The influence of screening of the polyion electrostatic potential on the counterion dynamics in polyelectrolyte solutions

NASA Astrophysics Data System (ADS)

Schipper, F. J. M.; Hollander, J. G.; Leyte, J. C.

1998-10-01

The self-diffusion coefficient of tetra-methylammonium counterion in solutions of polymethacrylic acid in 0953-8984/10/41/004/img1 has been measured over a broad polyion concentration range at a constant degree of neutralization and at different ratios of added monovalent or bivalent salt to polyions. A maximum counterion self-diffusion coefficient was observed as a function of polyion concentration. The value of the self-diffusion coefficient at the maximum did not depend on the valency of the added salt. The maximum was found at lower polymer concentrations and with a higher value, when the ratio of added salt to polyions was increased, as predicted by the Poisson-Boltzmann-Smoluchowski equation in the cylindrical cell model for polyelectrolytes. At higher polyion concentrations a maximum counterion self-diffusion coefficient against the ratio of added salt and polyions was observed, which has not been reported before. Upon increasing this ratio the electrostatic potential of the polyelectrolyte gets screened, leading to an increase of the counterion self-diffusion coefficient. Concentration effects of the added salt on the other hand ultimately lead to a decrease of the counterion self-diffusion coefficient, which explains the occurrence of a maximum.

Characteristics of DEAE-dextran-MMA graft copolymer as a nonviral gene carrier.

PubMed

Onishi, Yasuhiko; Eshita, Yuki; Murashita, Aya; Mizuno, Masaaki; Yoshida, Jun

2007-09-01

A stable and soapless latex of diethylaminoethyl-dextran-methyl methacrylate (DEAE-dextran-MMA) graft copolymer (DDMC) has been developed for nonviral gene delivery vectors that are possible to autoclave. DDMC relatively easily formed a polyion complex between DNA and DDMC by the hydrophobic force of graft poly(MMA) depending on its large positive entropy change (DeltaS). DDMC has been confirmed as having a high protection facility for DNase by DNase degradation test.Transfection activity was determined using the beta-galactosidase assay, and a higher value of 16 times or more was confirmed for the DDMC samples in comparison with one of the starting DEAE-dextran hydrochloride samples. The resulting DDMC, having an amphiphilic domain so as to form a polymer micelle, should become a stable latex with a hydrophilic-hydrophobic microseparated domain. The complex of DDMC and plasmid DNA may be formed on the spherical structure of the amphiphilic microseparated domain of DDMC and have a good affinity to the cell membrane. The infrared absorption spectrum shift to a high-energy direction at around 3450 cm(-1), because of the complexes between DNA and DDMC, may cause the formation of more compact structures, not only by a coulomb force between the phosphoric acid of DNA and the DEAE group of DEAE-dextran copolymer but also by a force from the multi-intermolecule hydrogen bond in the backbone polymer DEAE-dextran and a hydrophobic force from the graft poly(MMA) in DDMC. It is thus concluded that DNA condensation may possibly have a high transfection efficiency via DDMC. The high efficiency of this graft copolymer, which is sterilized by an autoclave, may thus make it a valuable tool for safe gene delivery.

Tuning the Size of Nanoassembles: A Hierarchical Transfer of Information from Dendrimers to Polyion Complexes.

PubMed

Amaral, Sandra P; Tawara, Maun H; Fernandez-Villamarin, Marcos; Borrajo, Erea; Martínez-Costas, José; Vidal, Anxo; Riguera, Ricardo; Fernandez-Megia, Eduardo

2018-05-04

The generation of dendrimers is a powerful tool in the control of the size and biodistribution of polyion complexes (PIC). Using a combinatorial screening of six dendrimers (18-243 terminal groups) and five oppositely charged PEGylated copolymers, a dendrimer-to-PIC hierarchical transfer of structural information was revealed with PIC diameters that increased from 80 to 500 nm on decreasing the dendrimer generation. This rise in size, which was also accompanied by a micelle-to-vesicle transition, is interpreted according to a cone- to rod-shaped progression in the architecture of the unit PIC (uPIC). This precise size tuning enabled dendritic PICs to act as nanorulers for controlled biodistribution. Overall, a domino-like control of the size and biological properties of PIC that is not attainable with linear polymers is feasible through dendrimer generation. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Interpretation with a Donnan-based concept of the influence of simple salt concentration on the apparent binding of divalent ions to the polyelectrolytes polystyrenesulfonate and dextran sulfate

USGS Publications Warehouse

Marinsky, J.A.; Baldwin, Robert F.; Reddy, M.M.

1985-01-01

It has been shown that the apparent enhancement of divalent metal ion binding to polyions such as polystyrenesulfonate (PSS) and dextran sulfate (DS) by decreasing the ionic strength of these mixed counterion systems (M2+, M+, X-, polyion) can be anticipated with the Donnan-based model developed by one of us (J.A.M.). Ion-exchange distribution methods have been employed to measure the removal by the polyion of trace divalent metal ion from simple salt (NaClO4)-polyion (NaPSS) mixtures. These data and polyion interaction data published earlier by Mattai and Kwak for the mixed counterion systems MgCl2-LiCl-DS and MgCl2-CsCl-DS have been shown to be amenable to rather precise analysis by this model. ?? 1985 American Chemical Society.

AIE-doped poly(ionic liquid) photonic spheres: a single sphere-based customizable sensing platform for the discrimination of multi-analytes† †Electronic supplementary information (ESI) available: Synthesis and characterization of the AIE luminogen, experimental details, response profiles and results of the multivariate analysis. See DOI: 10.1039/c7sc02409f Click here for additional data file.

PubMed Central

Zhang, Wanlin; Gao, Ning; Cui, Jiecheng; Wang, Chen; Wang, Shiqiang; Zhang, Guanxin; Dong, Xiaobiao

2017-01-01

By simultaneously exploiting the unique properties of ionic liquids and aggregation-induced emission (AIE) luminogens, as well as photonic structures, a novel customizable sensing system for multi-analytes was developed based on a single AIE-doped poly(ionic liquid) photonic sphere. It was found that due to the extraordinary multiple intermolecular interactions involved in the ionic liquid units, one single sphere could differentially interact with broader classes of analytes, thus generating response patterns with remarkable diversity. Moreover, the optical properties of both the AIE luminogen and photonic structure integrated in the poly(ionic liquid) sphere provide multidimensional signal channels for transducing the involved recognition process in a complementary manner and the acquisition of abundant and sufficient sensing information could be easily achieved on only one sphere sensor element. More importantly, the sensing performance of our poly(ionic liquid) photonic sphere is designable and customizable through a simple ion-exchange reaction and target-oriented multi-analyte sensing can be conveniently realized using a selective receptor species, such as counterions, showing great flexibility and extendibility. The power of our single sphere-based customizable sensing system was exemplified by the successful on-demand detection and discrimination of four multi-analyte challenge systems: all 20 natural amino acids, nine important phosphate derivatives, ten metal ions and three pairs of enantiomers. To further demonstrate the potential of our spheres for real-life application, 20 amino acids in human urine and their 26 unprecedented complex mixtures were also discriminated between by the single sphere-based array. PMID:28989662

Counterion self-diffusion in polyelectrolyte solutions

NASA Astrophysics Data System (ADS)

Schipper, F. J. M.; Hollander, J. G.; Leyte, J. C.

1997-12-01

The self-diffusion coefficient of 0953-8984/9/50/019/img1, tetra-methylammonium 0953-8984/9/50/019/img2, tetra-ethylammonium 0953-8984/9/50/019/img3, tetra-propylammonium 0953-8984/9/50/019/img4 and tetra-butylammonium 0953-8984/9/50/019/img5 in solutions of the weak polymethacrylic acid (PMA) were measured with PFG NMR. No additional salt was present in any of the experiments. The polyion concentration and degree of neutralization were varied. The maximum relative counterion self-diffusion coefficient against polyion concentration, that was reported earlier, was observed for both alkali and tetra-alkylammonium 0953-8984/9/50/019/img6 counterions. We propose that the maximum is due to the combination of the obstruction by the polyion and the changing counterion distribution at increasing polyion concentration. An explanation of this proposal is offered in terms of the Poisson - Boltzmann - Smoluchowski (PBS) model for polyelectrolytes. Qualitative agreement of this model with experiment was found for the dependence of the counterion self-diffusion coefficient on the degree of neutralization of the polyion, on counterion radius and on polyion concentration, over a concentration range from 0.01 to 1 0953-8984/9/50/019/img7. Adaption of the theoretical obstruction, to fit the self-diffusion data of the solvent, also greatly improves the model predictions on the counterion self-diffusion.

Potentiometric sensing of nuclease activities and oxidative damage of single-stranded DNA using a polycation-sensitive membrane electrode.

PubMed

Ding, Jiawang; Qin, Wei

2013-09-15

A simple, general and label-free potentiometric method to measure nuclease activities and oxidative DNA damage in a homogeneous solution using a polycation-sensitive membrane electrode is reported. Protamine, a linear polyionic species, is used as an indicator to report the cleavage of DNA by nucleases such as restriction and nonspecific nucleases, and the damage of DNA induced by hydroxyl radicals. Measurements can be done with a titration mode or a direct detection mode. For the potentiometric titration mode, the enzymatic cleavage dramatically affects the electrostatical interaction between DNA and protamine and thus shifts the response curve for the potentiometric titration of the DNA with protamine. Under the optimized conditions, the enzyme activities can be sensed potentiometrically with detection limits of 2.7×10(-4)U/µL for S1 nuclease, and of 3.9×10(-4)U/µL for DNase I. For the direct detection mode, a biocomplex between protamine and DNA is used as a substrate. The nuclease of interest cleaves the DNA from the protamine/DNA complex into smaller fragments, so that free protamine is generated and can be detected potentiometrically via the polycation-sensitive membrane electrode. Using a direct measurement, the nuclease activities could be rapidly detected with detection limits of 3.2×10(-4)U/µL for S1 nuclease, and of 4.5×10(-4)U/µL for DNase I. Moreover, the proposed potentiometric assays demonstrate the potential applications in the detection of hydroxyl radicals. It is anticipated that the present potentiometric strategy will provide a promising platform for high-throughput screening of nucleases, reactive oxygen species and the drugs with potential inhibition abilities. Copyright © 2013 Elsevier B.V. All rights reserved.

A Simple and Universal Gel Permeation Chromatography Technique for Precise Molecular Weight Characterization of Well-Defined Poly(ionic liquid)s

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

He, Hongkun; Zhong, Mingjiang; Adzima, Brian

2013-03-20

Poly(ionic liquid)s (PILs) are an important class of technologically relevant materials. However, characterization of well-defined polyionic materials remains a challenge. Herein, we have developed a simple and versatile gel permeation chromatography (GPC) methodology for molecular weight (MW) characterization of PILs with a variety of anions. PILs with narrow MW distributions were synthesized via atom transfer radical polymerization, and the MWs obtained from GPC were further confirmed via nuclear magnetic resonance end group analysis.

Poly(1-Vinyl-1,2,4-triazolium) Poly(Ionic Liquid)s: Synthesis and the Unique Behavior in Loading Metal Ions.

PubMed

Zhang, Weiyi; Yuan, Jiayin

2016-07-01

Herein, the synthesis of a series of poly(4-alkyl-1-vinyl-1,2,4-triazolium) poly(ionic liquid)s is reported either via straightforward free radical polymerization of their corresponding ionic liquid monomers or via anion metathesis of the polymer precursors bearing halide as counter anion. The ionic liquid monomers are first prepared via N-alkylation reaction of commercially available 1-vinyl-1,2,4-triazole with alkyl iodides, followed by anion metathesis with targeted fluorinated anions. The thermal properties and solubilities of these poly(ionic liquid)s have been systematically investigated. Interestingly, it is found that the poly(4-ethyl-1-vinyl-1,2,4-triazolium) poly(ionic liquid) exhibited an improved loading capacity of transition metal ions in comparison with its imidazolium counterpart. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mass transport in polyelectrolyte solutions

NASA Astrophysics Data System (ADS)

Schipper, F. J. M.; Leyte, J. C.

1999-02-01

The self-diffusion coefficients of the three components of a salt-free heavy-water solution of polymethacrylic acid, completely neutralized with tetra-methylammonium hydroxide, were measured over a broad concentration range. Three concentration regions were observed for the self-diffusion of both the polyions and the counterions. At polyion concentrations below 0.01 mol monomer kg-1, the dilute concentration regime for the polymer, the polyion self-diffusion coefficient approaches the self-diffusion coefficient of a freely diffusing rod upon dilution. At polyelectrolyte concentrations above 0.1 mol monomer kg-1, the self-diffusion coefficients of the solvent, the counterions and the polymer decreased with concentration, suggesting that this decrease is due to a topological constraint on the motions of the components. In the intermediate-concentration region, the self-diffusion coefficients of the polyions and the counterions are independent of the concentration. The polyion dynamic behaviour is, in the intermediate- and high-concentration regions, reasonably well described by that of a hard sphere, with a radius of 3.7 nm. A correct prediction for the solvent dynamics is given by the obstruction effect of this hard sphere on the solvent. The relative counterion self-diffusion coefficient is predicted almost quantitatively over the entire concentration range with the Poisson-Boltzmann-Smoluchowski model for the spherical cell, provided that the sphere radius and the number of charges are chosen appropriately (approximately the number of charges in a persistence length). Using this model, the dependence of the counterion self-diffusion coefficient on the ionic strength, polyion concentration and counterion radius is calculated quantitatively over a large concentration range.

Development of polyion-complex hydrogels as an alternative approach for the production of bio-based polymers for food packaging applications: A review

USDA-ARS?s Scientific Manuscript database

Development of packaging materials from renewable resources has for a long time been desirable for sustainability reasons, but with the recent explosion in prices of petroleum products, this now becomes also more economically viable. This paper shows how fundamental chemistry underlying three forms ...

Harnessing Poly(ionic liquid)s for Sensing Applications.

PubMed

Guterman, Ryan; Ambrogi, Martina; Yuan, Jiayin

2016-07-01

The interest in poly(ionic liquid)s for sensing applications is derived from their strong interactions to a variety of analytes. By combining the desirable mechanical properties of polymers with the physical and chemical properties of ILs, new materials can be created. The tunable nature of both ionic liquids and polymers allows for incredible diversity, which is exemplified in their broad applicability. In this article we examine the new field of poly(ionic liquid) sensors by providing a detailed look at the current state-of-the-art sensing devices for solvents, gases, biomolecules, pH, and anions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Thermo- and electro-dual responsive poly(ionic liquid) electrolyte based smart windows.

PubMed

Chen, Fei; Ren, Yongyuan; Guo, Jiangna; Yan, Feng

2017-01-31

Thermo- and electro-dual responsive poly(ionic liquid) (PIL) based electrolytes were synthesized by co-polymerization of N-isopropylacrylamide (NIPAM) with (or without) 3-butyl-1-vinyl-imidazolium bromide ([BVIm][Br]) using diallyl-viologen (DAV) as both the cross-linking agent and electrochromic material.

Precise engineering of siRNA delivery vehicles to tumors using polyion complexes and gold nanoparticles.

PubMed

Kim, Hyun Jin; Takemoto, Hiroyasu; Yi, Yu; Zheng, Meng; Maeda, Yoshinori; Chaya, Hiroyuki; Hayashi, Kotaro; Mi, Peng; Pittella, Frederico; Christie, R James; Toh, Kazuko; Matsumoto, Yu; Nishiyama, Nobuhiro; Miyata, Kanjiro; Kataoka, Kazunori

2014-09-23

For systemic delivery of siRNA to solid tumors, a size-regulated and reversibly stabilized nanoarchitecture was constructed by using a 20 kDa siRNA-loaded unimer polyion complex (uPIC) and 20 nm gold nanoparticle (AuNP). The uPIC was selectively prepared by charge-matched polyionic complexation of a poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) copolymer bearing ∼40 positive charges (and thiol group at the ω-end) with a single siRNA bearing 40 negative charges. The thiol group at the ω-end of PEG-PLL further enabled successful conjugation of the uPICs onto the single AuNP through coordinate bonding, generating a nanoarchitecture (uPIC-AuNP) with a size of 38 nm and a narrow size distribution. In contrast, mixing thiolated PEG-PLLs and AuNPs produced a large aggregate in the absence of siRNA, suggesting the essential role of the preformed uPIC in the formation of nanoarchitecture. The smart uPIC-AuNPs were stable in serum-containing media and more resistant against heparin-induced counter polyanion exchange, compared to uPICs alone. On the other hand, the treatment of uPIC-AuNPs with an intracellular concentration of glutathione substantially compromised their stability and triggered the release of siRNA, demonstrating the reversible stability of these nanoarchitectures relative to thiol exchange and negatively charged AuNP surface. The uPIC-AuNPs efficiently delivered siRNA into cultured cancer cells, facilitating significant sequence-specific gene silencing without cytotoxicity. Systemically administered uPIC-AuNPs showed appreciably longer blood circulation time compared to controls, i.e., bare AuNPs and uPICs, indicating that the conjugation of uPICs onto AuNP was crucial for enhancing blood circulation time. Finally, the uPIC-AuNPs efficiently accumulated in a subcutaneously inoculated luciferase-expressing cervical cancer (HeLa-Luc) model and achieved significant luciferase gene silencing in the tumor tissue. These results demonstrate the strong potential of uPIC-AuNP nanoarchitectures for systemic siRNA delivery to solid tumors.

[Advances of poly (ionic liquid) materials in separation science].

PubMed

Liu, Cuicui; Guo, Ting; Su, Rina; Gu, Yuchen; Deng, Qiliang

2015-11-01

Ionic liquids, as novel ionization reagents, possess beneficial characteristics including good solubility, conductivity, thermal stability, biocompatibility, low volatility and non-flammability. Ionic liquids are attracting a mass of attention of analytical chemists. Poly (ionic liquid) materials have common performances of ionic liquids and polymers, and have been successfully applied in separation science area. In this paper, we discuss the interaction mechanisms between the poly(ionic liquid) materials and analytes including hydrophobic/hydrophilic interactions, hydrogen bond, ion exchange, π-π stacking and electrostatic interactions, and summarize the application advances of the poly(ionic liquid) materials in solid phase extraction, chromatographic separation and capillary electrophoresis. At last, we describe the future prospect of poly(ionic liquid) materials.

Dielectric dispersion of short single-stranded DNA in aqueous solutions with and without added salt.

PubMed

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

2007-01-01

Dielectric spectroscopy measurements were performed for aqueous solutions of short single-stranded DNA with 30 to 120 bases of thymine over a frequency range of 10;{5} to 10;{8}Hz . Dielectric dispersion was found to include two relaxation processes in the ranges from 10;{5} to 10;{6} and from 10;{6} to 10;{8}Hz , respectively, with the latter mainly discussed in this study. The dielectric increment and the relaxation time of the high-frequency relaxation of DNA in solutions without added salt exhibited concentration and polymer-length dependences eventually identical to those for dilute polyion solutions described in previous studies. For solutions with added salt, on the other hand, those dielectric parameters were independent of salt concentration up to a certain critical value and started to decrease with further increasing salt concentration. This critical behavior is well explained by our newly extended cell model that takes into account the spatial distribution of loosely bound counterions around DNA molecules as a function of salt concentration.

Functionalized graphene sheets with poly(ionic liquid)s and high adsorption capacity of anionic dyes

NASA Astrophysics Data System (ADS)

Zhao, Weifeng; Tang, Yusheng; Xi, Jia; Kong, Jie

2015-01-01

Graphene sheets were covalently functionalized with poly(1-vinylimidazole) (PVI) type poly(ionic liquid), by utilizing a diazonium addition reaction and the subsequent grafting of PVI polymers onto the graphene sheet surface by a quaternarization reaction. The resultant modified graphene sheets showed improved dispersion property when being dissolved in DMF and ethanol. FTIR, XPS, XRD and TEM observations confirmed the success of the covalent functionalization, and thermogravimetric analysis revealed that the grafting ratio of PVI was ∼12 wt%. The obtained PVI-functionalized graphene showed a high capability for removing anionic dyes such as methyl blue (MB) from water solution. The experimental data of isotherm fitted well with the Langmuir adsorption model. The adsorption capacity of 1910 mg g-1 for methyl blue (MB) dye was observed for functionalized graphene sheets with poly(ionic liquid)s, which was higher than that of unmodified graphene. The high adsorption capacity observed in this study emphasizes that poly(ionic liquid)s-modified graphene materials have a great potential for water purification as they are highly efficient and stable adsorbents for sustainability.

Target-induced formation of gold amalgamation on DNA-based sensing platform for electrochemical monitoring of mercury ion coupling with cycling signal amplification strategy.

PubMed

Chen, Jinfeng; Tang, Juan; Zhou, Jun; Zhang, Lan; Chen, Guonan; Tang, Dianping

2014-01-31

Heavy metal ion pollution poses severe risks in human health and environmental pollutant, because of the likelihood of bioaccumulation and toxicity. Driven by the requirement to monitor trace-level mercury ion (Hg(2+)), herein we construct a new DNA-based sensor for sensitive electrochemical monitoring of Hg(2+) by coupling target-induced formation of gold amalgamation on DNA-based sensing platform with gold amalgamation-catalyzed cycling signal amplification strategy. The sensor was simply prepared by covalent conjugation of aminated poly-T(25) oligonucleotide onto the glassy carbon electrode by typical carbodiimide coupling. Upon introduction of target analyte, Hg(2+) ion was intercalated into the DNA polyion complex membrane based on T-Hg(2+)-T coordination chemistry. The chelated Hg(2+) ion could induce the formation of gold amalgamation, which could catalyze the p-nitrophenol with the aid of NaBH4 and Ru(NH3)6(3+) for cycling signal amplification. Experimental results indicated that the electronic signal of our system increased with the increasing Hg(2+) level in the sample, and has a detection limit of 0.02nM with a dynamic range of up to 1000nM Hg(2+). The strategy afforded exquisite selectivity for Hg(2+) against other environmentally related metal ions. In addition, the methodology was evaluated for the analysis of Hg(2+) in spiked tap-water samples, and the recovery was 87.9-113.8%. Copyright © 2013 Elsevier B.V. All rights reserved.

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

PubMed

Raisin, Sophie; Morille, Marie; Bony, Claire; Noël, Danièle; Devoisselle, Jean-Marie; Belamie, Emmanuel

2017-08-22

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Facile synthesis of poly(ionic liquid)-bonded magnetic nanospheres as a high-performance sorbent for the pretreatment and determination of phenolic compounds in water samples.

PubMed

Bi, Wentao; Wang, Man; Yang, Xiaodi; Row, Kyung Ho

2014-07-01

Poly(ionic liquid)-bonded magnetic nanospheres were easily synthesized and applied to the pretreatment and determination of phenolic compounds in water samples, which have detrimental effects on water quality and the health of living beings. The high affinity of poly(ionic liquid)s toward the target compounds as well as the magnetic behavior of Fe3 O4 were combined in this material to provide an efficient and simple magnetic solid-phase extraction approach. The adsorption behavior of the poly(ionic liquid)-bonded magnetic nanospheres was examined to optimize the synthesis. Different parameters affecting the magnetic solid-phase extraction of phenolic compounds were assessed in terms of adsorption and recovery. Under the optimal conditions, the proposed method showed excellent detection sensitivity with limits of detection in the range of 0.3-0.8 ng/mL and precision in the range of 1.2-3.3%. This method was also applied successfully to the analysis of real water samples; good spiked recoveries over the range of 82.5-99.2% were obtained. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-Antibacterial Activity Relationships of Imidazolium-Type Ionic Liquid Monomers, Poly(ionic liquids) and Poly(ionic liquid) Membranes: Effect of Alkyl Chain Length and Cations.

PubMed

Zheng, Zhiqiang; Xu, Qiming; Guo, Jiangna; Qin, Jing; Mao, Hailei; Wang, Bin; Yan, Feng

2016-05-25

The structure-antibacterial activity relationship between the small molecular compounds and polymers are still elusive. Here, imidazolium-type ionic liquid (IL) monomers and their corresponding poly(ionic liquids) (PILs) and poly(ionic liquid) membranes were synthesized. The effect of chemical structure, including carbon chain length of substitution at the N3 position and charge density of cations (mono- or bis-imidazolium) on the antimicrobial activities against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was investigated by determination of minimum inhibitory concentration (MIC). The antibacterial activities of both ILs and PILs were improved with the increase of the alkyl chain length and higher charge density (bis-cations) of imidazolium cations. Moreover, PILs exhibited lower MIC values relative to the IL monomers. However, the antibacterial activities of PIL membranes showed no correlation to those of their analogous small molecule IL monomers and PILs, which increased with the charge density (bis-cations) while decreasing with the increase of alkyl chain length. The results indicated that antibacterial property studies on small molecules and homopolymers may not provide a solid basis for evaluating that in corresponding polymer membranes.

Polyion selective polymeric membrane-based pulstrode as a detector in flow-injection analysis.

PubMed

Bell-Vlasov, Andrea K; Zajda, Joanna; Eldourghamy, Ayman; Malinowska, Elzbieta; Meyerhoff, Mark E

2014-04-15

A method for the detection of polyions using fully reversible polyion selective polymeric membrane type pulstrodes as detectors in a flow-injection analysis (FIA) system is examined. The detection electrode consists of a plasticized polymeric membrane doped with 10 wt % of tridodecylmethylammonium-dinonylnaphthalene sulfonate (TDMA/DNNS) ion-exchanger salt. The pulse sequence used involves a short (1 s) galvanostatic pulse, an open-circuit pulse (0.5 s) during which the EMF of the cell is measured, and a longer (15 s) potentiostatic pulse to return the membrane to its original chemical composition. It is shown that total pulse sequence times can be optimized to yield reproducible real-time detection of injected samples of protamine and heparin at up to 20 samples/h. Further, it is shown that the same membrane detector can be employed for FIA detection of both polycations at levels ≥10 μg/mL and polyanions at levels of ≥40 μg/mL by changing the direction of the galvanostatic pulse. The methodology described may also be applicable in the detection of polyionic species at low levels in other flowing configurations, such as in liquid chromatography and capillary electrophoresis.

Fast Conversion of Ionic Liquids and Poly(Ionic Liquid)s into Porous Nitrogen-Doped Carbons in Air

PubMed Central

Men, Yongjun; Ambrogi, Martina; Han, Baohang; Yuan, Jiayin

2016-01-01

Ionic liquids and poly(ionic liquid)s have been successfully converted into nitrogen-doped porous carbons with tunable surface area up to 1200 m2/g at high temperatures in air. Compared to conventional carbonization process conducted under inert gas to produce nitrogen-doped carbons, the new production method was completed in a rather shorter time without noble gas protection. PMID:27070588

Fast Conversion of Ionic Liquids and Poly(Ionic Liquid)s into Porous Nitrogen-Doped Carbons in Air.

PubMed

Men, Yongjun; Ambrogi, Martina; Han, Baohang; Yuan, Jiayin

2016-04-08

Ionic liquids and poly(ionic liquid)s have been successfully converted into nitrogen-doped porous carbons with tunable surface area up to 1200 m²/g at high temperatures in air. Compared to conventional carbonization process conducted under inert gas to produce nitrogen-doped carbons, the new production method was completed in a rather shorter time without noble gas protection.

Plasmon-enhanced photocurrent generation from self-assembled monolayers of phthalocyanine by using gold nanoparticle films.

PubMed

Sugawa, Kosuke; Akiyama, Tsuyoshi; Kawazumi, Hirofumi; Yamada, Sunao

2009-04-09

The effect of localized electric fields on the photocurrent responses of phthalocyanine that was self-assembled on a gold nanoparticle film was investigated by comparing the conventional and the total internal reflection (TIR) experimental systems. In the case of photocurrent measurements, self-assembled monolayers (SAMs) of a thiol derivative of palladium phthalocyanine (PdPc) were prepared on the surface of gold-nanoparticle film that was fixed on the surface of indium-tin-oxide (ITO) substrate via a polyion (PdPc/AuP/polyion/ITO) or on the ITO surface (PdPc/ITO). Photocurrent action spectra from the two samples were compared by using the conventional spectrometer, and were found that PdPc/AuP/polyion/ITO gave considerably larger photocurrent signals than PdPc/ITO under the identical concentration of PdPc. In the case of the TIR experiments for the PdPc/AuP/polyion/ITO and the PdPc/AuP/Glass systems, incident-angle profiles of photocurrent and emission signals were correlated with each other, and they were different from that of the PdPc/ITO system. Accordingly, it was demonstrated that the photocurrent signals were certainly enhanced by the localized electric fields of the gold-nanoparticle film.

International Conference on Langmuir-Blodgett Films (5th) Held in Paris, France on 26-30 August 1991, Abstracts Booklet

DTIC Science & Technology

1991-08-01

Fojihh LN.. aIsetiki, LA. Bazaheu*o, .Kaimx BP30 CI Photo-amplified stroget n y with Incorporation of non-amphiphilic compounds polyion complexed p...donor- films of N-acyl-p-nitroaniline and its acceptor organic compounds homologous amphiphile D. Zhu, C. Yang, Yu. Liu, Y. Xu Y..Mixamo, K, Kauiu, T...R. Kenn, H. MChwald, K. Kjaer, J. LB films of polyheterocyclic compounds with Als-Nielsen long alkyl chains HLNAkabam K. Fukuda, M. Ikeda, K

Tumor microenvironment dual-responsive core-shell nanoparticles with hyaluronic acid-shield for efficient co-delivery of doxorubicin and plasmid DNA.

PubMed

Wang, Tianqi; Yu, Xiaoyue; Han, Leiqiang; Liu, Tingxian; Liu, Yongjun; Zhang, Na

2017-01-01

As the tumor microenvironment (TME) develops, it is critical to take the alterations of pH value, reduction and various enzymes of the TME into consideration when constructing the desirable co-delivery systems. Herein, TME pH and enzyme dual-responsive core-shell nanoparticles were prepared for the efficient co-delivery of chemotherapy drug and plasmid DNA (pDNA). A novel pH-responsive, positively charged drug loading material, doxorubicin (DOX)-4-hydrazinobenzoic acid (HBA)-polyethyleneimine (PEI) conjugate (DOX-HBA-PEI, DHP), was synthesized to fabricate positively charged polyion complex inner core DHP/DNA nanoparticles (DDN). Hyaluronic acid (HA) was an enzyme-responsive shell which could protect the core and enhance the co-delivery efficiency through CD44-mediated endocytosis. The HA-shielded pH and enzyme dual-responsive nanoparticles (HDDN) were spherical with narrow distribution. The particle size of HDDN was 148.3±3.88 nm and the zeta potential was changed to negative (-18.1±2.03 mV), which led to decreased cytotoxicity. The cumulative release of DOX from DHP at pH 5.0 (66.4%) was higher than that at pH 7.4 (30.1%), which indicated the pH sensitivity of DHP. The transfection efficiency of HDDN in 10% serum was equal to that in the absence of serum, while the transfection of DDN was significantly decreased in the presence of 10% serum. Furthermore, cellular uptake studies and co-localization assay showed that HDDN were internalized effectively through CD44-mediated endocytosis in the tumor cells. The efficient co-delivery of DOX and pEGFP was confirmed by fluorescent image taken by laser confocal microscope. It can be concluded that TME dual-responsive HA-shielded core-shell nanoparticles could be considered as a promising platform for the co-delivery of chemotherapy drug and pDNA.

Density-tunable conjugation of cyclic RGD ligands with polyion complex vesicles for the neovascular imaging of orthotopic glioblastomas

NASA Astrophysics Data System (ADS)

Kawamura, Wataru; Miura, Yutaka; Kokuryo, Daisuke; Toh, Kazuko; Yamada, Naoki; Nomoto, Takahiro; Matsumoto, Yu; Sueyoshi, Daiki; Liu, Xueying; Aoki, Ichio; Kano, Mitsunobu R.; Nishiyama, Nobuhiro; Saga, Tsuneo; Kishimura, Akihiro; Kataoka, Kazunori

2015-06-01

Introduction of ligands into 100 nm scaled hollow capsules has great potential for diagnostic and therapeutic applications in drug delivery systems. Polyethylene glycol-conjugated (PEGylated) polyion complex vesicles (PICsomes) are promising hollow nano-capsules that can survive for long periods in the blood circulation and can be used to deliver water-soluble macromolecules to target tissues. In this study, cyclic RGD (cRGD) peptide, which is specifically recognized by αVβ3 and αvβ5 integrins that are expressed at high levels in the neovascular system, was conjugated onto the distal end of PEG strands on PICsomes for active neovascular targeting. Density-tunable cRGD-conjugation was achieved using PICsomes with definite fraction of end-functionalized PEG, to substitute 20, 40, and 100% of PEG distal end of the PICsomes to cRGD moieties. Compared with control-PICsomes without cRGD, cRGD-PICsomes exhibited increased uptake into human umbilical vein endothelial cells. Intravital confocal laser scanning microscopy revealed that the 40%-cRGD-PICsomes accumulated mainly in the tumor neovasculature and remained in the perivascular region even after 24 h. Furthermore, we prepared superparamagnetic iron oxide (SPIO)-loaded cRGD-PICsomes for magnetic resonance imaging (MRI) and successfully visualized the neovasculature in an orthotopic glioblastoma model, which suggests that SPIO-loaded cRGD-PICsomes might be useful as a MRI contrast reagent for imaging of the tumor microenvironment, including neovascular regions that overexpress αVβ3 integrins.

Detecting Levels of Polyquaternium-10 (PQ-10) via Potentiometric Titration with Dextran Sulphate and Monitoring the Equivalence Point with a Polymeric Membrane-Based Polyion Sensor.

PubMed

Ferguson, Stephen A; Wang, Xuewei; Meyerhoff, Mark E

2016-08-07

Polymeric quaternary ammonium salts (polyquaterniums) have found increasing use in industrial and cosmetic applications in recent years. More specifically, polyquaternium-10 (PQ-10) is routinely used in cosmetic applications as a conditioner in personal care product formulations. Herein, we demonstrate the use of potentiometric polyion-sensitive polymeric membrane-based electrodes to quantify PQ-10 levels. Mixtures containing both PQ-10 and sodium lauryl sulfate (SLS) are used as model samples to illustrate this new method. SLS is often present in cosmetic samples that contain PQ-10 (e.g., shampoos, etc.) and this surfactant species interferes with the polyion sensor detection chemistry. However, it is shown here that SLS can be readily separated from the PQ-10/SLS mixture by use of an anion-exchange resin and that the PQ-10 can then be titrated with dextran sulphate (DS). This titration is monitored by potentiometric polyanion sensors to provide equivalence points that are directly proportional to PQ-10 concentrations.

Novel redox nanomedicine improves gene expression of polyion complex vector

NASA Astrophysics Data System (ADS)

Toh, Kazuko; Yoshitomi, Toru; Ikeda, Yutaka; Nagasaki, Yukio

2011-12-01

Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS) affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP) as an ROS scavenger. When polyethyleneimine (PEI)/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI)/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF)-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

CO2 Responsive Imidazolium-Type Poly(Ionic Liquid) Gels.

PubMed

Zhang, Jing; Xu, Dan; Guo, Jiangna; Sun, Zhe; Qian, Wenjing; Zhang, Ye; Yan, Feng

2016-07-01

Poly(ionic liquid) (PIL) gels with CO2 stimulus responsiveness have been synthesized through the copolymerization of an imidazolium-type ionic liquid monomer with 2-(dimethyl amino) ethyl methacrylate. Upon bubbling with CO2 gas, the prepared PIL solution is converted to a transparent and stable gel, which can be turned back to the initial solution state after N2 bubbling. The reversible sol-gel phase transition behavior is proved by the reversible values of viscosity and ionic conductivity. The possible mechanism for such a reversible sol-gel phase transition is demonstrated by NMR, conductivity, and rheological measurements. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Poly(ionic liquids)-coated stainless-steel wires packed into a polyether ether ketone tube for in-tube solid-phase microextraction.

PubMed

Feng, Juanjuan; Wang, Xiuqin; Tian, Yu; Luo, Chuannan; Sun, Min

2017-12-01

An in-tube solid-phase microextraction device was developed by packing poly(ionic liquids)-coated stainless-steel wires into a polyether ether ketone tube. An anion-exchange process was performed to enhance the extraction performance. Surface properties of poly(ionic liquids)-coated stainless-steel wires were characterized by scanning electron microscopy and energy dispersive X-ray spectrometry. The extraction device was connected to high-performance liquid chromatography equipment to build an online enrichment and analysis system. Ten polycyclic aromatic hydrocarbons were used as model analytes, and important conditions including extraction time and desorption time were optimized. The enrichment factors from 268 to 2497, linear range of 0.03-20 μg/L, detection limits of 0.010-0.020 μg/L, extraction and preparation repeatability with relative standard deviation less than 1.8 and 19%, respectively were given by the established online analysis method. It has been used to detect polycyclic aromatic hydrocarbons in environmental samples, with the relative recovery (5, 10 μg/L) in the range of 85.1-118.9%. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Encapsulation of rat bone marrow stromal cells using a poly-ion complex gel of chitosan and succinylated poly(Pro-Hyp-Gly).

PubMed

Kusumastuti, Yuni; Shibasaki, Yoshiaki; Hirohara, Shiho; Kobayashi, Mime; Terada, Kayo; Ando, Tsuyoshi; Tanihara, Masao

2017-03-01

Encapsulation of stem cells into a three-dimensional (3D) scaffold is necessary to achieve tissue regeneration. Prefabricated 3D scaffolds, such as fibres or porous sponges, have limitations regarding homogeneous cell distribution. Hydrogels that can encapsulate cells such as animal-derived collagen gels need adjustment of the pH and/or temperature upon cell mixing. In this report, we fabricated a poly-ion complex (PIC) hydrogel of chitosan and succinylated poly(Pro-Hyp-Gly) and assessed its effect on cell viability after encapsulation of rat bone marrow stromal cells. PIC hydrogels were obtained successfully with a concentration of each precursor as low as 3.0-3.8 mg/ml. The maximum gelation and swelling ratios were achieved with an equal molar ratio (1:1) of anionic and cationic groups. Using chitosan acetate as a cationic precursor produced a PIC hydrogel with both a significantly greater gelation ratio and a better swelling ratio than chitosan chloride. Ammonium succinylated poly(Pro-Hyp-Gly) as an anionic precursor gave similar gelation and swelling ratios to those of sodium succinylated poly(Pro-Hyp-Gly). Cell encapsulation was also achieved successfully by mixing rat bone marrow stromal cells with the PIC hydrogel simultaneously during its formation. The PIC hydrogel was maintained in the culture medium for 7 days at 37°C and the encapsulated cells survived and proliferated in it. Although it is necessary to improve its functionality, this PIC hydrogel has the potential to act as a 3D scaffold for cell encapsulation and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Frit inlet field-flow fractionation techniques for the characterization of polyion complex self-assemblies.

PubMed

Till, Ugo; Gaucher, Mireille; Amouroux, Baptiste; Gineste, Stéphane; Lonetti, Barbara; Marty, Jean-Daniel; Mingotaud, Christophe; Bria, Carmen R M; Williams, S Kim Ratanathanawongs; Violleau, Frédéric; Mingotaud, Anne-Françoise

2017-01-20

Polymer self-assemblies joining oppositely charged chains, known as polyion complexes (PICs), have been formed using poly(ethyleneoxide - b - acrylic acid)/poly(l-lysine), poly(ethyleneoxide-b-acrylic acid)/dendrigraft poly(l-lysine) and poly[(3-acrylamidopropyl) trimethylammonium chloride - b - N - isopropyl acrylamide]/poly(acrylic acid). The self-assemblies have been first characterized in batch by Dynamic Light Scattering. In a second step, their analysis by Flow Field-Flow Fractionation techniques (FlFFF) was examined. They were shown to be very sensitive to shearing, especially during the focus step of the fractionation, and this led to an incompatibility with asymmetrical FlFFF. On the other hand, Frit Inlet FlFFF proved to be very efficient to observe them, either in its symmetrical (FI-FlFFF) or asymmetrical version (FI-AsFlFFF). Conditions of elution were found to optimize the sample recovery in pure water. Spherical self-assemblies were detected, with a size range between 70-400nm depending on the polymers. Compared to batch DLS, FI-AsFlFFF clearly showed the presence of several populations in some cases. The influence of salt on poly(ethyleneoxide-b-acrylic acid) (PEO-PAA) 6000-3000/dendrigraft poly(l-lysine) (DGL 3) was also assessed in parallel in batch DLS and FI-AsFlFFF. Batch DLS revealed a first process of swelling of the self-assembly for low concentrations up to 0.8M followed by the dissociation. FI-AsFlFFF furthermore indicated a possible ejection of DGL3 from the PIC assembly for concentrations as low as 0.2M, which could not be observed in batch DLS. Copyright © 2016 Elsevier B.V. All rights reserved.

