shows a qualitative pattern of drug/DNA interaction in terms of band shift and broadening. UV-VIS electronic absorption spectra were employed to measure the affinity constants of drug/DNA binding using Scatchard analysis.

  15. Regulating DNA Self-assembly by DNA-Surface Interactions.

    Science.gov (United States)

    Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde

    2017-12-14

    DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Annonalide and derivatives: Semisynthesis, cytotoxic activities and studies on interaction of annonalide with DNA.

    Science.gov (United States)

    Marques, Ricardo A; Gomes, Akenaton O C V; de Brito, Maria V; Dos Santos, Ana L P; da Silva, Gladyane S; de Lima, Leandro B; Nunes, Fátima M; de Mattos, Marcos C; de Oliveira, Fátima C E; do Ó Pessoa, Cláudia; de Moraes, Manoel O; de Fátima, Ângelo; Franco, Lucas L; Silva, Marina de M; Dantas, Maria Dayanne de A; Santos, Josué C C; Figueiredo, Isis M; da Silva-Júnior, Edeíldo F; de Aquino, Thiago M; de Araújo-Júnior, João X; de Oliveira, Maria C F; Leslie Gunatilaka, A A

    2018-02-01

    The cytotoxic activity of the pimarane diterpene annonalide (1) and nine of its semisynthetic derivatives (2-10) was investigated against the human tumor cell lines HL-60 (leukemia), PC-3 (prostate adenocarcinoma), HepG2 (hepatocellular carcinoma), SF-295 (glioblastoma) and HCT-116 (colon cancer), and normal mouse fibroblast (L929) cells. The preparation of 2-10 involved derivatization of the side chain of 1 at C-13. Except for 2, all derivatives are being reported for the first time. Most of the tested compounds presented IC 50 s below 4.0 μM, being considered potential antitumor agents. The structures of all new compounds were elucidated by spectroscopic analyses including 2D NMR and HRMS. Additionally, the interaction of annonalide (1) with ctDNA was evaluated using spectroscopic techniques, and the formation of a supramolecular complex with the macromolecule was confirmed. Competition assays with fluorescent probes (Hoechst and ethidium bromide) and theoretical studies confirmed that 1 interacts preferentially via DNA intercalation with stoichiometric ratio of 1:1 (1:ctDNA). The ΔG value was calculated as -28.24 kJ mol -1 , and indicated that the interaction process occurs spontaneously. Docking studies revealed that van der Walls is the most important interaction in 1-DNA and EB-DNA complexes, and that both ligands (1 and EB) interact with the same DNA residues (DA6, DA17 and DT19). Copyright © 2018. Published by Elsevier B.V.

  17. Interaction and dynamics of homologous pairing protein 2 (HOP2) and DNA studied by MD simulation

    Science.gov (United States)

    Moktan, Hem; Pezza, Roberto; Zhou, Donghua

    2015-03-01

    The homologous pairing protein 2 (Hop2) plays an important role in meiosis and DNA repair. Together with protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. We recently determined the NMR structure of the N-terminal domain of Hop2 and proposed a model of Protein-DNA complex based on NMR chemical shift perturbations and mutagenesis studies (Moktan, J Biol Chem 2014 10.1074/jbc.M114.548180). However structure and dynamics of the complex have not been studied at the atomic level yet. Here, we used classical MD simulations to study the interactions between the N-terminal HOP2 and DNA. The simulated results indicate that helix3 (H3) interacts with DNA in major groove and wing1 (W1) interacts mostly in minor groove mainly via direct hydrogen bonds. Also it is found that binding leads to reduced fluctuations in both protein and DNA. Several water bridge interactions have been identified. The residue-wise contributions to the interaction energy were evaluated. Also the functional motion of the protein is analyzed using principal component analysis. The results confirmed the importance of H3 and W1 for the stability of the complex, which is consistent with our previous experimental studies.

  18. Energetic studies on DNA-peptide interaction in relation to the enthalpy-entropy compensation paradox.

    Science.gov (United States)

    Yang, Robin C K; Huang, Jonathan T B; Chien, Shih-Chuan; Huang, Roy; Jeng, Kee-Ching G; Chen, Yen-Chung; Liao, Mokai; Wu, Jia-Rong; Hung, Wei-Kang; Hung, Chia-Chun; Chen, Yu-Ling; Waring, Michael J; Sheh, Leung

    2013-01-07

    This study aims to interpret the energetic basis of complex DNA-peptide interactions according to a novel allosteric interaction network approach. In common with other designed peptides, five new conjugates incorporating the XPRK or XHypRK motif (Hyp = hydroxyproline) attached to a N-methylpyrrole (Py) tract with a basic tail have been found to display cooperative binding to DNA involving multiple monodentate as well as interstrand bidentate interactions. Using quantitative DNase I footprinting it appears that allosteric communication via cooperative binding to multiple sites on complementary DNA strands corresponds to two different types of DNA-peptide interaction network. Temperature variation experiments using a dodecapeptide RY-12 show that lower temperature (25 °C) favor a circuit type of allosteric interaction network, whereas higher temperatures (31 and 37 °C) afford only a partial-circuit type of network. Circular dichroism studies show that our five peptides induce significant local conformational changes in DNA via the minor groove, with apparently dimeric binding stoichiometry. Isothermal titration calorimetry reveals that these peptides, together with another seven for comparison, are strongly exothermic upon binding to a model 13-mer DNA duplex, characterized by ΔH ranging from -14.7 to -74.4 kcal mol(-1), and also high TΔS ranging from -6.5 to -65.9 kcal mol(-1). Multiple monodentate and bidentate interactions, as well as ionic forces that mediate positive cooperativity in sequence recognition, are consistent with a dramatic decrease in entropy and a 'tightening' effect of DNA conformation. Distinctive enthalpy-entropy compensation (EEC) relationships are demonstrated for the interaction of all twelve designed peptides with DNA, affording a straight line of slope close to unity when ΔH is plotted versus TΔS, with a y-axis intercept (average ΔG) corresponding to -8.5 kcal mol(-1), while the observed ΔG ranges from -8.2 to -9.1 kcal mol(-1) for

  19. physico-chemical studies on DNA-drugs interaction and their analytical applications

    International Nuclear Information System (INIS)

    Kandil, S.A

    2003-01-01

    The present thesis is devoted to study the interaction of some antibacterial agents i.e. fluoroquinolones . these agents include ciprofloxacin, norfloxacin , ofloxacin , pefloxacin and levofloxacin with DNA. voltammetric and spectrophotometric methods were used to carry out this study. Also the interaction of the suggested drugs with DNA at the surface of carbon electrode by cyclic voltammetry and differential pulse techniques is examined. The work comprises three chapters: (1) includes an introduction of voltammetry , differential pulse, drug-DNA interaction and fluoroquinoline- DNA interaction and literature survey on fluoroquinolones.Chapter (II) includes preparation of the solutions and instruments which were used for the measurements using the different techniques.Chapter(III) comprises three parts; (1) deals with the interaction of fluoroquinolones (ciprofloxacin, norfloxacin, ofloxacin, pefloxacin and levofloxacin) with DNA in solution have been investigated by means of voltammetry and spectroscopy . the results show that the values of binding constant of fluoroquinolne drugs with DNA obtained through the changes of the anodic peak current, and their values are, 30900,31000,32300,32000 and 32500 M -1 respectively. or changes of absorption and values are, 36000,30200.38300,36500 and 34400 M -1 receptively.(II) includes voltammetric behavior of fluoroquinolones on DNA-modified carbon paste electrode. (III)includes analytical application for proposed method for the determination of levofloxacin as a typical example for fluoroquinolones. Concentration in the range 5.0x10 -7 ∼ 5.0x10 -6 mol/L , with a detection limit of 1.0x10 -7 mol/L. direct and simple determination of levofloxacin in urine was established with no manipulation of urine sample other than dilution 1:10

  20. Electrochemical study of varenicline adsorptive behaviour and its interaction with DNA

    Directory of Open Access Journals (Sweden)

    Radulović Valentina

    2012-01-01

    Full Text Available The electrochemical behaviour of novel nicotinic α4β2 subtype receptor partial agonist varenicline (VAR which is used for smoking cessation, was investigated in Britton-Robinson buffers (pH 2.0-12.0 by cyclic, differential pulse and square wave voltammetry at a hanging mercury drop elctrode. The influence of pH, scan rate, concentration, accumulation potential and time on peak current and potential suggested that in alkaline media the redox process was adsorption controlled. Also, the experimental value of surface coverage, G = 1.03´10-10 mol cm-2, was used to determine the conditions when VAR was fully adsorbed at the electrode surface. Having in mind potential high toxicity of VAR due to the presence of quinoxaline structure, its interaction with DNA was postulated, and studied when both compounds were in the adsorbed state at modified HMDE. Using adsorptive transfer technique, the changes in potential and decrease in normalized peak currents were observed. The estimated value of the ratio of surface-binding constants indicated that the reduced form of VAR interacted with dsDNA more strongly than the oxidized form. Subtle DNA damage under conditions of direct DNA-VAR interaction at room temperature was observed. The proposed type of interaction was an intercalation. This study used simple electroanalytical methodology and showed the potential of DNA/HMDE biosensor for investigation of genotoxic effects.

  1. In vitro study on the interaction of 4,4-dimethylcurcumin with calf thymus DNA

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Bing-Mi, E-mail: liubingmi@163.com [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Bai, Chong-Liang [Centre for Molecular Science and Engineering, Northeastern University, Shenyang 110819 (China); Zhang, Jun; Liu, Yang; Dong, Bo-Yang; Zhang, Yi-Tong [Department of Pharmacy, Liaoning University, Shenyang 110036 (China); Liu, Bin, E-mail: liubinzehao@163.com [Department of Pharmacy, Liaoning University, Shenyang 110036 (China)

    2015-10-15

    The interaction of 4,4-dimethylcurcumin (DMCU), a synthesized analog of curcumin, with calf-thymus DNA (ct-DNA) was investigated using fluorescence, absorption, and circular dichroism (CD) spectroscopy, coupled with viscosity measurements and molecular docking techniques. DMCU was found to bind to ct-DNA with moderate binding affinity through groove binding as evidenced by a decrease in the absorption intensity in combination with no obvious change in the relative specific viscosity of ct-DNA and the CD spectrum. Thermodynamic analysis of the fluorescence data obtained at different temperatures suggested that the binding process was spontaneous and was primarily driven by hydrogen bonding and van der Waals forces. Furthermore, competitive binding experiments with ethidium bromide and 4′,6-diamidino-2-phenylindole as probes showed that DMCU could preferentially bind in the minor groove of double-stranded DNA. The results obtained from the molecular docking studies were consistent with these experimental results. This study explored the potential applicability of the spectroscopic properties of DMCU for studying its interactions with relevant biological or biomimicking targets. - Highlights: • 4,4-dimethylcurcumin (DMCU) has strong fluorescence characteristics. • DMCU could bind to DNA through groove binding. • Docking studies revealed that DMCU bound to the A–T region in the minor groove.

  2. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Research Article. A Qualitative and Quantitative Assay to Study. DNA/Drug Interaction Based on Sequence Selective. Inhibition of Restriction Endonucleases. Syed A Hassan1*, Lata Chauhan2, Ritu Barthwal2 and Aparna Dixit3. 1 Faculty of Computing and Information Technology, King Abdul Aziz University, Rabigh-21911 ...

  3. FT-Raman and QM/MM study of the interaction between histamine and DNA

    International Nuclear Information System (INIS)

    Ruiz-Chica, A.J.; Soriano, A.; Tunon, I.; Sanchez-Jimenez, F.M.; Silla, E.; Ramirez, F.J.

    2006-01-01

    The interaction between histamine and highly polymerized calf-thymus DNA has been investigated using FT-Raman spectroscopy and the hybrid QM/MM (quantum mechanics/molecular mechanics) methodology. Raman spectra of solutions containing histamine and calf-thymus DNA, at different molar ratios, were recorded. Solutions were prepared at physiological settings of pH and ionic strength, using both natural and heavy water as the solvent. The analysis of the spectral changes on the DNA Raman spectra when adding different concentrations of histamine allowed us to identify the reactive sites of DNA and histamine, which were used to built two minor groove and one intercalated binding models. They were further used as starting points of the QM/MM theoretical study. However, minimal energy points were only reached for the two minor groove models. For each optimized structure, we calculated analytical force constants of histamine molecule in order to perform the vibrational dynamics. Normal mode descriptions allowed us to compare calculated wavenumbers for DNA-interacting histamine to those measured in the Raman spectra of DNA-histamine solutions

  4. An Ultraviolet Resonance Raman Spectroscopic Study of Cisplatin and Transplatin Interactions with Genomic DNA.

    Science.gov (United States)

    Geng, Jiafeng; Aioub, Mena; El-Sayed, Mostafa A; Barry, Bridgette A

    2017-09-28

    Ultraviolet resonance Raman (UVRR) spectroscopy is a label-free method to define biomacromolecular interactions with anticancer compounds. Using UVRR, we describe the binding interactions of two Pt(II) compounds, cisplatin (cis-diamminedichloroplatinum(II)) and its isomer, transplatin, with nucleotides and genomic DNA. Cisplatin binds to DNA and other cellular components and triggers apoptosis, whereas transplatin is clinically ineffective. Here, a 244 nm UVRR study shows that purine UVRR bands are altered in frequency and intensity when mononucleotides are treated with cisplatin. This result is consistent with previous suggestions that purine N7 provides the cisplatin-binding site. The addition of cisplatin to DNA also causes changes in the UVRR spectrum, consistent with binding of platinum to purine N7 and disruption of hydrogen-bonding interactions between base pairs. Equally important is that transplatin treatment of DNA generates similar UVRR spectral changes, when compared to cisplatin-treated samples. Kinetic analysis, performed by monitoring decreases of the 1492 cm -1 band, reveals biphasic kinetics and is consistent with a two-step binding mechanism for both platinum compounds. For cisplatin-DNA, the rate constants (6.8 × 10 -5 and 6.5 × 10 -6 s -1 ) are assigned to the formation of monofunctional adducts and to bifunctional, intrastrand cross-linking, respectively. In transplatin-DNA, there is a 3.4-fold decrease in the rate constant of the slow phase, compared with the cisplatin samples. This change is attributed to generation of interstrand, rather than intrastrand, adducts. This longer reaction time may result in increased competition in the cellular environment and account, at least in part, for the lower pharmacological efficacy of transplatin.

  5. INTERACTION OF IRON(II MIXED-LIGAND COMPLEXES WITH DNA: BASE-PAIR SPECIFICITY AND THERMAL DENATURATION STUDIES

    Directory of Open Access Journals (Sweden)

    Mudasir Mudasir

    2010-06-01

    Full Text Available A research about base-pair specificity of the DNA binding of [Fe(phen3]2+, [Fe(phen2(dip]2+ and [Fe(phen(dip2]2+ complexes and the effect of calf-thymus DNA (ct-DNA binding of these metal complexes on thermal denaturation of ct-DNA has been carried out. This research is intended to evaluate the preferential binding of the complexes to the sequence of DNA (A-T or G-C sequence and to investigate the binding strength and mode upon their interaction with DNA. Base-pair specificity of the DNA binding of the complexes was determined by comparing the equilibrium binding constant (Kb of each complex to polysynthetic DNA that contain only A-T or G-C sequence. The Kb value of the interaction was determined by spectrophotometric titration and thermal denaturation temperature (Tm was determined by monitoring the absorbance of the mixture solution of each complex and ct-DNA at λ =260 nm as temperature was elevated in the range of 25 - 100 oC. Results of the study show that in general all iron(II complexes studied exhibit a base-pair specificity in their DNA binding to prefer the relatively facile A-T sequence as compared to the G-C one. The thermal denaturation experiments have demonstrated that Fe(phen3]2+ and [Fe(phen2(dip]2+ interact weakly with double helical DNA via electrostatic interaction as indicated by insignificant changes in melting temperature, whereas [Fe(phen2(dip]2+  most probably binds to DNA in mixed modes of interaction, i.e.: intercalation and electrostatic interaction. This conclusion is based on the fact that the binding of [Fe(phen2(dip]2+ to ct-DNA moderately increase the Tm value of ct- DNA   Keywords: DNA Binding, mixed-ligand complexes

  6. Studies on the arctiin and its interaction with DNA by spectral methods

    International Nuclear Information System (INIS)

    Sun Yantao; Zhang Hanqi; Bi Shuyun; Zhou Xiaofu; Wang Liang; Yan Yongsheng

    2011-01-01

    The emission spectra of arctiin were determined under various experimental conditions. In addition, a fluorescence method was developed to obtain the binding constants and sites of the interaction between arctiin and DNA. A competitive binding experiment and melting temperature mensuration were carried out to investigate the binding mechanism of arctiin and DNA. The experimental results showed that the interaction between arctiin and DNA belongs to a groove binding mode. - Highlights: → Determined the emission spectra of arctiin by fluorescence spectrometry. → Obtain the binding constants and sites of interaction between arctiin and DNA. → Calculate the binding parameters according an improved calculation method.

  7. In vitro fluorescence studies of transcription factor IIB-DNA interaction.

    Science.gov (United States)

    Górecki, Andrzej; Figiel, Małgorzata; Dziedzicka-Wasylewska, Marta

    2015-01-01

    General transcription factor TFIIB is one of the basal constituents of the preinitiation complex of eukaryotic RNA polymerase II, acting as a bridge between the preinitiation complex and the polymerase, and binding promoter DNA in an asymmetric manner, thereby defining the direction of the transcription. Methods of fluorescence spectroscopy together with circular dichroism spectroscopy were used to observe conformational changes in the structure of recombinant human TFIIB after binding to specific DNA sequence. To facilitate the exploration of the structural changes, several site-directed mutations have been introduced altering the fluorescence properties of the protein. Our observations showed that binding of specific DNA sequences changed the protein structure and dynamics, and TFIIB may exist in two conformational states, which can be described by a different microenvironment of W52. Fluorescence studies using both intrinsic and exogenous fluorophores showed that these changes significantly depended on the recognition sequence and concerned various regions of the protein, including those interacting with other transcription factors and RNA polymerase II. DNA binding can cause rearrangements in regions of proteins interacting with the polymerase in a manner dependent on the recognized sequences, and therefore, influence the gene expression.

  8. Cytogenetic evaluation and DNA interaction studies of the food colorants amaranth, erythrosine and tartrazine.

    Science.gov (United States)

    Mpountoukas, Panagiotis; Pantazaki, Anastasia; Kostareli, Efterpi; Christodoulou, Pantelitsa; Kareli, Dimitra; Poliliou, Stamatia; Mourelatos, Costas; Lambropoulou, Vasso; Lialiaris, Theodore

    2010-10-01

    Food coloring agents, amaranth, erythrosine and tartrazine have been tested at 0.02-8mM in human peripheral blood cells in vitro, in order to investigate their genotoxic, cytotoxic and cytostatic potential. Amaranth at the highest concentration (8mM) demonstrates high genotoxicity, cytostaticity and cytotoxicity. The frequency of SCEs/cell was increased 1.7 times over the control level. Additionally, erythrosine at 8, 4 and 2mM shows a high cytotoxicity and cytostaticity. Finally, tartrazine seems to be toxic at 8 and 4mM. No signs of genotoxicity were observed. Reversely, tartrazine showed cytotoxicity at 1 and 2mM. Furthermore, spectroscopic titration studies for the interaction of these food additives with DNA showed that these dyes bind to calf thymus DNA and distinct isosbestic points are observed clearly suggesting binding of the dyes to DNA. Additionally DNA electrophoretic mobility experiments showed that these colorants are obviously capable for strong binding to linear dsDNA causing its degradation. PCR amplification of all DNA fragments (which previously were pre-treated with three different concentrations of the colorants, extracted from agarose gel after separation and then purified), seems to be attenuated with a manner dye concentration-dependent reflecting in a delayed electrophoretic mobility due to the possible binding of some molecules of the dyes. Evaluation of the data and curves were obtained after quantitative and qualitative analysis of the lanes of the gel by an analyzer computer program. Our results indicate that these food colorants had a toxic potential to human lymphocytes in vitro and it seems that they bind directly to DNA. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  9. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage Phi29.

    Science.gov (United States)

    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J; Smith, Douglas E

    2014-06-20

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine(3+) causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation.

  10. An NMR study of the covalent and noncovalent interactions of CC-1065 and DNA

    International Nuclear Information System (INIS)

    Scahill, T.A.; Jensen, R.M.; Swenson, D.H.; Hatzenbuhler, N.T.; Petzold, G.; Wierenga, W.; Brahme, N.D.

    1990-01-01

    The binding of the antitumor drug CC-1065 has been studied with nuclear magnetic resonance (NMR) spectroscopy. This study involves two parts, the elucidation of the covalent binding site of the drug to DNA and a detailed investigation of the noncovalent interactions of CC-1065 with a DNA fragment through analysis of 2D NOE (NOESY) experiments. A CC-1065-DNA adduct was prepared, and an adenine adduct was released upon heating. NMR ( 1 H and 13 C) analysis of the adduct shows that the drug binds to N3 of adenine by reaction of its cyclopropyl group. The reaction pathway and product formed were determined by analysis of the 13 C DEPT spectra. An octamer duplex, d(CGATTAGC·GCTAATCG), was synthesized and used in the interaction study of CC-1065 and the oligomer. The duplex and the drug-octamer complex were both analyzed by 2D spectroscopy (COSY, NOESY). The relative intensity of the NOEs observed between the drug (CC-1065) and the octamer duplex shows conclusively that the drug is located in the minor groove, covalently attached to N3 of adenine 6 and positioned from the 3' → 5' end in relation to strand A [d(CGATTA 6 GC)]. A mechanism for drug binding and stabilization can be inferred from the NOE data and model-building studies

  11. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

    Energy Technology Data Exchange (ETDEWEB)

    Dusinska, Maria, E-mail: Maria.DUSINSKA@nilu.no [CEE-Health Effects Group, NILU - Norwegian Institute for Air Research, Kjeller (Norway); Staruchova, Marta; Horska, Alexandra [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia); Smolkova, Bozena [Laboratory of Cancer Genetics, Cancer Research Institute of the Slovak Academy of Sciences, Bratislava (Slovakia); Collins, Andrew [Department of Nutrition, Faculty of Medicine, University of Oslo (Norway); Bonassi, Stefano [Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome (Italy); Volkovova, Katarina [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia)

    2012-08-01

    Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

  12. Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

    International Nuclear Information System (INIS)

    Dusinska, Maria; Staruchova, Marta; Horska, Alexandra; Smolkova, Bozena; Collins, Andrew; Bonassi, Stefano; Volkovova, Katarina

    2012-01-01

    Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

  13. Spectroscopic study on the interaction of eugenol with salmon sperm DNA in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bi Shuyun, E-mail: sy_bi@sina.com [College of Chemistry, Changchun Normal University, Changchun 130032 (China); Yan Lili; Wang Yu; Pang Bong; Wang Tianjiao [College of Chemistry, Changchun Normal University, Changchun 130032 (China)

    2012-09-15

    Fluorescence spectra, absorption spectra, melting temperature, ionic strength effect, and viscosity experiments were described that characterize the interaction of eugenol with salmon sperm DNA in vitro. Eugenol was found to bind but weakly to DNA, with binding constants of 4.23 Multiplication-Sign 10{sup 3}, 3.62 Multiplication-Sign 10{sup 3} and 2.47 Multiplication-Sign 10{sup 3} L mol{sup -1} at 18, 28 and 38 Degree-Sign C respectively. The Stern-Volmer plots at different temperatures suggested that the quenching type of fluorescence of eugenol by DNA was a static quenching. Both the relative viscosity and the melting temperature of DNA were increased by the addition of eugenol. The changes of ionic strength had no affect on the binding. In addition, the binding constant of eugenol with single stranded DNA (ssDNA) was larger than that of eugenol with double stranded DNA (dsDNA). These results revealed that the binding mode of eugenol to DNA was intercalative binding. The thermodynamic parameters {Delta}H, {Delta}G and {Delta}S were also obtained according to the Van't Hoff equations, which suggested that hydrogen bond or van der Waals force might play an important role in a binding of eugenol to DNA. Based on the theory of the Foerster energy transference, the binding distance between DNA and eugenol was determined as 4.40 nm, indicating that the static fluorescence quenching of eugenol by DNA was also a non-radiation energy transfer process. - Highlights: Black-Right-Pointing-Pointer DNA quenched the fluorescence of eugenol. Black-Right-Pointing-Pointer Binding constant, binding site and binding force were determined. Black-Right-Pointing-Pointer Binding mode of eugenol to DNA was intercalative. Black-Right-Pointing-Pointer Energy transfer occurred between eugenol and DNA.

  14. Study of neutral red interaction with DNA by resolution of rank deficient multi-way fluorescence data

    DEFF Research Database (Denmark)

    Moghaddam, Fatemeh Ghasemi; Kompany Zare, Mohsen; Gholami, Somayeh

    2012-01-01

    The interaction of neutral red (NR) as an efficient anticancer drug with DNA was studied under physiological pH condition. Three-way data array were recorded by measuring excitation-emission fluorescence during the titration of neutral red with DNA at constant pH. The acid-base equilibrium constant...

  15. Spectroscopic studies of the interaction between pirimicarb and calf thymus DNA

    Science.gov (United States)

    Zhang, Guowen; Hu, Xing; Pan, Junhui

    2011-02-01

    The interaction between pirimicarb and calf thymus DNA in physiological buffer (pH 7.4) was investigated with the use of Neutral Red (NR) dye as a spectral probe by UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopy, as well as viscosity measurements and DNA melting techniques. The results revealed that an intercalation binding should be the interaction mode of pirimicarb to DNA. CD spectra indicated that pirimicarb induced conformational changes of DNA. The binding constants of pirimicarb with DNA were obtained by the fluorescence quenching method. The thermodynamic parameters, enthalpy change (Δ Hθ) and entropy change (Δ Sθ) were calculated to be -52.13 ± 2.04 kJ mol -1 and -108.8 ± 6.72 J mol -1 K -1 according to the van't Hoff equation, which suggested that hydrogen bonds and van der Waals forces might play a major role in the binding of pirimicarb to DNA. Further, the alternative least squares (ALS) method was applied to resolve a complex two-way array of the absorption spectra data, which provided simultaneously the concentration information for the three reaction components, pirimicarb, NR and DNA-NR. This ALS analysis indicated that the intercalation of pirimicarb into the DNA by substituting for NR in the DNA-NR complex.

  16. Spectroscopic studies of the interaction between pirimicarb and calf thymus DNA.

    Science.gov (United States)

    Zhang, Guowen; Hu, Xing; Pan, Junhui

    2011-02-01

    The interaction between pirimicarb and calf thymus DNA in physiological buffer (pH 7.4) was investigated with the use of Neutral Red (NR) dye as a spectral probe by UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopy, as well as viscosity measurements and DNA melting techniques. The results revealed that an intercalation binding should be the interaction mode of pirimicarb to DNA. CD spectra indicated that pirimicarb induced conformational changes of DNA. The binding constants of pirimicarb with DNA were obtained by the fluorescence quenching method. The thermodynamic parameters, enthalpy change (ΔHθ) and entropy change (ΔSθ) were calculated to be -52.13±2.04 kJ mol(-1) and -108.8±6.72 J mol(-1) K(-1) according to the van't Hoff equation, which suggested that hydrogen bonds and van der Waals forces might play a major role in the binding of pirimicarb to DNA. Further, the alternative least squares (ALS) method was applied to resolve a complex two-way array of the absorption spectra data, which provided simultaneously the concentration information for the three reaction components, pirimicarb, NR and DNA-NR. This ALS analysis indicated that the intercalation of pirimicarb into the DNA by substituting for NR in the DNA-NR complex. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. DNA interaction, antioxidant activity, and bioactivity studies of two ruthenium(II) complexes

    Science.gov (United States)

    Han, Bing-Jie; Jiang, Guang-Bin; Yao, Jun-Hua; Li, Wei; Wang, Ji; Huang, Hong-Liang; Liu, Yun-Jun

    2015-01-01

    Two new ruthenium(II) polypyridyl complexes [Ru(dmb)2(dcdppz)](ClO4)2 (1) and [Ru(bpy)2(dcdppz)](ClO4)2 (2) were prepared and characterized. The crystal structure of the complex 2 was solved by single crystal X-ray diffraction. The complex crystallizes in the monoclinic system, space group P21/n with a = 12.9622(14) Å, b = 17.1619(19) Å, c = 22.7210(3) Å, β = 100.930(2)°, R = 0.0536, Rω = 0.1111. The DNA-binding constants for complexes 1 and 2 were determined to be 1.92 × 105 (s = 1.72) and 2.24 × 105 (s = 1.86) M-1, respectively. The DNA-binding behaviors showed that complexes 1 and 2 interact with DNA by intercalative mode. The antioxidant activities of the ligand and the complexes were performed. Ligand, dcdppz, has no cytotoxicity against the selected cell lines. Complex 1 shows higher cytotoxicity than complex 2, but lower than cisplatin toward selected cell lines. The apoptosis and cell cycle arrest were investigated, and the apoptotic mechanism of BEL-7402 cells was studied by reactive oxygen species (ROS), mitochondrial membrane potential and western blot analysis. Complex 1 induces apoptosis in BEL-7402 cells through ROS-mediated mitochondrial dysfunction pathway and by regulating the expression of Bcl-2 family proteins.

  18. DNA interaction studies of sesamol (3,4-methylenedioxyphenol) food additive.

    Science.gov (United States)

    Kashanian, Soheila; Tahmasian Ghobadi, Ameneh; Roshanfekr, Hamideh; Shariati, Zohreh

    2013-02-01

    The interaction of native calf thymus DNA (CT-DNA) with sesamol (3,4-methylenedioxyphenol) in Tris-HCl buffer at neutral pH 7.4 was monitored by absorption spectrophotometry, viscometry and spectrofluorometry. It is found that sesamol molecules could interact with DNA outside and/or groove binding modes, as are evidenced by: hyperchromism in UV absorption band, very slow decrease in specific viscosity of DNA, and small increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of sesamol, which indicates that it is able to partially release the bound MB. Furthermore, the enthalpy and entropy of the reaction between sesamol and CT-DNA showed that the reaction is enthalpy-favored and entropy-disfavored (ΔH = -174.08 kJ mol(-1); ΔS = -532.92 J mol(-1) K(-1)). The binding constant was determined using absorption measurement and found to be 2.7 × 10(4) M(-1); its magnitude suggests that sesamol interacts to DNA with a high affinity.

  19. Spectroscopic studies on the interaction of mimosine with BSA and DNA

    Science.gov (United States)

    Baltazar, C. J.; Mun, R.; Tajmir-Riahi, H. A.; Bariyanga, J.

    2018-06-01

    Mimosine has shown antitumor activity towards cancer cells. It has also been found to inhibit deoxyribonucleic acid (DNA) but the interaction is not fully understood. Here we report the results of investigation of its interactions with bovine serum albumin (BSA) and DNA in aqueous solution (pH 7.4) using FTIR and UV spectroscopic methods. Mimosine was found to disrupt the conformation of BSA by reducing its α-helix component and promoting a partial unfolding of the protein. In addition, the results indicated that mimosine may bind to DNA by electrostatic attractions via phosphate groups and grooves. The overall binding constant of DNA -mimosine complex was 5 × 10 3 M-1.

  20. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage phi29

    OpenAIRE

    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J.; Smith, Douglas E.

    2014-01-01

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine3+ causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interacti...

  1. Dynamics of DNA conformations and DNA-protein interaction

    DEFF Research Database (Denmark)

    Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen

    2005-01-01

    Optical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA...... denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed...... in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent...

  2. Molecular spectroscopic and thermodynamic studies on the interaction of anti-platelet drug ticlopidine with calf thymus DNA

    Science.gov (United States)

    Afrin, Shumaila; Rahman, Yusra; Sarwar, Tarique; Husain, Mohammed Amir; Ali, Abad; Shamsuzzaman; Tabish, Mohammad

    2017-11-01

    Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV-visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 103 M- 1, which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results.

  3. A study on interaction of DNA molecules and carbon nanotubes for an effective ejection of the molecules

    International Nuclear Information System (INIS)

    Wu, N.; Wang, Q.

    2012-01-01

    The ejection of DNA molecules from carbon nanotubes is reported from interaction energy perspectives by molecular dynamics simulations. The critical ejection energy, which is to be applied to a DNA molecule for a successful ejection from a carbon nanotube, is investigated based on a study on the friction and binding energy between the DNA molecule and the tube. An effective ejection is realized by subjecting a kinetic energy on the DNA molecule that is larger than the solved critical ejection energy. In addition, the relationship between ejection energies and sizes of DNA molecules and carbon nanotubes is investigated. -- Highlights: ► Report the ejection of DNA molecules from CNTs from interaction energy perspectives. ► Develop a methodology for the critical energy of an effective ejection of a DNA molecule from a CNT. ► Present the relationship between critical ejection energies and sizes of DNA molecules and CNTs. ► Provide a general guidance on the ejection of encapsulated molecules from CNTs.

  4. Multi-spectroscopic and molecular docking studies on the interaction of darunavir, a HIV protease inhibitor with calf thymus DNA.

    Science.gov (United States)

    Shi, Jie-Hua; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi

    2018-03-15

    Molecular interaction of darunavir (DRV), a HIV protease inhibitor with calf thymus deoxyribonucleic acid (ct-DNA) was studied in physiological buffer (pH7.4) by multi-spectroscopic approaches hand in hand with viscosity measurements and molecular docking technique. The UV absorption and fluorescence results together revealed the formation of a DRV-ct-DNA complex having binding affinities of the order of 10 3 M -1 , which was more in keeping with the groove binding. The results that DRV bound to ct-DNA via groove binding mode was further evidenced by KI quenching studies, viscosity measurements, competitive binding investigations with EB and Rhodamine B and CD spectral analysis. The effect of ionic strength indicated the negligible involvement of electrostatic interaction between DRV and ct-DNA. The thermodynamic parameters regarding the binding interaction of DRV with ct-DNA in terms of enthalpy change (ΔH 0 ) and entropy change (ΔS 0 ) were -63.19kJ mol -1 and -141.92J mol -1 K -1 , indicating that hydrogen bonds and van der Waals forces played a predominant role in the binding process. Furthermore, molecular simulation studies suggested that DRV molecule was prone to bind in the A-T rich region of the minor groove of DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Experimental and molecular docking studies on DNA binding interaction of adefovir dipivoxil: Advances toward treatment of hepatitis B virus infections

    Science.gov (United States)

    Shahabadi, Nahid; Falsafi, Monireh

    The toxic interaction of adefovir dipivoxil with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove binding mode. The binding constant of UV-visible and the number of binding sites were 3.33 ± 0.2 × 104 L mol-1and 0.99, respectively. The fluorimetric studies showed that the reaction between the drug and CT-DNA is exothermic (ΔH = 34.4 kJ mol-1; ΔS = 184.32 J mol-1 K-1). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of CT-DNA in the presence of adefovir dipivoxil, which verified the groove binding mode. Furthermore, the drug induces detectable changes in its viscosity. The molecular modeling results illustrated that adefovir strongly binds to groove of DNA by relative binding energy of docked structure -16.83 kJ mol-1. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with bio macromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo.

  6. Thermodynamic study of rhodamine 123-calf thymus DNA interaction: determination of calorimetric enthalpy by optical melting study.

    Science.gov (United States)

    Masum, Abdulla Al; Chakraborty, Maharudra; Pandya, Prateek; Halder, Umesh Chandra; Islam, Md Maidul; Mukhopadhyay, Subrata

    2014-11-20

    In this paper, the interaction of rhodamine123 (R123) with calf thymus DNA has been studied using molecular modeling and other biophysical methods like UV-vis spectroscopy, fluoremetry, optical melting, isothermal titration calorimetry, and circular dichroic studies. Results showed that the binding energy is about -6 to -8 kcal/mol, and the binding process is favored by both negative enthalpy change and positive entropy change. A new method to determine different thermodynamic properties like calorimetric enthalpy and heat capacity change has been introduced in this paper. The obtained data has been crossed-checked by other methods. After dissecting the free-energy contribution, it was observed that the binding was favored by both negative hydrophobic free energy and negative molecular free energy which compensated for the positive free energies due to the conformational change loss of rotational and transitional freedom of the DNA helix.

  7. NMR studies of DNA oligomers and their interactions with minor groove binding ligands

    Energy Technology Data Exchange (ETDEWEB)

    Fagan, Patricia A. [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    The cationic peptide ligands distamycin and netropsin bind noncovalently to the minor groove of DNA. The binding site, orientation, stoichiometry, and qualitative affinity of distamycin binding to several short DNA oligomers were investigated by NMR spectroscopy. The oligomers studied contain A,T-rich or I,C-rich binding sites, where I = 2-desaminodeoxyguanosine. I•C base pairs are functional analogs of A•T base pairs in the minor groove. The different behaviors exhibited by distamycin and netropsin binding to various DNA sequences suggested that these ligands are sensitive probes of DNA structure. For sites of five or more base pairs, distamycin can form 1:1 or 2:1 ligand:DNA complexes. Cooperativity in distamycin binding is low in sites such as AAAAA which has narrow minor grooves, and is higher in sites with wider minor grooves such as ATATAT. The distamycin binding and base pair opening lifetimes of I,C-containing DNA oligomers suggest that the I,C minor groove is structurally different from the A,T minor groove. Molecules which direct chemistry to a specific DNA sequence could be used as antiviral compounds, diagnostic probes, or molecular biology tools. The author studied two ligands in which reactive groups were tethered to a distamycin to increase the sequence specificity of the reactive agent.

  8. DNA interaction studies of new nano metal based anticancer agent: validation by spectroscopic methods

    International Nuclear Information System (INIS)

    Tabassum, Sartaj; Chandra Sharma, Girish; Arjmand, Farukh; Azam, Ameer

    2010-01-01

    A new nano dimensional heterobimetallic Cu-Sn containing complex as a potential drug candidate was designed, synthesized and characterized by analytical and spectral methods. The electronic absorption and electron paramagnetic resonance parameters of the complex revealed that the Cu(II) ion exhibits a square pyramidal geometry with the two pyrazole nitrogen atoms, the amine nitrogen atom and the carboxylate oxygen of the phenyl glycine chloride ligand located at the equatorial sites and the coordinated chloride ion occupying an apical position. 119 Sn NMR spectral data showed a hexa-coordinated environment around the Sn(IV) metal ion. TEM, AFM and XRD measurements illustrate that the complex could induce the condensation of CT-DNA to a particulate nanostructure. The interaction of the Cu-Sn complex with CT-DNA was investigated by UV-vis absorption and emission spectroscopy, as well as cyclic voltammetric measurements. The results indicated that the complex interacts with DNA through an electrostatic mode of binding with an intrinsic binding constant K b = 8.42 x 10 4 M -1 . The Cu-Sn complex exhibits effective cleavage of pBR322 plasmid DNA by an oxidative cleavage mechanism, monitored at different concentrations both in the absence and in the presence of reducing agents.

  9. DNA interaction studies of new nano metal based anticancer agent: validation by spectroscopic methods

    Energy Technology Data Exchange (ETDEWEB)

    Tabassum, Sartaj; Chandra Sharma, Girish; Arjmand, Farukh [Department of Chemistry, Aligarh Muslim University, Aligarh-202002 (India); Azam, Ameer [Center of Excellence in Materials Science (Nanomaterials), Department of Applied Physics, Aligarh Muslim University, Aligarh 202002, UP (India)

    2010-05-14

    A new nano dimensional heterobimetallic Cu-Sn containing complex as a potential drug candidate was designed, synthesized and characterized by analytical and spectral methods. The electronic absorption and electron paramagnetic resonance parameters of the complex revealed that the Cu(II) ion exhibits a square pyramidal geometry with the two pyrazole nitrogen atoms, the amine nitrogen atom and the carboxylate oxygen of the phenyl glycine chloride ligand located at the equatorial sites and the coordinated chloride ion occupying an apical position. {sup 119}Sn NMR spectral data showed a hexa-coordinated environment around the Sn(IV) metal ion. TEM, AFM and XRD measurements illustrate that the complex could induce the condensation of CT-DNA to a particulate nanostructure. The interaction of the Cu-Sn complex with CT-DNA was investigated by UV-vis absorption and emission spectroscopy, as well as cyclic voltammetric measurements. The results indicated that the complex interacts with DNA through an electrostatic mode of binding with an intrinsic binding constant K{sub b} = 8.42 x 10{sup 4} M{sup -1}. The Cu-Sn complex exhibits effective cleavage of pBR322 plasmid DNA by an oxidative cleavage mechanism, monitored at different concentrations both in the absence and in the presence of reducing agents.

  10. Study of DNA interactions with bifenthrin by spectroscopic techniques and molecular modeling

    Science.gov (United States)

    Zhu, Pan; Zhang, Guowen; Ma, Yadi; Zhang, Yepeng; Miao, Hong; Wu, Yongning

    2013-08-01

    The interaction between bifenthrin (BF) and calf thymus DNA (ctDNA) in physiological buffer (pH 7.4) was investigated by UV-vis absorption, fluorescence, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy, coupled with viscosity measurements and molecular docking techniques. It was found that BF molecular could intercalate into the base pairs of ctDNA as evidenced by significant increases in absorption intensity, fluorescence polarization and relative viscosity of ctDNA, decrease in iodide quenching effect, and induced CD spectral changes. The association constant of BF with ctDNA was evaluated to be in the order of 104 L mol-1. Thermodynamic analysis of the binding data obtained at different temperatures suggested that the binding process was primarily driven by hydrogen bonds and van der Waals forces, as the values of the enthalpy change (ΔH) and the entropy change (ΔS) were calculated to be -31.13 ± 1.89 kJ mol-1 and -22.79 ± 1.21 J mol-1 K-1, respectively. The results of FT-IR spectra and molecular docking showed that a specific binding mainly existed between BF and adenine and guanine bases.

  11. Genotoxicity studies on DNA-interactive telomerase inhibitors with application as anti-cancer agents.

    Science.gov (United States)

    Harrington, Dean J; Cemeli, Eduardo; Carder, Joanna; Fearnley, Jamie; Estdale, Sian; Perry, Philip J; Jenkins, Terence C; Anderson, Diana

    2003-01-01

    Telomerase-targeted strategies have aroused recent interest in anti-cancer chemotherapy, because DNA-binding drugs can interact with high-order tetraplex rather than double-stranded (duplex) DNA targets in tumour cells. However, the protracted cell-drug exposure times necessary for clinical application require that telomerase inhibitory efficacy must be accompanied by both low inherent cytotoxicity and the absence of mutagenicity/genotoxicity. For the first time, the genotoxicity of a number of structurally diverse DNA-interactive telomerase inhibitors is examined in the Ames test using six Salmonella typhimurium bacterial strains (TA1535, TA1537, TA1538, TA98, TA100, and TA102). DNA damage induced by each agent was also assessed using the Comet assay with human lymphocytes. The two assay procedures revealed markedly different genotoxicity profiles that are likely to reflect differences in metabolism and/or DNA repair between bacterial and mammalian cells. The mutational spectrum for a biologically active fluorenone derivative, shown to be mutagenic in the TA100 strain, was characterised using a novel and rapid assay method based upon PCR amplification of a fragment of the hisG46 allele, followed by RFLP analysis. Preliminary analysis indicates that the majority (84%) of mutations induced by this compound are C --> A transversions at position 2 of the missense proline codon of the hisG46 allele. However, despite its genotoxic bacterial profile, this fluorenone agent gave a negative response in the Comet assay, and demonstrates how unwanted systemic effects (e.g., cytotoxicity and genotoxicity) can be prevented or ameliorated through suitable molecular fine-tuning of a candidate drug in targeted human tumour cells. Copyright 2003 Wiley-Liss, Inc.

  12. Carcinogen-DNA interaction study by base sequence footprinting. Final report, July 1, 1983-June 30, 1986

    International Nuclear Information System (INIS)

    Bases, R.

    1986-01-01

    Our previous studies on acetylaminofluorene (AAF) modified DNA demonstrated three kinds of structural changes in DNA of defined base sequence. For example, adduct formation by N-Aco-AAF was found at each guanine. We studied the interaction of IgG specific for AAF guanosine in an in vitro system using AAF modified phi X-174 rf DNA. We had expected to find protection against DNAase I digestion. Instead, when the DNA was immunobound to an inert matrix via the IgG, DNAase I digestion was enhanced 20 fold without changing the base sequence pattern of digestion. DNAase I hypersensitive sites are a necessary but not a sufficient condition for transcription. Moreover, some hypersensitive sites are stably propagated, independent of the continued presence of the inducer. Stability of these hypersensitive sites in the absence of their inducer suggests that they can be propagated. It appeared likely that distortion of DNA by a carcinogen adduct such as AAF, and the interaction of this modified DNA with a specific protein such as IgG or cellular proteins might inappropriately enhance the transcription of specific genes. That hypothesis will be tested; surprisingly, little is known about the early action of carcinogens on expression of specific genes. 34 refs., 2 figs., 1 tab

  13. Interaction studies of resistomycin from Streptomyces aurantiacus AAA5 with calf thymus DNA and bovine serum albumin

    Science.gov (United States)

    Vijayabharathi, R.; Sathyadevi, P.; Krishnamoorthy, P.; Senthilraja, D.; Brunthadevi, P.; Sathyabama, S.; Priyadarisini, V. Brindha

    2012-04-01

    Resistomycin, a secondary metabolite produced by Streptomyces aurantiacus AAA5. The binding interaction of resistomycin with calf thymus DNA (CT DNA) and bovine serum albumin (BSA) was investigated by spectrophotometry, spectrofluorimetry, circular dichroism (CD) and synchronous fluorescence techniques under physiological conditions in vitro. Absorption spectral studies along with the fluorescence competition with ethidium bromide measurements and circular dichroism clearly suggest that the resistomycin bind with CT DNA relatively strong via groove binding. BSA interaction results revealed that the drug was found to quench the fluorescence intensity of the protein through a static quenching mechanism. The number of binding sites 'n' and apparent binding constant 'K' calculated according to the Scatchard equation exhibit a good binding property to bovine serum albumin protein. In addition, the results observed from synchronous fluorescence measurements clearly demonstrate the occurrence of conformational changes of BSA upon addition of the test compound.

  14. Scanning Probe Optical Tweezers: a new tool to study DNA-protein interactions

    NARCIS (Netherlands)

    Huisstede, J.H.G.

    2006-01-01

    The main goal of the work described in this thesis is to construct a microscope in which OT and scanning probe microscopy (SPM) are combined, to be able to localize proteins while simultaneously controlling the tension within the DNA molecule. This apparatus enables the study of the effect of

  15. A Qualitative and Quantitative Assay to Study DNA/Drug Interaction ...

    African Journals Online (AJOL)

    Purpose: To explore the use of restriction inhibition assay (RIA) to study the binding specificity of some anticancer drugs. Methods: A 448 bp DNA fragment derived from pBCKS+ plasmid (harboring the polylinker region with multiple restriction endonuclease sites) was used as a template for sequence selective inhibition of ...

  16. From nonspecific DNA-protein encounter complexes to the prediction of DNA-protein interactions.

    Directory of Open Access Journals (Sweden)

    Mu Gao

    2009-03-01

    Full Text Available DNA-protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA-protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA-protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA-protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA-protein interaction modes exhibit some similarity to specific DNA-protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Calpha deviation from native is up to 5 A from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA-protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.

  17. Real-Time Study of the Interaction between G-Rich DNA Oligonucleotides and Lead Ion on DNA Tetrahedron-Functionalized Sensing Platform by Dual Polarization Interferometry.

    Science.gov (United States)

    Wang, Shuang; Lu, Shasha; Zhao, Jiahui; Huang, Jianshe; Yang, Xiurong

    2017-11-29

    G-quadruplex plays roles in numerous physiological and pathological processes of organisms. Due to the unique properties of G-quadruplex (e.g., forming G4/hemin complexes with catalytic activity and electron acceptability, binding with metal ions, proteins, fluorescent ligands, and so on), it has been widely applied in biosensing. But the formation process of G-quadruplex is not yet fully understood. Here, a DNA tetrahedron platform with higher reproducibility, regenerative ability, and time-saving building process was coupled with dual polarization interferometry technique for the real-time and label-free investigation of the specific interaction process of guanine-rich singled-stranded DNA (G-rich ssDNA) and Pb 2+ . The oriented immobilization of probes greatly decreased the spatial hindrance effect and improved the accessibility of the probes to the Pb 2+ ions. Through real-time monitoring of the whole formation process of the G-quadruplex, we speculated that the probes on the tetrahedron platform initially stood on the sensing surface with a random coil conformation, then the G-rich ssDNA preliminarily formed unstable G-quartets by H-bonding and cation binding, subsequently forming a completely folded and stable quadruplex structure through relatively slow strand rearrangements. On the basis of these studies, we also developed a novel sensing platform for the specific and sensitive determination of Pb 2+ and its chelating agent ethylenediaminetetraacetic acid. This study not only provides a proof-of-concept for conformational dynamics of G-quadruplex-related drugs and pathogenes, but also enriches the biosensor tools by combining nanomaterial with interfaces technique.

  18. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    Full Text Available A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  19. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Science.gov (United States)

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  20. Anthraquinone-chalcone hybrids: synthesis, preliminary antiproliferative evaluation and DNA-interaction studies.

    Science.gov (United States)

    Marković, Violeta; Debeljak, Nevena; Stanojković, Tatjana; Kolundžija, Branka; Sladić, Dušan; Vujčić, Miroslava; Janović, Barbara; Tanić, Nikola; Perović, Milka; Tešić, Vesna; Antić, Jadranka; Joksović, Milan D

    2015-01-07

    Novel anthraquinone based chalcone compounds were synthesized starting from 1-acetylanthraquinone in a Claisen-Schmidt reaction and evaluated for their anticancer potential against three human cancer cell lines. Compounds 4a, 4b and 4j showed promising activity in inhibition of HeLa cells with IC50 values ranging from 2.36 to 2.73 μM and low cytotoxicity against healthy MRC-5 cell lines. The effects that compounds produces on the cell cycle were investigated by flow cytometry. It was found that 4a, 4b and 4j cause the accumulation of cells in the S and G2/M phases in a dose-dependent manner and induce caspase-dependent apoptosis. All of three compounds exhibit calf thymus DNA-binding activity. The determined binding constants by absorption titrations (2.65 × 10(3) M(-1), 1.36 × 10(3) M(-1)and 2.51 × 10(3) M(-1) of 4a/CT-DNA, 4b/CT-DNA and 4j/CT-DNA, respectively) together with fluorescence displacement analysis designate 4a, 4b and 4j as strong minor groove binders, but no cleavage of plasmid DNA was observed. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. Electrochemical study of the interaction between dsDNA and copper(I) using carbon paste and hanging mercury drop electrode.

    Science.gov (United States)

    Stanić, Z; Girousi, S

    2008-06-30

    The interaction of copper(I) with double-stranded (ds) calf thymus DNA was studied in solution and at the electrode surface by means of transfer voltammetry using a carbon paste electrode (CPE) as working electrode in 0.2 M acetate buffer solution (pH 5.0). As a result of the interaction of Cu(I) between the base pairs of the dsDNA, the characteristic peaks of dsDNA, due to the oxidation of guanine and adenine, increased and after a certain concentration of Cu(I) a new peak at +1.37 V appeared, probably due to the formation of a purine-Cu(I) complex (dsDNA-Cu(I) complex). Accordingly, the interaction of copper(I) with calf thymus dsDNA was studied in solution as well as at the electrode surface using hanging mercury drop electrode (HMDE) by means of alternating current voltammetry (AC voltammetry) in 0.3 M NaCl and 50 mM sodium phosphate buffer (pH 8.5) as supporting electrolyte. Its interaction with DNA is shown to be time dependent. Significant changes in the characteristic peaks of dsDNA were observed after addition of higher concentration of Cu(I) to a solution containing dsDNA, as a result of the interaction between Cu(I) and dsDNA. All the experimental results indicate that Cu(I) can bind to DNA by electrostatic binding and form an association complex.

  2. Hair Dye–DNA Interaction: Plausible Cause of Mutation

    Directory of Open Access Journals (Sweden)

    Swati Maiti

    2015-09-01

    Full Text Available Hair dye is one of the most popular cosmetic products which are used more widely and frequently to improve an individual’s appearance. Although the genotoxic effects of dye ingredients are widely reported, hair dye in its usable form is not reported extensively. In this contribution, we report the possible mode of interaction of hair dye with DNA which leads to genotoxicity. The effect of dye DNA interaction was studied on the most popular and globally used hair dye with Calf Thymus DNA and plasmid DNA. This interaction of dye DNA was studied by spectroscopic analyses and gel electrophoresis. The result had shown positive interaction of dye with DNA. Gel electrophoresis study confirms the binding of dye with DNA which results in linearization and fragmentation of the plasmid DNA. Dye–DNA interaction causes fragmentation and oxidation of DNA in absence of any catalyst, implies high toxicity of commercial hair dyes. Thus, it can be deduced from the present studies that hair dye in its usable form may lead to its penetration through skin affecting genomic DNA possesses genotoxic property and can be treated as one of the most common mutagen.

  3. Spectroscopic and molecular modeling study on the interaction of ctDNA with 3′-deoxy-3′-azido doxorubicin

    International Nuclear Information System (INIS)

    Geng, Shaoguang; Cui, Yanrui; Liu, Qingfeng; Cui, Fengling; Zhang, Guisheng; Chi, Yanwei; Peng, Hao

    2013-01-01

    The method of synthesizing 3′-deoxy-3′-azido doxorubicin (ADOX) directly from doxorubicin has been developed. This study presents the interaction between ADOX and calf thymus deoxyribonucleic acid (ctDNA) by using spectroscopic methods and molecular modeling techniques. Iodide quenching, fluorescence polarization, viscosity and molecular modeling studies of ADOX–ctDNA interactions indicated that ADOX was an intercalator of ctDNA and preferentially bound to C–G rich regions of ctDNA. Simultaneously, spectroscopic results indicated that the quenching mechanism of ADOX–ctDNA was a static quenching. According to thermodynamic parameters, electrostatic force played roles in the interaction of ADOX with ctDNA. -- Highlights: ●An approach to 3′-deoxy-3′-azido doxorubicin (ADOX) from doxorubicin was developed. ●The quenching mechanism of ADOX with ctDNA was a static quenching type. ●The binding mode between ADOX and ctDNA was intercalative binding. ●The results of molecular docking corroborated results of spectra investigations

  4. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A

    OpenAIRE

    Stoltenburg, Regina; Schubert, Thomas; Strehlitz, Beate

    2015-01-01

    A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for moni...

  5. New dinuclear palladium(II) complexes: Studies of the nucleophilic substitution reactions, DNA/BSA interactions and cytotoxic activity.

    Science.gov (United States)

    Ćoćić, Dušan; Jovanović, Snežana; Nišavić, Marija; Baskić, Dejan; Todorović, Danijela; Popović, Suzana; Bugarčić, Živadin D; Petrović, Biljana

    2017-10-01

    Six new dinuclear Pd(II) complexes, [{Pd(2,2'-bipy)Cl} 2 (μ-pz)](ClO 4 ) 2 (Pd1), [{Pd(dach)Cl} 2 (μ-pz)](ClO 4 ) 2 (Pd2), [{Pd(en)Cl} 2 (μ-pz)](ClO 4 ) 2 (Pd3), [{Pd(2,2'-bipy)Cl} 2 (μ-4,4'-bipy)](ClO 4 ) 2 (Pd4), [{Pd(dach)Cl} 2 (μ-4,4'-bipy)](ClO 4 ) 2 (Pd5) and [{Pd(en)Cl} 2 (μ-4,4'-bipy)](ClO 4 ) 2 (Pd6) (where 2,2'-bipy=2,2'-bipyridyl, pz=pyrazine, dach=trans-(±)-1,2-diaminocyclohexane, en=ethylenediamine, 4,4'-bipy=4,4'-bipyridyl) have been synthesized and characterized by elemental microanalysis, IR, 1 H NMR and MALDI-TOF mass spectrometry. The pK a values of corresponding diaqua complexes were determined by spectrophotometric pH titration. Substitution reactions with thiourea (Tu), l-methionine (l-Met), l-cysteine (l-Cys), l-histidine (l-His) and guanosine-5'-monophosphate (5'-GMP) were studied under the pseudo-first order conditions at pH7.2. Reactions of Pd1 with Tu, l-Met and l-Cys were followed by decomposition of complexes, while structures of dinuclear complexes were preserved during the substitution with nitrogen donors. Interactions with calf-thymus DNA (CT-DNA) were followed by absorption spectroscopy and fluorescence quenching measurements. All complexes can bind to CT-DNA exhibiting high intrinsic binding constants (K b =10 4 -10 5 M -1 ). Competitive studies with ethidium bromide (EB) have shown that complexes can displace DNA-bound EB. High values of binding constants towards bovine serum albumin protein (BSA) indicate good binding affinity. Finally, all complexes showed moderate to high cytotoxic activity against HeLa (human cervical epithelial carcinoma cell lines) and MDA-MB-231 (human breast epithelial carcinoma cell lines) tumor cell lines inducing apoptotic type cell death, whereas normal fibroblasts were significantly less sensitive. The impact on cell cycle of these cells was distinctive, where Pd4, Pd5 and Pd6 showed the most prominent effect arresting MDA-MB-231 (human lung fibroblast cell lines) cell in G1/S phase of cell

  6. DNA Origami Reorganizes upon Interaction with Graphite: Implications for High-Resolution DNA Directed Protein Patterning

    Directory of Open Access Journals (Sweden)

    Masudur Rahman

    2016-10-01

    Full Text Available Although there is a long history of the study of the interaction of DNA with carbon surfaces, limited information exists regarding the interaction of complex DNA-based nanostructures with the important material graphite, which is closely related to graphene. In view of the capacity of DNA to direct the assembly of proteins and optical and electronic nanoparticles, the potential for combining DNA-based materials with graphite, which is an ultra-flat, conductive carbon substrate, requires evaluation. A series of imaging studies utilizing Atomic Force Microscopy has been applied in order to provide a unified picture of this important interaction of structured DNA and graphite. For the test structure examined, we observe a rapid destabilization of the complex DNA origami structure, consistent with a strong interaction of single-stranded DNA with the carbon surface. This destabilizing interaction can be obscured by an intentional or unintentional primary intervening layer of single-stranded DNA. Because the interaction of origami with graphite is not completely dissociative, and because the frustrated, expanded structure is relatively stable over time in solution, it is demonstrated that organized structures of pairs of the model protein streptavidin can be produced on carbon surfaces using DNA origami as the directing material.

  7. DNA Origami Reorganizes upon Interaction with Graphite: Implications for High-Resolution DNA Directed Protein Patterning

    Science.gov (United States)

    Rahman, Masudur; Neff, David; Green, Nathaniel; Norton, Michael L.

    2016-01-01

    Although there is a long history of the study of the interaction of DNA with carbon surfaces, limited information exists regarding the interaction of complex DNA-based nanostructures with the important material graphite, which is closely related to graphene. In view of the capacity of DNA to direct the assembly of proteins and optical and electronic nanoparticles, the potential for combining DNA-based materials with graphite, which is an ultra-flat, conductive carbon substrate, requires evaluation. A series of imaging studies utilizing Atomic Force Microscopy has been applied in order to provide a unified picture of this important interaction of structured DNA and graphite. For the test structure examined, we observe a rapid destabilization of the complex DNA origami structure, consistent with a strong interaction of single-stranded DNA with the carbon surface. This destabilizing interaction can be obscured by an intentional or unintentional primary intervening layer of single-stranded DNA. Because the interaction of origami with graphite is not completely dissociative, and because the frustrated, expanded structure is relatively stable over time in solution, it is demonstrated that organized structures of pairs of the model protein streptavidin can be produced on carbon surfaces using DNA origami as the directing material. PMID:28335324

  8. Baculovirus proteins IE-1, LEF-3, and P143 interact with DNA in vivo: a formaldehyde cross-linking study

    International Nuclear Information System (INIS)

    Ito, Emma; Sahri, Daniela; Knippers, Rolf; Carstens, Eric B.

    2004-01-01

    IE-1, LEF-3, and P143 are three of six proteins encoded by Autographa californica nucleopolyhedrovirus (AcMNPV) essential for baculovirus DNA replication in transient replication assays. IE-1 is the major baculovirus immediate early transcription regulator. LEF-3 is a single-stranded DNA binding protein (SSB) and P143 is a DNA helicase protein. To investigate their interactions in vivo, we treated AcMNPV-infected Spodoptera frugiperda cells with formaldehyde and separated soluble proteins from chromatin by cell fractionation and cesium chloride equilibrium centrifugation. Up to 70% of the total LEF-3 appeared in the fraction of soluble, probably nucleoplasmic proteins, while almost all P143 and IE-1 were associated with viral chromatin in the nucleus. This suggests that LEF-3 is produced in quantities that are higher than needed for the coverage of single stranded regions that arise during viral DNA replication and is consistent with the hypothesis that LEF-3 has other functions such as the localization of P143 to the nucleus. Using a chromatin immunoprecipitation procedure, we present the first direct evidence of LEF-3, P143, and IE-1 proteins binding to closely linked sites on viral chromatin in vivo, suggesting that they may form replication complexes on viral DNA in infected cells

  9. Microarrays in ecological research: A case study of a cDNA microarray for plant-herbivore interactions

    Directory of Open Access Journals (Sweden)

    Gase Klaus

    2004-09-01

    Full Text Available Abstract Background Microarray technology allows researchers to simultaneously monitor changes in the expression ratios (ERs of hundreds of genes and has thereby revolutionized most of biology. Although this technique has the potential of elucidating early stages in an organism's phenotypic response to complex ecological interactions, to date, it has not been fully incorporated into ecological research. This is partially due to a lack of simple procedures of handling and analyzing the expression ratio (ER data produced from microarrays. Results We describe an analysis of the sources of variation in ERs from 73 hybridized cDNA microarrays, each with 234 herbivory-elicited genes from the model ecological expression system, Nicotiana attenuata, using procedures that are commonly used in ecologic research. Each gene is represented by two independently labeled PCR products and each product was arrayed in quadruplicate. We present a robust method of normalizing and analyzing ERs based on arbitrary thresholds and statistical criteria, and characterize a "norm of reaction" of ERs for 6 genes (4 of known function, 2 of unknown with different ERs as determined across all analyzed arrays to provide a biologically-informed alternative to the use of arbitrary expression ratios in determining significance of expression. These gene-specific ERs and their variance (gene CV were used to calculate array-based variances (array CV, which, in turn, were used to study the effects of array age, probe cDNA quantity and quality, and quality of spotted PCR products as estimates of technical variation. Cluster analysis and a Principal Component Analysis (PCA were used to reveal associations among the transcriptional "imprints" of arrays hybridized with cDNA probes derived from mRNA from N. attenuata plants variously elicited and attacked by different herbivore species and from three congeners: N. quadrivalis, N. longiflora and N. clevelandii. Additionally, the PCA

  10. Interaction of E. coli DNA with tobacco mesophyll protoplasts

    International Nuclear Information System (INIS)

    Heyn, R.F.

    1975-01-01

    This chapter is part of a dissertation dealing with the interaction of DNA with protoplasts. Having established the length of time during which tobacco mesophyll protoplasts do not synthesize DNA following their isolation, it is important to know the extent of DNA uptake just before the onset of DNA synthesis (and possible integration) and to find optimal conditions for this uptake. Therefore, the association of E. coli DNA with tobacco protoplasts was studied. Care should be taken with the interpretation of ''uptake'' results: adsorption phenomena play a very important role and may do so at the plasmalemma of naked protoplasts. To solve the problems involved, the use of radiation-damaged DNA was attempted. With E. coli DNA possessing a large number of thymine containing pyrimidine dimers, the loss of dimers from DNA recovered from treated protoplasts was tested in order to obtain an indication of ''real'' uptake. The results are reported

  11. Synthesis and characterization of new unsymmetrical Schiff base Zn (II) and Co (II) complexes and study of their interactions with bovin serum albumin and DNA by spectroscopic techniques

    Science.gov (United States)

    Sedighipoor, Maryam; Kianfar, Ali Hossein; Sabzalian, Mohammad R.; Abyar, Fatemeh

    2018-06-01

    Two novel tetra-coordinated Cobalt(II) and Zinc (II) chelate series with the general formula of [Co (L)·2H2O] (1) and [Zn (L)] (2) [L = N-2-hydroxyacetophenon-N‧-2-hydroxynaphthaldehyde-1,2 phenylenediimine)] with biologically active Schiff base ligands were synthesized and recognized by elemental analysis and multi-nuclear spectroscopy (IR and 1H and 13C NMR); then, their biological activities including DNA and protein interactions were studied. The interaction of the synthesized compounds with bovine serum albumin (BSA) was investigated via fluorescence spectroscopy, showing the affinity of the complexes for these proteins with relatively high binding constant values and the changed secondary BSA structure in the presence of the complexes. The interaction of these compounds with CT-DNA was considered by UV-Vis technique, emission titration, viscosity measurements, helix melting methods, and circular dichroism (CD) spectroscopy, confirming that the complexes were bound to CT-DNA by the intercalation binding mode. Furthermore, the complexes had the capability to displace the DNA-bound MB, as shown by the competitive studies of these complexes with methylene blue (MB), thereby suggesting the intercalation mode for the competition. Finally, the theoretical studies carried out by the docking method were performed to calculate the binding constants and recognize the binding site of the BSA and DNA by the complexes. In addition, in vitro and in silico studies showed that the compounds were degradable by bacterial and fungal biodegradation activities.

  12. Multi-spectroscopic method study the interaction of anti-inflammatory drug ketoprofen and calf thymus DNA and its analytical application

    Science.gov (United States)

    Guo, Hongqin; Cai, Changqun; Gong, Hang; Chen, Xiaoming

    2011-06-01

    Interactions of the anti-inflammatory drug ketoprofen with calf thymus DNA (ctDNA) in aqueous solution have been studied by multi-spectroscopic method including resonance light scattering (RLS) technique, ultraviolet spectra (UV), 1H NMR, etc. The characteristics of RLS spectra, the effective factors and optimum conditions of the reaction have been unequivocally investigated. Mechanism investigations have shown that ketoprofen can bind to ctDNA by groove binding and form large particles, which resulted in the enhancement of RLS intensity. In Critic acid-Na 2HPO 4 buffer (pH = 6.5), ketoprofen has a maximum peak 451.5 nm and the RLS intensity is remarkably enhanced by trace amount of ctDNA due to the interaction between ketoprofen and ctDNA. The enhancement of RLS signal is directly proportional to the concentration of ctDNA in the range of 1.20 × 10 -6-1.0 × 10 -5 mol/L, and its detection limit (3 σ) is 1.33 × 10 -9 mol/L. The method is simple, rapid, practical and relatively free from interference generated by coexisting substance, and was applied to the determination of trace amounts of nucleic acid in synthetic samples with satisfactory results.

  13. Part I. Generation of tailored radio-frequency pulses for NMR. Part II. Deuterium NMR studies of oriented DNA, and its interaction with water

    International Nuclear Information System (INIS)

    Brandes, R.

    1988-01-01

    A novel method for generating tailored radio-frequency pulses for use in NMR is presented. For this purpose, an inexpensive device based on analog audio filters was built. As an application, the superior selectivity of this method is shown by comparing it with a soft pulse excitation. The theoretical response of the magnetization to these tailored rf pulses is also calculated. Deuterium NMR line shapes of 2 H-labeled purine bases in solid, uniaxially oriented Li- and Na-DNA have been obtained. The spectral densities of motion were determined for the Li-DNA samples to test a model for uncorrelated, restricted base motion. For the first time, a 2 H spectrum is reported for 2 H labeled DNA in the liquid crystalline state. A procedure is outlined to separate the base motion from the DNA axis motion. In addition to the studies of DNA itself, the interaction of water (D 2 O) with samples of uniaxially oriented Na- and Li-DNA have been studied by high resolution 2 H NMR

  14. Aryl-1H-imidazole[4,5f][1,10]phenanthroline Cu(II) complexes: Electrochemical and DNA interaction studies.

    Science.gov (United States)

    Rajebhosale, Bharati S; Dongre, Shivali N; Deshpande, Sameer S; Kate, Anup N; Kumbhar, Anupa A

    2017-10-01

    The reaction of aryl imidazo[4,5f] [1,10]phenanthrolines with Cu(NO 3 ) 2 lead to the formation of Cu(II) complexes of the type [Cu(L)(NO 3 ) 2 ] where L=PIP, 2-(phenyl) [4,5f] imidazo phenanthroline; HPIP=2-(2-hydroxyphenyl)imidazo [4,5f] phenanthroline and NIP=2-(naphthyl) [4,5f] imidazo phenanthroline. The interaction of these complexes with calf thymus DNA has been studied using viscosity measurements, UV-visible and fluorescence spectroscopy. Chemical nuclease activity of these complexes has also been investigated. All complexes cleave DNA via oxidative pathway involving singlet oxygen. Molecular docking studies revealed that these complexes bind to DNA through minor groove. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Synthesis, characterization, DNA/protein interaction and cytotoxicity studies of Cu(II) and Co(II) complexes derived from dipyridyl triazole ligands

    Science.gov (United States)

    Zhang, Wei; Yao, Di; Wei, Yi; Tang, Jie; Bian, He-Dong; Huang, Fu-Ping; Liang, Hong

    2016-06-01

    Four different transition metal complexes containing dipyridyl triazole ligands, namely [Cu(abpt)2Cl2]·2H2O (1), [Cu(abpt)2(ClO4)2] (2), [Co2(abpt)2(H2O)2Cl2]·Cl2·4H2O (3) and [Co2(Hbpt)2(CH3OH)2(NO3)2] (4) have been designed, synthesized and further structurally characterized by X-ray crystallography, ESI-MS, elemental analysis, IR and Raman spectroscopy. In these complexes, the both ligands act as bidentate ligands with N, N donors. DNA binding interactions with calf thymus DNA (ct-DNA) of the ligand and its complexes 1 ~ 4 were investigated via electronic absorption, fluorescence quenching, circular dichroism and viscosity measurements as well as confocal Laser Raman spectroscopy. The results show these complexes are able to bind to DNA via the non-covalent mode i.e. intercalation and groove binding or electrostatic interactions. The interactions with bovine serum albumin (BSA) were also studied using UV-Vis and fluorescence spectroscopic methods which indicated that fluorescence quenching of BSA by these compounds was the presence of both static and dynamic quenching. Moreover, the in vitro cytotoxic effects of the complexes against four cell lines SK-OV-3, HL-7702, BEL7404 and NCI-H460 showed the necessity of the coordination action on the biological properties on the respective complex and that all four complexes exhibited substantial cytotoxic activity.

  16. Preliminary study of mechanism of action of SN38 derivatives. Physicochemical data, evidence of interaction and alkylation of DNA octamer d(GCGATCGC)2.

    Science.gov (United States)

    Naumczuk, Beata; Kawęcki, Robert; Bocian, Wojciech; Bednarek, Elżbieta; Sitkowski, Jerzy; Kozerski, Lech

    2017-02-01

    The synthesis of water-soluble SN38 derivatives is presented, and their stability in solutions used during drug development studies has been investigated. A preliminary study of mechanism of action of 9-aminomethyl SN38 is presented. Using NMR techniques, the interaction of the oligomer d(GCGATCGC) 2 is studied, showing that the terminal GC base pairs are the main site of interaction. Using pulsed field gradient spin echo and mass spectroscopy, evidence of a spontaneous alkylation reaction of the DNA oligomer with SN38 derivatives is presented. A proposed mechanism of reaction is suggested. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Carcinogen-DNA interaction study by base sequence footprinting. Progress report, July 1, 1985-January 21, 1986

    International Nuclear Information System (INIS)

    Bases, R.

    1986-01-01

    Acetyl-aminofluorene (AAF) modified plasmid pSV 2 CAT is being studied to learn how the adducts influence expression of chloramphenicol acetyltransferase (CAT) genes. phi X-174 RF DNA exhibits specific base sequence abnormalities induced by the formation of AAF adducts. The DNAase I sensitive state of AAF modified DNA sequences could presumably lead to enhanced expression of genes since it is a well-known characteristic of active or potentially active derepressed genes. DNAase I hypersensitive sites are necessary but not sufficient for transcription. We observed enhanced expression of CAT genes in CV-1 cells after transfection with modified plasmids, using electroporation to introduce the plasmids into the cells. 34 refs., 2 figs

  18. Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

    International Nuclear Information System (INIS)

    Stefanovsky, V.Yu.; Dimitrov, S.I.; Angelov, D.; Pashev, I.G.

    1989-01-01

    The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA

  19. Single-Molecule Manipulation of Double-Stranded DNA Using Optical Tweezers: Interaction Studies of DNA with RecA and YOYO-1

    NARCIS (Netherlands)

    Bennink, Martin L.; Scharer, Orlando D.; Kanaar, Ronald; Sakata-Sogawa, Kumiko; Schins, J.M.; Kanger, Johannes S.; de Grooth, B.G.; Greve, Jan

    1999-01-01

    By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first

  20. Interaction of sulforaphane with DNA and RNA.

    Directory of Open Access Journals (Sweden)

    Farzaneh Abassi Joozdani

    Full Text Available Sulforaphane (SFN is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN-DNA and -RNA complexes by Fourier transform infrared (FTIR and UV-Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2, while RNA binding is through G, U, A bases with some degree of SFN-phosphate (PO2 interaction. Overall binding constants were estimated to be K(SFN-DNA=3.01 (± 0.035×10(4 M(-1 and K(SFN-RNA= 6.63 (±0.042×10(3 M(-1. At high SFN concentration (SFN/RNA = 1/1, DNA conformation changed from B to A occurred, while RNA remained in A-family structure.

  1. Interaction of nogalamycin and analogs with DNA and other biopolymers

    Energy Technology Data Exchange (ETDEWEB)

    Krueger, W C [Univ. of Minnesota, Minneapolis; Pschigoda, L M; Schpok, S L.F.; Moscowitz, A.; McGovren, J P; Neta, P; Merritt, M V; Li, L H

    1981-01-01

    The interaction with calf thymus DNA of the anthracycline antibiotics, nogalamycin and its analogs, was studied by electronic absorption, circular dichroism (CD), thermal denaturation, solvent partition and pulse radiolysis techniques. The Scatchard, thermal denaturation (..delta..T/sub m/), difference circular dichroism (..delta..CD) and solvent partition binding parameters gave the same order of relative binding on a given lot of DNA, but some parameters were DNA-lot-dependent. In general, molecules containing the sugar moiety nogalose at C-7 or those having the natural or dis stereochemistry of nogalamycin at C-7 bound more strongly to DNA than did the molecules lacking nogalose or those with the opposite configuration at C-7 (con stereochemistry). This stereochemical-binding correlation differs from that found for adriamycin which has the con stereochemistry, but which binds strongly to DNA. Scatchard binding parameters could not be obtained from the pulse radiolysis or solvent partition techniques because of solubility difficulties.

  2. Photosensitive interaction of RSU 1069 with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, D.I.; Knox, R.J.; Skolimowski, I.M.; Zahoor, A.; Knight, R.C.

    1984-08-01

    RSU 1069 is a 2-nitroimidazole radiosensitizer with an aziridine-containing side chain. In light (360 nm) the absorbance maximum of the nitro group at 325 nm disappears, which is accompanied by expulsion of the nitro group as the nitrite ion. This photosensitive effect was used to determine separately the damage of DNA induced by the reduced nitro group and the alkylating property of the aziridine. The aziridine-induced DNA damage is maximized in the dark when the nitro group is either absent (electrolytically reduced prior to the addition of DNA) or non functional (unreduced). In the light, damage is reduced. Typical DNA damage includes helix disruption leading to single strand breaks and the release of thymidine. Alkaline filter elution studies show evidence only for strand breakage and none for cross-linking indicating the drug is capable of mono-functional alkylation only.

  3. Photosensitive interaction of RSU 1069 with DNA

    International Nuclear Information System (INIS)

    Edwards, D.I.; Knox, R.J.; Skolimowski, I.M.; Zahoor, A.; Knight, R.C.

    1984-01-01

    RSU 1069 is a 2-nitroimidazole radiosensitizer with an aziridine-containing side chain. In light (360 nm) the absorbance maximum of the nitro group at 325 nm disappears, which is accompanied by expulsion of the nitro group as the nitrite ion. This photosensitive effect was used to determine separately the damage of DNA induced by the reduced nitro group and the alkylating property of the aziridine. The aziridine-induced DNA damage is maximized in the dark when the nitro group is either absent (electrolytically reduced prior to the addition of DNA) or non functional (unreduced). In the light, damage is reduced. Typical DNA damage includes helix disruption leading to single strand breaks and the release of thymidine. Alkaline filter elution studies show evidence only for strand breakage and none for cross-linking indicating the drug is capable of mono-functional alkylation only

  4. Complexes of DNA bases and Watson-Crick base pairs interaction with neutral silver Agn (n = 8, 10, 12) clusters: a DFT and TDDFT study.

    Science.gov (United States)

    Srivastava, Ruby

    2018-03-01

    We study the binding of the neutral Ag n (n = 8, 10, 12) to the DNA base-adenine (A), guanine (G) and Watson-Crick -adenine-thymine, guanine-cytosine pairs. Geometries of complexes were optimized at the DFT level using the hybrid B3LYP functional. LANL2DZ effective core potential was used for silver and 6-31 + G ** was used for all other atoms. NBO charges were analyzed using the Natural population analysis. The absorption properties of Ag n -A,G/WC complexes were also studied using time-dependent density functional theory. The absorption spectra for these complexes show wavelength in the visible region. It was revealed that silver clusters interact more strongly with WC pairs than with isolated DNA complexes. Furthermore, it was found that the electronic charge transferred from silver to isolated DNA clusters are less than the electronic charge transferred from silver to the Ag n -WC complexes. The vertical ionization potential, vertical electron affinity, hardness, and electrophilicity index of Ag n -DNA/WC complexes have also been discussed.

  5. Particle Interactions in DNA-laden Flows

    International Nuclear Information System (INIS)

    Bybee, M D; Miller, G H; Trebotich, D

    2005-01-01

    Microfluidic devices are becoming state-of-the-art in many significant applications including pathogen detection, continuous monitoring, and drug delivery. Numerical algorithms which can simulate flows of complex fluids within these devices are needed for their development and optimization. A method is being developed at LLNL by Trebotich et. al. [30] for simulations of DNA-laden flows in complex microscale geometries such as packed bed reactors and pillar chips. In this method an incompressible Newtonian fluid is discretized with Cartesian grid embedded boundary methods, and the DNA is represented by a bead-rod polymer model. The fluid and polymer are coupled through a body force. In its current state, polymer-surface interactions are treated as elastic collisions between beads and surface, and polymer-polymer interactions are neglected. Implementation of polymer-polymer interactions is the main objective of this work. It is achieved by two methods: (1) a rigid constraint whereby rods elastically bounce off one another, and (2) a smooth potential acting between rods. In addition, a smooth potential is also implemented for the polymer-surface interactions. Background information will also be presented as well as related work by other researchers

  6. Particle Interactions in DNA-laden Flows

    Energy Technology Data Exchange (ETDEWEB)

    Bybee, M D; Miller, G H; Trebotich, D

    2005-12-20

    Microfluidic devices are becoming state-of-the-art in many significant applications including pathogen detection, continuous monitoring, and drug delivery. Numerical algorithms which can simulate flows of complex fluids within these devices are needed for their development and optimization. A method is being developed at LLNL by Trebotich et. al. [30] for simulations of DNA-laden flows in complex microscale geometries such as packed bed reactors and pillar chips. In this method an incompressible Newtonian fluid is discretized with Cartesian grid embedded boundary methods, and the DNA is represented by a bead-rod polymer model. The fluid and polymer are coupled through a body force. In its current state, polymer-surface interactions are treated as elastic collisions between beads and surface, and polymer-polymer interactions are neglected. Implementation of polymer-polymer interactions is the main objective of this work. It is achieved by two methods: (1) a rigid constraint whereby rods elastically bounce off one another, and (2) a smooth potential acting between rods. In addition, a smooth potential is also implemented for the polymer-surface interactions. Background information will also be presented as well as related work by other researchers.

  7. DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

    Science.gov (United States)

    Keyamura, Kenji; Katayama, Tsutomu

    2011-08-19

    Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

  8. DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

    Science.gov (United States)

    Keyamura, Kenji; Katayama, Tsutomu

    2011-01-01

    Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

  9. DNA binding studies of tartrazine food additive.

    Science.gov (United States)

    Kashanian, Soheila; Zeidali, Sahar Heidary

    2011-07-01

    The interaction of native calf thymus DNA with tartrazine in 10 mM Tris-HCl aqueous solution at neutral pH 7.4 was investigated. Tartrazine is a nitrous derivative and may cause allergic reactions, with a potential of toxicological risk. Also, tartrazine induces oxidative stress and DNA damage. Its DNA binding properties were studied by UV-vis and circular dichroism spectra, competitive binding with Hoechst 33258, and viscosity measurements. Tartrazine molecules bind to DNA via groove mode as illustrated by hyperchromism in the UV absorption band of tartrazine, decrease in Hoechst-DNA solution fluorescence, unchanged viscosity of DNA, and conformational changes such as conversion from B-like to C-like in the circular dichroism spectra of DNA. The binding constants (K(b)) of DNA with tartrazine were calculated at different temperatures. Enthalpy and entropy changes were calculated to be +37 and +213 kJ mol(-1), respectively, according to the Van't Hoff equation, which indicated that the reaction is predominantly entropically driven. Also, tartrazine does not cleave plasmid DNA. Tartrazine interacts with calf thymus DNA via a groove interaction mode with an intrinsic binding constant of 3.75 × 10(4) M(-1).

  10. Energetics of the protein-DNA-water interaction

    Directory of Open Access Journals (Sweden)

    Marabotti Anna

    2007-01-01

    Full Text Available Abstract Background To understand the energetics of the interaction between protein and DNA we analyzed 39 crystallographically characterized complexes with the HINT (Hydropathic INTeractions computational model. HINT is an empirical free energy force field based on solvent partitioning of small molecules between water and 1-octanol. Our previous studies on protein-ligand complexes demonstrated that free energy predictions were significantly improved by taking into account the energetic contribution of water molecules that form at least one hydrogen bond with each interacting species. Results An initial correlation between the calculated HINT scores and the experimentally determined binding free energies in the protein-DNA system exhibited a relatively poor r2 of 0.21 and standard error of ± 1.71 kcal mol-1. However, the inclusion of 261 waters that bridge protein and DNA improved the HINT score-free energy correlation to an r2 of 0.56 and standard error of ± 1.28 kcal mol-1. Analysis of the water role and energy contributions indicate that 46% of the bridging waters act as linkers between amino acids and nucleotide bases at the protein-DNA interface, while the remaining 54% are largely involved in screening unfavorable electrostatic contacts. Conclusion This study quantifies the key energetic role of bridging waters in protein-DNA associations. In addition, the relevant role of hydrophobic interactions and entropy in driving protein-DNA association is indicated by analyses of interaction character showing that, together, the favorable polar and unfavorable polar/hydrophobic-polar interactions (i.e., desolvation mostly cancel.

  11. Studies of interaction between histone F2b and DNA from normal and exposed to X-radiation calf lymph nodes

    International Nuclear Information System (INIS)

    Bartkowiak, J.; Gaczynski, M.

    1978-01-01

    Affinity chromatography has been used to compare the specificity of interaction between DNA and histone F2b. Histone-sepharose gels were prepared by binding the F2b protein to CNBr-activated Sepharose 4B (Pharmacia, Sweden). DNA was applied to columns formed from the gels, and a linear gradient of concentration of NaCl used for elution. Absorption at 260 nm, and derivative melting points were measured for those samples of effluent containing DNA. The results indicated that the fractionation of DNA was not conditioned only by the composition of the DNA bases. There were significant differences in the interaction with DNA of gels prepared from histone F2b molecules from X-irradiated and normal lymph nodes. It is concluded that histone F2b remaining in lymph nodes after irradiation had properties which differed from protein in normal tissue. (U.K.)

  12. Interactions within the mammalian DNA methyltransferase family

    Directory of Open Access Journals (Sweden)

    Ehrenhofer-Murray Ann E

    2003-05-01

    Full Text Available Abstract Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase.

  13. Interactions within the mammalian DNA methyltransferase family

    Science.gov (United States)

    Margot, Jean B; Ehrenhofer-Murray, Ann E; Leonhardt, Heinrich

    2003-01-01

    Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase. PMID:12777184

  14. Interaction of carbon nano tubes with DNA segments

    International Nuclear Information System (INIS)

    Peressinotto, Valdirene Sullas Teixeira

    2007-01-01

    Single- and double-stranded DNA (deoxyribonucleic acid) molecules can strongly bind to single-walled carbon nanotubes (SWNT) via non-covalent interactions. Under certain conditions, the DNA molecule spontaneously self-assembles into a helical wrapping around the tubular structure of the carbon nanotubes to form DNA/SWNT hybrids, which are both stable and soluble in water. This system has recently received extensive attention, since, besides rendering SWNTs dispersible in water as individual tubes, the DNA hybrids are very promising candidates for many applications in nanotechnology and molecular biology. All the possible applications for DNA-SWNT hybrids require, however, a fully understanding of DNA-nanotube wrapping mechanism which is still lacking in the literature. In this context, the aim of this work was to investigate the non-covalent interaction in aqueous medium between SWNTs and synthetic DNA segments having a known nucleotide sequence. Initially, the study was focused on poly d(GT)n sequences (n = 10, 30 and 45) that contain a sequence of alternating guanine and thymine bases and for which the efficiency to disperse and separate carbon nanotubes has already been demonstrated. Besides the size of GT sequences, the effects of ionic strength and pH in the interaction were also investigated. Afterwards, we studied the interaction of SWNT with DNA molecules that contain only a single type of nitrogenous base (DNA homopolymers), which has not been reported in details in the literature. We investigated homopolymers of poly dA 20 , poly dT 20 , poly dC 20 and the duplex poly dA 20 :dT 20 . Most of the study was carried out with small-diameter HiPco SWNTs (with diameters between 0.7 and 1.2 nm). In some studies, SWNTs with diameter around 1.4 nm, synthesized via laser ablation and arc-discharge methods, were also investigated. The arc-discharge nanotubes used in this study were functionalized with carboxylic groups (-COOH) due to their purification using strong

  15. Binding and thermodynamics of REV peptide-ctDNA interaction.

    Science.gov (United States)

    Upadhyay, Santosh Kumar

    2017-03-01

    The thermodynamics of DNA-ligand binding is important as it provides useful information to understand the details of binding processes. HIV-1 REV response element (RRE) located in the env coding region of the viral genome is reported to be well conserved across different HIV-1 isolates. In this study, the binding characteristics of Calf thymus DNA (ctDNA) and REV peptide from HIV-1 were investigated using spectroscopic (UV-visible, fluorescence, and circular dichroism (CD)) and isothermal titration calorimetric (ITC) techniques. Thermal stability and ligand binding properties of the ctDNA revealed that native ctDNA had a T m of 75.5 °C, whereas the ctDNA-REV peptide complex exhibited an incremental shift in the T m by 8 °C, indicating thermal stability of the complex. CD data indicated increased ellipticity due to large conformational changes in ctDNA molecule upon binding with REV peptide and two binding stoichiometric modes are apparent. The ctDNA experienced condensation due to large conformational changes in the presence of REV peptide and positive B→Ψ transition was observed at higher molar charge ratios. Fluorescence studies performed at several ligand concentrations revealed a gradual decrease in the fluorescence intensity of EtBr-bound ctDNA in response to increasing ligand concentrations. The fluorescence data further confirmed two stoichiometric modes of binding for ctDNA-REV peptide complex as previously observed with CD studies. The binding enthalpies were determined using ITC in the temperature range of 293 K-308 K. The ITC binding isotherm was exothermic at all temperatures examined, with low ΔH values indicating that the ctDNA-REV peptide interaction is driven largely by entropy. The heat capacity change (ΔC p ) was insignificant, an unusual finding in the area of DNA-peptide interaction studies. The variation in the values obtained for ΔH, ΔS, and ΔG with temperature further suggests that ctDNA-REV peptide interaction is entropically

  16. Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage.

    Science.gov (United States)

    Mallik, Sarita; Popodi, Ellen M; Hanson, Andrew J; Foster, Patricia L

    2015-09-01

    Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence

  17. Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery.

    Directory of Open Access Journals (Sweden)

    Felipe Merino

    2015-06-01

    Full Text Available Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

  18. Ionizing radiation interactions with DNA: nanodosimetry

    International Nuclear Information System (INIS)

    Bug, Marion; Nettelbeck, Heidi; Hilgers, Gerhard; Rabus, Hans

    2011-01-01

    The metrology of ionizing radiation is based on measuring values that are averaged over macroscopic volume elements, for instance the energy dose is defined as ratio of the energy deposited on the absorber and the absorber mass. For biological or medical radiation effects the stochastic nature of radiation interaction id of main importance, esp. the interaction of ionizing radiation with the DNA as the genetic information carrier. For radiotherapy and risk evaluation purposes a comprehensive system of radiation weighing factors and other characteristics, like radiation quality or relative biological efficacy was developed. The nanodosimetry is aimed to develop a metrological basis relying on physical characteristics of the microscopic structure of ionizing radiation tracks. The article includes the development of experimental nanodosimetric methods, the respective calibration techniques, Monte-Carlo simulation of the particle track microstructure and the correlation nanodosimetry and biological efficiency.

  19. Spectroscopic studies on the interactions of 5-ethyl-6-phenyl-3,8-bis((3-aminoalkyl)propanamido)phenanthridin-5-ium derivatives with G-quadruplex DNA

    Science.gov (United States)

    Yalçın, Ergin; Duyar, Halil; Ihmels, Heiko; Seferoğlu, Zeynel

    2018-05-01

    An improved microwave-induced synthesis of five ethidium derivatives (Ethidium derivatives, 2a-d) is presented. As the derivatives 2a-d have been proposed previously to be telomerase inhibitors, the binding interactions of these ethidium derivatives with G-quadruplex DNA were evaluated by means of photometric and fluorimetric titration, thermal DNA denaturation, CD and 1H NMR spectroscopy. In particular, the compound bearing 3,8-bis(pyrrolidin-1-yl)propanamido substituent 2a exhibits high selectivity for G-quadruplex DNA relative to duplex DNA.

  20. Interactions of tetracationic porphyrins with DNA and their effects on DNA cleavage

    Science.gov (United States)

    Lebedeva, Natalya Sh.; Yurina, Elena S.; Gubarev, Yury A.; Syrbu, Sergey A.

    2018-06-01

    The interaction of tetracationic porphyrins with DNA was studied using UV-Vis absorption, fluorescence spectroscopy and viscometry, and the particle sizes were determined. Аs cationic porphyrins, two isomer porphyrins, 3,3‧,3″,3‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP3) and 4,4‧,4″,4‴-(5,10,15,20-Porphyrintetrayl)tetrakis(1-methylpyridinium) (TMPyP4), were studied. They differ in the position of NCH3+ group in phenyl ring of the porphyrins and hence, in degree of freedom of rotation of the phenyl rings about the central macrocycle. It was found that intercalated complexes are formed at DNA/porphyrin molar ratios (R) of 2.2 and 3.9 for TMPyP3 и TMPyP4, respectively. Decreasing R up to 0.4 and 0.8 for TMPyP3 и TMPyP4, respectively, leads mainly to formation of outside complexes due to π-π stacking between the porphyrin chromophores interacting electrostatically with phosphate framework of DNA. Each type of the obtained complexes was characterized using Scatchard approach. It was ascertained that the affinity of TMPyP4 to DNA is stronger than TMPyP3, meanwhile the wedge effect of the latter is higher. The differences between the porphyrin isomers become more evident at irradiation of their complexes with DNA. It was established that irradiation of the intercalated complexes results in DNA fragmentation. In the case of TMPyP4, DNA fragments of different size are formed. The irradiation of the outside DNA/porphyrin complexes leads to cleavage of DNA (TMPyP3 and TMPyP4) and partial destruction of the complex due to photolysis of the porphyrin (TMPyP3).

  1. RNA Study Using DNA Nanotechnology.

    Science.gov (United States)

    Tadakuma, Hisashi; Masubuchi, Takeya; Ueda, Takuya

    2016-01-01

    Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Surface-enhanced Raman spectroscopic study of DNA and 6-mercapto-1-hexanol interactions using large area mapping

    DEFF Research Database (Denmark)

    Frøhling, Kasper Bayer; Alstrøm, Tommy Sonne; Bache, Michael

    2016-01-01

    intensities and peak positions it is possible to directly inspect the interplay between DNA and 6-mercapto-1-hexanol on gold covered nanopillars. It is demonstrated that optimised functionalization parameters can be extracted from the Raman spectra directly. Using the peak-fitting approach it is possible...

  3. A QM/MM study of the absorption spectrum of harmane in water solution and interacting with DNA: the crucial role of dynamic effects.

    Science.gov (United States)

    Etienne, Thibaud; Very, Thibaut; Perpète, Eric A; Monari, Antonio; Assfeld, Xavier

    2013-05-02

    We present a time-dependent density functional theory computation of the absorption spectra of one β-carboline system: the harmane molecule in its neutral and cationic forms. The spectra are computed in aqueous solution. The interaction of cationic harmane with DNA is also studied. In particular, the use of hybrid quantum mechanics/molecular mechanics methods is discussed, together with its coupling to a molecular dynamics strategy to take into account dynamic effects of the environment and the vibrational degrees of freedom of the chromophore. Different levels of treatment of the environment are addressed starting from purely mechanical embedding to electrostatic and polarizable embedding. We show that a static description of the spectrum based on equilibrium geometry only is unable to give a correct agreement with experimental results, and dynamic effects need to be taken into account. The presence of two stable noncovalent interaction modes between harmane and DNA is also presented, as well as the associated absorption spectrum of harmane cation.

  4. 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...

  5. Helical chirality: a link between local interactions and global topology in DNA.

    Directory of Open Access Journals (Sweden)

    Youri Timsit

    Full Text Available DNA supercoiling plays a major role in many cellular functions. The global DNA conformation is however intimately linked to local DNA-DNA interactions influencing both the physical properties and the biological functions of the supercoiled molecule. Juxtaposition of DNA double helices in ubiquitous crossover arrangements participates in multiple functions such as recombination, gene regulation and DNA packaging. However, little is currently known about how the structure and stability of direct DNA-DNA interactions influence the topological state of DNA. Here, a crystallographic analysis shows that due to the intrinsic helical chirality of DNA, crossovers of opposite handedness exhibit markedly different geometries. While right-handed crossovers are self-fitted by sequence-specific groove-backbone interaction and bridging Mg(2+ sites, left-handed crossovers are juxtaposed by groove-groove interaction. Our previous calculations have shown that the different geometries result in differential stabilisation in solution, in the presence of divalent cations. The present study reveals that the various topological states of the cell are associated with different inter-segmental interactions. While the unstable left-handed crossovers are exclusively formed in negatively supercoiled DNA, stable right-handed crossovers constitute the local signature of an unusual topological state in the cell, such as the positively supercoiled or relaxed DNA. These findings not only provide a simple mechanism for locally sensing the DNA topology but also lead to the prediction that, due to their different tertiary intra-molecular interactions, supercoiled molecules of opposite signs must display markedly different physical properties. Sticky inter-segmental interactions in positively supercoiled or relaxed DNA are expected to greatly slow down the slithering dynamics of DNA. We therefore suggest that the intrinsic helical chirality of DNA may have oriented the early

  6. The free-energy cost of interaction between DNA loops.

    Science.gov (United States)

    Huang, Lifang; Liu, Peijiang; Yuan, Zhanjiang; Zhou, Tianshou; Yu, Jianshe

    2017-10-03

    From the viewpoint of thermodynamics, the formation of DNA loops and the interaction between them, which are all non-equilibrium processes, result in the change of free energy, affecting gene expression and further cell-to-cell variability as observed experimentally. However, how these processes dissipate free energy remains largely unclear. Here, by analyzing a mechanic model that maps three fundamental topologies of two interacting DNA loops into a 4-state model of gene transcription, we first show that a longer DNA loop needs more mean free energy consumption. Then, independent of the type of interacting two DNA loops (nested, side-by-side or alternating), the promotion between them always consumes less mean free energy whereas the suppression dissipates more mean free energy. More interestingly, we find that in contrast to the mechanism of direct looping between promoter and enhancer, the facilitated-tracking mechanism dissipates less mean free energy but enhances the mean mRNA expression, justifying the facilitated-tracking hypothesis, a long-standing debate in biology. Based on minimal energy principle, we thus speculate that organisms would utilize the mechanisms of loop-loop promotion and facilitated tracking to survive in complex environments. Our studies provide insights into the understanding of gene expression regulation mechanism from the view of energy consumption.

  7. Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.

    Science.gov (United States)

    Su'etsugu, Masayuki; Takata, Makoto; Kubota, Toshio; Matsuda, Yusaku; Katayama, Tsutomu

    2004-06-01

    In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. Copyright Blackwell Publishing Limited

  8. Synthesis and Structural Investigation of New Bio-Relevant Complexes of Lanthanides with 5-Hydroxyflavone: DNA Binding and Protein Interaction Studies

    Directory of Open Access Journals (Sweden)

    Alexandra-Cristina Munteanu

    2016-12-01

    Full Text Available In the present work, we attempted to develop new metal coordination complexes of the natural flavonoid 5-hydroxyflavone with Sm(III, Eu(III, Gd(III, Tb(III. The resultant hydroxo complexes have been characterized by a variety of spectroscopic techniques, including fluorescence, FT-IR, UV-Vis, EPR and mass spectral studies. The general chemical formula of the complexes is [Ln(C15H9O33(OH2(H2Ox]·nH2O, where Ln is the lanthanide cation and x = 0 for Sm(III, x = 1 for Eu(III, Gd(III, Tb(III and n = 0 for Sm(III, Gd(III, Tb(III, n = 1 for Eu(III, respectively. The proposed structures of the complexes were optimized by DFT calculations. Theoretical calculations and experimental determinations sustain the proposed structures of the hydroxo complexes, with two molecules of 5-hydroxyflavone acting as monoanionic bidentate chelate ligands. The interaction of the complexes with calf thymus DNA has been explored by fluorescence titration and UV-Vis absorption binding studies, and revealed that the synthesized complexes interact with DNA with binding constants (Kb ~ 104. Human serum albumin (HSA and transferrin (Tf binding studies have also been performed by fluorescence titration techniques (fluorescence quenching studies, synchronous fluorescence spectra. The apparent association constants (Ka and thermodynamic parameters have been calculated from the fluorescence quenching experiment at 299 K, 308 K, and 318 K. The quenching curves indicate that the complexes bind to HSA with smaller affinity than the ligand, but to Tf with higher binding affinities than the ligand.

  9. Estrogen receptor accessory proteins augment receptor-DNA interaction and DNA bending.

    Science.gov (United States)

    Landel, C C; Potthoff, S J; Nardulli, A M; Kushner, P J; Greene, G L

    1997-01-01

    Increasing evidence suggests that accessory proteins play an important role in the ability of the estrogen receptor (ER) and other nuclear hormone receptors to modulate transcription when bound to cis-acting hormone response elements in target genes. We have previously shown that four proteins, hsp70, protein disulfide isomerase (PDI) and two unknown proteins (p48 and p45), copurify with ER that has been isolated by site-specific DNA chromatography (BERE) and influence the interaction of ER with DNA in vitro. To better define the nature of these effects, we used filter binding and electrophoretic mobility shift assays to study the ability of these proteins to alter the kinetics of ER-DNA interaction and to influence the ability of ER to bend DNA when bound to an estrogen response element (ERE). The results of both assays indicate that ERE-purified ER, with its four associated proteins (hsp70, PDI, p48, p45), has a greater ability to bind to the vitellogenin A2 ERE than ER purified by estradiol-Sepharose chromatography in the absence (ESeph) or presence (EATP) of ATP, in which p48, p45 (ESeph) and hsp70 (EATP) are removed. Surprisingly, the rates of association and dissociation of ER and ERE were essentially the same for all three mixtures, suggesting that one or more ER-associated proteins, especially p45 and p48, may be required for ER to attain maximum DNA binding activity. In addition, circular permutation and phasing analyses demonstrated that the same ER-associated proteins produced higher order ER-DNA complexes that significantly increased the magnitude of DNA distortion, but did not alter the direction of the ER-induced bend of ERE-containing DNA fragments, which was toward the major groove of the DNA helix. These results suggest that p45 and/or p48 and possibly hsp70, play an important role both in the specific DNA binding and bending activities of ER and thus contribute to the overall stimulation of transcription in target genes that contain cis

  10. Authenticity in ancient DNA studies

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Willerslev, Eske

    2006-01-01

    Ancient DNA studies represent a powerful tool that can be used to obtain genetic insights into the past. However, despite the publication of large numbers of apparently successful ancient DNA studies, a number of problems exist with the field that are often ignored. Therefore, questions exist as ...

  11. Experimental study of oxidative DNA damage

    DEFF Research Database (Denmark)

    Loft, Steffen; Deng, Xin-Sheng; Tuo, Jingsheng

    1998-01-01

    Animal experiments allow the study of oxidative DNA damage in target organs and the elucidation of dose-response relationships of carcinogenic and other harmful chemicals and conditions as well as the study of interactions of several factors. So far the effects of more than 50 different chemical ...

  12. The importance of pKa in an analysis of the interaction of compounds with DNA.

    Science.gov (United States)

    Saha, Mouli; Nandy, Promita; Chakraborty, Mousumi; Das, Piyal; Das, Saurabh

    2018-05-01

    pK a of a compound is crucial for determining the contributions of different forms of it towards overall binding with DNA. Hence it is important to use correct pK a values in DNA interaction studies. This study takes a look at the importance of pK a values to realize binding of compounds with DNA. Since pK a of a compound determined in the presence of DNA is quite different from that determined in its absence hence, presence of different forms of a compound during interaction with DNA is different from that realized if the determination of pK a is done in normal aqueous solution in absence of DNA. Hence, calculations determining contributions of different forms of a compound interacting with DNA are affected accordingly. Two simple analogues of anthracyclines, alizarin and purpurin, were used to investigate the influence DNA has on pK a values. Indeed, they were different in presence of DNA than when determined in normal aqueous solution. pK a1 for alizarin and purpurin determined in the absence and presence of calf thymus DNA were used in equations that determine contributions of two forms (neutral and anionic) towards overall binding with DNA. The study concludes that correct pK a values, determined correctly i.e. under appropriate conditions, must be used for DNA binding experiments to evaluate contributions of individual forms. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. DNA hydration studied by neutron fiber diffraction

    Energy Technology Data Exchange (ETDEWEB)

    Fuller, W.; Forsyth, V.T.; Mahendrasingam, A.; Langan, P.; Pigram, W.J. [Keele Univ. (United Kingdom)] [and others

    1994-12-31

    The development of neutron high angle fiber diffraction to investigate the location of water around the deoxyribonucleic acid (DNA) double-helix is described. The power of the technique is illustrated by its application to the D and A conformations of DNA using the single crystal diffractometer, D19, at the Institute Laue-Langevin, Grenoble and the time of flight diffractometer, SXD, at the Rutherford Appleton ISIS Spallation Neutron Source. These studies show the existence of bound water closely associated with the DNA. The patterns of hydration in these two DNA conformations are quite distinct and are compared to those observed in X-ray single crystal studies of two-stranded oligodeoxynucleotides. Information on the location of water around the DNA double-helix from the neutron fiber diffraction studies is combined with that on the location of alkali metal cations from complementary X-ray high angle fiber diffraction studies at the Daresbury Laboratory SRS using synchrotron radiation. These analyses emphasize the importance of viewing DNA, water and ions as a single system with specific interactions between the three components and provide a basis for understanding the effect of changes in the concentration of water and ions in inducing conformations] transitions in the DNA double-helix.

  14. DNA hydration studied by neutron fiber diffraction

    International Nuclear Information System (INIS)

    Fuller, W.; Forsyth, V.T.; Mahendrasingam, A.; Langan, P.; Pigram, W.J.

    1994-01-01

    The development of neutron high angle fiber diffraction to investigate the location of water around the deoxyribonucleic acid (DNA) double-helix is described. The power of the technique is illustrated by its application to the D and A conformations of DNA using the single crystal diffractometer, D19, at the Institute Laue-Langevin, Grenoble and the time of flight diffractometer, SXD, at the Rutherford Appleton ISIS Spallation Neutron Source. These studies show the existence of bound water closely associated with the DNA. The patterns of hydration in these two DNA conformations are quite distinct and are compared to those observed in X-ray single crystal studies of two-stranded oligodeoxynucleotides. Information on the location of water around the DNA double-helix from the neutron fiber diffraction studies is combined with that on the location of alkali metal cations from complementary X-ray high angle fiber diffraction studies at the Daresbury Laboratory SRS using synchrotron radiation. These analyses emphasize the importance of viewing DNA, water and ions as a single system with specific interactions between the three components and provide a basis for understanding the effect of changes in the concentration of water and ions in inducing conformations] transitions in the DNA double-helix

  15. Probe DNA-Cisplatin Interaction with Solid-State Nanopores

    Science.gov (United States)

    Zhou, Zhi; Hu, Ying; Li, Wei; Xu, Zhi; Wang, Pengye; Bai, Xuedong; Shan, Xinyan; Lu, Xinghua; Nanopore Collaboration

    2014-03-01

    Understanding the mechanism of DNA-cisplatin interaction is essential for clinical application and novel drug design. As an emerging single-molecule technology, solid-state nanopore has been employed in biomolecule detection and probing DNA-molecule interactions. Herein, we reported a real-time monitoring of DNA-cisplatin interaction by employing solid-state SiN nanopores. The DNA-cisplatin interacting process is clearly classified into three stages by measuring the capture rate of DNA-cisplatin adducts. In the first stage, the negative charged DNA molecules were partially discharged due to the bonding of positive charged cisplatin and forming of mono-adducts. In the second stage, forming of DNA-cisplatin di-adducts with the adjacent bases results in DNA bending and softening. The capture rate increases since the softened bi-adducts experience a lower barrier to thread into the nanopores. In the third stage, complex structures, such as micro-loop, are formed and the DNA-cisplatin adducts are aggregated. The capture rate decreases to zero as the aggregated adduct grows to the size of the pore. The characteristic time of this stage was found to be linear with the diameter of the nanopore and this dynamic process can be described with a second-order reaction model. We are grateful to Laboratory of Microfabrication, Dr. Y. Yao, and Prof. R.C. Yu (Institute of Physics, Chinese Academy of Sciences) for technical assistance.

  16. Pt(II) and Pd(II) complexes with ibuprofen hydrazide: Characterization, theoretical calculations, antibacterial and antitumor assays and studies of interaction with CT-DNA

    Science.gov (United States)

    Manzano, Carlos M.; Bergamini, Fernando R. G.; Lustri, Wilton R.; Ruiz, Ana Lúcia T. G.; de Oliveira, Ellen C. S.; Ribeiro, Marcos A.; Formiga, André L. B.; Corbi, Pedro P.

    2018-02-01

    Palladium(II) and platinum(II) complexes with a hydrazide derivative of ibuprofen (named HIB) were synthesized and characterized by chemical and spectroscopic methods. Elemental and thermogravimetric analyses, as well as ESI-QTOF-MS studies for both complexes, confirmed a 1:2:2 metal/HIB/Cl- molar ratio. The crystal structure of the palladium(II) complex was solved by single crystal X-ray diffractometric analysis, which permitted identifying the coordination formula [PdCl2(HIB)2]. Crystallographic studies also indicate coordination of HIB to the metal by the NH2 group. Nuclear magnetic resonance and infrared spectroscopies reinforced the coordination observed in the crystal structure and suggested that the platinum(II) complex presents similar coordination modes and structure when compared with the Pd(II) complex. The complexes had their structures optimized with the aid of DFT methods. In vitro antiproliferative assays showed that the [PdCl2(HIB)2] complex is active over ovarian cancer cell line OVCAR-03, while biophysical studies indicated its capacity to interact with CT-DNA. The complexes were inactive over Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa bacterial strains.

  17. Analysis of DNA interactions using single-molecule force spectroscopy.

    Science.gov (United States)

    Ritzefeld, Markus; Walhorn, Volker; Anselmetti, Dario; Sewald, Norbert

    2013-06-01

    Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given.