The use of polyion complex micelles to enhance the oral delivery of salmon calcitonin and transport mechanism across the intestinal epithelial barrier.

PubMed

Li, Na; Li, Xin-Ru; Zhou, Yan-Xia; Li, Wen-Jing; Zhao, Yong; Ma, Shu-Jin; Li, Jin-Wen; Gao, Ya-Jie; Liu, Yan; Wang, Xing-Lin; Yin, Dong-Dong

2012-12-01

The objective of the present study was to demonstrate the effect of polyanionic copolymer mPEG-grafted-alginic acid (mPEG-g-AA)-based polyion complex (PIC) micelles on enhancing the oral absorption of salmon calcitonin (sCT) in vivo and in vitro and identify the transepithelial transport mechanism of PIC micelles across the intestinal barrier. mPEG-g-AA was first successfully synthesized and characterized in cytotoxicity. The PIC micelles were approximately of 72 nm in diameter with a narrow distribution. The extremely significant enhancement of hypocalcemia efficacy of sCT-loaded PIC micelles in rats was evidenced by intraduodenal administration in comparison with sCT solution. The presence of mPEG-grafted-chitosan in PIC micelles had no favorable effect on this action in the referred content. In the Caco-2 transport studies, PIC micelles could significantly increase the permeability of sCT across Caco-2 monolayers without significantly affecting transepithelial electrical resistance values during the transport study. No evident alterations in the F-actin cytoskeleton were detected by confocal microscope observation following treatment of the cell monolayers with PIC micelles, which further certified the incapacity of PIC micelles to open the intercellular tight junctions. In addition, TEM observations showed that the intact PIC micelles were transported across the everted gut sac. These suggested that the transport of PIC micelles across Caco-2 cell monolayers involve a predominant transcytosis mechanism via endocytosis rather than paracellular pathway. Furthermore, PIC micelles were localized in both the cytoplasm and the nuclei observed by CLSM. Therefore, PIC micelles might be a potentially applicable tool for enhancing the oral absorption of cationic peptide and protein drugs. Copyright © 2012 Elsevier Ltd. All rights reserved.

Frontiers in poly(ionic liquid)s: syntheses and applications.

PubMed

Qian, Wenjing; Texter, John; Yan, Feng

2017-02-20

We review recent works on the synthesis and application of poly(ionic liquid)s (PILs). Novel chemical structures, different synthetic strategies and controllable morphologies are introduced as a supplement to PIL systems already reported. The primary properties determining applications, such as ionic conductivity, aqueous solubility, thermodynamic stability and electrochemical/chemical durability, are discussed. Furthermore, the near-term applications of PILs in multiple fields, such as their use in electrochemical energy materials, stimuli-responsive materials, carbon materials, and antimicrobial materials, in catalysis, in sensors, in absorption and in separation materials, as well as several special-interest applications, are described in detail. We also discuss the limitations of PIL applications, efforts to improve PIL physics, and likely future developments.

Manual and Flow-Injection Detection/Quantification of Polyquaterniums via Fully Reversible Polyion-Sensitive Polymeric Membrane-Based Ion-Selective Electrodes.

PubMed

Ferguson, Stephen A; Meyerhoff, Mark E

2017-10-27

The detection of four different polyquaterniums (PQs) using a fully reversible potentiometric polyion sensor in three different detection modes is described. The polyion sensing "pulstrodes" serve as the detector for direct dose-response experiments, beaker titrations, and in a flow-injection analysis (FIA) system. Direct polycation response toward PQ-2, PQ-6, PQ-10, and poly(2-methacryloxyethyltrimethylammonium) chloride (PMETAC) yields characteristic information about each PQ species (e.g., relative charge densities, etc.) via syringe pump addition of each PQ species to a background electrolyte solution. Quantitative titrations are performed using a syringe pump to deliver heparin as the polyanion titrant to quantify all four PQs at μg/mL levels. Both the direct and indirect methods incorporate the use of a three-electrode system including counter, double junction reference, and working electrodes. The working electrode possesses a plasticized poly(vinyl chloride) (PVC) membrane containing the neutral lipophilic salt of dinonylnaphthalenesulfonate (DNNS - ) tridodecylmethylammonium (TDMA + ). Further, the titration method is shown to be useful to quantify PQ-6 levels in recreational swimming pool water collected in Ann Arbor, MI. Finally, a FIA system equipped with a pulstrode detector is used to demonstrate the ability to potentially quantify PQ levels via a more streamlined and semiautomated testing platform.

Chitosan/alginate complexes for vaginal delivery of chlorhexidine digluconate.

PubMed

Abruzzo, A; Bigucci, F; Cerchiara, T; Saladini, B; Gallucci, M C; Cruciani, F; Vitali, B; Luppi, B

2013-01-16

Chitosan/alginate complexes were prepared at different polycation/polyanion molar ratios and freeze-dried vaginal inserts were obtained for chlorhexidine digluconate local delivery in genital infections. Complex yield, FT-IR spectra, and TGA thermograms were studied to confirm the interaction between the two polyions. The influence of different complexes on physical handling, morphology, and drug distribution in the samples were evaluated by friability test, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS), respectively. In vitro water-uptake, mucoadhesion and release tests were performed as well as microbiological tests toward pathogenic vaginal microorganisms. The results showed that the selection of suitable chitosan/alginate molar ratio and drug loading allowed modulate insert ability to hydrate, adhere to the mucosa, and release chlorhexidine digluconate. The insert containing an excess of alginate was found to be the best performing formulation and showed good antimicrobial activity toward the pathogens Candida albicans and Escherichia coli. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mesoporous poly(ionic liquid) supported palladium(II) catalyst for oxidative coupling of benzene under atmospheric oxygen

NASA Astrophysics Data System (ADS)

Liu, Yangqing; Wang, Kai; Hou, Wei; Shan, Wanjian; Li, Jing; Zhou, Yu; Wang, Jun

2018-01-01

Multi-functional mesoporous poly(ionic liquid) (MPIL) containing pyridine-based ionic liquid (IL) moieties and adjacent double sbnd COOH groups was synthesized through the free radical copolymerization of IL monomer N-propane sulfonate-4-vinylpyridine, maleic anhydride and divinylbenzene. Palladium(II) species were anchored on this MPIL support, affording the first efficient heterogeneous catalyst for the oxidative coupling of benzene to biphenyl under atmospheric oxygen at low temperature. The biphenyl yield of 15.0% (selectivity: 98.5%, turnover number: 62) was even higher than the one over the homogeneous counterpart palladium acetate. The catalyst can be facilely separated and reused. The IL moiety in the polymeric framework endowed the formation of immobilized palladium(II) species with high electrophilicity, which responds to the high performance.

High-performance supercapacitors based on poly(ionic liquid)-modified graphene electrodes.

PubMed

Kim, Tae Young; Lee, Hyun Wook; Stoller, Meryl; Dreyer, Daniel R; Bielawski, Christopher W; Ruoff, Rodney S; Suh, Kwang S

2011-01-25

We report a high-performance supercapacitor incorporating a poly(ionic liquid)-modified reduced graphene oxide (PIL:RG-O) electrode and an ionic liquid (IL) electrolyte (specifically, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)amide or EMIM-NTf(2)). PIL:RG-O provides enhanced compatibility with the IL electrolyte, thereby increasing the effective electrode surface area accessible to electrolyte ions. The supercapacitor assembled with PIL:RG-O electrode and EMIM-NTf(2) electrolyte showed a stable electrochemical response up to 3.5 V operating voltage and was capable of yielding a maximum energy density of 6.5 W·h/kg with a power density of 2.4 kW/kg. These results demonstrate the potential of the PIL:RG-O material as an electrode in high-performance supercapacitors.

Multifunctional nanoparticulate polyelectrolyte complexes.

PubMed

Hartig, Sean M; Greene, Rachel R; DasGupta, Jayasri; Carlesso, Gianluca; Dikov, Mikhail M; Prokop, Ales; Davidson, Jeffrey M

2007-12-01

Water-soluble, biodegradable, polymeric, polyelectrolyte complex dispersions (PECs) have evolved because of the limitations, in terms of toxicity, of the currently available systems. These aqueous nanoparticulate architectures offer a significant advantage for products that may be used as drug delivery systems in humans. PECs are created by mixing oppositely charged polyions. Their hydrodynamic diameter, surface charge, and polydispersity are highly dependent on concentration, ionic strength, pH, and molecular parameters of the polymers that are used. In particular, the complexation between polyelectrolytes with significantly different molecular weights leads to the formation of water-insoluble aggregates. Several PEC characteristics are favorable for cellular uptake and colloidal stability, including hydrodynamic diameter less than 200 nm, surface charge of >30 mV or <-30 mV, spherical morphology, and polydispersity index (PDI) indicative of a homogeneous distribution. Maintenance of these properties is critical for a successful delivery vehicle. This review focuses on the development and potential applications of PECs as multi-functional, site-specific nanoparticulate drug/gene delivery and imaging devices.

Porous polycarbene-bearing membrane actuator for ultrasensitive weak-acid detection and real-time chemical reaction monitoring.

PubMed

Sun, Jian-Ke; Zhang, Weiyi; Guterman, Ryan; Lin, Hui-Juan; Yuan, Jiayin

2018-04-30

Soft actuators with integration of ultrasensitivity and capability of simultaneous interaction with multiple stimuli through an entire event ask for a high level of structure complexity, adaptability, and/or multi-responsiveness, which is a great challenge. Here, we develop a porous polycarbene-bearing membrane actuator built up from ionic complexation between a poly(ionic liquid) and trimesic acid (TA). The actuator features two concurrent structure gradients, i.e., an electrostatic complexation (EC) degree and a density distribution of a carbene-NH 3 adduct (CNA) along the membrane cross-section. The membrane actuator performs the highest sensitivity among the state-of-the-art soft proton actuators toward acetic acid at 10 -6  mol L -1 (M) level in aqueous media. Through competing actuation of the two gradients, it is capable of monitoring an entire process of proton-involved chemical reactions that comprise multiple stimuli and operational steps. The present achievement constitutes a significant step toward real-life application of soft actuators in chemical sensing and reaction technology.

Colloidal titration of aqueous zirconium solutions with poly(vinyl sulfate) by potentiometric endpoint detection using a toluidine blue selective electrode.

PubMed

Sakurada, Osamu; Kato, Yasutake; Kito, Noriyoshi; Kameyama, Keiichi; Hattori, Toshiaki; Hashiba, Minoru

2004-02-01

Zirconium oxy-salts were hydrolyzed to form positively charged polymer or cluster species in acidic solutions. The zirconium hydrolyzed polymer was found to react with a negatively charged polyelectrolyte, such as poly(vinyl sulfate), and to form a stoichiometric polyion complex. Thus, colloidal titration with poly(vinyl sulfate) was applied to measure the zirconium concentration in an acidic solution by using a Toluidine Blue selective plasticized poly(vinyl chloride) membrane electrode as a potentiometric end-point detecting device. The determination could be performed with 1% of the relative standard deviation. The colloidal titration stoichiometry at pH < or = 2 was one mol of zirconium per equivalent mol of poly(vinyl sulfate).

Electrowetting of Weak Polyelectrolyte-Coated Surfaces.

PubMed

Sénéchal, Vincent; Saadaoui, Hassan; Rodriguez-Hernandez, Juan; Drummond, Carlos

2017-05-23

Polymer coatings are commonly used to modify interfacial properties like wettability, lubrication, or biocompatibility. These properties are determined by the conformation of polymer molecules at the interface. Polyelectrolytes are convenient elementary bricks to build smart materials, given that polyion chain conformation is very sensitive to different environmental variables. Here we discuss the effect of an applied electric field on the properties of surfaces coated with poly(acrylic acid) brushes. By combining atomic force microscopy, quartz crystal microbalance, and contact angle experiments, we show that it is possible to precisely tune polyion chain conformation, surface adhesion, and surface wettability using very low applied voltages if the polymer grafting density and environmental conditions (pH and ionic strength) are properly formulated. Our results indicate that the effective ionization degree of the grafted weak polyacid can be finely controlled with the externally applied field, with important consequences for the macroscopic surface properties.

Redox responsive nanotubes from organometallic polymers by template assisted layer by layer fabrication

NASA Astrophysics Data System (ADS)

Song, Jing; Jańczewski, Dominik; Guo, Yuanyuan; Xu, Jianwei; Vancso, G. Julius

2013-11-01

Redox responsive nanotubes were fabricated by the template assisted layer-by-layer (LbL) assembly method and employed as platforms for molecular payload release. Positively and negatively charged organometallic poly(ferrocenylsilane)s (PFS) were used to construct the nanotubes, in combination with other polyions. During fabrication, multilayers of these polyions were deposited onto the inner pores of template porous membranes, followed by subsequent removal of the template. Anodized porous alumina and track-etched polycarbonate membranes were used as templates. The morphology, electrochemistry, composition and other properties of the obtained tubular structure were characterized by fluorescence microscopy, scanning (SEM) and transmission electron microscopy (TEM) and energy-dispersive X-ray (EDX) spectroscopy. Composite nanotubes, consisting of poly(acrylic acid) anions with PFS+ and nanoparticles including fluorophore labelled dextran and decorated quantum dots, with PFS polyelectrolytes were also fabricated, broadening the scope of the structures. Cyclic voltammograms of PFS containing nanotubes showed similar redox responsive behaviour to thin LbL assembled films. Redox triggered release of labelled macromolecules from these tubular structures demonstrated application potential in controlled molecular delivery.Redox responsive nanotubes were fabricated by the template assisted layer-by-layer (LbL) assembly method and employed as platforms for molecular payload release. Positively and negatively charged organometallic poly(ferrocenylsilane)s (PFS) were used to construct the nanotubes, in combination with other polyions. During fabrication, multilayers of these polyions were deposited onto the inner pores of template porous membranes, followed by subsequent removal of the template. Anodized porous alumina and track-etched polycarbonate membranes were used as templates. The morphology, electrochemistry, composition and other properties of the obtained tubular structure were characterized by fluorescence microscopy, scanning (SEM) and transmission electron microscopy (TEM) and energy-dispersive X-ray (EDX) spectroscopy. Composite nanotubes, consisting of poly(acrylic acid) anions with PFS+ and nanoparticles including fluorophore labelled dextran and decorated quantum dots, with PFS polyelectrolytes were also fabricated, broadening the scope of the structures. Cyclic voltammograms of PFS containing nanotubes showed similar redox responsive behaviour to thin LbL assembled films. Redox triggered release of labelled macromolecules from these tubular structures demonstrated application potential in controlled molecular delivery. Electronic supplementary information (ESI) available: Nanotube wall thickness determination protocol. See DOI: 10.1039/c3nr03927g

About the singular behavior of the ionic condensation of sodium chondroitin sulfate: Conductivity study in water and water dioxane mixture

NASA Astrophysics Data System (ADS)

M'halla, Jalel; Besbes, Rafik; Bouazzi, Ramzi; Boughammoura, Sondes

2006-01-01

In this work, we generalized the (Bjerrum-Debye-Fuoss-MSA) double layer model to an ellipsoidal polyion (chondroitin sulfate) of (∣ Zs∣ e) structural charge, Ls structural length, R minor axe and ( R2 + L2/4) 1/2 major axe. With L ⩽ Ls. Na + counter ions are distributed on the contact (or condensed) layer and on the Debye layer (ionic atmosphere). Both layers are ellipsoidal equipotentials of, respectively, R and d minor axes and are concentric to the polyion. With d = ( R + 1/2 Γ), Γ is the Debye-MSA screen parameter. The equilibrium distribution of Na + ions is derived from a "two states" statistical approach, leading to a general implicit expression for the rate of condensation (1 - α). The generality of this formula results from the fact that it takes into account the finite size of the polyion ( L ≠ ∞ and R ≠ 0) and allows to calculate α for different conformations of the polyion: (ellipsoidal L ≠ 0, cylindrical: L = Ls, spherical: L → 0, and Manning's model: RL-1 → 0). The main conclusion of this model is that, α obeys to the Ostwald's principle of dilution ( α → 1 when CNa+ → 0). This result is contrary to Manning's theory, for which α is a constant αM independent on the concentration Ci: αM = bS/(∣ Zi∣ Lb), with bS = Ls/∣ Zs∣ and Lb = e2/( ɛkT) is the Bjerrum length. However, our analysis shows that the rate of variation: (∂ α/∂ Ci) in a given range of concentration, depends on the structural parameter bS. Indeed, the critical Manning condition ( α-1αM = 1, ⇒(∂ α/∂ Ci) ≈ 0), is compatible with the general following "rod-like model" approximation: (1-α)≈|Zi|(πbS)[4πZi2Lb][αCiR2];withbS″=αM-1 only for some peculiar values of bS and Lb (i.e., dielectric constant: ɛ). In water at 25 °C ( ɛ = 78.3), this singular behavior occurs for a range of a relative low or moderate concentration for some polyelectrolytes of bS structural parameter of about 5.8 Å. This is the case of sodium chondroitin sulfate in water ( bS = 5.72 Å). The addition of dioxane increases Lb, consequently, α is shifted from its Manning's value. In order to verify this dioxane effect, we have compared experimental equivalent conductibilities Λexp of sodium chondroitin sulfate in water ( no shift) and water-dioxane (60 wt%) mixture ( positive shift), to their theoretical values ΛM, ΛcthandΛsth corresponding, respectively, to the Manning, cylindrical and spherical models. This comparison allows also, to explain the conformation "chosen" by the polyion, in order to minimizing the friction effects (due to: viscosity; ionic and dielectric relaxations) and therefore, to optimize its mobility by the shift of its rate of ionic condensation α.

Poly(ionic liquids) functionalized polypyrrole/graphene oxide nanosheets for electrochemical sensor to detect dopamine in the presence of ascorbic acid.

PubMed

Mao, Hui; Liang, Jiachen; Zhang, Haifeng; Pei, Qi; Liu, Daliang; Wu, Shuyao; Zhang, Yu; Song, Xi-Ming

2015-08-15

Novel poly(ionic liquids) functionalized polypyrrole/graphene oxide nanosheets (PILs/PPy/GO) were prepared by the polymerization of 1-vinyl-3-ethylimidazole bromide (VEIB) on the surface of N-vinyl imidazolium modified PPy/GO nanosheets. Due to the synergistic effects of GO with well-defined lamellar structures, conductive PPy and biocompatible PILs, PILs/PPy/GO modified glassy carbon electrode (GCE) presented the excellent electrochemical catalytic activity towards dopamine (DA) with good stability, high sensitivity and wide linear range in the present of ascorbic acid (AA) with high concentration. PILs played an essential role for the simultaneous determination of DA and AA in a mixture, whose existence effectively improved the transmission mode of electrons and resulted in the different electrocatalytic performance towards the oxidation of DA and AA. It is indicated that PILs/PPy/GO nanosheets can act as a good steady and sensitive electrode material for the development of improved DA sensors. Copyright © 2015 Elsevier B.V. All rights reserved.

Organic-inorganic hybrid polyionic liquid based polyoxometalate as nano porous material for selective oxidation of sulfides

NASA Astrophysics Data System (ADS)

Rafiee, Ezzat; Shahebrahimi, Shabnam

2017-07-01

Organic-inorganic hybrid nano porous materials based on poly(ionic liquid)-polyoxometalate (PIL-POM) were reported. These hybrid materials were synthesized by the reaction of 4-vinyl pyridine with 1,3-propanesultone, followed by the polymerization and also sulfonate-functionalized cross-linked poly(4-vinylpyridine) and combining these polymers with H5PMo10V2O40 (PMo10V2). Activity of prepared PIL-PMo10V2 hybrids were investigated as catalysts for oxidation of sulfides with H2O2 as oxidant. For understanding catalytic activities of the PIL-PMo10V2 hybrids in oxidation of sulfides, effect of catalyst composition, substrate, and reaction conditions were studied. The results show that the PIL-PMo10V2 hybrids are active as selective heterogeneous catalysts for oxidation of sulfides and can be recovered and reused. The catalyst was characterized by FT-IR, TGA-DSC, XRD, SEM/EDX, BET, CV and zeta potential measurement. Also, average molecular weight of prepared catalysts were measured.

Nitrogen-doped carbon capsules via poly(ionic liquid)-based layer-by-layer assembly.

PubMed

Zhao, Qiang; Fellinger, Tim-Patrick; Antonietti, Markus; Yuan, Jiayin

2012-07-13

Layer-by-layer (LbL) assembly technique is applied for the first time for the preparation of nitrogen-doped carbon capsules. This approach uses colloid silica as template and two polymeric deposition components, that is, poly(ammonium acrylate) and a poly (ionic liquid) poly(3-cyanomethyl-1-vinylimidazolium bromide), which acts as both the carbon precursor and nitrogen source. Nitrogen-doped carbon capsules are prepared successfully by polymer wrapping, subsequent carbonization and template removal. The as-synthesized carbon capsules contain ≈7 wt% of nitrogen and have a structured specific surface area of 423 m(2) g(-1). Their application as supercapacitor has been briefly introduced. This work proves that LbL assembly methodology is available for preparing carbon structures of complex morphology. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Free fatty acids electronically bridge the self-assembly of a three-component nanocomplex consisting of amylose, protein, and free fatty acids.

PubMed

Zhang, Genyi; Maladen, Michelle; Campanella, Osvaldo H; Hamaker, Bruce R

2010-08-25

The self-assembly of a ternary complex, which is formed through heating and cooling of a mixture of amylose (1.0 mg/mL), whey protein isolate (50 μg/mL), and free fatty acids (FFAs, 250 μg/mL) was investigated. High-performance size-exclusion chromatography-multi-angle laser light scattering (HPSEC-MALLS) analysis showed that the complex is a water-soluble supramolecule (Mw = 6-7 × 10(6)), with a radius of gyration of 20-100 nm, indicating a nanoscale complex. Experimental results using 1-monostearyl-rac-glycerol (MSG) or cetyl alcohol that is similar to FFA in structure (except the headgroup) indicate that FFAs are the bridge between thermodynamically incompatible amylose and protein molecules and their functional carboxyl group is essential to the formation of the complex. Additionally, the effects of pH and salt treatments suggest that electrostatic interactions between negatively charged carboxyl groups of FFAs and polyionic protein are the foundation for the self-assembly of the complex. The fact that FFA is one important component in the self-assembled complex with an estimated molar ratio of 6:1:192 (amylose/protein/FFA, ∼4-5% FFA) demonstrates that it might be used as a nanocarrier for the controlled release of lipophilic functional materials to maintain their stability, bioactivity, and more importantly water solubility.

Forward-Osmosis Desalination with Poly(Ionic Liquid) Hydrogels as Smart Draw Agents.

PubMed

Fan, Xuelin; Liu, Huili; Gao, Yating; Zou, Zhu; Craig, Vincent S J; Zhang, Guangzhao; Liu, Guangming

2016-06-01

The combination of high desalination efficiency, negligible draw-solute leakage, nontoxicity, ease of regeneration, and effective separation to produce liquid water makes the smart draw agents developed here highly suited for forward-osmosis desalination. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Curcumin-Combretastatin Nanocells as Breast Cancer Cytotoxic and Antiangiogenic Agent

DTIC Science & Technology

2008-09-01

network of polyionic (e.g. polyethylenimine , polyacrylic acid, poly-L-lysine etc.) and neutral polymer chains (e.g. PEG, Pluronic/Poloxamer...devices were then mounted back to device stations placed inside a CO2 incubator. The CI was automatically monitored by the RT-CES system. The results

Pharmacological performance of novel poly-(ionic liquid)-grafted chitosan-N-salicylidene Schiff bases and their complexes.

PubMed

Elshaarawy, Reda F M; Refaee, Ayaat A; El-Sawi, Emtithal A

2016-08-01

In our endeavor to develop a new class of pharmacological candidates with antimicrobial and anticancer efficacy, a series of biopolymeric chitosan Schiff bases bearing salicylidene ionic liquid (IL-Sal) brushes (ILCSB1-3, poly-(GlcNHAc-GlcNH2-(GlcN-Sal-IL)) was successfully synthesized by adopting efficient synthetic routes. Unfortunately, metalation trials of these biopolymeric Schiff bases afford the corresponding Ag(I)/M(II) complexes (where M=Co, Pd). These designed architectures were structurally characterized and pharmacologically evaluated for their in vitro antimicrobial, against common bacterial and fungal pathogens, and anticancer activities against human colon carcinoma (HCT-116) cell line. In conclusion functionalization of chitosan with IL-Sal brushes coupled with metalation of formed ILCSBs were synergistically enhanced its antimicrobial and antitumor properties to a great extent. Noteworthy, Ag-ILCSB2 (IC50=9.13μg/mL) was ca. 5-fold more cytotoxic against HCT-116 cell line than ILCSB2 (IC50=43.30μg/mL). Copyright © 2016. Published by Elsevier Ltd.

Visual indication of enviromental humidity by using poly(ionic liquid) photonic crystals.

PubMed

Huang, Jing; Tao, Cheng-an; An, Qi; Lin, Changxu; Li, Xuesong; Xu, Dan; Wu, Yiguang; Li, Xiaogang; Shen, Dezhong; Li, Guangtao

2010-06-21

The combination of poly (ionic liquid) and photonic structure affords a new class of self-reporting humidity sensory materials with excellent reversibility, which are able to rapidly, sensitively and visually indicate environmental humidity with colour change from blue to green, orange, and red, covering the whole visible range.

Supercapacitors based on modified graphene electrodes with poly(ionic liquid)

NASA Astrophysics Data System (ADS)

Trigueiro, João Paulo C.; Lavall, Rodrigo L.; Silva, Glaura G.

2014-06-01

The improved accessibility of the electrolyte to the surface of carbon nanomaterials is a challenge to be overcome in supercapacitors based on ionic liquid electrolytes. In this study, we report the preparation of supercapacitors based on reduced graphene oxide (RGO) electrodes and ionic liquid as the electrolyte (specifically, 1-methyl-1-propylpyrrolidinium bis(trifluoromethylsulfonyl)imide or [MPPy][TFSI]). Two types of electrodes were compared: the RGO-based electrode and a poly(ionic liquid)-modified RGO electrode (PIL:RGO). The supercapacitor produced with the PIL:RGO electrode and [MPPy][TFSI] showed an electrochemical stability of 3 V and provided a capacitance of 71.5 F g-1 at room temperature; this capacitance is 130% higher with respect to the RGO-based supercapacitor. The decrease of the specific capacitance after 2000 cycles is only 10% for the PIL:RGO-based device. The results revealed the potential of the PIL:RGO material as an electrode for supercapacitors. This composite electrode increases the compatibility with the ionic liquid electrolyte compared to an RGO electrode, promoting an increase in the effective surface area of the electrode accessible to the electrolyte ions.

Highly Swellable, Dual-Responsive Hydrogels Based on PNIPAM and Redox Active Poly(ferrocenylsilane) Poly(ionic liquid)s: Synthesis, Structure, and Properties.

PubMed

Feng, Xueling; Zhang, Kaihuan; Chen, Peng; Sui, Xiaofeng; Hempenius, Mark A; Liedberg, Bo; Vancso, G Julius

2016-12-01

Highly swellable, dual-responsive hydrogels, consisting of thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) and redox-responsive poly(ferrocenylsilane) (PFS) based poly(ionic liquid)s (PILs) are formed by photo-polymerization. PFS chains bearing cross-linkable vinylimidazolium (VIm) side groups are copolymerized with NIPAM in aqueous solutions under ultraviolet light (λ = 365 nm) in the presence of a photoinitiator. The PFS-PILs serve as a macro-cross-linker and also provide redox responsiveness. The swelling ratio, morphology, and lower critical solution temperature (LCST) of the hydrogels are studied as a function of the PNIPAM/PFS ratio. The value of the LCST is dependent on the choice of the counterion of the PIL and the PNIPAM/PFS ratio. The hydrogel is employed as a reducing environment for the in situ fabrication of gold nanoparticles (AuNPs), forming AuNP-hydrogel composites. The localized surface plasmon resonance peak of the as-synthesized Au nanoparticles inside the hydrogel could be tuned by altering the temperature. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Guar gum as biosourced building block to generate highly conductive and elastic ionogels with poly(ionic liquid) and ionic liquid.

PubMed

Zhang, Biao; Sudre, Guillaume; Quintard, Guilhem; Serghei, Anatoli; David, Laurent; Bernard, Julien; Fleury, Etienne; Charlot, Aurélia

2017-02-10

In this study, we report on the simple and straightforward preparation of ionogels arising from the addition of guar gum (a plant-based polysaccharide) in a solution of precisely-defined poly(ionic liquid) chains (PIL) in imidazolium-based ionic liquid (IL). The development of intermolecular polar interactions (mainly hydrogen bonds) and topologic chain entanglements induces the formation of physical biohybrid ionogels, whose elastic properties can be easily tuned by varying the composition (up to 30000Pa). The combined presence of guar gum and PIL confers excellent dimensional stability to the ionogels with no IL exudation combined with high thermal properties (up to 310°C). The resulting materials are shown to exhibit gel scattering profiles and high conductivities (> 10 -4 S/cm at 30°C). The benefit linked to the formation of guar/PIL associations in IL medium enables to find a good compromise between the mechanical cohesion and the mobility ensuring the ionic transport. Copyright © 2016 Elsevier Ltd. All rights reserved.

Linear and Star Poly(ionic liquid) Assemblies: Surface Monolayers and Multilayers.

PubMed

Erwin, Andrew J; Xu, Weinan; He, Hongkun; Matyjaszewski, Krzysztof; Tsukruk, Vladimir V

2017-04-04

The surface morphology and organization of poly(ionic liquid)s (PILs), poly[1-(4-vinylbenzyl)-3-butylimidazolium bis(trifluoromethylsulfonyl)imide] are explored in conjunction with their molecular architecture, adsorption conditions, and postassembly treatments. The formation of stable PIL Langmuir and Langmuir-Blodgett (LB) monolayers at the air-water and air-solid interfaces is demonstrated. The hydrophobic bis(trifluoromethylsulfonyl)imide (Tf 2 N - ) is shown to be a critical agent governing the assembly morphology, as observed in the reversible condensation of LB monolayers into dense nanodroplets. The PIL is then incorporated as an unconventional polyelectrolyte component in the layer-by-layer (LbL) films of hydrophobic character. We demonstrate that the interplay of capillary forces, macromolecular mobility, and structural relaxation of the polymer chains influence the dewetting mechanisms in the PIL multilayers, thereby enabling access to a diverse set of highly textured, porous, and interconnected network morphologies for PIL LbL films that would otherwise be absent in conventional LbL films. Their compartmentalized internal structure is relevant to molecular separation membranes, ultrathin hydrophobic coatings, targeted cargo delivery, and highly conductive films.

Macro-hydrogels versus nanoparticles by the controlled assembly of polysaccharides.

PubMed

Costalat, M; Alcouffe, P; David, L; Delair, T

2015-12-10

The controlled assembly of oppositely charged chitosan (CS, Mw ∼ 33 × 10(3) to 600 × 10(3)g mol(-1)) and dextran sulfate (DS, Mw = 1.3 × 10(6)g mol(-1)) or heparin (HP, Mw = 1.8 × 10(4)g mol(-1)) led either to nanoparticles or macro-hydrogels, at room temperature. The control over the electrostatic attractive interactions was achieved using 2 mol L(-1) NaCl in the polyion solutions and subsequent dialysis to let the assembly occur. Macrohydrogels formed with an excess of polyanion. In the presence of an excess of polycation, colloidal gels were exclusively obtained. At salt concentrations lower than 1 mol L(-1), the spontaneous gelation provided macro-hydrogels, whatever the polyion in excess. Rheology measurements showed a similar elastic behaviour for CS-DS and CS-HP hydrogels, though CS-HP hydrogels appeared less cohesive. SAXS experiments revealed an aggregate morphology with internal and surface structure depending on the degree of acetylation (DA) of chitosan. Copyright © 2015 Elsevier Ltd. All rights reserved.

Microphase separation and the formation of ion conductivity channels in poly(ionic liquid)s: A coarse-grained molecular dynamics study

NASA Astrophysics Data System (ADS)

Weyman, Alexander; Bier, Markus; Holm, Christian; Smiatek, Jens

2018-05-01

We study generic properties of poly(ionic liquid)s (PILs) via coarse-grained molecular dynamics simulations in bulk solution and under confinement. The influence of different side chain lengths on the spatial properties of the PIL systems and on the ionic transport mechanism is investigated in detail. Our results reveal the formation of apolar and polar nanodomains with increasing side chain length in good agreement with previous results for molecular ionic liquids. The ion transport numbers are unaffected by the occurrence of these domains, and the corresponding values highlight the potential role of PILs as single-ion conductors in electrochemical devices. In contrast to bulk behavior, a pronounced formation of ion conductivity channels in confined systems is initiated in close vicinity to the boundaries. We observe higher ion conductivities in these channels for increasing PIL side chain lengths in comparison with bulk values and provide an explanation for this effect. The appearance of these domains points to an improved application of PILs in modern polymer electrolyte batteries.

Microscopic Characterization of Individual Submicron Bubbles during the Layer-by-Layer Deposition: Towards Creating Smart Agents

NASA Astrophysics Data System (ADS)

Kato, Riku; Frusawa, Hiroshi

2015-07-01

We investigated the individual properties of various polyion-coated bubbles with a mean diameter ranging from 300 to 500 nm. Dark field microscopy allows one to track the individual particles of the submicron bubbles (SBs) encapsulated by the layer-by-layer (LbL) deposition of cationic and anionic polyelectrolytes (PEs). Our focus is on the two-step charge reversals of PE-SB complexes: the first is a reversal from negatively charged bare SBs with no PEs added to positive SBs encapsulated by polycations (monolayer deposition), and the second is overcharging into negatively charged PE-SB complexes due to the subsequent addition of polyanions (double-layer deposition). The details of these phenomena have been clarified through the analysis of a number of trajectories of various PE-SB complexes that experience either Brownian motion or electrophoresis. The contrasted results obtained from the analysis were as follows: an amount in excess of the stoichiometric ratio of the cationic polymers was required for the first charge-reversal, whereas the stoichiometric addition of the polyanions lead to the electrical neutralization of the PE-SB complex particles. The recovery of the stoichiometry in the double-layer deposition paves the way for fabricating multi-layered SBs encapsulated solely with anionic and cationic PEs, which provides a simple protocol to create smart agents for either drug delivery or ultrasound contrast imaging.

Microscopic Characterization of Individual Submicron Bubbles during the Layer-by-Layer Deposition: Towards Creating Smart Agents.

PubMed

Kato, Riku; Frusawa, Hiroshi

2015-07-08

We investigated the individual properties of various polyion-coated bubbles with a mean diameter ranging from 300 to 500 nm. Dark field microscopy allows one to track the individual particles of the submicron bubbles (SBs) encapsulated by the layer-by-layer (LbL) deposition of cationic and anionic polyelectrolytes (PEs). Our focus is on the two-step charge reversals of PE-SB complexes: the first is a reversal from negatively charged bare SBs with no PEs added to positive SBs encapsulated by polycations (monolayer deposition), and the second is overcharging into negatively charged PE-SB complexes due to the subsequent addition of polyanions (double-layer deposition). The details of these phenomena have been clarified through the analysis of a number of trajectories of various PE-SB complexes that experience either Brownian motion or electrophoresis. The contrasted results obtained from the analysis were as follows: an amount in excess of the stoichiometric ratio of the cationic polymers was required for the first charge-reversal, whereas the stoichiometric addition of the polyanions lead to the electrical neutralization of the PE-SB complex particles. The recovery of the stoichiometry in the double-layer deposition paves the way for fabricating multi-layered SBs encapsulated solely with anionic and cationic PEs, which provides a simple protocol to create smart agents for either drug delivery or ultrasound contrast imaging.

Remarks on Polyelectrolyte Conformation

NASA Astrophysics Data System (ADS)

de Gennes, P. G.; Pincus, P.; Velasco, R. M.; Brochard, F.

Nous discutons des conformations de polymères linéaires chargés en faisant les hypothèses suivantes : a) la chaĬne sans charge est flexible, b) la force éctrostatique domine les interactions monomère-monomère c) il n'y a pas de sels. 1) Pour le cas dilué (chaĬne non enchevetrees) en corrigeant le calcul self-consistant fait récemment par Richmond [1a], on trouve une taille des polyions égale a = R ND, qui est une fonction linéaire de l'indice de polymérisation N. Ce rèsultat est en accord avec les prècèdents travaux de Hermans et Overbeek [1b], Kuhn, Kunzle et Katchalsky [1c]. 2) Il existe un domaine pour des concentrations très petites c (c** < c < c*) oò les interactions èlectrostatiques entre les polyions sont supèrieures aux ènergies thermiques, il semble donc possible que les polyions puissent former un rèseau pèriodique à trois dimensions. Nèanmoins, il semble difficile de mettre en èvidence un rèseau si diluè. 3) Jusqu'ici toutes les expériences avec les polyélectrolytes sans sels ont été pratiquement faites à des concentrations c > c*, pour lesquelles les différentes cha.nes sont enchevêtrées. Pour discuter ce régime on s.intéresse uniquement au cas où la charge par unité de longueur est près du (ou audessus du) seuil de condensation, donc il existe une seule longueur ξ(c) caractérisant les corrélations; à trois dimensions 03BE a le même comportement que le rayon de Debye pour les contre-ions. On a considéré quelques conformations possibles : a) un réseau hexagonal de batonnets; b) un réseau cubique de batonnets; c) une phase isotrope de cha.nes partiellement flexibles. Les différentes structures formées de batonnets semblent avoir la même énergie électrostatique. Ce fait suggère que la phase isotrope peut être la plus favorable. On analyse cette dernière phase en utilisant les mêmes méthodes qui se sont révélées efficaces pour les solutions des polymères neutres. Dans le modèle isotrope chaque cha.ne a le comportement d.une succession des petites pelotes (blobs) de taille 03BE. Les effets électrostatiques sont importants à l.intérieur d'un blobs et analogues au cas (1). Mais ces interactions sont écrantées entre les blobs ; chaque cha.ne a un comportement idéal à grande échelle et son rayon est R(c) ~ c-¼ N½. Si on suppose que les effets dynamiques des enchevêtrements sont faibles on trouve une valeur pour la viscosité ηsp/c ~ Nc-½ We discuss the conformations of linear polyions assuming that a) the corresponding uncharged chain is flexible ; b) electrostatic forces dominate the monomer-monomer interactions; c) no salt is added. 1) For the dilute case (non overlapping chains) correcting a recent self-consistent calculation by Richmond [1a], we find an overall polyion size R = Nd which is a linear function of the polymerization index N in agreement with the early work of Hermans and Overbeek, [1b], Kuhn, Kunzle, and Katchalsky [1c]. 2) There is a range of very low concentration c (c** < c < c*) where the chains do not overlap (c < c*) but where the electrostatic interactions between polyions are much larger than thermal energies (c > c**) : here we expect that the polyions build up a 3-dimensional periodic lattice ; however, the detection of such an extremely dilute lattice appears difficult. 3) Practically all experiments on salt-free polyelectrolytes have been performed at concentrations c > c* where different chains overlap each other. To discuss this regime we restrict our attention to cases where the charge per unit length is near (or above) the condensation threshold : then a single length ξ(c) characterizes the correlation; in 3 dimensions ξ scales like the Debye radius associated with the counter ions. We consider several possible conformations : a) hexagonal lattice of rigid rods ; b) cubic lattice of rigid rods; c) isotropic phase of partially flexible chains. The various rigid rod structures appear to have very similar electrostatic energies. This suggests that the isotropic phase might possibly be the most favorable. We analyse this latter phase using the same scaling methods which have recently been helpful for neutral polymer solutions (2). In the isotropic model each chain behaves like a succession of segments of size. Inside one segment electrostatic effects are important and similar to case (1) above. Between segments the interactions are screened out, and tach chain is ideal on a large scale, with radius R(c) ~ c-¼ N½. If we (tentatively) assume that the dynamical effects of entanglements are weak, we are than led to a viscosity ηsp/c ~ Nc-½.