  18. DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific.

    Science.gov (United States)

    Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt V; Ferapontova, Elena E

    2010-06-01

    A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

  19. Synthesis, spectral characterization, antimicrobial, DNA interactions ...

    Indian Academy of Sciences (India)

    KUNCHE SUDEEPA

    2018-05-04

    May 4, 2018 ... structural aspects of FMBC and its Cu(II), Ni(II) and. Zn(II) complexes ... of DNA was down- loaded from protein data bank24 (www.rcsb.org) pdb id: ... the reaction mixture was refluxed on water bath for 4–8 h maintaining the ...

  20. Charge-transfer interactions of Cr species with DNA.

    Science.gov (United States)

    Nowicka, Anna M; Matysiak-Brynda, Edyta; Hepel, Maria

    2017-10-01

    Interactions of Cr species with nucleic acids in living organisms depend strongly on Cr oxidation state and the environmental conditions. As the effects of these interactions range from benign to pre-mutagenic to carcinogenic, careful assessment of the hazard they pose to human health is necessary. We have investigated methods that would enable quantifying the DNA damage caused by Cr species under varying environmental conditions, including UV, O 2 , and redox potential, using simple instrumental techniques which could be in future combined into a field-deployable instrumentation. We have employed electrochemical quartz crystal nanogravimetry (EQCN), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) to evaluate the extent of DNA damage expressed in terms of guanine oxidation yield (η) and changes in specific characteristics provided by these techniques. The effects of the interactions of Cr species with DNA were analyzed using a model calf thymus DNA (ctDNA) film on a gold electrode (Au@ctDNA) in different media, including: (i) Cr(VI), (ii) Cr(VI) reduced at -0.2V, (iii) Cr(III)+UV radiation+O 2 , and Cr(III), obtaining the η values: 7.4±1.4, 1.5±0.4, 1.1±0.31%, and 0%, respectively, thus quantifying the hazard posed. The EIS measurements have enabled utilizing the decrease in charge-transfer resistance (R ct ) for ferri/ferrocyanide redox probe at an Au@ctDNA electrode to assess the oxidative ctDNA damage by Cr(VI) species. In this case, circular dichroism indicates an extensive damage to the ctDNA hydrogen bonding. On the other hand, Cr(III) species have not induced any damage to ctDNA, although the EQCN measurements show an electrostatic binding to DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. DNA-psoralen interaction: a single molecule experiment.

    Science.gov (United States)

    Rocha, M S; Viana, N B; Mesquita, O N

    2004-11-15

    By attaching one end of a single lambda-DNA molecule to a microscope coverslip and the other end to a polystyrene microsphere trapped by an optical tweezers, we can study the entropic elasticity of the lambda-DNA by measuring force versus extension as we stretch the molecule. This powerful method permits single molecule studies. We are particularly interested in the effects of the photosensitive drug psoralen on the elasticity of the DNA molecule. We have illuminated the sample with different light sources, studying how the different wavelengths affect the psoralen-DNA linkage. To do this, we measure the persistence length of individual DNA-psoralen complexes.

  2. Electrochemical and calorimetric investigation of interaction of novel biscationic anticancer agents with DNA; Investigacao eletroquimica e calorimetrica da interacao de novos agentes antitumorais biscationicos com DNA

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Lauris Lucia da; Donnici, Claudio Luis; Lopes, Julio Cesar Dias, E-mail: cdonnici@terra.com.br [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Inst. de Ciencias Exatas. Dept. de Quimica; Goulart, Marilia Oliveira Fonseca; Abreu, Fabiane Caxico de; Paula, Francine Santos de [Universidade Federal de Alagoas (UFAL), Maceio, AL (Brazil). Campus A.C. Simoes. Inst. de Quimica e Biotecnologia; Bravo, Carlos E. Salas; Santoro, Marcelo Matos [Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil). Dept. de Bioquimica e Imunologia; Denadai, Angelo Marcio Leite [Centro Federal de Educacao Tecnologica, Timoteo, MG (Brazil). Campus VII; Santos, Alexandre Martins Costa [Universidade Federal do Espirito Santo, Vitoria, ES (Brazil). Dept. de Ciencias Fisiologicas; Montanari, Carlos Alberto [Universidade de Sao Paulo, Sao Carlos, SP (Brazil). Inst. de Quimica

    2012-07-01

    Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-{alpha}-{omega}-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered. (author)

  3. Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair

    International Nuclear Information System (INIS)

    Dowd, D.R.; Lloyd, R.S.

    1990-01-01

    Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance

  4. Correlation between energy deposition and molecular damage from Auger electrons: A case study of ultra-low energy (5–18 eV) electron interactions with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Rezaee, Mohammad, E-mail: Mohammad.Rezaee@USherbrooke.ca; Hunting, Darel J.; Sanche, Léon [Groupe en Sciences des Radiations, Département de Médecine Nucléaire et Radiobiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4 (Canada)

    2014-07-15

    Purpose: The present study introduces a new method to establish a direct correlation between biologically related physical parameters (i.e., stopping and damaging cross sections, respectively) for an Auger-electron emitting radionuclide decaying within a target molecule (e.g., DNA), so as to evaluate the efficacy of the radionuclide at the molecular level. These parameters can be applied to the dosimetry of Auger electrons and the quantification of their biological effects, which are the main criteria to assess the therapeutic efficacy of Auger-electron emitting radionuclides. Methods: Absorbed dose and stopping cross section for the Auger electrons of 5–18 eV emitted by{sup 125}I within DNA were determined by developing a nanodosimetric model. The molecular damages induced by these Auger electrons were investigated by measuring damaging cross section, including that for the formation of DNA single- and double-strand breaks. Nanoscale films of pure plasmid DNA were prepared via the freeze-drying technique and subsequently irradiated with low-energy electrons at various fluences. The damaging cross sections were determined by employing a molecular survival model to the measured exposure–response curves for induction of DNA strand breaks. Results: For a single decay of{sup 125}I within DNA, the Auger electrons of 5–18 eV deposit the energies of 12.1 and 9.1 eV within a 4.2-nm{sup 3} volume of a hydrated or dry DNA, which results in the absorbed doses of 270 and 210 kGy, respectively. DNA bases have a major contribution to the deposited energies. Ten-electronvolt and high linear energy transfer 100-eV electrons have a similar cross section for the formation of DNA double-strand break, while 100-eV electrons are twice as efficient as 10 eV in the induction of single-strand break. Conclusions: Ultra-low-energy electrons (<18 eV) substantially contribute to the absorbed dose and to the molecular damage from Auger-electron emitting radionuclides; hence, they should

  5. Mutant p53 interactions with supercoiled DNA

    Czech Academy of Sciences Publication Activity Database

    Brázdová, Marie; Němcová, Kateřina; Činčárová, Lenka; Šebest, Peter; Pivoňková, Hana; Brázda, Václav; Fojta, Miroslav; Paleček, Emil

    2007-01-01

    Roč. 24, č. 6 (2007), s. 639-640 ISSN 0739-1102. [Alban 2007: The 15th Conversation . 19.06.2007-23.06.2007, Albany] R&D Projects: GA MŠk(CZ) 1K04119; GA ČR(CZ) GP204/06/P369; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : mutant p53 * supercoiled DNA * cancer Subject RIV: BO - Biophysics

  6. Interaction between DNA Polymerase β and BRCA1.

    Directory of Open Access Journals (Sweden)

    Aya Masaoka

    Full Text Available The breast cancer 1 (BRCA1 protein is a tumor suppressor playing roles in DNA repair and cell cycle regulation. Studies of DNA repair functions of BRCA1 have focused on double-strand break (DSB repair pathways and have recently included base excision repair (BER. However, the function of BRCA1 in BER is not well defined. Here, we examined a BRCA1 role in BER, first in relation to alkylating agent (MMS treatment of cells and the BER enzyme DNA polymerase β (pol β. MMS treatment of BRCA1 negative human ovarian and chicken DT40 cells revealed hypersensitivity, and the combined gene deletion of BRCA1 and pol β in DT40 cells was consistent with these factors acting in the same repair pathway, possibly BER. Using cell extracts and purified proteins, BRCA1 and pol β were found to interact in immunoprecipitation assays, yet in vivo and in vitro assays for a BER role of BRCA1 were negative. An alternate approach with the human cells of immunofluorescence imaging and laser-induced DNA damage revealed negligible BRCA1 recruitment during the first 60 s after irradiation, the period typical of recruitment of pol β and other BER factors. Instead, 15 min after irradiation, BRCA1 recruitment was strong and there was γ-H2AX co-localization, consistent with DSBs and repair. The rapid recruitment of pol β was similar in BRCA1 positive and negative cells. However, a fraction of pol β initially recruited remained associated with damage sites much longer in BRCA1 positive than negative cells. Interestingly, pol β expression was required for BRCA1 recruitment, suggesting a partnership between these repair factors in DSB repair.

  7. Interactions between nucleic acids and antibodies to Z-DNA

    International Nuclear Information System (INIS)

    Leng, M.; Hartmann, B.; Malfoy, B.; Pilet, J.; Ramstein, J.; Sage, E.

    1983-01-01

    In this paper we report some properties of the antibodies to Z-DNA. To determine more precisely the antigenic determinant, the interactions between the antibodies and several polynucleotides have been studied. We found that the antibodies bind very weakly to poly(dI-br 5 dC).(dI-br 5 dC). This polynucleotide can adopt the Z conformation in high salt concentration, as we have demonstrated by infrared spectroscopy. Moreover, from the study of the exchange rate of the protons involved in hydrogen bonds in this polynucleotide and from previous studies on poly(dG-dC).poly(dG-dC) (Ramstein and Leng 1980; Pilet and Leng 1982), we propose a quantitative description of the dynamic structure of the Z form. 34 references, 5 figures, 3 tables

  8. Ru/Fe bimetallic complexes: Synthesis, characterization, cytotoxicity and study of their interactions with DNA/HSA and human topoisomerase IB.

    Science.gov (United States)

    Takarada, Jessica E; Guedes, Adriana P M; Correa, Rodrigo S; Silveira-Lacerda, Elisângela de P; Castelli, Silvia; Iacovelli, Federico; Deflon, Victor Marcelo; Batista, Alzir Azevedo; Desideri, Alessandro

    2017-12-15

    Three ruthenium/iron-based compounds, 1: [Ru(MIm)(bipy)(dppf)]PF 6 (MIm = 2-mercapto-1-methylimidazole anion), 2: [RuCl(Im)(bipy)(dppf)]PF 6 (Im = imidazole), and 3: [Ru(tzdt)(bipy)(dppf)]PF 6 (tzdt = 1,3-thiazolidine-2-thione anion) (dppf = 1,1'-bis(diphenylphosphine)ferrocene and bipy = 2,2'-bipyridine), were synthesized, and characterized by elemental analyses, conductivity, UV/Vis, IR, 1 H, 13 C and 31 P{1H} NMR spectroscopies, and by electrochemical technique. The complex 3 was also characterized by single-crystal X-ray. The three ruthenium(II) complexes show cytotoxicity against DU-145 (prostate carcinoma cells) and A549 (lung carcinoma cells) tumor cells. The free ligands do not exhibit any cytotoxic activity, such as evident by the IC 50 values higher than 200 μM. UV/Vis and viscosity experiments showed that the complexes interact weakly with the DNA molecule, via electrostatic forces. The interaction of the complexes 1-3 with the HSA is moderate, with K b values in range of 10 5 -10 7  M -1 , presenting a static mechanism of interaction stabilized by hydrophobic. Complexes 2 and 3 showed high affinity for the FA7 HSA site as evidenced by fluorescence spectroscopy and molecular docking. Complexes 1-3 were tested as potential human Topoisomerase IB inhibitors by analysing the different steps of the enzyme catalytic cycle. The results indicate that all compounds efficiently inhibit the DNA relaxation and the cleavage reaction, in which the effect increases upon pre-incubation. Complexes 1 and 2 are also able to slow down the religation reaction. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. DNA Encoding Training Using 3D Gesture Interaction.

    Science.gov (United States)

    Nicola, Stelian; Handrea, Flavia-Laura; Crişan-Vida, Mihaela; Stoicu-Tivadar, Lăcrămioara

    2017-01-01

    The work described in this paper summarizes the development process and presents the results of a human genetics training application, studying the 20 amino acids formed by the combination of the 3 nucleotides of DNA targeting mainly medical and bioinformatics students. Currently, the domain applications using recognized human gestures of the Leap Motion sensor are used in molecules controlling and learning from Mendeleev table or in visualizing the animated reactions of specific molecules with water. The novelty in the current application consists in using the Leap Motion sensor creating new gestures for the application control and creating a tag based algorithm corresponding to each amino acid, depending on the position in the 3D virtual space of the 4 nucleotides of DNA and their type. The team proposes a 3D application based on Unity editor and on Leap Motion sensor where the user has the liberty of forming different combinations of the 20 amino acids. The results confirm that this new type of study of medicine/biochemistry using the Leap Motion sensor for handling amino acids is suitable for students. The application is original and interactive and the users can create their own amino acid structures in a 3D-like environment which they could not do otherwise using traditional pen-and-paper.

  10. Interaction of carbon nano tubes with DNA segments; Interacao de nanotubos de carbono com segmentos de DNA

    Energy Technology Data Exchange (ETDEWEB)

    Peressinotto, Valdirene Sullas Teixeira

    2007-07-01

    Single- and double-stranded DNA (deoxyribonucleic acid) molecules can strongly bind to single-walled carbon nanotubes (SWNT) via non-covalent interactions. Under certain conditions, the DNA molecule spontaneously self-assembles into a helical wrapping around the tubular structure of the carbon nanotubes to form DNA/SWNT hybrids, which are both stable and soluble in water. This system has recently received extensive attention, since, besides rendering SWNTs dispersible in water as individual tubes, the DNA hybrids are very promising candidates for many applications in nanotechnology and molecular biology. All the possible applications for DNA-SWNT hybrids require, however, a fully understanding of DNA-nanotube wrapping mechanism which is still lacking in the literature. In this context, the aim of this work was to investigate the non-covalent interaction in aqueous medium between SWNTs and synthetic DNA segments having a known nucleotide sequence. Initially, the study was focused on poly d(GT)n sequences (n = 10, 30 and 45) that contain a sequence of alternating guanine and thymine bases and for which the efficiency to disperse and separate carbon nanotubes has already been demonstrated. Besides the size of GT sequences, the effects of ionic strength and pH in the interaction were also investigated. Afterwards, we studied the interaction of SWNT with DNA molecules that contain only a single type of nitrogenous base (DNA homopolymers), which has not been reported in details in the literature. We investigated homopolymers of poly dA{sub 20}, poly dT{sub 20}, poly dC{sub 20} and the duplex poly dA{sub 20}:dT{sub 20}. Most of the study was carried out with small-diameter HiPco SWNTs (with diameters between 0.7 and 1.2 nm). In some studies, SWNTs with diameter around 1.4 nm, synthesized via laser ablation and arc-discharge methods, were also investigated. The arc-discharge nanotubes used in this study were functionalized with carboxylic groups (-COOH) due to their

  11. In vitro DNA binding studies of Aspartame, an artificial sweetener.

    Science.gov (United States)

    Kashanian, Soheila; Khodaei, Mohammad Mehdi; Kheirdoosh, Fahimeh

    2013-03-05

    A number of small molecules bind directly and selectively to DNA, by inhibiting replication, transcription or topoisomerase activity. In this work the interaction of native calf thymus DNA (CT-DNA) with Aspartame (APM), an artificial sweeteners was studied at physiological pH. DNA binding study of APM is useful to understand APM-DNA interaction mechanism and to provide guidance for the application and design of new and safer artificial sweeteners. The interaction was investigated using spectrophotometric, spectrofluorometric competition experiment and circular dichroism (CD). Hypochromism and red shift are shown in UV absorption band of APM. A strong fluorescence quenching reaction of DNA to APM was observed and the binding constants (Kf) of DNA with APM and corresponding number of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy changes (ΔH) and entropy changes (ΔS) were calculated to be +181kJmol(-1) and +681Jmol(-1)K(-1) according to Van't Hoff equation, which indicated that reaction is predominantly entropically driven. Moreover, spectrofluorometric competition experiment and circular dichroism (CD) results are indicative of non-intercalative DNA binding nature of APM. We suggest that APM interacts with calf thymus DNA via groove binding mode with an intrinsic binding constant of 5×10(+4)M(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Interaction of Zn(II)bleomycin-A2 and Zn(II)peplomycin with a DNA hairpin containing the 5'-GT-3' binding site in comparison with the 5'-GC-3' binding site studied by NMR spectroscopy.

    Science.gov (United States)

    Follett, Shelby E; Ingersoll, Azure D; Murray, Sally A; Reilly, Teresa M; Lehmann, Teresa E

    2017-10-01

    Bleomycins are a group of glycopeptide antibiotics synthesized by Streptomyces verticillus that are widely used for the treatment of various neoplastic diseases. These antibiotics have the ability to chelate a metal center, mainly Fe(II), and cause site-specific DNA cleavage. Bleomycins are differentiated by their C-terminal regions. Although this antibiotic family is a successful course of treatment for some types of cancers, it is known to cause pulmonary fibrosis. Previous studies have identified that bleomycin-related pulmonary toxicity is linked to the C-terminal region of these drugs. This region has been shown to closely interact with DNA. We examined the binding of Zn(II)peplomycin and Zn(II)bleomycin-A 2 to a DNA hairpin of sequence 5'-CCAGTATTTTTACTGG-3', containing the binding site 5'-GT-3', and compared the results with those obtained from our studies of the same MBLMs bound to a DNA hairpin containing the binding site 5'-GC-3'. We provide evidence that the DNA base sequence has a strong impact in the final structure of the drug-target complex.

  13. DNA damage in human lymphocytes due to synergistic interaction between ionizing radiation and pesticide

    International Nuclear Information System (INIS)

    Kim, J. K.; Lee, K. H.; Lee, B. H.; Chun, K. J.

    2001-01-01

    Biological risks may arise from the possibility of the synergistic interaction between harmful factors such as ionizing radiation and pesticide. The effect of pesticide on radiation-induced DNA damage in human in human blood lymphocytes was evaluated by the single cell gel electrophoresis (SCGE) assay. The lymphocytes, with or without pretreatment of the pesticide, were exposed to 2.0 Gy of gamma ray. Significantly increased tail moment, which was a marker of DNA strand breaks in SCGE assay, showed an excellent dose-response relationship. The present study confirms that the pesticide has the cytotoxic effect on lymphocytes and that it interacts synergistically with ionizing radiationon DNA damage, as well

  14. Dynamics of interaction between complement-fixing antibody/dsDNA immune complexes and erythrocytes. In vitro studies and potential general applications to clinical immune complex testing

    International Nuclear Information System (INIS)

    Taylor, R.P.; Horgan, C.; Hooper, M.; Burge, J.

    1985-01-01

    Soluble antibody/ 3 H-double-stranded PM2 DNA (dsDNA) immune complexes were briefly opsonized with complement and then allowed to bind to human erythrocytes (via complement receptors). The cells were washed and subsequently a volume of autologous blood in a variety of media was added, and the release of the bound immune complexes from the erythrocytes was studied as a function of temperature and time. After 1-2 h, the majority of the bound immune complexes were not released into the serum during blood clotting at either 37 degrees C or room temperature, but there was a considerably greater release of the immune complexes into the plasma of blood that was anticoagulated with EDTA. Similar results were obtained using various conditions of opsonization and also using complexes that contained lower molecular weight dsDNA. Thus, the kinetics of release of these antibody/dsDNA immune complexes differed substantially from the kinetics of release of antibody/bovine serum albumin complexes that was reported by others. Studies using the solution phase C1q immune complex binding assay confirmed that in approximately half of the SLE samples that were positive for immune complexes, there was a significantly higher level of detectable immune complexes in plasma vs. serum. Freshly drawn erythrocytes from some SLE patients exhibiting this plasma/serum discrepancy had IgG antigen on their surface that was released by incubation in EDTA plasma. Thus, the higher levels of immune complexes observed in EDTA plasma vs. serum using the C1q assay may often reflect the existence of immune complexes circulating in vivo bound to erythrocytes

  15. Production of a full-length infectious GFP-tagged cDNA clone of Beet mild yellowing virus for the study of plant-polerovirus interactions.

    Science.gov (United States)

    Stevens, Mark; Viganó, Felicita

    2007-04-01

    The full-length cDNA of Beet mild yellowing virus (Broom's Barn isolate) was sequenced and cloned into the vector pLitmus 29 (pBMYV-BBfl). The sequence of BMYV-BBfl (5721 bases) shared 96% and 98% nucleotide identity with the other complete sequences of BMYV (BMYV-2ITB, France and BMYV-IPP, Germany respectively). Full-length capped RNA transcripts of pBMYV-BBfl were synthesised and found to be biologically active in Arabidopsis thaliana protoplasts following electroporation or PEG inoculation when the protoplasts were subsequently analysed using serological and molecular methods. The BMYV sequence was modified by inserting DNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene close to its 3' end. A. thaliana protoplasts electroporated with these RNA transcripts were biologically active and up to 2% of transfected protoplasts showed GFP-specific fluorescence. The exploitation of these cDNA clones for the study of the biology of beet poleroviruses is discussed.

  16. Protein and Drug Interactions in the Minor Groove of DNA

    Czech Academy of Sciences Publication Activity Database

    Morávek, Z.; Neidle, S.; Schneider, Bohdan

    2002-01-01

    Roč. 30, č. 5 (2002), s. 1182-1191 ISSN 0305-1048 R&D Projects: GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z4040901 Keywords : protein * DNA * interactions Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 7.051, year: 2002

  17. In Vitro Interactions between 17β-Estradiol and DNA Result in Formation of the Hormone-DNA Complexes

    Directory of Open Access Journals (Sweden)

    Zbynek Heger

    2014-07-01

    Full Text Available Beyond the role of 17β-estradiol (E2 in reproduction and during the menstrual cycle, it has been shown to modulate numerous physiological processes such as cell proliferation, apoptosis, inflammation and ion transport in many tissues. The pathways in which estrogens affect an organism have been partially described, although many questions still exist regarding estrogens’ interaction with biomacromolecules. Hence, the present study showed the interaction of four oligonucleotides (17, 20, 24 and/or 38-mer with E2. The strength of these interactions was evaluated using optical methods, showing that the interaction is influenced by three major factors, namely: oligonucleotide length, E2 concentration and interaction time. In addition, the denaturation phenomenon of DNA revealed that the binding of E2 leads to destabilization of hydrogen bonds between the nitrogenous bases of DNA strands resulting in a decrease of their melting temperatures (Tm. To obtain a more detailed insight into these interactions, MALDI-TOF mass spectrometry was employed. This study revealed that E2 with DNA forms non-covalent physical complexes, observed as the mass shifts for app. 270 Da (Mr of E2 to higher molecular masses. Taken together, our results indicate that E2 can affect biomacromolecules, as circulating oligonucleotides, which can trigger mutations, leading to various unwanted effects.

  18. Immunoglobulins acquire the ability to interact with DNA after chromatography on QAE-Sephadex

    International Nuclear Information System (INIS)

    Sulaeva, N.I.; Lekakh, I.V.; Poverennyi, A.M.

    1986-01-01

    It was established that IgG isolated from the sera of healthy humans contains a substantial number of antibodies that react with native DNA. Their ability to interact with DNA is manifested only after chromatography on an anion exchange resin, as a result of which IgG is divided into two portions - acid and basic immunoglobulins. The peculiarities of the interaction of both fractions with DNA and the specificity of this reaction were investigated. It was shown that the investigated IgG can react with native and denatured DNA, dextran sulfate, poly(G), and poly(I). The question of the possibility of the interaction of the antibodies studied with the charged structures of the cell and that of the role of these antibodies in the normal and pathological states are discussed

  19. Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses.

    Science.gov (United States)

    Nebbioso, Angela; Benedetti, Rosaria; Conte, Mariarosaria; Carafa, Vincenzo; De Bellis, Floriana; Shaik, Jani; Matarese, Filomena; Della Ventura, Bartolomeo; Gesuele, Felice; Velotta, Raffaele; Martens, Joost H A; Stunnenberg, Hendrik G; Altucci, Carlo; Altucci, Lucia

    2017-09-15

    Interactions between DNA and proteins are mainly studied through chemical procedures involving bi-functional reagents, mostly formaldehyde. Chromatin immunoprecipitation is used to identify the binding between transcription factors (TFs) and chromatin, and to evaluate the occurrence and impact of histone/DNA modifications. The current bottleneck in probing DNA-protein interactions using these approaches is caused by the fact that chemical crosslinkers do not discriminate direct and indirect bindings or short-lived chromatin occupancy. Here, we describe a novel application of UV laser-induced (L-) crosslinking and demonstrate that a combination of chemical and L-crosslinking is able to distinguish between direct and indirect DNA-protein interactions in a small number of living cells. The spatial and temporal dynamics of TF bindings to chromatin and their role in gene expression regulation may thus be assessed. The combination of chemical and L-crosslinking offers an exciting and unprecedented tool for biomedical applications.

  20. An experimental and theoretical study on the interaction of DNA and BSA with novel Ni2 +, Cu2 + and VO2 + complexes derived from vanillin bidentate Schiff base ligand

    Science.gov (United States)

    Dostani, Morteza; Kianfar, Ali Hossein; Mahmood, Wan Ahmad Kamil; Dinari, Mohammad; Farrokhpour, Hossein; Sabzalian, Mohammad R.; Abyar, Fatemeh; Azarian, Mohammad Hossein

    2017-06-01

    In this investigation, the structure of bidentate N,N-Schiff base ligand of vanillin, (E)-4-(((2-amino-5-nitrophenyl)imino)methyl)-2-methoxyphenol (HL) was determined by single crystal X-ray diffraction. The interaction of new [CuL2], [NiL2] and [VOL2] complexes with DNA and BSA was explored through UV-Vis and fluorescence spectroscopy. The electronic spectra changes displayed an isosbestic point for the complexes upon titration with DNA. The Kb values for the complexes [CuL2], [NiL2] and [VOL2] were 2.4 × 105, 1.9 × 105 and 4.2 × 104, respectively. [CuL2] complex was bound more toughly than [NiL2] and [VOL2] complexes. These complexes had a significant interaction with Bovine Serum Albumin (BSA) and the results demonstrated that the quenching mechanism was a static procedure. Also, the complexes interacted with BSA by more than one binding site (n > 1). Finally, the theoretical studies were performed using the docking method to calculate the binding constants and recognize the binding site of the DNA and BSA with the complexes. The ligand and complexes including Ni2 +, Cu2 + and VO2 + ions were colonized by fungal growth.

  1. Dynamics of bleomycin interaction with a strongly bound hairpin DNA substrate, and implications for cleavage of the bound DNA.

    Science.gov (United States)

    Bozeman, Trevor C; Nanjunda, Rupesh; Tang, Chenhong; Liu, Yang; Segerman, Zachary J; Zaleski, Paul A; Wilson, W David; Hecht, Sidney M

    2012-10-31

    Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe·BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B(2) binding to a strongly bound hairpin DNA, to define the effects of Fe(3+), salt, and temperature on BLM-DNA interaction. One strong primary DNA binding site, and at least one much weaker site, were documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus, enhanced binding to a given site does not necessarily result in increased DNA degradation at that site; i.e., for strongly bound DNAs, the facility of DNA cleavage must involve other parameters in addition to the intrinsic rate of C-4' H atom abstraction from DNA sugars.

  2. Multiplex single-molecule interaction profiling of DNA barcoded proteins

    Science.gov (United States)

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

    2014-01-01

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

  3. Synthesis, spectral characterization, theoretical, antimicrobial, DNA interaction and in vitro anticancer studies of Cu(II and Zn(II complexes with pyrimidine-morpholine based Schiff base ligand

    Directory of Open Access Journals (Sweden)

    M. Sankarganesh

    2018-05-01

    Full Text Available Novel Cu(II (1 and Zn(II (2 complexes with 4-(1-(4-morpholinophenylethylideneaminopyrimidine-5-carbonitrile (L have been synthesized and characterized by various spectroscopic and analytical techniques. DFT (density functional theory studies result confirms that, LMCT mechanism have been done between L and M(II ions. The antimicrobial studies indicate that the ligand L and complexes 1 & 2 exhibit higher activity against the E. coli bacteria and C. albicans fungi. The groove binding mode of ligand L and complexes 1 & 2 with CT-DNA have been confirmed by electronic absorption, competitive binding, viscometric and cyclic voltammetric studies. The electronic absorption titration of ligand L and complexes 1 & 2 with DNA have been carried out in different buffer solutions (pH 4.0, 7.0 & 10.0. The Kb values of ligand L and complexes 1 & 2 are higher in acidic buffer at pH 4.0 (Kb = 2.42 × 105, L; 2.8 × 105, 1; 2.65 × 105, 2 and the results revealed that, the interaction of synthesized compounds with DNA were higher in the acidic medium than basic and neutral medium. Furthermore, CT-DNA cleavage studies of ligand L and complexes 1 & 2 have been studied. The in vitro anticancer activities results show that complexes 1 & 2 have moderate cytotoxicity against cancer cell lines and low toxicity on normal cell line than ligand L. Keywords: Pyrimidine, Morpholine, DFT, Antimicrobial, DNA binding, Anticancer studies

  4. DNA requirements for interaction of the C-terminal region of Ku80 with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

    Science.gov (United States)

    Radhakrishnan, Sarvan Kumar; Lees-Miller, Susan P

    2017-09-01

    Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25bp dsDNA or 25bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35bp blunt ended dsDNA) or 25bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. The Importance of Electron Correlation on Stacking Interaction of Adenine-Thymine Base-Pair Step in B-DNA: A Quantum Monte Carlo Study.

    Science.gov (United States)

    Hongo, Kenta; Cuong, Nguyen Thanh; Maezono, Ryo

    2013-02-12

    We report fixed-node diffusion Monte Carlo (DMC) calculations of stacking interaction energy between two adenine(A)-thymine(T) base pairs in B-DNA (AA:TT), for which reference data are available, obtained from a complete basis set estimate of CCSD(T) (coupled-cluster with singles, doubles, and perturbative triples). We consider four sets of nodal surfaces obtained from self-consistent field calculations and examine how the different nodal surfaces affect the DMC potential energy curves of the AA:TT molecule and the resulting stacking energies. We find that the DMC potential energy curves using the different nodes look similar to each other as a whole. We also benchmark the performance of various quantum chemistry methods, including Hartree-Fock (HF) theory, second-order Møller-Plesset perturbation theory (MP2), and density functional theory (DFT). The DMC and recently developed DFT results of the stacking energy reasonably agree with the reference, while the HF, MP2, and conventional DFT methods give unsatisfactory results.

  6. Substrate interactions and promiscuity in a viral DNA packaging motor.

    Science.gov (United States)

    Aathavan, K; Politzer, Adam T; Kaplan, Ariel; Moffitt, Jeffrey R; Chemla, Yann R; Grimes, Shelley; Jardine, Paul J; Anderson, Dwight L; Bustamante, Carlos

    2009-10-01

    The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage varphi29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring of gene product 16 (gp16), involves two phases-an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps triggered by hydrolysis product release. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5'-3' strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily.

  7. Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

    Science.gov (United States)

    Smith, Rebecca J; Savoian, Matthew S; Weber, Lauren E; Park, Jeong Hyeon

    2016-11-04

    Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

  8. Non-Watson–Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

    Science.gov (United States)

    Tackett, Alan J.; Corey, David R.; Raney, Kevin D.

    2002-01-01

    Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications. PMID:11842106

  9. Synthesis and DNA interaction of a Sm(III) complex of a Schiff base ...

    African Journals Online (AJOL)

    The interaction between the Sm(III) complex of an ionic Schiff base [HL]-, derived from vanillin and L-tryptophan, and herring sperm DNA at physiological pH (7.40) has been studied by UV-Vis absorption, fluorescence and viscosity methods. The binding ratios nSm(III) : nK[HL] = 1:1 and nSm(III)L: nDNA =5:1 were confirmed ...

  10. Interaction of DNA/nuclear protein/polycation and the terplexes for gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Shen Yuan; Pan Shirong; Feng Min; Wen Yuting; Deng Jingjing; Luo Xin; Wu Chuanbin [School of Pharmaceutical Sciences, Sun Yat-sen University, Zhongshan II Road 74, Guangzhou 510080 (China); Peng Hui, E-mail: fengmin@mail.sysu.edu.cn [School of Zhongshan Medicine, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou 510080 (China)

    2010-01-29

    Nuclear transport of exogenous DNA is a major barrier to nonviral gene delivery that needs to be addressed in the design of new vectors. In this study, we prepared pDNA/HMGB1/PEG-PEI terplexes to promote nuclear import. HMGB1 in the terplexes was used to assist the transportation of pDNA into the nucleus of cells, since it contained nuclear localization signal (NLS); PEG chains were introduced to stabilize pDNA/vector terplexes and reduce the cytotoxicity. HMGB1/PEG-PEI combined vectors have been investigated specifically for their structure interaction by atomic force microscopy and circular dichroic spectroscopy. The results demonstrated that the HMGB1 molecule could bind with the pDNA chains, but not condense pDNA well. The PEG-PEI further compacted pDNA/HMGB1 complexes into nanosized spherical terplexes. The pDNA delivered by HMGB1/PEG-PEI combined vectors was significantly accumulated in the nucleus of cells, as observed by confocal laser scanning microscopy. The percentage of GFP-transfected cells and VEGF protein expression level induced by HMGB1/PEG-PEI were 2.6-4.9-fold and 1.4-2.8-fold higher, respectively, than that of a common cationic polymer PEI 25 kDa. Therefore, the HMGB1/PEG-PEI combined vector could be used as a versatile vector for promoting exogenous DNA nuclear localization, thereby enhancing its expression.

  11. DNA Self-Assembly and Computation Studied with a Coarse-grained Dynamic Bonded Model

    DEFF Research Database (Denmark)

    Svaneborg, Carsten; Fellermann, Harold; Rasmussen, Steen

    2012-01-01

    We utilize a coarse-grained directional dynamic bonding DNA model [C. Svaneborg, Comp. Phys. Comm. (In Press DOI:10.1016/j.cpc.2012.03.005)] to study DNA self-assembly and DNA computation. In our DNA model, a single nucleotide is represented by a single interaction site, and complementary sites can...

  12. Label-free detection of biomolecular interactionDNA — Antimicrobial peptide binding

    DEFF Research Database (Denmark)

    Fojan, Peter; Jensen, Kasper Risgaard; Gurevich, Leonid

    2011-01-01

    the molecule. In particular, surface plasmon resonance (SPR) sensors have been already demonstrated suitable for food-safety control, label-free screening for various disease markers in bodily fluids, as well as for real-time continuous monitoring of drug levels in intensive care environment. We envisage...... of plasmon based biosensors to the study of the interaction of Antimicrobial peptide IL4 and DNA. Our results indicate high affinity binding between IL4 and DNA thereby preventing DNA replication and eventually killing the affected cell. We speculate that this is common for a large class of Antimicrobial...

  13. Interaction of a copper (II) complex containing an artificial sweetener (aspartame) with calf thymus DNA.

    Science.gov (United States)

    Shahabadi, Nahid; Khodaei, Mohammad Mehdi; Kashanian, Soheila; Kheirdoosh, Fahimeh

    2014-01-01

    A copper (II) complex containing aspartame (APM) as ligand, Cu(APM)2Cl2⋅2H2O, was synthesized and characterized. In vitro binding interaction of this complex with native calf thymus DNA (CT-DNA) was studied at physiological pH. The interaction was studied using different methods: spectrophotometric, spectrofluorometric, competition experiment, circular dichroism (CD) and viscosimetric techniques. Hyperchromicity was observed in UV absorption band of Cu(APM)2Cl2⋅2H2O. A strong fluorescence quenching reaction of DNA to Cu(APM)2Cl2⋅2H2O was observed and the binding constants (Kf) and corresponding numbers of binding sites (n) were calculated at different temperatures. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be+89.3 kJ mol(-1) and+379.3 J mol(-1) K(-1) according to Van't Hoff equation which indicated that reaction is predominantly entropically driven. Experimental results from spectroscopic methods were comparable and further supported by viscosity measurements. We suggest that Cu(APM)2Cl2⋅2H2O interacts with calf thymus DNA via a groove interaction mode with an intrinsic binding constant of 8×10+4 M(-1). Binding of this copper complex to DNA was found to be stronger compared to aspartame which was studied recently. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Antimicrobial activity, cytotoxicity and DNA binding studies of carbon dots

    Science.gov (United States)

    Jhonsi, Mariadoss Asha; Ananth, Devanesan Arul; Nambirajan, Gayathri; Sivasudha, Thilagar; Yamini, Rekha; Bera, Soumen; Kathiravan, Arunkumar

    2018-05-01

    In recent years, quantum dots (QDs) are one of the most promising nanomaterials in life sciences community due to their unexploited potential in biomedical applications; particularly in bio-labeling and sensing. In the advanced nanomaterials, carbon dots (CDs) have shown promise in next generation bioimaging and drug delivery studies. Therefore the knowledge of the exact nature of interaction with biomolecules is of great interest to designing better biosensors. In this study, the interaction between CDs derived from tamarind and calf thymus DNA (ct-DNA) has been studied by vital spectroscopic techniques, which revealed that the CDs could interact with DNA via intercalation. The apparent association constant has been deduced from the absorption spectral changes of ct-DNA-CDs using the Benesi-Hildebrand equation. From the DNA induced emission quenching experiments the apparent DNA binding constant of the CDs (Kapp) have also been evaluated. Furthermore, we have analyzed the antibacterial and antifungal activity of CDs using disc diffusion assay method which exhibited excellent activity against E. coli and C. albicans with inhibition zone in the range of 7-12 mm. The biocompatible nature of CDs was confirmed by an in vitro cytotoxicity test on L6 normal rat myoblast cells by using MTT assay. The cell viability is not affected till the high dosage of CDs (200 μg/mL) for >48 h. As a consequence of the work, future development of CDs for microbial control and DNA sensing among the various biomolecules is possible in view of emerging biofields.

  15. DNA-binding, DNA cleavage and cytotoxicity studies of two anthraquinone derivatives.

    Science.gov (United States)

    Gholivand, M B; Kashanian, S; Peyman, H

    2012-02-15

    The interaction of native calf thymus DNA (CT-DNA) with two anthraquinones including quinizarin (1,4-dihydroxy anthraquinone) and danthron (1,8-dihydroxy anthraquinone) in a mixture of 0.04M Brittone-Robinson buffer and 50% of ethanol were studied at physiological pH by spectrofluorometric and cyclic voltammetry techniques. The former technique was used to calculate the binding constants of anthraquinones-DNA complexes at different temperatures. Thermodynamic study indicated that the reactions of both anthraquinone-DNA systems are predominantly entropically driven. Furthermore, the binding mechanisms on the reaction of the two anthraquinones with DNA and the effect of ionic strength on the fluorescence property of the system have also been investigated. The results of the experiments indicated that the binding modes of quinizarin and danthron with DNA were evaluated to be groove binding. Moreover, the cytotoxic activity of both compounds against human chronic myelogenous leukemia K562 cell line and DNA cleavage were investigated. The results indicated that these compounds slightly cleavage pUC18 plasmid DNA and showed minor antitumor activity against K562 (human chronic myeloid leukemia) cell line. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Molecular dynamics simulations of the DNA interaction with metallic nanoparticles and TiO2 surfaces

    International Nuclear Information System (INIS)

    Kholmurodov, Kh.T.; Krasavin, E.A.; Dushanov, E.B.; Hassan, H.K.; Galal, A.; ElHabashy, H.A.; Sweilam, N.H.; Yasuoka, K.

    2013-01-01

    The understanding of the mechanism of DNA interactions and binding with metallic nanoparticles (NPs) and surfaces represents a great interest in today's medicine applications due to diagnostic and treatment of oncology diseases. Recent experimental and simulation studies involve the DNA interaction with highly localized proton beams or metallic NPs (such as Ag, Au, etc.), aimed at targeted cancer therapy through the injection of metal micro- or nanoparticles into the tumor tissue with consequent local microwave or laser heating. The effects of mutational structure changes in DNA and protein structures could result in destroying of native chemical (hydrogen) bonds or, on the contrary, creating of new bonds that do not normally exist there. The cause of such changes might be the alteration of one or several nucleotides (in DNA) or the substitution of specific amino acid residues (in proteins) that can lead to the essential structural destabilization or unfolding. At the atomic or molecular level, the replacement of one nucleotide by another (in DNA double helices) or replacement of one amino acid residue by another (in proteins) cause essential modifications of the molecular force fields of the environment that break locally important hydrogen bonds underlying the structural stability of the biological molecules. In this work, the molecular dynamics(MD) simulations were performed for four DNA models and the flexibilities of the purine and pyrimidine nucleotides during the interaction process with the metallic NPs and TiO 2 surface were clarified

  17. eMethylsorb: electrochemical quantification of DNA methylation at CpG resolution using DNA-gold affinity interactions.

    Science.gov (United States)

    Sina, Abu Ali Ibn; Howell, Sidney; Carrascosa, Laura G; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

    2014-11-07

    We report a simple electrochemical method referred to as "eMethylsorb" for the detection of DNA methylation. The method relies on the base dependent affinity interaction of DNA with gold. The methylation status of DNA is quantified by monitoring the electrochemical current as a function of the relative adsorption level of bisulphite treated DNA samples onto a bare gold electrode. This method can successfully distinguish methylated and unmethylated epigenotypes at single CpG resolution.

  18. DNMT1-interacting RNAs block gene specific DNA methylation

    Science.gov (United States)

    Di Ruscio, Annalisa; Ebralidze, Alexander K.; Benoukraf, Touati; Amabile, Giovanni; Goff, Loyal A.; Terragni, Joylon; Figueroa, Maria Eugenia; De Figureido Pontes, Lorena Lobo; Alberich-Jorda, Meritxell; Zhang, Pu; Wu, Mengchu; D’Alò, Francesco; Melnick, Ari; Leone, Giuseppe; Ebralidze, Konstantin K.; Pradhan, Sriharsa; Rinn, John L.; Tenen, Daniel G.

    2013-01-01

    Summary DNA methylation was described almost a century ago. However, the rules governing its establishment and maintenance remain elusive. Here, we present data demonstrating that active transcription regulates levels of genomic methylation. We identified a novel RNA arising from the CEBPA gene locus critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extended the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene selective demethylation of therapeutic targets in disease. PMID:24107992

  19. Sequence-influenced interactions of oligoacridines with DNA detected by retarded gel electrophorectic migrations

    International Nuclear Information System (INIS)

    Nielsen, P.E.; Zhen, W.; Henriksen, U.; Buchardt, O.

    1988-01-01

    The authors have found that di-, tri-, tetra-, and hexa-9-acridinylamines are so efficiently associated with DNA during electrophoresis in polyacrylamide or agarose gels that they retard its migration. The retardation is roughly proportional to the reagent to base pair ratio, and the magnitude of the retardation indicates that a combined charge neutralization/helix extension mechanism is mainly responsible for the effect. Furthermore, DNA sequence dependent differences are observed. Thus, the pUC 19 restriction fragments (HaeIII or AluI), which in the native state comigrate upon gel electrophoretic analysis, could be separated in the presence of a diacridine, and specific DNA fragments responded differently to different diacridines. These results suggest that the effect also is due to a contribution from the DNA conformation and that the DNA conformation dynamics are influenced differently upon binding of different diacridines. They foresee three applications of this observation: (1) in analytical gel electrophoretic separation of otherwise comigrating DNA molecules, (2) in studies of polyintercalator-DNA interaction, and (3) in measurements of polyintercalator-induced DNA unwinding

  20. Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy.

    Directory of Open Access Journals (Sweden)

    Vladislav V Melekhov

    Full Text Available Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe2+ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.

  1. DNA repair studies in mammalian germ cells

    International Nuclear Information System (INIS)

    Sega, G.A.; Owens, J.G.

    1984-01-01

    In submammalian test systems, nitrosocarbamates (NEC) are 100-fold more mutagenic than are their corresponding nitrosourea homologues. To learn more about its interaction with germ-cell DNA in the mouse testis, male mice were given i.p. injections of NEC. Testicular injections of [ 3 H]dThd were given along with the NEC. Sixteen days after treatment, sperm were recovered from the caudal epididymides and assayed for an unscheduled-DNA-synthesis

  2. Interactions of quercetin-uranium complexes with biomembranes and DNA

    Energy Technology Data Exchange (ETDEWEB)

    Attia, Enas Mohammed Hassan

    2014-07-21

    has been also confirmed from the DFT calculations. Finally, interaction experiments of uranyl-quercetin complex with DNA have been performed to assess an alternative uranyl-trapping and photoreduction system. The data show that consecutive addition of quercetin and uranyl destabilizes DNA. However, a preformed uranyl quercetin complex has very little effect on DNA structure. On the other hand, quercetin and uranyl appear to bind to DNA as a preformed complex in the loop portion of hairpin DNA. Therefore, also HP DNA is expected to be a suitable but less effective trapping system for the uranyl quercetin complex and its potential photoproducts.

  3. Interactions of quercetin-uranium complexes with biomembranes and DNA

    International Nuclear Information System (INIS)

    Attia, Enas Mohammed Hassan

    2014-01-01

    has been also confirmed from the DFT calculations. Finally, interaction experiments of uranyl-quercetin complex with DNA have been performed to assess an alternative uranyl-trapping and photoreduction system. The data show that consecutive addition of quercetin and uranyl destabilizes DNA. However, a preformed uranyl quercetin complex has very little effect on DNA structure. On the other hand, quercetin and uranyl appear to bind to DNA as a preformed complex in the loop portion of hairpin DNA. Therefore, also HP DNA is expected to be a suitable but less effective trapping system for the uranyl quercetin complex and its potential photoproducts.

  4. Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

    Science.gov (United States)

    Patel, Meera J; Bhatia, Lavesh; Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

    2017-09-01

    DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. A new insight into the interaction of ZnO with calf thymus DNA through surface defects.

    Science.gov (United States)

    Das, Sumita; Chatterjee, Sabyasachi; Pramanik, Srikrishna; Devi, Parukuttyamma Sujatha; Kumar, Gopinatha Suresh

    2018-01-01

    Experimental evidences on the binding interaction of ZnO and Calf Thymus (CT) DNA using several biophysical techniques are the centre of interest of the present study. The interaction of ZnO with CT DNA has been investigated in detail by absorption spectral study, fluorescence titration, Raman analysis, zeta potential measurement, viscometric experiment along with thermal melting study and microscopic analysis. Steady-state fluorescence study revealed the quenching (48%) of the surface defect related peak intensity of ZnO on interaction with DNA. The optimized concentration of ZnO and DNA to obtain this level of quenching has been found to be 0.049mM and 1.027μM, respectively. Additional fluorescence study with 8-hydroxy-5-quinoline (HQ) as a fluorescence probe for Zn 2+ ruled out the dissolution effect of ZnO under the experimental conditions. DNA conjugation on the surface of ZnO was also supported by Raman study. The quantitative variation in conductivity as well as electrophoretic mobility indicated significant interaction of ZnO with the DNA molecule. Circular dichroism (CD) and viscometry titrations provided clear evidence in support of the conformational retention of the DNA on interaction with ZnO. The binding interaction was found to be predominantly entropy driven in nature. The bio-physical studies presented in this paper exploring ZnO-CT DNA interaction could add a new horizon to understand the interaction between metal oxide and DNA. Copyright © 2017. Published by Elsevier B.V.

  6. Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

    Science.gov (United States)

    Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

    2014-01-01

    HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

  7. Energetic and binding properties of DNA upon interaction with dodecyl trimethylammonium bromide.

    Science.gov (United States)

    Bathaie, S Z; Moosavi-Movahedi, A A; Saboury, A A

    1999-02-15

    The interaction of dodecyl trimethylammonium bromide (DTAB), a cationic surfactant, with calf thymus DNA has been studied by various methods, including potentiometric technique using DTAB-selective plastic membrane electrode at 27 and 37 degreesC, isothermal titration microcalorimetry and UV spectrophotometry at 27 degreesC using 0.05 M Tris buffer and 0.01 M NaCl at pH 7.4. The free energy is calculated from binding isotherms on the basis of Wyman binding potential theory and the enthalpy of binding according to van't Hoff relation. The enthalpy of unfolding has been determined by subtraction of the enthalpy of binding from the microcalorimetric enthalpy. The results show that, after the interaction of first DTAB molecule to DNA (base molarity) through the electrostatic interaction, the second DTAB molecule also binds to DNA through electrostatic interaction. At this stage, the predom-inant DNA conformational change occurs. Afterwards up to 20 DTAB molecules, below the critical micelle concentration of DTAB, bind through hydrophobic interactions.

  8. Mapping DNA damage-dependent genetic interactions in yeast via party mating and barcode fusion genetics.

    Science.gov (United States)

    Díaz-Mejía, J Javier; Celaj, Albi; Mellor, Joseph C; Coté, Atina; Balint, Attila; Ho, Brandon; Bansal, Pritpal; Shaeri, Fatemeh; Gebbia, Marinella; Weile, Jochen; Verby, Marta; Karkhanina, Anna; Zhang, YiFan; Wong, Cassandra; Rich, Justin; Prendergast, D'Arcy; Gupta, Gaurav; Öztürk, Sedide; Durocher, Daniel; Brown, Grant W; Roth, Frederick P

    2018-05-28

    Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1 , SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

  9. Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

    CERN Document Server

    Williams, Mark C

    2011-01-01

    This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

  10. Analysis of damaged DNA / proteins interactions: Methodological optimizations and applications to DNA lesions induced by platinum anticancer drugs

    International Nuclear Information System (INIS)

    Bounaix Morand du Puch, Ch

    2010-10-01

    DNA lesions contribute to the alteration of DNA structure, thereby inhibiting essential cellular processes. Such alterations may be beneficial for chemotherapies, for example in the case of platinum anticancer agents. They generate bulky adducts that, if not repaired, ultimately cause apoptosis. A better understanding of the biological response to such molecules can be obtained through the study of proteins that directly interact with the damages. These proteins constitute the DNA lesions interactome. This thesis presents the development of tools aiming at increasing the list of platinum adduct-associated proteins. Firstly, we designed a ligand fishing system made of damaged plasmids immobilized onto magnetic beads. Three platinum drugs were selected for our study: cisplatin, oxali-platin and satra-platin. Following exposure of the trap to nuclear extracts from HeLa cancer cells and identification of retained proteins by proteomics, we obtained already known candidates (HMGB1, hUBF, FACT complex) but also 29 new members of the platinated-DNA interactome. Among them, we noted the presence of PNUTS, TOX4 and WDR82, which associate to form the recently-discovered PTW/PP complex. Their capture was then confirmed with a second model, namely breast cancer cell line MDA MB 231, and the biological consequences of such an interaction now need to be elucidated. Secondly, we adapted a SPRi bio-chip to the study of platinum-damaged DNA/proteins interactions. Affinity of HMGB1 and newly characterized TOX4 for adducts generated by our three platinum drugs could be validated thanks to the bio-chip. Finally, we used our tools, as well as analytical chemistry and biochemistry methods, to evaluate the role of DDB2 (a factor involved in the recognition of UV-induced lesions) in the repair of cisplatin adducts. Our experiments using MDA MB 231 cells differentially expressing DDB2 showed that this protein is not responsible for the repair of platinum damages. Instead, it appears to act

  11. DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions.

    Science.gov (United States)

    Lambrughi, Matteo; De Gioia, Luca; Gervasio, Francesco Luigi; Lindorff-Larsen, Kresten; Nussinov, Ruth; Urani, Chiara; Bruschi, Maurizio; Papaleo, Elena

    2016-11-02

    Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA-protein interaction

    International Nuclear Information System (INIS)

    Kurita, Hirofumi; Yasuda, Hachiro; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

    2009-01-01

    We carried out an individual DNA manipulation using an optical trapping for a microbead. This manipulation system is based on a fluorescent microscopy equipped with an IR laser. Both ends of linear DNA molecule were labeled with a biotin and a thiol group, respectively. Then the biotinylated end was attached to a microbead, and the other was immobilized on a thiol-linkable glass surface. We controlled the form of an individual DNA molecule by moving the focal point of IR laser, which trapped the microbead. In addition, we applied single-molecule approach to analyze DNA hydrolysis. We also used microchannel for single-molecule observation of DNA hydrolysis. The shortening of DNA in length caused by enzymatic hydrolysis was observed in real-time. The single-molecule DNA manipulation should contribute to elucidate detailed mechanisms of DNA-protein interactions

  13. Dose calculation due to electrons interaction with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Mark, S; Orion, I; Shani, G [Ben-Gurion Univ. of the Negev, Beersheba (Israel). Dept. of Nuclear Engineering; Laster, B [Brookhaven National Lab., Upton, NY (United States)

    1996-12-01

    Experiments done with gadolinium loaded V79 Chinese Hamster cells, irradiated with thermal neutrons, showed that cells lethality increased by a factor of 1.8 compared to the case where the Gd atoms were located outside the cell.(l) It was obvious that the dramatic increase in cell lethality is due to the emission of Auger electrons following the {sup 157}Gd(n,{gamma}){sup 158}Gd reaction. Electrons of various energies from about 40 keV (very few) to less than 1 keV, are emitted. In the present work, energy absorbed in DNA was calculated, due to interaction of electron of different energies: 30, 15, 10, 8, 5 and 2 keV. The Monte Carlo code EGS4(2) was used for the calculations. The DNA was modeled as a series of alternative layers of sugar (phosphate - C{sub 5}O{sub 5}H{sub 7}P p=1.39gr cm{sup -1}) and water. The sugar layer thickness was assumed 2.5nm and the water layer thickness 10nm. An isotropic electron source was assumed to be located in a water layer and the electrons interactions (absorption and scattering) were calculated in the forward hemisphere. The energy absorbed in a group of 8 layers, (4 sugar and 4 water) was calculated for each one of the electron energies. An interesting fact found in those calculations; when the source electrons energy is 10 keV or more, most of the electrons are absorbed in the DNA-water system, are at energy about 2keV. There is no good explanation for this phenomenon except for assuming that when the electron`s energy reaches a low point of about 2keV, it cannot escape absorption in the medium. 10% of the 10 keV electrons deposit their entire energy in the 8 layers range (authors).

  14. Structure of uracil-DNA glycosylase from Mycobacterium tuberculosis: insights into interactions with ligands

    International Nuclear Information System (INIS)

    Kaushal, Prem Singh; Talawar, Ramappa K.; Varshney, Umesh; Vijayan, M.

    2010-01-01

    The molecule of uracil-DNA glycosylase from M. tuberculosis exhibits domain motion on binding to DNA or a proteinaceous inhibitor. The highly conserved DNA-binding region interacts with a citrate ion in the structure. Uracil N-glycosylase (Ung) is the most thoroughly studied of the group of uracil DNA-glycosylase (UDG) enzymes that catalyse the first step in the uracil excision-repair pathway. The overall structure of the enzyme from Mycobacterium tuberculosis is essentially the same as that of the enzyme from other sources. However, differences exist in the N- and C-terminal stretches and some catalytic loops. Comparison with appropriate structures indicate that the two-domain enzyme closes slightly when binding to DNA, while it opens slightly when binding to the proteinaceous inhibitor Ugi. The structural changes in the catalytic loops on complexation reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino-acid residues in the catalytic loops. The uracil-binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil, in addition to providing insights into other possible interactions that inhibitors could be involved in

  15. Palladium polypyridyl complexes: synthesis, characterization, DNA interaction and biological activity on Leishmania (L.) mexicana

    Energy Technology Data Exchange (ETDEWEB)

    Navarro, Maribel [Instituto Venezolano de Investigaciones Cientificas, Caracas (Venezuela). Centro de Quimica; Betancourt, Adelmo [Universidad de Carabobo, Valencia (Venezuela). Facultad Experimental de Ciencia y Tecnologia. Dept. de Quimica; Hernandez, Clara [Universidad de Carabobo Sede Aragua, Maracay (Venezuela). Facultad de Ciencias de la Salud. Dept. de Ciencias Basicas; Marchan, Edgar [Universidad de Oriente, Cumana (Venezuela). Inst. de Investigaciones en Biomedicina y Ciencias Aplicadas. Nucleo de Sucre

    2008-07-01

    This paper describes the search for new potential chemotherapeutic agents based on transition metal complexes with planar ligands. In this study, palladium polypyridyl complexes were synthesized and characterized by elemental analysis, NMR, UV-VIS and IR spectroscopies. The interaction of the complexes with DNA was also investigated by spectroscopic methods. All metal-to-ligand charge transfer (MLCT) bands of the palladium polypyridyl complexes exhibited hypochromism and red shift in the presence of DNA. The binding constant and viscosity data suggested that the complexes [PdCl{sub 2}(phen)] and [PdCl{sub 2}(phendiamine)] interact with DNA by electrostatic forces. Additionally, these complexes induced an important leishmanistatic effect on L. (L.) mexicana promastigotes at the final concentration of 10 {mu}mol L{sup -1} in 48 h. (author)

  16. Palladium polypyridyl complexes: synthesis, characterization, DNA interaction and biological activity on Leishmania (L.) mexicana

    International Nuclear Information System (INIS)

    Navarro, Maribel; Betancourt, Adelmo; Hernandez, Clara; Marchan, Edgar

    2008-01-01

    This paper describes the search for new potential chemotherapeutic agents based on transition metal complexes with planar ligands. In this study, palladium polypyridyl complexes were synthesized and characterized by elemental analysis, NMR, UV-VIS and IR spectroscopies. The interaction of the complexes with DNA was also investigated by spectroscopic methods. All metal-to-ligand charge transfer (MLCT) bands of the palladium polypyridyl complexes exhibited hypochromism and red shift in the presence of DNA. The binding constant and viscosity data suggested that the complexes [PdCl 2 (phen)] and [PdCl 2 (phendiamine)] interact with DNA by electrostatic forces. Additionally, these complexes induced an important leishmanistatic effect on L. (L.) mexicana promastigotes at the final concentration of 10 μmol L -1 in 48 h. (author)

  17. Molecular dynamics simulations revealed structural differences among WRKY domain-DNA interaction in barley (Hordeum vulgare).

    Science.gov (United States)

    Pandey, Bharati; Grover, Abhinav; Sharma, Pradeep

    2018-02-12

    The WRKY transcription factors are a class of DNA-binding proteins involved in diverse plant processes play critical roles in response to abiotic and biotic stresses. Genome-wide divergence analysis of WRKY gene family in Hordeum vulgare provided a framework for molecular evolution and functional roles. So far, the crystal structure of WRKY from barley has not been resolved; moreover, knowledge of the three-dimensional structure of WRKY domain is pre-requisites for exploring the protein-DNA recognition mechanisms. Homology modelling based approach was used to generate structures for WRKY DNA binding domain (DBD) and its variants using AtWRKY1 as a template. Finally, the stability and conformational changes of the generated model in unbound and bound form was examined through atomistic molecular dynamics (MD) simulations for 100 ns time period. In this study, we investigated the comparative binding pattern of WRKY domain and its variants with W-box cis-regulatory element using molecular docking and dynamics (MD) simulations assays. The atomic insight into WRKY domain exhibited significant variation in the intermolecular hydrogen bonding pattern, leading to the structural anomalies in the variant type and differences in the DNA-binding specificities. Based on the MD analysis, residual contribution and interaction contour, wild-type WRKY (HvWRKY46) were found to interact with DNA through highly conserved heptapeptide in the pre- and post-MD simulated complexes, whereas heptapeptide interaction with DNA was missing in variants (I and II) in post-MD complexes. Consequently, through principal component analysis, wild-type WRKY was also found to be more stable by obscuring a reduced conformational space than the variant I (HvWRKY34). Lastly, high binding free energy for wild-type and variant II allowed us to conclude that wild-type WRKY-DNA complex was more stable relative to variants I. The results of our study revealed complete dynamic and structural information

  18. Study of DNA reconstruction enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Sekiguchi, M [Kyushu Univ., Fukuoka (Japan). Faculty of Science

    1976-12-01

    Description was made of the characteristics and mechanism of 3 reconstructive enzymes which received from M. luteus or E. coli or T4, and of which natures were clarified as reconstructive enzymes of DNA irradiated with ultraviolet rays. As characteristics, the site of breaking, reaction, molecular weight, electric charge in the neutrality and a specific adhesion to DNA irradiated with ultraviolet rays were mentioned. As to mutant of ultraviolet ray sensitivity, hereditary control mechanism of removal and reconstruction by endo-nuclease activation was described, and suggestion was referred to removal and reconstruction of cells of xedoderma pigmentosum which is a hereditary disease of human. Description was also made as to the mechanism of exonuclease activation which separates dimer selectively from irradiated DNA.

  19. Predicting Variation of DNA Shape Preferences in Protein-DNA Interaction in Cancer Cells with a New Biophysical Model.

    Science.gov (United States)

    Batmanov, Kirill; Wang, Junbai

    2017-09-18

    DNA shape readout is an important mechanism of transcription factor target site recognition, in addition to the sequence readout. Several machine learning-based models of transcription factor-DNA interactions, considering DNA shape features, have been developed in recent years. Here, we present a new biophysical model of protein-DNA interactions by integrating the DNA shape properties. It is based on the neighbor dinucleotide dependency model BayesPI2, where new parameters are restricted to a subspace spanned by the dinucleotide form of DNA shape features. This allows a biophysical interpretation of the new parameters as a position-dependent preference towards specific DNA shape features. Using the new model, we explore the variation of DNA shape preferences in several transcription factors across various cancer cell lines and cellular conditions. The results reveal that there are DNA shape variations at FOXA1 (Forkhead Box Protein A1) binding sites in steroid-treated MCF7 cells. The new biophysical model is useful for elucidating the finer details of transcription factor-DNA interaction, as well as for predicting cancer mutation effects in the future.

  20. Sequence Dependent Interactions Between DNA and Single-Walled Carbon Nanotubes

    Science.gov (United States)

    Roxbury, Daniel

    It is known that single-stranded DNA adopts a helical wrap around a single-walled carbon nanotube (SWCNT), forming a water-dispersible hybrid molecule. The ability to sort mixtures of SWCNTs based on chirality (electronic species) has recently been demonstrated using special short DNA sequences that recognize certain matching SWCNTs of specific chirality. This thesis investigates the intricacies of DNA-SWCNT sequence-specific interactions through both experimental and molecular simulation studies. The DNA-SWCNT binding strengths were experimentally quantified by studying the kinetics of DNA replacement by a surfactant on the surface of particular SWCNTs. Recognition ability was found to correlate strongly with measured binding strength, e.g. DNA sequence (TAT)4 was found to bind 20 times stronger to the (6,5)-SWCNT than sequence (TAT)4T. Next, using replica exchange molecular dynamics (REMD) simulations, equilibrium structures formed by (a) single-strands and (b) multiple-strands of 12-mer oligonucleotides adsorbed on various SWCNTs were explored. A number of structural motifs were discovered in which the DNA strand wraps around the SWCNT and 'stitches' to itself via hydrogen bonding. Great variability among equilibrium structures was observed and shown to be directly influenced by DNA sequence and SWCNT type. For example, the (6,5)-SWCNT DNA recognition sequence, (TAT)4, was found to wrap in a tight single-stranded right-handed helical conformation. In contrast, DNA sequence T12 forms a beta-barrel left-handed structure on the same SWCNT. These are the first theoretical indications that DNA-based SWCNT selectivity can arise on a molecular level. In a biomedical collaboration with the Mayo Clinic, pathways for DNA-SWCNT internalization into healthy human endothelial cells were explored. Through absorbance spectroscopy, TEM imaging, and confocal fluorescence microscopy, we showed that intracellular concentrations of SWCNTs far exceeded those of the incubation

  1. Interaction of Proliferating Cell Nuclear Antigen With DNA at the Single Molecule Level

    KAUST Repository

    Raducanu, Vlad-Stefan

    2016-05-01

    Proliferating cell nuclear antigen (PCNA) is a key factor involved in Eukaryotic DNA replication and repair, as well as other cellular pathways. Its importance comes mainly from two aspects: the large numbers of interacting partners and the mechanism of facilitated diffusion along the DNA. The large numbers of interacting partners makes PCNA a necessary factor to consider when studying DNA replication, either in vitro or in vivo. The mechanism of facilitated diffusion along the DNA, i.e. sliding along the duplex, reduces the six degrees of freedom of the molecule, three degrees of freedom of translation and three degrees of freedom of rotation, to only two, translation along the duplex and rotational tracking of the helix. Through this mechanism PCNA can recruit its partner proteins and localize them to the right spot on the DNA, maybe in the right spatial orientation, more effectively and in coordination with other proteins. Passive loading of the closed PCNA ring on the DNA without free ends is a topologically forbidden process. Replication factor C (RFC) uses energy of ATP hydrolysis to mechanically open the PCNA ring and load it on the dsDNA. The first half of the introduction gives overview of PCNA and RFC and the loading mechanism of PCNA on dsDNA. The second half is dedicated to a diffusion model and to an algorithm for analyzing PCNA sliding. PCNA and RFC were successfully purified, simulations and a mean squared displacement analysis algorithm were run and showed good stability and experimental PCNA sliding data was analyzed and led to parameters similar to the ones in literature.

  2. Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

    Science.gov (United States)

    Maltseva, E A; Krasikova, Y S; Naegeli, H; Lavrik, O I; Rechkunova, N I

    2014-06-01

    Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E > K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

  3. WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

    Energy Technology Data Exchange (ETDEWEB)

    Rangaraj, K.; Cooper, P.K.; Trego, K.S.

    2009-01-01

    The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of

  4. Perturbations in DNA structure upon interaction with porphyrins revealed by chemical probes, DNA footprinting and molecular modelling.

    Science.gov (United States)

    Ford, K G; Neidle, S

    1995-06-01

    The interactions of several porphyrins with a 74 base-pair DNA sequence have been examined by footprinting and chemical protection methods. Tetra-(4-N-methyl-(pyridyl)) porphyrin (TMPy), two of its metal complexes and tetra-(4-trimethylanilinium) porphyrin (TMAP) bind to closely similar AT-rich sequences. The three TMPy ligands produce modest changes in DNA structure and base accessibility on binding, in contrast to the large-scale conformational changes observed with TMAP. Molecular modelling studies have been performed on TMPy and TMAP bound in the AT-rich minor groove of an oligonucleotide. These have shown that significant structural change is needed to accommodate the bulky trimethyl substituent groups of TMAP, in contrast to the facile minor groove fit of TMPy.

  5. Balancing the Interactions of Ions, Water, and DNA in the Drude Polarizable Force Field

    OpenAIRE

    Savelyev, Alexey; MacKerell, Alexander D.

    2014-01-01

    Recently we presented a first-generation all-atom Drude polarizable force field for DNA based on the classical Drude oscillator model, focusing on optimization of key dihedral angles followed by extensive validation of the force field parameters. Presently, we describe the procedure for balancing the electrostatic interactions between ions, water, and DNA as required for development of the Drude force field for DNA. The proper balance of these interactions is shown to impact DNA stability and...

  6. Use of Drosophila to study DNA repair

    International Nuclear Information System (INIS)

    Boyd, J.B.; Harris, P.V.; Sakaguchi, K.

    1988-01-01

    This paper discusses Drosophila, the premier metazoan organism for analyzing many fundamental features of eukaryotic gene regulation. The authors present adaptations of several approaches for studying DNA repair to an analysis of repair-defective mutants in Drosophila. A current understanding of Drosophila DNA repair is described

  7. Identification of coupling DNA motif pairs on long-range chromatin interactions in human K562 cells

    KAUST Repository

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin

    2015-01-01

    Motivation: The protein-DNA interactions between transcription factors (TFs) and transcription factor binding sites (TFBSs, also known as DNA motifs) are critical activities in gene transcription. The identification of the DNA motifs is a vital task for downstream analysis. Unfortunately, the long-range coupling information between different DNA motifs is still lacking. To fill the void, as the first-of-its-kind study, we have identified the coupling DNA motif pairs on long-range chromatin interactions in human. Results: The coupling DNA motif pairs exhibit substantially higher DNase accessibility than the background sequences. Half of the DNA motifs involved are matched to the existing motif databases, although nearly all of them are enriched with at least one gene ontology term. Their motif instances are also found statistically enriched on the promoter and enhancer regions. Especially, we introduce a novel measurement called motif pairing multiplicity which is defined as the number of motifs that are paired with a given motif on chromatin interactions. Interestingly, we observe that motif pairing multiplicity is linked to several characteristics such as regulatory region type, motif sequence degeneracy, DNase accessibility and pairing genomic distance. Taken into account together, we believe the coupling DNA motif pairs identified in this study can shed lights on the gene transcription mechanism under long-range chromatin interactions. © The Author 2015. Published by Oxford University Press.

  8. Design, synthesis and DNA interactions of a chimera between a platinum complex and an IHF mimicking peptide.

    Science.gov (United States)

    Rao, Harita; Damian, Mariana S; Alshiekh, Alak; Elmroth, Sofi K C; Diederichsen, Ulf

    2015-12-28

    Conjugation of metal complexes with peptide scaffolds possessing high DNA binding affinity has shown to modulate their biological activities and to enhance their interaction with DNA. In this work, a platinum complex/peptide chimera was synthesized based on a model of the Integration Host Factor (IHF), an architectural protein possessing sequence specific DNA binding and bending abilities through its interaction with a minor groove. The model peptide consists of a cyclic unit resembling the minor grove binding subdomain of IHF, a positively charged lysine dendrimer for electrostatic interactions with the DNA phosphate backbone and a flexible glycine linker tethering the two units. A norvaline derived artificial amino acid was designed to contain a dimethylethylenediamine as a bidentate platinum chelating unit, and introduced into the IHF mimicking peptides. The interaction of the chimeric peptides with various DNA sequences was studied by utilizing the following experiments: thermal melting studies, agarose gel electrophoresis for plasmid DNA unwinding experiments, and native and denaturing gel electrophoresis to visualize non-covalent and covalent peptide-DNA adducts, respectively. By incorporation of the platinum metal center within the model peptide mimicking IHF we have attempted to improve its specificity and DNA targeting ability, particularly towards those sequences containing adjacent guanine residues.

  9. Identification of coupling DNA motif pairs on long-range chromatin interactions in human K562 cells

    KAUST Repository

    Wong, Ka-Chun

    2015-09-27

    Motivation: The protein-DNA interactions between transcription factors (TFs) and transcription factor binding sites (TFBSs, also known as DNA motifs) are critical activities in gene transcription. The identification of the DNA motifs is a vital task for downstream analysis. Unfortunately, the long-range coupling information between different DNA motifs is still lacking. To fill the void, as the first-of-its-kind study, we have identified the coupling DNA motif pairs on long-range chromatin interactions in human. Results: The coupling DNA motif pairs exhibit substantially higher DNase accessibility than the background sequences. Half of the DNA motifs involved are matched to the existing motif databases, although nearly all of them are enriched with at least one gene ontology term. Their motif instances are also found statistically enriched on the promoter and enhancer regions. Especially, we introduce a novel measurement called motif pairing multiplicity which is defined as the number of motifs that are paired with a given motif on chromatin interactions. Interestingly, we observe that motif pairing multiplicity is linked to several characteristics such as regulatory region type, motif sequence degeneracy, DNase accessibility and pairing genomic distance. Taken into account together, we believe the coupling DNA motif pairs identified in this study can shed lights on the gene transcription mechanism under long-range chromatin interactions. © The Author 2015. Published by Oxford University Press.

  10. An atomistic geometrical model of the B-DNA configuration for DNA-radiation interaction simulations

    Science.gov (United States)

    Bernal, M. A.; Sikansi, D.; Cavalcante, F.; Incerti, S.; Champion, C.; Ivanchenko, V.; Francis, Z.

    2013-12-01

    In this paper, an atomistic geometrical model for the B-DNA configuration is explained. This model accounts for five organization levels of the DNA, up to the 30 nm chromatin fiber. However, fragments of this fiber can be used to construct the whole genome. The algorithm developed in this work is capable to determine which is the closest atom with respect to an arbitrary point in space. It can be used in any application in which a DNA geometrical model is needed, for instance, in investigations related to the effects of ionizing radiations on the human genetic material. Successful consistency checks were carried out to test the proposed model. Catalogue identifier: AEPZ_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEPZ_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 1245 No. of bytes in distributed program, including test data, etc.: 6574 Distribution format: tar.gz Programming language: FORTRAN. Computer: Any. Operating system: Multi-platform. RAM: 2 Gb Classification: 3. Nature of problem: The Monte Carlo method is used to simulate the interaction of ionizing radiation with the human genetic material in order to determine DNA damage yields per unit absorbed dose. To accomplish this task, an algorithm to determine if a given energy deposition lies within a given target is needed. This target can be an atom or any other structure of the genetic material. Solution method: This is a stand-alone subroutine describing an atomic-resolution geometrical model of the B-DNA configuration. It is able to determine the closest atom to an arbitrary point in space. This model accounts for five organization levels of the human genetic material, from the nucleotide pair up to the 30 nm chromatin fiber. This subroutine carries out a series of coordinate transformations

  11. Antibacterial effect of cationic porphyrazines and anionic phthalocyanine and their interaction with plasmid DNA

    Science.gov (United States)

    Hassani, Leila; Hakimian, Fatemeh; Safaei, Elham; Fazeli, Zahra

    2013-11-01

    Resistance to antibiotics is a public health issue and identification of new antibacterial agents is one of the most important goals of pharmacological research. Among the novel developed antibacterial agents, porphyrin complexes and their derivatives are ideal candidates for use in medical applications. Phthalocyanines differ from porphyrins by having nitrogen atoms link the individual pyrrol units. The aza analogues of the phthalocyanines (azaPcs) such as tetramethylmetalloporphyrazines are heterocyclic Pc analogues. In this investigation, interaction of an anionic phthalocyanine (Cu(PcTs)) and two cationic tetrapyridinoporphyrazines including [Cu(2,3-tmtppa)]4+ and [Cu(3,4-tmtppa)]4+ complexes with plasmid DNA was studied using spectroscopic and gel electrophoresis methods. In addition, antibacterial effect of the complexes against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria was investigated using dilution test method. The results indicated that both porphyrazines have significant antibacterial properties, but Cu(PcTs) has weak antibacterial effect. Compairing the binding of the phthalocyanine and the porphyrazines to DNA demonstrated that the interaction of cationic porphyrazines is stronger than the anionic phthalocyanine remarkably. The extent of hypochromicity and red shift of absorption spectra indicated preferential intercalation of the two porphyrazine into the base pairs of DNA helix. Gel electrophoresis result implied Cu(2,3-tmtppa) and Cu(3,4-tmtppa) are able to perform cleavage of the plasmid DNA. Consequently, DNA binding and cleavage might be one of the antibacterial mechanisms of the complexes.

  12. RADX interacts with single-stranded DNA to promote replication fork stability

    DEFF Research Database (Denmark)

    Schubert, Lisa; Ho, Teresa; Hoffmann, Saskia

    2017-01-01

    Single-stranded DNA (ssDNA) regions form as an intermediate in many DNA-associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide-binding (OB) fold domain. The heterotrimeric, multi-OB fold domain-containing Replication Protein A (RPA) complex...... ssDNA-binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA-binding proteins....

  13. In and out of the minor groove: interaction of an AT-rich DNA with the drug CD27

    Energy Technology Data Exchange (ETDEWEB)

    Acosta-Reyes, Francisco J. [Universitat Politécnica de Catalunya, Diagonal 647, 08028 Barcelona (Spain); Dardonville, Christophe [Instituto de Química Médica, IQM–CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Koning, Harry P. de; Natto, Manal [University of Glasgow, 120 University Place, Glasgow G12 8TA, Scotland (United Kingdom); Subirana, Juan A.; Campos, J. Lourdes, E-mail: lourdes.campos@upc.edu [Universitat Politécnica de Catalunya, Diagonal 647, 08028 Barcelona (Spain)

    2014-06-01

    New features of an antiprotozoal DNA minor-groove binding drug, which acts as a cross-linking agent, are presented. It also fills the minor groove of DNA completely and prevents the access of proteins. These features are also expected for other minor-groove binding drugs when associated with suitable DNA targets. The DNA of several pathogens is very rich in AT base pairs. Typical examples include the malaria parasite Plasmodium falciparum and the causative agents of trichomoniasis and trypanosomiases. This fact has prompted studies of drugs which interact with the minor groove of DNA, some of which are used in medical practice. Previous studies have been performed almost exclusively with the AATT sequence. New features should be uncovered through the study of different DNA sequences. In this paper, the crystal structure of the complex of the DNA duplex d(AAAATTTT){sub 2} with the dicationic drug 4, 4′-bis(imidazolinylamino)diphenylamine (CD27) is presented. The drug binds to the minor groove of DNA as expected, but it shows two new features that have not previously been described: (i) the drugs protrude from the DNA and interact with neighbouring molecules, so that they may act as cross-linking agents, and (ii) the drugs completely cover the whole minor groove of DNA and displace bound water. Thus, they may prevent the access to DNA of proteins such as AT-hook proteins. These features are also expected for other minor-groove binding drugs when associated with all-AT DNA. These findings allow a better understanding of this family of compounds and will help in the development of new, more effective drugs. New data on the biological interaction of CD27 with the causative agent of trichomoniasis, Trichomonas vaginalis, are also reported.

  14. In and out of the minor groove: interaction of an AT-rich DNA with the drug CD27

    International Nuclear Information System (INIS)

    Acosta-Reyes, Francisco J.; Dardonville, Christophe; Koning, Harry P. de; Natto, Manal; Subirana, Juan A.; Campos, J. Lourdes

    2014-01-01

    New features of an antiprotozoal DNA minor-groove binding drug, which acts as a cross-linking agent, are presented. It also fills the minor groove of DNA completely and prevents the access of proteins. These features are also expected for other minor-groove binding drugs when associated with suitable DNA targets. The DNA of several pathogens is very rich in AT base pairs. Typical examples include the malaria parasite Plasmodium falciparum and the causative agents of trichomoniasis and trypanosomiases. This fact has prompted studies of drugs which interact with the minor groove of DNA, some of which are used in medical practice. Previous studies have been performed almost exclusively with the AATT sequence. New features should be uncovered through the study of different DNA sequences. In this paper, the crystal structure of the complex of the DNA duplex d(AAAATTTT) 2 with the dicationic drug 4, 4′-bis(imidazolinylamino)diphenylamine (CD27) is presented. The drug binds to the minor groove of DNA as expected, but it shows two new features that have not previously been described: (i) the drugs protrude from the DNA and interact with neighbouring molecules, so that they may act as cross-linking agents, and (ii) the drugs completely cover the whole minor groove of DNA and displace bound water. Thus, they may prevent the access to DNA of proteins such as AT-hook proteins. These features are also expected for other minor-groove binding drugs when associated with all-AT DNA. These findings allow a better understanding of this family of compounds and will help in the development of new, more effective drugs. New data on the biological interaction of CD27 with the causative agent of trichomoniasis, Trichomonas vaginalis, are also reported

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  16. Topoisomerase I tyrosine phosphorylation site and the DNA-interactive site

    International Nuclear Information System (INIS)

    Roll, D.; Durban, E.

    1986-01-01

    Phosphorylation of topoisomerase I (topo I) at serine by NII kinase is accompanied by stimulation of enzymatic activity. In contrast, phosphorylation at tyrosine by tyrosine kinase seems to inhibit enzymatic activity. This inhibition may be caused by interference of the phosphorylated tyrosine residue with the interaction of topo I with DNA. To test this, topo I was labeled with crude membrane fraction enriched for EGF-receptor kinase in presence of γ-P32-ATP and electrophoresed on SDS-polyacrylamide gels. Stained topo I bands were excised, dried, digested with trypsin and analyzed on a C18 reverse-phase HPLC column. One major peak of radioactivity eluted at fraction 23 with 20% acetonitrile. To obtain the DNA-interactive site, topo I was incubated with pBR322 DNA labeled by nick-translation followed by DNase I treatment, and electrophoresis on SDS-polyacrylamide gels. Tryptic peptides were generated and analyzed by reverse-phase HPLC. A major peak of radioactivity eluted at fraction 16-18 with 15.5-17% acetonitrile. Studies are in progress to resolve whether (a) the two peptides are different, i.e. the tyrosine-P site and DNA-tyrosine interactive site are localized at different regions of the topo I or (b) the peptide sequences are identical but the covalent attachment of deoxynucleotides altered the peptide's elution from the HPLC column

  17. Alecto 2 - interaction studies

    International Nuclear Information System (INIS)

    Brunet, J.P.; Clouet d'Orval, Ch.; Mougniot, J.C.; Penet, F.

    1965-01-01

    Weak interactions were experimentally studies with the tank of the critical assembly Alecto II and one, two or three bottles containing solutions of various concentrations. In particular, was studied the validity of certain classical assumptions, shielding effects, screening and semi-reflexion effects, importance of thermal coupling. The method of the 'k eff , solid angle' is shown to apply to such a system. The determination by divergence and pulsed neutron technique of the reactivity related to a millimeter of solution level affords the obtention of critical heights in terms of reactivity. (authors) [fr

  18. DNA-nuclear matrix interactions and ionizing radiation sensitivity

    International Nuclear Information System (INIS)

    Schwartz, J.L.; Vaughan, A.T.M.

    1993-01-01

    The association between inherent ionizing radiation sensitivity and DNA supercoil unwinding in mammalian cells suggests that the organization of the DNA in chromosomes plays an important role in radiation responses. In this paper, a model is proposed which suggests that these DNA unwinding alterations reflect differences in the attachment of DNA to the nuclear matrix. In radioresistant cells, the MAR structure might exist in a more stable, open configuration, limiting DNA unwinding following strand break induction and influencing the rate and nature of DNA double-strand break rejoining

  19. DNA effects upon the reaction between acetonitrile pentacyanoferrate (II) and ruthenium pentammine pyrazine: Kinetic and thermodynamic evidence of the interaction of DNA with anionic species

    International Nuclear Information System (INIS)

    Grueso, E.; Prado-Gotor, R.; Lopez, M.; Gomez-Herrera, C.; Sanchez, F.

    2005-01-01

    The kinetics of the reaction between ruthenium pentaammine pyrazine and acetonitrile pentacyanoferrate (II) to obtain the binuclear anionic complex [Fe(CN) 5 pzRu(NH 3 ) 5 ] - , and the reverse (dissociation) process, have been studied in solutions containing DNA. The results corresponding to this reaction and those corresponding to the reverse (dissociation) process show a clear influence of DNA on their kinetics. The results can be interpreted using a modified Pseudophase Model. From the results obtained for the dissociation reaction one can conclude that the binuclear anionic complex [Fe(CN) 5 pzRu(NH 3 ) 5 ] - interacts with DNA

  20. Biflorin induces cytotoxicity by DNA interaction in genetically different human melanoma cell lines.

    Science.gov (United States)

    Ralph, Ana Carolina Lima; Calcagno, Danielle Queiroz; da Silva Souza, Luciana Gregório; de Lemos, Telma Leda Gomes; Montenegro, Raquel Carvalho; de Arruda Cardoso Smith, Marília; de Vasconcellos, Marne Carvalho

    2016-08-01

    Cancer is a public health problem and the second leading cause of death worldwide. The incidence of cutaneous melanoma has been notably increasing, resulting in high aggressiveness and poor survival rates. Taking into account the antitumor activity of biflorin, a substance isolated from Capraria biflora L. roots that is cytotoxic in vitro and in vivo, this study aimed to demonstrate the action of biflorin against three established human melanoma cell lines that recapitulate the molecular landscape of the disease in terms of genetic alterations and mutations, such as the TP53, NRAS and BRAF genes. The results presented here indicate that biflorin reduces the viability of melanoma cell lines by DNA interactions. Biflorin causes single and double DNA strand breaks, consequently inhibiting cell cycle progression, replication and DNA repair and promoting apoptosis. Our data suggest that biflorin could be considered as a future therapeutic option for managing melanoma. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. DNA Replication and Cell Cycle Progression Regulatedby Long Range Interaction between Protein Complexes bound to DNA.

    Science.gov (United States)

    Matsson, L

    2001-12-01

    A nonstationary interaction that controlsDNA replication and the cell cycle isderived from many-body physics in achemically open T cell. The model predictsa long range force F'(ξ) =- (κ/2) ξ(1 - ξ)(2 - ξ)between thepre-replication complexes (pre-RCs) boundby the origins in DNA, ξ = ϕ/N being the relativedisplacement of pre-RCs, ϕ the number of pre-RCs, N the number of replicons to be replicated,and κ the compressibilitymodulus in the lattice of pre-RCs whichbehaves dynamically like an elasticallybraced string. Initiation of DNAreplication is induced at the thresholdϕ = N by a switch ofsign of F''(ξ), fromattraction (-) and assembly in the G(1) phase (0force at ϕ = 2N, from repulsion inS phase back to attraction in G(2), when all primed replicons havebeen duplicated once. F'(0) = 0corresponds to a resting cell in theabsence of driving force at ϕ= 0. The model thus ensures that the DNAcontent in G(2) cells is exactlytwice that of G(1) cells. The switch of interaction at the R-point, at which N pre-RCs have been assembled, starts the release of Rb protein thus also explaining the shift in the Rb phosphorylation from mitogen-dependent cyclinD to mitogen-independent cyclin E.Shape,slope and scale of the response curvesderived agree well with experimental datafrom dividing T cells and polymerising MTs,the variable length of which is due to anonlinear dependence of the growthamplitude on the initial concentrations oftubulin dimers and guanosine-tri-phosphate(GTP). The model also explains the dynamic instabilityin growing MTs.

  2. A Novel Open Tubular Capillary Electrochromatographic Method for Differentiating the DNA Interaction Affinity of Environmental Contaminants.

    Directory of Open Access Journals (Sweden)

    Lucia D'Ulivo

    Full Text Available The interaction of chemicals with DNA may lead to genotoxicity, mutation or carcinogenicity. A simple open tubular capillary electrochromatographic method is proposed to rapidly assess the interaction affinity of three environmental contaminants (1,4-phenylenediamine, pyridine and 2,4-diaminotoluene to DNA by measuring their retention in the capillaries coated with DNA probes. DNA oligonucleotide probes were immobilized on the inner wall of a fused silica capillary that was first derivatized with 3-(aminopropyl-triethoxysilane (APTES. The difference in retention times and factors was considered as the difference in interaction affinity of the contaminants to the DNA probes. The interaction of the contaminants with both double-stranded (dsDNA and single-stranded DNA (ssDNA coatings was compared. Retention factors of 1,4-phenylenediamine, pyridine and 2,4-diaminotoluene in the capillary coated with ssDNA probe were 0.29, 0.42, and 0.44, respectively. A similar trend was observed in the capillary coated with dsDNA, indicating that 2,4-diaminotoluene has the highest affinity among the three contaminants. The relative standard deviation (RSD for the retention factors was in the range of 0.05-0.69% (n = 3. The results demonstrated that the developed technique could be applied for preliminary screening purpose to provide DNA interaction affinity information of various environmental contaminants.

  3. Interaction of organophosphorus pesticides with DNA nucleotides on a Boron-doped diamond electrode

    Energy Technology Data Exchange (ETDEWEB)

    Garbellini, Gustavo S.; Uliana, Carolina V.; Yamanaka, Hideko, E-mail: gustgarb@yahoo.com.br [Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Bauru, SP (Brazil). Dept. de Quimica Analitica

    2013-12-01

    Diamond electrode was used to evaluate the interaction of the nucleotides guanosine monophosphate (GMP) and adenosine monophosphate (AMP) with the pesticides chlorpyrifos, methamidophos and monocrotophos. Changes were observed in the currents and peak potentials of the nucleotide voltammograms in the presence of the pesticides, with dependence on the pesticide concentration (from 5.0 Multiplication-Sign 10{sup -7} to 5.0 Multiplication-Sign 10{sup -5} mol L{sup -1}) and the interaction time (from 1 min to 4 h). This is probably due to binding of the pesticides to the nitrogenous bases present in the nucleotides, which could lead to problems in the DNA replication and biological functions of nucleotides. The pesticides showed stronger interaction with AMP than with GMP. Studies of the interaction of 50 Micro-Sign g mL{sup -1} DNA with the pesticides (from 30 min to 4 h and from 1.0 Multiplication-Sign 10{sup -6} to 6.0 Multiplication-Sign 10{sup -5} mol L{sup -1}) did not reveal any peaks relating to double helix opening or DNA unwinding. (author)

  4. HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.

    Science.gov (United States)

    Guillen-Ahlers, Hector; Rao, Prahlad K; Levenstein, Mark E; Kennedy-Darling, Julia; Perumalla, Danu S; Jadhav, Avinash Y L; Glenn, Jeremy P; Ludwig-Kubinski, Amy; Drigalenko, Eugene; Montoya, Maria J; Göring, Harald H; Anderson, Corianna D; Scalf, Mark; Gildersleeve, Heidi I S; Cole, Regina; Greene, Alexandra M; Oduro, Akua K; Lazarova, Katarina; Cesnik, Anthony J; Barfknecht, Jared; Cirillo, Lisa A; Gasch, Audrey P; Shortreed, Michael R; Smith, Lloyd M; Olivier, Michael

    2016-06-01

    Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of Saccharomyces cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Highly functionalized piperidines: Free radical scavenging, anticancer activity, DNA interaction and correlation with biological activity

    OpenAIRE

    Suvankar Das; Cristiane J. da Silva; Marina de M. Silva; Maria Dayanne de A. Dantas; Ângelo de Fátima; Ana Lúcia T. Góis Ruiz; Cleiton M. da Silva; João Ernesto de Carvalho; Josué C.C. Santos; Isis M. Figueiredo; Edeildo F. da Silva-Júnior; Thiago M. de Aquino; João X. de Araújo-Júnior; Goutam Brahmachari; Luzia Valentina Modolo

    2018-01-01

    Twenty-five piperidines were studied as potential radical scavengers and antitumor agents. Quantitative interaction of compounds with ctDNA using spectroscopic techniques was also evaluated. Our results demonstrate that the evaluated piperidines possesses different abilities to scavenge the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the anion radical superoxide (·O2−). The piperidine 19 was the most potent radical DPPH scavenger, while the most effective to ·O2− scavenger was piperidine...

  6. DNA-nuclear matrix interactions and ionizing radiation sensitivity

    International Nuclear Information System (INIS)

    Schwartz, J.L.; Chicago Univ., IL; Vaughan, A.T.M.

    1993-01-01

    The association between inherent ionizing radiation sensitivity and DNA supercoil unwinding in mammalian cells suggests that the DNA-nuclear matrix attachment region (MAR) plays an important role in radiation response. In radioresistant cells, the MAR structure may exist in a more stable, open configuration, limiting DNA unwinding following strand break induction and maintaining DNA ends in close proximity for more rapid and accurate rejoining. In addition, the open configuration at these matrix attachment sites may serve to facilitate rapid DNA processing of breaks by providing (1) sites for repair proteins to collect and (2) energy to drive enzymatic reactions

  7. Comparison on the interaction of Al3+/nano-Al13 with calf thymus DNA /salmon sperm DNA

    Science.gov (United States)

    Ma, Fei; Ma, Yue; Du, Changwen; Yang, Xiaodi; Shen, Renfang

    2015-11-01

    The conformation change, binding mode and binding site between Al3+/nano-Al13 and calf thymus DNA/salmon sperm DNA were investigated by UV-vis absorption, FTIR spectra, Raman spectroscopy and CD spectra, as well as melting curves measurement. The UV-vis spectra and circular dichroism spectra results suggested that the phosphate group structure was changed when Al3+ interacted with DNA, while the double-helix was distorted when nano-Al13 interacted with DNA. The FTIR and Raman spectroscopy revealed that the binding sites were Al3+ … PO2, Al3+ … N7/guanine PO2 … Al13 … N7-C8/guanine with calf thymus DNA, and Al3+ … N3-O2/cytosine, Al3+ … N7-C8/guanine, PO2 … Al13 … N7-C8/guanine, PO2 … Al13 … N1/adenine with salmon sperm DNA, respectively. The electrostatic binding was existed between Al3+ and DNA, and the electrostatic binding and complexing were found between nano-Al13 and DNA.

  8. Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori.

    Directory of Open Access Journals (Sweden)

    Tara Kashav

    2009-10-01

    Full Text Available Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD of H. pylori DnaB (HpDnaB helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

  9. Anticancer drug-DNA interactions measured using a photoinduced electron-transfer mechanism based on luminescent quantum dots.

    Science.gov (United States)

    Yuan, Jipei; Guo, Weiwei; Yang, Xiurong; Wang, Erkang

    2009-01-01

    A sensing system based on the photoinduced electron transfer of quantum dots (QDs) was designed to measure the interaction of anticancer drug and DNA, taking mitoxantrone (MTX) as a model drug. MTX adsorbed on the surface of QDs can quench the photoluminescence (PL) of QDs through the photoinduced electron-transfer process; and then the addition of DNA will bring the restoration of QDs PL intensity, as DNA can bind with MTX and remove it from QDs. Sensitive detection of MTX with the detection limit of 10 nmol L(-1) and a linear detection range from 10 nmol L(-1) to 4.5 micromol L(-1) was achieved. The dependence of PL intensity on DNA amount was successfully utilized to investigate the interactions between MTX and DNA. Both the binding constants and the sizes of binding site of MTX-DNA interactions were calculated based on the equations deduced for the PL recovery process. The binding constant obtained in our experiment was generally consistent with previous reports. The sensitive and speedy detection of MTX as well as the avoidance of modification or immobilization process made this system suitable and promising in the drug-DNA interaction studies.

  10. An AP endonuclease 1-DNA polymerase beta complex: theoretical prediction of interacting surfaces.

    Directory of Open Access Journals (Sweden)

    Alexej Abyzov

    2008-04-01

    Full Text Available Abasic (AP sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1 cleaves the phosphodiester backbone 5' to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-beta. While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-beta, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-beta based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-beta located downstream of APEX1 (3' to the damaged site and three with pol-beta located upstream of APEX1 (5' to the damaged site. Molecular dynamics (MD simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (approximately -10.0 kcal/mol to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-beta at the 3'-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-beta in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-beta in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3' side. The method described here can be used for analysis in

  11. DNA-interactive properties of crotamine, a cell-penetrating polypeptide and a potential drug carrier.

    Directory of Open Access Journals (Sweden)

    Pei-Chun Chen

    Full Text Available Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss or (ds double stranded molecules. The affinities of the protein for ss- vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of ∼3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more

  12. Interaction of a non-histone chromatin protein (high-mobility group protein 2) with DNA

    International Nuclear Information System (INIS)

    Goodwin, G.H.; Shooter, K.V.; Johns, E.W.

    1975-01-01

    The interaction with DNA of the calf thymus chromatin non-histone protein termed the high-mobility group protein 2 has been studied by sedimentation analysis in the ultracentrifuge and by measuring the binding of the 125 I-labelled protein to DNA. The results have been compared with those obtained previously by us [Eur. J. Biochem. (1974) 47, 263-270] for the interaction of high-mobility group protein 1 with DNA. Although the binding parameters are similar for these two proteins, high-mobility group protein 2 differs from high-mobility group protein 1 in that the former appears to change the shape of the DNA to a more compact form. The molecular weight of high-mobility group protein 2 has been determined by equilibrium sedimentation and a mean value of 26,000 was obtained. A low level of nuclease activity detected in one preparation of high-mobility group protein 2 has been investigated. (orig.) [de

  13. Programmable display of DNA-protein chimeras for controlling cell-hydrogel interactions via reversible intermolecular hybridization.

    Science.gov (United States)

    Zhang, Zhaoyang; Li, Shihui; Chen, Niancao; Yang, Cheng; Wang, Yong

    2013-04-08

    Extensive studies have been recently carried out to achieve dynamic control of cell-material interactions primarily through physicochemical stimulation. The purpose of this study was to apply reversible intermolecular hybridization to program cell-hydrogel interactions in physiological conditions based on DNA-antibody chimeras and complementary oligonucleotides. The results showed that DNA oligonucleotides could be captured to and released from the immobilizing DNA-functionalized hydrogels with high specificity via DNA hybridization. Accordingly, DNA-antibody chimeras were captured to the hydrogels, successfully inducing specific cell attachment. The cell attachment to the hydrogels reached the plateau at approximately half an hour after the functionalized hydrogels and the cells were incubated together. The attached cells were rapidly released from the bound hydrogels when triggering complementary oligonucleotides were introduced to the system. However, the capability of the triggering complementary oligonucleotides in releasing cells was affected by the length of intermolecular hybridization. The length needed to be at least more than 20 base pairs in the current experimental setting. Notably, because the procedure of intermolecular hybridization did not involve any harsh condition, the released cells maintained the same viability as that of the cultured cells. The functionalized hydrogels also exhibited the potential to catch and release cells repeatedly. Therefore, this study demonstrates that it is promising to regulate cell-material interactions dynamically through the DNA-programmed display of DNA-protein chimeras.

  14. DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair.

    Science.gov (United States)

    Serrano, M A; Li, Z; Dangeti, M; Musich, P R; Patrick, S; Roginskaya, M; Cartwright, B; Zou, Y

    2013-05-09

    Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR.

  15. A model system for DNA repair studies

    International Nuclear Information System (INIS)

    Lange, C.S.; Perlmutter, E.

    1984-01-01

    The search for the ''lethal lesion:'' which would yield a molecular explanation of biological survival curves led to attempts to correlate unrepaired DNA lesions with loss of reproductive integrity. Such studies have shown the crucial importance of DNA repair systems. The unrepaired DSB has been sought for such correlation, but in such study the DNA was too large, polydisperse, and/or structurally complex to permit precise measurement of break induction and repair. Therefore, an analog of higher order systems but with a genome of readily measurable size, is needed. Bacteriophage T4 is such an analog. Both its biological (PFU) and molecular (DNA) survival curves are exponentials. Its aerobic /sub PFU/D/sub 37///sub DNA/D/sub 37/ ratio, (410 +- 4.5Gy/540 +- 25 Gy) indicates that 76 +- 4% of lethality at low multiplicity infection (moi 1) the survival is greater than can be explained if the assumption of no parental DSB repair were valid. Both T4 and its host have DSB repair systems which can be studied by the infectious center method. Results of such studies are discussed

  16. WHY DOES 5-METHYL CHRYSENE INTERACT WITH DNA LIKE BOTH A PLANAR AND A NON-PLANAR POLYCYCLIC AROMATIC HYDROCARBON? QUANTUM MECHANICAL STUDIES

    Science.gov (United States)

    Polycyclic aromatic hydrocarbons are a large class of anthropogenic chemicals found in the environment. Some class members are potent animal carcinogens while other similar class members show little carcinogenic activity. When considering a series of in vitro studies of the int...

  17. Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

    Science.gov (United States)

    Liberek, K; Osipiuk, J; Zylicz, M; Ang, D; Skorko, J; Georgopoulos, C

    1990-02-25

    The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

  18. Neutron Reflectometry Investigations of the Interaction of DNA-PAMAM Dendrimers with Model Biological Membranes

    International Nuclear Information System (INIS)

    Ainalem, M.L.; Rennie, A.R.; Campbell, Richard; Edler, Karen; Nylander, Tommy

    2009-01-01

    The systemic delivery of DNA for gene therapy requires control of DNA compaction by an agent, such a lipid, surfactant or a polymer (e.g. cationic dendrimers) as well as understanding of how this complex interacts with a biological membrane. Poly (amido amine) (PAMAM) dendrimers have been reported to be a promising synthetic gene-transfection agent. We have studied the structure of the complexes formed between DNA and PAMAM dendrimers with SANS, dynamic light scattering and cryo-TEM. Here we noted that the structure of the complex formed strongly depends on the generation of the dendrimer. The results of the adsorption of generation 2 (G2) and 4 (G4) PAMAM dendrimers to surface deposited bilayers, consisting of palmitoyl oleoyl phosphatidyl choline on silicon surface, have been studied using neutron reflectometry (NR). The NR data shows that the dendrimers are able to penetrate the bilayer. However, the complex is less able to penetrate the bilayer, but rather stays on the top of the bilayer. The dendrimers appear slightly flattened on the surface in comparison with their size in bulk as determined by light scattering. We will also report on the interfacial behavior of the DNA-PAMAM complexes at other types of studies of interfaces, important for biomedical applications, where NR has allowed us to determine the layer structure and composition. (author)

  19. Studying DNA Looping by Single-Molecule FRET

    OpenAIRE

    Le, Tung T.; Kim, Harold D.

    2014-01-01

    Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can als...

  20. Disruption of PCNA-lamins A/C interactions by prelamin A induces DNA replication fork stalling.

    Science.gov (United States)

    Cobb, Andrew M; Murray, Thomas V; Warren, Derek T; Liu, Yiwen; Shanahan, Catherine M

    2016-09-02

    The accumulation of prelamin A is linked to disruption of cellular homeostasis, tissue degeneration and aging. Its expression is implicated in compromised genome stability and increased levels of DNA damage, but to date there is no complete explanation for how prelamin A exerts its toxic effects. As the nuclear lamina is important for DNA replication we wanted to investigate the relationship between prelamin A expression and DNA replication fork stability. In this study we report that the expression of prelamin A in U2OS cells induced both mono-ubiquitination of proliferating cell nuclear antigen (PCNA) and subsequent induction of Pol η, two hallmarks of DNA replication fork stalling. Immunofluorescence microscopy revealed that cells expressing prelamin A presented with high levels of colocalisation between PCNA and γH2AX, indicating collapse of stalled DNA replication forks into DNA double-strand breaks. Subsequent protein-protein interaction assays showed prelamin A interacted with PCNA and that its presence mitigated interactions between PCNA and the mature nuclear lamina. Thus, we propose that the cytotoxicity of prelamin A arises in part, from it actively competing against mature lamin A to bind PCNA and that this destabilises DNA replication to induce fork stalling which in turn contributes to genomic instability.

  1. The Isolation of DNA by Polycharged Magnetic Particles: An Analysis of the Interaction by Zeta Potential and Particle Size.

    Science.gov (United States)

    Haddad, Yazan; Xhaxhiu, Kledi; Kopel, Pavel; Hynek, David; Zitka, Ondrej; Adam, Vojtech

    2016-04-20

    Magnetic isolation of biological targets is in major demand in the biotechnology industry today. This study considers the interaction of four surface-modified magnetic micro- and nanoparticles with selected DNA fragments. Different surface modifications of nanomaghemite precursors were investigated: MAN37 (silica-coated), MAN127 (polyvinylpyrrolidone-coated), MAN158 (phosphate-coated), and MAN164 (tripolyphosphate-coated). All particles were positive polycharged agglomerated monodispersed systems. Mean particle sizes were 0.48, 2.97, 2.93, and 3.67 μm for MAN37, MAN127, MAN164, and MAN158, respectively. DNA fragments exhibited negative zeta potential of -0.22 mV under binding conditions (high ionic strength, low pH, and dehydration). A decrease in zeta potential of particles upon exposure to DNA was observed with exception of MAN158 particles. The measured particle size of MAN164 particles increased by nearly twofold upon exposure to DNA. Quantitative PCR isolation of DNA with a high retrieval rate was observed by magnetic particles MAN127 and MAN164. Interaction between polycharged magnetic particles and DNA is mediated by various binding mechanisms such as hydrophobic and electrostatic interactions. Future development of DNA isolation technology requires an understanding of the physical and biochemical conditions of this process.

  2. Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

    Science.gov (United States)

    Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

    2017-05-01

    Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

  3. Interactions between low energy electrons and DNA: a perspective from first-principles simulations

    Science.gov (United States)

    Kohanoff, Jorge; McAllister, Maeve; Tribello, Gareth A.; Gu, Bin

    2017-09-01

    DNA damage caused by irradiation has been studied for many decades. Such studies allow us to better assess the dangers posed by radiation, and to increase the efficiency of the radiotherapies that are used to combat cancer. A full description of the irradiation process involves multiple size and time scales. It starts with the interaction of radiation—either photons or swift ions—and the biological medium, which causes electronic excitation and ionisation. The two main products of ionising radiation are thus electrons and radicals. Both of these species can cause damage to biological molecules, in particular DNA. In the long run, this molecular level damage can prevent cells from replicating and can hence lead to cell death. For a long time it was assumed that the main actors in the damage process were the radicals. However, experiments in a seminal paper by the group of Leon Sanche in 2000 showed that low-energy electrons (LEE), such as those generated when ionising biological targets, can also cause bond breaks in biomolecules, and strand breaks in plasmid DNA in particular (Boudaiffa et al 2000 Science 287 1658-60). These results prompted a significant amount of experimental and theoretical work aimed at elucidating the role played by LEE in DNA damage. In this Topical Review we provide a general overview of the problem. We discuss experimental findings and theoretical results hand in hand with the aim of describing the physics and chemistry that occurs during the process of radiation damage, from the initial stages of electronic excitation, through the inelastic propagation of electrons in the medium, the interaction of electrons with DNA, and the chemical end-point effects on DNA. A very important aspect of this discussion is the consideration of a realistic, physiological environment. The role played by the aqueous solution and the amino acids from the histones in chromatin must be considered. Moreover, thermal fluctuations must be incorporated when

  4. Quantum Mechanical Studies of DNA and LNA

    DEFF Research Database (Denmark)

    Koch, Troels; Shim, Irene; Lindow, Morten

    2014-01-01

    Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies of the e......Quantum mechanical (QM) methodology has been employed to study the structure activity relations of DNA and locked nucleic acid (LNA). The QM calculations provide the basis for construction of molecular structure and electrostatic surface potentials from molecular orbitals. The topologies...

  5. Demonstrating Interactions of Transcription Factors with DNA by Electrophoretic Mobility Shift Assay.

    Science.gov (United States)

    Yousaf, Nasim; Gould, David

    2017-01-01

    Confirming the binding of a transcription factor with a particular DNA sequence may be important in characterizing interactions with a synthetic promoter. Electrophoretic mobility shift assay is a powerful approach to demonstrate the specific DNA sequence that is bound by a transcription factor and also to confirm the specific transcription factor involved in the interaction. In this chapter we describe a method we have successfully used to demonstrate interactions of endogenous transcription factors with sequences derived from endogenous and synthetic promoters.

  6. A Study on Spectro-Analytical Aspects, DNA - Interaction, Photo-Cleavage, Radical Scavenging, Cytotoxic Activities, Antibacterial and Docking Properties of 3 - (1 - (6 - methoxybenzo [d] thiazol - 2 - ylimino) ethyl) - 6 - methyl - 3H - pyran - 2, 4 - dione and its Metal Complexes.

    Science.gov (United States)

    Ravi, Mudavath; Chennam, Kishan Prasad; Ushaiah, B; Eslavath, Ravi Kumar; Perugu, Shyam; Ajumeera, Rajanna; Devi, Ch Sarala

    2015-09-01

    The focus of the present work is on the design, synthesis, characterization, DNA-interaction, photo-cleavage, radical scavenging, in-vitro cytotoxicity, antimicrobial, docking and kinetic studies of Cu (II), Cd (II), Ce (IV) and Zr (IV) metal complexes of an imine derivative, 3 - (1 - (6 - methoxybenzo [d] thiazol - 2 - ylimino) ethyl) - 6 - methyl - 3H - pyran - 2, 4 - dione. The investigation of metal ligand interactions for the determination of composition of metal complexes, corresponding kinetic studies and antioxidant activity in solution was carried out by spectrophotometric methods. The synthesized metal complexes were characterized by EDX analysis, Mass, IR, (1)H-NMR, (13)C-NMR and UV-Visible spectra. DNA binding studies of metal complexes with Calf thymus (CT) DNA were carried out at room temperature by employing UV-Vis electron absorption, fluorescence emission and viscosity measurement techniques. The results revealed that these complexes interact with DNA through intercalation. The results of in vitro antibacterial studies showed the enhanced activity of chelating agent in metal chelated form and thus inferring scope for further development of new therapeutic drugs. Cell viability experiments indicated that all complexes showed significant dose dependent cytotoxicity in selected cell lines. The molecular modeling and docking studies were carried out with energy minimized structures of metal complexes to identify the receptor to metal interactions.