Surface and Electrochemical Properties of Polymer Brush-Based Redox Poly(Ionic Liquid).

PubMed

Bui-Thi-Tuyet, Van; Trippé-Allard, Gaëlle; Ghilane, Jalal; Randriamahazaka, Hyacinthe

2016-10-26

Redox-active poly(ionic liquid) poly(3-(2-methacryloyloxy ethyl)-1-(N-(ferrocenylmethyl) imidazolium bis(trifluoromethylsulfonyl)imide deposited onto electrode surfaces has been prepared using surface-initiated atom transfer radical polymerization SI-ATRP. The process starts by electrochemical immobilization of initiator layer, and then methacrylate monomer carrying ferrocene and imidazolium units is polymerized in ionic liquid media via SI-ATRP process. The surfaces analyses of the polymer exhibit a well-defined polymer brushlike structure and confirm the presence of ferrocene and ionic moieties within the film. Furthermore, the electrochemical investigations of poly(redox-active ionic liquid) in different media demonstrate that the electron transfer is not restricted by the rate of counterion migration into/out of the polymer. The attractive electrochemical performance of these materials is further demonstrated by performing electrochemical measurement, of poly(ferrocene ionic liquid), in solvent-free electrolyte. The facile synthesis of such highly ordered electroactive materials based ionic liquid could be useful for the fabrication of nanostructured electrode suitable for performing electrochemistry in solvent free electrolyte. We also demonstrate possible applications of the poly(FcIL) as electrochemically reversible surface wettability system and as electrochemical sensor for the catalytic activity toward the oxidation of tyrosine.

Ionic Liquids and Poly(ionic liquid)s for Morphosynthesis of Inorganic Materials.

PubMed

Gao, Min-Rui; Yuan, Jiayin; Antonietti, Markus

2017-04-24

Ionic liquids (ILs) are new, innovative ionic solvents with rich physicochemical properties and intriguing pre-organized solvent structures; these materials offer great potential to impact across versatile areas of scientific research, for example, synthetic inorganic chemistry. Recent use of ILs as precursors, templates, and solvents has led to inorganic materials with tailored sizes, dimensionalities, morphologies, and functionalities that are difficult to obtain, or even not accessible, by using conventional solvents. Poly(ionic liquid)s (PILs) polymerized from IL monomers also raise the prospect of modifying nucleation, growth, and crystallization of inorganic objects, shedding light on the synthesis of a wide range of new materials. Here we survey recent key progress in using ILs and PILs in the field of synthetic inorganic chemistry. As well as highlighting the unique features of ILs and PILs that enable advanced synthesis, the effects of adding other solvents to the final products, along with the emerging applications of the created inorganic materials will be discussed. We finally provide an outlook on several development opportunities that could lead to new advancements of this exciting research field. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Poly(Ionic Liquid) Semi-Interpenetrating Network Multi-Responsive Hydrogels

PubMed Central

Tudor, Alexandru; Florea, Larisa; Gallagher, Simon; Burns, John; Diamond, Dermot

2016-01-01

Herein we describe poly(ionic liquid) hydrogel actuators that are capable of responding to multiple stimuli, namely temperature, ionic strength and white light irradiation. Using two starting materials, a crosslinked poly ionic liquid (PIL) and a linear poly(N-isopropylacrylamide-co-spiropyran-co-acrylic acid), several semi-interpenetrating (sIPN) hydrogels were synthesised. The dimensions of hydrogels discs were measured before and after applying the stimuli, to quantify their response. Samples composed of 100% crosslinked PIL alone showed an average area reduction value of ~53% when the temperature was raised from 20 °C to 70 °C, ~24% when immersed in 1% w/w NaF salt solution and no observable photo-response. In comparison, sIPNs containing 300% w/w linear polymer showed an average area reduction of ~45% when the temperature was raised from 20 °C to 70 °C, ~36% when immersed in 1% NaF w/w salt solution and ~10% after 30 min exposure to white light irradiation, respectively. Moreover, by varying the content of the linear component, fine-control over the photo-, thermo- and salt response, swelling-deswelling rate and mechanical properties of the resulting sIPN was achieved. PMID:26861339

Hydrogels with a Memory: Dual-Responsive, Organometallic Poly(ionic liquid)s with Hysteretic Volume-Phase Transition

PubMed Central

2017-01-01

We report on the synthesis and structure–property relations of a novel, dual-responsive organometallic poly(ionic liquid) (PIL), consisting of a poly(ferrocenylsilane) backbone of alternating redox-active, silane-bridged ferrocene units and tetraalkylphosphonium sulfonate moieties in the side groups. This PIL is redox responsive due to the presence of ferrocene in the backbone and also exhibits a lower critical solution temperature (LCST)-type thermal responsive behavior. The LCST phase transition originates from the interaction between water molecules and the ionic substituents and shows a concentration-dependent, tunable transition temperature in aqueous solution. The PIL’s LCST-type transition temperature can also be influenced by varying the redox state of ferrocene in the polymer main chain. As the polymer can be readily cross-linked and is easily converted into hydrogels, it represents a new dual-responsive materials platform. Interestingly, the as-formed hydrogels display an unusual, strongly hysteretic volume-phase transition indicating useful thermal memory properties. By employing the dispersing abilities of this cationic PIL, CNT-hydrogel composites were successfully prepared. These hybrid conductive composite hydrogels showed bi-stable states and tunable resistance in heating–cooling cycles. PMID:28654756

Installation of a reactive site for covalent wiring onto an intrinsically conductive poly(ionic liquid)

DOE PAGES

Brombosz, Scott M.; Lee, Sungwon; Firestone, Millicent A.

2014-11-04

We describe post-polymerization radical bromination of a nanostructured poly(ionic liquid) that selectively introduces a reactive bromo-group onto the polyalkylthiophene backbone. Raman and FT-IR spectroscopy proves that the bromine is successfully introduced at the 3-methyl position of the thiophene and that the molecular structure of the polymer remains largely intact with only minimal chain scission detected. FT-IR and Vis-NIR spectroscopy indicates that incorporation of the bromine induces twisting (loss of co-planarity) of the polythiophene backbone. WAXS confirms retention of an ordered lamellar structure with minor lattice spacing contraction. Cyclic voltammetry confirms spectroscopic findings that the bromination reaction yields a stable p-dopedmore » polymer. The installed bromine is susceptible to nucleophilic displacement permitting the covalent attachment of other functional molecules, such as a dialkylphosphonate. Elemental analysis of such a transformation established that 100 % functionalization can be achieved. These results collectively demonstrate that post-modification of a π-conjugated polymer can be used to both tune electronic and photonic properties, as well as install a chemoselective attachment point for the covalent wiring of other molecules.« less

Development of chitosan-pullulan composite nanoparticles for nasal delivery of vaccines: in vivo studies.

PubMed

Cevher, Erdal; Salomon, Stefan K; Somavarapu, Satyanarayana; Brocchini, Steve; Alpar, H Oya

2015-01-01

Here, we aimed at developing chitosan/pullulan composite nanoparticles and testing their potential as novel systems for the nasal delivery of diphtheria toxoid (DT). All the chitosan derivatives [N-trimethyl (TMC), chloride and glutamate] and carboxymethyl pullulan (CMP) were synthesised and antigen-loaded composites were prepared by polyion complexation of chitosan and pullulan derivatives (particle size: 239-405 nm; surface charge: +18 and +27 mV). Their immunological effects after intranasal administration to mice were compared to intramuscular route. Composite nanoparticles induced higher levels of IgG responses than particles formed with chitosan derivative and antigen. Nasally administered TMC-pullulan composites showed higher DT serum IgG titre when compared with the other composites. Co-encapsulation of CpG ODN within TMC-CMP-DT nanoparticles resulted in a balanced Th1/Th2 response. TMC/pullulan composite nanoparticles also induced highest cytokine levels compared to those of chitosan salts. These findings demonstrated that TMC-CMP-DT composite nanoparticles are promising delivery system for nasal vaccination.

Effect of salt and surfactant concentration on the structure of polyacrylate gel/surfactant complexes.

PubMed

Nilsson, Peter; Unga, Johan; Hansson, Per

2007-09-20

Small-angle X-ray scattering was used to elucidate the structure of crosslinked polyacrylate gel/dodecyltrimethylammonium bromide complexes equilibrated in solutions of varying concentrations of surfactant and sodium bromide (NaBr). Samples were swollen with no ordering (micelle free), or they were collapsed with either several distinct peaks (cubic Pm3n) or one broad correlation peak (disordered micellar). The main factor determining the structure of the collapsed complexes was found to be the NaBr concentration, with the cubic structure existing up to approximately 150 mM NaBr and above which only the disordered micellar structure was found. Increasing the salt concentration decreases the polyion mediated attractive forces holding the micelles together causing swelling of the gel. At sufficiently high salt concentration the micelle-micelle distance in the gel becomes too large for the cubic structure to be retained, and it melts into a disordered micellar structure. As most samples were above the critical micelle concentration, the bulk of the surfactant was in the form of micelles in the solution and the surfactant concentration thereby had only a minor influence on the structure. However, in the region around 150 mM NaBr, increasing the surfactant concentration, at constant NaBr concentration, was found to change the structure from disordered micellar to ordered cubic and back to disordered again.

FOR STIMUL-RESPONSIVE POLYMERS WITH ENHANCED EFFICIENCY IN RESERVOIR RECOVERY PROCESSES

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

Charles McCormick; Roger Hester

This report contains a series of terpolymers containing acrylic acid, methacrylamide and a twin-tailed hydrophobic monomer that were synthesized using micellar polymerization methods. These polymer systems were characterized using light scattering, viscometry, and fluorescence methods. Viscosity studies indicate that increasing the nonpolar character of the hydrophobic monomer (longer chain length or twin tailed vs. single tailed) results in enhanced viscosity in aqueous solutions. The interactions of these polymers with surfactants were investigated. These surfactants include sodium dodecyl sulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), Triton X-100. Viscosity measurements of DiC{sub 6}AM and DiC{sub 8}AM mixtures indicate little interaction with SDS,more » gelation with CTAB, and hemimicelle formation followed by polymer hydrophobe solubilization with Triton X-100. The DiC{sub 10}Am terpolymer shows similar interaction behavior with CTAB and Triton X-100. However, the enhanced hydrophobic nature of the DiC{sub 10} polymer allows complex formation with SDS as confirmed by surface tensiometry. Fluorescence measurements performed on a dansyl labeled DiC{sub 10}Am terpolymer in the presence of increasing amounts of each of the surfactant indicate relative interaction strengths to be CTAB>Triton X-100>SDS. A modified model based on Yamakawa-Fujii and Odjik-Skolnick-Fixman theories was found to describe the contribution of electrostatic forces to the excluded volume of a polyelectrolyte in solution. The model was found to be valid for flexible polymer coils in aqueous salt solutions where intermolecular interactions are minimal. The model suggested that a dimensionless group of parameters termed the dimensionless viscosity should be proportional to the dimensionless ratio of solution screening length to polyion charge spacing. Several sets of experimental data from the literature and from our laboratory have been analyzed according to the model and the results suggest that the two dimensionless groups are indeed related by a universal constant. This model has identified the parameters that are important to fluid mobility, thereby revealing methods to enhance solution performance when using polyions solutions as displacing fluids in oil reservoirs.« less

Innovative Poly(Ionic Liquid)s by the Polymerization of Deep Eutectic Monomers.

PubMed

Isik, Mehmet; Ruiperez, Fernando; Sardon, Haritz; Gonzalez, Alba; Zulfiqar, Sonia; Mecerreyes, David

2016-07-01

The incorporation of ionic liquid (IL) chemistry into functional polymers has extended the properties and applications of polyelectrolytes. However, ILs are expensive due to the presence of fluorinated anions or complicated synthetic steps which limit their technological viability. Here, we show a new family of poly(ionic liquid)s (PILs) which are based in cheap and renewable chemicals and involves facile synthetic approaches. Thus, deep eutectic monomers (DEMs) are prepared for the first time by using quaternary ammonium compounds and various hydrogen bond donors such as citric acid, terephthalic acid or an amidoxime. The deep eutectic formation is made through a simple mixing of the ingredients. Differential scanning calorimetry, nuclear magnetic resonance (NMR) and computational studies reveal the formation of the DEMs due to the ionic interactions. The resulting DEMs are liquid which facilitates their polymerization using mild photopolymerization or polycondensation strategies. Spectroscopic characterizations reveal the successful formation of the polymers. By this way, a new family of PILs can be synthesized which can be used for different applications. As an example, the polymers show promising performance as solid CO2 sorbents. Altogether the deep eutectic monomer route can lead to non-toxic, cheap and easy-to-prepare alternatives to current PILs for different applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Formation of wormlike aggregates of fluorocarbon-hydrocarbon hybrid surfactant by Langmuir-Blodgett transfer and alignment of gold nanoparticles.

PubMed

Kondo, Yukishige; Fukuoka, Hiroshi; Nakano, Shuichi; Hayashi, Kohei; Tsukagoshi, Tatsuya; Matsumoto, Mutsuyoshi; Yoshino, Norio

2007-05-22

A novel anionic fluorocarbon-hydrocarbon hybrid surfactant (SS-Hyb-Na+) with a disulfide group has been synthesized from 11-bromo-1-undecanal and perfluorohexylethyl iodide via three steps. The Langmuir-Blodgett (LB) transfer of the 1:100 (mol/mol) mixed monolayer of SS-Hyb-Na+ and stearyl alcohol (C18OH) formed on an aqueous solution containing a cationic polymer, poly(diallyldimethylammonium chloride) (PDDA+Cl-) onto a hydrophobic silicon wafer yields the formation of wormlike aggregates consisting of SS-Hyb-/PDDA+ polyion complexes. It is found that the aggregates align along the withdrawal direction of the wafer substrate. When the wafer on which the wormlike aggregates exist is immersed into the dispersion of gold nanoparticles (Au NPs) prepared by the citrate reduction method, Au NPs align along the wormlike structures. Even though the surface of the wafer is placed either vertical or parallel to the monolayer compression direction during the LB transfer, the one-dimensional (1D) array of Au NPs is observed along the withdrawal direction of the wafer. This indicates that the wormlike aggregates of SS-Hyb-/PDDA+ complexes are aligned during the LB transfer, and the aligned aggregates behave as a scaffold in the 1D array of Au NPs.

Secure and effective gene delivery system of plasmid DNA coated by polynucleotide.

PubMed

Kodama, Yukinobu; Ohkubo, Chikako; Kurosaki, Tomoaki; Egashira, Kanoko; Sato, Kayoko; Fumoto, Shintaro; Nishida, Koyo; Higuchi, Norihide; Kitahara, Takashi; Nakamura, Tadahiro; Sasaki, Hitoshi

2015-01-01

Polynucleotides are anionic macromolecules which are expected to transfer into the targeted cells through specific uptake mechanisms. So, we developed polynucleotides coating complexes of plasmid DNA (pDNA) and polyethylenimine (PEI) for a secure and efficient gene delivery system and evaluated their usefulness. Polyadenylic acid (polyA), polyuridylic acid (polyU), polycytidylic acid (polyC), and polyguanylic acid (polyG) were examined as the coating materials. pDNA/PEI/polyA, pDNA/PEI/polyU, and pDNA/PEI/polyC complexes formed nanoparticles with a negative surface charge although pDNA/PEI/polyG was aggregated. The pDNA/PEI/polyC complex showed high transgene efficiency in B16-F10 cells although there was little efficiency in pDNA/PEI/polyA and pDNA/PEI/polyU complexes. An inhibition study strongly indicated the specific uptake mechanism of pDNA/PEI/polyC complex. Polynucleotide coating complexes had lower cytotoxicity than pDNA/PEI complex. The pDNA/PEI/polyC complex showed high gene expression selectively in the spleen after intravenous injection into mice. The pDNA/PEI/polyC complex showed no agglutination with erythrocytes and no acute toxicity although these were observed in pDNA/PEI complex. Thus, we developed polynucleotide coating complexes as novel vectors for clinical gene therapy, and the pDNA/PEI/polyC complex as a useful candidate for a gene delivery system.

Proton and metal ion binding to natural organic polyelectrolytes-I. Studies with synthetic model compounds

USGS Publications Warehouse

Marinsky, J.A.; Reddy, M.M.

1984-01-01

A unified physico-chemical model, based on a modified Henderson-Hasselbalch equation, for the analysis of ion complexation reactions involving charged polymeric systems is presented and verified. In this model pH = pKa+p(??Ka) + log(??/1 - ??) where Ka is the intrinsic acid dissociation constant of the ionizable functional groups on the polymer, ??Ka is the deviation of the intrinsic constant due to electrostatic interaction between the hydrogen ion and the polyanion, and alpha (??) is the polyacid degree of ionization. Using this approach pKa values for repeating acidic units of polyacrylic (PAA) and polymethacrylic (PMA) acids were found to be 4.25 ?? 0.03 and 4.8 ?? 0.1, respectively. The polyion electrostatic deviation term derived from the potentiometric titration data (i.e. p(??Ka)) is used to calculate metal ion concentration at the complexation site on the surface of the polyanion. Intrinsic cobalt-polycarboxylate binding constants (7.5 for PAA and 5.6 for PMA), obtained using this procedure, are consistent with the range of published binding constants for cobalt-monomer carboxylate complexes. In two phase systems incorporation of a Donnan membrane potential term allows determination of the intrinsic pKa of a cross-linked PMA gel, pKa = 4.83, in excellent agreement with the value obtained for the linear polyelectrolyte and the monomer. Similarly, the intrinsic stability constant for cobalt ion binding to a PMA-gel (??CoPMA+ = 11) was found to be in agreement with the linear polyelectrolyte analogue and the published data for cobalt-carboxylate monodentate complexes. ?? 1984.

Significance of the DNA-Histone Complex Level as a Predictor of Major Adverse Cardiovascular Events in Hemodialysis Patients: The Effect of Uremic Toxin on DNA-Histone Complex Formation.

PubMed

Jeong, Jong Cheol; Kim, Ji-Eun; Gu, Ja-Yoon; Yoo, Hyun Ju; Ryu, Ji Won; Kim, Dong Ki; Joo, Kwon Wook; Kim, Hyun Kyung

2016-01-01

Neutrophils can release the DNA-histone complex into circulation following exposure to inflammatory stimuli. This prospective study investigated whether the DNA-histone complex and other biomarkers could predict major cardiovascular adverse events (MACEs) in hemodialysis (HD) patients. The levels of circulating DNA-histone complexes, cell-free DNA, interleukin (IL)-6, and neutrophil elastase were measured in 60 HD patients and 28 healthy controls. MACE was assessed at 24 months. Uremic toxin-induced neutrophil released contents were measured in vitro. Compared with controls, HD patients showed higher levels of DNA-histone complexes and IL-6. The DNA-histone complex level was inversely associated with the Kt/V. In a multivariable Cox analysis, the high level of DNA-histone complexes was a significant independent predictor of MACE. The uremic toxins induced DNA-histone complex formation in normal neutrophils in vitro. The DNA-histone complex is a potentially useful marker to predict MACE in HD patients. Uremic toxins induced DNA-histone complex formation in vitro. © 2015 S. Karger AG, Basel.

Heterogeneous conversion of CO2 into cyclic carbonates at ambient pressure catalyzed by ionothermal-derived meso-macroporous hierarchical poly(ionic liquid)s† †Electronic supplementary information (ESI) available: Experimental section, details of the IR, 13C NMR and XPS spectra, characterization (1H NMR, 13C NMR, 13C CP-MAS NMR, TG, XRD, SEM, elemental analysis), N2 adsorption–desorption isotherms, pore size distribution, recycling test of the catalyst, comparison of the literature catalytic activity of different heterogeneous catalysts. See DOI: 10.1039/c5sc02050f

PubMed Central

Wang, Xiaochen; Zhou, Yu; Guo, Zengjing; Chen, Guojian; Li, Jing; Shi, Yuming; Liu, Yangqing

2015-01-01

Meso-macroporous hierarchical poly(ionic liquid)s (MPILs) with extremely high ionic site densities and tunable pore structures were ionothermally synthesized through the free radical self-polymerization of our newly designed rigid bis-vinylimidazolium salt monomer. The synthesis avoided the use of any templates, gave a high yield (>99%) and allowed recycling of the IL solvent; thus it is facile, atom-efficient, environmentally friendly and sustainable. The synthesized MPILs possessed distinctive features of polycation matrices, abundant halogen anions, and large surface areas. They not only presented enhanced CO2 capture, but led to breakthroughs in the heterogeneous catalytic conversion of CO2 into cyclic carbonates: (1) unprecedented high activity at atmospheric pressure and low temperature; (2) good substrate compatibility, even being active towards the extremely inert aliphatic long carbon-chain alkyl epoxides. This result renders the first occasion of a metal–solvent–additive free recyclable heterogeneous cycloaddition of CO2 at such mild conditions. PMID:29861930

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes.

PubMed

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.

Design of Bioactive Organic-inorganic Hybrid Materials with Self-setting Ability

NASA Astrophysics Data System (ADS)

Miyazaki, T.; Machida, S.; Morita, Y.; Ishida, E.

2011-10-01

Paste-like materials with ability of self-setting are attractive for bone substitutes, since they can be injected from the small hole with minimized invasion to the patient. Although bone cements which set as apatite are clinically used, there is limitation on clinical applications due to their mechanical properties such as high brittleness and low fracture toughness. To overcome this problem, organic-inorganic hybrids based on a flexible polymer are attractive. We have obtained an idea for design of self-setting hybrids using polyion complex fabricated by ionic interaction of anionic and cationic polymers. We aimed at preparation of organic-inorganic hybrids exhibiting self-setting ability and bioactivity. The liquid component was prepared from cationic chitosan aqueous solution. The powder component was prepared by mixing various carrageenans with α-tricalcium phosphate (α-TCP). The obtained cements set within 1 day. Compressive strength showed tendency to increase with increase in α-TCP content in the powder component. The prepared cements formed the apatite in simulated body fluid within 3 days. Novel self-setting materials based on organic-inorganic hybrid can be designed utilizing ionic interaction of polysaccharide.

Solution Structures of 2 : 1 And 1 : 1 DNA Polymerase - DNA Complexes Probed By Ultracentrifugation And Small-Angle X-Ray Scattering

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

Tang, K.H.; /Ohio State U.; Niebuhr, M.

2009-04-30

We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase {beta} (Pol {beta}) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol {beta}-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol {beta}-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDamore » 5{prime}-dRP lyase domain of the second Pol {beta} molecule with the active site of the 1 : 1 Pol {beta}-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5{prime}-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5{prime}-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol {beta}-DNA complex enhances the function of Pol {beta}.« less

DNA synthesis involving a complexes form of DNA polymerase I in extracts of Escherichia coli.

PubMed Central

Hendler, R W; Pereira, M; Scharff, R

1975-01-01

DNA polymerase I (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) has been recovered as a complex of about 390,000 molecular weight. The complex displays an ATP-stimulated DNA-synthesizing activity that prefers native to heat-denatured DNA. Genetic evidence indicates that the recBC enzyme is associated with the polymerase in the complex. Preliminary evidence for complexes involving DNA polymerases II and III is also presented. PMID:1094453

STATIC AND KINETIC SITE-SPECIFIC PROTEIN-DNA PHOTOCROSSLINKING: ANALYSIS OF BACTERIAL TRANSCRIPTION INITIATION COMPLEXES

PubMed Central

Naryshkin, Nikolai; Druzhinin, Sergei; Revyakin, Andrei; Kim, Younggyu; Mekler, Vladimir; Ebright, Richard H.

2009-01-01

Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking--involving rapid-quench-flow mixing and pulsed-laser irradiation--permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes. PMID:19378179

Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

PubMed

Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

2016-12-13

Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

An isolated Hda-clamp complex is functional in the regulatory inactivation of DnaA and DNA replication.

PubMed

Kawakami, Hironori; Su'etsugu, Masayuki; Katayama, Tsutomu

2006-10-01

In Escherichia coli, a complex consisting of Hda and the DNA-loaded clamp-subunit of the DNA polymerase III holoenzyme promotes hydrolysis of DnaA-ATP. The resultant ADP-DnaA is inactive for initiation of chromosomal DNA replication, thereby repressing excessive initiations. As the cellular content of the clamp is 10-100 times higher than that of Hda, most Hda molecules might be complexed with the clamp in vivo. Although Hda predominantly forms irregular aggregates when overexpressed, in the present study we found that co-overexpression of the clamp with Hda enhances Hda solubility dramatically and we efficiently isolated the Hda-clamp complex. A single molecule of the complex appears to consist of two Hda molecules and a single clamp. The complex is competent in DnaA-ATP hydrolysis and DNA replication in the presence of DNA and the clamp deficient subassembly of the DNA polymerase III holoenzyme (pol III*). These findings indicate that the clamp contained in the complex is loaded onto DNA through an interaction with the pol III* and that the Hda activity is preserved in these processes. The complex consisting of Hda and the DNA-unloaded clamp may play a specific role in a process proceeding to the DnaA-ATP hydrolysis in vivo.

Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein.

PubMed

Osipiuk, J; Georgopoulos, C; Zylicz, M

1993-03-05

It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site. The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site. The first step of this disassembly reaction is the binding of DnaK to the lambda P protein. In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex. We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE. Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex. Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone. Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK. However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein. These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

Interaction of DNA with Simple and Mixed Ligand Copper(II) Complexes of 1,10-Phenanthrolines as Studied by DNA-Fiber EPR Spectroscopy

PubMed Central

Chikira, Makoto; Ng, Chew Hee; Palaniandavar, Mallayan

2015-01-01

The interaction of simple and ternary Cu(II) complexes of 1,10-phenanthrolines with DNA has been studied extensively because of their various interesting and important functions such as DNA cleavage activity, cytotoxicity towards cancer cells, and DNA based asymmetric catalysis. Such functions are closely related to the DNA binding modes of the complexes such as intercalation, groove binding, and electrostatic surface binding. A variety of spectroscopic methods have been used to study the DNA binding mode of the Cu(II) complexes. Of all these methods, DNA-fiber electron paramagnetic resonance (EPR) spectroscopy affords unique information on the DNA binding structures of the complexes. In this review we summarize the results of our DNA-fiber EPR studies on the DNA binding structure of the complexes and discuss them together with the data accumulated by using other measurements. PMID:26402668

In vitro and in vivo gene delivery using chitosan/hyaluronic acid nanoparticles: Influences of molecular mass of hyaluronic acid and lyophilization on transfection efficiency.

PubMed

Sato, Toshinori; Nakata, Mitsuhiro; Yang, Zhihong; Torizuka, Yu; Kishimoto, Satoko; Ishihara, Masayuki

2017-08-01

Lyophilization is an effective method for preserving nonviral gene vectors. To improve the stability and transgene expression of lyophilized plasmid DNA (pDNA) complexes, we coated the surfaces of pDNA/chitosan complexes with hyaluronic acid (HA) of varying molecular masses. The transgene expression of pDNA/chitosan/HA ternary complexes was characterized in vitro and in vivo. pDNA complexes were lyophilized overnight and the resultant products with spongy, porous consistencies were stored at -30, 4 or 25°C for 2 weeks. Rehydrated complexes were characterized using gel retardation assays, aiming to confirm complex formation, measure particle size and evaluate zeta potential, as well as conduct luciferase gene reporter assays. The anti-tumor effects of pDNA ternary complexes were evaluated using suicide gene (pTK) coding thymidine kinase in Huh7-implanted mice. Transfection efficiencies of pDNA/chitosan/HA ternary complexes were dependent on the average molecular masses of HA. The coating of pDNA/chitosan complexes with HA maintained the cellular transfection efficiencies of lyophilized pDNA ternary complexes. Furthermore, intratumoral injection of lyophilized, rehydrated pDNA ternary complexes into tumor-bearing mice showed a significant suppression of tumor growth. The coating of pDNA/chitosan complexes with high-molecular-weight HA augmented the stability and cellular transfection ability of the complexes after lyophilization-rehydration. Copyright © 2017 John Wiley & Sons, Ltd.

Immunodetection of human topoisomerase I-DNA covalent complexes

PubMed Central

Patel, Anand G.; Flatten, Karen S.; Peterson, Kevin L.; Beito, Thomas G.; Schneider, Paula A.; Perkins, Angela L.; Harki, Daniel A.; Kaufmann, Scott H.

2016-01-01

A number of established and investigational anticancer drugs slow the religation step of DNA topoisomerase I (topo I). These agents induce cytotoxicity by stabilizing topo I-DNA covalent complexes, which in turn interact with advancing replication forks or transcription complexes to generate lethal lesions. Despite the importance of topo I-DNA covalent complexes, it has been difficult to detect these lesions within intact cells and tumors. Here, we report development of a monoclonal antibody that specifically recognizes covalent topo I-DNA complexes, but not free topo I or DNA, by immunoblotting, immunofluorescence or flow cytometry. Utilizing this antibody, we demonstrate readily detectable topo I-DNA covalent complexes after treatment with camptothecins, indenoisoquinolines and cisplatin but not nucleoside analogues. Topotecan-induced topo I-DNA complexes peak at 15–30 min after drug addition and then decrease, whereas indotecan-induced complexes persist for at least 4 h. Interestingly, simultaneous staining for covalent topo I-DNA complexes, phospho-H2AX and Rad51 suggests that topotecan-induced DNA double-strand breaks occur at sites distinct from stabilized topo I-DNA covalent complexes. These studies not only provide new insight into the action of topo I-directed agents, but also illustrate a strategy that can be applied to study additional topoisomerases and their inhibitors in vitro and in vivo. PMID:26917015

DNA-Binding Interaction Studies of Microwave Assisted Synthesized Sulfonamide Substituted 8-Hydroxyquinoline Derivatives.

PubMed

Dixit, Ritu B; Patel, Tarosh S; Vanparia, Satish F; Kunjadiya, Anju P; Keharia, Harish R; Dixit, Bharat C

2011-01-01

Sulfonamide substituted 8-hydroxyquinoline derivatives were prepared using a microwave synthesizer. The interaction of sulfonamide substituted 8-hydroxyquinoline derivatives and their transition metal complexes with Plasmid (pUC 19) DNA and Calf Thymus DNA were investigated by UV spectroscopic studies and gel electrophoresis measurements. The interaction between ligand/metal complexes and DNA was carried out by increasing the concentration of DNA from 0 to 12 μl in UV spectroscopic study, while the concentration of DNA in gel electrophoresis remained constant at 10 μl. These studies supported the fact that, the complex binds to DNA by intercalation via ligand into the base pairs of DNA. The relative binding efficacy of the complexes to DNA was much higher than the binding efficacy of ligands, especially the complex of Cu-AHQMBSH had the highest binding ability to DNA. The mobility of the bands decreased as the concentration of the complex was increased, indicating that there was increase in the interaction between the metal ion and DNA. Complexes of AHQMBSH were excellent for DNA binding as compared to HQMABS.

Splenic gene delivery system using self-assembling nano-complex with phosphatidylserine analog.

PubMed

Kurosaki, Tomoaki; Nakasone, Chihiro; Kodama, Yukinobu; Egashira, Kanoko; Harasawa, Hitomi; Muro, Takahiro; Nakagawa, Hiroo; Kitahara, Takashi; Higuchi, Norihide; Nakamura, Tadahiro; Sasaki, Hitoshi

2015-01-01

The recognition of phosphatidylserine on the erythrocyte membrane mediates erythrophagocytosis by resident spleen macrophages. The application of phosphatidylserine to a gene vector may be a novel approach for splenic drug delivery. Therefore, we chose 1,2-dioleoyl-sn-glycero-3-phospho-L-serin (DOPS) as an analogue of phosphatidylserine for splenic gene delivery of plasmid DNA (pDNA). In the present study, we successfully prepared a stable pDNA ternary complex using DOPS and polyethyleneimine (PEI) and evaluated its efficacy and safety. The pDNA/PEI complex had a positive charge and showed high transgene efficacy, although it caused cytotoxicity and agglutination. The addition of DOPS changed the ζ-potential of the pDNA/PEI complex to negative. It is known that anionic complexes are not taken up well by cells. Surprisingly, however, the pDNA/PEI/DOPS complex showed relatively high transgene efficacy in vitro. Fluorescence microscope observation revealed that the pDNA/PEI/DOPS complex internalized the cells while maintaining the complex formation. The injection of the pDNA/PEI complex killed most mice within 24 h at high doses, although all mice in the pDNA/PEI/DOPS complex group survived. The ternary complex with DOPS showed markedly better safety compared with the pDNA/PEI complex. The pDNA/PEI/DOPS complex showed high gene expression selectively in the spleen after intravenous injection into mice. Thus the ternary complex with DOPS can be used to deliver pDNA to the spleen, in which immune cells are abundant. It appears to have an excellent safety level, although further study to determine the mechanism of action is necessary.

Formation and enzymatic degradation of poly-l-arginine/fucoidan multilayer films.

PubMed

Webber, Jessie L; Benbow, Natalie L; Krasowska, Marta; Beattie, David A

2017-11-01

A polyelectrolyte multilayer (PEM) system based on biopolymers has been constructed and studied in its formation and enzymatic breakdown. The multilayer is composed of fucoidan (a proven antimicrobial/anti-inflammatory seaweed-based polysaccharide) and poly-l-arginine (a polypeptide that can be readily degraded with trypsin to yield arginine, a known NO donor), thus making the multilayer a potential dual action surface treatment for wound dressings. Studies on the formation of the multilayer revealed that the film built-up in the expected stepwise manner with consistent reversal of the zeta potential upon the adsorption of each subsequent polyion. The completed film (8 bilayers) was seen to have low hydration (30% water), as determined by H 2 O/D 2 O solvent replacement studies using the quartz crystal microbalance, with an adsorbed mass (without hydration water) of approx. 4.8μgcm -2 , as determined by quantitative attenuated total reflectance Fourier transform infrared (ATR FTIR) spectroscopy. The enzymatic breakdown of the film in response to exposure to trypsin was also investigated, and the film was seen to release both polymers over time, with a projected complete film removal period of approximately 24h. Critically, this information was determined using ATR FTIR spectroscopy experiments, which allowed unambiguous deconvolution of the removal rates of the two polyions, which is information that cannot be obtained from other methodologies used to study enzymatic breakdown of surface films. Copyright © 2017 Elsevier B.V. All rights reserved.

Analytical methods to determine the comparative DNA binding studies of curcumin-Cu(II) complexes

NASA Astrophysics Data System (ADS)

Rajesh, Jegathalaprathaban; Rajasekaran, Marichamy; Rajagopal, Gurusamy; Athappan, Periakaruppan

2012-11-01

DNA interaction studies of two mononuclear [1:1(1); 1:2(2)] copper(II) complexes of curcumin have been studied. The interaction of these complexes with CT-DNA has been explored by physical methods to propose modes of DNA binding of the complexes. Absorption spectral titrations of complex 1 with CT-DNA shows a red-shift of 3 nm with the DNA binding affinity of Kb, 5.21 × 104 M-1 that are higher than that obtained for 2 (red-shift, 2 nm; Kb, 1.73 × 104 M-1) reveal that the binding occurs in grooves as a result of the interaction is via exterior phosphates. The CD spectra of these Cu(II) complexes show a red shift of 3-10 nm in the positive band with increase in intensities. This spectral change of induced CD due to the hydrophobic interaction of copper complexes with DNA is the characteristic of B to A conformational change. The EB displacement assay also reveals the same trend as observed in UV-Vis spectral titration. The addition of complexes 1 and 2 to the DNA bound ethidium bromide (EB) solutions causes an obvious reduction in emission intensities indicating that these complexes competitively bind to DNA with EB. The positive shift of both the Epc and E0' accompanied by reduction of peak currents in differential pulse voltammogram (DPV), upon adding different concentrations of DNA to the metal complexes, are obviously in favor of strong binding to DNA. The super coiled plasmid pUC18 DNA cleavage ability of Cu(II) complexes in the presence of reducing agent reveals the single strand DNA cleavage (ssDNA) is observed. The hydroxyl radical (HOrad ) and the singlet oxygen are believed to be the reactive species responsible for the cleavage.

Intercalation of a Zn(II) complex containing ciprofloxacin drug between DNA base pairs.

PubMed

Shahabadi, Nahid; Asadian, Ali Ashraf; Mahdavi, Mryam

2017-11-02

In this study, an attempt has been made to study the interaction of a Zn(II) complex containing an antibiotic drug, ciprofloxacin, with calf thymus DNA using spectroscopic methods. It was found that Zn(II) complex could bind with DNA via intercalation mode as evidenced by: hyperchromism in UV-Vis spectrum; these spectral characteristics suggest that the Zn(II) complex interacts with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA. DNA binding constant (K b = 1.4 × 10 4 M -1 ) from spectrophotometric studies of the interaction of Zn(II) complex with DNA is comparable to those of some DNA intercalative polypyridyl Ru(II) complexes 1.0 -4.8 × 10 4 M -1 . CD study showed stabilization of the right-handed B form of DNA in the presence of Zn(II) complex as observed for the classical intercalator methylene blue. Thermodynamic parameters (ΔH < 0 and ΔS < 0) indicated that hydrogen bond and Van der Waals play main roles in this binding prose. Competitive fluorimetric studies with methylene blue (MB) dye have shown that Zn(II) complex exhibits the ability of this complex to displace with DNA-MB, indicating that it binds to DNA in strong competition with MB for the intercalation.

Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

PubMed

Osipiuk, J; Zylicz, M

1991-01-01

Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

Nucleoprotein Complexes Containing Replicating Simian Virus 40 DNA: Comparison with Polyoma Nucleoprotein Complexes

PubMed Central

Hall, Mark R.; Meinke, William; Goldstein, David A.

1973-01-01

Procedures for isolating nucleoprotein complexes containing replicating polyoma DNA from infected mouse cells were used to prepare short-lived nucleoprotein complexes (r-SV40 complexes) containing replicating simian virus 40 (SV40) DNA from infected monkey cells. Like the polyoma complexes, r-SV40 complexes were only partially released from nuclei by cell lysis but could be extracted from nuclei by prolonged treatment with solutions containing Triton X-100. r-SV40 complexes sedimented faster than complexes containing SV40 supercoiled DNA (SV40 complex) in sucrose gradients, and both types of SV40 nucleoprotein complexes sedimented ahead of polyoma complexes containing supercoiled polyoma DNA (py complex). The sedimentation rates of py complex and SV40 complex were 56 and 61S, respectively, based on the sedimentation rate of the mouse large ribosomal subunit as a marker. r-SV40 complexes sedimented as multiple peaks between 56 and 75S. Sedimentation and buoyant density measurements indicated that protein is bound to all forms of SV40 DNA at about the same ratio of protein to DNA (1-2/1) as was reported for polyoma nucleoproteins. PMID:4359958

Synthesis of trimethoprim metal complexes: Spectral, electrochemical, thermal, DNA-binding and surface morphology studies.

PubMed

Demirezen, Nihat; Tarınç, Derya; Polat, Duygu; Ceşme, Mustafa; Gölcü, Ayşegül; Tümer, Mehmet

2012-08-01

Complexes of trimethoprim (TMP), with Cu(II), Zn(II), Pt(II), Ru(III) and Fe(III) have been synthesized. Then, these complexes have been characterized by spectroscopic techniques involving UV-vis, IR, mass and (1)H NMR. CHN elemental analysis, electrochemical and thermal behavior of complexes have also been investigated. The electrochemical properties of all complexes have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV spectroscopy and cyclic voltammetry. UV studies of the interaction of the complexes with DNA have shown that these compounds can bind to CT DNA. The binding constants of the complexes with CT DNA have also been calculated. The cyclic voltammograms of the complexes in the presence of CT DNA have shown that the complexes can bind to CT DNA by both the intercalative and the electrostatic binding mode. The antimicrobial activity of these complexes has been evaluated against three Gram-positive and four Gram-negative bacteria. Antifungal activity against two different fungi has been evaluated and compared with the reference drug TMP. Almost all types of complexes show excellent activity against all type of bacteria and fungi. The morphology of the CT DNA, TMP, metal ions and metal complexes has been investigated by scanning electron microscopy (SEM). To get the SEM images, the interaction of compounds with CT DNA has been studied by means of differential pulse voltammetry (DPV) at CT DNA modified pencil graphite electrode (PGE). The decrease in intensity of the guanine oxidation signals has been used as an indicator for the interaction mechanism. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

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

Tully, D.B.; Cidlowski, J.A.