  7. GANP regulates the choice of DNA repair pathway by DNA-PKcs interaction in AID-dependent IgV region diversification.

    Science.gov (United States)

    Eid, Mohammed Mansour Abbas; Maeda, Kazuhiko; Almofty, Sarah Ameen; Singh, Shailendra Kumar; Shimoda, Mayuko; Sakaguchi, Nobuo

    2014-06-15

    RNA export factor germinal center-associated nuclear protein (GANP) interacts with activation-induced cytidine deaminase (AID) and shepherds it from the cytoplasm to the nucleus and toward the IgV region loci in B cells. In this study, we demonstrate a role for GANP in the repair of AID-initiated DNA damage in chicken DT40 B cells to generate IgV region diversity by gene conversion and somatic hypermutation. GANP plays a positive role in IgV region diversification of DT40 B cells in a nonhomologous end joining-proficient state. DNA-PKcs physically interacts with GANP, and this interaction is dissociated by dsDNA breaks induced by a topoisomerase II inhibitor, etoposide, or AID overexpression. GANP affects the choice of DNA repair mechanism in B cells toward homologous recombination rather than nonhomologous end joining repair. Thus, GANP presumably plays a critical role in protection of the rearranged IgV loci by favoring homologous recombination of the DNA breaks under accelerated AID recruitment. Copyright © 2014 by The American Association of Immunologists, Inc.

  8. Investigation of hyperfine interactions in DNA nitrogenous bases using perturbed angular correlation spectroscopy

    International Nuclear Information System (INIS)

    Silva, Andreia dos Santos; Carbonari, Artur Wilson; Lapolli, Andre Luis; Saxena, Rajendra Narain; Saitovitch, Henrique

    2013-01-01

    Perturbed γγ angular correlations (PAC) spectroscopy has been used to study the DNA nitrogenous bases (adenine, cytosine, guanine, thymine), using 111 In→ 111 Cd and 111m Cd→ 111 Cd probe nuclei. One of the advantages of applying PAC technique to biological molecules is that the experiments can be carried out on molecules in aqueous solution [1], approaching the function of molecules under conditions that are close to in vivo conditions. The measurements were carried out for DNA nitrogenous bases molecules at 295 K and 77 K in order to investigate dynamic and static hyperfine interactions, respectively. The interpretation of the results was based on the measurements of dynamic interaction characterized by the decay constant from which valuable information on the macroscopic behavior of the molecules was obtained [2; 3]. On the other hand, PAC measurements at low temperature showed interaction frequency (ν Q ), asymmetry parameter (η) and the distribution of the quadrupole frequency (δ). These parameters provide a local microscopic description of the chemical environment in the neighborhood of the probe nuclei. Results showed differences in the hyperfine interactions of probe nuclei bound to the studied biomolecules. Such differences were observed by variations in the hyperfine parameters, which depended on the type of biomolecule and the results also showed that the probe nuclei bounded at the molecules in some cases and at others did not. (author)

  9. Synthesis of furan-based DNA binders and their interaction with DNA

    International Nuclear Information System (INIS)

    Voege, Andrea; Hoffmann, Sascha; Gabel, Detlef

    2006-01-01

    In recent years, many substances, based on naturally occurring DNA-binding molecules have been developed for the use in cancer therapy and as virostatica. Most of these substances are binding specifically to A-T rich sequences in the DNA minor groove. Neutral and positively charged DNA-binders are known. BNCT is most effective, which the boron is directly located in the cellular nucleus, so that the intercation with thermal neutrons can directly damage the DNA. To reach this aim, we have connected ammonioundecahydrododecaborate(1-) to DNA-binding structures such as 2,5-bis(4-formylphenyl)furan via a Schiff-Base reaction followed by a reduction of the imine to a secondary amine. In a following step the amine can be alkylated to insert positive charges to prevent repulsion between the compounds and the negatively charged sugar-phosphate-backbone of the DNA. (author)

  10. 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.

  11. Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

    International Nuclear Information System (INIS)

    Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

    1991-01-01

    Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

  12. Investigation on the toxic interaction of typical plasticizers with calf thymus DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Xiaojing [School of Environmental Science and Engineering, China–America CRC for Environment & Health, Shandong University, 27# Shanda South Road, Jinan 250100, Shandong Province (China); Zong, Wansong, E-mail: gaocz@sdu.edu.cn [College of Population, Resources and Environment, Shandong Normal University, 88# East Wenhua Road, Jinan 250014 (China); Liu, Chunguang; Liu, Yang [School of Environmental Science and Engineering, China–America CRC for Environment & Health, Shandong University, 27# Shanda South Road, Jinan 250100, Shandong Province (China); Gao, Canzhu, E-mail: rutaoliu@sdu.edu.cn [School of Environmental Science and Engineering, China–America CRC for Environment & Health, Shandong University, 27# Shanda South Road, Jinan 250100, Shandong Province (China); Liu, Rutao [School of Environmental Science and Engineering, China–America CRC for Environment & Health, Shandong University, 27# Shanda South Road, Jinan 250100, Shandong Province (China)

    2015-05-15

    The interactions of typical plasticizers dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP) with calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopic techniques and molecular modeling. Experimental results indicated that the characteristic fluorescence intensity of phthalic acid rose with the increase of DNA concentration; while the characteristic fluorescence intensities of plasticizers decreased with the increase of DNA concentration. Experiments on native and denatured DNA determined that plasticizers interacted with DNA both in groove and electrostatic binding mode. The molecular modeling results further illustrated that there is groove binding between them; hydrogen bonding and Van der Waals interactions were the main forces. With the extension of branched-chains, the binding effects between plasticizers and DNA were weakened, which could be related to the increased steric hindrance. - Highlights: • This work established the binding mode of plasticizers with DNA on molecular level. • The mechanism was explored by fluorescence spectroscopic and molecular docking methods. • There are two kinds of binding mode between DMP, DEP, DBP and DNA, electrostatic and groove. • With the branched chain extension, the binding effect of plasticizers and DNA has been weakened.

  13. Lichen secondary metabolites as DNA-interacting agents

    Czech Academy of Sciences Publication Activity Database

    Plšíková, J.; Štěpánková, Jana; Kašpárková, Jana; Brabec, Viktor; Bačkor, M.; Kozurková, M.

    2014-01-01

    Roč. 28, č. 2 (2014), s. 182-186 ISSN 0887-2333 Institutional support: RVO:68081707 Keywords : Lichens * DNA-binding * Topoisomerase I, II Subject RIV: BO - Biophysics Impact factor: 2.903, year: 2014

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

    Science.gov (United States)

    Awate, Sanket; Brosh, Robert M

    2017-06-08

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

  15. Interaction of gold nanoparticles with Pfu DNA polymerase and effect on polymerase chain reaction.

    Science.gov (United States)

    Sun, L-P; Wang, S; Zhang, Z-W; Ma, Y-Y; Lai, Y-Q; Weng, J; Zhang, Q-Q

    2011-03-01

    The interaction of gold nanoparticles with Pfu DNA polymerase has been investigated by a number of biological, optical and electronic spectroscopic techniques. Polymerase chain reaction was performed to show gold nanoparticles' biological effect. Ultraviolet-visible and circular dichroism spectra analysis were applied to character the structure of Pfu DNA polymerase after conjugation with gold nanoparticles. X-ray photoelectron spectroscopy was used to investigate the bond properties of the polymerase-gold nanoparticles complex. The authors demonstrate that gold nanoparticles do not affect the amplification efficiency of polymerase chain reaction using Pfu DNA polymerase, and Pfu DNA polymerase displays no significant changes of the secondary structure upon interaction with gold nanoparticles. The adsorption of Pfu DNA polymerase to gold nanoparticles is mainly through Au-NH(2) bond and electrostatic interaction. These findings may have important implications regarding the safety issue as gold nanoparticles are widely used in biomedical applications.

  16. Localized surface plasmon resonance (LSPR) study of DNA hybridization at single nanoparticle transducers

    International Nuclear Information System (INIS)

    Schneider, T.; Jahr, N.; Jatschka, J.; Csaki, A.; Stranik, O.; Fritzsche, W.

    2013-01-01

    The effect of DNA–DNA interaction on the localized surface plasmon resonance of single 80 nm gold nanoparticles is studied. Therefore, both the attachment of the capture DNA strands at the particle surface and the sequence-specific DNA binding (hybridization) of analyte DNA to the immobilized capture DNA is subject of investigations. The influence of substrate attachment chemistry, the packing density of DNA as controlled by an assisting layer of smaller molecules, and the distance as increased by a linker on the LSPR efficiency is investigated. The resulting changes in signal can be related to a higher hybridization efficiency of the analyte DNA to the immobilized capture DNA. The subsequent attachment of additional DNA strands to this system is studied, which allows for a multiple step detection of binding and an elucidation of the resulting resonance shifts. The detection limit was determined for the utilized DNA system by incubation with various concentration of analyte DNA. Although the method allows for a marker-free detection, we show that additional markers such as 20 nm gold particle labels increase the signal and thereby the sensitivity significantly. The study of resonance shift for various DNA lengths revealed that the resonance shift per base is stronger for shorter DNA molecules (20 bases) as compared to longer ones (46 bases).

  17. 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

  18. Interaction of zinc and cobalt with dipeptides and their DNA binding ...

    Indian Academy of Sciences (India)

    Unknown

    Concentrated CT DNA stock solutions were pre- pared in tris .... the SH, NH and NH2 hydrogen exchange with D2O the changes in ... which is an index of the interaction between the. DNA and ... Since ESI-MS is a powerful new approach for.

  19. DNA interaction with platinum-based cytostatics revealed by DNA sequencing.

    Science.gov (United States)

    Smerkova, Kristyna; Vaculovic, Tomas; Vaculovicova, Marketa; Kynicky, Jindrich; Brtnicky, Martin; Eckschlager, Tomas; Stiborova, Marie; Hubalek, Jaromir; Adam, Vojtech

    2017-12-15

    The main mechanism of action of platinum-based cytostatic drugs - cisplatin, oxaliplatin and carboplatin - is the formation of DNA cross-links, which restricts the transcription due to the disability of DNA to enter the active site of the polymerase. The polymerase chain reaction (PCR) was employed as a simplified model of the amplification process in the cell nucleus. PCR with fluorescently labelled dideoxynucleotides commonly employed for DNA sequencing was used to monitor the effect of platinum-based cytostatics on DNA in terms of decrease in labeling efficiency dependent on a presence of the DNA-drug cross-link. It was found that significantly different amounts of the drugs - cisplatin (0.21 μg/mL), oxaliplatin (5.23 μg/mL), and carboplatin (71.11 μg/mL) - were required to cause the same quenching effect (50%) on the fluorescent labelling of 50 μg/mL of DNA. Moreover, it was found that even though the amounts of the drugs was applied to the reaction mixture differing by several orders of magnitude, the amount of incorporated platinum, quantified by inductively coupled plasma mass spectrometry, was in all cases at the level of tenths of μg per 5 μg of DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Interaction of water with oriented DNA in the A- and B-form conformations

    International Nuclear Information System (INIS)

    Brandes, R.; Rupprecht, A.; Kearns, D.R.

    1989-01-01

    High resolution 2 H nuclear magnetic resonance (NMR) was used to investigate the interaction of D 2 O with solid samples of uniaxially oriented Li-DNA (B-form DNA) and Na-DNA (A- and B-form DNA). At low levels of hydration, 0 approximately 4 D 2 O/nucleotide, the 2 H spectra shows a very weak (due to short T2) broad single resonance, suggestive of unrestricted rotational diffusion of the water. At approximately 5 or more D 2 O/nucleotide, the Li-DNA (B-form) spectra suddenly exhibit a large doublet splitting, characteristic of partially ordered water. With increasing hydration, the general trend is a decrease of this splitting. From our analysis we show that the DNA water structure reorganizes as the DNA is progressively hydrated. The D 2 O interaction with Na-DNA is rather different than with Li-DNA. Below 10 D 2 O/nucleotide Na-DNA is normally expected to be in the A-form, and a small, or negligible splitting is observed. In the range 9-19 D 2 O/nucleotide, the splitting increases with increasing hydration. Above approximately 20 D 2 O/nucleotide Na-DNA converts entirely to the B-form and the D 2 O splittings are then similar to those found in Li-DNA. We show that the complex Na-DNA results obtained in the range 0-20 D 2 O/nucleotide are caused by a mixture of A- and B-DNA in those samples

  1. Discovering approximate-associated sequence patterns for protein-DNA interactions

    KAUST Repository

    Chan, Tak Ming; Wong, Ka Chun; Lee, Kin Hong; Wong, Man Hon; Lau, Chi Kong; Tsui, Stephen Kwok Wing; Leung, Kwong Sak

    2010-01-01

    Motivation: The bindings between transcription factors (TFs) and transcription factor binding sites (TFBSs) are fundamental protein-DNA interactions in transcriptional regulation. Extensive efforts have been made to better understand the protein

  2. Interaction of Proliferating Cell Nuclear Antigen With DNA at the Single Molecule Level

    KAUST Repository

    Raducanu, Vlad-Stefan

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) is a key factor involved in Eukaryotic DNA replication and repair, as well as other cellular pathways. Its importance comes mainly from two aspects: the large numbers of interacting partners

  3. Characterization of protein-DNA interactions in trypanosomes.

    OpenAIRE

    Ricardo Pariona Llanos

    2014-01-01

    O T. cruzi, é o agente causador da doença de Chagas. O estado redox NAD+/NADH intracelular é fundamental na manutenção do metabolismo celular. A GAPDH apresenta a função de proteção do telômero em mamíferos contra degradação, isto por causa de ligar se ao telômero. Aqui, mostramos que a GAPDH recombinante de T. cruzi (rTcGAPDH) interage com o DNA telomérico. A rTcGAPDH liga ao DNA de simples fita. Mostramos que a GAPDH liga ao DNA telomérico in vivo em células epimastigotas, onde a [NADH] é m...

  4. Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

    Science.gov (United States)

    Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

    2003-08-01

    Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

  5. The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA.

    Science.gov (United States)

    Su'etsugu, Masayuki; Harada, Yuji; Keyamura, Kenji; Matsunaga, Chika; Kasho, Kazutoshi; Abe, Yoshito; Ueda, Tadashi; Katayama, Tsutomu

    2013-12-01

    DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  6. 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

  7. Thermodynamics of the Interaction between Alzheimer's Disease Related Tau Protein and DNA

    Science.gov (United States)

    Camero, Sergio; Benítez, María J.; Cuadros, Raquel; Hernández, Félix; Ávila, Jesús; Jiménez, Juan S.

    2014-01-01

    Tau hyperphosphorylation can be considered as one of the hallmarks of Alzheimer's disease and other tauophaties. Besides its well-known role as a microtubule associated protein, Tau displays a key function as a protector of genomic integrity in stress situations. Phosphorylation has been proven to regulate multiple processes including nuclear translocation of Tau. In this contribution, we are addressing the physicochemical nature of DNA-Tau interaction including the plausible influence of phosphorylation. By means of surface plasmon resonance (SPR) we measured the equilibrium constant and the free energy, enthalpy and entropy changes associated to the Tau-DNA complex formation. Our results show that unphosphorylated Tau binding to DNA is reversible. This fact is in agreement with the protective role attributed to nuclear Tau, which stops binding to DNA once the insult is over. According to our thermodynamic data, oscillations in the concentration of dephosphorylated Tau available to DNA must be the variable determining the extent of Tau binding and DNA protection. In addition, thermodynamics of the interaction suggest that hydrophobicity must represent an important contribution to the stability of the Tau-DNA complex. SPR results together with those from Tau expression in HEK cells show that phosphorylation induces changes in Tau protein which prevent it from binding to DNA. The phosphorylation-dependent regulation of DNA binding is analogous to the Tau-microtubules binding inhibition induced by phosphorylation. Our results suggest that hydrophobicity may control Tau location and DNA interaction and that impairment of this Tau-DNA interaction, due to Tau hyperphosphorylation, could contribute to Alzheimer's pathogenesis. PMID:25126942

  8. DNA oligonucleotide conformations: high resolution NMR studies

    International Nuclear Information System (INIS)

    Mellema, J.-R.

    1984-01-01

    The present work describes a DNA double-helix model, which is well comparable with the models derived from fibre-diffraction studies. The model has a mononucleotide repeat with torsion angles in accordance with average geometries as derived from 1 H NMR studies. Special attention was paid to reduce the number of short H-H nonbonding contacts, which are abundantly present in the 'classical' fibre-diffraction models. Chapter 3 describes the first complete assignment of a 1 H NMR spectrum of a DNA tetramer, d(TAAT). Preliminary conformational data derived from the spectral parameters recorded at 27 0 C are given. A more detailed analysis employing temperature-dependence studies is given in Chapter 4. (Auth.)

  9. Cationic liposome/DNA complexes: from structure to interactions with cellular membranes.

    Science.gov (United States)

    Caracciolo, Giulio; Amenitsch, Heinz

    2012-10-01

    Gene-based therapeutic approaches are based upon the concept that, if a disease is caused by a mutation in a gene, then adding back the wild-type gene should restore regular function and attenuate the disease phenotype. To deliver the gene of interest, both viral and nonviral vectors are used. Viruses are efficient, but their application is impeded by detrimental side-effects. Among nonviral vectors, cationic liposomes are the most promising candidates for gene delivery. They form stable complexes with polyanionic DNA (lipoplexes). Despite several advantages over viral vectors, the transfection efficiency (TE) of lipoplexes is too low compared with those of engineered viral vectors. This is due to lack of knowledge about the interactions between complexes and cellular components. Rational design of efficient lipoplexes therefore requires deeper comprehension of the interactions between the vector and the DNA as well as the cellular pathways and mechanisms involved. The importance of the lipoplex structure in biological function is revealed in the application of synchrotron small-angle X-ray scattering in combination with functional TE measurements. According to current understanding, the structure of lipoplexes can change upon interaction with cellular membranes and such changes affect the delivery efficiency. Recently, a correlation between the mechanism of gene release from complexes, the structure, and the physical and chemical parameters of the complexes has been established. Studies aimed at correlating structure and activity of lipoplexes are reviewed herein. This is a fundamental step towards rational design of highly efficient lipid gene vectors.

  10. In vivo protein-DNA interactions at the β-globin gene locus

    International Nuclear Information System (INIS)

    Tohru Ikuta; Yuet Wai Kan

    1991-01-01

    The authors have investigated in vivo protein-DNA interactions in the β-globin gene locus by dimethyl sulfate (DMS) footprinting in K562 cells, which express var-epsilon- and γ-globin but not β-globin. In the locus control region, hypersensitive site 2 (HS-2) exhibited footprints in several putative protein binding motifs. HS-3 was not footprinted. The β promoter was also not footprinted, while extensive footprints were observed in the promoter of the active γ-globin gene. No footprints were seen in the A γ and β3' enhancers. With several motifs, additional protein interactions and alterations in binding patterns occurred with hemin induction. In HeLa cells, some footprints were observed in some of the motifs in HS-2, compatible with the finding that HS-2 has some enhancer function in HeLa cells, albeit much weaker than its activity in K562 cells. No footprint was seen in B lymphocytes. In vivo footprinting is a useful method for studying relevant protein-DNA interactions in erythroid cells

  11. DNMT1-interacting RNAs block gene-specific DNA methylation

    Czech Academy of Sciences Publication Activity Database

    Di Ruscio, A.; Ebralidze, A.; Benoukraf, T.; Amabile, G.; Goff, L.A.; Terragni, J.; Figueroa, M.E.; Pontes, L.L.D.; Alberich-Jorda, Meritxell; Zhang, P.; Wu, M.C.; D´Alo, F.; Melnick, A.; Leone, G.; Ebralidze, K.K.; Pradhan, S.; Rinn, J.L.; Tenen, D.G.

    2013-01-01

    Roč. 503, č. 7476 (2013), s. 371-376 ISSN 0028-0836 R&D Projects: GA MŠk LK21307 Institutional support: RVO:68378050 Keywords : DNA methylation * non-coding RNA * DNMT1 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 42.351, year: 2013

  12. Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

    Science.gov (United States)

    Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

    2013-01-01

    Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. © 2013. Published by Elsevier B.V. All rights reserved.

  13. Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

    OpenAIRE

    Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

    2004-01-01

    In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation...

  14. Investigations on the interactions of diclofenac sodium with HSA and ctDNA using molecular modeling and multispectroscopic methods

    Science.gov (United States)

    Cui, Yanrui; Hao, Erjun; Hui, Guangquan; Guo, Wei; Cui, Fengling

    2013-06-01

    A tentative study on interaction of diclofenac sodium (DF-Na) with human serum albumin (HSA) and calf thymus DNA (ctDNA) was conducted by using multi-spectroscopic and molecular modeling techniques under simulative physiological conditions. The results of spectroscopic measurements suggested that the quenching mechanisms were static quenching. Three-dimensional fluorescence spectroscopy clearly demonstrated the occurrence of conformational changes of HSA with addition of DF-Na. In addition, competitive studies with ethidium bromide (EB) have shown that DF-Na can bind to ctDNA relatively strong via groove binding. Based on the values of thermodynamic parameters and the results of molecular modeling, it was confirmed that hydrophobic forces and hydrogen bond were the mainly binding forces in DF-Na-HSA and DF-Na-DNA systems. The binding distance between DF-Na and HSA was also determined using the theory of the Förster energy transference.

  15. A theoretical study on interaction of proline with gold cluster

    Indian Academy of Sciences (India)

    with Au3 (Pakiari and Jamshidi 2007) and interaction of. ∗. Author for correspondence (harjinder.singh@iiit.ac.in) small gold clusters with xDNA base pairs (Sharma et al. 2009) have motivated us to carry out a theoretical study on interaction of proline with gold nanoparticles. Proline is unique among the natural amino acids ...

  16. A force-based, parallel assay for the quantification of protein-DNA interactions.

    Science.gov (United States)

    Limmer, Katja; Pippig, Diana A; Aschenbrenner, Daniela; Gaub, Hermann E

    2014-01-01

    Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA), parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

  17. A force-based, parallel assay for the quantification of protein-DNA interactions.

    Directory of Open Access Journals (Sweden)

    Katja Limmer

    Full Text Available Analysis of transcription factor binding to DNA sequences is of utmost importance to understand the intricate regulatory mechanisms that underlie gene expression. Several techniques exist that quantify DNA-protein affinity, but they are either very time-consuming or suffer from possible misinterpretation due to complicated algorithms or approximations like many high-throughput techniques. We present a more direct method to quantify DNA-protein interaction in a force-based assay. In contrast to single-molecule force spectroscopy, our technique, the Molecular Force Assay (MFA, parallelizes force measurements so that it can test one or multiple proteins against several DNA sequences in a single experiment. The interaction strength is quantified by comparison to the well-defined rupture stability of different DNA duplexes. As a proof-of-principle, we measured the interaction of the zinc finger construct Zif268/NRE against six different DNA constructs. We could show the specificity of our approach and quantify the strength of the protein-DNA interaction.

  18. DNA:DNA hybridization studies on the pink-pigmented facultative methylotrophs.

    Science.gov (United States)

    Hood, D W; Dow, C S; Green, P N

    1987-03-01

    The genomic relatedness among 36 strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) was estimated by determination of DNA base composition and by DNA:DNA hybridization studies. A reproducible hybridization system was developed for the rapid analysis of multiple DNA samples. Results indicated that the PPFMs comprise four major and several minor homology groups, and that they should remain grouped in a single genus, Methylobacterium.

  19. Highly functionalized piperidines: Free radical scavenging, anticancer activity, DNA interaction and correlation with biological activity

    Directory of Open Access Journals (Sweden)

    Suvankar Das

    2018-01-01

    Full Text Available Twenty-five piperidines were studied as potential radical scavengers and antitumor agents. Quantitative interaction of compounds with ctDNA using spectroscopic techniques was also evaluated. Our results demonstrate that the evaluated piperidines possesses different abilities to scavenge the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH and the anion radical superoxide (·O2−. The piperidine 19 was the most potent radical DPPH scavenger, while the most effective to ·O2− scavenger was piperidine 10. In general, U251, MCF7, NCI/ADR-RES, NCI-H460 and HT29 cells were least sensitive to the tested compounds and all compounds were considerably more toxic to the studied cancer cell lines than to the normal cell line HaCaT. The binding mode of the compounds and ctDNA was preferably via intercalation. In addition, these results were confirmed based on theoretical studies. Finally, a linear and exponential correlation between interaction constant (Kb and GI50 for several human cancer cell was observed.

  20. A Theoretical and Experimental Study of DNA Self-assembly

    Science.gov (United States)

    Chandran, Harish

    The control of matter and phenomena at the nanoscale is fast becoming one of the most important challenges of the 21st century with wide-ranging applications from energy and health care to computing and material science. Conventional top-down approaches to nanotechnology, having served us well for long, are reaching their inherent limitations. Meanwhile, bottom-up methods such as self-assembly are emerging as viable alternatives for nanoscale fabrication and manipulation. A particularly successful bottom up technique is DNA self-assembly where a set of carefully designed DNA strands form a nanoscale object as a consequence of specific, local interactions among the different components, without external direction. The final product of the self-assembly process might be a static nanostructure or a dynamic nanodevice that performs a specific function. Over the past two decades, DNA self-assembly has produced stunning nanoscale objects such as 2D and 3D lattices, polyhedra and addressable arbitrary shaped substrates, and a myriad of nanoscale devices such as molecular tweezers, computational circuits, biosensors and molecular assembly lines. In this dissertation we study multiple problems in the theory, simulations and experiments of DNA self-assembly. We extend the Turing-universal mathematical framework of self-assembly known as the Tile Assembly Model by incorporating randomization during the assembly process. This allows us to reduce the tile complexity of linear assemblies. We develop multiple techniques to build linear assemblies of expected length N using far fewer tile types than previously possible. We abstract the fundamental properties of DNA and develop a biochemical system, which we call meta-DNA, based entirely on strands of DNA as the only component molecule. We further develop various enzyme-free protocols to manipulate meta-DNA systems and provide strand level details along with abstract notations for these mechanisms. We simulate DNA circuits by

  1. Line narrowing spectroscopic studies of DNA-carcinogen adducts and DNA-dye complexes

    Energy Technology Data Exchange (ETDEWEB)

    Suh, Myungkoo [Iowa State Univ., Ames, IA (United States)

    1995-12-06

    Laser-induced fluorescence line narrowing and non-line narrowing spectroscopic methods were applied to conformational studies of stable DNA adducts of the 7β, 8α-dihydoxy-9α, l0α-epoxy-7,8,9, 10-tetrahydrobenzo[α]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, (-)-trans-, (+)-cis- and (-)-cis adducts of anti-BPDE bound to exocyclic amino group of the central guanine in an 11-mer oligonucleotide, exist in a mixture of conformations in frozen aqueous buffer matrices. The (+)-trans adduct adopts primarily an external conformation with a smaller fraction ( ~25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant π-π stacking interactions between the pyrenyl residues and the bases. Conformations of the trans-adduct of (+)-anti -BPDE in 11-mer oligonucleotides were studied as a function of flanking bases. In single stranded form the adduct at G2 or G3 (5 ft-flanking, base guanine) adopts a conformation with strong, interaction with the bases. In contrast, the adduct with a 5ft-flanking, thymine exists in a primarily helixexternal conformation. Similar differences were observed in the double stranded oligonucleotides. The nature of the 3ft-flanking base has little influence on the conformational equilibrium of the (+)-trans-anti BPDE-dG adduct. The formation and repair of BPDE-N2-dG in DNA isolated from the skin of mice treated topically with benzo[α]pyrene (BP) was studied. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N-dG, and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N2-dG.

  2. Line narrowing spectroscopic studies of DNA-carcinogen adducts and DNA-dye complexes

    International Nuclear Information System (INIS)

    Suh, Myungkoo.

    1995-01-01

    Laser-induced fluorescence line narrowing and non-line narrowing spectroscopic methods were applied to conformational studies of stable DNA adducts of the 7β, 8α-dihydoxy-9α, l0α-epoxy-7,8,9, 10-tetrahydrobenzo[α]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, (-)-trans-, (+)-cis- and (-)-cis adducts of anti-BPDE bound to exocyclic amino group of the central guanine in an 11-mer oligonucleotide, exist in a mixture of conformations in frozen aqueous buffer matrices. The (+)-trans adduct adopts primarily an external conformation with a smaller fraction ( ∼ 25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant π-π stacking interactions between the pyrenyl residues and the bases. Conformations of the trans-adduct of (+)-anti -BPDE in 11-mer oligonucleotides were studied as a function of flanking bases. In single stranded form the adduct at G 2 or G 3 (5 ft-flanking, base guanine) adopts a conformation with strong, interaction with the bases. In contrast, the adduct with a 5ft-flanking, thymine exists in a primarily helixexternal conformation. Similar differences were observed in the double stranded oligonucleotides. The nature of the 3ft-flanking base has little influence on the conformational equilibrium of the (+)-trans-anti BPDE-dG adduct. The formation and repair of BPDE-N 2 -dG in DNA isolated from the skin of mice treated topically with benzo[α]pyrene (BP) was studied. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N-dG, and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N 2 -dG

  3. DNA binding studies of Sunset Yellow FCF using spectroscopy, viscometry and electrochemical techniques

    Science.gov (United States)

    Asaadi, Sara; Hajian, Reza

    2017-10-01

    Color is one of the important factors in food industry. All food companies use synthetic pigments to improve the aesthetic of products. Studies on the interaction between deoxyribonucleic acid (DNA) and food dye molecules is important because DNA is responsible for some processes including replication and transcription of cells, mutations, genetic diseases, and some synthetic chemical nucleases. In this study, the molecular interaction between Sunset Yellow FCF (SY) as a common food coloring additive and calf thymus DNA (ct-DNA) has been studied using UV-Vis spectrophotometry, spectrofluorometry, Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry and viscometry techniques. The binding constant between ct-DNA and SY in phosphate buffer solution (pH 7.4) was calculated as 2.09 × 103 L mol-1. The non-electrostatic bonding constant (K0t) was almost consistent and the ratio of K0t/Kb increased by increasing the ionic strength in the range of 0.01-0.1 mol L-1 of KCl. This observation shows that, the molecular bonding of SY to ct-DNA is a combination of electrostatic and intercalation interactions. In the electrochemical studies, an oxidation peak at 0.71 V and a reduction peak at about 0.63 V was observed with the peak potential difference (ΔEp) of 0.08 V, showing a reversible process. The oxidation and reduction peaks were significantly decreased in the presence of ct-DNA and the reduction peak current shifted to negative values. In spectrofluorometric study, the fluorescence intensity of SY increased dramatically after successive addition of DNA due to the increasing of molecular surface area and decreasing of impact frequency between solvent and SY-DNA adduct. Moreover, viscometric study shows that the increasing of viscosity for SY solution in the presence of DNA is due to the intercalation mechanism with double strand DNA (ds-DNA).

  4. Quantitative analysis of TALE-DNA interactions suggests polarity effects.

    Science.gov (United States)

    Meckler, Joshua F; Bhakta, Mital S; Kim, Moon-Soo; Ovadia, Robert; Habrian, Chris H; Zykovich, Artem; Yu, Abigail; Lockwood, Sarah H; Morbitzer, Robert; Elsäesser, Janett; Lahaye, Thomas; Segal, David J; Baldwin, Enoch P

    2013-04-01

    Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ≈ NN > NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 10(3)-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches 'standard' RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5'-T. Another surprising observation was that base mismatches at the 5' end of the target site had more disruptive effects on affinity than those at the 3' end, particularly in designed TALEs. These results provide evidence that TALE-DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

  5. A selective chemosensor for fluoride ion and its interaction with Calf Thymus DNA.

    Science.gov (United States)

    Ghosh, Soumen; Al Masum, Abdulla; Ganguly, Aniruddha; Islam, Md Maidul; Alam, Md Akhtarul; Guchhait, Nikhil

    2017-05-05

    The amido-Schiff base 1 (N 1 , N 3 -bis (2-nitrobenzylidene)benzene-1,3-dicabohydrazide) containing a CONH group and CHN linkage has been synthesized by the condensation between isophthalic acid dihydrazide and o-nitrobenzaldehyde. This molecule can act as a fluoride ion sensor with high selectivity and sensitivity. Presence of nitro group in the phenyl ring may be responsible for the detection of fluoride ion visually with a dramatic color change from colorless to deep red in aqueous dimethyl sulphoxide solution. This Schiff base can be used as test kit for sensing of fluoride ion in the solid state. Compound 1 can detect fluoride also in commercially available toothpaste. As the compound has adequate solubility in DMSO-water mixture (7:93, v/v) and having some hydrogen bond donor and acceptor centers, we have investigated its nature of binding with Calf Thymus-DNA (CT-DNA) using theoretical molecular modelling and other experimental methods like UV-vis spectroscopy, circular dichroic and thermal melting studies. Thermodynamic parameters have been obtained using the well known Van't Hoff's equation. From both theoretical and experimental findings it has been observed that it can interact effectively with CT-DNA with binding energy -7.55kcal/mol to -7.50kcal/mol. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. DNA methylation analysis from saliva samples for epidemiological studies.

    Science.gov (United States)

    Nishitani, Shota; Parets, Sasha E; Haas, Brian W; Smith, Alicia K

    2018-06-18

    Saliva is a non-invasive, easily accessible tissue, which is regularly collected in large epidemiological studies to examine genetic questions. Recently, it is becoming more common to use saliva to assess DNA methylation. However, DNA extracted from saliva is a mixture of both bacterial and human DNA derived from epithelial and immune cells in the mouth. Thus, there are unique challenges to using salivary DNA in methylation studies that can influence data quality. This study assesses: (1) quantification of human DNA after extraction; (2) delineation of human and bacterial DNA; (3) bisulfite conversion (BSC); (4) quantification of BSC DNA; (5) PCR amplification of BSC DNA from saliva and; (6) quantitation of DNA methylation with a targeted assay. The framework proposed will allow saliva samples to be more widely used in targeted epigenetic studies.

  7. A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

    Directory of Open Access Journals (Sweden)

    Konstantin A. Tsetsarkin

    2016-08-01

    Full Text Available An arthropod-borne virus, Zika virus (ZIKV, has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics.

  8. Correlation between the thermodynamic stability of DNA duplexes and the interaction and solvation energies of DNA building blocks

    Czech Academy of Sciences Publication Activity Database

    Řezáč, Jan; Hobza, Pavel

    2008-01-01

    Roč. 73, č. 2 (2008), s. 161-174 ISSN 0010-0765 R&D Projects: GA MŠk LC512; GA ČR GA203/05/0009; GA ČR(CZ) GD203/05/H001 Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA duplex * interaction energy * stability Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.784, year: 2008

  9. Fluorescence Correlation Spectroscopy of Spermine-DNA Interactions - Nanostructure and Physical Supramolecular Chemistry of DNA Condensation

    Czech Academy of Sciences Publication Activity Database

    Kral, Teresa; Langner, M.; Hof, Martin; Adjimatera, N.; Blagbrough, I. S.

    2004-01-01

    Roč. 98, Supplement (2004), s22-s23 ISSN 0009-2770 Institutional research plan: CEZ:AV0Z4040901 Keywords : fluorescence * nanostructure * DNA condensation Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.348, year: 2004

  10. Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

    Science.gov (United States)

    Sugitani, Norie; Voehler, Markus W; Roh, Michelle S; Topolska-Woś, Agnieszka M; Chazin, Walter J

    2017-10-13

    Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, Aishwarya; Moharana, Kedar; Wallace, Susan S.; Doublié, Sylvie

    2016-12-19

    The base excision repair (BER) pathway repairs oxidized lesions in the DNA that result from reactive oxygen species generated in cells. If left unrepaired, these damaged DNA bases can disrupt cellular processes such as replication. NEIL1 is one of the 11 human DNA glycosylases that catalyze the first step of the BER pathway, i.e. recognition and excision of DNA lesions. NEIL1 interacts with essential replication proteins such as the ring-shaped homotrimeric proliferating cellular nuclear antigen (PCNA). We isolated a complex formed between NEIL1 and PCNA (±DNA) using size exclusion chromatography (SEC). This interaction was confirmed using native gel electrophoresis and mass spectrometry. Stokes radii measured by SEC hinted that PCNA in complex with NEIL1 (±DNA) was no longer a trimer. Height measurements and images obtained by atomic force microscopy also demonstrated the dissociation of the PCNA homotrimer in the presence of NEIL1 and DNA, while small-angle X-ray scattering analysis confirmed the NEIL1 mediated PCNA trimer dissociation and formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex. Furthermore, ab initio shape reconstruction provides insights into the solution structure of this previously unreported complex. Together, these data point to a potential mechanistic switch between replication and BER.

  12. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    International Nuclear Information System (INIS)

    Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

    2015-01-01

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA

  13. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  14. Alpha Particles and X Rays Interact in Inducing DNA Damage in U2OS Cells.

    Science.gov (United States)

    Sollazzo, Alice; Brzozowska, Beata; Cheng, Lei; Lundholm, Lovisa; Haghdoost, Siamak; Scherthan, Harry; Wojcik, Andrzej

    2017-10-01

    Survivors of the atomic bombings of Hiroshima and Nagasaki are monitored for health effects within the Life Span Study (LSS). The LSS results represent the most important source of data about cancer effects from ionizing radiation exposure, which forms the foundation for the radiation protection system. One uncertainty connected to deriving universal risk factors from these results is related to the problem of mixed radiation qualities. The A-bomb explosions generated a mixed beam of the sparsely ionizing gamma radiation and densely ionizing neutrons. However, until now the possible interaction of the two radiation types of inducing biological effects has not been taken into consideration. The existence of such interaction would suggest that the application of risk factors derived from the LSS to predict cancer effects after pure gamma-ray irradiation (such as in the Fukushima prefecture) leads to an overestimation of risk. To analyze the possible interaction of radiation types, a mixed-beam exposure facility was constructed where cells can be exposed to sparsely ionizing X rays and densely ionizing alpha particles. U2OS cells were used, which are stably transfected with a plasmid coding for the DNA repair gene 53BP1 coupled to a gene coding for the green fluorescent protein (GFP). The induction and repair of DNA damage, which are known to be related to cancer induction, were analyzed. The results suggest that alpha particles and X rays interact, leading to cellular and possibly cancer effects, which cannot be accurately predicted based on assuming simple additivity of the individual mixed-beam components.

  15. DNA builds and strengthens the extracellular matrix in Myxococcus xanthus biofilms by interacting with exopolysaccharides.

    Directory of Open Access Journals (Sweden)

    Wei Hu

    Full Text Available One intriguing discovery in modern microbiology is the extensive presence of extracellular DNA (eDNA within biofilms of various bacterial species. Although several biological functions have been suggested for eDNA, including involvement in biofilm formation, the detailed mechanism of eDNA integration into biofilm architecture is still poorly understood. In the biofilms formed by Myxococcus xanthus, a Gram-negative soil bacterium with complex morphogenesis and social behaviors, DNA was found within both extracted and native extracellular matrices (ECM. Further examination revealed that these eDNA molecules formed well organized structures that were similar in appearance to the organization of exopolysaccharides (EPS in ECM. Biochemical and image analyses confirmed that eDNA bound to and colocalized with EPS within the ECM of starvation biofilms and fruiting bodies. In addition, ECM containing eDNA exhibited greater physical strength and biological stress resistance compared to DNase I treated ECM. Taken together, these findings demonstrate that DNA interacts with EPS and strengthens biofilm structures in M. xanthus.

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

    Science.gov (United States)

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

    2015-06-05

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

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

    2015-01-01

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

  18. Non-covalent interactions of cadmium sulphide and gold nanoparticles with DNA

    Science.gov (United States)

    Atay, Z.; Biver, T.; Corti, A.; Eltugral, N.; Lorenzini, E.; Masini, M.; Paolicchi, A.; Pucci, A.; Ruggeri, G.; Secco, F.; Venturini, M.

    2010-08-01

    Mercaptoethanol-capped CdS nanoparticles (CdSnp) and monohydroxy-(1-mercaptoundec-11-yl)tetraethylene-glycol-capped Au nanoparticles (Aunp) were synthesised, characterised and their interactions with DNA were investigated. Aunp are stable in different aqueous solvents, whereas CdSnp do precipitate in 0.1 M NaCl and form two different cluster types in 0.1 M NaNO3. As regards the CdSnp/DNA interaction, absorbance and fluorescence titrations, ethidium bromide displacement assays and gel electrophoresis experiments indicate that a non-covalent interaction between DNA and the CdSnp external surface does take place. The binding constant was evaluated to be equal to (2.2 ± 0.5) × 105 M-1. On the contrary, concerning Aunp, no direct interaction with DNA could be observed. Possible interaction with serum albumin was also checked, but no effects could be observed for either CdSnp or Aunp. Finally, short-time exposure of cultured cells to nanoparticles revealed the ability of CdSnp to enter the cells and allocate both in cytosol and nucleus, thus promoting cell proliferation at low concentration ( p resulted in a significant inhibition of cell growth, accompanied by apoptotic cell death. Aunp neither enter the cells, nor do affect cell proliferation. In conclusion, our data indicate that CdSnp can strongly interact with living cells and nucleic acid while no effects or interactions were observed for Aunp.

  19. The interaction of taurine-salicylaldehyde Schiff base copper(II) complex with DNA and the determination of DNA using the complex as a fluorescence probe

    Science.gov (United States)

    Zhang, Xiaoyan; Wang, Yong; Zhang, Qianru; Yang, Zhousheng

    2010-09-01

    The interaction of taurine-salicylaldehyde Schiff base copper(II) (Cu(TSSB) 22+) complex with DNA was explored by using UV-vis, fluorescence spectrophotometry, and voltammetry. In pH 7.4 Tris-HCl buffer solution, the binding constant of the Cu(TSSB) 22+ complex interaction with DNA was 3.49 × 10 4 L mol -1. Moreover, due to the fluorescence enhancing of Cu(TSSB) 22+ complex in the presence of DNA, a method for determination of DNA with Cu(TSSB) 22+ complex as a fluorescence probe was developed. The fluorescence spectra indicated that the maximum excitation and emission wavelength were 389 nm and 512 nm, respectively. Under optimal conditions, the calibration graphs are linear over the range of 0.03-9.03 μg mL -1 for calf thymus DNA (CT-DNA), 0.10-36 μg mL -1 for yeast DNA and 0.01-10.01 μg mL -1 for salmon DNA (SM-DNA), respectively. The corresponding detection limits are 7 ng mL -1 for CT-DNA, 3 ng mL -1 for yeast DNA and 3 ng mL -1 for SM-DNA. Using this method, DNA in synthetic samples was determined with satisfactory results.

  20. Role of minor groove width and hydration pattern on amsacrine interaction with DNA.

    Directory of Open Access Journals (Sweden)

    Deepak K Jangir

    Full Text Available Amsacrine is an anilinoacridine derivative anticancer drug, used to treat a wide variety of malignancies. In cells, amsacrine poisons topoisomerase 2 by stabilizing DNA-drug-enzyme ternary complex. Presence of amsacrine increases the steady-state concentration of these ternary complexes which in turn hampers DNA replication and results in subsequent cell death. Due to reversible binding and rapid slip-out of amsacrine from DNA duplex, structural data is not available on amsacrine-DNA complexes. In the present work, we designed five oligonucleotide duplexes, differing in their minor groove widths and hydration pattern, and examined their binding with amsacrine using attenuated total reflection Fourier transform infrared (ATR-FTIR spectroscopy. Complexes of amsacrine with calf thymus DNA were also evaluated for a comparison. Our results demonstrate for the first time that amsacrine is not a simple intercalator; rather mixed type of DNA binding (intercalation and minor groove takes place between amsacrine and DNA. Further, this binding is highly sensitive towards the geometries and hydration patterns of different minor grooves present in the DNA. This study shows that ligand binding to DNA could be very sensitive to DNA base composition and DNA groove structures. Results demonstrated here could have implication for understanding cytotoxic mechanism of aminoacridine based anticancer drugs and provide directions to modify these drugs for better efficacy and few side-effects.

  1. Interactions between ionic liquid surfactant [C12mim]Br and DNA in dilute brine.

    Science.gov (United States)

    He, Yunfei; Shang, Yazhuo; Liu, Zhenhai; Shao, Shuang; Liu, Honglai; Hu, Ying

    2013-01-01

    Interactions between ionic liquid surfactant [C(12)mim]Br and DNA in dilute brine were investigated in terms of various experimental methods and molecular dynamics (MD) simulation. It was shown that the aggregation of [C(12)mim]Br on DNA chains is motivated not only by electrostatic attractions between DNA phosphate groups and [C(12)mim]Br headgroups but also by hydrophobic interactions among [C(12)mim]Br alkyl chains. Isothermal titration calorimetry analysis indicated that the [C(12)mim]Br aggregation in the presence and absence of DNA are both thermodynamically favored driven by enthalpy and entropy. DNA undergoes size transition and conformational change induced by [C(12)mim]Br, and the charges of DNA are neutralized by the added [C(12)mim]Br. Various microstructures were observed such as DNA with loose coil conformation in nature state, necklace-like structures, and compact spherical aggregates. MD simulation showed that the polyelectrolyte collapses upon the addition of oppositely charged surfactants and the aggregation of surfactants around the polyelectrolyte was reaffirmed. The simulation predicted the gradual neutralization of the negatively charged polyelectrolyte by the surfactant, consistent with the experimental results. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Microbial interactions chapter: binding and entry of DNA in bacterial transformation

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1977-01-01

    Genetic transformation of bacteria by DNA released from cells of a related strain is discussed. The mechanism by which the giant information-bearing molecules of DNA are transported into the bacterial cell was investigated. It was concluded that the overall process of DNA uptake consists of two main steps, binding of donor DNA to the outside of the cell and entry of the bound DNA into the cell. Each step is discussed in detail. Inasmuch as these phenomena occur at the cell surface, they are related to structures and functions of the cell wall and membrane. In addition, the development of competence, that is the formation of cell surface structures allowing DNA uptake, is examined from both a physiological and evolutionary point of view. Genetic transfer mediated by free DNA is an obvious and important form of cellular interaction. The development of competence involves another, quite distinct system of interaction between bacterial cells. Streptococcus pneumoniae, Bacillus subtilis, and Hemophilus influenzae were used as the test organisms. 259 references.

  3. Impedance analysis of DNA and DNA-drug interactions on thin mercury film electrodes

    Czech Academy of Sciences Publication Activity Database

    Hasoň, Stanislav; Dvořák, Jakub; Jelen, František; Vetterl, Vladimír

    2002-01-01

    Roč. 32, č. 2 (2002), s. 167-179 ISSN 1040-8347 R&D Projects: GA AV ČR IAA4004901; GA AV ČR IAA4004002; GA AV ČR IBS5004107 Grant - others:GA FRVŠ(XC) G40583; GA FRVŠ(XC) F40564 Institutional research plan: CEZ:AV0Z5004920 Keywords : electrochemical impedance spectroscopy * intercalators * DNA at electrode surface Subject RIV: BO - Biophysics Impact factor: 2.074, year: 2002

  4. Structure and decoy-mediated inhibition of the SOX18/Prox1-DNA interaction.

    Science.gov (United States)

    Klaus, Miriam; Prokoph, Nina; Girbig, Mathias; Wang, Xuecong; Huang, Yong-Heng; Srivastava, Yogesh; Hou, Linlin; Narasimhan, Kamesh; Kolatkar, Prasanna R; Francois, Mathias; Jauch, Ralf

    2016-05-05

    The transcription factor (TF) SOX18 drives lymphatic vessel development in both embryogenesis and tumour-induced neo-lymphangiogenesis. Genetic disruption of Sox18 in a mouse model protects from tumour metastasis and established the SOX18 protein as a molecular target. Here, we report the crystal structure of the SOX18 DNA binding high-mobility group (HMG) box bound to a DNA element regulating Prox1 transcription. The crystals diffracted to 1.75Å presenting the highest resolution structure of a SOX/DNA complex presently available revealing water structure, structural adjustments at the DNA contact interface and non-canonical conformations of the DNA backbone. To explore alternatives to challenging small molecule approaches for targeting the DNA-binding activity of SOX18, we designed a set of five decoys based on modified Prox1-DNA. Four decoys potently inhibited DNA binding of SOX18 in vitro and did not interact with non-SOX TFs. Serum stability, nuclease resistance and thermal denaturation assays demonstrated that a decoy circularized with a hexaethylene glycol linker and terminal phosphorothioate modifications is most stable. This SOX decoy also interfered with the expression of a luciferase reporter under control of a SOX18-dependent VCAM1 promoter in COS7 cells. Collectively, we propose SOX decoys as potential strategy for inhibiting SOX18 activity to disrupt tumour-induced neo-lymphangiogenesis. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos

    International Nuclear Information System (INIS)

    Yu, Shuangying; Tang, Song; Mayer, Gregory D.; Cobb, George P.; Maul, Jonathan D.

    2015-01-01

    Highlights: • Interactive effects of UVB radiation-pesticide co-exposures were examined in frogs. • Responses included induction of DNA photo-adducts and DNA damage and repair genes. • Elevated DNA adduct levels occurred for co-exposures compared to UVB alone. • One mechanism is that pesticides may alter nuclear excision repair gene expression. - Abstract: Pesticide use and ultraviolet-B (UVB) radiation have both been suggested to adversely affect amphibians; however, little is known about their interactive effects. One potential adverse interaction could involve pesticide-induced dysregulation of DNA repair pathways, resulting in greater numbers of DNA photo-adducts from UVB exposure. In the present study, we investigated the interactive effects of UVB radiation and two common pesticides (endosulfan and α-cypermethrin) on induction of DNA photo-adducts and expression of DNA damage and repair related genes in African clawed frog (Xenopus laevis) embryos. We examined 13 genes that are, collectively, involved in stress defense, cell cycle arrest, nucleotide excision repair (NER), base excision repair, mismatch repair, DNA repair regulation, and apoptosis. We exposed X. laevis embryos to 0, 25, and 50 μg/L endosulfan or 0, 2.5, and 5.0 μg/L α-cypermethrin for 96 h, with environmentally relevant exposures of UVB radiation during the last 7 h of the 96 h exposure. We measured the amount of cyclobutane pyrimidine dimers (CPDs) and mRNA abundance of the 13 genes among treatments including control, pesticide only, UVB only, and UVB and pesticide co-exposures. Each of the co-exposure scenarios resulted in elevated CPD levels compared to UVB exposure alone, suggesting an inhibitory effect of endosulfan and α-cypermethrin on CPD repair. This is attributed to results indicating that α-cypermethrin and endosulfan reduced mRNA abundance of XPA and HR23B, respectively, to levels that may affect the initial recognition of DNA lesions. In contrast, both pesticides

  6. Interdependence of pyrene interactions and tetramolecular G4-DNA assembly.

    Science.gov (United States)

    Doluca, Osman; Withers, Jamie M; Loo, Trevor S; Edwards, Patrick J B; González, Carlos; Filichev, Vyacheslav V

    2015-03-28

    Controlling the arrangement of organic chromophores in supramolecular architectures is of primary importance for the development of novel functional molecules. Insertion of a twisted intercalating nucleic acid (TINA) moiety, containing phenylethynylpyren-1-yl derivatives, into a G-rich DNA sequence alters G-quadruplex folding, resulting in supramolecular structures with defined pyrene arrangements. Based on CD, NMR and ESI-mass-spectra, as well as TINA excited dimer (excimer) fluorescence emission we propose that insertion of the TINA monomer in the middle of a dTG4T sequence (i.e. dTGGXGGT, where X is TINA) converts a parallel tetramolecular G-quadruplex into an assembly composed of two identical antiparallel G-quadruplex subunits stacked via TINA-TINA interface. Kinetic analysis showed that TINA-TINA association controls complex formation in the presence of Na(+) but barely competes with guanine-mediated association in K(+) or in the sequence with the longer G-run (dTGGGXGGGT). These results demonstrate new perspectives in the design of molecular entities that can kinetically control G-quadruplex formation and show how tetramolecular G-quadruplexes can be used as a tuneable scaffold to control the arrangement of organic chromophores.

  7. DNA cards: determinants of DNA yield and quality in collecting genetic samples for pharmacogenetic studies.

    Science.gov (United States)

    Mas, Sergi; Crescenti, Anna; Gassó, Patricia; Vidal-Taboada, Jose M; Lafuente, Amalia

    2007-08-01

    As pharmacogenetic studies frequently require establishment of DNA banks containing large cohorts with multi-centric designs, inexpensive methods for collecting and storing high-quality DNA are needed. The aims of this study were two-fold: to compare the amount and quality of DNA obtained from two different DNA cards (IsoCode Cards or FTA Classic Cards, Whatman plc, Brentford, Middlesex, UK); and to evaluate the effects of time and storage temperature, as well as the influence of anticoagulant ethylenediaminetetraacetic acid on the DNA elution procedure. The samples were genotyped by several methods typically used in pharmacogenetic studies: multiplex PCR, PCR-restriction fragment length polymorphism, single nucleotide primer extension, and allelic discrimination assay. In addition, they were amplified by whole genome amplification to increase genomic DNA mass. Time, storage temperature and ethylenediaminetetraacetic acid had no significant effects on either DNA card. This study reveals the importance of drying blood spots prior to isolation to avoid haemoglobin interference. Moreover, our results demonstrate that re-isolation protocols could be applied to increase the amount of DNA recovered. The samples analysed were accurately genotyped with all the methods examined herein. In conclusion, our study shows that both DNA cards, IsoCode Cards and FTA Classic Cards, facilitate genetic and pharmacogenetic testing for routine clinical practice.

  8. The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions.

    Directory of Open Access Journals (Sweden)

    Vladislav Kljashtorny

    Full Text Available The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD. Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP, albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

  9. Applications of DNA-Stable Isotope Probing in Bioremediation Studies

    Science.gov (United States)

    Chen, Yin; Vohra, Jyotsna; Murrell, J. Colin

    DNA-stable isotope probing, a method to identify active microorganisms without the prerequisite of cultivation, has been widely applied in the study of microorganisms involved in the degradation of environmental pollutants. Recent advances and technique considerations in applying DNA-SIP in bioremediation are highlighted. A detailed protocol of a DNA-SIP experiment is provided.

  10. Mechanisms of radiation interaction with DNA: Potential implications for radiation protection

    International Nuclear Information System (INIS)

    1988-01-01

    The Office of Health and Environmental Research (OHER) of the US Department of Energy conducts a broad multidisciplinary research program which includes basic biophysics, biophysical chemistry, molecular and cellular biology as well as experimental animal studies and opportunistic human studies. This research is directed at understanding how low levels of radiation of various qualities produce the spectrum of biological effects that are seen for such exposures. This workshop was entitled ''Mechanisms of Radiation Interaction with DNA: Potential Implications for Radiation Protection.'' It ws jointly sponsored by the Department of Energy and the Commission of European Communities. The aim of the workshop was to review the base of knowledge in the area of mechanisms of radiation action at the DNA level, and to explore ways in which this information can be applied to the development of scientifically sound concepts and procedures for use in the field of radiation protection. The overview of research provided by this multidisciplinary group will be helpful to the Office in program planning. This report includes a summary of the presentations, extended abstracts, the meeting agenda, research recommendations, and a list of participants. Individual papers are processed separately for the data base

  11. Mechanisms of radiation interaction with DNA: Potential implications for radiation protection

    Energy Technology Data Exchange (ETDEWEB)

    1988-01-01

    The Office of Health and Environmental Research (OHER) of the US Department of Energy conducts a broad multidisciplinary research program which includes basic biophysics, biophysical chemistry, molecular and cellular biology as well as experimental animal studies and opportunistic human studies. This research is directed at understanding how low levels of radiation of various qualities produce the spectrum of biological effects that are seen for such exposures. This workshop was entitled ''Mechanisms of Radiation Interaction with DNA: Potential Implications for Radiation Protection.'' It ws jointly sponsored by the Department of Energy and the Commission of European Communities. The aim of the workshop was to review the base of knowledge in the area of mechanisms of radiation action at the DNA level, and to explore ways in which this information can be applied to the development of scientifically sound concepts and procedures for use in the field of radiation protection. The overview of research provided by this multidisciplinary group will be helpful to the Office in program planning. This report includes a summary of the presentations, extended abstracts, the meeting agenda, research recommendations, and a list of participants. Individual papers are processed separately for the data base.

  12. Anticancer drug mithramycin interacts with core histones: An additional mode of action of the DNA groove binder

    Directory of Open Access Journals (Sweden)

    Amrita Banerjee

    2014-01-01

    Full Text Available Mithramycin (MTR is a clinically approved DNA-binding antitumor antibiotic currently in Phase 2 clinical trials at National Institutes of Health for treatment of osteosarcoma. In view of the resurgence in the studies of this generic antibiotic as a human medicine, we have examined the binding properties of MTR with the integral component of chromatin – histone proteins – as a part of our broad objective to classify DNA-binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode or both histones and chromosomal DNA (dual binding mode. The present report shows that besides DNA, MTR also binds to core histones present in chromatin and thus possesses the property of dual binding in the chromatin context. In contrast to the MTR–DNA interaction, association of MTR with histones does not require obligatory presence of bivalent metal ion like Mg2+. As a consequence of its ability to interact with core histones, MTR inhibits histone H3 acetylation at lysine 18, an important signature of active chromatin, in vitro and ex vivo. Reanalysis of microarray data of Ewing sarcoma cell lines shows that upon MTR treatment there is a significant down regulation of genes, possibly implicating a repression of H3K18Ac-enriched genes apart from DNA-binding transcription factors. Association of MTR with core histones and its ability to alter post-translational modification of histone H3 clearly indicates an additional mode of action of this anticancer drug that could be implicated in novel therapeutic strategies.

  13. Interaction Mode between Inclusion Complex of Vitamin K3 with γ- Cyclodextrin and Herring-Sperm DNA.

    Science.gov (United States)

    Tang, Yan; Cai, Li; Xue, Kang; Wang, Chunling; Xiong, Xiaoli

    2016-05-03

    Methods including spectroscopy, electronic chemistry and thermodynamics were used to study the inclusion effect between γ-cyclodextrin (CD) and vitamin K3(K3), as well as the interaction mode between herring-sperm DNA (hsDNA) and γ-CD-K3 inclusion complex. The results from ultraviolet spectroscopic method indicated that VK3 and γ-CD formed 1:1 inclusion complex, with the inclusion constant Kf = 1.02 × 10(4) L/mol, which is based on Benesi-Hildebrand's viewpoint. The outcomes from the probe method and Scatchard methods suggested that the interaction mode between γ-CD-K3 and DNA was a mixture mode, which included intercalation and electrostatic binding effects. The binding constants were K (θ)25°C = 2.16 × 10(4) L/mol, and K(θ)37°C = 1.06 × 10(4) L/mol. The thermodynamic functions of the interaction between γ-CD-K3 and DNA were ΔrHm(θ) = -2.74 × 10(4) J/mol, ΔrSm(θ) = 174.74 J·mol(-1)K(-1), therefore, both ΔrHm(θ) (enthalpy) and ΔrSm(θ) (entropy) worked as driven forces in this action.

  14. Studying DNA looping by single-molecule FRET.

    Science.gov (United States)

    Le, Tung T; Kim, Harold D

    2014-06-28

    Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

  15. Studies on DNA repair in Bacillus subtilis

    International Nuclear Information System (INIS)

    Inoue, Tadashi; Kada, Tsuneo

    1977-01-01

    An enzyme which enhances the priming activity of γ-irradiated DNA for type I DNA polymerase (EC 2.7.7.7) was identified and partially purified from extracts of Bacillus subtilis cells. The enzyme preferentially degraded γ-irradiated DNA into acid-soluble materials. DNA preparations treated with heat, ultraviolet light, pancreatic DNAase (EC 3.1.4.5) or micrococcal DNAase (EC 3.1.4.7) were not susceptible to the enzyme. However, sonication rendered DNA susceptible to the enzyme to some extent. From these results, it is supposed that this enzyme may function by 'cleaning' damaged terminals produced by γ-irradiation to serve as effective primer of sites for repair synthesis by the type I DNA polymerase

  16. Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA

    NARCIS (Netherlands)

    Ellens, D.J.; Sussenbach, J.S.; Jansz, H.S.

    1974-01-01

    Replicating Ad5 DNA was isolated from nuclei of infected KB cells and studied by electron microscopy. Branched as well as unbranched linear intermediates were observed containing extended regions of single-stranded DNA. The relationship between the branched and unbranched structures was studied

  17. Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

    Science.gov (United States)

    Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

    2004-01-01

    In Escherichia coli, interactions between the replication initiation protein DnaA, the β subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and β proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with β in vitro. A new β-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified β-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind β. A 10-amino-acid peptide containing the E. coli Hda β-binding motif was shown to compete with Hda for binding to β in an Hda-β interaction assay. These results establish that the interaction of Hda with β is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication. PMID:15150238

  18. Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein.

    Science.gov (United States)

    Kurz, Mareike; Dalrymple, Brian; Wijffels, Gene; Kongsuwan, Kritaya

    2004-06-01

    In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP. This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication. However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved. Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro. A new beta-binding motif, a hexapeptide with the consensus sequence QL[SP]LPL, related to the previously identified beta-binding pentapeptide motif (QL[SD]LF) was found in the amino terminus of the Hda protein. Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta. A 10-amino-acid peptide containing the E. coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay. These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence. We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication.

  19. Exploring the Interactions of the Dietary Plant Flavonoids Fisetin and Naringenin with G-Quadruplex and Duplex DNA, Showing Contrasting Binding Behavior: Spectroscopic and Molecular Modeling Approaches.

    Science.gov (United States)

    Bhattacharjee, Snehasish; Chakraborty, Sandipan; Sengupta, Pradeep K; Bhowmik, Sudipta

    2016-09-01

    Guanine-rich sequences have the propensity to fold into a four-stranded DNA structure known as a G-quadruplex (G4). G4 forming sequences are abundant in the promoter region of several oncogenes and become a key target for anticancer drug binding. Here we have studied the interactions of two structurally similar dietary plant flavonoids fisetin and naringenin with G4 as well as double stranded (duplex) DNA by using different spectroscopic and modeling techniques. Our study demonstrates the differential binding ability of the two flavonoids with G4 and duplex DNA. Fisetin more strongly interacts with parallel G4 structure than duplex DNA, whereas naringenin shows stronger binding affinity to duplex rather than G4 DNA. Molecular docking results also corroborate our spectroscopic results, and it was found that both of the ligands are stacked externally in the G4 DNA structure. C-ring planarity of the flavonoid structure appears to be a crucial factor for preferential G4 DNA recognition of flavonoids. The goal of this study is to explore the critical effects of small differences in the structure of closely similar chemical classes of such small molecules (flavonoids) which lead to the contrasting binding properties with the two different forms of DNA. The resulting insights may be expected to facilitate the designing of the highly selective G4 DNA binders based on flavonoid scaffolds.

  20. Low-energy-electron interactions with DNA: approaching cellular conditions with atmospheric experiments

    International Nuclear Information System (INIS)

    Alizadeh, E.; Sanche, L.

    2014-01-01

    A novel technique has been developed to investigate low energy electron (LEE)-DNA interactions in the presence of small biomolecules (e.g., N 2 , O 2 , H 2 O) found near DNA in the cell nucleus, in order to simulate cellular conditions. In this technique, LEEs are emitted from a metallic surface exposed by soft X-rays and interact with DNA thin films at standard ambient temperature and pressure (SATP). Whereas atmospheric N 2 had little effect on the yields of LEE-induced single and double strand breaks, both O 2 and H 2 O considerably modified and increased such damage. The highest yields were obtained when DNA is embedded in a combined O 2 and H 2 O atmosphere. In this case, the amount of additional double strand breaks was supper-additive. The effect of modifying the chemical and physical stability of DNA by platinum-based chemotherapeutic agents (Pt-drugs) including cisplatin, carboplatin and oxaliplatin was also investigated with this technique. The results obtained provide information on the role played by subexcitation-energy electrons and dissociative electron attachment in the radiosensitization of DNA by Pt-drugs, which is an important step to unravel the mechanisms of radiosensitization of these agents in chemo-radiation cancer therapy. (authors)

  1. Low-energy-electron interactions with DNA: approaching cellular conditions with atmospheric experiments

    Science.gov (United States)

    Alizadeh, Elahe; Sanche, Léon

    2014-04-01

    A novel technique has been developed to investigate low energy electron (LEE)-DNA interactions in the presence of small biomolecules (e.g., N2, O2, H2O) found near DNA in the cell nucleus, in order to simulate cellular conditions. In this technique, LEEs are emitted from a metallic surface exposed by soft X-rays and interact with DNA thin films at standard ambient temperature and pressure (SATP). Whereas atmospheric N2 had little effect on the yields of LEE-induced single and double strand breaks, both O2 and H2O considerably modified and increased such damage. The highest yields were obtained when DNA is embedded in a combined O2 and H2O atmosphere. In this case, the amount of additional double strand breaks was supper-additive. The effect of modifying the chemical and physical stability of DNA by platinum-based chemotherapeutic agents (Pt-drugs) including cisplatin, carboplatin and oxaliplatin was also investigated with this technique. The results obtained provide information on the role played by subexcitation-energy electrons and dissociative electron attachment in the radiosensitization of DNA by Pt-drugs, which is an important step to unravel the mechanisms of radiosensitisation of these agents in chemoradiation cancer therapy.

  2. DNA base dimers are stabilized by hydrogen-bonding interactions including non-Watson-Crick pairing near graphite surfaces.

    Science.gov (United States)

    Shankar, Akshaya; Jagota, Anand; Mittal, Jeetain

    2012-10-11

    Single- and double-stranded DNA are increasingly being paired with surfaces and nanoparticles for numerous applications, such as sensing, imaging, and drug delivery. Unlike the majority of DNA structures in bulk that are stabilized by canonical Watson-Crick pairing between Ade-Thy and Gua-Cyt, those adsorbed on surfaces are often stabilized by noncanonical base pairing, quartet formation, and base-surface stacking. Not much is known about these kinds of interactions. To build an understanding of the role of non-Watson-Crick pairing on DNA behavior near surfaces, one requires basic information on DNA base pair stacking and hydrogen-bonding interactions. All-atom molecular simulations of DNA bases in two cases--in bulk water and strongly adsorbed on a graphite surface--are conducted to study the relative strengths of stacking and hydrogen bond interactions for each of the 10 possible combinations of base pairs. The key information obtained from these simulations is the free energy as a function of distance between two bases in a pair. We find that stacking interactions exert the dominant influence on the stability of DNA base pairs in bulk water as expected. The strength of stability for these stacking interactions is found to decrease in the order Gua-Gua > Ade-Gua > Ade-Ade > Gua-Thy > Gua-Cyt > Ade-Thy > Ade-Cyt > Thy-Thy > Cyt-Thy > Cyt-Cyt. On the other hand, mutual interactions of surface-adsorbed base pairs are stabilized mostly by hydrogen-bonding interactions in the order Gua-Cyt > Ade-Gua > Ade-Thy > Ade-Ade > Cyt-Thy > Gua-Gua > Cyt-Cyt > Ade-Cyt > Thy-Thy > Gua-Thy. Interestingly, several non-Watson-Crick base pairings, which are commonly ignored, have similar stabilization free energies due to interbase hydrogen bonding as Watson-Crick pairs. This clearly highlights the importance of non-Watson-Crick base pairing in the development of secondary structures of oligonucleotides near surfaces.

  3. Spectrophotometric analysis of flavonoid-DNA binding interactions at physiological conditions

    Science.gov (United States)

    Janjua, Naveed Kausar; Siddiqa, Asima; Yaqub, Azra; Sabahat, Sana; Qureshi, Rumana; Haque, Sayed ul

    2009-12-01

    Mode of interactions of three flavonoids [morin (M), quercetin (Q), and rutin (R)] with chicken blood ds.DNA (ck.DNA) has been investigated spectrophotometrically at different temperatures including body temperature (310 K) and at two physiological pH values, i.e. 7.4 (human blood pH) and 4.7 (stomach pH). The binding constants, Kf, evaluated using Benesi-Hildebrand equation showed that the flavonoids bind effectively through intercalation at both pH values and body temperature. Quercetin, somehow, showed greater binding capabilities with DNA. The free energies of flavonoid-DNA complexes indicated the spontaneity of their binding. The order of binding constants of three flavonoids at both pH values were found to be Kf(Q) > Kf(R) > Kf(M) and at 310 K.

  4. Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions.

    Science.gov (United States)

    Silvas, Tania V; Hou, Shurong; Myint, Wazo; Nalivaika, Ellen; Somasundaran, Mohan; Kelch, Brian A; Matsuo, Hiroshi; Kurt Yilmaz, Nese; Schiffer, Celia A

    2018-05-14

    The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.

  5. Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex.

    Science.gov (United States)

    Witosch, Justine; Wolf, Eva; Mizuno, Naoko

    2014-11-10

    The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. GEMC1 is a TopBP1-interacting protein required for chromosomal DNA replication.

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-05-01

    Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

  7. GEMC1 is a TopBP1 interacting protein required for chromosomal DNA replication

    Science.gov (United States)

    Balestrini, Alessia; Cosentino, Claudia; Errico, Alessia; Garner, Elizabeth; Costanzo, Vincenzo

    2010-01-01

    Many factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication in complex multicellular organisms is poorly understood. Here, we report the identification of GEMC1, a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus egg extract we show that Xenopus GEMC1 (xGEMC1) binds to checkpoint and replication factor TopBP1, which promotes xGEMC1 binding to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 directly interacts with replication factors such as Cdc45 and Cdk2-CyclinE by which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication whereas depletion of xGEMC1 prevents DNA replication onset due to impairment of Cdc45 loading onto chromatin. Likewise, inhibition of GEMC1 expression by morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in higher eukaryotes by mediating TopBP1 and Cdk2 dependent recruitment of Cdc45 onto replication origins. PMID:20383140

  8. DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination.

    Science.gov (United States)

    Qin, Weihua; Wolf, Patricia; Liu, Nan; Link, Stephanie; Smets, Martha; La Mastra, Federica; Forné, Ignasi; Pichler, Garwin; Hörl, David; Fellinger, Karin; Spada, Fabio; Bonapace, Ian Marc; Imhof, Axel; Harz, Hartmann; Leonhardt, Heinrich

    2015-08-01

    DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.

  9. Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA

    Science.gov (United States)

    Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

    2016-01-01

    A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging.

  10. Isolate extended state in the DNA molecular transistor with surface interaction

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Le, E-mail: wang_le917@gs.zzu.edu.cn; Qin, Zhi-Jie

    2016-02-01

    The field effect characteristic of a DNA molecular device is investigated in a tight binding model with binary disorder and side site correlation. Using the transfer-matrix method and Landauer–Büttiker theory, we find that the system has isolated extended state that is irrespective of the DNA sequence and can be modulated by the gate voltage. When the gate voltage reaches some proper value, the isolated extended state appears at the Fermi level of the system and the long range charge transport is greatly enhanced. We attribute this phenomenon to the combination of the external field, the surface interaction, and the intrinsic disorder of DNA. The result is a generic feature of the nanowire with binary disorder and surface interaction.

  11. Architecture of the bacteriophage T4 activator MotA/promoter DNA interaction during sigma appropriation.

    Science.gov (United States)

    Hsieh, Meng-Lun; James, Tamara D; Knipling, Leslie; Waddell, M Brett; White, Stephen; Hinton, Deborah M

    2013-09-20

    Gene expression can be regulated through factors that direct RNA polymerase to the correct promoter sequence at the correct time. Bacteriophage T4 controls its development in this way using phage proteins that interact with host RNA polymerase. Using a process called σ appropriation, the T4 co-activator AsiA structurally remodels the σ(70) subunit of host RNA polymerase, while a T4 activator, MotA, engages the C terminus of σ(70) and binds to a DNA promoter element, the MotA box. Structures for the N-terminal (NTD) and C-terminal (CTD) domains of MotA are available, but no structure exists for MotA with or without DNA. We report the first molecular map of the MotA/DNA interaction within the σ-appropriated complex, which we obtained by using the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). We conjugated surface-exposed, single cysteines in MotA with FeBABE and performed cleavage reactions in the context of stable transcription complexes. The DNA cleavage sites were analyzed using ICM Molsoft software and three-dimensional physical models of MotA(NTD), MotA(CTD), and the DNA to investigate shape complementarity between the protein and the DNA and to position MotA on the DNA. We found that the unusual "double wing" motif present within MotA(CTD) resides in the major groove of the MotA box. In addition, we have used surface plasmon resonance to show that MotA alone is in a very dynamic equilibrium with the MotA element. Our results demonstrate the utility of fine resolution FeBABE mapping to determine the architecture of protein-DNA complexes that have been recalcitrant to traditional structure analyses.

  12. The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

    Science.gov (United States)

    Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

    2009-01-01

    We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

  13. Influence of anticancer drugs on interactions of tumor suppressor protein p53 with DNA

    Czech Academy of Sciences Publication Activity Database

    Pivoňková, Hana; Němcová, Kateřina; Brázdová, Marie; Kašpárková, Jana; Brabec, Viktor; Fojta, Miroslav

    2005-01-01

    Roč. 272, Suppl. 1 (2005), s. 562 ISSN 1474-3833. [FEBS Congress /30./ and IUBMB Conference /9./. 02.07.2005-07.07.2005, Budapest] R&D Projects: GA MZd(CZ) NC7574 Institutional research plan: CEZ:AV0Z50040507 Keywords : tumour suppressor protein p53 * anticancer drugs * interaction with DNA Subject RIV: BO - Biophysics

  14. A discriminatory function for prediction of protein-DNA interactions based on alpha shape modeling.

    Science.gov (United States)

    Zhou, Weiqiang; Yan, Hong

    2010-10-15

    Protein-DNA interaction has significant importance in many biological processes. However, the underlying principle of the molecular recognition process is still largely unknown. As more high-resolution 3D structures of protein-DNA complex are becoming available, the surface characteristics of the complex become an important research topic. In our work, we apply an alpha shape model to represent the surface structure of the protein-DNA complex and developed an interface-atom curvature-dependent conditional probability discriminatory function for the prediction of protein-DNA interaction. The interface-atom curvature-dependent formalism captures atomic interaction details better than the atomic distance-based method. The proposed method provides good performance in discriminating the native structures from the docking decoy sets, and outperforms the distance-dependent formalism in terms of the z-score. Computer experiment results show that the curvature-dependent formalism with the optimal parameters can achieve a native z-score of -8.17 in discriminating the native structure from the highest surface-complementarity scored decoy set and a native z-score of -7.38 in discriminating the native structure from the lowest RMSD decoy set. The interface-atom curvature-dependent formalism can also be used to predict apo version of DNA-binding proteins. These results suggest that the interface-atom curvature-dependent formalism has a good prediction capability for protein-DNA interactions. The code and data sets are available for download on http://www.hy8.com/bioinformatics.htm kenandzhou@hotmail.com.

  15. Interaction of spermine with DNA, vitamin C and bovine serum albumin in the unirradiated and gamma irradiated states

    International Nuclear Information System (INIS)

    Upadhyay, S.N.; Lal, C.; Bhardwaj, R.; Chaturvedi, S.; Chaudhury, N.K.

    2006-01-01

    Structural deformability of spermine with radiation dose (maximum 10 Gy) has been proved. Complex formation of spermine with DNA, vitamin C and BSA took place. Calibration and radiation-induced absorption changes in spermine by ninhydrin reagent has been followed quantitatively. Interaction of vitamin C with DNA and their radiation-induced changes have been reported. Interaction of spermine with DNA in the unirradiated and gamma irradiated states in 10 -3 M phosphate buffer and water have been compared. Addition of spermine and vitamin C to DNA makes DNA structure more condensed. Bovine serum albumin also binds with spermine and protects it from radiation-induced degradation. (author)

  16. Structure of DNA toroids and electrostatic attraction of DNA duplexes

    International Nuclear Information System (INIS)

    Cherstvy, A G

    2005-01-01

    DNA-DNA electrostatic attraction is considered as the driving force for the formation of DNA toroids in the presence of DNA condensing cations. This attraction comes from the DNA helical charge distribution and favours hexagonal toroidal cross-sections. The latter is in agreement with recent cryo-electron microscopy studies on DNA condensed with cobalt hexammine. We treat the DNA-DNA interactions within the modern theory of electrostatic interaction between helical macromolecules. The size and thickness of the toroids is calculated within a simple model; other models of stability of DNA toroids are discussed and compared

  17. Non-covalent interactions of cadmium sulphide and gold nanoparticles with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Atay, Z. [Bogazici University, Department of Chemistry (Turkey); Biver, T., E-mail: tarita@dcci.unipi.i [Universita di Pisa, Dipartimento di Chimica e Chimica Industriale (Italy); Corti, A. [Universita di Pisa, Dipartimento di Patologia Sperimentale BMIE (Italy); Eltugral, N. [Universita di Pisa, Dipartimento di Chimica e Chimica Industriale (Italy); Lorenzini, E.; Masini, M.; Paolicchi, A. [Universita di Pisa, Dipartimento di Patologia Sperimentale BMIE (Italy); Pucci, A.; Ruggeri, G.; Secco, F.; Venturini, M. [Universita di Pisa, Dipartimento di Chimica e Chimica Industriale (Italy)

    2010-08-15

    Mercaptoethanol-capped CdS nanoparticles (CdS{sub np}) and monohydroxy-(1-mercaptoundec-11-yl)tetraethylene-glycol-capped Au nanoparticles (Au{sub np}) were synthesised, characterised and their interactions with DNA were investigated. Au{sub np} are stable in different aqueous solvents, whereas CdS{sub np} do precipitate in 0.1 M NaCl and form two different cluster types in 0.1 M NaNO{sub 3}. As regards the CdS{sub np}/DNA interaction, absorbance and fluorescence titrations, ethidium bromide displacement assays and gel electrophoresis experiments indicate that a non-covalent interaction between DNA and the CdS{sub np} external surface does take place. The binding constant was evaluated to be equal to (2.2 {+-} 0.5) x 10{sup 5} M{sup -1}. On the contrary, concerning Au{sub np}, no direct interaction with DNA could be observed. Possible interaction with serum albumin was also checked, but no effects could be observed for either CdS{sub np} or Au{sub np}. Finally, short-time exposure of cultured cells to nanoparticles revealed the ability of CdS{sub np} to enter the cells and allocate both in cytosol and nucleus, thus promoting cell proliferation at low concentration (p < 0.005), while longer-time exposure resulted in a significant inhibition of cell growth, accompanied by apoptotic cell death. Au{sub np} neither enter the cells, nor do affect cell proliferation. In conclusion, our data indicate that CdS{sub np} can strongly interact with living cells and nucleic acid while no effects or interactions were observed for Au{sub np}.

  18. NMR studies concerning base-base interactions in oligonucleotides

    International Nuclear Information System (INIS)

    Hoogen, Y.T. van den.

    1988-01-01

    Two main subjects are treated in the present thesis. The firsst part principally deals with the base-base interactions in single-stranded oligoribonucleotides. The second part presents NMR and model-building studies of DNA and RNA duplexes containing an unpaired base. (author). 242 refs.; 26 figs.; 24 tabs

  19. [Verification of a decrease in the rigidity of the phage lambda DNA polymeric chain in low ionic strength aqueous solutions by testing the polymer-polymer interlink interactions].

    Science.gov (United States)

    Arutiunian, A V; Ivanova, M A; Kurliand, D I; Kapshin, Iu S; Landa, S B; Poshekhonov, S T; Drobchenko, E A; Shevelev, I V

    2011-01-01

    Changes in the rigidity of the polymetric chain of phage lambda double-strand DNA have been studied by laser correlation spectroscopy. It was shown that, as the ionic strength increases, the effect of the screening of the hydrodynamic interaction of the links of the polymeric chain specific for polymeric coils arises in a DNA solution. It is assumed that the screening occurs when the threshold of the overlapping of DNA coils is achieved. The overlapping of coils is the result of a previously observed significant rise of DNA coil size from abnormally small DNA coils in low ionic strength buffers (about 10(-2) M Na+ or less) to maximum possible large coils in the 5SSC and 5SSC-like buffers. Further analysis of the far interlink interactions in linear lambda phage DNA coils in similar buffers at pH 7 and 4 confirms the earlier proposal about the role of H+ ions in the appearance of abnormally small DNA coils. The abnormal decrease in the DNA coil size in low ionic strength buffers is not a specific feature of lambda phage DNA only.

  20. Interactions of DNA with graphene and sensing applications of graphene field-effect transistor devices: A review

    Energy Technology Data Exchange (ETDEWEB)

    Green, Nathaniel S.; Norton, Michael L., E-mail: norton@marshall.edu

    2015-01-01

    Highlights: • The interaction of DNA, including DNA nanostructures, and graphene is reviewed. • Comparison of DNA graphene field-effect transistor (GFET) with other detection methods. • Discussion of challenges present in the detection mechanism of GFETs. • Use of DNA aptamer GFET sensors for the detection of small molecules and proteins. - Abstract: Graphene field-effect transistors (GFET) have emerged as powerful detection platforms enabled by the advent of chemical vapor deposition (CVD) production of the unique atomically thin 2D material on a large scale. DNA aptamers, short target-specific oligonucleotides, are excellent sensor moieties for GFETs due to their strong affinity to graphene, relatively short chain-length, selectivity, and a high degree of analyte variability. However, the interaction between DNA and graphene is not fully understood, leading to questions about the structure of surface-bound DNA, including the morphology of DNA nanostructures and the nature of the electronic response seen from analyte binding. This review critically evaluates recent insights into the nature of the DNA graphene interaction and its affect on sensor viability for DNA, small molecules, and proteins with respect to previously established sensing methods. We first discuss the sorption of DNA to graphene to introduce the interactions and forces acting in DNA based GFET devices and how these forces can potentially affect the performance of increasingly popular DNA aptamers and even future DNA nanostructures as sensor substrates. Next, we discuss the novel use of GFETs to detect DNA and the underlying electronic phenomena that are typically used as benchmarks for characterizing the analyte response of these devices. Finally, we address the use of DNA aptamers to increase the selectivity of GFET sensors for small molecules and proteins and compare them with other, state of the art, detection methods.

  1. Modulation of the genotoxicity of bleomycin by amines through noncovalent DNA interactions and alteration of physiological conditions in yeast

    International Nuclear Information System (INIS)

    Hoffmann, George R.; Gessner, Gabrielle S.; Hughes, Jennifer F.; Ronan, Matthew V.; Sylvia, Katelyn E.; Willett, Christine J.

    2007-01-01

    The effects of amines on the induction of mitotic gene conversion by bleomycin (BLM) were studied at the trp5 locus in Saccharomyces cerevisiae strain D7. BLM induces double-strand breaks in DNA and is a potent recombinagen in this assay. The polyamine spermidine causes concentration-dependent protection against the genotoxicity of BLM, reducing the convertant frequency by over 90% under the most protective conditions. Spermine, diethylenetriamine, ethylenediamine, putrescine, and ethylamine were also antigenotoxic in combined treatments with BLM. There was a general correspondence between the protective effect and the number of amino groups, suggesting that more strongly cationic amines tend to be stronger antirecombinagens. Electrostatic association of the amines with DNA probably hinders BLM access to the 4' position of deoxyribose where it generates a free radical. Other amines interact with BLM differently from these unbranched aliphatic amines. The aminothiol cysteamine inhibits the genotoxicity of BLM under hypoxic conditions but increases it under euoxic conditions. In contrast, pargyline potentiates the genotoxicity of BLM under hypoxic conditions but not under euoxic conditions. The antirecombinagenic effect of cysteamine apparently involves DNA binding and depletion of oxygen needed for BLM activity, whereas its potentiation of BLM entails its serving as an electron source for the activation of BLM. Pargyline may enhance BLM indirectly by preventing the depletion of oxygen by monoamine and polyamine oxidase. The planar 9-aminoacridine weakly induces gene conversion in strain D7, but it is strongly synergistic with BLM. Enhancement of BLM activity by this compound and by the related nitroacridine Entozon is apparently mediated by intercalation of the acridine ring system into DNA. Thus, the influence of amines on the genotoxicity of BLM in yeast encompasses antigenotoxic, potentiating, and synergistic interactions. The underlying mechanisms involve

  2. Modulation of the genotoxicity of bleomycin by amines through noncovalent DNA interactions and alteration of physiological conditions in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, George R. [Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610-2395 (United States)], E-mail: ghoffmann@holycross.edu; Gessner, Gabrielle S.; Hughes, Jennifer F.; Ronan, Matthew V.; Sylvia, Katelyn E.; Willett, Christine J. [Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610-2395 (United States)

    2007-10-01

    The effects of amines on the induction of mitotic gene conversion by bleomycin (BLM) were studied at the trp5 locus in Saccharomyces cerevisiae strain D7. BLM induces double-strand breaks in DNA and is a potent recombinagen in this assay. The polyamine spermidine causes concentration-dependent protection against the genotoxicity of BLM, reducing the convertant frequency by over 90% under the most protective conditions. Spermine, diethylenetriamine, ethylenediamine, putrescine, and ethylamine were also antigenotoxic in combined treatments with BLM. There was a general correspondence between the protective effect and the number of amino groups, suggesting that more strongly cationic amines tend to be stronger antirecombinagens. Electrostatic association of the amines with DNA probably hinders BLM access to the 4' position of deoxyribose where it generates a free radical. Other amines interact with BLM differently from these unbranched aliphatic amines. The aminothiol cysteamine inhibits the genotoxicity of BLM under hypoxic conditions but increases it under euoxic conditions. In contrast, pargyline potentiates the genotoxicity of BLM under hypoxic conditions but not under euoxic conditions. The antirecombinagenic effect of cysteamine apparently involves DNA binding and depletion of oxygen needed for BLM activity, whereas its potentiation of BLM entails its serving as an electron source for the activation of BLM. Pargyline may enhance BLM indirectly by preventing the depletion of oxygen by monoamine and polyamine oxidase. The planar 9-aminoacridine weakly induces gene conversion in strain D7, but it is strongly synergistic with BLM. Enhancement of BLM activity by this compound and by the related nitroacridine Entozon is apparently mediated by intercalation of the acridine ring system into DNA. Thus, the influence of amines on the genotoxicity of BLM in yeast encompasses antigenotoxic, potentiating, and synergistic interactions. The underlying mechanisms involve

  3. Raman spectroscopic study of plasma-treated salmon DNA

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Geon Joon; Kim, Yong Hee; Choi, Eun Ha [Plasma Bioscience Research Center, Kwangwoon University, Seoul 139-701 (Korea, Republic of); Kwon, Young-Wan [Department of Chemistry, Korea University, Seoul 136-701 (Korea, Republic of)

    2013-01-14

    In this research, we studied the effect of plasma treatment on the optical/structural properties of the deoxyribonucleic acid (DNA) extracted from salmon sperm. DNA-cetyltrimethylammonium (CTMA) films were obtained by complexation of DNA with CTMA. Circular dichroism (CD) and Raman spectra indicated that DNA retained its double helical structure in the solid film. The Raman spectra exhibited several vibration modes corresponding to the nuclear bases and the deoxyribose-phosphate backbones of the DNA, as well as the alkylchains of CTMA. Dielectric-barrier-discharge (DBD) plasma treatment induced structural modification and damage to the DNA, as observed by changes in the ultraviolet-visible absorption, CD, and Raman spectra. The optical emission spectra of the DBD plasma confirmed that DNA modification was induced by plasma ions such as reactive oxygen species and reactive nitrogen species.

  4. Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo.

    Science.gov (United States)

    Komar, Dorota N; Mouriz, Alfonso; Jarillo, José A; Piñeiro, Manuel

    2016-01-14

    Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

  5. A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response

    DEFF Research Database (Denmark)

    Blasius, Melanie; Wagner, Sebastian A; Choudhary, Chuna Ram

    2014-01-01

    RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome...

  6. A Hard Constraint Algorithm to Model Particle Interactions in DNA-laden Flows

    Energy Technology Data Exchange (ETDEWEB)

    Trebotich, D; Miller, G H; Bybee, M D

    2006-08-01

    We present a new method for particle interactions in polymer models of DNA. The DNA is represented by a bead-rod polymer model and is fully-coupled to the fluid. The main objective in this work is to implement short-range forces to properly model polymer-polymer and polymer-surface interactions, specifically, rod-rod and rod-surface uncrossing. Our new method is based on a rigid constraint algorithm whereby rods elastically bounce off one another to prevent crossing, similar to our previous algorithm used to model polymer-surface interactions. We compare this model to a classical (smooth) potential which acts as a repulsive force between rods, and rods and surfaces.

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

    Science.gov (United States)

    Kunkel, Thomas A; Burgers, Peter M J

    2017-08-01

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

  8. Copper(II Complexes Based on Aminohydroxamic Acids: Synthesis, Structures, In Vitro Cytotoxicities and DNA/BSA Interactions

    Directory of Open Access Journals (Sweden)

    Jia Zhang

    2018-05-01

    Full Text Available Four complexes, [Cu2(glyha(bpy2(H2O]·2ClO4·H2O (1, [Cu2(glyha(phen2]·2ClO4 (2, [Cu2(alaha(bpy2Cl]·Cl·4H2O (3, and [{Cu2(alaha(phen2}{Cu2(alaha(phen2(NO3}]·3NO3 (4 (glyha2− = dianion glycinehydroxamic acid, alaha2− = dianion alaninehydroxamic acid, bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline have been successfully synthesized and characterized by X-ray single crystal diffraction. The interactions of these complexes with calf thymus DNA (CT-DNA were studied through UV spectroscopy, fluorescence spectroscopy, and circular dichroism. The results revealed that complexes 1–4 could interact with CT-DNA through intercalation. Interactions of all complexes with bovine serum albumin (BSA were confirmed by the docking study to quench the intrinsic fluorescence of BSA in a static quenching process. Furthermore, the in vitro cytotoxic effect of the complexes was also examined on four tumor cell lines, including human lung carcinoma cell line (A549, human colon carcinoma cell line (HCT-116, human promyelocytic leukemia cell (HL-60 and cervical cancer cell line (HeLa. All complexes exhibited different antitumor activities.

  9. Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    Science.gov (United States)

    Wang, Chao; Lv, Yangyong; Wang, Bin; Yin, Chao; Lin, Ying; Pan, Li

    2015-01-01

    The genome-scale delineation of in vivo protein–DNA interactions is key to understanding genome function. Only ∼5% of transcription factors (TFs) in the Aspergillus genus have been identified using traditional methods. Although the Aspergillus oryzae genome contains >600 TFs, knowledge of the in vivo genome-wide TF-binding sites (TFBSs) in aspergilli remains limited because of the lack of high-quality antibodies. We investigated the landscape of in vivo protein–DNA interactions across the A. oryzae genome through coupling the DNase I digestion of intact nuclei with massively parallel sequencing and the analysis of cleavage patterns in protein–DNA interactions at single-nucleotide resolution. The resulting map identified overrepresented de novo TF-binding motifs from genomic footprints, and provided the detailed chromatin remodeling patterns and the distribution of digital footprints near transcription start sites. The TFBSs of 19 known Aspergillus TFs were also identified based on DNase I digestion data surrounding potential binding sites in conjunction with TF binding specificity information. We observed that the cleavage patterns of TFBSs were dependent on the orientation of TF motifs and independent of strand orientation, consistent with the DNA shape features of binding motifs with flanking sequences. PMID:25883143

  10. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    Science.gov (United States)

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

  11. Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

    Science.gov (United States)

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

    2014-11-27

    In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

  12. RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair.

    Science.gov (United States)

    Krasikova, Yuliya S; Rechkunova, Nadejda I; Maltseva, Ekaterina A; Lavrik, Olga I

    2018-01-01

    Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

  13. RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair.

    Directory of Open Access Journals (Sweden)

    Yuliya S Krasikova

    Full Text Available Replication protein A (RPA and the xeroderma pigmentosum group A (XPA protein are indispensable for both pathways of nucleotide excision repair (NER. Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

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

    Science.gov (United States)

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

    2014-05-28

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

  15. Single Molecule Study of DNA Organization and Recombination

    Science.gov (United States)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  16. Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions

    Science.gov (United States)

    Sprengel, Andreas; Lill, Pascal; Stegemann, Pierre; Bravo-Rodriguez, Kenny; Schöneweiß, Elisa-C.; Merdanovic, Melisa; Gudnason, Daniel; Aznauryan, Mikayel; Gamrad, Lisa; Barcikowski, Stephan; Sanchez-Garcia, Elsa; Birkedal, Victoria; Gatsogiannis, Christos; Ehrmann, Michael; Saccà, Barbara

    2017-02-01

    The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.

  17. Mechanisms of radiation interaction with DNA: Potential implications for radiation protection

    International Nuclear Information System (INIS)

    Sinclair, W.K.; Fry, R.J.M.

    1987-01-01

    An overview of presentations and discussions which took place at the US Department of Energy/Commission of European Communities (DOE/CEC) workshop on ''Mechanisms of Radiation Interaction with DNA: Potential Implications for Radiation Protection,'' held at San Diego, California, January 21-22, 1987, is provided. The Department has traditionally supported fundamental research on interactions of ionizing radiation with different biological systems and at all levels of biological organization. The aim of this workshop was to review the base of knowledge in the area of mechanisms of radiation action at the DNA level, and to explore ways in which this information can be applied to the development of scientifically sound concepts and procedures for use in the field of radiation protection

  18. The interaction of DNA gyrase with the bacterial toxin CcdB

    DEFF Research Database (Denmark)

    Kampranis, S C; Howells, A J; Maxwell, A

    1999-01-01

    CcdB is a bacterial toxin that targets DNA gyrase. Analysis of the interaction of CcdB with gyrase reveals two distinct complexes. An initial complex (alpha) is formed by direct interaction between GyrA and CcdB; this complex can be detected by affinity column and gel-shift analysis, and has...... of this initial complex with ATP in the presence of GyrB and DNA slowly converts it to a second complex (beta), which has a lower rate of ATP hydrolysis and is unable to catalyse supercoiling. The efficiency of formation of this inactive complex is dependent on the concentrations of ATP and CcdB. We suggest...

  19. NMR studies of DNA structures in solution

    International Nuclear Information System (INIS)

    Rinkel, L.J.

    1987-01-01

    This thesis describes the conformational analysis of some polynucleotides in aqueous solution. A new graphical method is presented as an aid in the pseudorotational analysis of the sugar rings in DNA by means of sums of proton-proton coupling constants. (Auth.)

  20. A positron annihilation study of hydrated DNA

    DEFF Research Database (Denmark)

    Warman, J. M.; Eldrup, Morten Mostgaard

    1986-01-01

    Positron annihilation measurements are reported for hydrated DNA as a function of water content and as a function of temperature (20 to -180.degree. C) for samples containing 10 and 50% wt of water. The ortho-positronium mean lifetime and its intensity show distinct variations with the degree...

  1. Antimalarial, antimicrobial, cytotoxic, DNA interaction and SOD like activities of tetrahedral copper(II) complexes

    Science.gov (United States)

    Mehta, Jugal V.; Gajera, Sanjay B.; Patel, Mohan N.

    2015-02-01

    The mononuclear copper(II) complexes with P, O-donor ligand and different fluoroquinolones have been synthesized and characterized by elemental analysis, electronic spectra, TGA, EPR, FT-IR and LC-MS spectroscopy. An antimicrobial efficiency of the complexes has been tested against five different microorganisms in terms of minimum inhibitory concentration (MIC) and displays very good antimicrobial activity. The binding strength and binding mode of the complexes with Herring Sperm DNA (HS DNA) have been investigated by absorption titration and viscosity measurement studies. The studies suggest the classical intercalative mode of DNA binding. Gel electrophoresis assay determines the ability of the complexes to cleave the supercoiled form of pUC19 DNA. Synthesized complexes have been tested for their SOD mimic activity using nonenzymatic NBT/NADH/PMS system and found to have good antioxidant activity. All the complexes show good cytotoxic and in vitro antimalarial activities.

  2. Interactive measurement and characterization of DNA molecules by analysis of AFM images

    Czech Academy of Sciences Publication Activity Database

    Marek, J.; Demjénová, E.; Tomori, Z.; Janáček, Jiří; Zolotová, I.; Valle, F.; Favre, M.; Dietler, G.

    2005-01-01

    Roč. 63, č. 2 (2005), s. 87-93 ISSN 1552-4922 Grant - others:VEGA(SK) 5048; VEGA(SK) 2185; CZ-SK(CZ) KONTAKT 139; Swiss National Science Foundation(CH) 2100-063746.00/1 Institutional research plan: CEZ:AV0Z5011922 Keywords : DNA * atomic force microscopy * interactive image analysis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.115, year: 2005

  3. 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

  4. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

    Science.gov (United States)

    Friso, Simonetta; Choi, Sang-Woon; Girelli, Domenico; Mason, Joel B.; Dolnikowski, Gregory G.; Bagley, Pamela J.; Olivieri, Oliviero; Jacques, Paul F.; Rosenberg, Irwin H.; Corrocher, Roberto; Selhub, Jacob

    2002-01-01

    DNA methylation, an essential epigenetic feature of DNA that modulates gene expression and genomic integrity, is catalyzed by methyltransferases that use the universal methyl donor S-adenosyl-l-methionine. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of 5-methyltetrahydrofolate (5-methylTHF), the methyl donor for synthesis of methionine from homocysteine and precursor of S-adenosyl-l-methionine. In the present study we sought to determine the effect of folate status on genomic DNA methylation with an emphasis on the interaction with the common C677T mutation in the MTHFR gene. A liquid chromatography/MS method for the analysis of nucleotide bases was used to assess genomic DNA methylation in peripheral blood mononuclear cell DNA from 105 subjects homozygous for this mutation (T/T) and 187 homozygous for the wild-type (C/C) MTHFR genotype. The results show that genomic DNA methylation directly correlates with folate status and inversely with plasma homocysteine (tHcy) levels (P < 0.01). T/T genotypes had a diminished level of DNA methylation compared with those with the C/C wild-type (32.23 vs.62.24 ng 5-methylcytosine/μg DNA, P < 0.0001). When analyzed according to folate status, however, only the T/T subjects with low levels of folate accounted for the diminished DNA methylation (P < 0.0001). Moreover, in T/T subjects DNA methylation status correlated with the methylated proportion of red blood cell folate and was inversely related to the formylated proportion of red blood cell folates (P < 0.03) that is known to be solely represented in those individuals. These results indicate that the MTHFR C677T polymorphism influences DNA methylation status through an interaction with folate status. PMID:11929966

  5. Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines

    Directory of Open Access Journals (Sweden)

    Santosh Podder

    2015-01-01

    Full Text Available Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF at 5 mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5 mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50 μM or 1 mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5 mM NaF together with radiation (1 Gy induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

  6. Additional mitochondrial DNA influences the interactions between the nuclear and mitochondrial genomes in a bovine embryo model of nuclear transfer.

    Science.gov (United States)

    Srirattana, Kanokwan; St John, Justin C

    2018-05-08

    We generated cattle embryos using mitochondrial supplementation and somatic cell nuclear transfer (SCNT), named miNT, to determine how additional mitochondrial DNA (mtDNA) modulates the nuclear genome. To eliminate any confounding effects from somatic cell mtDNA in intraspecies SCNT, donor cell mtDNA was depleted prior to embryo production. Additional oocyte mtDNA did not affect embryo development rates but increased mtDNA copy number in blastocyst stage embryos. Moreover, miNT-derived blastocysts had different gene expression profiles when compared with SCNT-derived blastocysts. Additional mtDNA increased expression levels of genes involved in oxidative phosphorylation, cell cycle and DNA repair. Supplementing the embryo culture media with a histone deacetylase inhibitor, Trichostatin A (TSA), had no beneficial effects on the development of miNT-derived embryos, unlike SCNT-derived embryos. When compared with SCNT-derived blastocysts cultured in the presence of TSA, additional mtDNA alone had beneficial effects as the activity of glycolysis may increase and embryonic cell death may decrease. However, these beneficial effects were not found with additional mtDNA and TSA together, suggesting that additional mtDNA alone enhances reprogramming. In conclusion, additional mtDNA increased mtDNA copy number and expression levels of genes involved in energy production and embryo development in blastocyst stage embryos emphasising the importance of nuclear-mitochondrial interactions.

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

    International Nuclear Information System (INIS)

    Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Audemard, Eric; Montermini, Laura; Meehan, Brian; Rak, Janusz

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Tae Hoon; Chennakrishnaiah, Shilpa [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Audemard, Eric [McGill University and Genome Quebec Innovation Centre, Montreal, Quebec (Canada); Montermini, Laura; Meehan, Brian [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Rak, Janusz, E-mail: janusz.rak@mcgill.ca [Montreal Children’s Hospital, Research Institute of McGill University Health Centre, McGill University, Montreal, Quebec (Canada)

    2014-08-22

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

  9. Study of the interaction of cysteamine and cystamine with the dipalmitoyl phosphatidylcholine

    International Nuclear Information System (INIS)

    Berleur, F.; Roman, V.; Jaskierowicz, D.; Audet, M.; Fatome, M.

    1984-10-01

    The thermodynamic study of the liposome-radioprotector system brings information on this interaction. The interaction is essentially electrostatic between the phosphate sites and the radioprotector cationic groups and this study could bring further information about the interaction between cysteamine and polyelectrolytic structures, DNA for example, and about the radioprotective properties of this drug [fr

  10. Metal based pharmacologically active complexes of Cu(II), Ni(II) and Zn(II): synthesis, spectral, XRD, antimicrobial screening, DNA interaction and cleavage investigation.

    Science.gov (United States)

    Raman, Natarajan; Mahalakshmi, Rajkumar; Arun, T; Packianathan, S; Rajkumar, R

    2014-09-05

    The present contribution reports a thorough characterization of newly obtained metallointercalators incorporating Schiff bases, formed by the condensation of N-acetoacetyl-o-toluidine with 1-amino-4-nitrobenzene (L(1))/1-amino-4-chlorobenzene (L(2)) as main ligand and 1,10-phenanthroline as co-ligand respectively. The characterization of newly formed metallointercalators has been done by (1)H NMR, UV-Vis, IR, EPR spectroscopy and molar conductivity studies. X-ray powder diffraction illustrates that they are crystalline nature. Binding interaction of these complexes with calf thymus (CT-DNA) has been investigated by emission, absorption, viscosity, cyclic voltammetry and differential pulse voltammetry. DNA binding experiments results reveal that the synthesized complexes interact with DNA through intercalative mode. The in vitro antibacterial and antifungal assay indicate that these complexes are good antimicrobial agents against various pathogens. The DNA cleavage exhibits that they act as efficient cleaving agents. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Multispectroscopic investigation of the interaction of BSA and DNA with the anticancer drug, N-(6-ferrocenyl-2-naphthoyl)-gamma-amino butyric acid methyl ester

    Science.gov (United States)

    Rajina, S. R.; Sudhi, Geethu; Austin, P.; Praveen, S. G.; Xavier, T. S.; Kenny, Peter T. M.; Binoy, J.

    2018-05-01

    The interaction of a drug with DNA and BSA play a great role in studying anti cancer activity and drug transport properties, which can be effectively, investigated using vibrational spectroscopy, UV visible spectroscopy and Fluorescence spectroscopy. The present work reports the structural features of N-(6-ferrocenyl-2-naphthoyl)-gamma-amino butyric acid Methyl ester (FNGABME) based on FTIR and FTRaman spectroscopy. The absorption and fluorescence spectroscopic methods were used to study the efficiency of the interaction of the compound FNGABME with BSA and DNA and also molecular docking were performed computationally to validate the results which shows that the title compound may exhibit inhibitory activity against the cancer cells.

  12. DNA-binding studies of valrubicin as a chemotherapy drug using spectroscopy and electrochemical techniques

    Directory of Open Access Journals (Sweden)

    Reza Hajian

    2017-06-01

    Full Text Available In this study, the molecular interactions between valrubicin, an anticancer drug, and fish sperm DNA have been studied in phosphate buffer solution (pH 7.4 using UV–Vis spectrophotometry and cyclic voltammetry techniques. Valrubicin intercalated into double stranded DNA under a weak displacement reaction with methylene blue (MB molecule in a competitive reaction. The binding constant (kb of valrubicin-DNA was determined as 1.75×103 L/mol by spectrophotometric titration. The value of non-electrostatic binding constant (kt0 was almost constant at different ionic strengths while the ratio of kt0/kb increased from 4.51% to 23.77%. These results indicate that valrubicin binds to ds-DNA via electrostatic and intercalation modes. Thermodynamic parameters including ΔH0, ΔS0 and ΔG0 for valrubicin-DNA interaction were determined as −25.21×103 kJ/mol, 1.55×102 kJ/mol K and −22.03 kJ/mol, respectively. Cyclic voltammetry study shows a pair of redox peaks for valrubicin at 0.45 V and 0.36 V (vs. Ag/AgCl. The peak currents decreased and peak positions shifted to positive direction in the presence of DNA, showing intercalation mechanism due to the variation in formal potential.

  13. Chiral halogenated Schiff base compounds: green synthesis, anticancer activity and DNA-binding study

    Science.gov (United States)

    Ariyaeifar, Mahnaz; Amiri Rudbari, Hadi; Sahihi, Mehdi; Kazemi, Zahra; Kajani, Abolghasem Abbasi; Zali-Boeini, Hassan; Kordestani, Nazanin; Bruno, Giuseppe; Gharaghani, Sajjad

    2018-06-01

    Eight enantiomerically pure halogenated Schiff base compounds were synthesized by reaction of halogenated salicylaldehydes with 3-Amino-1,2-propanediol (R or S) in water as green solvent at ambient temperature. All compounds were characterized by elemental analyses, NMR (1H and 13C), circular dichroism (CD) and FT-IR spectroscopy. FS-DNA binding studies of these compounds carried out by fluorescence quenching and UV-vis spectroscopy. The obtained results revealed that the ligands bind to DNA as: (Rsbnd ClBr) > (Rsbnd Cl2) > (Rsbnd Br2) > (Rsbnd I2) and (Ssbnd ClBr) > (Ssbnd Cl2) > (Ssbnd Br2) > (Ssbnd I2), indicating the effect of halogen on binding constant. In addition, DNA-binding constant of the Ssbnd and R-enantiomers are different from each other. The ligands can form halogen bonds with DNA that were confirmed by molecular docking. This method was also measured the bond distances and bond angles. The study of obtained data can have concluded that binding affinity of the ligands to DNA depends on strength of halogen bonds. The potential anticancer activity of ligands were also evaluated on MCF-7 and HeLa cancer cell lines by using MTT assay. The results showed that the anticancer activity and FS-DNA interaction is significantly dependent on the stereoisomers of Schiff base compounds as R-enantiomers displayed significantly higher activity than S-enantiomers. The molecular docking was also used to illustrate the specific DNA-binding of synthesized compounds and groove binding mode of DNA interaction was proposed for them. In addition, molecular docking results indicated that there are three types of bonds (Hsbnd and X-bond and hX-bond) between synthesized compounds and base pairs of DNA.

  14. Mycobacterium tuberculosis class II apurinic/apyrimidinic-endonuclease/3'-5' exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β-clamp.

    Science.gov (United States)

    Khanam, Taran; Rai, Niyati; Ramachandran, Ravishankar

    2015-10-01

    The class-II AP-endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β-clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel 239 QLRFPKK245 motif in the DNA-binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding-groove (PBG) and the C-terminal of β-clamp located on different domains interact with XthA. The β-clamp-XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β-clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β-clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β-clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β-clamp is important for interactions with XthA, while the C-terminal domain predominantly mediates functional interactions in the substrate's presence. © 2015 John Wiley & Sons Ltd.

  15. The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site.

    Science.gov (United States)

    Claveria-Gimeno, Rafael; Lanuza, Pilar M; Morales-Chueca, Ignacio; Jorge-Torres, Olga C; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

    2017-01-31

    Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities.