1989-03-07

Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins priormore » to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.« less

Volumetric and calorimetric properties of aqueous ionene solutions

PubMed Central

Lukšič, Miha; Hribar-Lee, Barbara

2016-01-01

The volumetric (partial and apparent molar volumes) and calorimetric properties (apparent heat capacities) of aqueous cationic polyelectrolyte solutions – ionenes – were studied using the oscillating tube densitometer and differential scanning calorimeter. The polyion’s charge density and the counterion properties were considered as variables. The special attention was put to evaluate the contribution of electrostatic and hydrophobic effects to the properties studied. The contribution of the CH2 group of the polyion’s backbone to molar volumes and heat capacities was estimated. Synergistic effect between polyion and counterions was found. PMID:28503012

CryoEM and image sorting for flexible protein/DNA complexes.

PubMed

Villarreal, Seth A; Stewart, Phoebe L

2014-07-01

Intrinsically disordered regions of proteins and conformational flexibility within complexes can be critical for biological function. However, disorder, flexibility, and heterogeneity often hinder structural analyses. CryoEM and single particle image processing techniques offer the possibility of imaging samples with significant flexibility. Division of particle images into more homogenous subsets after data acquisition can help compensate for heterogeneity within the sample. We present the utility of an eigenimage sorting analysis for examining two protein/DNA complexes with significant conformational flexibility and heterogeneity. These complexes are integral to the non-homologous end joining pathway, and are involved in the repair of double strand breaks of DNA. Both complexes include the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and biotinylated DNA with bound streptavidin, with one complex containing the Ku heterodimer. Initial 3D reconstructions of the two DNA-PKcs complexes resembled a cryoEM structure of uncomplexed DNA-PKcs without additional density clearly attributable to the remaining components. Application of eigenimage sorting allowed division of the DNA-PKcs complex datasets into more homogeneous subsets. This led to visualization of density near the base of the DNA-PKcs that can be attributed to DNA, streptavidin, and Ku. However, comparison of projections of the subset structures with 2D class averages indicated that a significant level of heterogeneity remained within each subset. In summary, image sorting methods allowed visualization of extra density near the base of DNA-PKcs, suggesting that DNA binds in the vicinity of the base of the molecule and potentially to a flexible region of DNA-PKcs. Copyright © 2013 Elsevier Inc. All rights reserved.

The Mini-Chromosome Maintenance (Mcm) Complexes Interact with DNA Polymerase α-Primase and Stimulate Its Ability to Synthesize RNA Primers

PubMed Central

You, Zhiying; De Falco, Mariarosaria; Kamada, Katsuhiko; Pisani, Francesca M.; Masai, Hisao

2013-01-01

The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes. PMID:23977294

Interaction study of some macrocyclic inorganic schiff base complexes with calf thymus DNA using spectroscopic and voltammetric methods

NASA Astrophysics Data System (ADS)

Bordbar, Maryam; Tavoosi, Fariba; Yeganeh-Faal, Ali; Zebarjadian, Mohammad Hasan

2018-01-01

The interaction of Cd(II), Zn(II) and Mn(II)-L (4,8-bis(2-pyridylmethyl)-4,8-diazaundecane-1,11-diamine) transition metal complexes with calf thymus DNA (CT-DNA) has been investigated using electronic, fluorescence and circular dichroism (CD) spectroscopy, thermal denaturation and cyclic voltammetry (CV). Based on the UV-Vis study, binding constants of the complexes with CT-DNA were calculated. Changes in the band of the CD spectrum, DNA melting temperature and in the ipa and ipc of the complexes in the presenceCT-DNA, overall, showed that the studied complex exhibited good DNA interaction ability with partial intercalation mode.

Carboxymethyl chitosan-poly(amidoamine) dendrimer core-shell nanoparticles for intracellular lysozyme delivery.

PubMed

Zhang, Xiaoyang; Zhao, Jun; Wen, Yan; Zhu, Chuanshun; Yang, Jun; Yao, Fanglian

2013-11-06

Intracellular delivery of native, active proteins is challenging due to the fragility of most proteins. Herein, a novel polymer/protein polyion complex (PIC) nanoparticle with core-shell structure was prepared. Carboxymethyl chitosan-grafted-terminal carboxyl group-poly(amidoamine) (CM-chitosan-PAMAM) dendrimers were synthesized by amidation and saponification reactions. (1)H NMR was used to characterize CM-chitosan-PAMAM dendrimers. The TEM images and results of lysozyme loading efficiency indicated that CM-chitosan-PAMAM dendrimers could self-assemble into core-shell nanoparticles, and lysozyme was efficiently encapsulated inside the core of CM-chitosan-PAMAM dendrimer nanoparticles. Activity of lysozyme was completely inhibited by CM-chitosan-PAMAM Dendrimers at physiological pH, whereas it was released into the medium and exhibited a significant enzymatic activity in an acidic intracellular environment. Moreover, the CM-chitosan-PAMAM dendrimer nanoparticles did not exhibit significant cytotoxicity in the range of concentrations below 3.16 mg/ml. The results indicated that these CM-chitosan-PAMAM dendrimers have excellent properties as highly potent and non-toxic intracellular protein carriers, which would create opportunities for novel applications in protein delivery. Copyright © 2013 Elsevier Ltd. All rights reserved.

FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5′-DNA end

PubMed Central

Sato, Koichi; Shimomuki, Mayo; Katsuki, Yoko; Takahashi, Daisuke; Kobayashi, Wataru; Ishiai, Masamichi; Miyoshi, Hiroyuki; Takata, Minoru; Kurumizaka, Hitoshi

2016-01-01

The FANCI-FANCD2 (I-D) complex is considered to work with RAD51 to protect the damaged DNA in the stalled replication fork. However, the means by which this DNA protection is accomplished have remained elusive. In the present study, we found that the I-D complex directly binds to RAD51, and stabilizes the RAD51-DNA filament. Unexpectedly, the DNA binding activity of FANCI, but not FANCD2, is explicitly required for the I-D complex-mediated RAD51-DNA filament stabilization. The RAD51 filament stabilized by the I-D complex actually protects the DNA end from nucleolytic degradation by an FA-associated nuclease, FAN1. This DNA end protection is not observed with the RAD51 mutant from FANCR patient cells. These results clearly answer the currently enigmatic question of how RAD51 functions with the I-D complex to prevent genomic instability at the stalled replication fork. PMID:27694619

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

PubMed Central

Witosch, Justine; Wolf, Eva; Mizuno, Naoko

2014-01-01

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. PMID:25348395

Incorporation of Deoxyribonucleic Acid Precursors by T4 Deoxyribonucleic Acid-Protein Complexes Retained on Glass Fiber Filters

PubMed Central

Miller, Robert C.; Kozinski, Andrzej W.

1970-01-01

Bacteriophage T4 deoxyribonucleic acid (DNA)-protein complexes were retained preferentially on glass fiber filters. DNA polymerase activity in the complex was detected through the incorporation of 3H-labeled DNA precursors. The primer-product DNA hybridized with both phage and Escherichia coli DNA. Density labeling experiments showed that about 30% of incorporated 3H-deoxyadenosine triphosphate was found in DNA which hybridized with phage DNA; this DNA was found to be covalently attached to the primer DNA. PMID:5497903

Adsorption of Nucleic Acid/Protein Supramolecular Complexes on Goethite: The Influence of Solution Interactions on Behavior at the Solution-Mineral Interface

NASA Astrophysics Data System (ADS)

Schmidt, M.; Martinez, C. E.

2017-12-01

Adsorption of biomolecule rich supramolecular complexes onto mineral surfaces plays an important role in the development of organo-mineral associations in soils. In this study, a series of supramolecular complexes of a model nucleic acid (deoxyribonucleic acid (DNA)) and protein (bovine serum albumin (BSA)) are synthesized, characterized and exposed to goethite to probe their adsorption behavior. To synthesize DNA/BSA complexes, a fixed DNA concentration (0.1 mg/mL) was mixed with a range of BSA concentrations (0.025-0.5 mg/mL) in 5 mM KCl at pH=5.0. Circular dichroism spectroscopy demonstrates strong, cooperative, Hill-type binding between DNA and BSA (Ka= 4.74 x 105 M-1) with DNA saturation achieved when BSA concentration reaches 0.4 mg/mL. Dynamic light scattering measurements of DNA/BSA complexes suggest binding accompanies disruption of DNA-DNA intermolecular electrostatic repulsion, resulting in a decrease of the DNA slow relaxation mode with increasing amount of BSA. Zeta potential measurements show increasing amounts of BSA lead to a reduction of negative charge on DNA/BSA complexes, in line with light scattering results. In situ attenuated total reflectance Fourier transform infrared spectroscopic studies of adsorption of DNA/BSA complexes onto goethite show that complexation of BSA with DNA appears to hinder direct coordination of DNA backbone phosphodiester groups with goethite, relative to DNA by itself. Furthermore, increasing amount of BSA (up to 0.4 mg/mL) in DNA/BSA complexes enhances DNA adsorption, possibly as a result of reduced repulsion between adsorbed DNA helices. When BSA concentration exceeds 0.4 mg/mL, a decrease in adsorbed DNA is observed. We hypothesize that this discrepancy in behavior between systems with BSA concentrations below and above saturation of DNA is caused by initial fast adsorption of loosely associated BSA on goethite, restricting access to goethite surface sites. Overall, these results highlight the impact of solution interaction between biomolecules on subsequent behavior at mineral surfaces. This work represents a bridge between model experiments with individual biomolecules and more complex natural systems, yielding a fundamental viewpoint of the formation of organo-mineral associations in soils.

Biosynthesis of edeine: II. Localization of edeine synthetase within Bacillus brevis Vm4.

PubMed

Kurylo-Borowska, Z

1975-07-14

Edeine-synthesizing polyenzymes, associated with a complex of sytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from dells intensively synthesizing edeines (18--20 h culture) contained edeine B. Edeine B was found to be bound covalently t o the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1--0.3 mumol/mg protein, depending on the age of cells. Detachment of deeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH4)2-SO4 at 45--55% saturation or by DEAE-cellulose column fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to two protein fractions of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16--22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine B of specific activity: 80 units/mjmol was released. The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associated with this complex did not effect the DNA-synthesizing activity.

Structure, stability, and thermodynamics of lamellar DNA-lipid complexes.

PubMed Central

Harries, D; May, S; Gelbart, W M; Ben-Shaul, A

1998-01-01

We develop a statistical thermodynamic model for the phase evolution of DNA-cationic lipid complexes in aqueous solution, as a function of the ratios of charged to neutral lipid and charged lipid to DNA. The complexes consist of parallel strands of DNA intercalated in the water layers of lamellar stacks of mixed lipid bilayers, as determined by recent synchrotron x-ray measurements. Elastic deformations of the DNA and the lipid bilayers are neglected, but DNA-induced spatial inhomogeneities in the bilayer charge densities are included. The relevant nonlinear Poisson-Boltzmann equation is solved numerically, including self-consistent treatment of the boundary conditions at the polarized membrane surfaces. For a wide range of lipid compositions, the phase evolution is characterized by three regions of lipid to DNA charge ratio, rho: 1) for low rho, the complexes coexist with excess DNA, and the DNA-DNA spacing in the complex, d, is constant; 2) for intermediate rho, including the isoelectric point rho = 1, all of the lipid and DNA in solution is incorporated into the complex, whose inter-DNA distance d increases linearly with rho; and 3) for high rho, the complexes coexist with excess liposomes (whose lipid composition is different from that in the complex), and their spacing d is nearly, but not completely, independent of rho. These results can be understood in terms of a simple charging model that reflects the competition between counterion entropy and inter-DNA (rho < 1) and interbilayer (rho > 1) repulsions. Finally, our approach and conclusions are compared with theoretical work by others, and with relevant experiments. PMID:9649376

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

PubMed

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

2013-06-14

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

Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1.

PubMed

Schneider, G J; Geiduschek, E P

1990-06-25

The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.

StructAlign, a Program for Alignment of Structures of DNA-Protein Complexes.

PubMed

Popov, Ya V; Galitsyna, A A; Alexeevski, A V; Karyagina, A S; Spirin, S A

2015-11-01

Comparative analysis of structures of complexes of homologous proteins with DNA is important in the analysis of DNA-protein recognition. Alignment is a necessary stage of the analysis. An alignment is a matching of amino acid residues and nucleotides of one complex to residues and nucleotides of the other. Currently, there are no programs available for aligning structures of DNA-protein complexes. We present the program StructAlign, which should fill this gap. The program inputs a pair of complexes of DNA double helix with proteins and outputs an alignment of DNA chains corresponding to the best spatial fit of the protein chains.

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

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

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Looping and clustering model for the organization of protein-DNA complexes on the bacterial genome

NASA Astrophysics Data System (ADS)

Walter, Jean-Charles; Walliser, Nils-Ole; David, Gabriel; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Wingreen, Ned S.; Broedersz, Chase P.

2018-03-01

The bacterial genome is organized by a variety of associated proteins inside a structure called the nucleoid. These proteins can form complexes on DNA that play a central role in various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, which forms an essential component of the segregation machinery in many bacteria. ChIP-Seq experiments show that ParB proteins localize around centromere-like parS sites on the DNA to which ParB binds specifically, and spreads from there over large sections of the chromosome. Recent theoretical and experimental studies suggest that DNA-bound ParB proteins can interact with each other to condense into a coherent 3D complex on the DNA. However, the structural organization of this protein-DNA complex remains unclear, and a predictive quantitative theory for the distribution of ParB proteins on DNA is lacking. Here, we propose the looping and clustering model, which employs a statistical physics approach to describe protein-DNA complexes. The looping and clustering model accounts for the extrusion of DNA loops from a cluster of interacting DNA-bound proteins that is organized around a single high-affinity binding site. Conceptually, the structure of the protein-DNA complex is determined by a competition between attractive protein interactions and loop closure entropy of this protein-DNA cluster on the one hand, and the positional entropy for placing loops within the cluster on the other. Indeed, we show that the protein interaction strength determines the ‘tightness’ of the loopy protein-DNA complex. Thus, our model provides a theoretical framework for quantitatively computing the binding profiles of ParB-like proteins around a cognate (parS) binding site.

Polystyrene-block-Poly(ionic liquid) Copolymers as Work Function Modifiers in Inverted Organic Photovoltaic Cells.

PubMed

Park, Jong Baek; Isik, Mehmet; Park, Hea Jung; Jung, In Hwan; Mecerreyes, David; Hwang, Do-Hoon

2018-02-07

Interfacial layers play a critical role in building up the Ohmic contact between electrodes and functional layers in organic photovoltaic (OPV) solar cells. These layers are based on either inorganic oxides (ZnO and TiO 2 ) or water-soluble organic polymers such as poly[(9,9-dioctyl-2,7-fluorene)-alt-(9,9-bis(3'-(N,N-dimethylamino)propyl)-2,7-fluorene)] and polyethylenimine ethoxylated (PEIE). In this work, we have developed a series of novel poly(ionic liquid) nonconjugated block copolymers for improving the performance of inverted OPV cells by using them as work function modifiers of the indium tin oxide (ITO) cathode. Four nonconjugated polyelectrolytes (n-CPEs) based on polystyrene and imidazolium poly(ionic liquid) (PSImCl) were synthesized by reversible addition-fragmentation chain transfer polymerization. The ratio of hydrophobic/hydrophilic block copolymers was varied depending on the ratio of polystyrene to the PSImCl block. The ionic density, which controls the work function of the electrode by forming an interfacial dipole between the electrode and the block copolymers, was easily tuned by simply changing the PSImCl molar ratio. The inverted OPV device with the ITO/PS 29 -b-PSImCl 60 cathode achieved the best power conversion efficiency (PCE) of 7.55% among the synthesized block copolymers, exhibiting an even higher PCE than that of the reference OPV device with PEIE (7.30%). Furthermore, the surface properties of the block copolymers films were investigated by contact angle measurements to explore the influence of the controlled hydrophobic/hydrophilic characters on the device performances.

An asymmetric structure of the Bacillus subtilis replication terminator protein in complex with DNA.

PubMed

Vivian, J P; Porter, C J; Wilce, J A; Wilce, M C J

2007-07-13

In Bacillus subtilis, the termination of DNA replication via polar fork arrest is effected by a specific protein:DNA complex formed between the replication terminator protein (RTP) and DNA terminator sites. We report the crystal structure of a replication terminator protein homologue (RTP.C110S) of B. subtilis in complex with the high affinity component of one of its cognate DNA termination sites, known as the TerI B-site, refined at 2.5 A resolution. The 21 bp RTP:DNA complex displays marked structural asymmetry in both the homodimeric protein and the DNA. This is in contrast to the previously reported complex formed with a symmetrical TerI B-site homologue. The induced asymmetry is consistent with the complex's solution properties as determined using NMR spectroscopy. Concomitant with this asymmetry is variation in the protein:DNA binding pattern for each of the subunits of the RTP homodimer. It is proposed that the asymmetric "wing" positions, as well as other asymmetrical features of the RTP:DNA complex, are critical for the cooperative binding that underlies the mechanism of polar fork arrest at the complete terminator site.

Green Tea Catechin-Based Complex Micelles Combined with Doxorubicin to Overcome Cardiotoxicity and Multidrug Resistance

PubMed Central

Cheng, Tangjian; Liu, Jinjian; Ren, Jie; Huang, Fan; Ou, Hanlin; Ding, Yuxun; Zhang, Yumin; Ma, Rujiang; An, Yingli; Liu, Jianfeng; Shi, Linqi

2016-01-01

Chemotherapy for cancer treatment has been demonstrated to cause some side effects on healthy tissues and multidrug resistance of the tumor cells, which greatly limits therapeutic efficacy. To address these limitations and achieve better therapeutic efficacy, combination therapy based on nanoparticle platforms provides a promising approach through delivering different agents simultaneously to the same destination with synergistic effect. In this study, a novel green tea catechin-based polyion complex (PIC) micelle loaded with doxorubicin (DOX) and (-)-Epigallocatechin-3-O-gallate (EGCG) was constructed through electrostatic interaction and phenylboronic acid-catechol interaction between poly(ethylene glycol)-block-poly(lysine-co-lysine-phenylboronic acid) (PEG-PLys/PBA) and EGCG. DOX was co-loaded in the PIC micelles through π-π stacking interaction with EGCG. The phenylboronic acid-catechol interaction endowed the PIC micelles with high stability under physiological condition. Moreover, acid cleavability of phenylboronic acid-catechol interaction in the micelle core has significant benefits for delivering EGCG and DOX to same destination with synergistic effects. In addition, benefiting from the oxygen free radicals scavenging activity of EGCG, combination therapy with EGCG and DOX in the micelle core could protect the cardiomyocytes from DOX-mediated cardiotoxicity according to the histopathologic analysis of hearts. Attributed to modulation of EGCG on P-glycoprotein (P-gp) activity, this kind of PIC micelles could effectively reverse multidrug resistance of cancer cells. These results suggested that EGCG based PIC micelles could effectively overcome DOX induced cardiotoxicity and multidrug resistance. PMID:27375779

Green Tea Catechin-Based Complex Micelles Combined with Doxorubicin to Overcome Cardiotoxicity and Multidrug Resistance.

PubMed

Cheng, Tangjian; Liu, Jinjian; Ren, Jie; Huang, Fan; Ou, Hanlin; Ding, Yuxun; Zhang, Yumin; Ma, Rujiang; An, Yingli; Liu, Jianfeng; Shi, Linqi

2016-01-01

Chemotherapy for cancer treatment has been demonstrated to cause some side effects on healthy tissues and multidrug resistance of the tumor cells, which greatly limits therapeutic efficacy. To address these limitations and achieve better therapeutic efficacy, combination therapy based on nanoparticle platforms provides a promising approach through delivering different agents simultaneously to the same destination with synergistic effect. In this study, a novel green tea catechin-based polyion complex (PIC) micelle loaded with doxorubicin (DOX) and (-)-Epigallocatechin-3-O-gallate (EGCG) was constructed through electrostatic interaction and phenylboronic acid-catechol interaction between poly(ethylene glycol)-block-poly(lysine-co-lysine-phenylboronic acid) (PEG-PLys/PBA) and EGCG. DOX was co-loaded in the PIC micelles through π-π stacking interaction with EGCG. The phenylboronic acid-catechol interaction endowed the PIC micelles with high stability under physiological condition. Moreover, acid cleavability of phenylboronic acid-catechol interaction in the micelle core has significant benefits for delivering EGCG and DOX to same destination with synergistic effects. In addition, benefiting from the oxygen free radicals scavenging activity of EGCG, combination therapy with EGCG and DOX in the micelle core could protect the cardiomyocytes from DOX-mediated cardiotoxicity according to the histopathologic analysis of hearts. Attributed to modulation of EGCG on P-glycoprotein (P-gp) activity, this kind of PIC micelles could effectively reverse multidrug resistance of cancer cells. These results suggested that EGCG based PIC micelles could effectively overcome DOX induced cardiotoxicity and multidrug resistance.

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

PubMed

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.

Synthesis, characterization and DNA-binding studies of mono and heterobimetallic complexes Cu sbnd Sn 2/Zn sbnd Sn 2 and their DNA cleavage activity

NASA Astrophysics Data System (ADS)

Arjmand, Farukh; Sayeed, Fatima

2010-02-01

Heterobimetallic complexes C 6H 24N 4O 6CuSn 2Cl 63, C 6H 24N 4O 6ZnSn 2Cl 64 have been synthesized from their monometallic analogs C 6H 16N 4O 2CuCl 21, C 6H 16N 4O 2ZnCl 22, and were characterized by various spectroscopic and analytical methods. The complexes 1-4 reveal an octahedral geometry for both central metal ions Cu/Zn as well as for Sn metal ion. The interaction of complexes 1-4 with CT-DNA, were investigated by using absorption, emission, cyclic voltammetry, viscometry and DNA cleavage studies. The emission quenching of 3 and 4 by [Fe(CN) 6] 4- depressed greatly when bound to CT-DNA. The results of spectroscopic, viscometric and cyclic voltammetry of complexes 3 and 4 revealed electrostatic mode of binding of the complexes with CT-DNA. These results revealed that 4 bind more avidly in comparison to 3 with CT-DNA. Gel electrophoresis of DNA with complexes 3 and 4 demonstrated that the complexes exhibit excellent cleavage activity under physiological conditions.

[DNA complexes, formed on aqueous phase surfaces: new planar polymeric and composite nanostructures].

PubMed

Antipina, M N; Gaĭnutdinov, R V; Rakhnianskaia, A A; Sergeev-Cherenkov, A N; Tolstikhina, A L; Iurova, T V; Kislov, V V; Khomutov, G B

2003-01-01

The formation of DNA complexes with Langmuir monolayers of the cationic lipid octadecylamine (ODA) and the new amphiphilic polycation poly-4-vinylpyridine with 16% of cetylpyridinium groups (PVP-16) on the surface of an aqueous solution of native DNA of low ionic strength was studied. Topographic images of Langmuir-Blodgett films of DNA/ODA and DNA/PVP-16 complexes applied to micaceous substrates were investigated by the method of atomic force microscopy. It was found that films of the amphiphilic polycation have an ordered planar polycrystalline structure. The morphology of planar DNA complexes with the amphiphilic cation substantially depended on the incubation time and the phase state of the monolayer on the surface of the aqueous DNA solution. Complex structures and individual DNA molecules were observed on the surface of the amphiphilic monolayer. Along with quasi-linear individual bound DNA molecules, characteristic extended net-like structures and quasi-circular toroidal condensed conformations of planar DNA complexes were detected. Mono- and multilayer films of DNA/PVP-16 complexes were used as templates and nanoreactors for the synthesis of inorganic nanostructures via the binding of metal cations from the solution and subsequent generation of the inorganic phase. As a result, ultrathin polymeric composite films with integrated DNA building blocks and quasi-linear arrays of inorganic semiconductor (CdS) and iron oxide nanoparticles and nanowires were obtained. The nanostructures obtained were characterized by scanning probe microscopy and transmission electron microscopy techniques. The methods developed are promising for investigating the mechanisms of structural organization and transformation in DNA and polyelectrolyte complexes at the gas-liquid interface and for the design of new extremely thin highly ordered planar polymeric and composite materials, films, and coatings with controlled ultrastructure for applications in nanoelectronics and nanobiotechnology.

Nickel-quinolones interaction. Part 4. Structure and biological evaluation of nickel(II)-enrofloxacin complexes compared to zinc(II) analogues.

PubMed

Skyrianou, Kalliopi C; Psycharis, Vassilis; Raptopoulou, Catherine P; Kessissoglou, Dimitris P; Psomas, George

2011-01-01

The nickel(II) complexes with the second-generation quinolone antibacterial agent enrofloxacin in the presence or absence of the nitrogen-donor heterocyclic ligands 1,10-phenanthroline, 2,2'-bipyridine or pyridine have been synthesized and characterized. Enrofloxacin acts as bidentate ligand coordinated to Ni(II) ion through the ketone oxygen and a carboxylato oxygen. The crystal structure of (1,10-phenanthroline)bis(enrofloxacinato)nickel(II) has been determined by X-ray crystallography. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA and bis(pyridine)bis(enrofloxacinato)nickel(II) exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes have shown that in the presence of CT DNA the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the complexes have been evaluated in comparison to the corresponding Zn(II) enrofloxacinato complexes as well as Ni(II) complexes with the first-generation quinolone oxolinic acid. Copyright © 2010 Elsevier Inc. All rights reserved.

[Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

PubMed

Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

2016-01-01

Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

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

PubMed

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.

Hairpin-shaped tetranuclear palladium(II) complex: synthesis, crystal structure, DNA binding and cytotoxicity activity studies.

PubMed

Gao, En-Jun; Wang, Ke-Hua; Zhu, Ming-Chang; Liu, Lei

2010-07-01

A novel tetranuclear palladium(II) complex [Pd(4)(phen)(4) (micro-pydc)(4)].10H(2)O (phen = 1,10-phenanthroline, pydc = pyridine-3,4-dicarboxylate) has been synthesized and characterized. In the tetranuclear complex, two pairs of dipalladated [Pd(phen)] moieties are bridged together by four pydc, presenting a hairpin molecular shape. The binding of the title complex with fish sperm DNA (FS-DNA) has been investigated by UV spectrum and fluorescence spectrum. All the results indicate that the complex bind to DNA in an intercalative mode and considerating the molecular shape and size, the dipalladated phenanthroline moieties bisintercalate to the base pairs of DNA. Agarose gel electrophoresis assay demonstrates the ability of the complex to cleave the pBR322 plasmid DNA. Cytotoxic activity studies show the complex exhibited good cytotoxic activity against four different cancer cell lines. Crown Copyright (c) 2010. Published by Elsevier Masson SAS. All rights reserved.

A core hSSB1–INTS complex participates in the DNA damage response

PubMed Central

Zhang, Feng; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Human single-stranded DNA-binding protein 1 (hSSB1) plays an important role in the DNA damage response and the maintenance of genomic stability. It has been shown that the core hSSB1 complex contains hSSB1, INTS3 and C9orf80. Using protein affinity purification, we have identified integrator complex subunit 6 (INTS6) as a major subunit of the core hSSB1 complex. INTS6 forms a stable complex with INTS3 and hSSB1 both in vitro and in vivo. In this complex, INTS6 directly interacts with INTS3. In response to the DNA damage response, along with INTS3 and hSSB1, INTS6 relocates to the DNA damage sites. Moreover, the hSSB1–INTS complex regulates the accumulation of RAD51 and BRCA1 at DNA damage sites and the correlated homologous recombination. PMID:23986477

Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP.

PubMed

Lin, C H; Patel, D J

1997-11-01

Structural studies by nuclear magnetic resonance (NMR) of RNA and DNA aptamer complexes identified through in vitro selection and amplification have provided a wealth of information on RNA and DNA tertiary structure and molecular recognition in solution. The RNA and DNA aptamers that target ATP (and AMP) with micromolar affinity exhibit distinct binding site sequences and secondary structures. We report below on the tertiary structure of the AMP-DNA aptamer complex in solution and compare it with the previously reported tertiary structure of the AMP-RNA aptamer complex in solution. The solution structure of the AMP-DNA aptamer complex shows, surprisingly, that two AMP molecules are intercalated at adjacent sites within a rectangular widened minor groove. Complex formation involves adaptive binding where the asymmetric internal bubble of the free DNA aptamer zippers up through formation of a continuous six-base mismatch segment which includes a pair of adjacent three-base platforms. The AMP molecules pair through their Watson-Crick edges with the minor groove edges of guanine residues. These recognition G.A mismatches are flanked by sheared G.A and reversed Hoogsteen G.G mismatch pairs. The AMP-DNA aptamer and AMP-RNA aptamer complexes have distinct tertiary structures and binding stoichiometries. Nevertheless, both complexes have similar structural features and recognition alignments in their binding pockets. Specifically, AMP targets both DNA and RNA aptamers by intercalating between purine bases and through identical G.A mismatch formation. The recognition G.A mismatch stacks with a reversed Hoogsteen G.G mismatch in one direction and with an adenine base in the other direction in both complexes. It is striking that DNA and RNA aptamers selected independently from libraries of 10(14) molecules in each case utilize identical mismatch alignments for molecular recognition with micromolar affinity within binding-site pockets containing common structural elements.

Exposure of DNA bases induced by the interaction of DNA and calf thymus DNA helix-destabilizing protein.

PubMed Central

Kohwi-Shigematsu, T; Enomoto, T; Yamada, M A; Nakanishi, M; Tsuboi, M

1978-01-01

The reaction of chloroacetaldehyde with adenine bases in DNA to give a fluorescent product was used to study the availability to intermolecular reaction of positions 1 and 6 of adenine in DNA complexes with calf thymus DNA helix-destabilizing protein. No inhibition of this reaction was observed when heat-denatured DNA was complexed with the protein at a protein/DNA weight ratio of 10:1, compared to free DNA. On the contrary, the same reaction was inhibited markedly for denatured DNA in the presence of calf thymus histone HI at protein/DNA weight ratio of 2:1. Furthermore, the exchange rate for hydrogens of amino and imide groups of DNA bases in DNA strands with deuterium in the solvent was totally unaffected upon complexing of DNA with the DNA helix-destabilizing protein as examined by stopped-flow ultraviolet spectroscopy. These results indicate that the DNA helix-destabilizing protein forms a complex with single-stranded DNA, leaving DNA bases uncovered by the protein. The fluorescence intensity of DNA pretreated with chloroacetaldehyde was amplified by nearly 3-fold upon addition of the DNA helix-destabilizing protein. The possibility of "unstacking" of DNA bases induced by the protein is discussed. PMID:216994

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

NASA Astrophysics Data System (ADS)

Wang, Feng; Liu, Juewen

2014-05-01

Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials.Metal containing polymers have expanded the property of polymers by involving covalently associated metal complexes. DNA is a special block copolymer. While metal ions are known to influence DNA, little is explored on its polymer property when strong metal complexes are associated. In this work, we study cisplatin modified DNA as a new polymer and probe. Out of the complexes formed between cisplatin-A15, HAuCl4-A15, Hg2+-T15 and Ag+-C15, only the cisplatin adduct is stable under the denaturing gel electrophoresis condition. Each Pt-nucleobase bond gives a positive charge and thus makes DNA a zwitterionic polymer. This allows ultrafast adsorption of DNA by graphene oxide (GO) and the adsorbed complex is highly stable. Non-specific DNA, protein, surfactants and thiolated compounds cannot displace platinated DNA from GO, while non-modified DNA is easily displaced in most cases. The stable GO/DNA conjugate is further tested for surface hybridization. This is the first demonstration of using metallated DNA as a polymeric material for interfacing with nanoscale materials. Electronic supplementary information (ESI) available: Methods, additional gels, kinetics, mass spectrum. See DOI: 10.1039/c4nr00867g

Novel strategies to construct complex synthetic vectors to produce DNA molecular weight standards.

PubMed

Chen, Zhe; Wu, Jianbing; Li, Xiaojuan; Ye, Chunjiang; Wenxing, He

2009-05-01

DNA molecular weight standards (DNA markers, nucleic acid ladders) are commonly used in molecular biology laboratories as references to estimate the size of various DNA samples in electrophoresis process. One method of DNA marker production is digestion of synthetic vectors harboring multiple DNA fragments of known sizes by restriction enzymes. In this article, we described three novel strategies-sequential DNA fragment ligation, screening of ligation products by polymerase chain reaction (PCR) with end primers, and "small fragment accumulation"-for constructing complex synthetic vectors and minimizing the mass differences between DNA fragments produced from restrictive digestion of synthetic vectors. The strategy could be applied to construct various complex synthetic vectors to produce any type of low-range DNA markers, usually available commercially. In addition, the strategy is useful for single-step ligation of multiple DNA fragments for construction of complex synthetic vectors and other applications in molecular biology field.

Biophysical and transfection studies of the diC(14)-amidine/DNA complex.

PubMed Central

Cherezov, Vadim; Qiu, Hong; Pector, Veronique; Vandenbranden, Michel; Ruysschaert, Jean-Marie; Caffrey, Martin

2002-01-01

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection. PMID:12023234

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

PubMed

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

2016-11-01

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

Antibacterial characteristics of newly developed amphiphilic lipids and DNA-lipid complexes against bacteria.

PubMed

Inoue, Y; Fukushima, T; Hayakawa, T; Takeuchi, H; Kaminishi, H; Miyazaki, K; Okahata, Y

2003-05-01

The purpose of this study was to investigate the antibacterial activity of newly developed amphiphilic lipids and DNA/lipid complexes against two types of oral bacteria and two types of hospital infection bacteria. Nine amphiphilic lipids were quantitatively prepared from the reaction of n-alkyl alcohol, alpha-amino acids, and p-toluenesulfonic acid. Nine DNA-lipid complexes were prepared by the simple mixing of DNA and amphiphilic lipids. The DNA-lipid complexes were insoluble in water. The antibacterial activity of lipids and DNA-lipid complexes against Porphyromonas gingivalis, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa were evaluated by the disk-diffusion method. Seven artificial lipids showed antibacterial behavior; in particular, the lipids prepared from n-decyl alcohol and glycine and from n-decyl alcohol and L-alanine showed antibacterial activity against the four bacterial strains used in this study. On the other hand, the lipids of glutamic acid derivatives did not show any antibacterial activity against the four bacteria strains except for the lipid with an n-octyl group. Five DNA-lipid complexes also had an antibacterial effect. The complex prepared from DNA and glycine decyl ester p-toluenesulfonic acid salt exhibited antibacterial activity against the four types of bacteria strains. In this study it was found that lipids and DNA-lipid complexes with a mono-decyl group or a mono-dodecyl group have more favorable antibacterial activity. Copyright 2003 Wiley Periodicals, Inc.

Thin Films Formed from Conjugated Polymers with Ionic, Water-Soluble Backbones.

PubMed

Voortman, Thomas P; Chiechi, Ryan C

2015-12-30

This paper compares the morphologies of films of conjugated polymers in which the backbone (main chain) and pendant groups are varied between ionic/hydrophilic and aliphatic/hydrophobic. We observe that conjugated polymers in which the pendant groups and backbone are matched, either ionic-ionic or hydrophobic-hydrophobic, form smooth, structured, homogeneous films from water (ionic) or tetrahydrofuran (hydrophobic). Mismatched conjugated polymers, by contrast, form inhomogeneous films with rough topologies. The polymers with ionic backbone chains are conjugated polyions (conjugated polymers with closed-shell charges in the backbone), which are semiconducting materials with tunable bad-gaps, not unlike uncharged conjugated polymers.

DNA condensing effects and sequence selectivity of DNA binding of antitumor noncovalent polynuclear platinum complexes.

PubMed

Malina, Jaroslav; Farrell, Nicholas P; Brabec, Viktor

2014-02-03

The noncovalent analogues of antitumor polynuclear platinum complexes represent a structurally discrete class of platinum drugs. Their chemical and biological properties differ significantly from those of most platinum chemotherapeutics, which bind to DNA in a covalent manner by formation of Pt-DNA adducts. In spite of the fact that these noncovalent polynuclear platinum complexes contain no leaving groups, they have been shown to bind to DNA with high affinity. We report here on the DNA condensation properties of a series of noncovalent analogues of antitumor polynuclear platinum complexes described by biophysical and biochemical methods. The results demonstrate that these polynuclear platinum compounds are capable of inducing DNA condensation at more than 1 order of magnitude lower concentrations than conventional spermine. Atomic force microscopy studies of DNA condensation confined to a mica substrate have revealed that the DNA morphologies become more compact with increasing concentration of the platinum complexes. Moreover, we also found that the noncovalent polynuclear platinum complex [{Pt(NH3)3}2-μ-{trans-Pt(NH3)2(NH2(CH2)6NH2)2}](6+) (TriplatinNC-A) binds to DNA in a sequence-dependent manner, namely, to A/T-rich sequences and A-tract regions, and that noncovalent polynuclear platinum complexes protect DNA from enzymatic cleavage by DNase I. The results suggest that mechanisms of antitumor and cytotoxic activities of these complexes may be associated with their unique ability to condense DNA along with their sequence-specific DNA binding. Owing to their high cellular accumulation, it is also reasonable to suggest that their mechanism of action is based on the competition with naturally occurring DNA condensing agents, such as polyamines spermine, spermidine, and putrescine, for intracellular binding sites, resulting in the disturbance of the correct binding of regulatory proteins initiating the onset of apoptosis.

The MBD7 complex promotes expression of methylated transgenes without significantly altering their methylation status

PubMed Central

Li, Dongming; Palanca, Ana Marie S; Won, So Youn; Gao, Lei; Feng, Ying; Vashisht, Ajay A; Liu, Li; Zhao, Yuanyuan; Liu, Xigang; Wu, Xiuyun; Li, Shaofang; Le, Brandon; Kim, Yun Ju; Yang, Guodong; Li, Shengben; Liu, Jinyuan; Wohlschlegel, James A; Guo, Hongwei; Mo, Beixin; Chen, Xuemei; Law, Julie A

2017-01-01

DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in Arabidopsis thaliana that suppresses the transcriptional silencing of two LUCIFERASE (LUC) reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased LUC expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing. DOI: http://dx.doi.org/10.7554/eLife.19893.001 PMID:28452714

Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.

PubMed Central

Kramers, C; Hylkema, M N; van Bruggen, M C; van de Lagemaat, R; Dijkman, H B; Assmann, K J; Smeenk, R J; Berden, J H

1994-01-01

Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis. Images PMID:8040312

Quaternary complexes composed of plasmid DNA/protamine/fish sperm DNA/stearic acid grafted chitosan oligosaccharide micelles for gene delivery.

PubMed

Du, Yong-Zhong; Lu, Ping; Yuan, Hong; Zhou, Jian-Ping; Hu, Fu-Qiang

2011-01-01

Quaternary complexes with condensed core of plasmid DNA, protamine, fish sperm DNA and shell of stearic acid grafted chitosan oligosaccharide (CSO-SA), were prepared. The CSO-SA could self-assemble to form nano-sized micelles in aqueous solution and demonstrated excellent internalization ability of tumor cells. Dynamic light scattering (DLS) measurement and transmission electrostatic microscope (TEM) images showed that quaternary complexes had spherical shape with about 25 nm number average diameter, and the size of quaternary complexes was smaller than that of CSO-SA micelles and CSO-SA micelles/plasmid DNA binary complexes. The transfection efficiencies of quaternary complexes on HEK293 and MCF-7 cells increased with incubation time, and were significantly higher than that of CSO-SA micelles/plasmid DNA binary complexes. The optimal transfection efficiency of quaternary complexes on HEK293 and MCF-7 cells measured by flow cytometer after 96 h was 23.82% and 41.43%, respectively. Whereas, the transfection efficiency of Lipofectamine™ 2000 on HEK293 and MCF-7 cells after 96 h was 32.45% and 33.23%, respectively. The data of luciferease activity measurement showed that the optimal ratio of plasmid DNA:fish sperm DNA:protamine:CSO-SA was 1:1:5:5. The results indicated that the present quaternary complexes were potential non-viral gene delivery system. Copyright © 2010 Elsevier B.V. All rights reserved.