  16. Molecular interactions and residues involved in force generation in the T4 viral DNA packaging motor.

    Science.gov (United States)

    Migliori, Amy D; Smith, Douglas E; Arya, Gaurav

    2014-12-12

    Many viruses utilize molecular motors to package their genomes into preformed capsids. A striking feature of these motors is their ability to generate large forces to drive DNA translocation against entropic, electrostatic, and bending forces resisting DNA confinement. A model based on recently resolved structures of the bacteriophage T4 motor protein gp17 suggests that this motor generates large forces by undergoing a conformational change from an extended to a compact state. This transition is proposed to be driven by electrostatic interactions between complementarily charged residues across the interface between the N- and C-terminal domains of gp17. Here we use atomistic molecular dynamics simulations to investigate in detail the molecular interactions and residues involved in such a compaction transition of gp17. We find that although electrostatic interactions between charged residues contribute significantly to the overall free energy change of compaction, interactions mediated by the uncharged residues are equally if not more important. We identify five charged residues and six uncharged residues at the interface that play a dominant role in the compaction transition and also reveal salt bridging, van der Waals, and solvent hydrogen-bonding interactions mediated by these residues in stabilizing the compact form of gp17. The formation of a salt bridge between Glu309 and Arg494 is found to be particularly crucial, consistent with experiments showing complete abrogation in packaging upon Glu309Lys mutation. The computed contributions of several other residues are also found to correlate well with single-molecule measurements of impairments in DNA translocation activity caused by site-directed mutations. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Spectroscopic profiling and computational study of the binding of tschimgine: A natural monoterpene derivative, with calf thymus DNA

    Science.gov (United States)

    Khajeh, Masoumeh Ashrafi; Dehghan, Gholamreza; Dastmalchi, Siavoush; Shaghaghi, Masoomeh; Iranshahi, Mehrdad

    2018-03-01

    DNA is a major target for a number of anticancer substances. Interaction studies between small molecules and DNA are essential for rational drug designing to influence main biological processes and also introducing new probes for the assay of DNA. Tschimgine (TMG) is a monoterpene derivative with anticancer properties. In the present study we tried to elucidate the interaction of TMG with calf thymus DNA (CT-DNA) using different spectroscopic methods. UV-visible absorption spectrophotometry, fluorescence and circular dichroism (CD) spectroscopies as well as molecular docking study revealed formation of complex between TMG and CT-DNA. Binding constant (Kb) between TMG and DNA was 2.27 × 104 M- 1, that is comparable to groove binding agents. The fluorescence spectroscopic data revealed that the quenching mechanism of fluorescence of TMG by CT-DNA is static quenching. Thermodynamic parameters (ΔH TMG with CT-DNA. Competitive binding assay with methylene blue (MB) and Hoechst 33258 using fluorescence spectroscopy displayed that TMG possibly binds to the minor groove of CT-DNA. These observations were further confirmed by CD spectral analysis, viscosity measurements and molecular docking.

  18. Discovering approximate-associated sequence patterns for protein-DNA interactions

    KAUST Repository

    Chan, Tak Ming

    2010-12-30

    Motivation: The bindings between transcription factors (TFs) and transcription factor binding sites (TFBSs) are fundamental protein-DNA interactions in transcriptional regulation. Extensive efforts have been made to better understand the protein-DNA interactions. Recent mining on exact TF-TFBS-associated sequence patterns (rules) has shown great potentials and achieved very promising results. However, exact rules cannot handle variations in real data, resulting in limited informative rules. In this article, we generalize the exact rules to approximate ones for both TFs and TFBSs, which are essential for biological variations. Results: A progressive approach is proposed to address the approximation to alleviate the computational requirements. Firstly, similar TFBSs are grouped from the available TF-TFBS data (TRANSFAC database). Secondly, approximate and highly conserved binding cores are discovered from TF sequences corresponding to each TFBS group. A customized algorithm is developed for the specific objective. We discover the approximate TF-TFBS rules by associating the grouped TFBS consensuses and TF cores. The rules discovered are evaluated by matching (verifying with) the actual protein-DNA binding pairs from Protein Data Bank (PDB) 3D structures. The approximate results exhibit many more verified rules and up to 300% better verification ratios than the exact ones. The customized algorithm achieves over 73% better verification ratios than traditional methods. Approximate rules (64-79%) are shown statistically significant. Detailed variation analysis and conservation verification on NCBI records demonstrate that the approximate rules reveal both the flexible and specific protein-DNA interactions accurately. The approximate TF-TFBS rules discovered show great generalized capability of exploring more informative binding rules. © The Author 2010. Published by Oxford University Press. All rights reserved.

  19. Conformational dynamism for DNA interaction in the Salmonella RcsB response regulator.

    Science.gov (United States)

    Casino, Patricia; Miguel-Romero, Laura; Huesa, Juanjo; García, Pablo; García-Del Portillo, Francisco; Marina, Alberto

    2018-01-09

    The RcsCDB phosphorelay system controls an extremely large regulon in Enterobacteriaceae that involves processes such as biofilm formation, flagella production, synthesis of extracellular capsules and cell division. Therefore, fine-tuning of this system is essential for virulence in pathogenic microorganisms of this group. The final master effector of the RcsCDB system is the response regulator (RR) RcsB, which activates or represses multiple genes by binding to different promoter regions. This regulatory activity of RcsB can be done alone or in combination with additional transcriptional factors in phosphorylated or dephosphorylated states. The capacity of RcsB to interact with multiple promoters and partners, either dephosphorylated or phosphorylated, suggests an extremely conformational dynamism for this RR. To shed light on the activation mechanism of RcsB and its implication on promoter recognition, we solved the crystal structure of full-length RcsB from Salmonella enterica serovar Typhimurium in the presence and absence of a phosphomimetic molecule BeF3-. These two novel structures have guided an extensive site-directed mutagenesis study at the structural and functional level that confirms RcsB conformational plasticity and dynamism. Our data allowed us to propose a β5-T switch mechanism where phosphorylation is coupled to alternative DNA binding ways and which highlights the conformational dynamism of RcsB to be so pleiotropic. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Different modes of interaction by TIAR and HuR with target RNA and DNA.

    Science.gov (United States)

    Kim, Henry S; Wilce, Matthew C J; Yoga, Yano M K; Pendini, Nicole R; Gunzburg, Menachem J; Cowieson, Nathan P; Wilson, Gerald M; Williams, Bryan R G; Gorospe, Myriam; Wilce, Jacqueline A

    2011-02-01

    TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U-rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2'-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.

  1. Mitochondrial DNA mapping of social-biological interactions in Brazilian Amazonian African-descendant populations

    Directory of Open Access Journals (Sweden)

    Bruno Maia Carvalho

    2008-01-01

    Full Text Available The formation of the Brazilian Amazonian population has historically involved three main ethnic groups, Amerindian, African and European. This has resulted in genetic investigations having been carried out using classical polymorphisms and molecular markers. To better understand the genetic variability and the micro-evolutionary processes acting in human groups in the Brazilian Amazon region we used mitochondrial DNA to investigate 159 maternally unrelated individuals from five Amazonian African-descendant communities. The mitochondrial lineage distribution indicated a contribution of 50.2% from Africans (L0, L1, L2, and L3, 46.6% from Amerindians (haplogroups A, B, C and D and a small European contribution of 1.3%. These results indicated high genetic diversity in the Amerindian and African lineage groups, suggesting that the Brazilian Amazonian African-descendant populations reflect a possible population amalgamation of Amerindian women from different Amazonian indigenous tribes and African women from different geographic regions of Africa who had been brought to Brazil as slaves. The present study partially mapped the historical biological and social interactions that had occurred during the formation and expansion of Amazonian African-descendant communities.

  2. Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4.

    Science.gov (United States)

    Lin, H; Rao, V B; Black, L W

    1999-06-04

    Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites. In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro. The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly

  3. Synthesis, structure, DNA/BSA binding and antibacterial studies of NNO tridentate Schiff base metal complexes

    Science.gov (United States)

    Sakthi, Marimuthu; Ramu, Andy

    2017-12-01

    A new salicylaldehyde derived 2,4-diiodo-6-((2-phenylaminoethylimino)methyl)phenol Schiff base(L) and its transition metal complexes of the type MLCl where, M = Cu(II), Ni(II), Co(II), Mn(II) and Zn(II) have been synthesized. The coordination mode of Schiff base holding NNO donor atoms with metal ions was well investigated by elemental analysis, ESI-mass as well as IR, UV-vis, CV and NMR spectral studies. The binding efficiency and mode of these complexes with biological macromolecules viz., herring sperm DNA (HS- DNA) and bovine serum albumin (BSA) have been explored through various spectroscopic techniques. The characteristic changes in absorption, emission and, circular dichroism spectra of the complexes with DNA indicate the noticeable interaction between them. From the all spectral information complexes could interact with DNA via non-intercalation mode of binding. The hyperchromisim in absorption band and hypochromisim in emission intensity of BSA with different complex concentrations shown significant information, and the binding affinity value has been predicted from Stern-Volmer plots. Further, all the complexes could cleave the circular plasmid pUC19 DNA efficiently by using an activator H2O2. The ligand and all metal(II) complexes showed good antibacterial activities. The molecular docking studies of the complexes with DNA were performed in order to make a comparison and conclusion with spectral technic results.

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

    Science.gov (United States)

    van Loon, Barbara; Samson, Leona D

    2013-03-01

    Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair. Copyright © 2012. Published by Elsevier B.V.

  5. [Mechanistic modelling allows to assess pathways of DNA lesion interactions underlying chromosome aberration formation].

    Science.gov (United States)

    Eĭdel'man, Iu A; Slanina, S V; Sal'nikov, I V; Andreev, S G

    2012-12-01

    The knowledge of radiation-induced chromosomal aberration (CA) mechanisms is required in many fields of radiation genetics, radiation biology, biodosimetry, etc. However, these mechanisms are yet to be quantitatively characterised. One of the reasons is that the relationships between primary lesions of DNA/chromatin/chromosomes and dose-response curves for CA are unknown because the pathways of lesion interactions in an interphase nucleus are currently inaccessible for direct experimental observation. This article aims for the comparative analysis of two principally different scenarios of formation of simple and complex interchromosomal exchange aberrations: by lesion interactions at chromosome territories' surface vs. in the whole space of the nucleus. The analysis was based on quantitative mechanistic modelling of different levels of structures and processes involved in CA formation: chromosome structure in an interphase nucleus, induction, repair and interactions of DNA lesions. It was shown that the restricted diffusion of chromosomal loci, predicted by computational modelling of chromosome organization, results in lesion interactions in the whole space of the nucleus being impossible. At the same time, predicted features of subchromosomal dynamics agrees well with in vivo observations and does not contradict the mechanism of CA formation at the surface of chromosome territories. On the other hand, the "surface mechanism" of CA formation, despite having certain qualities, proved to be insufficient to explain high frequency of complex exchange aberrations observed by mFISH technique. The alternative mechanism, CA formation on nuclear centres is expected to be sufficient to explain frequent complex exchanges.

  6. Engineering DNA Backbone Interactions Results in TALE Scaffolds with Enhanced 5-Methylcytosine Selectivity.

    Science.gov (United States)

    Rathi, Preeti; Witte, Anna; Summerer, Daniel

    2017-11-08

    Transcription activator-like effectors (TALEs) are DNA major-groove binding proteins widely used for genome targeting. TALEs contain an N-terminal region (NTR) and a central repeat domain (CRD). Repeats of the CRD selectively recognize each one DNA nucleobase, offering programmability. Moreover, repeats with selectivity for 5-methylcytosine (5mC) and its oxidized derivatives can be designed for analytical applications. However, both TALE domains also nonspecifically interact with DNA phosphates via basic amino acids. To enhance the 5mC selectivity of TALEs, we aimed to decrease the nonselective binding energy of TALEs. We substituted basic amino acids with alanine in the NTR and identified TALE mutants with increased selectivity. We then analysed conserved, DNA phosphate-binding KQ diresidues in CRD repeats and identified further improved mutants. Combination of mutations in the NTR and CRD was highly synergetic and resulted in TALE scaffolds with up to 4.3-fold increased selectivity in genomic 5mC analysis via affinity enrichment. Moreover, transcriptional activation in HEK293T cells by a TALE-VP64 construct based on this scaffold design exhibited a 3.5-fold increased 5mC selectivity. This provides perspectives for improved 5mC analysis and for the 5mC-conditional control of TALE-based editing constructs in vivo.

  7. RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.

    Science.gov (United States)

    Theriot, Corey A; Hegde, Muralidhar L; Hazra, Tapas K; Mitra, Sankar

    2010-06-04

    The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome. Copyright 2010 Elsevier B.V. All rights reserved.

  8. In vitro DNA binding studies of lenalidomide using spectroscopic in combination with molecular docking techniques

    Science.gov (United States)

    Xu, Liang; Hu, Yan-Xi; Li, Yan-Cheng; Zhang, Li; Ai, Hai-Xin; Liu, Yu-Feng; Liu, Hong-Sheng

    2018-02-01

    In the present work, the binding interaction between lenalidomide (LEN) and calf thymus DNA (ct-DNA) was systematically studied by using fluorescence, ultraviolet-visible (UV-vis) absorption, circular dichroism (CD) spectroscopies under imitated physiological conditions (pH = 7.4) coupled with molecular docking. It was found that LEN was bound to ct-DNA with high binding affinity (Ka = 2.308 × 105 M-1 at 283 K) through groove binding as evidenced by a slight decrease in the absorption intensity in combination with CD spectra. Thermodynamic parameters (ΔG 0 and ΔS interaction. Furthermore, competitive binding experiments with ethidium bromide and 4‧, 6-dia-midino-2-phenylindoleas probes showed that LEN could preferentially bind in the minor groove of double-stranded DNA. The average lifetime of LEN was calculated to be 7.645 ns. The φ of LEN was measured as 0.09 and non-radiation energy transfer between LEN and DNA had occurred. The results of the molecular docking were consistent with the experimental results. This study explored the potential applicability of the spectroscopic properties of LEN and also investigated its interactions with relevant biological targets. In addition, it will provide some theoretical references for the deep research of simultaneous administration of LEN with other drugs.

  9. ChIP-exo interrogation of Crp, DNA, and RNAP holoenzyme interactions

    DEFF Research Database (Denmark)

    Latif, Haythem; Federowicz, Stephen; Ebrahim, Ali

    2018-01-01

    Numerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo...

  10. Scanning tunneling spectroscopy study of DNA conductivity

    Czech Academy of Sciences Publication Activity Database

    Kratochvílová, Irena; Král, Karel; Bunček, M.; Nešpůrek, Stanislav; Todorciuc, Tatiana; Weiter, M.; Navrátil, J.; Schneider, Bohdan; Pavluch, J.

    2008-01-01

    Roč. 6, č. 3 (2008), s. 422-426 ISSN 1895-1082 R&D Projects: GA AV ČR KAN401770651; GA MŠk OC 137; GA AV ČR KAN400720701; GA AV ČR KAN200100801 Institutional research plan: CEZ:AV0Z10100520; CEZ:AV0Z40500505; CEZ:AV0Z40550506 Keywords : molecular electronics * DNA * scanning tunneling microscopy * conductivity * charge carrier transport Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 0.448, year: 2008

  11. Coulomb and CH-π interactions in (6-4) photolyase-DNA complex dominate DNA binding and repair abilities.

    Science.gov (United States)

    Terai, Yuma; Sato, Ryuma; Yumiba, Takahiro; Harada, Ryuhei; Shimizu, Kohei; Toga, Tatsuya; Ishikawa-Fujiwara, Tomoko; Todo, Takeshi; Iwai, Shigenori; Shigeta, Yasuteru; Yamamoto, Junpei

    2018-05-14

    (6-4) Photolyases ((6-4)PLs) are flavoenzymes that repair the carcinogenic UV-induced DNA damage, pyrimidine(6-4)pyrimidone photoproducts ((6-4)PPs), in a light-dependent manner. Although the reaction mechanism of DNA photorepair by (6-4)PLs has been intensively investigated, the molecular mechanism of the lesion recognition remains obscure. We show that a well-conserved arginine residue in Xenopus laevis (6-4)PL (Xl64) participates in DNA binding, through Coulomb and CH-π interactions. Fragment molecular orbital calculations estimated attractive interaction energies of -80-100 kcal mol-1 for the Coulomb interaction and -6 kcal mol-1 for the CH-π interaction, and the loss of either of them significantly reduced the affinity for (6-4)PP-containing oligonucleotides, as well as the quantum yield of DNA photorepair. From experimental and theoretical observations, we formulated a DNA binding model of (6-4)PLs. Based on the binding model, we mutated this Arg in Xl64 to His, which is well conserved among the animal cryptochromes (CRYs), and found that the CRY-type mutant exhibited reduced affinity for the (6-4)PP-containing oligonucleotides, implying the possible molecular origin of the functional diversity of the photolyase/cryptochrome superfamily.

  12. Synthesis, characterization and interaction of N,N'-dipyridoxyl (1,4-butanediamine) Co(III) salen complex with DNA and HSA

    Science.gov (United States)

    Janati Fard, F.; Mashhadi Khoshkhoo, Z.; Mirtabatabaei, H.; Housaindokht, M. R.; Jalal, R.; Eshtiagh Hosseini, H.; Bozorgmehr, M. R.; Esmaeili, A. A.; Javan Khoshkholgh, M.

    2012-11-01

    Co(III) salen complex with N,N'-dipyridoxyl (1,4-butanediamine) Schiff-base ligand as tetradentate ligand was synthesized and characterized by the elemental and spectroscopic analysis. The interaction of this complex with calf thymus DNA (ct DNA) has been investigated in vitro using UV absorption, fluorescence spectroscopy, thermal denaturation and gel electrophoresis techniques. The binding constant has been estimated to be 1 × 104 M-1 using UV absorption. The addition of ct DNA to Co(III) salen solution resulted in a fluorescence quenching. The binding constant and site size binding have been calculated in connection with other experimental observations show that the interactive model between Co(III) salen and ct DNA is an intercalative one. The interaction between plasmid DNA (pTZ57R DNA) and this complex is confirmed by gel electrophoresis studies. Furthermore, the interaction between HSA and Co(III) salen complex was investigated by UV absorption, fluorescence spectroscopy and molecular modeling. The binding constant for the interaction of this complex with HSA were found to be 3.854 × 104 M-1 using UV absorption, which was in good agreement with the binding constant obtained from fluorescence method (3.866 × 104 M-1). The binding distance between HSA and this complex was estimated to be 2.48 nm according to Förster theory of non-radioactive energy transfer. Molecular modeling studies suggested that hydrophobic interaction was the predominant intermolecular forces stabilizing Co(III) complex-HSA system.

  13. Probe Microscopic Studies of DNA Molecules on Carbon Nanotubes

    Directory of Open Access Journals (Sweden)

    Kazuo Umemura

    2016-10-01

    Full Text Available Hybrids of DNA and carbon nanotubes (CNTs are promising nanobioconjugates for nanobiosensors, carriers for drug delivery, and other biological applications. In this review, nanoscopic characterization of DNA-CNT hybrids, in particular, characterization by scanning probe microscopy (SPM, is summarized. In many studies, topographical imaging by atomic force microscopy has been performed. However, some researchers have demonstrated advanced SPM operations in order to maximize its unique and valuable functions. Such sophisticated approaches are attractive and will have a significant impact on future studies of DNA-CNT hybrids.

  14. Theoretical studies of molecular interactions

    Energy Technology Data Exchange (ETDEWEB)

    Lester, W.A. Jr. [Univ. of California, Berkeley (United States)

    1993-12-01

    This research program is directed at extending fundamental knowledge of atoms and molecules including their electronic structure, mutual interaction, collision dynamics, and interaction with radiation. The approach combines the use of ab initio methods--Hartree-Fock (HF) multiconfiguration HF, configuration interaction, and the recently developed quantum Monte Carlo (MC)--to describe electronic structure, intermolecular interactions, and other properties, with various methods of characterizing inelastic and reaction collision processes, and photodissociation dynamics. Present activity is focused on the development and application of the QMC method, surface catalyzed reactions, and reorientation cross sections.

  15. Electrochemical monitoring of the interaction between mitomycin C and DNA at chitosan--carbon nanotube composite modified electrodes

    OpenAIRE

    CANAVAR, Pembe Ece; EKŞİN, Ece; ERDEM, Arzum

    2015-01-01

    Single-walled carbon nanotube (CNT) and chitosan composite (chitosan*CNT) based sensors were developed as DNA biosensors, and then they were applied for electrochemical investigation of the interaction between the anticancer drug mitomycin C (MC) and DNA. The oxidation signals of MC and guanine were monitored before and after the interaction process by differential pulse voltammetry (DPV). The DPV results were in good agreement with those of electrochemical impedance spectroscopy (EIS)....

  16. Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

    Science.gov (United States)

    Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

    2015-07-01

    The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Platinum(II Complexes with Tetradentate Schiff Bases as Ligands: Synthesis, Characterization and Detection of DNA Interaction by Differential Pulse Voltammetry

    Directory of Open Access Journals (Sweden)

    Lijun Li

    2012-01-01

    Full Text Available Five sterically hindered platinum(II complexes with tetradentate schiff bases as ligands, [Pt(L] (L= N,N′-bisalicylidene-1,2-ethylenediamine (L1, N,N′-bisalicylidene-1,2-cyclohexanediamine (L2, N,N′-bis(5-hydroxyl-salicylidene-1,2-cyclohexanediamine (L3, N,N′-bisalicylidene-1,2-diphenyl-ethylenediamine (L4 and N,N′-bis(3-tert-butyl-5-methyl-salicylidene-1,2-diphenylethylenediamine (L5 have been synthesized and characterized by IR spectroscopy and elemental analysis. The sterical hindrance of antitumor drug candidates potentially makes them less susceptible to deactivation by sulphur containing proteins and helping to overcome resistance mechanisms. The interaction of these metal complexes with fish sperm single-stranded DNA (ssDNA was studied electrochemically based on the oxidation signals of guanine and adenine. Differential pulse voltammetry was employed to monitor the DNA interaction in solution by using renewable pencil graphite electrode. The results indicate that ligands with different groups can strongly affect the interaction between [Pt(L] complexes and ssDNA due to sterical hindrances and complex [Pt(L1] has the best interaction with DNA among the five complexes.

  18. Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

    International Nuclear Information System (INIS)

    Tao Weitao; Budd, Martin; Campbell, Judith L.

    2003-01-01

    We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1Δ double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Δ alone. However, surprisingly, the dna2-2 sgs1Δ lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1Δ lethality is only partially suppressed by deletion of rad51Δ. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones

  19. Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

    Energy Technology Data Exchange (ETDEWEB)

    Tao Weitao; Budd, Martin; Campbell, Judith L

    2003-11-27

    We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1{delta} double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1{delta} alone. However, surprisingly, the dna2-2 sgs1{delta} lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1{delta} lethality is only partially suppressed by deletion of rad51{delta}. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.

  20. Synthesis and characterization of 6,6'-bis(2-hydroxyphenyl)-2,2'-bipyridine ligand and its interaction with ct-DNA

    Science.gov (United States)

    Selamat, Norhidayah; Heng, Lee Yook; Hassan, Nurul Izzaty; Karim, Nurul Huda Abd

    2015-09-01

    The tetradentate ligand with four donor atoms OONN was synthesized. Bis(phenoxy)bipyridine ligand was prepared by Suzuki coupling reaction between 6,6'-dibromo-2,2'-bipyridyl and 2-hydroxyphenylboronic acid with presence of palladium (II) acetate. Bis(phenoxy)bipyridine ligand was also synthesized by demethylating of 6,6'-bis(2-methoxyphenyl)-2,2'-bipyridyl ligand through solvent free reaction using pyridine hydrocloride. The formation of both phenoxy and methoxy ligands was confirmed by 1H, 2D cosy and 13C NMR spectroscopy, ESI-MS spectrometry, FTIR spectroscopy. The purity of the ligand was confirmed by melting point. Binding studies of small molecules with DNA are useful to understand the reaction mechanism and to provide guidance for the application and design of new and more efficient drugs targeted to DNA. In this study, the binding interaction between the synthesized ligand with calf thymus-DNA (ct-DNA) has been investigated by UV/Vis DNA titration study. From the UV/Vis DNA study, it shows that bis(phenoxy)bipyridine ligand bind with ct-DNA via outside binding with binding contant Kb = 1.19 × 103 ± 0.08 M-1.

  1. Synthesis and characterization of 6,6’-bis(2-hydroxyphenyl)-2,2’-bipyridine ligand and its interaction with ct-DNA

    International Nuclear Information System (INIS)

    Selamat, Norhidayah; Heng, Lee Yook; Hassan, Nurul Izzaty; Karim, Nurul Huda Abd

    2015-01-01

    The tetradentate ligand with four donor atoms OONN was synthesized. Bis(phenoxy)bipyridine ligand was prepared by Suzuki coupling reaction between 6,6’-dibromo-2,2’-bipyridyl and 2-hydroxyphenylboronic acid with presence of palladium (II) acetate. Bis(phenoxy)bipyridine ligand was also synthesized by demethylating of 6,6’-bis(2-methoxyphenyl)-2,2’-bipyridyl ligand through solvent free reaction using pyridine hydrocloride. The formation of both phenoxy and methoxy ligands was confirmed by 1 H, 2D cosy and 13 C NMR spectroscopy, ESI-MS spectrometry, FTIR spectroscopy. The purity of the ligand was confirmed by melting point. Binding studies of small molecules with DNA are useful to understand the reaction mechanism and to provide guidance for the application and design of new and more efficient drugs targeted to DNA. In this study, the binding interaction between the synthesized ligand with calf thymus-DNA (ct-DNA) has been investigated by UV/Vis DNA titration study. From the UV/Vis DNA study, it shows that bis(phenoxy)bipyridine ligand bind with ct-DNA via outside binding with binding contant K b = 1.19 × 10 3 ± 0.08 M −1

  2. Synthesis and characterization of 6,6’-bis(2-hydroxyphenyl)-2,2’-bipyridine ligand and its interaction with ct-DNA

    Energy Technology Data Exchange (ETDEWEB)

    Selamat, Norhidayah; Heng, Lee Yook; Hassan, Nurul Izzaty; Karim, Nurul Huda Abd [School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43650 Bangi, Selangor (Malaysia)

    2015-09-25

    The tetradentate ligand with four donor atoms OONN was synthesized. Bis(phenoxy)bipyridine ligand was prepared by Suzuki coupling reaction between 6,6’-dibromo-2,2’-bipyridyl and 2-hydroxyphenylboronic acid with presence of palladium (II) acetate. Bis(phenoxy)bipyridine ligand was also synthesized by demethylating of 6,6’-bis(2-methoxyphenyl)-2,2’-bipyridyl ligand through solvent free reaction using pyridine hydrocloride. The formation of both phenoxy and methoxy ligands was confirmed by {sup 1}H, 2D cosy and {sup 13}C NMR spectroscopy, ESI-MS spectrometry, FTIR spectroscopy. The purity of the ligand was confirmed by melting point. Binding studies of small molecules with DNA are useful to understand the reaction mechanism and to provide guidance for the application and design of new and more efficient drugs targeted to DNA. In this study, the binding interaction between the synthesized ligand with calf thymus-DNA (ct-DNA) has been investigated by UV/Vis DNA titration study. From the UV/Vis DNA study, it shows that bis(phenoxy)bipyridine ligand bind with ct-DNA via outside binding with binding contant K{sub b} = 1.19 × 10{sup 3} ± 0.08 M{sup −1}.

  3. Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing

    International Nuclear Information System (INIS)

    Liang Liang; Dou Peng; Dong Mingming; Ke Xiaokang; Bian Ningsheng; Liu Zhen

    2009-01-01

    Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05 ± 0.83 x 10 -3 s -1 . The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.

  4. Time-resolved studies of direct effects of radiation on DNA

    International Nuclear Information System (INIS)

    Fielden, E.M.; O'Neill, P.; Al-Kazwini, A.

    1987-01-01

    The biological changes induced by ionising radiation are a consequence of radiation-induced chemical events taking place at times <1s. These events are strongly influenced by the presence of chemical modifiers. Since DNA is a principle target for radiation-induced cell killing, DNA-free radicals are generated by direct ionisation of DNA moieties (direct effect) and by reaction with hydroxyl radicals formed by radiolysis of the water which is in the vicinity of the DNA (indirect effect). In order to study the 'direct' effects of radiation on DNA the following model approaches are discussed:- 1) Use of the technique of pulse radiolysis to investigate in aqueous solution the interactions of deoxynucleosides with SO/sub 4//sup .-/ whereby one-electron oxidised species of the bases are generated; and 2) time resolved, radiation-induced changes to solid DNA and related macromolecules (e.g. radiation-induced luminescence) in order to obtain an understanding of charge/energy migration as a result of ionisation of DNA. The influence of chemical modifiers and of environment is discussed in terms of the properties of the radiation-induced species produced. Since the properties of base radicals produced by SO/sub 4//sup .-/ are similar to those of the base OH-adducts oxidising properties, potential similarities between the 'direct' and 'indirect' effects of radiation are presented

  5. Physical interaction between components of DNA mismatch repair and nucleotide excision repair

    International Nuclear Information System (INIS)

    Bertrand, P.; Tishkoff, D.X.; Filosi, N.; Dasgupta, R.; Kolodner, R.D.

    1998-01-01

    Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as 'bait,' and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes

  6. Magnesium-DNA interactions and the possible relation of magnesium to carcinogenesis. Irradiation and free radicals.

    Science.gov (United States)

    Anastassopoulou, J; Theophanides, T

    2002-04-01

    Magnesium deficiency causes renal complications. The appearance of several diseases is related to its depletion in the human body. In radiotherapy, as well as in chemotherapy, especially in treatment of cancers with cis-platinum, hypomagnesaemia is observed. The site effects of chemotherapy that are due to hypomagnesaemia are decreased using Mg supplements. The role of magnesium in DNA stabilization is concentration dependent. At high concentrations there is an accumulation of Mg binding, which induces conformational changes leading to Z-DNA, while at low concentration there is deficiency and destabilization of DNA. The biological and clinical consequences of abnormal concentrations are DNA cleavage leading to diseases and cancer. Carcinogenesis and cell growth are also magnesium-ion concentration dependent. Several reports point out that the interaction of magnesium in the presence of other metal ions showed that there is synergism with Li and Mn, but there is magnesium antagonism in DNA binding with the essential metal ions in the order: Zn>Mg>Ca. In the case of toxic metals such as Cd, Ga and Ni there is also antagonism for DNA binding. It was found from radiolysis of deaerated aqueous solutions of the nucleoside 5'-guanosine monophosphate (5'-GMP) in the presence as well as in the absence of magnesium ions that, although the addition of hydroxyl radicals (*OH) has been increased by 2-fold, the opening of the imidazole ring of the guanine base was prevented. This effect was due to the binding of Mg2+ ions to N7 site of the molecule by stabilizing the five-member ring imitating cis-platinum. It was also observed using Fourier Transform Infrared spectroscopy, Raman spectroscopy and Fast Atom Bombardment mass spectrometry that *OH radicals subtract H atoms from the C1', C4' and C5' sites of the nucleotide. Irradiation of 5'-GMP in the presence of oxygen (2.5 x 10(-4) M) shows that magnesium is released from the complex. There is spectroscopic evidence that

  7. Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates

    Directory of Open Access Journals (Sweden)

    Lavrik O. I.

    2012-06-01

    Full Text Available Aim. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ using 5-formyluridine (5-foU containing DNA duplexes which are imitating the intermediates during replication of the leading DNA strand, and to study the influence of replication factors hRPA and hPCNA on this activity. Methods. The EMSA and the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis across 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i both proteins are able to catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate enzyme; (ii hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol at the nucleotide incorporation directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage displaced into the duplex; (iii hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.

  8. Development of a defined-sequence DNA system for use in DNA misrepair studies

    International Nuclear Information System (INIS)

    Sutton, S.; Tobias, C.A.

    1984-01-01

    The authors have developed a system that allows them to study cellular DNA repair processes at the molecular level. In particular, the authors are using this system to examine the consequences of a misrepair of radiation-induced DNA damage, as a function of dose. The cells being used are specially engineered haploid yeast cells. Maintained in the cells, at one copy per cell, is a cen plasmid, a plasmid that behaves like a functional chromosome. This plasmid carries a small defined sequence of DNA from the E. coli lac z gene. It is this lac z region (called the alpha region) that serves as the target for radiation damage. Two copies of the complimentary portion of the lac z gene are integrated into the yeast genome. Irradiated cells are screened for possible mutation in the alpha region by testing the cells' ability to hydrolyze xgal, a lactose substrate. The DNA of interest is then extracted from the cells, sequenced, and the sequence is compared to that of the control. Unlike the usual defined-sequence DNA systems, theirs is an in vivo system. A disadvantage is the relatively high background mutation rate. Results achieved with this system, as well as future applications, are discussed

  9. Binding studies of guggulsterone-E to calf thymus DNA by multi-spectroscopic, calorimetric and molecular docking studies

    Science.gov (United States)

    Ikhlas, Shoeb; Ahmad, Masood

    2018-02-01

    Guggulsterone, a sterol found in plants is used as an ayurvedic medicine for many diseases such as obesity, internal tumors, ulcers etc. E and Z are two isoforms of guggulsterone, wherein guggulsterone-E (GUGE) has also been shown to have anticancer potential. Most of the anticancer drugs target nucleic acids. Therefore, we studied the mode of interaction between ctDNA and GUGE using UV-Vis, fluorescence and CD spectroscopy, isothermal calorimetry along with molecular docking studies. Hoechst 3325, ethidium bromide and rhodamine-B displacement experiments confirms that GUGE binds in the minor groove of DNA. ITC results further suggest these interactions to be feasible and spontaneous with hydrogen bond formation and van der waals interactions. Lastly, molecular docking also suggests GUGE to be a minor groove binder interacting through a single hydrogen bond formation between OH group of GUGE and nitrogen (N3) of adenosine (A6).

  10. Novel essential residues of Hda for interaction with DnaA in the regulatory inactivation of DnaA: unique roles for Hda AAA Box VI and VII motifs.

    Science.gov (United States)

    Nakamura, Kenta; Katayama, Tsutomu

    2010-04-01

    Escherichia coli ATP-DnaA initiates chromosomal replication. For preventing extra-initiations, a complex of ADP-Hda and the DNA-loaded replicase clamp promotes DnaA-ATP hydrolysis, yielding inactive ADP-DnaA. However, the Hda-DnaA interaction mode remains unclear except that the Hda Box VII Arg finger (Arg-153) and DnaA sensor II Arg-334 within each AAA(+) domain are crucial for the DnaA-ATP hydrolysis. Here, we demonstrate that direct and functional interaction of ADP-Hda with DnaA requires the Hda residues Ser-152, Phe-118 and Asn-122 as well as Hda Arg-153 and DnaA Arg-334. Structural analyses suggest intermolecular interactions between Hda Ser-152 and DnaA Arg-334 and between Hda Phe-118 and the DnaA Walker B motif region, in addition to an intramolecular interaction between Hda Asn-122 and Arg-153. These interactions likely sustain a specific association of ADP-Hda and DnaA, promoting DnaA-ATP hydrolysis. Consistently, ATP-DnaA and ADP-DnaA interact with the ADP-Hda-DNA-clamp complex with similar affinities. Hda Phe-118 and Asn-122 are contained in the Box VI region, and their hydrophobic and electrostatic features are basically conserved in the corresponding residues of other AAA(+) proteins, suggesting a conserved role for Box VI. These findings indicate novel interaction mechanisms for Hda-DnaA as well as a potentially fundamental mechanism in AAA(+) protein interactions.

  11. Large-scale symmetry-adapted perturbation theory computations via density fitting and Laplace transformation techniques: investigating the fundamental forces of DNA-intercalator interactions.

    Science.gov (United States)

    Hohenstein, Edward G; Parrish, Robert M; Sherrill, C David; Turney, Justin M; Schaefer, Henry F

    2011-11-07

    Symmetry-adapted perturbation theory (SAPT) provides a means of probing the fundamental nature of intermolecular interactions. Low-orders of SAPT (here, SAPT0) are especially attractive since they provide qualitative (sometimes quantitative) results while remaining tractable for large systems. The application of density fitting and Laplace transformation techniques to SAPT0 can significantly reduce the expense associated with these computations and make even larger systems accessible. We present new factorizations of the SAPT0 equations with density-fitted two-electron integrals and the first application of Laplace transformations of energy denominators to SAPT. The improved scalability of the DF-SAPT0 implementation allows it to be applied to systems with more than 200 atoms and 2800 basis functions. The Laplace-transformed energy denominators are compared to analogous partial Cholesky decompositions of the energy denominator tensor. Application of our new DF-SAPT0 program to the intercalation of DNA by proflavine has allowed us to determine the nature of the proflavine-DNA interaction. Overall, the proflavine-DNA interaction contains important contributions from both electrostatics and dispersion. The energetics of the intercalator interaction are are dominated by the stacking interactions (two-thirds of the total), but contain important contributions from the intercalator-backbone interactions. It is hypothesized that the geometry of the complex will be determined by the interactions of the intercalator with the backbone, because by shifting toward one side of the backbone, the intercalator can form two long hydrogen-bonding type interactions. The long-range interactions between the intercalator and the next-nearest base pairs appear to be negligible, justifying the use of truncated DNA models in computational studies of intercalation interaction energies.

  12. TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site.

    Science.gov (United States)

    Azhibek, Dulat; Skvortsov, Dmitry; Andreeva, Anna; Zatsepin, Timofei; Arutyunyan, Alexandr; Zvereva, Maria; Dontsova, Olga

    2016-06-01

    Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G-rich sequence, and with noncoding RNA, Telomeric repeat-containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2'-OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G-quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate - the 3'-end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3'-end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  13. Synthetic models related to DNA-intercalating molecules. Interactions between 8-alkoxypsoralen and adenine

    International Nuclear Information System (INIS)

    Decout, J.L.; Lhomme, J.

    1983-01-01

    To investigate the interactions and the photoreactions between furocoumarins and adenine, compounds in which a psoralen molecule is linked by different polymethylene bridges have been synthesised. Ring-ring intramolecular interactions are observed by UV spectroscopy. Thermodynamic parameters of these hydrophobic interactions are determined by the study of the variation of the hypochromic effect with temperature. (author)

  14. Cooperative assembly of Co-Smad4 MH1 with R-Smad1/3 MH1 on DNA: a molecular dynamics simulation study.

    Directory of Open Access Journals (Sweden)

    Guihong Wang

    Full Text Available Smads, the homologs of Sma and MAD proteins, play a key role in gene expression regulation in the transforming growth factor-β (TGF-β signaling pathway. Recent experimental studies have revealed that Smad4/R-Smad heterodimers bound on DNA are energetically more favorable than homodimeric R-Smad/R-Smad complexes bound on DNA, which indicates that Smad4 might act as binding vehicle to cooperatively assemble with activated R-Smads on DNA in the nucleus. However, the details of interaction mechanism for cooperative recruitment of Smad4 protein to R-Smad proteins on DNA, and allosteric communication between the Smad4-DNA and R-Smad-DNA interfaces via DNA mediating are not yet clear so far.In the present work, we have constructed a series of Smadn+DNA+Smadn (n = 1, 3, 4 models and carried out molecular dynamics simulations, free energy calculations and DNA dynamics analysis for them to study the interaction properties of Smadn (n = 1, 3, 4 with DNA molecule.The results revealed that the binding of Smad4 protein to DNA molecule facilitates energetically the formation of the heteromeric Smad4+DNA+Smad1/3 complex by increasing the affinity of Smad1/3 with DNA molecule. Further investigations through the residue/base motion correlation and DNA dynamics analyses predicted that the binding of Smad4 protein to DNA molecule in the heteromeric Smad4+DNA+Smad1/3 model induces an allosteric communication from the Smad4-DNA interface to Smad1/Smad3-DNA interface via DNA base-pair helical motions, surface conformation changes and new hydrogen bond formations. The present work theoretically explains the mechanism of cooperative recruitment of Smad4 protein to Smad1/3 protein via DNA-mediated indirect readout mode in the nucleus.

  15. Synergy of irofulven in combination with other DNA damaging agents: synergistic interaction with altretamine, alkylating, and platinum-derived agents in the MV522 lung tumor model.

    Science.gov (United States)

    Kelner, Michael J; McMorris, Trevor C; Rojas, Rafael J; Estes, Leita A; Suthipinijtham, Pharnuk

    2008-12-01

    Irofulven (MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product present in the Omphalotus illudins (Jack O'Lantern) mushroom. This novel agent produces DNA damage, that in contrast to other agents, is predominately ignored by the global genome repair pathway of the nucleotide excision repair (NER)(2) system. The aim of this study was to determine the antitumor activity of irofulven when administered in combination with 44 different DNA damaging agents, whose damage is in general detected and repaired by the genome repair pathway. The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with different DNA damaging agents. Two main classes of DNA damaging agents, platinum-derived agents, and select bifunctional alkylating agents, demonstrated in vivo synergistic or super-additive interaction with irofulven. DNA helicase inhibiting agents also demonstrated synergy in vitro, but an enhanced interaction with irofulven could not be demonstrated in vivo. There was no detectable synergistic activity between irofulven and agents capable of inducing DNA cleavage or intercalating into DNA. These results indicate that the antitumor activity of irofulven is enhanced when combined with platinum-derived agents, altretamine, and select alkylating agents such as melphalan or chlorambucil. A common factor between these agents appears to be the production of intrastrand DNA crosslinks. The synergistic interaction between irofulven and other agents may stem from the nucleotide excision repair system being selectively overwhelmed at two distinct points in the pathway, resulting in prolonged stalling of transcription forks, and subsequent initiation of apoptosis.

  16. Synthesis of schiff bases of pyridine-4-carbaldehyde and their antioxidant and DNA binding studies

    International Nuclear Information System (INIS)

    Shamim, S.; Murtaza, S.; Nazar, M.F.

    2016-01-01

    A series of Schiff bases of pyridine-4-carbaldehyde with 3-aminobenzoic acid, 2-aminobenzoic acid, 4-aminobenzoic acid, 1,3-phenylenediamine, 1,2-phenylenediamine, 2-aminothiophenol, 4-aminoantipyrene, 2-aminophenol and naphthalene-1-amine was synthesized and compounds were characterized by FTIR, NMR and mass spectrometry. The synthesized compounds were evaluated for their antioxidant and DNA binding interaction studies. DPPH scavenging method was used to evaluate the antioxidant activities of synthesized Schiff bases at six gradually increasing concentrations of 0.5-5mg/ml. 2-((pyridin-4-ylmethylidene)amino)phenol came out to be the most efficient antioxidant at a concentration of 4mg/ml with 74% inhibition of free radicals generated by DPPH. The DNA binding interaction of the synthesized Schiff bases was determined using UV-Vis absorption titration method. Both the hypochromic and hyperchromic effects were observed along the series. The values for the binding constant (K) and free energy change (G) were calculated and most of the Schiff bases have high positive K values which indicate the efficient binding of Schiff bases with DNA. Molecular docking studies as carried out using PatchDock molecular algorithm software also indicated the high values for geometrical shape complementarity score suggesting the stabilities of Schiff bases/DNA complex. Docking studies also suggested the minor groove binding of the Schiff bases with DNA. Drug-likeness of the synthesized compounds was also tested in silico and the results are accordingly discussed. (author)

  17. Study on DNA damages induced by UV radiation

    International Nuclear Information System (INIS)

    Doan Hong Van; Dinh Ba Tuan; Tran Tuan Anh; Nguyen Thuy Ngan; Ta Bich Thuan; Vo Thi Thuong Lan; Tran Minh Quynh; Nguyen Thi Thom

    2015-01-01

    DNA damages in Escherichia coli (E. coli) exposed to UV radiation have been investigated. After 30 min of exposure to UV radiation of 5 mJ/cm"2, the growth of E. coli in LB broth medium was about only 10% in compared with non-irradiated one. This results suggested that the UV radiation caused the damages for E. coli genome resulted in reduction in its growth and survival, and those lesions can be somewhat recovered. For both solutions of plasmid DNAs and E. coli cells containing plasmid DNA, this dose also caused the breakage on single and double strands of DNA, shifted the morphology of DNA plasmid from supercoiled to circular and linear forms. The formation of pyrimidine dimers upon UV radiation significantly reduced when the DNA was irradiated in the presence of Ganoderma lucidum extract. Thus, studies on UV-induced DNA damage at molecular level are very essential to determine the UV radiation doses corresponding to the DNA damages, especially for creation and selection of useful radiation-induced mutants, as well as elucidation the protective effects of the specific compounds against UV light. (author)

  18. Interaction between insulin and calf thymus DNA, and quantification of insulin and calf thymus DNA by a resonance Rayleigh scattering method

    International Nuclear Information System (INIS)

    Kong, L.; Liu, Z.; Hu, X.; Liu, S.; Li, W.

    2012-01-01

    The interaction of insulin with calf thymus deoxyribonucleic acid (ctDNA) leads to a complex that displays remarkably enhanced resonance Rayleigh scattering (RRS). The complex and its formation were investigated by atomic force microscopy and by absorption, fluorescence and circular dichroism spectroscopies. We show that the Tyr B16, Tyr B26 and Phe B24 amino acids near the active center (Phe B25) were influenced by the interaction, whereas Tyr A14, Tyr A19 and Phe B1 (which are located far away from the active center) were less influenced. The interaction provide a way in the quantitation of both ctDNA and insulin with high sensitivity. When ctDNA is used as a probe to quantify insulin, the detection limit (3σ) is 6.0 ng mL -1 . If, inversely, insulin is used as a probe to quantify ctDNA, the detection limit (3σ) is 7.2 ng mL -1 . The analysis of synthetic DNA samples and an insulin infection sample provided satisfactory results. (author)

  19. Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases

    Czech Academy of Sciences Publication Activity Database

    Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

    2016-01-01

    Roč. 24, č. 6 (2016), s. 1268-1276 ISSN 0968-0896 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61388963 Keywords : nucleosides * nucleotides * pyrimidines * DNA methyltransferases * DNA polymerases Subject RIV: CC - Organic Chemistry Impact factor: 2.930, year: 2016

  20. Single-molecule studies of DNA transcription using atomic force microscopy

    International Nuclear Information System (INIS)

    Billingsley, Daniel J; Crampton, Neal; Thomson, Neil H; Bonass, William A; Kirkham, Jennifer

    2012-01-01

    Atomic force microscopy (AFM) can detect single biomacromolecules with a high signal-to-noise ratio on atomically flat biocompatible support surfaces, such as mica. Contrast arises from the innate forces and therefore AFM does not require imaging contrast agents, leading to sample preparation that is relatively straightforward. The ability of AFM to operate in hydrated environments, including humid air and aqueous buffers, allows structure and function of biological and biomolecular systems to be retained. These traits of the AFM are ensuring that it is being increasingly used to study deoxyribonucleic acid (DNA) structure and DNA–protein interactions down to the secondary structure level. This report focuses in particular on reviewing the applications of AFM to the study of DNA transcription in reductionist single-molecule bottom-up approaches. The technique has allowed new insights into the interactions between ribonucleic acid (RNA) polymerase to be gained and enabled quantification of some aspects of the transcription process, such as promoter location, DNA wrapping and elongation. More recently, the trend is towards studying the interactions of more than one enzyme operating on a single DNA template. These methods begin to reveal the mechanics of gene expression at the single-molecule level and will enable us to gain greater understanding of how the genome is transcribed and translated into the proteome. (topical review)

  1. Ionizing radiation interactions with DNA: nanodosimetry; Ionisierende Strahlungswechselwirkung mit der DNS. Nanodosimetrie

    Energy Technology Data Exchange (ETDEWEB)

    Bug, Marion; Nettelbeck, Heidi [Physikalisch-Technische Bundesanstalt (PTB), Braunschweig (Germany). Arbeitsgruppe ' Biologische Wirksamkeit ionisierender Strahlung' ; Hilgers, Gerhard [Physikalisch-Technische Bundesanstalt (PTB), Braunschweig (Germany). Arbeitsgruppe ' Nanodosimetrie' ; Rabus, Hans [Physikalisch-Technische Bundesanstalt (PTB), Braunschweig (Germany). Fachbereich ' Grundlagen der Dosimetrie'

    2011-06-15

    The metrology of ionizing radiation is based on measuring values that are averaged over macroscopic volume elements, for instance the energy dose is defined as ratio of the energy deposited on the absorber and the absorber mass. For biological or medical radiation effects the stochastic nature of radiation interaction is of main importance, esp. the interaction of ionizing radiation with the DNA as the genetic information carrier. For radiotherapy and risk evaluation purposes a comprehensive system of radiation weighing factors and other characteristics, like radiation quality or relative biological efficacy was developed. The nanodosimetry is aimed to develop a metrological basis relying on physical characteristics of the microscopic structure of ionizing radiation tracks. The article includes the development of experimental nanodosimetric methods, the respective calibration techniques, Monte-Carlo simulation of the particle track microstructure and the correlation nanodosimetry and biological efficiency.

  2. Cutting edge: Antimalarial drugs inhibit IFN-β production through blockade of cyclic GMP-AMP synthase-DNA interaction.

    Science.gov (United States)

    An, Jie; Woodward, Joshua J; Sasaki, Tomikazu; Minie, Mark; Elkon, Keith B

    2015-05-01

    Type I IFN is strongly implicated in the pathogenesis of systemic autoimmune diseases, such as lupus, and rare monogenic IFNopathies, including Aicardi-Goutières syndrome. Recently, a new DNA-activated pathway involving the enzyme cyclic GMP-AMP synthase (cGAS) was described and potentially linked to Aicardi-Goutières syndrome. To identify drugs that could potentially inhibit cGAS activity, we performed in silico screening of drug libraries. By computational analysis, we identified several antimalarial drugs (AMDs) that were predicted to interact with the cGAS/dsDNA complex. Our studies validated that several AMDs were effective inhibitors of IFN-β production and that they functioned by inhibiting dsDNA stimulation of cGAS. Because AMDs have been widely used in human diseases and have an excellent safety profile, our findings suggest new therapeutic strategies for the treatment of severe debilitating diseases associated with type I IFNs due to cGAS activation. Copyright © 2015 by The American Association of Immunologists, Inc.

  3. Modeling techniques and fluorescence imaging investigation of the interactions of an anthraquinone derivative with HSA and ctDNA.

    Science.gov (United States)

    Fu, Zheng; Cui, Yanrui; Cui, Fengling; Zhang, Guisheng

    2016-01-15

    A new anthraquinone derivative (AORha) was synthesized. Its interactions with human serum albumin (HSA) and calf thymus DNA (ctDNA) were investigated by fluorescence spectroscopy, UV-visible absorption spectroscopy and molecular modeling. Cell viability assay and cell imaging experiment were performed using cervical cancer cells (HepG2 cells). The fluorescence results revealed that the quenching mechanism was static quenching. At different temperatures (290, 300, 310 K), the binding constants (K) and the number of binding sites (n) were determined, respectively. The positive ΔH and ΔS values showed that the binding of AORha with HSA was hydrophobic force, which was identical with the molecular docking result. Studying the fluorescence spectra, UV spectra and molecular modeling also verified that the binding mode of AORha and ctDNA might be intercalative. When HepG2 cells were treated with AORha, the fluorescence became brighter and turned green, which could be used for bioimaging. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Spectroscopic investigation on interaction and sonodynamic damage of Riboflavin to DNA under ultrasonic irradiation by using Methylene Blue as fluorescent probe

    Science.gov (United States)

    Wang, Qi; Wu, Qiong; Wang, Jun; Chen, Dandan; Fan, Ping; Wang, Baoxin

    2014-01-01

    In this paper, the Riboflavin (RF) as a sonosensitizer and Methylene Blue (MB) as a fluorescent probe were used to study the interaction and sonodynamic damage to Deoxyribonucleic Acid (DNA) by fluorescence and UV-vis spectroscopy. The results showed that the RF could efficiently bind to DNA in aqueous solution and exchange with the MB through competing reaction. And then, under ultrasonic irradiation, the RF could obviously damage the DNA. In addition, the influencing factors such as ultrasonic irradiation time and RF concentration on the sonodynamic damage to DNA were also considered. The experimental results showed that the sonodynamic damage degree increase with the increase of ultrasonic irradiation time and RF concentration. Perhaps, this paper may offer some important subjects for broadening the application of RF in sonodynamic therapy (SDT) technologies for tumor treatment.

  5. Studies of DNA supercoiling in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Cook, D.N.

    1990-10-01

    This thesis describes a number of diverse experiments whose common theme is to elaborate some aspect of DNA supercoiling. The torsion elastic constant of DNA is measure as a function of superhelix density using the technique of picosecond Time Resolved Fluorescence Polarization Anisotropy (FPA) of intercalated ethidium bromide. The results agree with theories which predict that the anisotropy decay should vary with the square root of the relative viscosity. This experiment furthermore demonstrates a sensitivity of FPA to a change in torsion elastic constant of less than 10%. A number of covalently closed DNA samples, ranging in superhelix density from = [minus]0.123 to [plus]0.042, are then examined. A novel method for measuring changes in local supercoiling on a large PNA molecule which is sensitive to changes in supercoiling of regions of chromosomal DNA as short as 1 kilobase in length is presented. Study of chromosomal supercoiling regulating anaerobic gene expression in the facultative photosynthetic bacterium, Rhodobacter capsulatus showed that no stable change in chromosomal supercoiling upon a shift from aerobic respiratory growth to anaerobic photosynthetic conditions. Studies to detect transient changes in DNA supercoiling indicate that DNA downstream from heavily transcribed genes for the photosynthetic reaction center are relaxed or perhaps overwound upon the induction of photosynthetic metabolism. These results are interpreted in terms of the twin domain model of transcriptional supercoiling.

  6. Studies of DNA supercoiling in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Cook, David Nelson [Univ. of California, Berkeley, CA (United States)

    1990-10-01

    This thesis describes a number of diverse experiments whose common theme is to elaborate some aspect of DNA supercoiling. The torsion elastic constant of DNA is measure as a function of superhelix density using the technique of picosecond Time Resolved Fluorescence Polarization Anisotropy (FPA) of intercalated ethidium bromide. The results agree with theories which predict that the anisotropy decay should vary with the square root of the relative viscosity. This experiment furthermore demonstrates a sensitivity of FPA to a change in torsion elastic constant of less than 10%. A number of covalently closed DNA samples, ranging in superhelix density from = -0.123 to +0.042, are then examined. A novel method for measuring changes in local supercoiling on a large PNA molecule which is sensitive to changes in supercoiling of regions of chromosomal DNA as short as 1 kilobase in length is presented. Study of chromosomal supercoiling regulating anaerobic gene expression in the facultative photosynthetic bacterium, Rhodobacter capsulatus showed that no stable change in chromosomal supercoiling upon a shift from aerobic respiratory growth to anaerobic photosynthetic conditions. Studies to detect transient changes in DNA supercoiling indicate that DNA downstream from heavily transcribed genes for the photosynthetic reaction center are relaxed or perhaps overwound upon the induction of photosynthetic metabolism. These results are interpreted in terms of the twin domain model of transcriptional supercoiling.