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

PubMed

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

2017-05-30

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

An anti-DNA antibody prefers damaged dsDNA over native.

PubMed

Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

2017-01-01

DNA-protein interactions, including DNA-antibody complexes, have both fundamental and practical significance. In particular, antibodies against double-stranded DNA play an important role in the pathogenesis of autoimmune diseases. Elucidation of structural mechanisms of an antigen recognition and interaction of anti-DNA antibodies provides a basis for understanding the role of DNA-containing immune complexes in human pathologies and for new treatments. Here we used Molecular Dynamic simulations of bimolecular complexes of a segment of dsDNA with a monoclonal anti-DNA antibody's Fab-fragment to obtain detailed structural and physical characteristics of the dynamic intermolecular interactions. Using a computationally modified crystal structure of a Fab-DNA complex (PDB: 3VW3), we studied in silico equilibrium Molecular Dynamics of the Fab-fragment associated with two homologous dsDNA fragments, containing or not containing dimerized thymine, a product of DNA photodamage. The Fab-fragment interactions with the thymine dimer-containing DNA was thermodynamically more stable than with the native DNA. The amino acid residues constituting a paratope and the complementary nucleotide epitopes for both Fab-DNA constructs were identified. Stacking and electrostatic interactions were shown to play the main role in the antibody-dsDNA contacts, while hydrogen bonds were less significant. The aggregate of data show that the chemically modified dsDNA (containing a covalent thymine dimer) has a higher affinity toward the antibody and forms a stronger immune complex. These findings provide a mechanistic insight into formation and properties of the pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus, associated with skin photosensibilization and DNA photodamage.

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex.

PubMed

Guo, Tai Wei; Bartesaghi, Alberto; Yang, Hui; Falconieri, Veronica; Rao, Prashant; Merk, Alan; Eng, Edward T; Raczkowski, Ashleigh M; Fox, Tara; Earl, Lesley A; Patel, Dinshaw J; Subramaniam, Sriram

2017-10-05

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition. Published by Elsevier Inc.

Diagnostic value of succinate ubiquinone reductase activity in the identification of patients with mitochondrial DNA depletion.

PubMed

Hargreaves, P; Rahman, S; Guthrie, P; Taanman, J W; Leonard, J V; Land, J M; Heales, S J R

2002-02-01

Mitochondrial DNA (mtDNA) depletion syndrome (McKusick 251880) is characterized by a progressive quantitative loss of mtDNA resulting in severe mitochondrial dysfunction. A diagnosis of mtDNA depletion can only be confirmed after Southern blot analysis of affected tissue. Only a limited number of centres have the facilities to offer this service, and this is frequently on an irregular basis. There is therefore a need for a test that can refine sample selection as well as complementing the molecular analysis. In this study we compared the activities of the nuclear-encoded succinate ubiquinone reductase (complex II) to the activities of the combined mitochondrial and nuclear-encoded mitochondrial electron transport chain (ETC) complexes; NADH:ubiquinone reductase (complex I), ubiquinol-cytochrome-c reductase (complex III), and cytochrome-c oxidase (complex IV), in skeletal muscle biopsies from 7 patients with confirmed mtDNA depletion. In one patient there was no evidence of an ETC defect. However, the remaining 6 patients exhibited reduced complex I and IV activities. Five of these patients also displayed reduced complex II-III (succinate:cytochrome-c reductase) activity. Individual measurement of complex II and complex III activities demonstrated normal levels of complex II activity compared to complex III, which was reduced in the 5 biopsies assayed. These findings suggest a possible diagnostic value for the detection of normal levels of complex II activity in conjunction with reduced complex I, III and IV activity in the identification of likely candidates for mtDNA depletion syndrome

Investigation of the electrostatic and hydration properties of DNA minor groove-binding by a heterocyclic diamidine by osmotic pressure.

PubMed

Erlitzki, Noa; Huang, Kenneth; Xhani, Suela; Farahat, Abdelbasset A; Kumar, Arvind; Boykin, David W; Poon, Gregory M K

2017-12-01

Previous investigations of sequence-specific DNA binding by model minor groove-binding compounds showed that the ligand/DNA complex was destabilized in the presence of compatible co-solutes. Inhibition was interpreted in terms of osmotic stress theory as the uptake of significant numbers of excess water molecules from bulk solvent upon complex formation. Here, we interrogated the AT-specific DNA complex formed with the symmetric heterocyclic diamidine DB1976 as a model for minor groove DNA recognition using both ionic (NaCl) and non-ionic cosolutes (ethylene glycol, glycine betaine, maltose, nicotinamide, urea). While the non-ionic cosolutes all destabilized the ligand/DNA complex, their quantitative effects were heterogeneous in a cosolute- and salt-dependent manner. Perturbation with NaCl in the absence of non-ionic cosolute showed that preferential hydration water was released upon formation of the DB1976/DNA complex. As salt probes counter-ion release from charged groups such as the DNA backbone, we propose that the preferential hydration uptake in DB1976/DNA binding observed in the presence of osmolytes reflects the exchange of preferentially bound cosolute with hydration water in the environs of the bound DNA, rather than a net uptake of hydration waters by the complex. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular dynamics simulations show altered secondary structure of clawless in binary complex with DNA providing insights into aristaless-clawless-DNA ternary complex formation.

PubMed

Kachhap, Sangita; Priyadarshini, Pragya; Singh, Balvinder

2017-05-01

Aristaless (Al) and clawless (Cll) homeodomains that are involved in leg development in Drosophila melanogaster are known to bind cooperatively to 5'-(T/C)TAATTAA(T/A)(T/A)G-3' DNA sequence, but the mechanism of their binding to DNA is unknown. Molecular dynamics (MD) studies have been carried out on binary, ternary, and reconstructed protein-DNA complexes involving Al, Cll, and DNA along with binding free energy analysis of these complexes. Analysis of MD trajectories of Cll-3A01, binary complex reveals that C-terminal end of helixIII of Cll, unwind in the absence of Al and remains so in reconstructed ternary complex, Cll-3A01-Al. In addition, this change in secondary structure of Cll does not allow it to form protein-protein interactions with Al in the ternary reconstructed complex. However, secondary structure of Cll and its interactions are maintained in other reconstructed ternary complex, Al-3A01-Cll where Cll binds to Al-3A01, binary complex to form ternary complex. These interactions as observed during MD simulations compare well with those observed in ternary crystal structure. Thus, this study highlights the role of helixIII of Cll and protein-protein interactions while proposing likely mechanism of recognition in ternary complex, Al-Cll-DNA.

Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

NASA Technical Reports Server (NTRS)

Cannone, Jaime J.; Barnes, Cindy L.; Achari, Aniruddha; Kundrot, Craig E.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

Sample preparation for SFM imaging of DNA, proteins, and DNA-protein complexes.

PubMed

Ristic, Dejan; Sanchez, Humberto; Wyman, Claire

2011-01-01

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate, and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nanometer resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA-bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA, and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

PubMed

Foti, M; Omichinski, J G; Stahl, S; Maloney, D; West, J; Schweitzer, B I

1999-02-05

We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site.

Fluoroquinolone-Gyrase-DNA Complexes

PubMed Central

Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin; Kurepina, Natalia; Luan, Gan; Oppegard, Lisa M.; Hiasa, Hiroshi; Marks, Kevin R.; Kerns, Robert J.; Berger, James M.; Drlica, Karl

2014-01-01

DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys466 gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly81 and GyrB-Glu466 residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases. PMID:24497635

Molecular dynamics simulations of DNA-polycation complexes

NASA Astrophysics Data System (ADS)

Ziebarth, Jesse; Wang, Yongmei

2008-03-01

A necessary step in the preparation of DNA for use in gene therapy is the packaging of DNA with a vector that can condense DNA and provide protection from degrading enzymes. Because of the immunoresponses caused by viral vectors, there has been interest in developing synthetic gene therapy vectors, with polycations emerging as promising candidates. Molecular dynamics simulations of the DNA duplex CGCGAATTCGCG in the presence of 20 monomer long sequences of the polycations, poly-L-lysine (PLL) and polyethyleneimine (PEI), with explicit counterions and TIP3P water, are performed to provide insight into the structure and formation of DNA polyplexes. After an initial separation of approximately 50 å, the DNA and polycation come together and form a stable complex within 10 ns. The DNA does not undergo any major structural changes upon complexation and remains in the B-form. In the formed complex, the charged amine groups of the polycation mainly interact with DNA phosphate groups, and rarely occupy electronegative sites in either the major or minor grooves. Differences between complexation with PEI and PLL will be discussed.

Controlled assembly of artificial protein-protein complexes via DNA duplex formation.

PubMed

Płoskoń, Eliza; Wagner, Sara C; Ellington, Andrew D; Jewett, Michael C; O'Reilly, Rachel; Booth, Paula J

2015-03-18

DNA-protein conjugates have found a wide range of applications. This study demonstrates the formation of defined, non-native protein-protein complexes via the site specific labeling of two proteins of interest with complementary strands of single-stranded DNA in vitro. This study demonstrates that the affinity of two DNA-protein conjugates for one another may be tuned by the use of variable lengths of DNA allowing reversible control of complex formation.

Multispectroscopic DNA-Binding studies and antimicrobial evaluation of new mixed-ligand Silver(I) complex and nanocomplex: A comparative study

NASA Astrophysics Data System (ADS)

Movahedi, Elaheh; Rezvani, Ali Reza

2018-05-01

A novel mixed-ligand Ag(I) complex, , has been synthesized and characterized by the elemental analysis, IR spectroscopy and 1HNMR. In the formula, dian and phen are N-(4,5-diazafluoren-9-ylidene)aniline and 1,10-phenanthroline, respectively. This complex also has been prepared at nano size by sonochemical technique and characterized by the FTIR and scanning electron microscopy (SEM). To evaluate the biological preferences of the Ag(I) complex and nanocomplex and verify the relationships between the structure and biological function, in vitro DNA binding and antibacterial experiments have been carried out. DNA-complex interaction has been pursued by electronic absorption titration, luminescence titration, competitive binding experiment, effect of ionic strength, thermodynamic studies, viscometric evaluation and circular dichroism spectroscopy in the physiological pH. Each compound displays significant binding trend to the CT-DNA. The mode of binding to the CT-DNA probably is a moderate intercalation mode with the partial insertion of the planar ligands between the base stacks of double-stranded DNA. The relative viscosities and circular dichroism spectra of the CT-DNA with the complex solutions, confirm the intense interactions of the Ag(I) complex and nanocomplex with DNA. An in vitro antibacterial test of the complex and nanocomplex on a series of the Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis) and the Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) shows a remarkable antibacterial feature of the Ag(I) complex. The MIC values (minimum inhibitory concentration) of the compounds compare with silver nitrate and silver sulfadiazine. The bacterial inhibitions of the Ag(I) complex and nanocomplex are agreed to their DNA binding affinities.

Induction of DNA-protein cross-links by platinum compounds.

PubMed

Woźniak, K; Walter, Z

2000-01-01

The differences between cis- and trans-diamminedichloroplatinum II (DDP) in forming DNA-protein cross-links in isolated human lymphocytes were investigated. Both cis- and trans-DDP can induce DNA-protein cross-links. We show that cis-DDP forms complexes between DNA and proteins faster than trans-DDP. This results from an increase in the quantity of DNA and platinum together with an increase in drug concentration. Under the same conditions trans-DDP causes a decrease in DNA-forming complexes with proteins. After a 12 h incubation of lymphocytes we observe a similar level of DNA in DNA-protein cross-links induced by DDP isomers, but more platinum appears in complexes induced by trans-DDP. The results obtained demonstrate that the antitumor drug - cis-DDP and the clinically ineffective trans-DDP induce links between DNA and proteins in a different manner. We suggest that the therapeutic activity of cis-DDP can in part arise from rapidly forming DNA-protein complexes which can destroy the most important cellular processes, such as replication and transcription.

Re-visiting protein-centric two-tier classification of existing DNA-protein complexes

PubMed Central

2012-01-01

Background Precise DNA-protein interactions play most important and vital role in maintaining the normal physiological functioning of the cell, as it controls many high fidelity cellular processes. Detailed study of the nature of these interactions has paved the way for understanding the mechanisms behind the biological processes in which they are involved. Earlier in 2000, a systematic classification of DNA-protein complexes based on the structural analysis of the proteins was proposed at two tiers, namely groups and families. With the advancement in the number and resolution of structures of DNA-protein complexes deposited in the Protein Data Bank, it is important to revisit the existing classification. Results On the basis of the sequence analysis of DNA binding proteins, we have built upon the protein centric, two-tier classification of DNA-protein complexes by adding new members to existing families and making new families and groups. While classifying the new complexes, we also realised the emergence of new groups and families. The new group observed was where β-propeller was seen to interact with DNA. There were 34 SCOP folds which were observed to be present in the complexes of both old and new classifications, whereas 28 folds are present exclusively in the new complexes. Some new families noticed were NarL transcription factor, Z-α DNA binding proteins, Forkhead transcription factor, AP2 protein, Methyl CpG binding protein etc. Conclusions Our results suggest that with the increasing number of availability of DNA-protein complexes in Protein Data Bank, the number of families in the classification increased by approximately three fold. The folds present exclusively in newly classified complexes is suggestive of inclusion of proteins with new function in new classification, the most populated of which are the folds responsible for DNA damage repair. The proposed re-visited classification can be used to perform genome-wide surveys in the genomes of interest for the presence of DNA-binding proteins. Further analysis of these complexes can aid in developing algorithms for identifying DNA-binding proteins and their family members from mere sequence information. PMID:22800292

Re-visiting protein-centric two-tier classification of existing DNA-protein complexes.

PubMed

Malhotra, Sony; Sowdhamini, Ramanathan

2012-07-16

Precise DNA-protein interactions play most important and vital role in maintaining the normal physiological functioning of the cell, as it controls many high fidelity cellular processes. Detailed study of the nature of these interactions has paved the way for understanding the mechanisms behind the biological processes in which they are involved. Earlier in 2000, a systematic classification of DNA-protein complexes based on the structural analysis of the proteins was proposed at two tiers, namely groups and families. With the advancement in the number and resolution of structures of DNA-protein complexes deposited in the Protein Data Bank, it is important to revisit the existing classification. On the basis of the sequence analysis of DNA binding proteins, we have built upon the protein centric, two-tier classification of DNA-protein complexes by adding new members to existing families and making new families and groups. While classifying the new complexes, we also realised the emergence of new groups and families. The new group observed was where β-propeller was seen to interact with DNA. There were 34 SCOP folds which were observed to be present in the complexes of both old and new classifications, whereas 28 folds are present exclusively in the new complexes. Some new families noticed were NarL transcription factor, Z-α DNA binding proteins, Forkhead transcription factor, AP2 protein, Methyl CpG binding protein etc. Our results suggest that with the increasing number of availability of DNA-protein complexes in Protein Data Bank, the number of families in the classification increased by approximately three fold. The folds present exclusively in newly classified complexes is suggestive of inclusion of proteins with new function in new classification, the most populated of which are the folds responsible for DNA damage repair. The proposed re-visited classification can be used to perform genome-wide surveys in the genomes of interest for the presence of DNA-binding proteins. Further analysis of these complexes can aid in developing algorithms for identifying DNA-binding proteins and their family members from mere sequence information.

A Ubiquitin-Proteasome Pathway for the Repair of Topoisomerase I-DNA Covalent Complexes*S⃞

PubMed Central

Lin, Chao-Po; Ban, Yi; Lyu, Yi Lisa; Desai, Shyamal D.; Liu, Leroy F.

2008-01-01

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged DNA, UV-irradiated DNA, alkylated DNA, uracil-substituted DNA, mismatched DNA, gapped and nicked DNA, and DNA with abasic sites). Top1 cleavage complexes arrest transcription and trigger transcription-dependent degradation of Top1, a phenomenon termed Top1 down-regulation. In the current study, we have investigated the role of Top1 down-regulation in the repair of Top1 cleavage complexes. Using quiescent (serum-starved) human WI-38 cells, camptothecin (CPT) was shown to induce Top1 down-regulation, which paralleled the induction of DNA single-strand breaks (SSBs) (assayed by comet assays) and ATM autophosphorylation (at Ser-1981). Interestingly, Top1 down-regulation, induction of DNA SSBs and ATM autophosphorylation were all abolished by the proteasome inhibitor MG132. Furthermore, studies using immunoprecipitation and dominant-negative ubiquitin mutants have suggested a specific requirement for the assembly of Lys-48-linked polyubiquitin chains for CPT-induced Top1 down-regulation. In contrast to the effect of proteasome inhibition, inactivation of PARP1 was shown to increase the amount of CPT-induced SSBs and the level of ATM autophosphorylation. Together, these results support a model in which Top1 cleavage complexes arrest transcription and activate a ubiquitin-proteasome pathway leading to the degradation of Top1 cleavage complexes. Degradation of Top1 cleavage complexes results in the exposure of Top1-concealed SSBs for repair through a PARP1-dependent process. PMID:18515798

Visualization of DNA and Protein-DNA Complexes with Atomic Force Microscopy

PubMed Central

Lyubchenko, Yuri L.; Gall, Alexander A.; Shlyakhtenko, Luda S.

2014-01-01

This article describes sample preparation techniques for AFM imaging of DNA and protein–DNA complexes. The approach is based on chemical functionalization of the mica surface with aminopropyl silatrane (APS) to yield an APS-mica surface. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations, and in a broad range of pH. The chapter describes the methodologies for the preparation of APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purifi cation of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein–DNA complexes. PMID:24357372

Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein

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

Chakraborty, Kaushik; Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in

The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitablemore » statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4–DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3–DNA complex.« less

Polyelectrolytes and Their Biological Interactions

PubMed Central

Katchalsky, A.

1964-01-01

Polyelectrolytes are water-soluble electrically charged polymers. Their properties are determined by the interplay of the electrical forces, the Brownian motion of the macromolecular chain, and intermolecular Van der Waals forces. Charged polyacids or polybases are stretched by the electrostatic forces, as evidenced by increase in solution viscosity, or by the stretching of polyelectrolyte gels. The electrical field of the polyions is neutralized by a dense atmosphere of counter-ions. The counter-ion attraction to the polyions is expressed by a reduction of the osmotic activity of the polyion—the osmotic pressure being only 15 to 20 per cent of the ideal in highly charged polyelectrolytes neutralized by monovalent counter-ions, and as low as 1 to 3 per cent of the ideal for polyvalent counter-ions. Since the ionic atmosphere is only slightly dependent on added low molecular salt, the osmotic pressure of polyelectrolyte salt mixtures is approximately equal to the sum of the osmotic pressure of polyelectrolyte and salt alone. Acidic and basic polyelectrolytes interact electrostatically with precipitation at the point of polymeric electroneutrality. At higher salt concentrations the interaction is inhibited by the screening of polymeric fixed charges. The importance of these interactions in enzymatic processes is discussed. The electrical double layer is polarizable as may be deduced from dielectric and conductometric studies. The polarizability leads to strong dipole formation in an electrical field. These macromolecular dipoles may play a role in the adsorption of polyelectrolytes on charged surfaces. The final part of the paper is devoted to interactions of polyelectrolytes with cell membranes and the gluing of cells to higher aggregates by charged biocolloids. ImagesFigure 17Figure 18Figure 19Figure 20 PMID:14104085

An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex

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

Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K.

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). But, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcsmore » (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Our collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.« less

An Intrinsically Disordered APLF Links Ku, DNA-PKcs, and XRCC4-DNA Ligase IV in an Extended Flexible Non-homologous End Joining Complex

DOE PAGES

Hammel, Michal; Yu, Yaping; Radhakrishnan, Sarvan K.; ...

2016-11-14

DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) in human cells is initiated by Ku heterodimer binding to a DSB, followed by recruitment of core NHEJ factors including DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4-like factor (XLF), and XRCC4 (X4)-DNA ligase IV (L4). Ku also interacts with accessory factors such as aprataxin and polynucleotide kinase/phosphatase-like factor (APLF). But, how these factors interact to tether, process, and ligate DSB ends while allowing regulation and chromatin interactions remains enigmatic. Here, small angle X-ray scattering (SAXS) and mutational analyses show APLF is largely an intrinsically disordered protein that binds Ku, Ku/DNA-PKcsmore » (DNA-PK), and X4L4 within an extended flexible NHEJ core complex. X4L4 assembles with Ku heterodimers linked to DNA-PKcs via flexible Ku80 C-terminal regions (Ku80CTR) in a complex stabilized through APLF interactions with Ku, DNA-PK, and X4L4. Our collective results unveil the solution architecture of the six-protein complex and suggest cooperative assembly of an extended flexible NHEJ core complex that supports APLF accessibility while possibly providing flexible attachment of the core complex to chromatin. The resulting dynamic tethering furthermore, provides geometric access of L4 catalytic domains to the DNA ends during ligation and of DNA-PKcs for targeted phosphorylation of other NHEJ proteins as well as trans-phosphorylation of DNA-PKcs on the opposing DSB without disrupting the core ligation complex. Overall the results shed light on evolutionary conservation of Ku, X4, and L4 activities, while explaining the observation that Ku80CTR and DNA-PKcs only occur in a subset of higher eukaryotes.« less

An overview of the structures of protein-DNA complexes

PubMed Central

Luscombe, Nicholas M; Austin, Susan E; Berman , Helen M; Thornton, Janet M

2000-01-01

On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes. PMID:11104519

Isolation and characterization of a virus-specific ribonucleoprotein complex from reticuloendotheliosis virus-transformed chicken bone marrow cells.

PubMed Central

Wong, T C; Kang, C Y

1978-01-01

Chicken bone marrow cells transformed by reticuloendotheliosis virus (REV) produce in the cytoplasm a ribonucleoprotein (RNP) complex which has a sedimentation value of approximately 80 to 100S and a density of 1.23 g/cm3. This RNP complex is not derived from the mature virion. An endogenous RNA-directed DNA polymerase activity is associated with the RNP complex. The enzyme activity was completely neutralized by anti-REV DNA polymerase antibody but not by anti-avian myeloblastosis virus DNA polymerase antibody. The DNA product from the endogenous RNA-directed DNA polymerase reaction of the RNP complex hybridized to REV RNA but not to avian leukosis virus RNA. The RNA extracted from the RNP hybridized only to REV-specific complementary DNA synthesized from an endogenous DNA polymerase reaction of purified REV. The size of the RNA in the RNP is 30 to 35S, which represents the subunit size of the genomic RNA. No 60S mature genomic RNA was found within the RNP complex. The significance of finding the endogenous DNA polymerase activity in the viral RNP in infected cells and the maturation process of 60S virion RNA of REV are discussed. PMID:81319

Downstream DNA Tension Regulates the Stability of the T7 RNA Polymerase Initiation Complex

PubMed Central

Skinner, Gary M.; Kalafut, Bennett S.; Visscher, Koen

2011-01-01

Gene transcription by the enzyme RNA polymerase is tightly regulated. In many cases, such as in the lac operon in Escherichia coli, this regulation is achieved through the action of protein factors on DNA. Because DNA is an elastic polymer, its response to enzymatic processing can lead to mechanical perturbations (e.g., linear stretching and supercoiling) that can affect the operation of other DNA processing complexes acting elsewhere on the same substrate molecule. Using an optical-tweezers assay, we measured the binding kinetics between single molecules of bacteriophage T7 RNA polymerase and DNA, as a function of tension. We found that increasing DNA tension under conditions that favor formation of the open complex results in destabilization of the preinitiation complex. Furthermore, with zero ribonucleotides present, when the closed complex is favored, we find reduced tension sensitivity, implying that it is predominantly the open complex that is sensitive. This result strongly supports the “scrunching” model for T7 transcription initiation, as the applied tension acts against the movement of the DNA into the scrunched state, and introduces linear DNA tension as a potential regulatory quantity for transcription initiation. PMID:21320448

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

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

Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in

2015-07-28

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

Features of the damage produced by proflavine on transforming deoxyribonucleic acid.

PubMed

Cabrera-Juárez, E; Sánchez-Rincón, D A

1979-03-01

Proflavine formed a complex with transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae, with optimal formation at a ratio of proflavine to DNA of 0.06. The rate of dissociation of the complex by dialysis increased in the order: native, denatured, renatured DNA. The transforming activity of the DNA was reduced by its interaction with proflavine. This inactivation was dependent on the physical state of the DNA, the proflavine concentration, and the temperature. DNA that had been denatured and renatured was most sensitive; native DNA was much less sensitive. The inactivation remained after dialysis and was stable to prolonged storage. It is concluded that the inactivation of transforming DNA by proflavine takes place by a mechanism different from that of DNA-proflavine complex formation.

Nuclear magnetic resonance-based model of a TF1/HmU-DNA complex.

PubMed

Silva, M V; Pasternack, L B; Kearns, D R

1997-12-15

Transcription factor 1 (TF1), a type II DNA-binding protein encoded by the Bacillus subtilis bacteriophage SPO1, has the capacity for sequence-selective DNA binding and a preference for 5-hydroxymethyl-2'-deoxyuridine (HmU)-containing DNA. In NMR studies of the TF1/HmU-DNA complex, intermolecular NOEs indicate that the flexible beta-ribbon and C-terminal alpha-helix are involved in the DNA-binding site of TF1, placing it in the beta-sheet category of DNA-binding proteins proposed to bind by wrapping two beta-ribbon "arms" around the DNA. Intermolecular and intramolecular NOEs were used to generate an energy-minimized model of the protein-DNA complex in which both DNA bending and protein structure changes are evident.

Single-Molecule Interactions of a Monoclonal Anti-DNA Antibody with DNA

PubMed Central

Nevzorova, Tatiana A.; Zhao, Qingze; Lomakin, Yakov A.; Ponomareva, Anastasia A.; Mukhitov, Alexander R.; Purohit, Prashant K.; Weisel, John W.; Litvinov, Rustem I.

2017-01-01

Interactions of DNA with proteins are essential for key biological processes and have both a fundamental and practical significance. In particular, DNA binding to anti-DNA antibodies is a pathogenic mechanism in autoimmune pathology, such as systemic lupus erythematosus. Here we measured at the single-molecule level binding and forced unbinding of surface-attached DNA and a monoclonal anti-DNA antibody MRL4 from a lupus erythematosus mouse. In optical trap-based force spectroscopy, a microscopic antibodycoated latex bead is trapped by a focused laser beam and repeatedly brought into contact with a DNA-coated surface. After careful discrimination of non-specific interactions, we showed that the DNA-antibody rupture force spectra had two regimes, reflecting formation of weaker (20–40 pN) and stronger (>40 pN) immune complexes that implies the existence of at least two bound states with different mechanical stability. The two-dimensional force-free off-rate for the DNA-antibody complexes was ~2.2 × 10−3 s−1, the transition state distance was ~0.94 nm, the apparent on-rate was ~5.26 s−1, and the stiffness of the DNA-antibody complex was characterized by a spring constant of 0.0021 pN/nm, suggesting that the DNA-antibody complex is a relatively stable, but soft and deformable macromolecular structure. The stretching elasticity of the DNA molecules was characteristic of single-stranded DNA, suggesting preferential binding of the MRL4 antibody to one strand of DNA. Collectively, the results provide fundamental characteristics of formation and forced dissociation of DNA-antibody complexes that help to understand principles of DNA-protein interactions and shed light on the molecular basis of autoimmune diseases accompanied by formation of anti-DNA antibodies. PMID:29104846

Antimalarial, antimicrobial, cytotoxic, DNA interaction and SOD like activities of tetrahedral copper(II) complexes

NASA Astrophysics Data System (ADS)

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.

Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif

PubMed Central

Ghosh, Supratim; Salsbury, Freddie R.; Horita, David A.; Gmeiner, William H.

2011-01-01

We report, based on semi-empirical calculations, that Zn2+ binds duplex DNA containing consecutive FdU–dA base pairs in the major groove with distorted trigonal bipyramidal geometry. In this previously uncharacterized binding motif, O4 and F5 on consecutive FdU are axial ligands while three water molecules complete the coordination sphere. NMR spectroscopy confirmed Zn2+ complexation occurred with maintenance of base pairing while a slight hypsochromic shift in circular dichroism (CD) spectra indicated moderate structural distortion relative to B-form DNA. Zn2+ complexation inhibited ethidium bromide (EtBr) intercalation and stabilized FdU-substituted duplex DNA (ΔTm > 15°C). Mg2+ neither inhibited EtBr complexation nor had as strong of a stabilizing effect. DNA sequences that did not contain consecutive FdU were not stabilized by Zn2+. A lipofectamine preparation of the Zn2+–DNA complex displayed enhanced cytotoxicity toward prostate cancer cells relative to the individual components prepared as lipofectamine complexes indicating the potential utility of Zn2+–DNA complexes for cancer treatment. PMID:21296761

Synthesis, structural characterization and DNA interaction of zinc complex from 2,6-diacetylpyridine dihydrazone and {4-[(2E)-2-(hydroxyimino)acetyl]phenoxy} acetic acid.

PubMed

Gup, Ramazan; Gökçe, Cansu; Dilek, Nefise

2015-03-01

A new water soluble zinc complex has been prepared and structurally characterized. The Zn(II) complex was synthesized by the reaction of 2,6-diacetylpyridine dihydrazone (dph) with {4-[(2E)-2-(hydroxyimino)acetyl]phenoxy} acetic acid (H₂L) in the presence of zinc(II) acetate. Single crystal X-ray diffraction study revealed that the zinc ion is situated in distorted trigonal-bipyramidal environment where the equatorial position is occupied by the nitrogen atom of pyridine ring and the oxygen atoms of acetate groups of two oxime ligands (H₂L) whereas the axial positions of the zinc complex are occupied by the imine nitrogen atoms of dph ligand. Characterization of the complex with FTIR, (1)H and (13)C NMR, UV-vis and elemental analysis also confirmed the proposed structure. Interaction of the Zn(II) complex with calf-thymus DNA (CT-DNA) was investigated through UV-vis spectroscopy and viscosity measurements. The results suggest that the complex preferably bind to DNA through the groove binding mode. The zinc complex cleaves plasmid pBR 322 DNA in the presence and absence of an oxidative agent (H₂O₂), possibly through a hydrolytic pathway which is also supported by DNA cleave experiments in the presence of different radical scavengers. The nuclease activity of the zinc complex significantly depends on concentration of the complex and incubation time both in the presence and absence of H₂O₂. DNA cleave activity is inhibited in the presence of methyl green indicating that the zinc complex seems to bind the major groove of DNA. Copyright © 2015 Elsevier B.V. All rights reserved.

Binding affinities of Schiff base Fe(II) complex with BSA and calf-thymus DNA: Spectroscopic investigations and molecular docking analysis

NASA Astrophysics Data System (ADS)

Rudra, Suparna; Dasmandal, Somnath; Patra, Chiranjit; Kundu, Arjama; Mahapatra, Ambikesh

2016-09-01

The binding interaction of a synthesized Schiff base Fe(II) complex with biological macromolecules viz., bovine serum albumin (BSA) and calf thymus(ct)-DNA have been investigated using different spectroscopic techniques coupled with viscosity measurements at physiological pH and 298 K. Regular amendments in emission intensities of BSA upon the action of the complex indicate significant interaction between them, and the binding interaction have been characterized by Stern Volmer plots and thermodynamic binding parameters. On the basis of this quenching technique one binding site with binding constant (Kb = (7.6 ± 0.21) × 105) between complex and protein have been obtained at 298 K. Time-resolved fluorescence studies have also been encountered to understand the mechanism of quenching induced by the complex. Binding affinities of the complex to the fluorophores of BSA namely tryptophan (Trp) and tyrosine (Tyr) have been judged by synchronous fluorescence studies. Secondary structural changes of BSA rooted by the complex has been revealed by CD spectra. On the other hand, hypochromicity of absorption spectra of the complex with the addition of ct-DNA and the gradual reduction in emission intensities of ethidium bromide bound ct-DNA in presence of the complex indicate noticeable interaction between ct-DNA and the complex with the binding constant (4.2 ± 0.11) × 106 M- 1. Life-time measurements have been studied to determine the relative amplitude of binding of the complex to ct-DNA base pairs. Mode of binding interaction of the complex with ct-DNA has been deciphered by viscosity measurements. CD spectra have also been used to understand the changes in ct-DNA structure upon binding with the metal complex. Density functional theory (DFT) and molecular docking analysis have been employed in highlighting the interactive phenomenon and binding location of the complex with the macromolecules.

Synthesis, structure, DNA/BSA binding and antibacterial studies of NNO tridentate Schiff base metal complexes

NASA Astrophysics Data System (ADS)

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.

Effects of cytotoxic cis- and trans-diammine monochlorido platinum(II) complexes on selenium-dependent redox enzymes and DNA.

PubMed

Lemmerhirt, Heidi; Behnisch, Steven; Bodtke, Anja; Lillig, Christopher H; Pazderova, Lucia; Kasparkova, Jana; Brabec, Viktor; Bednarski, Patrick J

2018-01-01

Here we present the preparation of 14 pairs of cis- and trans-diammine monochlorido platinum(II) complexes, coordinated to heterocycles (i.e., imidazole, 2-methylimidazole and pyrazole) and linked to various acylhydrazones, which were designed as potential inhibitors of the selenium-dependent enzymes glutathione peroxidase 1 (GPx-1) and thioredoxin reductase 1 (TrxR-1). However, no inhibition of bovine GPx-1 and only weak inhibition of murine TrxR-1 was observed in in vitro assays. Nonetheless, the cis configured diammine monochlorido Pt(II) complexes exhibited cytotoxic and apoptotic properties on various human cancer cell lines, whereas the trans configured complexes generally showed weaker potency with a few exceptions. On the other hand, the trans complexes were generally more likely to lack cross-resistance to cisplatin than the cis analogues. Platinum was found bound to the nuclear DNA of cancer cells treated with representative Pt complexes, suggesting that DNA might be a possible target. Thus, detailed in vitro binding experiments with DNA were conducted. Interactions of the compounds with calf thymus DNA were investigated, including Pt binding kinetics, circular dichroism (CD) spectral changes, changes in DNA melting temperatures, unwinding of supercoiled plasmids and ethidium bromide displacement in DNA. The CD results indicate that the most active cis configured pyrazole-derived complex causes unique structural changes in the DNA compared to the other complexes as well as to those caused by cisplatin, suggesting a denaturation of the DNA structure. This may be important for the antiproliferative activity of this compound in the cancer cells. Copyright © 2017. Published by Elsevier Inc.

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

NASA Astrophysics Data System (ADS)

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).

Phosphorylation of human INO80 is involved in DNA damage tolerance

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

Kato, Dai; Waki, Mayumi; Umezawa, Masaki

Highlights: Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced PCNA ubiquitination. Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. Black-Right-Pointing-Pointer Western blot analyses showed phosphorylated hINO80 C-terminus. Black-Right-Pointing-Pointer Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in themore » DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.« less

Direct Imaging of Gene-Carrier Complexes in Animal Cells

NASA Astrophysics Data System (ADS)

Lin, Alison J.; Slack, Nelle L.; Ahmad, Ayesha; Matsumoto, Brian; Safinya, Cyrus R.

1998-03-01

Cationic lipids are promising gene carriers for DNA transfection. Establishing the correlations between structures of cationic lipid/DNA complexes (CL-DNA) and pathways of transfection will greatly aid us in achieving the optimal CL-DNA transfections. Our first step is to determine the uptake mechanism of DNA by studying the interactions and structures of DNA and cationic lipids. X-ray diffraction shows that the CL-DNA undergoes structural phase transitions from lamellar( J. Raedler, I. Koltover, T. Salditt, C. R. Safinya, Science 275, 810 (1997).) to inverted hexagonal self-assemblies as we change the lipid composition. X-ray diffraction and optical microscopy techniques are used to directly image the progress of the CL-DNA in mouse L-cells and unravel the complex structure in-situ. Fluorescence and confocal optical microscopy techniques allow us to monitor the interactions between the complexes and different organelles in the cell cytoplasm. Current results indicate that once inside cells, complexes containing DOPE follow a different pathway from those containing DOPC. This research is funded by NSF-DMR-9624091, PRF-31352-AC7, and Los Alamos-STB/UC:96-108.

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

PubMed

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2012-08-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

Targeted DNA delivery to cancer cells using a biotinylated chitosan carrier.

PubMed

Darvishi, Mohammad H; Nomani, Alireza; Hashemzadeh, Hadi; Amini, Mohsen; Shokrgozar, Mohammad A; Dinarvand, Rassoul

2017-05-01

A novel biotinylated chitosan-graft-polyethyleneimine (Bio-Chi-g-PEI) copolymer was synthesized and evaluated as a nonviral gene delivery carrier for improvement of the transfection efficiency, endosomal escape, and targeted gene delivery of a plasmid encoding green fluorescent protein N1 (pEGFP-N1) into two different biotin-overexpressing cell lines including HeLa and OVCAR-3 cells. The structure of the obtained copolymers was confirmed by 1 H nuclear magnetic resonance ( 1 H NMR) and Fourier transform infrared spectroscopy. Physicochemical properties of the Bio-Chi-g-PEI/plasmid DNA (pDNA) complexes such as complex stability, size, zeta potential, and their morphology were investigated at various weight ratios of copolymer to pDNA. Bio-Chi-g-PEI copolymers could effectively condense pDNA into small particles with average diameters less than 164 nm and the zeta potential of +34.8 mV at the N/P ratio of 40/1. As revealed by flow cytometry, Bio-Chi-g-PEI/pDNA complexes had lower cytotoxicity than that of PEI 25 kDa/pDNA complexes in both cell lines. In vitro experiments revealed that the Bio-Chi-gPEI/pDNA complexes not only had much lower cytotoxicity, but also displayed higher transfection efficiency than that of PEI 25kDa/pDNA complexes. High percentage of cancer cells was successfully transfected by Bio-Chi-g-PEI/pDNA and properly expressed GFP protein. This study indicates that this copolymer complex can be a promising gene delivery carrier. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

In vitro transfection of plasmid DNA by amine derivatives of gelatin accompanied with ultrasound irradiation.

PubMed

Hosseinkhani, Hossein; Aoyama, Ternyoshi; Yamamoto, Shingo; Ogawa, Osamu; Tabata, Yasuhiko

2002-10-01

The purpose of this study is to examine the ultrasound (US)-enhanced gene expression by the complexes of a plasmid DNA with gelatin derivatives of aminization. Gelatin derivatives with different introduced extents of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were prepared with a water-soluble carbodiimide. The molecular size and zeta potential of the gelatin derivatives before and after complexation with the plasmid DNA were examined. After incubation with the complexes with or without US exposure, the DNA expression of rat gastric mucosal cells was measured to evaluate the effect of the type of gelatin derivatives on their gene expression. The cell uptake of the complexes, the cell viability, and the buffering effect of gelatin derivatives were examined. The apparent molecular size and zeta potential of gelatin derivatives became larger as their aminization extent increased although the Sm gelatin derivative of higher aminization showed a larger value than other corresponding derivatives. Irrespective of the type of gelatin derivatives, the apparent molecular size of plasmid DNA was reduced by increasing the gelatin-DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin-DNA complexes showed the zeta potential of 10-15 mV. The cells incubated with the complex exhibited significantly stronger luciferase activities than free plasmid DNA, and the activity was further enhanced by US irradiation. The enhancement was significant for the Sm derivative compared with the corresponding Ed and Sd derivatives. The amount of plasmid DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative, whereas US irradiation did not significantly increase the DNA internalization. US irradiation had no effect on the viability of cells incubated with every gelatin derivative-plasmid DNA complex, although the viability was decreased by the complex incubation. The buffering capacity of Sm derivative was higher than that of Ed and Sd derivatives and comparable with that of polyethylene amine. Among amine derivatives of gelatin, the Sm derivative enabled the plasmid DNA to induce the US-enhanced gene expression of cells in vitro most effectively because of the superior buffering effect.

Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

PubMed Central

Yeung, A T; Mattes, W B; Grossman, L

1986-01-01

An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction. PMID:3960727

DNA assisted self-assembly of PAMAM dendrimers.

PubMed

Mandal, Taraknath; Kumar, Mattaparthi Venkata Satish; Maiti, Prabal K

2014-10-09

We report DNA assisted self-assembly of polyamidoamine (PAMAM) dendrimers using all atom Molecular Dynamics (MD) simulations and present a molecular level picture of a DNA-linked PAMAM dendrimer nanocluster, which was first experimentally reported by Choi et al. (Nano Lett., 2004, 4, 391-397). We have used single stranded DNA (ssDNA) to direct the self-assembly process. To explore the effect of pH on this mechanism, we have used both the protonated (low pH) and nonprotonated (high pH) dendrimers. In all cases studied here, we observe that the DNA strand on one dendrimer unit drives self-assembly as it binds to the complementary DNA strand present on the other dendrimer unit, leading to the formation of a DNA-linked dendrimer dimeric complex. However, this binding process strongly depends on the charge of the dendrimer and length of the ssDNA. We observe that the complex with a nonprotonated dendrimer can maintain a DNA length dependent inter-dendrimer distance. In contrast, for complexes with a protonated dendrimer, the inter-dendrimer distance is independent of the DNA length. We attribute this observation to the electrostatic complexation of a negatively charged DNA strand with the positively charged protonated dendrimer.

Site-directed DNA crosslinking of large multisubunit protein-DNA complexes.

PubMed

Persinger, Jim; Bartholomew, Blaine

2009-01-01

Several methods have been developed to site-specifically incorporate photoreactive nucleotide analogs into DNA for the purpose of identifying the proteins and their domains that are in contact with particular regions of DNA. The synthesis of several deoxynucleotide analogs that have a photoreactive group tethered to the nucleotide base and the incorporation of these analogs into DNA are described. In a second approach, oligonucleotide with a photoreactive group attached to the phosphate backbone is chemically synthesized. The photoreactive oligonucleotide is then enzymatically incorporated into DNA by annealing it to a complementary DNA template and extending with DNA polymerase. Both approaches have been effectively used to map protein-DNA interactions in large multisubunit complexes such as the eukaryotic transcription or ATP-dependent chromatin remodeling complexes. Not only do these techniques map the binding sites of the various subunits in these complexes, but when coupled with peptide mapping also determine the protein domain that is in close proximity to the different DNA sites. The strength of these techniques is the ability to scan a large number of potential sites by making combinations of different DNA probes and is facilitated by using an immobilized DNA template for synthesis.

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP–DnaA-specific initiation complexes

PubMed Central

Keyamura, Kenji; Fujikawa, Norie; Ishida, Takuma; Ozaki, Shogo; Su’etsugu, Masayuki; Fujimitsu, Kazuyuki; Kagawa, Wataru; Yokoyama, Shigeyuki; Kurumizaka, Hitoshi; Katayama, Tsutomu

2007-01-01

Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 Å resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP–DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP–DnaA, and stimulated the assembly of inactive ADP–DnaA–oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP–DnaA-specific–oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP–DnaA–oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner. PMID:17699754

Effect of Experimental Parameters on Alginate/Chitosan Microparticles for BCG Encapsulation

PubMed Central

Caetano, Liliana A.; Almeida, António J.; Gonçalves, Lídia M.D.

2016-01-01

The aim of the present study was to develop novel Mycobacterium bovis bacille Calmette-Guérin (BCG)-loaded polymeric microparticles with optimized particle surface characteristics and biocompatibility, so that whole live attenuated bacteria could be further used for pre-exposure vaccination against Mycobacterium tuberculosis by the intranasal route. BCG was encapsulated in chitosan and alginate microparticles through three different polyionic complexation methods by high speed stirring. For comparison purposes, similar formulations were prepared with high shear homogenization and sonication. Additional optimization studies were conducted with polymers of different quality specifications in a wide range of pH values, and with three different cryoprotectors. Particle morphology, size distribution, encapsulation efficiency, surface charge, physicochemical properties and biocompatibility were assessed. Particles exhibited a micrometer size and a spherical morphology. Chitosan addition to BCG shifted the bacilli surface charge from negative zeta potential values to strongly positive ones. Chitosan of low molecular weight produced particle suspensions of lower size distribution and higher stability, allowing efficient BCG encapsulation and biocompatibility. Particle formulation consistency was improved when the availability of functional groups from alginate and chitosan was close to stoichiometric proportion. Thus, the herein described microparticulate system constitutes a promising strategy to deliver BCG vaccine by the intranasal route. PMID:27187418

Tough and Conductive Hybrid Hydrogels Enabling Facile Patterning.

PubMed

Zhu, Fengbo; Lin, Ji; Wu, Zi Liang; Qu, Shaoxing; Yin, Jun; Qian, Jin; Zheng, Qiang

2018-04-25

Conductive polymer hydrogels (CPHs) that combine the unique properties of hydrogels and electronic properties of conductors have shown their great potentials in wearable/implantable electronic devices, where materials with remarkable mechanical properties, high conductivity, and easy processability are demanding. Here, we have developed a new type of polyion complex/polyaniline (PIC/PAni) hybrid hydrogels that are tough, conductive, and can be facilely patterned. The incorporation of conductive phase (PAni) into PIC matrix through phytic acid resulted in hybrid gels with ∼65 wt % water; high conductivity while maintaining the key viscoelasticity of the tough matrix. The gel prepared from 1 M aniline (Ani) exhibited the breaking strain, fracture stress, tensile modulus, and electrical conductivity of 395%, 1.15 MPa, 5.31 MPa, and 0.7 S/m, respectively, superior to the most existing CPHs. The mechanical and electrical performance of PIC/PAni hybrid hydrogels exhibited pronounced rate-dependent and self-recovery behaviors. The hybrid gels can effectively detect subtle human motions as strain sensors. Alternating conductive/nonconductive patterns can be readily achieved by selective Ani polymerization using stencil masks. This facile patterning method based on PIC/PAni gels can be readily scaled up for fast fabrication of wavy gel circuits and multichannel sensor arrays, enabling real-time monitoring of the large-extent and large-area deformations with various sensitivities.

Exploration of cellular DNA lesion, DNA-binding and biocidal ordeal of novel curcumin based Knoevenagel Schiff base complexes incorporating tryptophan: Synthesis and structural validation

NASA Astrophysics Data System (ADS)

Chandrasekar, Thiravidamani; Raman, Natarajan

2016-07-01

A few novel Schiff base transition metal complexes of general formula [MLCl] (where, L = Schiff base, obtained by the condensation reaction of Knoevenagel condensate of curcumin, L-tryptophan and M = Cu(II), Ni(II), Co(II), and Zn(II)), were prepared by stencil synthesis. They were typified using UV-vis, IR, EPR spectral techniques, micro analytical techniques, magnetic susceptibility and molar conductivity. Geometry of the metal complexes was examined and recognized as square planar. DNA binding and viscosity studies revealed that the metal(II) complexes powerfully bound via an intercalation mechanism with the calf thymus DNA. Gel-electrophoresis technique was used to investigate the DNA cleavage competence of the complexes and they establish to approve the cleavage of pBR322 DNA in presence of oxidant H2O2. This outcome inferred that the synthesized complexes showed better nuclease activity. Moreover, the complexes were monitored for antimicrobial activities. The results exposed that the synthesized compounds were forceful against all the microbes under exploration.

New modulated design, docking and synthesis of carbohydrate-conjugate heterobimetallic CuII-SnIV complex as potential topoisomerase II inhibitor: in vitro DNA binding, cleavage and cytotoxicity against human cancer cell lines.

PubMed

Tabassum, Sartaj; Afzal, Mohd; Arjmand, Farukh

2014-03-03

New carbohydrate-conjugate heterobimetallic complexes [C₂₂H₅₀N₆O₁₃CuSnCl₂] (3) and [C₂₂H₅₈N₆O₁₇NiSnCl₂] (4) were synthesized from their monometallic analogs [C₂₂H₅₂N₆O₁₃Cu] (1) and [C₂₂H₆₀N₆O₁₇Ni] (2) containing N-glycoside ligand (L). In vitro DNA binding studies of L and complexes (1-4) with CT DNA were carried out by employing various biophysical and molecular docking techniques which revealed that heterobimetallic complex 3 strongly binds to DNA in comparison to 4, monometallic complexes (1 and 2) and the free ligand. Complex 3 cleaves pBR322 DNA via hydrolytic pathway (confirmed by T4 DNA ligase assay) and inhibited Topo-II activity in a dose-dependent manner. Furthermore, complex 3 was docked into the ATPase domain of human-Topo-II in order to probe the possible mechanism of inhibition. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway

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

Joo, Woo; Xu, Guozhou; Persky, Nicole S.

2011-08-29

Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each proteinmore » has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.« less

Structure of the FANCI-FANCD2 Complex: Insights into the Fanconi Anemia DNA Repair Pathway

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

W Joo; G Xu; n Persky

2011-12-31

Fanconi anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand cross-links (ICLs). Central to this pathway is the Fanconi anemia I-Fanconi anemia D2 (FANCI-FANCD2) (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 angstrom crystal structure of the {approx}300 kilodalton ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex and that they may serve to stabilize I-D heterodimerization. The 7.8 angstrom electron-density map of FANCI-DNA crystals and in vitro data show that each proteinmore » has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.« less

Synergistic effect of ATP for RuvA-RuvB-Holliday junction DNA complex formation.

PubMed

Iwasa, Takuma; Han, Yong-Woon; Hiramatsu, Ryo; Yokota, Hiroaki; Nakao, Kimiko; Yokokawa, Ryuji; Ono, Teruo; Harada, Yoshie

2015-12-14

The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA-RuvB-Holliday junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA-Holliday junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA-Holliday junction DNA complex in the following order: no nucleotide, ADP, ATPγS, and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA-RuvB-Holliday junction DNA complex formation.

Concerted formation of macromolecular Suppressor–mutator transposition complexes

PubMed Central

Raina, Ramesh; Schläppi, Michael; Karunanandaa, Balasulojini; Elhofy, Adam; Fedoroff, Nina

1998-01-01

Transposition of the maize Suppressor–mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range. PMID:9671711

DNA-binding and oxidative properties of cationic phthalocyanines and their dimeric complexes with anionic phthalocyanines covalently linked to oligonucleotides.

PubMed

Kuznetsova, A A; Lukyanets, E A; Solovyeva, L I; Knorre, D G; Fedorova, O S

2008-12-01

Design of chemically modified oligonucleotides for regulation of gene expression has attracted considerable attention over the past decades. One actively pursued approach involves antisense or antigene oligonucleotide constructs carrying reactive groups, many of these based on transition metal complexes. The complexes of Fe(II) and Co(II) with phthalocyanines are extremely good catalysts of oxidation of organic compounds with molecular oxygen and hydrogen peroxide. The binding of positively charged Fe(II) and Co(II) phthalocyanines with single- and double-stranded DNA was investigated. It was shown that these phthalocyanines interact with nucleic acids through an outside binding mode. The site-directed modification of single-stranded DNA by O2 and H2O2 in the presence of dimeric complexes of negatively and positively charged Fe(II) and Co(II) phthalocyanines was investigated. These complexes were formed directly on single-stranded DNA through interaction between negatively charged phthalocyanine in conjugate and positively charged phthalocyanine in solution. The resulting oppositely charged phthalocyanine complexes showed significant increase of catalytic activity compared with monomeric forms of phthalocyanines Fe(II) and Co(II). These complexes catalyzed the DNA oxidation with high efficacy and led to direct DNA strand cleavage. It was determined that oxidation of DNA by molecular oxygen catalyzed by complex of Fe(II)-phthalocyanines proceeds with higher rate than in the case of Co(II)-phthalocyanines but the latter led to a greater extent of target DNA modification.

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

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

Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert

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

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

DOE PAGES

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

2015-06-02

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

Features of the damage produced by proflavine on transforming deoxyribonucleic acid.

PubMed Central

Cabrera-Juárez, E; Sánchez-Rincón, D A

1979-01-01

Proflavine formed a complex with transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae, with optimal formation at a ratio of proflavine to DNA of 0.06. The rate of dissociation of the complex by dialysis increased in the order: native, denatured, renatured DNA. The transforming activity of the DNA was reduced by its interaction with proflavine. This inactivation was dependent on the physical state of the DNA, the proflavine concentration, and the temperature. DNA that had been denatured and renatured was most sensitive; native DNA was much less sensitive. The inactivation remained after dialysis and was stable to prolonged storage. It is concluded that the inactivation of transforming DNA by proflavine takes place by a mechanism different from that of DNA-proflavine complex formation. PMID:312284

Combined effects of metal complexation and size expansion in the electronic structure of DNA base pairs

NASA Astrophysics Data System (ADS)

Brancolini, Giorgia; Di Felice, Rosa

2011-05-01

Novel DNA derivatives have been recently investigated in the pursuit of modified DNA duplexes to tune the electronic structure of DNA-based assemblies for nanotechnology applications. Size-expanded DNAs (e.g., xDNA) and metalated DNAs (M-DNA) may enhance stacking interactions and induce metallic conductivity, respectively. Here we explore possible ways of tailoring the DNA electronic structure by combining the aromatic size expansion with the metal-doping. We select the salient structures from our recent study on natural DNA pairs complexed with transition metal ions and consider the equivalent model configurations for xDNA pairs. We present the results of density functional theory electronic structure calculations of the metalated expanded base-pairs with various localized basis sets and exchange-correlation functionals. Implicit solvent and coordination water molecules are also included. Our results indicate that the effect of base expansion is largest in Ag-xGC complexes, while Cu-xGC complexes are the most promising candidates for nanowires with enhanced electron transfer and also for on-purpose modification of the DNA double-helix for signal detection.

Activation of the DnaK-ClpB Complex is Regulated by the Properties of the Bound Substrate.

PubMed

Fernández-Higuero, Jose Angel; Aguado, Alejandra; Perales-Calvo, Judit; Moro, Fernando; Muga, Arturo

2018-04-11

The chaperone ClpB in bacteria is responsible for the reactivation of aggregated proteins in collaboration with the DnaK system. Association of these chaperones at the aggregate surface stimulates ATP hydrolysis, which mediates substrate remodeling. However, a question that remains unanswered is whether the bichaperone complex can be selectively activated by substrates that require remodeling. We find that large aggregates or bulky, native-like substrates activates the complex, whereas a smaller, permanently unfolded protein or extended, short peptides fail to stimulate it. Our data also indicate that ClpB interacts differently with DnaK in the presence of aggregates or small peptides, displaying a higher affinity for aggregate-bound DnaK, and that DnaK-ClpB collaboration requires the coupled ATPase-dependent remodeling activities of both chaperones. Complex stimulation is mediated by residues at the β subdomain of DnaK substrate binding domain, which become accessible to the disaggregase when the lid is allosterically detached from the β subdomain. Complex activation also requires an active NBD2 and the integrity of the M domain-ring of ClpB. Disruption of the M-domain ring allows the unproductive stimulation of the DnaK-ClpB complex in solution. The ability of the DnaK-ClpB complex to discrimínate different substrate proteins might allow its activation when client proteins require remodeling.

Requirement of the Mre11 complex and exonuclease 1 for activation of the Mec1 signaling pathway.

PubMed

Nakada, Daisuke; Hirano, Yukinori; Sugimoto, Katsunori

2004-11-01

The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

Design, synthesis, DNA-binding affinity, cytotoxicity, apoptosis, and cell cycle arrest of Ru(II) polypyridyl complexes.

PubMed

Venkat Reddy, Putta; Reddy, Mallepally Rajender; Avudoddi, Srishailam; Praveen Kumar, Yata; Nagamani, Chintakuntla; Deepika, Nancherla; Nagasuryaprasad, K; Singh, Surya Satyanarayana; Satyanarayana, Sirasani

2015-09-15

A novel polypyridyl ligand CNPFIP (CNPFIP=2-(5(4-chloro-2-nitrophenyl)furan-2-yl)-1H-imidazo[4,5f][1,10]phenanthroline) and its mononuclear Ru(II) polypyridyl complexes of [Ru(phen)2CNPFIP](2+)(1) (phen=1,10-phenanthroline), [Ru(bpy)2CNPFIP](2+)(2) (bpy=2,2'-bipyridine), and [Ru(dmb)2CNPFIP](2+)(3) (dmb=4,4'-dimethyl-2,2'-bipyridine) have been synthesized successfully and characterized thoroughly by elemental analysis, UV/Vis, IR, NMR, and ESI-MS. The interaction of the Ru(II) complexes with calf thymus DNA (CT-DNA) was investigated by absorption titration, fluorescence, viscosity measurements. The experimental results suggest that three complexes bind to CT-DNA through an intercalative mode and the DNA-binding affinity of complex 1 is greater than that of complexes 2 and 3. The photocleavage of plasmid pBR322 DNA by ruthenium complexes 1, 2, and 3 was investigated. We have also tested three complexes for their antimicrobial activity against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria. The in vitro cytotoxicity of these complexes was evaluated by MTT assay, and complex 1 shows higher cytotoxicity than 2 and 3 on HeLa cells. The induced apoptosis and cell cycle arrest of HeLa cells were investigated by flow cytometry for 24h. The molecular docking of ruthenium complexes 1, 2, and 3 with the active site pocket residues of human DNA TOP1 was performed using LibDock. Copyright © 2015 Elsevier Inc. All rights reserved.

Fluoroquinolone-gyrase-DNA complexes: two modes of drug binding.

PubMed

Mustaev, Arkady; Malik, Muhammad; Zhao, Xilin; Kurepina, Natalia; Luan, Gan; Oppegard, Lisa M; Hiasa, Hiroshi; Marks, Kevin R; Kerns, Robert J; Berger, James M; Drlica, Karl

2014-05-02

DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have the fluoroquinolone C-7 ring system facing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modified chloroacetyl derivative of ciprofloxacin (Cip-AcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys(466) gyrase as evidenced by resistance to reversal by both EDTA and thermal treatments. Surprisingly, cross-linking was also readily seen with complexes formed by mutant GyrA-G81C gyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinolone-resistant GyrA-G81C variant of Escherichia coli and its Mycobacterium smegmatis equivalent (GyrA-G89C). Cip-AcCl-mediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinolone C-7 ring and both GyrA and GyrB. Because the GyrA-Gly(81) and GyrB-Glu(466) residues are far apart (17 Å) in the crystal structure of cleaved complexes, two modes of quinolone binding must exist. The presence of two binding modes raises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNA topoisomerases.

Using AFM to probe the complexation of DNA with anionic lipids mediated by Ca(2+): the role of surface pressure.

PubMed

Luque-Caballero, Germán; Martín-Molina, Alberto; Sánchez-Treviño, Alda Yadira; Rodríguez-Valverde, Miguel A; Cabrerizo-Vílchez, Miguel A; Maldonado-Valderrama, Julia

2014-04-28

Complexation of DNA with lipids is currently being developed as an alternative to classical vectors based on viruses. Most of the research to date focuses on cationic lipids owing to their spontaneous complexation with DNA. Nonetheless, recent investigations have revealed that cationic lipids induce a large number of adverse effects on DNA delivery. Precisely, the lower cytotoxicity of anionic lipids accounts for their use as a promising alternative. However, the complexation of DNA with anionic lipids (mediated by cations) is still in early stages and is not yet well understood. In order to explore the molecular mechanisms underlying the complexation of anionic lipids and DNA we proposed a combined methodology based on the surface pressure-area isotherms, Gibbs elasticity and Atomic Force Microscopy (AFM). These techniques allow elucidation of the role of the surface pressure in the complexation and visualization of the interfacial aggregates for the first time. We demonstrate that the DNA complexes with negatively charged model monolayers (DPPC/DPPS 4 : 1) only in the presence of Ca(2+), but is expelled at very high surface pressures. Also, according to the Gibbs elasticity plot, the complexation of lipids and DNA implies a whole fluidisation of the monolayer and a completely different phase transition map in the presence of DNA and Ca(2+). AFM imaging allows identification for the first time of specific morphologies associated with different packing densities. At low surface coverage, a branched net like structure is observed whereas at high surface pressure fibers formed of interfacial aggregates appear. In summary, Ca(2+) mediates the interaction between DNA and negatively charged lipids and also the conformation of the ternary system depends on the surface pressure. Such observations are important new generic features of the interaction between DNA and anionic lipids.

Visualization of complex DNA damage along accelerated ions tracks

NASA Astrophysics Data System (ADS)

Kulikova, Elena; Boreyko, Alla; Bulanova, Tatiana; Ježková, Lucie; Zadneprianetc, Mariia; Smirnova, Elena

2018-04-01

The most deleterious DNA lesions induced by ionizing radiation are clustered DNA double-strand breaks (DSB). Clustered or complex DNA damage is a combination of a few simple lesions (single-strand breaks, base damage etc.) within one or two DNA helix turns. It is known that yield of complex DNA lesions increases with increasing linear energy transfer (LET) of radiation. For investigation of the induction and repair of complex DNA lesions, human fibroblasts were irradiated with high-LET 15N ions (LET = 183.3 keV/μm, E = 13MeV/n) and low-LET 60Co γ-rays (LET ≈ 0.3 keV/μm) radiation. DNA DSBs (γH2AX and 53BP1) and base damage (OGG1) markers were visualized by immunofluorecence staining and high-resolution microscopy. The obtained results showed slower repair kinetics of induced DSBs in cells irradiated with accelerated ions compared to 60Co γ-rays, indicating induction of more complex DNA damage. Confirming previous assumptions, detailed 3D analysis of γH2AX/53BP1 foci in 15N ions tracks revealed more complicated structure of the foci in contrast to γ-rays. It was shown that proteins 53BP1 and OGG1 involved in repair of DNA DSBs and modified bases, respectively, were colocalized in tracks of 15N ions and thus represented clustered DNA DSBs.

Complexes formed between DNA and poly(amido amine) dendrimers of different generations--modelling DNA wrapping and penetration.

PubMed

Qamhieh, Khawla; Nylander, Tommy; Black, Camilla F; Attard, George S; Dias, Rita S; Ainalem, Marie-Louise

2014-07-14

This study deals with the build-up of biomaterials consisting of biopolymers, namely DNA, and soft particles, poly(amido amine) (PAMAM) dendrimers, and how to model their interactions. We adopted and applied an analytical model to provide further insight into the complexation between DNA (4331 bp) and positively charged PAMAM dendrimers of generations 1, 2, 4, 6 and 8, previously studied experimentally. The theoretical models applied describe the DNA as a semiflexible polyelectrolyte that interacts with dendrimers considered as either hard (impenetrable) spheres or as penetrable and soft spheres. We found that the number of DNA turns around one dendrimer, thus forming a complex, increases with the dendrimer size or generation. The DNA penetration required for the complex to become charge neutral depends on dendrimer generation, where lower generation dendrimers require little penetration to give charge neutral complexes. High generation dendrimers display charge inversion for all considered dendrimer sizes and degrees of penetration. Consistent with the morphologies observed experimentally for dendrimer/DNA aggregates, where highly ordered rods and toroids are found for low generation dendrimers, the DNA wraps less than one turn around the dendrimer. Disordered globular structures appear for high generation dendrimers, where the DNA wraps several turns around the dendrimer. Particularly noteworthy is that the dendrimer generation 4 complexes, where the DNA wraps about one turn around the dendrimers, are borderline cases and can form all types of morphologies. The net-charges of the aggregate have been estimated using zeta potential measurements and are discussed within the theoretical framework.

Molecular architecture of the human GINS complex

PubMed Central

Boskovic, Jasminka; Coloma, Javier; Aparicio, Tomás; Zhou, Min; Robinson, Carol V; Méndez, Juan; Montoya, Guillermo

2007-01-01

Chromosomal DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of these is the GINS complex, which is required for initiation and elongation phases in eukaryotic DNA replication. The GINS complex consists of four paralogous subunits. At the G1/S transition, GINS is recruited to the origins of replication where it assembles with cell-division cycle protein (Cdc)45 and the minichromosome maintenance mutant (MCM)2–7 to form the Cdc45/Mcm2–7/GINS (CMG) complex, the presumed replicative helicase. We isolated the human GINS complex and have shown that it can bind to DNA. By using single-particle electron microscopy and three-dimensional reconstruction, we obtained a medium-resolution volume of the human GINS complex, which shows a horseshoe shape. Analysis of the protein interactions using mass spectrometry and monoclonal antibody mapping shows the subunit organization within the GINS complex. The structure and DNA-binding data suggest how GINS could interact with DNA and also its possible role in the CMG helicase complex. PMID:17557111

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

PubMed Central

Buczek, Pawel; Horvath, Martin P.

2009-01-01

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

25th anniversary article: "Cooking carbon with salt": carbon materials and carbonaceous frameworks from ionic liquids and poly(ionic liquid)s.

PubMed

Fellinger, Tim-Patrick; Thomas, Arne; Yuan, Jiayin; Antonietti, Markus

2013-11-06

This review surveys recent work on the use of ionic liquids (ILs) and polymerized ionic liquids (PILs) as precursors to synthesize functional carbon materials. As solvents or educts with negligible vapour pressure, these systems enable simple processing, composition, and structural control of the resulting carbons under rather simple and green synthesis conditions. Recent applications of the resulting nanocarbons across a multitude of fields, such as fuel cells, energy storage in batteries and supercapacitors, catalysis, separation, and sorption materials are highlighted. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lightweight, Room-Temperature CO2 Gas Sensor Based on Rare-Earth Metal-Free Composites-An Impedance Study.

PubMed

Willa, Christoph; Schmid, Alexander; Briand, Danick; Yuan, Jiayin; Koziej, Dorota

2017-08-02

We report a light, flexible, and low-power poly(ionic liquid)/alumina composite CO 2 sensor. We monitor the direct-current resistance changes as a function of CO 2 concentration and relative humidity and demonstrate fast and reversible sensing kinetics. Moreover, on the basis of the alternating-current impedance measurements we propose a sensing mechanism related to proton conduction and gas diffusion. The findings presented herein will promote the development of organic/inorganic composite CO 2 gas sensors. In the future, such sensors will be useful for numerous practical applications ranging from indoor air quality control to the monitoring of manufacturing processes.

The two radii of a charged particle.

PubMed

Michov, B M

1989-01-01

The existence of two radii of each charged particle-a geometric and electrokinetic radii, is supposed. The mathematical relationship between them in the four possible combinations of an ion and its counterion is analyzed: (i) at equal geometric radii and, in absolute values, equal valencies; (ii) at equal geometric radii and, in absolute values, different valencies; (iii) at different geometric radii and, in absolute values, equal valencies; (iv) at different geometric radii and, in absolute values, different valencies. One of the equations worked out can be used to define the relationship between the geometric and electrokinetic radii of a polyion. All the equations are used in working out precise calculations.

Electrostatic self-assembly of polyions on charged substrates

NASA Astrophysics Data System (ADS)

Campbell, A.; Adams, W. W.; Bunning, T. J.; Visser, D.; Bliznyuk, V. N.; Tsukruk, V. V.

1997-03-01

The kinetics of formation of self-assembled monolayers is studied for polystyrene sulfonate(PSS) adsorbed on oppositely charged surfaces of amine terminated self-assembled monolayers(SAM) and polyallylamine(PAA). During the early stages of deposition in both cases, an inhomogeneous deposition is noted as measured by atomic force and friction force microscopy. Island formation of unperturbed PSS coils on defect sites is observed during the initial stage of deposition. Longer deposition times result in an equilibration of the polymer layers into highly flattened macromolecular chains. AFM and FFM measurements are combined with ellipsometer and X-ray reflectivity results to quantitate the layer thicknesses and roughness with time.

Transcription-Coupled Repair and Complex Biology.

PubMed

Portman, James R; Strick, Terence R

2018-05-04

All active living organisms mitigate DNA damage via DNA repair, and the so-called nucleotide excision repair pathway (NER) represents a functionally major part of the cell's DNA repair repertoire [1]. In this pathway, the damaged strand of DNA is incised and removed before being resynthesized. This form of DNA repair requires a multitude of proteins working in a complex choreography. Repair thus typically involves detection of a DNA lesion; validation of that detection event; search for an appropriate incision site and subsequent DNA incision; DNA unwinding/removal; and DNA resynthesis and religation. These activities are ultimately the result of molecules randomly diffusing and bumping into each other and acting in succession. It is also true however that repair components are often assembled into functional complexes which may be more efficient or regular in their mode of action. Studying DNA repair complexes for their mechanisms of assembly, action, and disassembly can help address fundamental questions such as whether DNA repair pathways are branched or linear; whether for instance they tolerate fluctuations in numbers of components; and more broadly how search processes between macromolecules take place or can be enhanced. Copyright © 2018. Published by Elsevier Ltd.

The centromeric nucleosome-like CENP–T–W–S–X complex induces positive supercoils into DNA

PubMed Central

Takeuchi, Kozo; Nishino, Tatsuya; Mayanagi, Kouta; Horikoshi, Naoki; Osakabe, Akihisa; Tachiwana, Hiroaki; Hori, Tetsuya; Kurumizaka, Hitoshi; Fukagawa, Tatsuo

2014-01-01

The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP–A. In addition to CENP–A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP–T–W–S–X complex. However, it is unclear how the CENP–T–W–S–X complex associates with centromere chromatin. Here, we demonstrate that the CENP–T–W–S–X complex binds preferentially to ∼100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP–T–W–S–X complex primarily binds to DNA as a (CENP–T–W–S–X)2 structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP–T–W–S–X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP–T or CENP–W, but not CENP–S or CENP–X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP–T–W–S–X complex. In summary, our work reveals the structural features and properties of the CENP–T–W–S–X complex for its localization to centromeres. PMID:24234442

Crystal structure of the Msx-1 homeodomain/DNA complex.

PubMed

Hovde, S; Abate-Shen, C; Geiger, J H

2001-10-09

The Msx-1 homeodomain protein plays a crucial role in craniofacial, limb, and nervous system development. Homeodomain DNA-binding domains are comprised of 60 amino acids that show a high degree of evolutionary conservation. We have determined the structure of the Msx-1 homeodomain complexed to DNA at 2.2 A resolution. The structure has an unusually well-ordered N-terminal arm with a unique trajectory across the minor groove of the DNA. DNA specificity conferred by bases flanking the core TAAT sequence is explained by well ordered water-mediated interactions at Q50. Most interactions seen at the TAAT sequence are typical of the interactions seen in other homeodomain structures. Comparison of the Msx-1-HD structure to all other high resolution HD-DNA complex structures indicate a remarkably well-conserved sphere of hydration between the DNA and protein in these complexes.

Synthesis, structure, DNA/protein binding, and cytotoxic activity of a rhodium(III) complex with 2,6-bis(2-benzimidazolyl)pyridine.

PubMed

Esteghamat-Panah, Roya; Hadadzadeh, Hassan; Farrokhpour, Hossein; Simpson, Jim; Abdolmaleki, Amir; Abyar, Fatemeh

2017-02-15

A new mononuclear rhodium(III) complex, [Rh(bzimpy)Cl 3 ] (bzimpy = 2,6-bis(2-benzimidazolyl)pyridine), was synthesized and characterized by elemental analysis and spectroscopic methods. The molecular structure of the complex was confirmed by single-crystal X-ray crystallography. The interaction of the complex with fish sperm DNA (FS-DNA) was investigated by UV spectroscopy, emission titration, and viscosity measurement in order to evaluate the possible DNA-binding mode and to calculate the corresponding DNA-binding constant. The results reveal that the Rh(III) complex interacts with DNA through groove binding mode with a binding affinity on the order of 10 4 . In addition, the binding of the Rh(III) complex to bovine serum albumin (BSA) was monitored by UV-Vis and fluorescence emission spectroscopy at different temperatures. The mechanism of the complex interaction was found to be static quenching. The thermodynamic parameters (ΔH, ΔS, and ΔG) obtained from the fluorescence spectroscopy data show that van der Waals interactions and hydrogen bonds play a major role in the binding of the Rh(III) complex to BSA. For the comparison of the DNA- and BSA-binding affinities of the free bzimpy ligand with its Rh(III) complex, the absorbance titration and fluorescence quenching experiments of the free bzimpy ligand with DNA and BSA were carried out. Competitive experiments using eosin Y and ibuprofen as site markers indicated that the complex was mainly located in the hydrophobic cavity of site I of the protein. These experimental results were confirmed by the results of molecular docking. Finally, the in vitro cytotoxicity properties of the Rh(III) complex against the MCF-7, K562, and HT-29 cell lines were evaluated and compared with those of the free ligand (bzimpy). It was found that the complexation process improved the anticancer activity significantly. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

[Ru(phen)2DPPZ]2+ is in contact with DNA bases when it forms a luminescent complex with single-stranded oligonucleotides.

PubMed

Moon, Seok Joon; Kim, Jong Moon; Choi, Ji Youn; Kim, Seog K; Lee, Je Seung; Jang, Ho G

2005-05-01

The luminescence intensity of the Delta- and Lambda-enantiomer of [Ru(phen)2DPPZ]2+ ([Ru(phenanthroline)2 dipyrido[3,2-a:2',3'-c]phenazine]2+) complex enhanced upon binding to double stranded DNA, which has been known as "light switch effect". The enhancement of the luminescence required the intercalation of the large ligand between DNA base pairs. In this study, we report the enhancement in the luminescence intensity when the metal complexes bind to single stranded oligonucleotides, indicating that the "light switch effect" does not require intercalation of the large DPPZ ligand. Oligonucleotides may provide a hydrophobic cavity for the [Ru(phen)2DPPZ]2+ complex to prevent the quenching by the water molecule. In the cavity, the metal complex is in contact with DNA bases as is evidenced by the observation that the excited energy of the DNA bases transfer to the bound metal complex. However, the contact of the metal complex with DNA bases is different from the stacking of DPPZ in the intercalation pocket. In addition to the normal two luminescence lifetimes, a short lifetime in the range of 1-2 ns was found for both the delta- and lambda-enantiomer of [Ru(phen)2DPPZ]2+ when complexed with single stranded oligonucleotides, which may be assigned to the metal complex that is outside of the cavity, interacting with phosphate groups of DNA.

Formation of Stable Cationic Lipid/DNA Complexes for Gene Transfer

NASA Astrophysics Data System (ADS)

Hofland, Hans E. J.; Shephard, Lee; Sullivan, Sean M.

1996-07-01

Stable cationic lipid/DNA complexes were formed by solubilizing cationic liposomes with 1% octylglucoside and complexing a DNA plasmid with the lipid in the presence of detergent. Removal of the detergent by dialysis yielded a lipid/DNA suspension that was able to transfect tissue culture cells up to 90 days after formation with no loss in activity. Similar levels of gene transfer were obtained by mixing the cationic lipid in a liposome form with DNA just prior to cell addition. However, expression was completely lost 24 hr after mixing. The transfection efficiency of the stable complex in 15% fetal calf serum was 30% of that obtained in the absence of serum, whereas the transient complex was completely inactivated with 2% fetal calf serum. A 90-day stability study comparing various storage conditions showed that the stable complex could be stored frozen or as a suspension at 4 degrees C with no loss in transfection efficiency. Centrifugation of the stable complex produced a pellet that contained approximately 90% of the DNA and 10% of the lipid. Transfection of cells with the resuspended pellet and the supernatant showed that the majority of the transfection activity was in the pellet and all the toxicity was in the supernatant. Formation of a stable cationic lipid/DNA complex has produced a transfection vehicle that can be stored indefinitely, can be concentrated with no loss in transfection efficiency, and the toxicity levels can be greatly reduced when the active complex is isolated from the uncomplexed lipid.

Structural Confirmation of a Bent and Open Model for the Initiation Complex of T7 RNA Polymerase

PubMed Central

Turingan, Rosemary S.; Liu, Cuihua; Hawkins, Mary E.; Martin, Craig T.

2008-01-01

T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein. Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the C-terminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. FRET measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model’s orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogs incorporated at various positions in the DNA we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7–8 bases and is sufficient to allow synthesis of a 3 base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes. PMID:17253774

Electrochemical DNA biosensor for detection of porcine oligonucleotides using ruthenium(II) complex as intercalator label redox

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

Halid, Nurul Izni Abdullah; Hasbullah, Siti Aishah; Heng, Lee Yook

2014-09-03

A DNA biosensor detection of oligonucleotides via the interactions of porcine DNA with redox active complex based on the electrochemical transduction is described. A ruthenium(II) complex, [Ru(bpy){sub 2}(PIP)]{sup 2+}, (bpy = 2,2′bipyridine, PIP = 2-phenylimidazo[4,5-f[[1,10-phenanthroline]) as DNA label has been synthesized and characterized by 1H NMR and mass spectra. The study was carried out by covalent bonding immobilization of porcine aminated DNA probes sequences on screen printed electrode (SPE) modified with succinimide-acrylic microspheres and [Ru(bpy){sub 2}(PIP)]{sup 2+} was used as electrochemical redox intercalator label to detect DNA hybridization event. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulsemore » voltammetry (DPV) over the potential range where the ruthenium (II) complex was active. The results indicate that the interaction of [Ru(bpy){sub 2}(PIP)]{sup 2+} with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.« less

Regulation of a Viral Proteinase by a Peptide and DNA in One-dimensional Space

PubMed Central

Blainey, Paul C.; Graziano, Vito; Pérez-Berná, Ana J.; McGrath, William J.; Flint, S. Jane; San Martín, Carmen; Xie, X. Sunney; Mangel, Walter F.

2013-01-01

Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase (AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 106 bp2/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA. PMID:23043138

Structure of the EndoMS-DNA Complex as Mismatch Restriction Endonuclease.

PubMed

Nakae, Setsu; Hijikata, Atsushi; Tsuji, Toshiyuki; Yonezawa, Kouki; Kouyama, Ken-Ichi; Mayanagi, Kouta; Ishino, Sonoko; Ishino, Yoshizumi; Shirai, Tsuyoshi

2016-11-01

Archaeal NucS nuclease was thought to degrade the single-stranded region of branched DNA, which contains flapped and splayed DNA. However, recent findings indicated that EndoMS, the orthologous enzyme of NucS, specifically cleaves double-stranded DNA (dsDNA) containing mismatched bases. In this study, we determined the structure of the EndoMS-DNA complex. The complex structure of the EndoMS dimer with dsDNA unexpectedly revealed that the mismatched bases were flipped out into binding sites, and the overall architecture most resembled that of restriction enzymes. The structure of the apo form was similar to the reported structure of Pyrococcus abyssi NucS, indicating that movement of the C-terminal domain from the resting state was required for activity. In addition, a model of the EndoMS-PCNA-DNA complex was preliminarily verified with electron microscopy. The structures strongly support the idea that EndoMS acts in a mismatch repair pathway. Copyright © 2016 Elsevier Ltd. All rights reserved.

The fractal based analysis of human face and DNA variations during aging.

PubMed

Namazi, Hamidreza; Akrami, Amin; Hussaini, Jamal; Silva, Osmar N; Wong, Albert; Kulish, Vladimir V

2017-01-16

Human DNA is the main unit that shapes human characteristics and features such as behavior. Thus, it is expected that changes in DNA (DNA mutation) influence human characteristics and features. Face is one of the human features which is unique and also dependent on his gen. In this paper, for the first time we analyze the variations of human DNA and face simultaneously. We do this job by analyzing the fractal dimension of DNA walk and face during human aging. The results of this study show the human DNA and face get more complex by aging. These complexities are mapped on fractal exponents of DNA walk and human face. The method discussed in this paper can be further developed in order to investigate the direct influence of DNA mutation on the face variations during aging, and accordingly making a model between human face fractality and the complexity of DNA walk.

Creating complex molecular topologies by configuring DNA four-way junctions

NASA Astrophysics Data System (ADS)

Liu, Di; Chen, Gang; Akhter, Usman; Cronin, Timothy M.; Weizmann, Yossi

2016-10-01

The realization of complex topologies at the molecular level represents a grand challenge in chemistry. This necessitates the manipulation of molecular interactions with high precision. Here we show that single-stranded DNA (ssDNA) knots and links can be created by utilizing the inherent topological properties that pertain to the DNA four-way junction, at which the two helical strands form a node and can be configured conveniently and connected for complex topological construction. Using this strategy, we produced series of ssDNA topoisomers with the same sequences. By finely designing the curvature and torsion, double-stranded DNA knots were accessed by hybridizing and ligating the complementary strands with the knotted ssDNA templates. Furthermore, we demonstrate the use of a constructed ssDNA knot both to probe the topological conversion catalysed by DNA topoisomerase and to study the DNA replication under topological constraint.

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

PubMed

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.