  7. A feature-based approach to modeling protein-DNA interactions.

    Directory of Open Access Journals (Sweden)

    Eilon Sharon

    Full Text Available Transcription factor (TF binding to its DNA target site is a fundamental regulatory interaction. The most common model used to represent TF binding specificities is a position specific scoring matrix (PSSM, which assumes independence between binding positions. However, in many cases, this simplifying assumption does not hold. Here, we present feature motif models (FMMs, a novel probabilistic method for modeling TF-DNA interactions, based on log-linear models. Our approach uses sequence features to represent TF binding specificities, where each feature may span multiple positions. We develop the mathematical formulation of our model and devise an algorithm for learning its structural features from binding site data. We also developed a discriminative motif finder, which discovers de novo FMMs that are enriched in target sets of sequences compared to background sets. We evaluate our approach on synthetic data and on the widely used TF chromatin immunoprecipitation (ChIP dataset of Harbison et al. We then apply our algorithm to high-throughput TF ChIP data from mouse and human, reveal sequence features that are present in the binding specificities of mouse and human TFs, and show that FMMs explain TF binding significantly better than PSSMs. Our FMM learning and motif finder software are available at http://genie.weizmann.ac.il/.

  8. Electronic structure of an anticancer drug DC81 and its interaction with DNA base pairs

    Energy Technology Data Exchange (ETDEWEB)

    Tiwari, Gargi, E-mail: gargi.tiwari@rediffmail.com; Sharma, Dipendra, E-mail: d-11sharma@rediffmail.com; Dwivedi, K. K., E-mail: dwivedikarunesh4@gmail.com [Department of Physics, DDU Gorakhpur University, Gorakhpur (India); Dwivedi, M. K., E-mail: dwivedi-ji@rediffmail.com [Department of Physics, Banaras Hindu University, Varanasi (India)

    2016-05-06

    The drug, 8-Hydroxy-7-methoxy-pyrrolo-[2,1-c][1,4] benzodiazepine-5-one, commonly christened as DC81 belongs to the pyrrolo-[2,1-c][1,4]benzodiazepine (PBDs) family. It is a member of the group of naturally occurring antitumour antibiotics produced by various Streptomyces species. The antitumour activity of DC81 is attributed to its sequence specific interaction with G-C rich DNA region in particular, for Pu-G-Pu motifs. In the present paper, physico-chemical properties DC81 have been carried out using an ab-initio method, HF/6-31G(d,p) with GAMESS program. MEP, HOMO and LUMO surfaces have been scanned. Ionization potential, electron affinity, electronegativity, global hardness and softness of the drug have been calculated. Further, drug-DNA interactions have been examined using modified second order perturbation theory along with multicentred-multipole expansion technique. Results have been discussed in the light of other theoretical and experimental observations. Efforts have been made to elucidate the binding patterns and thereby biological properties of the drug.

  9. Ni(II) complexes of arginine Schiff-bases and its interaction with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sallam, S.A., E-mail: shehabsallam@yahoo.com [Chemistry Department, Faculty of Science, Suez Canal University, Isamilia (Egypt); Abbas, A.M. [Chemistry Department, Faculty of Science, Suez Canal University, Isamilia (Egypt)

    2013-04-15

    Ni(II) complexes with Schiff-bases obtained by condensation of arginine with salicylaldehyde; 2,3-; 2,4-; 2,5-dihydroxybenzaldehyde and o-hydroxynaphthaldehyde have been synthesized using the template method in ethanol or ammonia media. They were characterized by elemental analyses, conductivity measurements, magnetic moment, UV, IR and {sup 1}H NMR spectra as well as thermal analysis (TG, DTG and DTA). The Schiff-bases are dibasic tridentate donors and the complexes have diamagnetic square planar and octahedral structures. The complexes decompose in three steps where kinetic and thermodynamic parameters of the decomposition steps were computed. The interactions of the formed complexes with FM-DNA were monitored by UV and fluorescence spectroscopy. -- Highlights: ► Arginine Schiff-bases and their nickel(II) complexes have been synthesized. ► Magnetic and spectral data show diamagnetic square planar and octahedral complexes. ► The complexes thermally decompose in three stages. Interaction with FM-DNA shows hyperchromism with blue shift.

  10. Ni(II) complexes of arginine Schiff-bases and its interaction with DNA

    International Nuclear Information System (INIS)

    Sallam, S.A.; Abbas, A.M.

    2013-01-01

    Ni(II) complexes with Schiff-bases obtained by condensation of arginine with salicylaldehyde; 2,3-; 2,4-; 2,5-dihydroxybenzaldehyde and o-hydroxynaphthaldehyde have been synthesized using the template method in ethanol or ammonia media. They were characterized by elemental analyses, conductivity measurements, magnetic moment, UV, IR and 1 H NMR spectra as well as thermal analysis (TG, DTG and DTA). The Schiff-bases are dibasic tridentate donors and the complexes have diamagnetic square planar and octahedral structures. The complexes decompose in three steps where kinetic and thermodynamic parameters of the decomposition steps were computed. The interactions of the formed complexes with FM-DNA were monitored by UV and fluorescence spectroscopy. -- Highlights: ► Arginine Schiff-bases and their nickel(II) complexes have been synthesized. ► Magnetic and spectral data show diamagnetic square planar and octahedral complexes. ► The complexes thermally decompose in three stages. Interaction with FM-DNA shows hyperchromism with blue shift

  11. A Penalty Method to Model Particle Interactions in DNA-laden Flows

    International Nuclear Information System (INIS)

    Trebotich, D; Miller, G H; Bybee, M D

    2006-01-01

    We present a hybrid fluid-particle algorithm to simulate flow and transport of DNA-laden fluids in microdevices. Relevant length scales in microfluidic systems range from characteristic channel sizes of millimeters to micron scale geometric variation (e.g., post arrays) to 10 nanometers for the length of a single rod in a bead-rod polymer representation of a biological material such as DNA. The method is based on a previous fluid-particle algorithm in which long molecules are represented as a chain of connected rods, but in which the physically unrealistic behavior of rod crossing occurred. We have extended this algorithm to include screened Coulombic forces between particles by implementing a Debye-Hueckel potential acting between rods. In the method an unsteady incompressible Newtonian fluid is discretized with a second-order finite difference method in the interior of the Cartesian grid domain; an embedded boundary volume-of-fluid formulation is used near boundaries. The bead-rod polymer model is fully coupled to the solvent through body forces representing hydrodynamic drag and stochastic thermal fluctuations. While intrapolymer interactions are modeled by a soft potential, polymer-structure interactions are treated as perfectly elastic collisions. We demonstrate this method on flow and transport of a polymer through a post array microchannel in 2D where the polymer incorporates more realistic physical parameters of DNA, and compare to previous simulations where rods are allowed to cross. We also show that the method is capable of simulating 3D flow in a packed bed micro-column

  12. A Penalty Method to Model Particle Interactions in DNA-laden Flows

    Energy Technology Data Exchange (ETDEWEB)

    Trebotich, D; Miller, G H; Bybee, M D

    2006-10-06

    We present a hybrid fluid-particle algorithm to simulate flow and transport of DNA-laden fluids in microdevices. Relevant length scales in microfluidic systems range from characteristic channel sizes of millimeters to micron scale geometric variation (e.g., post arrays) to 10 nanometers for the length of a single rod in a bead-rod polymer representation of a biological material such as DNA. The method is based on a previous fluid-particle algorithm in which long molecules are represented as a chain of connected rods, but in which the physically unrealistic behavior of rod crossing occurred. We have extended this algorithm to include screened Coulombic forces between particles by implementing a Debye-Hueckel potential acting between rods. In the method an unsteady incompressible Newtonian fluid is discretized with a second-order finite difference method in the interior of the Cartesian grid domain; an embedded boundary volume-of-fluid formulation is used near boundaries. The bead-rod polymer model is fully coupled to the solvent through body forces representing hydrodynamic drag and stochastic thermal fluctuations. While intrapolymer interactions are modeled by a soft potential, polymer-structure interactions are treated as perfectly elastic collisions. We demonstrate this method on flow and transport of a polymer through a post array microchannel in 2D where the polymer incorporates more realistic physical parameters of DNA, and compare to previous simulations where rods are allowed to cross. We also show that the method is capable of simulating 3D flow in a packed bed micro-column.

  13. DNA Methylation Variants at HIF3A Locus, B-Vitamin Intake, and Long-term Weight Change: Gene-Diet Interactions in Two U.S. Cohorts.

    Science.gov (United States)

    Huang, Tao; Zheng, Yan; Qi, Qibin; Xu, Min; Ley, Sylvia H; Li, Yanping; Kang, Jae H; Wiggs, Janey; Pasquale, Louis R; Chan, Andrew T; Rimm, Eric B; Hunter, David J; Manson, JoAnn E; Willett, Walter C; Hu, Frank B; Qi, Lu

    2015-09-01

    The first epigenome-wide association study of BMI identified DNA methylation at an HIF3A locus associated with BMI. We tested the hypothesis that DNA methylation variants are associated with BMI according to intake of B vitamins. In two large cohorts, we found significant interactions between the DNA methylation-associated HIF3A single nucleotide polymorphism (SNP) rs3826795 and intake of B vitamins on 10-year changes in BMI. The association between rs3826795 and BMI changes consistently increased across the tertiles of total vitamin B2 and B12 intake (all P for interaction vitamin B2 intake and -0.10 (SE 0.06), -0.01 (SE 0.06), and 0.10 (SE 0.07) within subgroups defined by increasing tertiles of total vitamin B12 intake. In two independent cohorts, a DNA methylation variant in HIF3A was associated with BMI changes through interactions with total or supplemental vitamin B2, vitamin B12, and folate. These findings suggest a potential causal relation between DNA methylation and adiposity. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  14. The preliminary study on the inductory signal triggering the error-prone DNA repair function in mammalian cells

    International Nuclear Information System (INIS)

    Su Zaozhong; Luo Zuyu

    1989-01-01

    The nature of the signal triggering error-prone DNA repair function in mammalian cells was studied from two notions: (1) Does the inducing signal result from the direct hitting the cellular targets by DNA-damaging agents? (2) Is inhibition of DNA replication a prerequisite condition for the triggering effect? Thus, the ultraviolet (UV)-irradiated exogenous DNAs were introduced into human and rat cells by transfection. The results showed that this transfection was able to induce the error-prone repair as efficient as direct UV-irradiation to cells. Moreover, the two inductory treaetments expressed similar kinetics and dose-responses. No matter whether the introduced DNAs initiated replication, they exhibited the incuctory activity. Therefore, it can be considered that DNA lesions itself, not the direct interaction of DNA-damaging agents with specific cellular targets, serve as a triggering signal for the inductory process. Inhibition of DNA replication is not a prerequisite for the inductory signal

  15. Interaction between the Chlamydia trachomatis histone H1-like protein (Hc1) and DNA

    DEFF Research Database (Denmark)

    Christiansen, G; Pedersen, Lotte Bang; Koehler, J E

    1993-01-01

    maintained its DNA-binding capacity and was able at high concentrations to form condensed aggregates with DNA (one molecule of Hc1 per base pair) independently of the form or size of the DNA but with a slight preference for supercoiled DNA. Hc1 alone is thus able to package DNA into condensed spherical...

  16. Fluorescence Microscopy of Nanochannel-Confined DNA.

    Science.gov (United States)

    Westerlund, Fredrik; Persson, Fredrik; Fritzsche, Joachim; Beech, Jason P; Tegenfeldt, Jonas O

    2018-01-01

    Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level and both the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments and analyze the data.

  17. Time of flight Laue fiber diffraction studies of perdeuterated DNA

    Energy Technology Data Exchange (ETDEWEB)

    Forsyth, V.T.; Whalley, M.A.; Mahendrasingam, A.; Fuller, W. [Keele Univ. (United Kingdom)] [and others

    1994-12-31

    The diffractometer SXD at the Rutherford Appleton Laboratory ISIS pulsed neutron source has been used to record high resolution time-of-flight Laue fiber diffraction data from DNA. These experiments, which are the first of their kind, were undertaken using fibers of DNA in the A conformation and prepared using deuterated DNA in order to minimis incoherent background scattering. These studies complement previous experiments on instrument D19 at the Institute Laue Langevin using monochromatic neutrons. Sample preparation involved drawing large numbers of these deuterated DNA fibers and mounting them in a parallel array. The strategy of data collection is discussed in terms of camera design, sample environment and data collection. The methods used to correct the recorded time-of-flight data and map it into the final reciprocal space fiber diffraction dataset are also discussed. Difference Fourier maps showing the distribution of water around A-DNA calculated on the basis of these data are compared with results obtained using data recorded from hydrogenated A-DNA on D19. Since the methods used for sample preparation, data collection and data processing are fundamentally different for the monochromatic and Laue techniques, the results of these experiments also afford a valuable opportunity to independently test the data reduction and analysis techniques used in the two methods.

  18. DNA hosted and aligned in aqueous interstitia of a lamellar liquid crystal – a membrane–biomacromolecule interaction model system

    KAUST Repository

    Carlsson, Nils; Jonsson, Fabian; Wilhelmsson, L. Marcus; Nordé n, Bengt; Å kerman, Bjö rn

    2013-01-01

    We report that DNA molecules can be intercalated and macroscopically oriented in the aqueous interstitia of a lyotropic lamellar liquid crystal. Using UV-vis linear dichroism and fluorescence spectroscopy we show that double-stranded oligonucleotides (25 base pairs) in the water-octanoate-decanol system remain base-paired in the B conformation and are confined in two dimensions, with the helix axis preferentially parallel to the lipid bilayer surfaces but free to rotate within this plane. The degree of helix confinement and the corresponding 2-D orientation can be improved by decreasing the thickness of the water interstitia via the fraction of water in the ternary mixture. Not surprisingly, the corresponding single-stranded oligonucleotides are not aligned, with their persistence length being short in comparison to the lamellar interstitium thickness. We propose this as a model system for studying interactions of DNA-ligand complexes near a lipid bilayer membrane which we demonstrate by using dye probes that are either covalently attached to one end of the oligonucleotide or reversibly bound by intercalation between the base pairs. Three cationic dyes, all strongly bound by intercalation to DNA when free in solution, are found to not bind to DNA but to prefer the membrane surface. The covalently attached Cy5 also binds to the bilayer while Cy3 tends to end-stack to the oligonucleotide duplex. The orientation of Cy5 parallel to the membrane indicates that electrostatic surface binding predominates over insertion into the hydrophobic interior of the membrane. Anionic and zwitterionic dyes (FAM and ROX) are found to remain randomly oriented in the water between the lipid bilayer surfaces. © The Royal Society of Chemistry.

  19. Fast repair of oxidizing OH adducts of DNA by hydroxycinnamic acid derivatives. A pulse radiolytic study

    International Nuclear Information System (INIS)

    Yue Jiang; Lin Weizhen; Yao Side; Lin Nianyun; Zhu Dayuan

    1999-01-01

    Using pulse radiolytic techniques, it has been demonstrated that the interactions of oxidizing OH adducts of DNA (ssDNA and dsDNA), polyA and polyG with hydroxycinnamic acid derivatives proceed via an electron transfer process (k=5-30x10 8 dm 3 mol -1 s -1 ). In addition, the rates for fast repair of OH adducts of dAMP, polyA and DNA (ssDNA and dsDNA) are slower than the corresponding rates for the rest OH adducts of DNA constituents. The slower rates for repair of oxidizing OH adducts of dAMP may be the rate determining step during the interaction of hydroxycinnamic acid derivatives with OH adducts of DNA containing the varieties of OH adducts of DNA constituents

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

    Energy Technology Data Exchange (ETDEWEB)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L. (UW-MED); (UCB)

    2015-04-22

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

  1. Differential Nuclear and Mitochondrial DNA Preservation in Post-Mortem Teeth with Implications for Forensic and Ancient DNA Studies

    Science.gov (United States)

    Higgins, Denice; Rohrlach, Adam B.; Kaidonis, John; Townsend, Grant; Austin, Jeremy J.

    2015-01-01

    Major advances in genetic analysis of skeletal remains have been made over the last decade, primarily due to improvements in post-DNA-extraction techniques. Despite this, a key challenge for DNA analysis of skeletal remains is the limited yield of DNA recovered from these poorly preserved samples. Enhanced DNA recovery by improved sampling and extraction techniques would allow further advancements. However, little is known about the post-mortem kinetics of DNA degradation and whether the rate of degradation varies between nuclear and mitochondrial DNA or across different skeletal tissues. This knowledge, along with information regarding ante-mortem DNA distribution within skeletal elements, would inform sampling protocols facilitating development of improved extraction processes. Here we present a combined genetic and histological examination of DNA content and rates of DNA degradation in the different tooth tissues of 150 human molars over short-medium post-mortem intervals. DNA was extracted from coronal dentine, root dentine, cementum and pulp of 114 teeth via a silica column method and the remaining 36 teeth were examined histologically. Real time quantification assays based on two nuclear DNA fragments (67 bp and 156 bp) and one mitochondrial DNA fragment (77 bp) showed nuclear and mitochondrial DNA degraded exponentially, but at different rates, depending on post-mortem interval and soil temperature. In contrast to previous studies, we identified differential survival of nuclear and mtDNA in different tooth tissues. Futhermore histological examination showed pulp and dentine were rapidly affected by loss of structural integrity, and pulp was completely destroyed in a relatively short time period. Conversely, cementum showed little structural change over the same time period. Finally, we confirm that targeted sampling of cementum from teeth buried for up to 16 months can provide a reliable source of nuclear DNA for STR-based genotyping using standard

  2. Dielectric behavior of irradiated and nonirradiadiated deoxyribonucleic acid (DNA)-crotonic acid interaction in 5% dextrose solution

    International Nuclear Information System (INIS)

    Erginun, M.

    1980-01-01

    Deoxyribonucleic acid (DNA), ex. thymus, dissolved in 5% dextrose, was exposed to gamma radiation at doses between 0-5000 Rads. Crotonic acid dissolved in 5% dextrose was added to this irradiated DNA at t=0 and t=24 hrs after irradiation, in concentrations between 0-1.000 mg/ml. The dielectric behavior of the DNA-irradiation-crotonic acid interaction was investigated at T=20 0 C by pH, permittivity (dielectric constant) and conductivity measurements. The pH, permittivity and conductivity measurements exhibit that the effective and critical conditions for the DNA-irradiation-crotonic acid interaction are; low doses of irradiation (350 Rad.), low concentrations of crotonic acid (0.05-0.100 mg/ml) and the addition of crotonic acid 24 hours after the irradiation. These results support and are in good agreement with those results observed with mammalian cells and laboratory animals when the chemical carcinogens are given in conjunction with radiation

  3. Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

    Directory of Open Access Journals (Sweden)

    Eveson J Paige

    2006-08-01

    Full Text Available Abstract Background Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ can be estimated by determining the rate of decline. Results The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λpredator = 0.0106 per nucleotide; mean λprey = 0.0176 per nucleotide. This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. Conclusion We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will

  4. In vitro study of DNA Adduct 8-OHdG Formation by using Bisphenol A in Calf Thymus DNA and 2’-Deoxyguanosine

    Science.gov (United States)

    Budiawan; Cahaya Dani, Intan; Bakri, Ridla; Handayani, Sri; Ratna Dewi, Evi

    2018-01-01

    The in vitro study of DNA Adduct 8-OHdG Formation due to BisphenolA (BPA) as xenobiotics has been conducted by using calf thymus DNA and 2’deoxyguanosine. The method of study was conducted by incubating calf thymus DNA and 2’dG with compounds trigger to radicals in the variation of pH (7.4 and 8.4), temperature (37°C and 60°C), and BPA concentrations (2 ppm and 10 ppm). To represent the work of CYP 450 enzyme in metabolic process of xenobiotics in the body and the effect of metal presence to the formation of radicals that can lead to 8-OHdG formation, we used iron(II) solution and also fenton reagent (Fe(II) and H2O2). The DNA used has 1.8 purity ratio (checked at λ260/λ280 by using Spectrophotometry UV-Vis). The results by using HPLC method showed that BPA could interact with DNA and DNA base (represent as calf thymus and 2’dG) and potentially induced 8-OHdG formation. The presence of iron(II) metal and Fenton reagent also induced the higher 8-OHdG formation. The higher of pH, temperature and concentrations also lead to 8-OHdG formation (ranger between 4 - 70 ppb).

  5. Photoluminescence studies of a Terbium(III) complex as a fluorescent probe for DNA detection

    Energy Technology Data Exchange (ETDEWEB)

    Khorasani-Motlagh, Mozhgan, E-mail: mkhorasani@chem.usb.ac.ir; Noroozifar, Meissam; Niroomand, Sona; Moodi, Asieh

    2013-11-15

    The photoluminescence properties of a Tb(III) complex of the form [Tb(phen){sub 2}Cl{sub 3}·OH{sub 2}] (phen=1,10-phenanthroline) in different solvents are presented. It shows the characteristic luminescence of the corresponding Ln{sup 3+} ion in the visible region. The emission intensity of this complex in coordinating solvent is higher than non-coordinating one. The suggested mechanism for the energy transfer between the ligand and Tb{sup 3+} ion is the intramolecular energy transfer mechanism. The interactions of the Tb(III) complex with fish salmon DNA are studied by fluorescence spectroscopy, circular dichroism study and viscosity measurements. The results of fluorescence titration reveal that DNA strongly quenches the intrinsic fluorescence of the complex through a static quenching procedure. The binding constant (K{sub b}) of the above metal complex at 25 °C is determined by the fluorescence titration method and it is found to be (8.06±0.01)×10{sup 3} M{sup −1}. The thermodynamic parameters (ΔH{sup 0}>0, ΔS{sup 0}>0 and ΔG{sup 0}<0) indicate that the hydrophobic interactions play a major role in DNA–Tb complex association. The results support the claim that the title complex bonds to FS-DNA by a groove mode. -- Highlights: • Photoluminescence of [Tb(phen){sub 2}Cl{sub 3}·OH{sub 2}] in different solvents are studied. • Tb(III) complex shows good binding affinity to FS DNA with K{sub b}=(8.06±0.01)×10{sup 3} M{sup −1}. • Viscosity of DNA almost unchanged by increasing amount of Tb complex. • CD spectrum of DNA has a little change with increasing amount of Tb complex. • Thermodynamic parameters indicate that the binding reaction is entropically driven.

  6. Interaction of an Fe derivative of TMAP (Fe(TMAP)OAc) with DNA in comparison with free-base TMAP.

    Science.gov (United States)

    Ghaderi, Masoumeh; Bathaie, S Zahra; Saboury, Ali-Akbar; Sharghi, Hashem; Tangestaninejad, Shahram

    2007-07-01

    We investigated the interaction of meso-tetrakis (N-para-methylanilium) porphyrin (TMAP) in its free base and Fe(II) form (Fe(TMAP)OAc) as a new derivative, with high molecular weight DNA at different ionic strengths, using various spectroscopic methods and microcalorimetry. The data obtained by spectrophotometery, circular dichroism (CD), fluorescence quenching and resonance light scattering (RLS) have demonstrated that TMAP association with DNA is via outside binding with self-stacking manner, which is accompanied with the "end-on" type complex formation in low ionic strength. However, in the case of Fe(TMAP)OAc, predominant mode of interaction is groove binding and after increasing in DNA concentration, unstable stacking-type aggregates are formed. In addition, isothermal titration calorimetric measurements have indicated the exothermic process of porphyrins binding to DNA, but the exothermisity in metal derivative of porphyrin is less than the free base. It confirmed the formation of a more organized aggregate of TMAP on DNA surface. Interactions of both porphyrins with DNA show high sensitivity to ionic strength. By addition of salt, the downfield CD signal of TMAP aggregates is shifted to a higher wavelength, which indicates some changes in the aggregates position. In the case of Fe(TMAP)OAc, addition of salt leads to changes in the mode of binding from groove binding to outside binding with self-stacking, which is accompanied with major changes in CD spectra, possibly indicating the formation of "face-on" type complex.

  7. An immunochemical approach to the study of DNA damage and repair

    International Nuclear Information System (INIS)

    Wallace, S.S.; Erlanger, B.F.

    1992-05-01

    The overall objective of this project has been to develop immunochemical methods to quantitate unique DNA base damages in order to facilitate studies on radiation-induced damage production and repair. Specifically, we have been using antibodies raised to damaged bases to quantitate unique lesions in model systems in order to evaluate their potential biological consequences. Our approach has been to synthesize modified nucleotides or nucleosides, conjugate them to protein carriers, and use the conjugates as immunogens in rabbits or to prepare monoclonal antibodies. We have been studying damages that are stable radiolysis products found in X-irradiated DNA and thus of potential biological consequence. Our aim is to build an in vitro and in vivo data base on the interactions between model DNA lesions and such cellular enzymes as DNA polymerases and repair endonucleases. Initial studies have focused on pyrimidine ring saturation products (thymine glycol.and dihydrothymine), products resulting from ring fragmentation or base loss (urea, Β-ureidoisobutyric acid, abasic sites), 7-hydro-8-oxopurines, and more recently, cytosine radiolysis products. These modified bases serve as useful models for examining the potential lethal and/or mutagenic (carcinogenic) effects of the products of DNA radiolysis

  8. NMR studies of a new family of DNA binding proteins: the THAP proteins

    International Nuclear Information System (INIS)

    Gervais, Virginie; Campagne, Sébastien; Durand, Jade; Muller, Isabelle; Milon, Alain

    2013-01-01

    The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

  9. NMR studies of a new family of DNA binding proteins: the THAP proteins

    Energy Technology Data Exchange (ETDEWEB)

    Gervais, Virginie, E-mail: virginie.gervais@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France); Campagne, Sebastien [ETH Zurich (Switzerland); Durand, Jade; Muller, Isabelle; Milon, Alain, E-mail: alain.milon@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France)

    2013-05-15

    The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

  10. Interaction between prenatal pesticide exposure and a common polymorphism in the PON1 gene on DNA methylation in genes associated with cardio-metabolic disease risk-an exploratory study

    DEFF Research Database (Denmark)

    Declerck, Ken; Remy, Sylvie; Wohlfahrt-Veje, Christine

    2017-01-01

    BACKGROUND: Prenatal environmental conditions may influence disease risk in later life. We previously found a gene-environment interaction between the paraoxonase 1 (PON1) Q192R genotype and prenatal pesticide exposure leading to an adverse cardio-metabolic risk profile at school age. However...... was observed in prenatally pesticide exposed children carrying the PON1 192R-allele. Differentially methylated genes were enriched in several neuroendocrine signaling pathways including dopamine-DARPP32 feedback (appetite, reward pathways), corticotrophin releasing hormone signaling, nNOS, neuregulin signaling...

  11. Identification of DNA polymerase molecules repairing DNA irradiated damage and molecular biological study on modified factors of mutation rate

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Koichi; Inoue, Shuji [National Inst. of Healthand Nutrition, Tokyo (Japan)

    1999-02-01

    DNA repairing polymerase has not been identified in human culture cells because the specificities of enzyme inhibitors used in previous studies were not so high. In this study, anti-sense oligonucleotides were transfected into human fibroblast cells by electroporation and several clones selected by geneticin treatment were found to express the RNA of the incorporated DNA. However, the expression was not significant and its reproducibility was poor. Then, a study on repairing mechanism was made using XP30 RO and XP 115 LO cells which are variant cells of xeroderma pigmentosum, a human hereditary disease aiming to identify the DNA polymerase related to the disease. There were abnormalities in DNA polymerase subunit {delta} or {epsilon} which consists DNA replication complex. Thus, it was suggested that the DNA replication of these mutant cells might terminate at the site containing such abnormality. (M.N.)

  12. Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction.

    Science.gov (United States)

    Hicks, Matt N; Gunasekara, Sanjiva; Serate, Jose; Park, Jin; Mosharaf, Pegah; Zhou, Yue; Lee, Jin-Won; Youn, Hwan

    2017-10-01

    The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP's recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.

  13. The Inner Membrane Protein PilG Interacts with DNA and the Secretin PilQ in Transformation.

    Directory of Open Access Journals (Sweden)

    Stephan A Frye

    Full Text Available Expression of type IV pili (Tfp, filamentous appendages emanating from the bacterial surface, is indispensable for efficient neisserial transformation. Tfp pass through the secretin pore consisting of the membrane protein PilQ. PilG is a polytopic membrane protein, conserved in Gram-positive and Gram-negative bacteria, that is required for the biogenesis of neisserial Tfp. PilG null mutants are devoid of pili and non-competent for transformation. Here, recombinant full-length, truncated and mutated variants of meningococcal PilG were overexpressed, purified and characterized. We report that meningococcal PilG directly binds DNA in vitro, detected by both an electromobility shift analysis and a solid phase overlay assay. PilG DNA binding activity was independent of the presence of the consensus DNA uptake sequence. PilG-mediated DNA binding affinity was mapped to the N-terminus and was inactivated by mutation of residues 43 to 45. Notably, reduced meningococcal transformation of DNA in vivo was observed when PilG residues 43 to 45 were substituted by alanine in situ, defining a biologically significant DNA binding domain. N-terminal PilG also interacted with the N-terminal region of PilQ, which previously was shown to bind DNA. Collectively, these data suggest that PilG and PilQ in concert bind DNA during Tfp-mediated transformation.

  14. Immobilization of DNA at Glassy Ccarbon Electrodes: A Critical Study of Adsorbed Layer

    Directory of Open Access Journals (Sweden)

    G. A. Rivas

    2005-11-01

    Full Text Available In this work we present a critical study of the nucleic acid layer immobilized atglassy carbon electrodes. Different studies were performed in order to assess the nature of theinteraction between DNA and the electrode surface. The adsorption and electrooxidation of DNAdemonstrated to be highly dependent on the surface and nature of the glassy carbon electrode. TheDNA layer immobilized at a freshly polished glassy carbon electrode was very stable even afterapplying highly negative potentials. The electron transfer of potassium ferricyanide, catechol anddopamine at glassy carbon surfaces modified with thin (obtained by adsorption under controlledpotential conditions and thick (obtained by casting the glassy carbon surface with highly concentratedDNA solutions DNA layers was slower than that at the bare glassy carbon electrode, although thiseffect was dependent on the thickness of the layer and was not charge selective. Raman experimentsshowed an important decrease of the vibrational modes assigned to the nucleobases residues,suggesting a strong interaction of these residues with the electrode surface. The hybridization ofoligo(dG21 and oligo(dC21 was evaluated from the guanine oxidation signal and the reduction of theredox indicator Co(phen33+ . In both cases the chronopotentiometric response indicated that thecompromise of the bases in the interaction of DNA with the electrode surface is too strong, preventingfurther hybridization. In summary, glassy carbon is a useful electrode material to detect DNA in adirect and very sensitive way, but not to be used for the preparation of biorecognition layers by directadsorption of the probe sequence on the electrode surface for detecting the hybridization event.

  15. Synthesis and DNA-binding study of imidazole linked thiazolidinone derivatives.

    Science.gov (United States)

    War, Javeed Ahmad; Srivastava, Santosh Kumar; Srivastava, Savitri Devi

    2017-02-01

    A novel series of imidazole-linked thiazolidinone hybrid molecules were designed and synthesized through a feasible synthetic protocol. The molecules were characterized with Fourier transform infrared (FT-IR), 1 H nuclear magnetic resonance (NMR), 13 C NMR and high-resolution mass spectrometry (HRMS) techniques. In vitro susceptibility tests against Gram-positive (S. aureus and B. subtilis) and Gram-negative bacteria (E. coli and P. aeruginosa) gave highly promising results. The most active molecule (3e) gave a minimal inhibitory concentration (MIC) value of 3.125 μg/mL which is on par with the reference drug streptomycin. Structure-activity relationships revealed activity enhancement by nitro and chloro groups when they occupied meta position of the arylidene ring in 2-((3-(imidazol-1-yl)propyl)amino)-5-benzylidenethiazolidin-4-ones. DNA-binding study of the most potent molecule 3e with salmon milt DNA (sm-DNA) under simulated physiological pH was probed with UV-visible absorption, fluorescence quenching, gel electrophoresis and molecular docking techniques. These studies established that compound 3e has a strong affinity towards DNA and binds at DNA minor groove with a binding constant (K b ) 0.18 × 10 2  L mol -1 . Molecular docking simulations predicted strong affinity of 3e towards DNA with a binding affinity (ΔG) -8.5 kcal/mol. Van der Waals forces, hydrogen bonding and hydrophobic interactions were predicted as the main forces of interaction. The molecule 3e exhibited specific affinity towards adenine-thiamine base pairs. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  16. Ferrocene-based guanidine derivatives: in vitro antimicrobial, DNA binding and docking supported urease inhibition studies.

    Science.gov (United States)

    Gul, Rukhsana; Rauf, Muhammad Khawar; Badshah, Amin; Azam, Syed Sikander; Tahir, Muhammad Nawaz; Khan, Azim

    2014-10-06

    Some novel ferrocenyl guanidines 1-8 were synthesized and characterized by different spectroscopic methods, elemental analysis and single crystal X-rays diffraction techniques. The crystallographic studies revealed that the existence of the strong non-bonding interactions facilitate these molecules to interact with biological macro-molecules like DNA that described to inherit good biological activities. The DNA interaction studies carried out by cyclic voltammetry (CV) and UV-visible spectroscopy are in close agreement with the binding constants (K) (0.79-5.4) × 10(5) (CV) and (0.72-5.1) × 10(5) (UV-vis). The shift in peak potential, current and absorption maxima of the studied ferrocenyl guanidines in the presence of DNA revealed that CV coupled with UV-vis spectroscopy could provide an opportune to characterize metal-based compounds-DNA interaction mechanism, a prerequisite for the design of new anticancer agents and understanding the molecular basis of their action. The compounds 1-8 have been screened for their antibacterial, antifungal and urease inhibition potency. A concurrent in silico study has also been applied on ferrocene moiety impregnated guanidines 1-8 to identify most active compounds having for inhibiting the activity of urease (pdb id 3LA4). Most of the compounds were found as potent inhibitors of urease and the compound 1 was found to be the most active with an IC50 of 16.83 ± 0.03 μM. The docking scores are in close agreement with the in vitro obtained IC50 values of inhibitors 1-8. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  17. Tyr120Asp mutation alters domain flexibility and dynamics of MeCP2 DNA binding domain leading to impaired DNA interaction: Atomistic characterization of a Rett syndrome causing mutation.

    Science.gov (United States)

    D'Annessa, Ilda; Gandaglia, Anna; Brivio, Elena; Stefanelli, Gilda; Frasca, Angelisa; Landsberger, Nicoletta; Di Marino, Daniele

    2018-05-01

    Mutations in the X-linked MECP2 gene represent the main origin of Rett syndrome, causing a profound intellectual disability in females. MeCP2 is an epigenetic transcriptional regulator containing two main functional domains: a methyl-CpG binding domain (MBD) and a transcription repression domain (TRD). Over 600 pathogenic mutations were reported to affect the whole protein; almost half of missense mutations affect the MBD. Understanding the impact of these mutations on the MBD structure and interaction with DNA will foster the comprehension of their pathogenicity and possibly genotype/phenotype correlation studies. Herein, we use molecular dynamics simulations to obtain a detailed view of the dynamics of WT and mutated MBD in the presence and absence of DNA. The pathogenic mutation Y120D is used as paradigm for our studies. Further, since the Y120 residue was previously found to be a phosphorylation site, we characterize the dynamic profile of the MBD also in the presence of Y120 phosphorylation (pY120). We found that addition of a phosphate group to Y120 or mutation in aspartic acid affect domain mobility that samples an alternative conformational space with respect to the WT, leading to impaired ability to interact with DNA. Experimental assays showing a significant reduction in the binding affinity between the mutated MBD and the DNA confirmed our predictions. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Pulse radiolysis studies on DNA-Binding radioprotectors

    International Nuclear Information System (INIS)

    Anderson, R.F.

    1996-01-01

    Full text: Hoechst 33342 and newly-synthesised analogues exhibit radioprotective activity in cultured cells and in vivo, as described in accompanying abstracts. These minor groove binding ligands bind at discreet sites in DNA, characterised by 3 to 4 consecutive AT base pairs, and DNA sequencing studies have shown focussed radioprotection at these binding sites. There is evidence that the bound ligands also confer more 'global' protection including the intervening DNA between the binding sites. The observed focussed radioprotection could be explained by H-atom donation from the ligand to radiation-induced carbon-centred deoxyribosyl radicals, but this mechanism is unlikely to account for the global radioprotection. We now report pulse radiolysis studies on another possible mechanism, namely reduction of transient radiation-induced oxidising species on DNA by the ligand, which is consistent with the report of reduction of G + by TMPD. Oxidation of deoxyguanosine (dG) by Br 2 - , produced by radiolysis of Br- in N 2 0-saturated solutions, in the presence of Hoechst 33342 results in the appearance of a transient ligand species which is kinetically resolvable from that obtained from direct oxidation of Hoechst 33342 by Br 2 - . A plot of reaction rate versus ligand concentration indicates that the rate constant for reduction of G + is approximately 3 x 10 8 dm 3 M -1 sec -1 . Similar experiments with DNA, rather than dG, also revealed a transient species corresponding to oxidation of the ligand, but the absolute rate of oxidation was considerably slower for the DNA-bound ligand compared to that for oxidation of the free ligand by G+. These results are clearly consistent with the proposed mechanism of radioprotection by Hoechst 33342 and its analogues, moreover, pulse radiolysis may provide a very useful endpoint for screening new analogues, as a preliminary to radiobiological evaluation

  19. Specificity of interactions among the DNA-packaging machine components of T4-related bacteriophages.

    Science.gov (United States)

    Gao, Song; Rao, Venigalla B

    2011-02-04

    Tailed bacteriophages use powerful molecular motors to package the viral genome into a preformed capsid. Packaging at a rate of up to ∼2000 bp/s and generating a power density twice that of an automobile engine, the phage T4 motor is the fastest and most powerful reported to date. Central to DNA packaging are dynamic interactions among the packaging components, capsid (gp23), portal (gp20), motor (gp17, large "terminase"), and regulator (gp16, small terminase), leading to precise orchestration of the packaging process, but the mechanisms are poorly understood. Here we analyzed the interactions between small and large terminases of T4-related phages. Our results show that the gp17 packaging ATPase is maximally stimulated by homologous, but not heterologous, gp16. Multiple interaction sites are identified in both gp16 and gp17. The specificity determinants in gp16 are clustered in the diverged N- and C-terminal domains (regions I-III). Swapping of diverged region(s), such as replacing C-terminal RB49 region III with that of T4, switched ATPase stimulation specificity. Two specificity regions, amino acids 37-52 and 290-315, are identified in or near the gp17-ATPase "transmission" subdomain II. gp16 binding at these sites might cause a conformational change positioning the ATPase-coupling residues into the catalytic pocket, triggering ATP hydrolysis. These results lead to a model in which multiple weak interactions between motor and regulator allow dynamic assembly and disassembly of various packaging complexes, depending on the functional state of the packaging machine. This might be a general mechanism for regulation of the phage packaging machine and other complex molecular machines.

  20. Characterization of Ofloxacin Interaction with Mutated (A91V) Quinolone Resistance Determining Region of DNA Gyrase in Mycobacterium Leprae through Computational Simulation.

    Science.gov (United States)

    Nisha, J; Shanthi, V

    2018-06-01

    Mycobacterium leprae, the causal agent of leprosy is non-cultivable in vitro. Thus, the assessment of antibiotic activity against Mycobacterium leprae depends primarily upon the time-consuming mouse footpad system. The GyrA protein of Mycobacterium leprae is the target of the antimycobacterial drug, Ofloxacin. In recent times, the GyrA mutation (A91V) has been found to be resistant to Ofloxacin. This phenomenon has necessitated the development of new, long-acting antimycobacterial compounds. The underlying mechanism of drug resistance is not completely known. Currently, experimentally crystallized GyrA-DNA-OFLX models are not available for highlighting the binding and mechanism of Ofloxacin resistance. Hence, we employed computational approaches to characterize the Ofloxacin interaction with both the native and mutant forms of GyrA complexed with DNA. Binding energy measurements obtained from molecular docking studies highlights hydrogen bond-mediated efficient binding of Ofloxacin to Asp47 in the native GyrA-DNA complex in comparison with that of the mutant GyrA-DNA complex. Further, molecular dynamics studies highlighted the stable binding of Ofloxacin with native GyrA-DNA complex than with the mutant GyrA-DNA complex. This mechanism provided a plausible reason for the reported, reduced effect of Ofloxacin to control leprosy in individuals with the A91V mutation. Our report is the first of its kind wherein the basis for the Ofloxacin drug resistance mechanism has been explored with the help of ternary Mycobacterium leprae complex, GyrA-DNA-OFLX. These structural insights will provide useful information for designing new drugs to target the Ofloxacin-resistant DNA gyrase.

  1. Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

    KAUST Repository

    Ghosh, Sharmistha; Hamdan, Samir; Richardson, Charles C.

    2010-01-01

    The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

    KAUST Repository

    Ghosh, Sharmistha

    2010-04-06

    The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Detailed kinetic analysis of the interaction between the FOXO4–DNA-binding domain and DNA

    Czech Academy of Sciences Publication Activity Database

    Vácha, P.; Zusková, Iva; Bumba, Ladislav; Večeř, J.; Obšilová, Veronika; Obšil, T.

    2013-01-01

    Roč. 184, DEC 31 (2013), s. 68-78 ISSN 0301-4622 R&D Projects: GA ČR(CZ) GAP207/11/0717 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : binding kinetics * DNA-binding domain * FOXO4 forkhead transcription factor Subject RIV: BO - Biophysics; CE - Biochemistry (MBU-M) Impact factor: 2.319, year: 2013

  4. MAGNETIC TWEEZERS FOR THE STUDY OF DNA TRACKING MOTORS

    Science.gov (United States)

    Manosas, Maria; Meglio, Adrien; Spiering, Michelle M.; Ding, Fangyuan; Benkovic, Stephen J.; Barre, François-Xavier; Saleh, Omar A.; Allemand, Jean François; Bensimon, David; Croquette, Vincent

    2011-01-01

    Single-molecule manipulation methods have opened a new vista on the study of molecular motors. Here we describe the use of magnetic traps for the investigation of the mechanism of DNA based motors, in particular helicases and translocases. PMID:20627163

  5. Radioautographic DNA synthesis study on mice Mus musculus gingival epithelium

    International Nuclear Information System (INIS)

    Silveira Tarelho, Z.V. da; Hetem, S.

    1984-01-01

    The DNA-synthetizing cells frequency in the gingival epithelium basal layer of the first lower molar region in young and adult mice were studied. The 3H-thymidine and radioautography were used. The labeled cells frequency was determined by calculating their proportions. The data were statiscally analysed. (M.A.C.) [pt

  6. Xeroderma pigmentosum: recent studies on the DNA repair defects

    International Nuclear Information System (INIS)

    Friedberg, E.C.

    1978-01-01

    Xeroderma pigmentosum is a recessive autosomal disease of humans that is characterized by a high prevalence of skin cancers. Results of studies on cells from such patients indicate a defect in the repair of DNA damage associated with exposure to ultraviolet radiation. Since this observation was reported, a large amount of information on this disease has accumulated in the literature

  7. SSR marker based DNA fingerprinting and diversity study in rice ...

    African Journals Online (AJOL)

    The genetic diversity and DNA fingerprinting of 15 elite rice genotypes using 30 SSR primers on chromosome numbers 7-12 was investigated. The results revealed that all the primers showed distinct polymorphism among the cultivars studied indicating the robust nature of microsatellites in revealing polymorphism. Cluster ...

  8. Molecular dynamics simulation studies of radiation damaged DNA. Molecules and repair enzymes

    International Nuclear Information System (INIS)

    Pinak, Miroslav

    2004-12-01

    Molecular dynamics (MD) studies on several radiation damages to DNA and their recognition by repair enzymes are introduced in order to describe the stepwise description of molecular process observed at radiation lesion sites. MD studies were performed on pyrimidine (thymine dimer, thymine glycol) and purine (8-oxoguanine) lesions using an MD simulation code AMBER 5.0. The force field was modified for each lesion. In all cases the significant structural changes in the DNA double helical structure were observed; a) the breaking of hydrogen bond network between complementary bases and resulting opening of the double helix (8-oxoguanine); b) the sharp bending of the DNA helix centered at the lesion site (thymine dimer, thymine glycol); and c) the flipping-out base on the strand complementary to the lesion (8-oxoguanine). These changes were related to the overall collapsing double helical structure around the lesion and might facilitate the docking of the repair enzyme into the DNA and formation of DNA-enzyme complex. In addition to the structural changes, at lesion sites there were found electrostatic interaction energy values different from those at native sites (thymine dimer -10 kcal/mol, thymine glycol -26 kcal/mol, 8-oxoguanine -48 kcal/mol). These values of electrostatic energy may discriminate lesion from values at native sites (thymine 0 kcal/mol, guanine -37 kcal/mol) and enable a repair enzyme to recognize a lesion during scanning DNA surface. The observed specific structural conformation and energetic properties at the lesions sites are factors that guide a repair enzyme to discriminate lesions from non-damaged native DNA segments. (author)

  9. Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques

    Directory of Open Access Journals (Sweden)

    Zhongjie Xu

    2016-06-01

    Full Text Available Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT exhibits significant antitumor activity. However, the mechanism of its pharmacological interaction with human serum albumin (HSA and DNA remains poorly understood. Here, we aimed to elucidate the interactions of Dp44mT with HSA and DNA using MTT assays, spectroscopic methods, and molecular docking analysis. Our results indicated that addition of HSA at a ratio of 1:1 did not alter the cytotoxicity of Dp44mT, but did affect the cytotoxicity of the Dp44mT-Cu complex. Data from fluorescence quenching and UV-VIS absorbance measurements demonstrated that Dp44mT could bind to HSA with a moderate affinity (Ka = approximately 104 M−1. CD spectra revealed that Dp44mT could slightly disrupt the secondary structure of HSA. Dp44mT could also interact with Ct-DNA, but had a moderate binding constant (KEB = approximately 104 M−1. Docking studies indicated that the IB site of HSA, but not the IIA and IIIA sites, could be favorable for Dp44mT and that binding of Dp44mT to HSA involved hydrogen bonds and hydrophobic force, consistent with thermodynamic results from spectral investigations. Thus, the moderate binding affinity of Dp44mT with HSA and DNA partially contributed to its antitumor activity and may be preferable in drug design approaches.

  10. Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

    Science.gov (United States)

    Kachhap, Sangita; Singh, Balvinder

    2015-01-01

    In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

  11. The MTHFR 677TT genotype and folate intake interact to lower global leukocyte DNA methylation in young Mexican American women.

    OpenAIRE

    Axume, Juan; Smith, Steven S; Pogribny, Igor P; Moriarty, David J.; Caudill., Marie A.

    2007-01-01

    DNA methylation is an epigenetic feature that is associated with X chromosome inactivation, genomic imprinting, transcriptional silencing of genes and genomic stability. Folate provides a labile source of methyl groups which may be used for cellular methylation reactions including DNA methylation. The methylenetetrahydrofolate reductase (MTHFR) 677C→T variant is an important determinant of folate nutriture and may influence DNA methylation. This study sought to assess the influence of the MTH...

  12. Binding of Bisphenol-F, a bisphenol analogue, to calf thymus DNA by multi-spectroscopic and molecular docking studies.

    Science.gov (United States)

    Usman, Afia; Ahmad, Masood

    2017-08-01

    BPF (Bisphenol-F), a member of the bisphenol family, having a wide range of industrial applications is gradually replacing Bisphenol-A. It is a recognized endocrine disrupting chemical (EDC). EDCs have been implicated in increased incidences of breast, prostate and testis cancers besides diabetes, obesity and decreased fertility. Due to the adverse effects of EDCs on human health, attempts have been directed towards their mechanism of toxicity especially at the molecular level. Hence, to understand the mechanism at the DNA level, interaction of BPF with calf thymus DNA was studied employing multi-spectroscopic, voltammetric and molecular docking techniques. Fluorescence spectra, cyclic voltammetry (CV), circular dichroism (CD) and molecular docking studies of BPF with DNA were suggestive of minor groove binding of BPF. UV-visible absorption and fluorescence spectra suggested static quenching due to complex formation between BPF and ctDNA. Hoechst 33258 (HO) and ethidium bromide (EB) displacement studies further confirmed such mode of BPF interaction. Thermodynamic and molecular docking parameters revealed the mechanism of binding of BPF with ctDNA to be favorable and spontaneous due to negative ΔG and occurring through hydrogen bonds and van der waals interactions. BPF induced DNA cleavage under in vitro conditions by plasmid nicking assay suggested it to be genotoxic. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    Directory of Open Access Journals (Sweden)

    Yuxin Feng

    Full Text Available Estrogen receptor alpha (ERα, a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

  14. Structural dynamics and interactions of Xeroderma pigmentosum complementation group A (XPA98-210) with damaged DNA.

    Science.gov (United States)

    Pradhan, Sushmita; Mattaparthi, Venkata Satish Kumar

    2017-10-25

    Nucleotide excision repair (NER) in higher organisms repair massive DNA abrasions caused by ultraviolet rays, and various mutagens, where Xeroderma pigmentosum group A (XPA) protein is known to be involved in damage recognition step. Any mutations in XPA cause classical Xeroderma pigmentosum disease. The extent to which XPA is required in the NER is still unclear. Here, we present the comparative study on the structural and conformational changes in globular DNA binding domain of XPA 98-210 in DNA bound and DNA free state. Atomistic molecular dynamics simulation was carried out for both XPA 98-210 systems using AMBER force fields. We observed that XPA 98-210 in presence of damaged DNA exhibited more structural changes compared to XPA 98-210 in its free form. When XPA is in contact with DNA, we found marked stability of the complex due to the formation of characteristic longer antiparallel β-sheets consisting mainly lysine residues.

  15. 1-Amino-4-hydroxy-9,10-anthraquinone - An analogue of anthracycline anticancer drugs, interacts with DNA and induces apoptosis in human MDA-MB-231 breast adinocarcinoma cells: Evaluation of structure-activity relationship using computational, spectroscopic and biochemical studies.

    Science.gov (United States)

    Mondal, Palash; Roy, Sanjay; Loganathan, Gayathri; Mandal, Bitapi; Dharumadurai, Dhanasekaran; Akbarsha, Mohammad A; Sengupta, Partha Sarathi; Chattopadhyay, Shouvik; Guin, Partha Sarathi

    2015-12-01

    The X-ray diffraction and spectroscopic properties of 1-amino-4-hydroxy-9,10-anthraquinone (1-AHAQ), a simple analogue of anthracycline chemotherapeutic drugs were studied by adopting experimental and computational methods. The optimized geometrical parameters obtained from computational methods were compared with the results of X-ray diffraction analysis and the two were found to be in reasonably good agreement. X-ray diffraction study, Density Functional Theory (DFT) and natural bond orbital (NBO) analysis indicated two types of hydrogen bonds in the molecule. The IR spectra of 1-AHAQ were studied by Vibrational Energy Distribution Analysis (VEDA) using potential energy distribution (PED) analysis. The electronic spectra were studied by TDDFT computation and compared with the experimental results. Experimental and theoretical results corroborated each other to a fair extent. To understand the biological efficacy of 1-AHAQ, it was allowed to interact with calf thymus DNA and human breast adino-carcinoma cell MDA-MB-231. It was found that the molecule induces apoptosis in this adinocarcinoma cell, with little, if any, cytotoxic effect in HBL-100 normal breast epithelial cell.

  16. Assessing SNP-SNP interactions among DNA repair, modification and metabolism related pathway genes in breast cancer susceptibility.

    Directory of Open Access Journals (Sweden)

    Yadav Sapkota

    Full Text Available Genome-wide association studies (GWASs have identified low-penetrance common variants (i.e., single nucleotide polymorphisms, SNPs associated with breast cancer susceptibility. Although GWASs are primarily focused on single-locus effects, gene-gene interactions (i.e., epistasis are also assumed to contribute to the genetic risks for complex diseases including breast cancer. While it has been hypothesized that moderately ranked (P value based weak single-locus effects in GWASs could potentially harbor valuable information for evaluating epistasis, we lack systematic efforts to investigate SNPs showing consistent associations with weak statistical significance across independent discovery and replication stages. The objectives of this study were i to select SNPs showing single-locus effects with weak statistical significance for breast cancer in a GWAS and/or candidate-gene studies; ii to replicate these SNPs in an independent set of breast cancer cases and controls; and iii to explore their potential SNP-SNP interactions contributing to breast cancer susceptibility. A total of 17 SNPs related to DNA repair, modification and metabolism pathway genes were selected since these pathways offer a priori knowledge for potential epistatic interactions and an overall role in breast carcinogenesis. The study design included predominantly Caucasian women (2,795 cases and 4,505 controls from Alberta, Canada. We observed two two-way SNP-SNP interactions (APEX1-rs1130409 and RPAP1-rs2297381; MLH1-rs1799977 and MDM2-rs769412 in logistic regression that conferred elevated risks for breast cancer (P(interaction<7.3 × 10(-3. Logic regression identified an interaction involving four SNPs (MBD2-rs4041245, MLH1-rs1799977, MDM2-rs769412, BRCA2-rs1799943 (P(permutation = 2.4 × 10(-3. SNPs involved in SNP-SNP interactions also showed single-locus effects with weak statistical significance, while BRCA2-rs1799943 showed stronger statistical significance (P

  17. The role of DNA-protein interaction in the UV damage of T7 bacteriophage at high fluences

    International Nuclear Information System (INIS)

    Fekete, A.; Ronto, G.

    1980-01-01

    The influence of higher fluences (0.5-10 kJm -2 ) and that of phage protein coat on the UV (lambda = 254 nm) damage of T7 DNA were studied by UV difference spectroscopy. Beside the pyrimidine dimers and adducts produced also in isolated DNA in the case of intact phages and fluences exceeding 0.5 kJ m -2 other photoproducts, probably DNA-protein cross-links were identified as well. Phages deprived of their protein coat by a thermal treatment show similar UV damage to that of isolated DNA. (author)

  18. IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

    Science.gov (United States)

    Liu, Mingming; Ba, Zhaoqing; Costa-Nunes, Pedro; Wei, Wei; Li, Lanxia; Kong, Fansi; Li, Yan; Chai, Jijie; Pontes, Olga; Qi, Yijun

    2017-03-01

    Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find tha