Spacer length controlled lamello-columnar to oblique-columnar mesophase transition in liquid crystalline DNA - discotic cationic lipid complexes

NASA Astrophysics Data System (ADS)

Zhu, Lei; Cui, Li; Miao, Jianjun

2006-03-01

A series of asymmetric triphenylene imidazolium salts with different spacer lengths (C5, C8, and C11) were synthesized and their ionic complexes with double-strand DNA were prepared in aqueous solution. The molecular composition of the complexes was determined by FTIR analysis. The liquid crystalline morphology was characterized by polarized light microscopy, X-ray diffraction (XRD), and transmission electron microscope. 2D XRD results indicated an oblique columnar phase for the complex with a short spacer length of C5, while lamello-columnar phases for those with longer spacer lengths (C8 and C11). Thin film circular dichroism results showed the disappearing of any helical conformation in the DNA in all the complexes. Instead, the complexation between single-strand RNA and discotic cationic lipids did not show columnar morphology; therefore, the columnar liquid crystalline morphology in the DNA-discotic cationic lipid complexes was attributed to the DNA double-strand chain rigidity.

Design, synthesis, spectral characterization, DNA interaction and biological activity studies of copper(II), cobalt(II) and nickel(II) complexes of 6-amino benzothiazole derivatives

NASA Astrophysics Data System (ADS)

Daravath, Sreenu; Kumar, Marri Pradeep; Rambabu, Aveli; Vamsikrishna, Narendrula; Ganji, Nirmala; Shivaraj

2017-09-01

Two novel Schiff bases, L1 = (2-benzo[d]thiazol-6-ylimino)methyl)-4,6-dichlorophenol), L2 = (1-benzo[d]thiazol-6-ylimino)methyl)-6-bromo-4-chlorophenol) and their bivalent transition metal complexes [M(L1)2] and [M(L2)2], where M = Cu(II), Co(II) and Ni(II) were synthesized and characterized by elemental analysis, NMR, IR, UV-visible, mass, magnetic moments, ESR, TGA, SEM, EDX and powder XRD. Based on the experimental data a square planar geometry around the metal ion is assigned to all the complexes (1a-2c). The interaction of synthesized metal complexes with calf thymus DNA was explored using UV-visible absorption spectra, fluorescence and viscosity measurements. The experimental evidence indicated that all the metal complexes strongly bound to CT-DNA through an intercalation mode. DNA cleavage experiments of metal(II) complexes with supercoiled pBR322 DNA have also been explored by gel electrophoresis in the presence of H2O2 as well as UV light, and it is found that the Cu(II) complexes cleaved DNA more effectively compared to Co(II), Ni(II) complexes. In addition, the ligands and their metal complexes were screened for antimicrobial activity and it is found that all the metal complexes were more potent than free ligands.

EM structure of a helicase-loader complex depicting a 6:2 binding sub-stoichiometry from Geobacillus kaustophilus HTA426

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

Lin, Yen-Chen; Naveen, Vankadari; Molecular Cell Biology, Taiwan International Graduate Program, Institute of Molecular Biology, Academia Sinica, and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan

During DNA replication, bacterial helicase is recruited as a complex in association with loader proteins to unwind the parental duplex. Previous structural studies have reported saturated 6:6 helicase-loader complexes with different conformations. However, structural information on the sub-stoichiometric conformations of these previously-documented helicase-loader complexes remains elusive. Here, with the aid of single particle electron-microscopy (EM) image reconstruction, we present the Geobacillus kaustophilus HTA426 helicase-loader (DnaC-DnaI) complex with a 6:2 binding stoichiometry in the presence of ATPγS. In the 19 Å resolution EM map, the undistorted and unopened helicase ring holds a robust loader density above the C-terminal RecA-like domain. Meanwhile, themore » path of the central DNA binding channel appears to be obstructed by the reconstructed loader density, implying its potential role as a checkpoint conformation to prevent the loading of immature complex onto DNA. Our data also reveals that the bound nucleotides and the consequently induced conformational changes in the helicase hexamer are essential for active association with loader proteins. These observations provide fundamental insights into the formation of the helicase-loader complex in bacteria that regulates the DNA replication process. - Highlights: • Helicase-loader complex structure with 6:2 sub-stoichiometry is resolved by EM. • Helicase hexamer in 6:2 sub-stoichiometry is constricted and un-opened. • 6:2 binding ratio of helicase-loader complex could act as a DNA loading checkpoint. • Nucleotides stabilize helicase-loader complex at low protein concentrations.« less

Molecular determinants of the interactions between proteins and ssDNA.

PubMed

Mishra, Garima; Levy, Yaakov

2015-04-21

ssDNA binding proteins (SSBs) protect ssDNA from chemical and enzymatic assault that can derail DNA processing machinery. Complexes between SSBs and ssDNA are often highly stable, but predicting their structures is challenging, mostly because of the inherent flexibility of ssDNA and the geometric and energetic complexity of the interfaces that it forms. Here, we report a newly developed coarse-grained model to predict the structure of SSB-ssDNA complexes. The model is successfully applied to predict the binding modes of six SSBs with ssDNA strands of lengths of 6-65 nt. In addition to charge-charge interactions (which are often central to governing protein interactions with nucleic acids by means of electrostatic complementarity), an essential energetic term to predict SSB-ssDNA complexes is the interactions between aromatic residues and DNA bases. For some systems, flexibility is required from not only the ssDNA but also, the SSB to allow it to undergo conformational changes and the penetration of the ssDNA into its binding pocket. The association mechanisms can be quite varied, and in several cases, they involve the ssDNA sliding along the protein surface. The binding mechanism suggests that coarse-grained models are appropriate to study the motion of SSBs along ssDNA, which is expected to be central to the function carried out by the SSBs.

Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

PubMed

Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

2016-08-01

The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

Substrate-mediated delivery of gene complex nanoparticles via polydopamine coating for enhancing competitiveness of endothelial cells.

PubMed

Li, Bo-Chao; Chang, Hao; Ren, Ke-Feng; Ji, Jian

2016-11-01

Substrate-mediated delivery of functional plasmid DNA (pDNA) has been proven to be a promising strategy to promote competitiveness of endothelial cells (ECs) over smooth muscle cells (SMCs), which is beneficial to inducing fast endothelialization of implanted vascular devices. Thus, it is of great importance to develop universal approaches with simplicity and easiness to immobilize DNA complex nanoparticles on substrates. In this study, the bioinspired polydopamine (PDA) coating was employed in immobilization of DNA complex nanoparticles, which were composed of protamine (PrS) and plasmid DNA encoding with hepatocyte growth factor (HGF-pDNA) gene. We demonstrated that the DNA complex nanoparticles can be successfully immobilized onto the PDA surface. Consequently, the HGF expression of both ECs and SMCs were significantly improved when they cultured on the DNA complex nanoparticles-immobilized substrates. Furthermore, EC proliferation was specifically promoted due to bioactivity of HGF, leading to an enhancement of EC competitiveness over SMCs. Our findings demonstrated the substrate-mediated functional gene nanoparticle delivery through PDA coating as a simple and efficient approach. It may hold great potential in the field of interventional cardiovascular implants. Copyright © 2016 Elsevier B.V. All rights reserved.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication

PubMed Central

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-01

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. PMID:27679476

The In-Situ Structure of Cationic Lipid/DNA Complexes in Animal Cells: Applications to Gene Therapy

NASA Astrophysics Data System (ADS)

Lin, Alison J.; Slack, Nelle L.; Idziak, S. H. J.; George, C. X.; Samuel, C. E.; Safinya, C. R.

1997-03-01

Gene therapy has been the focus of many recent investigations. One promising technique is to use cationic lipids as vectors for DNA transfection. However, the exact mechanism of DNA uptake is unknown, due to a lack of knowledge regarding interactions and structures of DNA and cationic lipids. We are developing x-ray and optical microscopy techniques to directly image the temporal and spatial distribution of cationic lipid/DNA complexes (CL-DNA) during the various stages of transfection in mouse L-cells. The structure of these complexes in water have been shown by x-ray studies to consist of alternating lipid bilayers and DNA monolayers.(J. Radler, I. Koltover, T. Salditt, C. R. Safinya, Science (January 1997)) We demonstrate the feasibility of in-situ x-ray diffraction studies of CL-DNA complexes in L-cells. The x-ray data implies that complexes are taken up by endocytosis and DOPE destabilizes the endosomal membrane. Results from optical microscopy studies and X-Gal staining of transfected cells support the x-ray data. Funded in part by NSF grant DMR-9624091, PRF (No. 31352-AC7), Los Alamos CULAR grant No. STB/UC: 96-118.

Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

PubMed Central

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2013-01-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1

PubMed Central

Butryn, Agata; Schuller, Jan M; Stoehr, Gabriele; Runge-Wollmann, Petra; Förster, Friedrich; Auble, David T; Hopfner, Karl-Peter

2015-01-01

Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex. DOI: http://dx.doi.org/10.7554/eLife.07432.001 PMID:26258880

DNA-lipid complexes: stability of honeycomb-like and spaghetti-like structures.

PubMed Central

May, S; Ben-Shaul, A

1997-01-01

A molecular level theory is presented for the thermodynamic stability of two (similar) types of structural complexes formed by (either single strand or supercoiled) DNA and cationic liposomes, both involving a monolayer-coated DNA as the central structural unit. In the "spaghetti" complex the central unit is surrounded by another, oppositely curved, monolayer, thus forming a bilayer mantle. The "honeycomb" complex is a bundle of hexagonally packed DNA-monolayer units. The formation free energy of these complexes, starting from a planar cationic/neutral lipid bilayer and bare DNA, is expressed as a sum of electrostatic, bending, mixing, and (for the honeycomb) chain frustration contributions. The electrostatic free energy is calculated using the Poisson-Boltzmann equation. The bending energy of the mixed lipid layers is treated in the quadratic curvature approximation with composition-dependent bending rigidity and spontaneous curvature. Ideal lipid mixing is assumed within each lipid monolayer. We found that the most stable monolayer-coated DNA units are formed when the charged/neutral lipid composition corresponds (nearly) to charge neutralization; the optimal monolayer radius corresponds to close DNA-monolayer contact. These conclusions are also valid for the honeycomb complex, as the chain frustration energy is found to be negligible. Typically, the stabilization energies for these structures are on the order of 1 k(B)T/A of DNA length, reflecting mainly the balance between the electrostatic and bending energies. The spaghetti complexes are less stable due to the additional bending energy of the external monolayer. A thermodynamic analysis is presented for calculating the equilibrium lipid compositions when the complexes coexist with excess bilayer. PMID:9370436

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

PubMed Central

Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

1999-01-01

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

Light amplification and lasing from dyes doped in DNA-complex thin films prepared by soaking method

NASA Astrophysics Data System (ADS)

Kawabe, Yutaka; Suzuki, Takemasa; Iisaka, You

2014-08-01

An alternative fabrication method for dye-doped DNA-surfactant complex films was developed and amplified spontaneous emission (ASE) and lasing under low energy optical pumping were demonstrated. In this new preparation technique, thin DNA-cethyltrimethylammonium (CTMA) complex films made by a spin coating method were stained with a hemicyanine dye by soaking them in acetone solution of the dye for one day. Molar ratio of the dye to DNA base pair for the final products was estimated to be 0.2, the value was much higher than those achieved via usual mixing method. ASE threshold value under pumping of a pulsed frequency-doubled YAG laser was about 0.3 mJ/cm2. Laser emission was also attained under the excitation with two interfering beams forming a dynamic grating of gain coefficient. Durability test indicated that 70% of their initial performance was maintained after 1 hour of continuous pumping. The technique was applied to water soluble dyes because the DNA complex was insoluble to water as well as acetone. We employed anionic Eosin Y dye, succeeding in sample formation and ASE emission. Different types of surfactants were also complexed with DNA, showing variation of emission peak wavelength. These results give a clue about the structure of the complex or interaction modes between DNA and surfactants, strongly suggesting that dye molecules are not intercalated into nor bound to DNA double strand directly, but are incorporated in the complex system via ion-exchange process or aggregating with cationic surfactants.

Imaging of DNA and Protein by SFM and Combined SFM-TIRF Microscopy.

PubMed

Grosbart, Małgorzata; Ristić, Dejan; Sánchez, Humberto; Wyman, Claire

2018-01-01

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nm resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

Improved stability and electrophoretic properties of preformed fluorescent cationic dye-DNA complexes in a taps-tetrapentylammonium buffer in agarose slab gels.

PubMed

Zeng, Z; Clark, S M; Mathies, R A; Glazer, A N

1997-10-01

High-resolution capillary electrophoresis sizing of preformed complexes of bis-intercalating fluorescent dyes with double-stranded DNA has been demonstrated using hydroxyethylcellulose and 3-[tris-(hydroxymethyl) methylamino]-1-propanesulfonic acid-tetrapentylammonium (Taps-NPe+4) buffers (S. M. Clark and R. A. Mathies, Anal. Chem. 69, 1355-1363, 1997). Such capillary electrophoresis separations were unattainable in conventional buffers containing other cations such as Tris+, Na+, and NH+4. We report here the behavior of preformed double-stranded DNA-dye complexes on agarose slab gel electrophoresis in 40 mM Taps-NPe+4, 1 mM H2EDTA, pH 8.2. Upon electrophoresis in this buffer (a) complexes formed at DNA base pairs:dye ratios ranging from 100:1 to 5:1 show the same mobility; (b) the half-lives of DNA-dye complexes with monointercalators are two- to threefold longer than those in commonly used Tris buffers; (c) there is little dye transfer between labeled and unlabeled DNA molecules; and (d) precise two-color sizing of preformed restriction fragment-dye complexes with fluorescent bisintercalators is achieved.

NMR and computational methods applied to the 3- dimensional structure determination of DNA and ligand-DNA complexes in solution

NASA Astrophysics Data System (ADS)

Smith, Jarrod Anson

2D homonuclear 1H NMR methods and restrained molecular dynamics (rMD) calculations have been applied to determining the three-dimensional structures of DNA and minor groove-binding ligand-DNA complexes in solution. The structure of the DNA decamer sequence d(GCGTTAACGC)2 has been solved both with a distance-based rMD protocol and an NOE relaxation matrix backcalculation-based protocol in order to probe the relative merits of the different refinement methods. In addition, three minor groove binding ligand-DNA complexes have been examined. The solution structure of the oligosaccharide moiety of the antitumor DNA scission agent calicheamicin γ1I has been determined in complex with a decamer duplex containing its high affinity 5'-TCCT- 3' binding sequence. The structure of the complex reinforces the belief that the oligosaccharide moiety is responsible for the sequence selective minor-groove binding activity of the agent, and critical intermolecular contacts are revealed. The solution structures of both the (+) and (-) enantiomers of the minor groove binding DNA alkylating agent duocarmycin SA have been determined in covalent complex with the undecamer DNA duplex d(GACTAATTGTC).d(GAC AATTAGTC). The results support the proposal that the alkylation activity of the duocarmycin antitumor antibiotics is catalyzed by a binding-induced conformational change in the ligand which activates the cyclopropyl group for reaction with the DNA. Comparisons between the structures of the two enantiomers covalently bound to the same DNA sequence at the same 5'-AATTA-3 ' site have provided insight into the binding orientation and site selectivity, as well as the relative rates of reactivity of these two agents.

Synthesis, spectroscopic, molecular orbital calculation, cytotoxic, molecular docking of DNA binding and DNA cleavage studies of transition metal complexes with N-benzylidene-N'-salicylidene-1,1-diaminopropane

NASA Astrophysics Data System (ADS)

Al-Mogren, Muneerah M.; Alaghaz, Abdel-Nasser M. A.; Elbohy, Salwa A. H.

2013-10-01

Eight mononuclear chromium(III), manganese(II), iron(III), cobalt(II), nickel(II), copper(II), zinc(II) and cadmium(II) complexes of Schiff's base ligand were synthesized and determined by different physical techniques. The complexes are insoluble in common organic solvents but soluble in DMF and DMSO. The measured molar conductance values in DMSO indicate that the complexes are non-electrolytic in nature. All the eight metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The analytical data helped to elucidate the structure of the metal complexes. The Schiff base is found to act as tridentate ligand using N2O donor set of atoms leading to an octahedral geometry for the complexes around all the metal ions. Quantum chemical calculations were performed with semi-empirical method to find the optimum geometry of the ligand and its complexes. Additionally in silico, the docking studies and the calculated pharmacokinetic parameters show promising futures for application of the ligand and complexes as high potency agents for DNA binding activity. The interaction of the complexes with calf thymus DNA (CT-DNA) has been investigated by UV absorption method, and the mode of CT-DNA binding to the complexes has been explored. Furthermore, the DNA cleavage activity by the complexes was performed. The Schiff base and their complexes have been screened for their antibacterial activity against bacterial strains [Staphylococcus aureus (RCMB010027), Staphylococcus epidermidis (RCMB010024), Bacillis subtilis (RCMB010063), Proteous vulgaris (RCMB 010085), Klebsiella pneumonia (RCMB 010093) and Shigella flexneri (RCMB 0100542)] and fungi [(Aspergillus fumigates (RCMB 02564), Aspergillus clavatus (RCMB 02593) and Candida albicans (RCMB05035)] by disk diffusion method. All the metal complexes have potent biocidal activity than the free ligand.

Mechanism for priming DNA synthesis by yeast DNA Polymerase α

PubMed Central

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

2013-01-01

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

Eukaryotic Replicative Helicase Subunit Interaction with DNA and Its Role in DNA Replication

PubMed Central

Martinez, Matthew P.; Wacker, Amanda L.; Bruck, Irina; Kaplan, Daniel L.

2017-01-01

The replicative helicase unwinds parental double-stranded DNA at a replication fork to provide single-stranded DNA templates for the replicative polymerases. In eukaryotes, the replicative helicase is composed of the Cdc45 protein, the heterohexameric ring-shaped Mcm2-7 complex, and the tetrameric GINS complex (CMG). The CMG proteins bind directly to DNA, as demonstrated by experiments with purified proteins. The mechanism and function of these DNA-protein interactions are presently being investigated, and a number of important discoveries relating to how the helicase proteins interact with DNA have been reported recently. While some of the protein-DNA interactions directly relate to the unwinding function of the enzyme complex, other protein-DNA interactions may be important for minichromosome maintenance (MCM) loading, origin melting or replication stress. This review describes our current understanding of how the eukaryotic replicative helicase subunits interact with DNA structures in vitro, and proposed models for the in vivo functions of replicative helicase-DNA interactions are also described. PMID:28383499

DNA ends alter the molecular composition and localization of Ku multicomponent complexes.

PubMed

Adelmant, Guillaume; Calkins, Anne S; Garg, Brijesh K; Card, Joseph D; Askenazi, Manor; Miron, Alex; Sobhian, Bijan; Zhang, Yi; Nakatani, Yoshihiro; Silver, Pamela A; Iglehart, J Dirk; Marto, Jarrod A; Lazaro, Jean-Bernard

2012-08-01

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

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

PubMed

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.

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

PubMed Central

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

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

PubMed

Li, Caroline M; Miao, Yunan; Lingeman, Robert G; Hickey, Robert J; Malkas, Linda H

2016-01-01

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Atomistic Simulations of Complex DNA DSBs and the Interactions with Ku70/80 Heterodimer

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2011-01-01

Compared to DNA with simple DSBs, the complex lesions can enhance the hydrogen bonds opening rate at the DNA terminus, and increase the mobility of the whole duplex. Binding of Ku drastically reduces the structural disruption and flexibility caused by the complex lesions. In all complex DSBs systems, the binding of DSB terminus with Ku70 is softened while the binding of the middle duplex with Ku80 is tightened. Binding of Ku promotes the rigidity of DNA duplexes, due to the clamp structure of the inner surface of the rings of Ku70/80.

On the origin of the decrease in stability of the DNA hairpin d(GCGAAGC) on complexation with aromatic drugs.

PubMed

Kostjukov, V V; Lantushenko, A O; Davies, D B; Evstigneev, M P

2007-08-01

Molecular dynamics simulations of drug-DNA complexes have been carried out in order to explain the experimentally observed decrease in thermal stability of the DNA hairpin d(GCGAAGC) on binding the aromatic drug molecules, daunomycin, ethidium bromide, novantrone and proflavine. This complexation behavior is in contrast to the stabilizing effect of the same aromatic drug molecules on DNA duplexes. Analysis of the energy parameters and the hydration properties of the complexes shows that the main factor correlating with the decrease in melting temperatures of the drug-hairpin complexes is the number of water bridges, with a reduction of at least 40% on ligand binding.

Structural Analysis of HMGD-DNA Complexes Reveal Influence of Intercalation on Sequence Selectivity and DNA Bending

PubMed Central

Churchill, Mair E.A.; Klass, Janet; Zoetewey, David L.

2010-01-01

The ubiquitous eukaryotic High-Mobility-Group-Box (HMGB) chromosomal proteins promote many chromatin-mediated cellular activities through their non-sequence-specific binding and bending of DNA. Minor groove DNA binding by the HMG box results in substantial DNA bending toward the major groove owing to electrostatic interactions, shape complementarity and DNA intercalation that occurs at two sites. Here, the structures of the complexes formed with DNA by a partially DNA intercalation-deficient mutant of Drosophila melanogaster HMGD have been determined by X-ray crystallography at a resolution of 2.85 Å. The six proteins and fifty base pairs of DNA in the crystal structure revealed a variety of bound conformations. All of the proteins bound in the minor groove, bridging DNA molecules, presumably because these DNA regions are easily deformed. The loss of the primary site of DNA intercalation decreased overall DNA bending and shape complementarity. However, DNA bending at the secondary site of intercalation was retained and most protein-DNA contacts were preserved. The mode of binding resembles the HMGB1-boxA-cisplatin-DNA complex, which also lacks a primary intercalating residue. This study provides new insights into the binding mechanisms used by HMG boxes to recognize varied DNA structures and sequences as well as modulate DNA structure and DNA bending. PMID:20800069

Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes

PubMed Central

Brabec, Viktor; Kasparkova, Jana; Kostrhunova, Hana; Farrell, Nicholas P.

2016-01-01

Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes. PMID:27574114

Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes.

PubMed

Brabec, Viktor; Kasparkova, Jana; Kostrhunova, Hana; Farrell, Nicholas P

2016-08-30

Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes.

Porcine endothelium induces DNA-histone complex formation in human whole blood: a harmful effect of histone on coagulation and endothelial activation.

PubMed

Yoo, Hyun Ju; Kim, Ji-Eun; Gu, Ja Yoon; Lee, Sae Bom; Lee, Hyun Joo; Hwang, Ho Young; Hwang, Yoohwa; Kim, Young Tae; Kim, Hyun Kyung

2016-11-01

Neutrophils play a role in xenograft rejection. When neutrophils are stimulated, they eject the DNA-histone complex into the extracellular space, called neutrophil extracellular traps (NET). We investigated whether NET formation actively occurs in the xenograft and contributes to coagulation and endothelial activation. Human whole blood was incubated with porcine aortic endothelial cells (pEC) from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. In the supernatant plasma from human blood, the level of the DNA-histone complex was measured by ELISA, and thrombin generation was measured using a calibrated automated thrombogram. Histone-induced tissue factor and adhesion molecule expression were measured by flow cytometry. pEC from both wild-type and GTKO pigs significantly induced DNA-histone complex formation in human whole blood. The DNA-histone complex produced shortened the thrombin generation time and clotting time. Histone alone dose-dependently induced tissue factor and adhesion molecule expression in pEC. Aurintricarboxylic acid pretreatment partially inhibited pEC-induced DNA-histone complex formation. DNA-histone complex actively generated upon xenotransplantation is a novel target to inhibit coagulation and endothelial activation. To prevent tissue factor and adhesion molecule expression, a strategy to block soluble histone may be required in xenotransplantation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multiply Intercalator-Substituted Cu(II) Cyclen Complexes as DNA Condensers and DNA/RNA Synthesis Inhibitors.

PubMed

Hormann, Jan; Malina, Jaroslav; Lemke, Oliver; Hülsey, Max J; Wedepohl, Stefanie; Potthoff, Jan; Schmidt, Claudia; Ott, Ingo; Keller, Bettina G; Brabec, Viktor; Kulak, Nora

2018-05-07

Many drugs that are applied in anticancer therapy such as the anthracycline doxorubicin contain DNA-intercalating 9,10-anthraquinone (AQ) moieties. When Cu(II) cyclen complexes were functionalized with up to three (2-anthraquinonyl)methyl substituents, they efficiently inhibited DNA and RNA synthesis resulting in high cytotoxicity (selective for cancer cells) accompanied by DNA condensation/aggregation phenomena. Molecular modeling suggests an unusual bisintercalation mode with only one base pair between the two AQ moieties and the metal complex as a linker. A regioisomer, in which the AQ moieties point in directions unfavorable for such an interaction, had a much weaker biological activity. The ligands alone and corresponding Zn(II) complexes (used as redox inert control compounds) also exhibited lower activity.

Development and physico-chemical characterization of cyclodextrin DNA complexes loaded liposomes

NASA Astrophysics Data System (ADS)

Tavares, Guilherme D.; Viana, Cristiane M.; Araújo, José G. V. C.; Ramaldes, Gilson A.; Carvalho, Wânia S.; Pesquero, Jorge L.; Vilela, José M. C.; Andrade, Margareth S.; de Oliveira, Mônica C.

2006-10-01

In the present study, anionic and pH-sensitive liposomes containing DNA were developed and characterized. These liposomes were obtained by binding the DNA with 6-monodeoxy-6-monoamine-β-cyclodextrin (Am-β-CD). This complex was encapsulated into the liposomes, which were characterized by encapsulation rate, diameter, zeta potential, and atomic force microscopy. The binding between Am-β-CD and the DNA was higher as of the +/- charge ratio. The amount of DNA encapsulated was approximately 10-14 μg/mL. The mean diameter and zeta potential were 186.0 nm and -56 mV, respectively. Liposomes which did not contain the complex were more prone to collapse over the mica surface. The vesicles containing the complex presented a narrower size distribution.

Evidence of an inverted hexagonal phase in self-assembled phospholipid-DNA-metal complexes

NASA Astrophysics Data System (ADS)

Francescangeli, O.; Pisani, M.; Stanic, V.; Bruni, P.; Weiss, T. M.

2004-08-01

We report the first observation of an inverted hexagonal phase of phospholipid-DNA-metal complexes. These ternary complexes are formed in a self-assembled manner when water solutions of neutral lipid dioleoylphosphatidylethanolamine (DOPE), DNA and divalent metal cations (Me2+; Me=Fe, Co, Mg, Mn) are mixed, which represents a striking example of supramolecular chemistry. The structure, derived from synchrotron X-ray diffraction, consists of cylindrical DNA strands coated by neutral lipid monolayers and arranged on a two-dimensional hexagonal lattice (HIIc). Besides the fundamental aspects, DOPE-DNA-Me2+ complexes may be of great interest as efficient nonviral delivery systems in gene therapy applications because of the low inherent cytotoxicity and the potential high transfection efficiency.

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair.

PubMed

LeBlanc, Sharonda; Wilkins, Hunter; Li, Zimeng; Kaur, Parminder; Wang, Hong; Erie, Dorothy A

2017-01-01

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein-protein and protein-DNA interactions. It can be used to capture snapshots of protein-DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein-protein and protein-DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein-protein and protein-DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein-DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein-protein and protein-nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure-function relationships that govern DNA repair processes. © 2017 Elsevier Inc. All rights reserved.

Lesion search and recognition by thymine DNA glycosylase revealed by single molecule imaging

PubMed Central

Buechner, Claudia N.; Maiti, Atanu; Drohat, Alexander C.; Tessmer, Ingrid

2015-01-01

The ability of DNA glycosylases to rapidly and efficiently detect lesions among a vast excess of nondamaged DNA bases is vitally important in base excision repair (BER). Here, we use single molecule imaging by atomic force microscopy (AFM) supported by a 2-aminopurine fluorescence base flipping assay to study damage search by human thymine DNA glycosylase (hTDG), which initiates BER of mutagenic and cytotoxic G:T and G:U mispairs in DNA. Our data reveal an equilibrium between two conformational states of hTDG–DNA complexes, assigned as search complex (SC) and interrogation complex (IC), both at target lesions and undamaged DNA sites. Notably, for both hTDG and a second glycosylase, hOGG1, which recognizes structurally different 8-oxoguanine lesions, the conformation of the DNA in the SC mirrors innate structural properties of their respective target sites. In the IC, the DNA is sharply bent, as seen in crystal structures of hTDG lesion recognition complexes, which likely supports the base flipping required for lesion identification. Our results support a potentially general concept of sculpting of glycosylases to their targets, allowing them to exploit the energetic cost of DNA bending for initial lesion sensing, coupled with continuous (extrahelical) base interrogation during lesion search by DNA glycosylases. PMID:25712093

Detection of DNA via the fluorescence quenching of Mn-doped ZnSe D-dots/doxorubicin/DNA ternary complexes system.

PubMed

Gao, Xue; Niu, Lu; Su, Xingguang

2012-01-01

This manuscript reports a method for the detection of double-stranded DNA, based on Mn:ZnSe d-dots and intercalating agent doxorubicin (DOX). DOX can quench the photoluminescence (PL) of Mn:ZnSe d-dots through photoinduced electron transfer process, after binding with Mn:ZnSe d-dots. The addition of DNA can result in the formation of the Mn:ZnSe d-dots-DOX-DNA ternary complexes, the fluorescence of the Mn:ZnSe d-dots-DOX complexes would be further quenched by the addition of DNA, thus allowing the detection of DNA. The formation mechanism of the Mn:ZnSe d-dots-DOX-DNA ternary complexes was studied in detail in this paper. Under optimal conditions, the quenched fluorescence intensity of Mn:ZnSe d-dots-DOX system are perfectly described by Stern-Volmer equation with the concentration of hsDNA ranging from 0.006 μg mL(-1) to 6.4 μg mL(-1). The detection limit (S/N = 3) for hsDNA is 0.5 ng mL(-1). The proposed method was successfully applied to the detection of DNA in synthetic samples and the results were satisfactory.

DNA incision evaluation, binding investigation and biocidal screening of Cu(II), Ni(II) and Co(II) complexes with isoxazole Schiff bases.

PubMed

Ganji, Nirmala; Chityala, Vijay Kumar; Marri, Pradeep Kumar; Aveli, Rambabu; Narendrula, Vamsikrishna; Daravath, Sreenu; Shivaraj

2017-10-01

Two new series of binary metal complexes [M(L 1 ) 2 ] and [M(L 2 ) 2 ] where, M=Cu(II), Ni(II) & Co(II) and L 1 =4-((3,4-dimethylisoxazol-5-ylimino)methyl)benzene-1,3-diol; L 2 =2-((3,4-dimethylisoxazol-5-ylimino)methyl)-5-methoxyphenol were synthesized and characterized by elemental analysis, 1 H NMR, 13 C NMR, FT-IR, ESI mass, UV-Visible, magnetic moment, ESR, SEM and powder XRD studies. Based on these results, a square planar geometry is assigned for all the metal complexes where the Schiff base acts as uninegatively charged bidentate chelating agent via the hydroxyl oxygen and azomethine nitrogen atoms. DNA binding studies of all the complexes with calf thymus DNA have been comprehensively investigated using electronic absorption spectroscopy, fluorescence quenching and viscosity studies. The oxidative and photo cleavage affinity of metal complexes towards supercoiled pBR322 DNA has been ascertained by agarose gel electrophoresis assay. From the results, it is observed that all the metal complexes bind effectively to CT-DNA via an intercalative mode of binding and also cleave pBR322 DNA in a promising manner. Further the Cu(II) complexes have shown better binding and cleavage properties towards DNA. The antimicrobial activities of the Schiff bases and their metal complexes were studied on bacterial and fungal strains and the results denoted that the complexes are more potent than their Schiff base ligands. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time

PubMed Central

Williams, Gareth J.; Hammel, Michal; Radhakrishnan, Sarvan Kumar; Ramsden, Dale; Lees-Miller, Susan P.; Tainer, John A.

2014-01-01

Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: 1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), 2) lesion processing, and 3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation. PMID:24656613

[Single-molecule detection and characterization of DNA replication based on DNA origami].

PubMed

Wang, Qi; Fan, Youjie; Li, Bin

2014-08-01

To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

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

PubMed

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.

Degradable polymeric carrier for the delivery of IL-10 plasmid DNA to prevent autoimmune insulitis of NOD mice.

PubMed

Koh, J J; Ko, K S; Lee, M; Han, S; Park, J S; Kim, S W

2000-12-01

Recently, we have reported that biodegradable poly [alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) can condense and protect plasmid DNA from DNase I. In this study, we investigated whether the systemic administration of pCAGGS mouse IL-10 (mIL-10) expression plasmid complexed with PAGA can reduce the development of insulitis in non-obese diabetic (NOD) mice. PAGA/mIL-10 plasmid complexes were stable for more than 60 min, but the naked DNA was destroyed within 10 min by DNase I. The PAGA/DNA complexes were injected into the tail vein of 3-week-old NOD mice. Serum mIL-10 level peaked at 5 days after injection, and could be detected for more than 9 weeks. The prevalence of severe insulitis on 12-week-old NOD mice was markedly reduced by the intravenous injection of PAGA/DNA complex (15.7%) compared with that of naked DNA injection (34.5%) and non-treated controls (90.9%). In conclusion, systemic administration of pCAGGS mIL-10 plasmid/PAGA complexes can reduce the severity of insulitis in NOD mice. This study shows that the PAGA/DNA complex has the potential for the prevention of autoimmune diabetes mellitus. Gene Therapy (2000) 7, 2099-2104.

Structure of homeodomain-leucine zipper/DNA complexes studied using hydroxyl radical cleavage of DNA and methylation interference.

PubMed

Tron, Adriana E; Comelli, Raúl N; Gonzalez, Daniel H

2005-12-27

Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.

Structure determination of uracil-DNA N-glycosylase from Deinococcus radiodurans in complex with DNA.

PubMed

Pedersen, Hege Lynum; Johnson, Kenneth A; McVey, Colin E; Leiros, Ingar; Moe, Elin

2015-10-01

Uracil-DNA N-glycosylase (UNG) is a DNA-repair enzyme in the base-excision repair (BER) pathway which removes uracil from DNA. Here, the crystal structure of UNG from the extremophilic bacterium Deinococcus radiodurans (DrUNG) in complex with DNA is reported at a resolution of 1.35 Å. Prior to the crystallization experiments, the affinity between DrUNG and different DNA oligonucleotides was tested by electrophoretic mobility shift assays (EMSAs). As a result of this analysis, two 16 nt double-stranded DNAs were chosen for the co-crystallization experiments, one of which (16 nt AU) resulted in well diffracting crystals. The DNA in the co-crystal structure contained an abasic site (substrate product) flipped into the active site of the enzyme, with no uracil in the active-site pocket. Despite the high resolution, it was not possible to fit all of the terminal nucleotides of the DNA complex into electron density owing to disorder caused by a lack of stabilizing interactions. However, the DNA which was in contact with the enzyme, close to the active site, was well ordered and allowed detailed analysis of the enzyme-DNA interaction. The complex revealed that the interaction between DrUNG and DNA is similar to that in the previously determined crystal structure of human UNG (hUNG) in complex with DNA [Slupphaug et al. (1996). Nature (London), 384, 87-92]. Substitutions in a (here defined) variable part of the leucine loop result in a shorter loop (eight residues instead of nine) in DrUNG compared with hUNG; regardless of this, it seems to fulfil its role and generate a stabilizing force with the minor groove upon flipping out of the damaged base into the active site. The structure also provides a rationale for the previously observed high catalytic efficiency of DrUNG caused by high substrate affinity by demonstrating an increased number of long-range electrostatic interactions between the enzyme and the DNA. Interestingly, specific interactions between residues in the N-terminus of a symmetry-related molecule and the complementary DNA strand facing away from the active site were also observed which seem to stabilize the enzyme-DNA complex. However, the significance of this observation remains to be investigated. The results provide new insights into the current knowledge about DNA damage recognition and repair by uracil-DNA glycosylases.

[Synthesis and study on the interaction of rare earth complexes of N', N-bis(2-pyridinecarboxamide)-1, 2-ethane with DNA].

PubMed

Lu, Xiao-Hong; Lin, Qiu-Yue; Hu, Rui-Ding; Liu, Wei-Dong; Feng, Jie

2007-06-01

Four rare earth complexes of N', N-bis(2-pyridinecarboxamide)-1, 2-ethane were synthesized and characterized by elemental analysis, conductivity measurement, thermal studies, IR and electronic spectra. The composition of the four complexes is [Ln(H2L)(NO3)2](NO3) x 3H2O (Ln=Sm, Eu, Gd, Tb). Results of spectral measurements indicate that the oxygen of carbonyl and the nitrogen of pyridyl coordinate with Ln(III) respectively, and the NO3- shows bidentate coordination. So the four complexes are 1 : 1 chelated complexes. The interaction between [Sm(H2L) (NO3)2](NO3) x 3H2O and DNA was studied by employing UV-Visible (UV-Vis) spectra, fluorescence spectra and SERS spectra. Experimental results show that with the incremental addition of DNA, the bands at 265 nm show hypochromism accompanied by a small red shift and the binding constant Kb Obtained is 1.24 x 10(5). Meanwhile fluorescence spectra show that the addition of [Sm(H2L) (NO3)2] (NO3) x 3H2O to DNA pretreated with EB causes an appreciable reduction in fluorescence intensity, indicating that the complex competes with ethidium bromide in binding to DNA, and free ethidium bromide increases. The addition of DNA causes the SERS signals of the complex to weaken and the band at 1 282 cm(-1) to disappear, which suggests that the planar pyridine molecule of the ligand may partly be inserted into the double-stranded helix plane in DNA, making pi electronic density of aromatic rings in complex change. The above phenomena indicate that [Sm(H2L) (NO3)] (NO3) x 3H2O interacts intensively with DNA.

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

PubMed Central

Chand, Mahesh Kumar; Nirwan, Neha; Diffin, Fiona M.; van Aelst, Kara; Kulkarni, Manasi; Pernstich, Christian; Szczelkun, Mark D.; Saikrishnan, Kayarat

2015-01-01

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission. PMID:26389736

Biochemical investigation of yttrium(III) complex containing 1,10-phenanthroline: DNA binding and antibacterial activity.

PubMed

Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Moodi, Asieh; Niroomand, Sona

2013-03-05

Characterization of the interaction between yttrium(III) complex containing 1,10-phenanthroline as ligand, [Y(phen)2Cl(OH2)3]Cl2⋅H2O, and DNA has been carried out by UV absorption, fluorescence spectra and viscosity measurements in order to investigate binding mode. The experimental results indicate that the yttrium(III) complex binds to DNA and absorption is decreasing in charge transfer band with the increase in amount of DNA. The binding constant (Kb) at different temperatures as well as thermodynamic parameters, enthalpy change (ΔH°) and entropy change (ΔS°), were calculated according to relevant fluorescent data and Vant' Hoff equation. The results of interaction mechanism studies, suggested that groove binding plays a major role in the binding of the complex and DNA. The activity of yttrium(III) complex against some bacteria was tested and antimicrobial screening tests shown growth inhibitory activity in the presence of yttrium(III) complex. Copyright © 2013 Elsevier B.V. All rights reserved.

Thin film DNA-complex-based dye lasers fabricated by immersion and conventional processes

NASA Astrophysics Data System (ADS)

Kawabe, Yutaka; Suzuki, Yuki

2017-08-01

DNA based thin film dye laser is one of promising optical devices for future technology. Laser oscillation and amplified spontaneous emission (ASE) were demonstrated by hemicyanine-doped DNA complex films prepared with `immersion method' as well as those made by a conventional way. In the immersion process, DNA-surfactant complex films were stained by immersion into an acetone solution including the dyes. In this study, three types of hemicyanines were incorporated with both methods, and laser oscillation was achieved with optically induced population grating formed in all of the complex films. The laser threshold values for six cases ranged in 0.07 - 0.18 mJ/cm2 , which was close to the best values made in DNA complex matrices. Continual pumping showed that laser oscillation persisted for 4 - 10 minutes. Immersion process gave superior laser capability especially for output efficiency over the conventional counterparts.

Synthesis and structure elucidation of a copper(II) Schiff-base complex: in vitro DNA binding, pBR322 plasmid cleavage and HSA binding studies.

PubMed

Tabassum, Sartaj; Ahmad, Musheer; Afzal, Mohd; Zaki, Mehvash; Bharadwaj, Parimal K

2014-11-01

New copper(II) complex with Schiff base ligand 4-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-benzoic acid (H₂L) was synthesized and characterized by spectroscopic and analytical and single crystal X-ray diffraction studies which revealed that the complex 1 exist in a distorted octahedral environment. In vitro CT-DNA binding studies were performed by employing different biophysical technique which indicated that the 1 strongly binds to DNA in comparison to ligand via electrostatic binding mode. Complex 1 cleaves pBR322 DNA via hydrolytic pathway and recognizes minor groove of DNA double helix. The HSA binding results showed that ligand and complex 1 has ability to quench the fluorescence emission intensity of Trp 214 residue available in the subdomain IIA of HSA. Copyright © 2014 Elsevier B.V. All rights reserved.

Single-stranded DNA Binding by the Helix-Hairpin-Helix Domain of XPF Protein Contributes to the Substrate Specificity of the ERCC1-XPF Protein Complex*

PubMed Central

Das, Devashish; Faridounnia, Maryam; Kovacic, Lidija; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E.

2017-01-01

The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble. PMID:28028171

Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy

PubMed Central

Forget, Anthony L.; Dombrowski, Christopher C.; Amitani, Ichiro; Kowalczykowski, Stephen C.

2015-01-01

In this Protocol, we describe a procedure to generate ‘DNA-dumbbells’ — single molecules of DNA with a microscopic bead attached at each end — and techniques for manipulating individual DNA-dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA-dumbbells and to visualize individual protein–DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free ‘reservoir’. The reservoir provides the means to examine formation of DNA–protein complexes in solution in the absence of external flow forces, while still maintaining a predetermined end-to-end extension of the DNA. These features facilitate examination of the role of three-dimensional DNA conformation and dynamics in protein–DNA interactions. Preparation of flow cells and reagents requires two days each; in situ DNA-dumbbell assembly and imaging of single protein–DNA complexes requires another day. PMID:23411634

EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele

PubMed Central

Rein, Katrin; Yanez, Diana A.; Terré, Berta; Palenzuela, Lluís; Aivio, Suvi; Wei, Kaichun; Edelmann, Winfried; Stark, Jeremy M.; Stracker, Travis H.

2015-01-01

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5′-3′ exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations. PMID:26160886

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes.

PubMed

Balasubramaniam, Muthukumar; Davids, Benem; Addai, Amma B; Pandhare, Jui; Dash, Chandravanu

2017-02-22

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

PubMed

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-09

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Study of base pair mutations in proline-rich homeodomain (PRH)-DNA complexes using molecular dynamics.

PubMed

Jalili, Seifollah; Karami, Leila; Schofield, Jeremy

2013-06-01

Proline-rich homeodomain (PRH) is a regulatory protein controlling transcription and gene expression processes by binding to the specific sequence of DNA, especially to the sequence 5'-TAATNN-3'. The impact of base pair mutations on the binding between the PRH protein and DNA is investigated using molecular dynamics and free energy simulations to identify DNA sequences that form stable complexes with PRH. Three 20-ns molecular dynamics simulations (PRH-TAATTG, PRH-TAATTA and PRH-TAATGG complexes) in explicit solvent water were performed to investigate three complexes structurally. Structural analysis shows that the native TAATTG sequence forms a complex that is more stable than complexes with base pair mutations. It is also observed that upon mutation, the number and occupancy of the direct and water-mediated hydrogen bonds decrease. Free energy calculations performed with the thermodynamic integration method predict relative binding free energies of 0.64 and 2 kcal/mol for GC to AT and TA to GC mutations, respectively, suggesting that among the three DNA sequences, the PRH-TAATTG complex is more stable than the two mutated complexes. In addition, it is demonstrated that the stability of the PRH-TAATTA complex is greater than that of the PRH-TAATGG complex.

Binding mode and thermodynamic studies on the interaction of the anticancer drug dacarbazine and dacarbazine-Cu(II) complex with single and double stranded DNA.

PubMed

Temerk, Yassien; Ibrahim, Hossieny

2014-07-01

The binding mode and thermodynamic characteristics of the anticancer drug dacarbazine (Dac) with double and single stranded DNA were investigated in the absence and presence of Cu(II) using cyclic voltammetry, square wave voltammetry and fluorescence spectroscopy. The interaction of Dac and Dac-Cu(II) complex with dsDNA indicated their intercalation into the base stacking domain of dsDNA double helix and the strength of interaction is independent on the ionic strength. The interaction of Dac with dsDNA in the presence of Cu(II) leads to a much stronger intercalation. The interaction mode of Dac molecules with ssDNA is electrostatic attraction via negative phosphate on the exterior of the ssDNA with Dac. The binding constants, stoichiometric coefficients and thermodynamic parameters of Dac and Dac-Cu(II) complex with dsDNA and ssDNA were evaluated. Comparison of the mode interaction of Dac with dsDNA and ssDNA was discussed. The decrease of peak current of Dac was proportional to DNA concentration, which was applied for determination of dsDNA and ssDNA concentration. Copyright © 2014 Elsevier B.V. All rights reserved.

Formation of Clustered DNA Damage after High-LET Irradiation: A Review

NASA Technical Reports Server (NTRS)

Hada, Megumi; Georgakilas, Alexandros G.

2008-01-01

Radiation can cause as well as cure cancer. The risk of developing radiation-induced cancer has traditionally been estimated from cancer incidence among survivors of the atomic bombs in Hiroshima and Nagasaki. These data provide the best estimate of human cancer risk over the dose range for low linear energy transfer (LET) radiations, such as X- or gamma-rays. The situation of estimating the real biological effects becomes even more difficult in the case of high LET particles encountered in space or as the result of domestic exposure to particles from radon gas emitters or other radioactive emitters like uranium-238. Complex DNA damage, i.e., the signature of high-LET radiations comprises by closely spaced DNA lesions forming a cluster of DNA damage. The two basic groups of complex DNA damage are double strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions (OCDL). Theoretical analysis and experimental evidence suggest there is increased complexity and severity of complex DNA damage with increasing LET (linear energy transfer) and a high mutagenic or carcinogenic potential. Data available on the formation of clustered DNA damage (DSBs and OCDL) by high-LET radiations are often controversial suggesting a variable response to dose and type of radiation. The chemical nature and cellular repair mechanisms of complex DNA damage have been much less characterized than those of isolated DNA lesions like an oxidized base or a single strand break especially in the case of high-LET radiation. This review will focus on the induction of clustered DNA damage by high-LET radiations presenting the earlier and recent relative data.

Study of intermolecular contacts in the proline-rich homeodomain (PRH)-DNA complex using molecular dynamics simulations.

PubMed

Jalili, Seifollah; Karami, Leila

2012-03-01

The proline-rich homeodomain (PRH)-DNA complex consists of a protein with 60 residues and a 13-base-pair DNA. The PRH protein is a transcription factor that plays a key role in the regulation of gene expression. PRH is a significant member of the Q50 class of homeodomain proteins. The homeodomain section of PRH is essential for binding to DNA and mediates sequence-specific DNA binding. Three 20-ns molecular dynamics (MD) simulations (free protein, free DNA and protein-DNA complex) in explicit solvent water were performed to elucidate the intermolecular contacts in the PRH-DNA complex and the role of dynamics of water molecules forming water-mediated contacts. The simulation provides a detailed explanation of the trajectory of hydration water molecules. The simulations show that some water molecules in the protein-DNA interface exchange with bulk waters. The simulation identifies that most of the contacts consisted of direct interactions between the protein and DNA including specific and non-specific contacts, but several water-mediated polar contacts were also observed. The specific interaction between Gln50 and C18 and water-mediated hydrogen bond between Gln50 and T7 were found to be present during almost the entire time of the simulation. These results show good consistency with experimental and previous computational studies. Structural properties such as root-mean-square deviations (RMSD), root-mean-square fluctuations (RMSF) and secondary structure were also analyzed as a function of time. Analyses of the trajectories showed that the dynamic fluctuations of both the protein and the DNA were lowered by the complex formation.

Combining H/D exchange mass spectroscopy and computational docking reveals extended DNA-binding surface on uracil-DNA glycosylase

PubMed Central

Roberts, Victoria A.; Pique, Michael E.; Hsu, Simon; Li, Sheng; Slupphaug, Geir; Rambo, Robert P.; Jamison, Jonathan W.; Liu, Tong; Lee, Jun H.; Tainer, John A.; Ten Eyck, Lynn F.; Woods, Virgil L.

2012-01-01

X-ray crystallography provides excellent structural data on protein–DNA interfaces, but crystallographic complexes typically contain only small fragments of large DNA molecules. We present a new approach that can use longer DNA substrates and reveal new protein–DNA interactions even in extensively studied systems. Our approach combines rigid-body computational docking with hydrogen/deuterium exchange mass spectrometry (DXMS). DXMS identifies solvent-exposed protein surfaces; docking is used to create a 3-dimensional model of the protein–DNA interaction. We investigated the enzyme uracil-DNA glycosylase (UNG), which detects and cleaves uracil from DNA. UNG was incubated with a 30 bp DNA fragment containing a single uracil, giving the complex with the abasic DNA product. Compared with free UNG, the UNG–DNA complex showed increased solvent protection at the UNG active site and at two regions outside the active site: residues 210–220 and 251–264. Computational docking also identified these two DNA-binding surfaces, but neither shows DNA contact in UNG–DNA crystallographic structures. Our results can be explained by separation of the two DNA strands on one side of the active site. These non-sequence-specific DNA-binding surfaces may aid local uracil search, contribute to binding the abasic DNA product and help present the DNA product to APE-1, the next enzyme on the DNA-repair pathway. PMID:22492624

Design of copper DNA intercalators with leishmanicidal activity.

PubMed

Navarro, Maribel; Cisneros-Fajardo, Efrén José; Sierralta, Aníbal; Fernández-Mestre, Mercedes; Silva, Pedro; Arrieche, Dwight; Marchán, Edgar

2003-04-01

The complexes [Cu(dppz)(NO(3))]NO(3) (1), [Cu(dppz)(2)(NO(3))]NO(3) (2), [Cu(dpq)(NO(3))]NO(3) (3), and [Cu(dpq)(2)(NO(3))]NO(3) (4) were synthesized and characterized by elemental analysis, FAB-mass spectrometry, EPR, UV, and IR spectroscopies, and molar conductivity. DNA interaction studies showed that intercalation is an important way of interacting with DNA for these complexes. The biological activity of these copper complexes was evaluated on Leishmania braziliensis promastigotes, and the results showed leishmanicidal activity. Preliminary ultrastructural studies with the most active complex (2) at 1 h revealed parasite swelling and binucleated cells. This finding suggests that the leishmanicidal activity of the copper complexes could be associated with their interaction with the parasitic DNA.

Minireview: DNA Replication in Plant Mitochondria

PubMed Central

Cupp, John D.; Nielsen, Brent L.

2014-01-01

Higher plant mitochondrial genomes exhibit much greater structural complexity as compared to most other organisms. Unlike well-characterized metazoan mitochondrial DNA (mtDNA) replication, an understanding of the mechanism(s) and proteins involved in plant mtDNA replication remains unclear. Several plant mtDNA replication proteins, including DNA polymerases, DNA primase/helicase, and accessory proteins have been identified. Mitochondrial dynamics, genome structure, and the complexity of dual-targeted and dual-function proteins that provide at least partial redundancy suggest that plants have a unique model for maintaining and replicating mtDNA when compared to the replication mechanism utilized by most metazoan organisms. PMID:24681310

Synthesis of novel Eu(III) luminescent probe based on 9- acridinecarboxylic acid skelton for sensing of ds-DNA.

PubMed

Azab, Hassan A; Hussein, Belal H M; El-Falouji, Abdullah I

2012-03-01

Eu(III)-9-acridinecarboxylate (9-ACA) complex was synthesized and characterized by elemental analysis, conductivity measurement, IR spectroscopy, thermal analysis, mass spectroscopy, (1)H-NMR, fluorescence and ultraviolet spectra. The results indicated that the composition of this complex is [Eu(III)-(9-ACA)(2)(NCS)(C(2)H(5)OH)(2)] 2.5 H(2)O and the oxygen of the carbonyl group coordinated to Eu(III). The interaction between the complex with nucleotides guanosine 5'- monophosphate (5'-GMP), adenosine 5'-diphosphates (5'-ADP), inosine (5'-IMP) and CT-DNA was studied by fluorescence spectroscopy. The fluorescence intensity of Eu(III)-9-acridinecarboxylate complex was enhanced with the addition of CT-DNA. The effect of pH values on the fluorescence intensity of Eu(III) complex was investigated. Under experimental conditions, the linear range was 9-50 ng mL(-1) for calf thymus DNA (CT- DNA) and the corresponding detection limit was 5 ng mL(-1). The results showed that Eu(III)-(9-ACA)(2) complex binds to CT-DNA with stability constant of 2.41 × 10(4) M.

Comparative study of DNA encapsulation into PLGA microparticles using modified double emulsion methods and spray drying techniques.

PubMed

Oster, C G; Kissel, T

2005-05-01

Recently, several research groups have shown the potential of microencapsulated DNA as adjuvant for DNA immunization and in tissue engineering approaches. Among techniques generally used for microencapsulation of hydrophilic drug substances into hydrophobic polymers, modified WOW double emulsion method and spray drying of water-in-oil dispersions take a prominent position. The key parameters for optimized microspheres are particle size, encapsulation efficiency, continuous DNA release and stabilization of DNA against enzymatic and mechanical degradation. This study investigates the possibility to encapsulate DNA avoiding shear forces which readily degrade DNA during this microencapsulation. DNA microparticles were prepared with polyethylenimine (PEI) as a complexation agent for DNA. Polycations are capable of stabilizing DNA against enzymatic, as well as mechanical degradation. Further, complexation was hypothesized to facilitate the encapsulation by reducing the size of the macromolecule. This study additionally evaluated the possibility of encapsulating lyophilized DNA and lyophilized DNA/PEI complexes. For this purpose, the spray drying and double emulsion techniques were compared. The size of the microparticles was characterized by laser diffractometry and the particles were visualized by scanning electron microscopy (SEM). DNA encapsulation efficiencies were investigated photometrically after complete hydrolysis of the particles. Finally, the DNA release characteristics from the particles were studied. Particles with a size of <10 microm which represent the threshold for phagocytic uptake could be prepared with these techniques. The encapsulation efficiency ranged from 100-35% for low theoretical DNA loadings. DNA complexation with PEI 25?kDa prior to the encapsulation process reduced the initial burst release of DNA for all techniques used. Spray-dried particles without PEI exhibited high burst releases, whereas double emulsion techniques showed continuous release rates.

Sequence-dependent DNA flexibility mediates DNase I cleavage.

PubMed

Heddi, Brahim; Abi-Ghanem, Josephine; Lavigne, Marc; Hartmann, Brigitte

2010-01-08

Understanding the preference of nonspecific proteins for certain DNA structural features requires an accurate description of the properties of free DNA, especially regarding their possible predisposition to adopt a conformation that favors the formation of a complex. Exploiting previous exhaustive NMR studies performed on free DNA oligomers, we investigated the molecular basis of DNase I sensitivity under conditions where DNase I binding limits the probability of cleavage. We showed that cleavage intensity was correlated with adjacent 3' phosphate linkage flexibility, monitored by (31)P chemical shifts. Examining NMR-refined DNA structures highlighted that sequence-dependent flexible phosphates were associated with large minor groove variations that may promote the affinity of DNase I, according to relevant DNA-protein complexes. In sum, this work demonstrates that specificity in DNA-DNase I interaction is mediated by DNA flexibility, which influences the induced-fit transitions required to form productive complexes.

Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination*

PubMed Central

Arai, Naoto; Kagawa, Wataru; Saito, Kengo; Shingu, Yoshinori; Mikawa, Tsutomu; Kurumizaka, Hitoshi; Shibata, Takehiko

2011-01-01

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a “recombination mediator” to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing. PMID:21454474

Drug-DNA interactions at single molecule level: A view with optical tweezers

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan

Studies of small molecule--DNA interactions are essential for developing new drugs for challenging diseases like cancer and HIV. The main idea behind developing these molecules is to target and inhibit the reproduction of the tumor cells and infected cells. We mechanically manipulate single DNA molecule using optical tweezers to investigate two molecules that have complex and multiple binding modes. Mononuclear ruthenium complexes have been extensively studied as a test for rational drug design. Potential drug candidates should have high affinity to DNA and slow dissociation kinetics. To achieve this, motifs of the ruthenium complexes are altered. Our collaborators designed a dumb-bell shaped binuclear ruthenium complex that can only intercalate DNA by threading through its bases. Studying the binding properties of this complex in bulk studies took hours. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to thread and make it fast compared to the bulk experiments. Stretching single DNA molecules with different concentration of drug molecules and holding it at a constant force allows the binding to reach equilibrium. By this we can obtain the equilibrium fractional ligand binding and length of DNA at saturated binding. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics of this complex process. The second complex discussed in this study is Actinomycin D (ActD), a well studied anti-cancer agent that is used as a prototype for developing new generations of drugs. However, the biophysical basis of its activity is still unclear. Because ActD is known to intercalate double stranded DNA (dsDNA), it was assumed to block replication by stabilizing dsDNA in front of the replication fork. However, recent studies have shown that ActD binds with even higher affinity to imperfect duplexes and some sequences of single stranded DNA (ssDNA). We directly measure the on and off rates by stretching the DNA molecule to a certain force and holding it at constant force while adding the drug and then while washing off the drug. Our finding resolves the long lasting controversy of ActD binding modes, clearly showing that both the dsDNA binding and ssDNA binding converge to the same single mode. The result supports the hypothesis that the primary characteristic of ActD that contributes to its biological activity is its ability to inhibit cellular replication by binding to transcription bubbles and causing cell death.

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

PubMed

Upadhya, Archana; Sangave, Preeti C

2016-10-01

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R 8 ) and CHK 6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH 3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

PARACEST Properties of a Dinuclear Neodymium(III) Complex Bound to DNA or Carbonate

PubMed Central

Nwe, Kido; Andolina, Christopher M.; Huang, Ching-Hui; Morrow, Janet R.

2009-01-01

A dinuclear Nd(III) macrocyclic complex of 1 (1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-p-xylene) and mononuclear complexes of 1,4,7-tris-1,4,7,10-tetraazacyclododecane 2, and 1,4,7-tris[(N-N-diethyl)carbamoylmethyl]-1,4,7,10-tetraazacyclododecane, 3, are prepared. Complexes of 1 and 2 give rise to a PARACEST (paramagnetic chemical exchange saturation transfer) peak from exchangeable amide protons that resonate approximately 12 ppm downfield from the bulk water proton resonance. The dinuclear Nd(III) complex is promising as a PARACEST contrast agent for MRI applications because it has an optimal pH of 7.5 and the rate constant for amide proton exchange (2700 s−1) is nearly as large as it can be within slow exchange conditions with bulk water. Dinuclear Ln2(1) complexes (Ln(III) = Nd(III), Eu(III)) bind tightly to anionic ligands including carbonate, diethylphosphate and DNA. The CEST amide peak of Nd2(1) is enhanced by certain DNA sequences that contain hairpin loops, but decreases in the presence of diethyl phosphate or carbonate. Direct excitation luminescence studies of Eu2(1) show that double-stranded and hairpin loop DNA sequences displace one water ligand on each Eu(III) center. DNA displaces carbonate ion despite the low dissociation constant for the Eu2(1) carbonate complex (Kd = 15 µM). Enhancement of the CEST effect of a lanthanide complex by binding to DNA is a promising step toward the preparation of PARACEST agents containing DNA scaffolds. PMID:19555071

PARACEST properties of a dinuclear neodymium(III) complex bound to DNA or carbonate.

PubMed

Nwe, Kido; Andolina, Christopher M; Huang, Ching-Hui; Morrow, Janet R

2009-07-01

A dinuclear Nd(III) macrocyclic complex of 1 (1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-p-xylene) and mononuclear complexes of 1,4,7-tris-1,4,7,10-tetraazacyclododecane, 2, and 1,4,7-tris[(N-N-diethyl)carbamoylmethyl]-1,4,7,10-tetraazacyclododecane, 3, are prepared. Complexes of 1 and 2 give rise to a PARACEST (paramagnetic chemical exchange saturation transfer) peak from exchangeable amide protons that resonate approximately 12 ppm downfield from the bulk water proton resonance. The dinuclear Nd(III) complex is promising as a PARACEST contrast agent for MRI applications, because it has an optimal pH of 7.5 and the rate constant for amide proton exchange (2700 s(-1)) is nearly as large as it can be within slow exchange conditions with bulk water. Dinuclear Ln(2)(1) complexes (Ln(III) = Nd(III), Eu(III)) bind tightly to anionic ligands including carbonate, diethyl phosphate, and DNA. The CEST amide peak of Nd(2)(1) is enhanced by certain DNA sequences that contain hairpin loops, but decreases in the presence of diethyl phosphate or carbonate. Direct excitation luminescence studies of Eu(2)(1) show that double-stranded and hairpin-loop DNA sequences displace one water ligand on each Eu(III) center. DNA displaces carbonate ion despite the low dissociation constant for the Eu(2)(1) carbonate complex (K(d) = 15 microM). Enhancement of the CEST effect of a lanthanide complex by binding to DNA is a promising step toward the preparation of PARACEST agents containing DNA scaffolds.

Genetics Home Reference: mitochondrial complex III deficiency

MedlinePlus

... DNA packaged in chromosomes within the cell nucleus (nuclear DNA). It is not clear why the severity ... deficiency Genetic Testing Registry: Mitochondrial complex III deficiency, nuclear type 2 Genetic Testing Registry: Mitochondrial complex III ...

Heteroleptic Copper(I) Complexes of "Scorpionate" Bis-pyrazolyl Carboxylate Ligand with Auxiliary Phosphine as Potential Anticancer Agents: An Insight into Cytotoxic Mode.

PubMed

Khan, Rais Ahmad; Usman, Mohammad; Dhivya, Rajakumar; Balaji, Perumalsamy; Alsalme, Ali; AlLohedan, Hamad; Arjmand, Farukh; AlFarhan, Khalid; Akbarsha, Mohammad Abdulkader; Marchetti, Fabio; Pettinari, Claudio; Tabassum, Sartaj

2017-03-24

New copper(I) complexes [CuCl(PPh 3 )(L)] (1: L = L A  = 4-carboxyphenyl)bis(3,5-dimethylpyrazolyl)methane; (2: L = L B  = 3-carboxyphenyl)bis(3,5-dimethylpyrazolyl)methane) were prepared and characterised by elemental analysis and various spectroscopic techniques such as FT-IR, NMR, UV-Vis, and ESI-MS. The molecular structures of complexes 1 and 2 were analyzed by theoretical B3LYP/DFT method. Furthermore, in vitro DNA binding studies were carried out to check the ability of complexes 1 and 2 to interact with native calf thymus DNA (CT-DNA) using absorption titration, fluorescence quenching and circular dichroism, which is indicative of more avid binding of the complex 1. Moreover, DNA mobility assay was also conducted to study the concentration-dependent cleavage pattern of pBR322 DNA by complex 1, and the role of ROS species to have a mechanistic insight on the cleavage pattern, which ascertained substantial roles by both hydrolytic and oxidative pathways. Additionally, we analyzed the potential of the interaction of complex 1 with DNA and enzyme (Topo I and II) with the aid of molecular modeling. Furthermore, cytotoxic activity of complex 1 was tested against HepG2 cancer cell lines. Thus, the potential of the complex 1 is promising though further in vivo investigations may be required before subjecting it to clinical trials.

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

PubMed

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.

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.

PubMed

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

Substitutional impact on biological activity of new water soluble Ni(II) complexes: Preparation, spectral characterization, X-ray crystallography, DNA/protein binding, antibacterial activity and in vitro cytotoxicity.

PubMed

Umadevi, C; Kalaivani, P; Puschmann, H; Murugan, S; Mohan, P S; Prabhakaran, R

2017-02-01

A series of new water soluble nickel(II) complexes containing triphenylphosphine and 4-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones were synthesized and characterized. Crystallographic investigations confirmed the structure of the complexes (1-4) having the general structure [Ni(4-Msal-Rtsc)(PPh 3 )] (Where R=H (1); CH 3 (2); C 2 H 5 (3); C 6 H 5 (4)) which showed that thiosemicarbazone ligands coordinated to nickel(II) ion as ONS tridentate bibasic donor. DNA/BSA protein binding ability of the ligands and their new complexes were studied by taking calf-thymus DNA (CT-DNA) and Bovine serum albumin (BSA) through absorption and emission titrations. Ethidium bromide (EB) displacement study showed the intercalative binding trend of the complexes to DNA. From the albumin binding studies, the mechanism of quenching was found as static and the alterations in the secondary structure of BSA by the compounds were confirmed with synchronous spectral studies. The binding affinity of the complexes to CT-DNA and BSA has the order of [Ni(4-Msal-etsc)(PPh 3 )] (3) >[Ni(4-Msal-mtsc)(PPh 3 )] (2) >[Ni(4-Msal-tsc)(PPh 3 )] (1) >[Ni(4-Msal-ptsc)(PPh 3 )] (4). In vitro cytotoxicity of the complexes was tested on human lung cancer cells (A549), human cervical cancer cells (HeLa), human liver carcinoma cells (Hep G2). All the complexes exhibited significant activity against three cancer cells. Among them, complex 4 exhibited almost 2.5 fold activity than cisplatin in A549 and HepG2 cell lines. In HeLa cell line, the complexes exhibited significant activity which is less than cisplatin. While comparing the activity of the complexes in A549 and HepG2 cell lines it falls in the order 4>1>2>3>cisplatin. The results obtained from DNA, protein binding and cytotoxicity studies, it is concluded that the cytotoxicity of the complexes as determined by MTT assay were not unduly influenced by the complexes having different binding efficiency with DNA and protein. The complexes exhibited good spectrum of antibacterial activity against four pathogenic bacteria such as E. faecalis (gram +ve), S. aureus (gram +ve), E. coli (gram -ve) and P. aeruginosa (gram -ve). Copyright © 2016 Elsevier B.V. All rights reserved.

Studies of interaction of emodin and DNA in the presence of ethidium bromide by spectroscopic method

NASA Astrophysics Data System (ADS)

Bi, Shuyun; Zhang, Hanqi; Qiao, Chunyu; Sun, Ying; Liu, Chunming

2008-01-01

Emodin interacting with deoxyribonucleic acid (DNA) has been studied by different spectroscopic techniques, such as fluorescence, ultraviolet and visible (UV-vis), and fourier transform infared (FT-IR) spectroscopies, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA-EB system on addition of emodin shows that the fluorescence quenching of DNA-EB complex by emodin occurs. The binding constants of emodin with DNA in the presence of EB are 6.02 × 10 4, 9.20 × 10 4 and 1.17 × 10 5 L mol -1 at 20, 35 and 50 °C, respectively. FT-IR spectrum further suggests that both the phosphate groups and the bases of DNA react with emodin. The reaction of DNA with emodin in the presence of EB is affected by ionic strength and temperature. The values of melting temperature ( Tm) of DNA-EB complex and emodin-DNA-EB complexes were determined, respectively. From the experiment evidences, the major binding mode of emodin with DNA should be the groove binding.

Influence of the DNA structure on the free radical induction due to proflavine and light treatment.

PubMed

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

1979-04-30

Induction of peroxide free radicals (detected by Electron Paramagnetic Resonance at 77 K) due to the photodynamic activity of proflavine was measured on bacteriophage phi X174 DNA either single-stranded (ss) as isolated from the virion, or double-stranded supercoiled (RFI) as isolated from the infected bacteria. Comparison was made with calf thymus DNA photosensitization. In order to use equivalent DNA-proflavine complexes, binding of the dye to the three DNA's was first determined under those conditions of high ionic strength favourable to the photodynamic reaction. Free radical induction was maximal for definite amounts of bound proflavine (which varied depending upon the DNA substrate) and at an ionic strength value of 0.5. The level of the maximal reaction increased in the following order: from phi Xss DNA to calf thymus DNA and finally to phi XRFI DNA. The conformation of the proflavine-DNA complex was thus a determinant for the efficiency of the photodynamic process. The ionic strength effect could not be explained by the evolution of the proflavine triplet state in irradiated proflavine-calf thymus DNA complexes.

On the binding of indeno[1,2-c]isoquinolines in the DNA-topoisomerase I cleavage complex.

PubMed

Xiao, Xiangshu; Antony, Smitha; Pommier, Yves; Cushman, Mark

2005-05-05

An ab initio quantum mechanics calculation is reported which predicts the orientation of indenoisoquinoline 4 in the ternary cleavage complex formed from DNA and topoisomerase I (top1). The results of this calculation are consistent with the hypothetical structures previously proposed for the indenoisoquinoline-DNA-top1 ternary complexes based on molecular modeling, the crystal structure of a recently reported ternary complex, and the biological results obtained with a pair of diaminoalkyl-substituted indenoisoquinoline enantiomers. The results of these studies indicate that the pi-pi stacking interactions between the indenoisoquinolines and the neighboring DNA base pairs play a major role in determining binding orientation. The calculation of the electrostatic potential surface maps of the indenoisoquinolines and the adjacent DNA base pairs shows electrostatic complementarity in the observed binding orientation, leading to the conclusion that electrostatic attraction between the intercalators and the base pairs in the cleavage complex plays a major stabilizing role. On the other hand, the calculation of LUMO and HOMO energies of indenoisoquinoline 13b and neighboring DNA base pairs in conjunction with NBO analysis indicates that charge transfer complex formation plays a relatively minor role in stabilizing the ternary complexes derived from indenoisoquinolines, DNA, and top1. The results of these studies are important in understanding the existing structure-activity relationships for the indenoisoquinolines as top1 inhibitors and as anticancer agents, and they will be important in the future design of indenoisoquinoline-based top1 inhibitors.

Peptides containing antigenic and cationic domains have enhanced, multivalent immunogenicity when bound to DNA vaccines.

PubMed

Riedl, Petra; Reimann, Jörg; Schirmbeck, Reinhold

2004-02-01

We explored strategies to codeliver DNA- and peptide-based vaccines in a way that enhances the immunogenicity of both components of the combination vaccine for T cells. Specific CD8(+) T cell responses to an antigenic peptide are primed when the peptide is fused to a cationic peptide domain that is bound to plasmid DNA or oligonucleotides (ODN; with or without CpG motifs). Plasmid DNA mixed with antigenic/cationic peptides or histones forms large complexes with different biological properties depending on the molar ratios of peptide/protein and polynucleotide. Complexes containing high (but not low) molar ratios of cationic peptide to DNA facilitate transfection (DNA uptake and expression of the plasmid-encoded product) of cells. In contrast, complexes containing low (but not high) molar ratios of cationic peptide to DNA prime potent multispecific T cell responses after a single intramuscular injection of the complexes. The general validity of this observation was confirmed mixing different antigenic/cationic peptides with different DNA vaccines. In these vaccine formulations, multispecific CD8(+) T cell responses specific for epitopes of the peptide- as well as the DNA-based vaccine were efficiently coprimed, together with humoral antibody responses to conformational determinants of large viral antigens encoded by the DNA vaccine. The data indicate that mixtures of DNA vaccines with antigenic, cationic peptides are immunogenic vaccine formulations particularly suited for the induction of multispecific T cell responses.

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

PubMed Central

Kedinger, C; Brison, O; Perrin, F; Wilhelm, J

1978-01-01

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure. Images PMID:207893

Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei.

PubMed

Kedinger, C; Brison, O; Perrin, F; Wilhelm, J

1978-05-01

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 1 (Ad2)-infected HeLa cell nuclei were shown by electron microscopy to contain filaments much thicker (about 200 A [20 nm]) than double-stranded DNA (about 20 A [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated, and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000-dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes have been visualized by electron microscopy. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of one thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Altogether, our results establish the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. In addition, our results indicate that, late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.

TFIIA changes the conformation of the DNA in TBP/TATA complexes and increases their kinetic stability.

PubMed

Hieb, Aaron R; Halsey, Wayne A; Betterton, Meredith D; Perkins, Thomas T; Kugel, Jennifer F; Goodrich, James A

2007-09-21

Eukaryotic mRNA transcription by RNA polymerase II is a highly regulated complex reaction involving numerous proteins. In order to control tissue and promoter specific gene expression, transcription factors must work in concert with each other and with the promoter DNA to form the proper architecture to activate the gene of interest. The TATA binding protein (TBP) binds to TATA boxes in core promoters and bends the TATA DNA. We have used quantitative solution fluorescence resonance energy transfer (FRET) and gel-based FRET (gelFRET) to determine the effect of TFIIA on the conformation of the DNA in TBP/TATA complexes and on the kinetic stability of these complexes. Our results indicate that human TFIIA decreases the angle to which human TBP bends consensus TATA DNA from 104 degrees to 80 degrees when calculated using a two-kink model. The kinetic stability of TBP/TATA complexes was greatly reduced by increasing the KCl concentration from 50 mM to 140 mM, which is more physiologically relevant. TFIIA significantly enhanced the kinetic stability of TBP/TATA complexes, thereby attenuating the effect of higher salt concentrations. We also found that TBP bent non-consensus TATA DNA to a lesser degree than consensus TATA DNA and complexes between TBP and a non-consensus TATA box were kinetically unstable even at 50 mM KCl. Interestingly, TFIIA increased the calculated bend angle and kinetic stability of complexes on a non-consensus TATA box, making them similar to those on a consensus TATA box. Our data show that TFIIA induces a conformational change within the TBP/TATA complex that enhances its stability under both in vitro and physiological salt conditions. Furthermore, we present a refined model for the effect that TFIIA has on DNA conformation that takes into account potential changes in bend angle as well as twist angle.

DNA purification by triplex-affinity capture and affinity capture electrophoresis

DOEpatents

Cantor, Charles R.; Ito, Takashi; Smith, Cassandra L.

1996-01-01

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel.

Super-resolution imaging identifies PARP1 and the Ku complex acting as DNA double-strand break sensors

PubMed Central

Yang, Guang; Liu, Chao; Chen, Shih-Hsun; Kassab, Muzaffer A; Hoff, J Damon; Yu, Xiaochun

2018-01-01

Abstract DNA double-strand breaks (DSBs) are fatal DNA lesions and activate a rapid DNA damage response. However, the earliest stage of DSB sensing remains elusive. Here, we report that PARP1 and the Ku70/80 complex localize to DNA lesions considerably earlier than other DSB sensors. Using super-resolved fluorescent particle tracking, we further examine the relocation kinetics of PARP1 and the Ku70/80 complex to a single DSB, and find that PARP1 and the Ku70/80 complex are recruited to the DSB almost at the same time. Notably, only the Ku70/80 complex occupies the DSB exclusively in the G1 phase; whereas PARP1 competes with the Ku70/80 complex at the DSB in the S/G2 phase. Moreover, in the S/G2 phase, PARP1 removes the Ku70/80 complex through its enzymatic activity, which is further confirmed by in vitro DSB-binding assays. Taken together, our results reveal PARP1 and the Ku70/80 complex as critical DSB sensors, and suggest that PARP1 may function as an important regulator of the Ku70/80 complex at the DSBs in the S/G2 phase. PMID:29447383

Polymer-induced DNA Condensation in the Lamellar Phase of DNA-Lipid Complexes

NASA Astrophysics Data System (ADS)

Martin, Ana; Lin, Alison J.; Schulze, Uwe; Safinya, Cyrus R.; Schmidt, Hans-Werner

2000-03-01

The lamellar phase of cationic lipid-DNA complexes (CL-DNA)[1,2] is a model system for the study of a polymer induced condensation in two dimensions. Measurements of X-ray diffraction show DNA condensation with the addition of cationic poly(ethylene glycol) PEG-lipid to the membrane of the CL-DNA complexes, revealing the existence of two different behaviors as a function of the PEG length. For shorter PEG the DNA condensation can be described by considering the charge increase on the membrane due to the incorporation of the cationic polymeric chains. For longer PEG a deviation from the predicted electrostatic distance between DNA chains is observed. This higher condensation is caused by a novel depletion-attraction interaction between DNA chains in two dimensions. This work is supported by NSF-DMR9972246 and a fellowship of the Education Ministry of Spain. [1] Rädler, JO; Koltover, I; Salditt, T; Safinya, CR., Science 275, 810 (1997). [2] Koltover, I; Salditt, T; Safinya, CR., Biophys. J. 77, 915 (1999).

Binding regularities in complexes of transcription factors with operator DNA: homeodomain family.

PubMed

Chirgadze, Yu N; Zheltukhin, E I; Polozov, R V; Sivozhelezov, V S; Ivanov, V V

2009-06-01

In order to disclose general regularities of binding in homeodomain-DNA complexes we considered five of them and extended the observed regularities over the entire homeodomain family. The five complexes have been selected by similarity of protein structures and patterns of contacting residues. Their long range interactions and interfaces were compared. The long-range stage of the recognition process was characterized by electrostatic potentials about 5 Angstrom away from molecular surfaces of protein or DNA. For proteins, clear positive potential is displayed only at the side contacting the DNA. The double-chained DNA molecule displays a rather strong negative potential, especially in their grooves. Thus, a functional role of electrostatics is a guiding of the protein into the DNA major groove, so the protein and DNA could form a loose non-specific complex. At the close-range stage, neutralization of the phosphate charges by positively charged residues is necessary for decreasing the strong electrostatic potential of DNA, allowing nucleotide bases to participate in the formation of protein-DNA atomic contacts in the interface. The recognizing alpha-helix of protein was shown to form both invariant and variable groups of contacts with DNA by means of certain specific side groups. The invariant contacts included highly specific protein-DNA hydrogen bonds between asparagine and adenine, nonpolar contacts of hydrophobic amino acids serving as a stereochemical barrier for fixing the protein factor on DNA, and an interface cluster of water molecules providing local conformational mobility necessary for the dissociation process. There is a unique water molecule within the interface that is conservative and located at the interface center. Invariant contacts of the proteins are mostly formed with the TAAT motif of the promoter DNA forward strand. While the invariant contacts specify the family of homeodomains, the variable contacts that are formed with the reverse strand of DNA provide specificity of individual complexes within the homeodomain family.

Lightweight, Room-Temperature CO2 Gas Sensor Based on Rare-Earth Metal-Free Composites—An Impedance Study

PubMed Central

2017-01-01

We report a light, flexible, and low-power poly(ionic liquid)/alumina composite CO2 sensor. We monitor the direct-current resistance changes as a function of CO2 concentration and relative humidity and demonstrate fast and reversible sensing kinetics. Moreover, on the basis of the alternating-current impedance measurements we propose a sensing mechanism related to proton conduction and gas diffusion. The findings presented herein will promote the development of organic/inorganic composite CO2 gas sensors. In the future, such sensors will be useful for numerous practical applications ranging from indoor air quality control to the monitoring of manufacturing processes. PMID:28726384

Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

PubMed Central

Ho, Dominik; Dose, Christian; Albrecht, Christian H.; Severin, Philip; Falter, Katja; Dervan, Peter B.; Gaub, Hermann E.

2009-01-01

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA·ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA·ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA·ligand complexes. PMID:19486688

Dissolving microneedles for DNA vaccination: Improving functionality via polymer characterization and RALA complexation

PubMed Central

Cole, Grace; McCaffrey, Joanne; Ali, Ahlam A.; McBride, John W.; McCrudden, Cian M.; Vincente-Perez, Eva M.; Donnelly, Ryan F.; McCarthy, Helen O.

2017-01-01

ABSTRACT DNA vaccination holds the potential to treat or prevent nearly any immunogenic disease, including cancer. To date, these vaccines have demonstrated limited immunogenicity in vivo due to the absence of a suitable delivery system which can protect DNA from degradation and improve transfection efficiencies in vivo. Recently, microneedles have been described as a novel physical delivery technology to enhance DNA vaccine immunogenicity. Of these devices, dissolvable microneedles promise a safe, pain-free delivery system which may simultaneously improve DNA stability within a solid matrix and increase DNA delivery compared to solid arrays. However, to date little work has directly compared the suitability of different dissolvable matrices for formulation of DNA-loaded microneedles. Therefore, the current study examined the ability of 4 polymers to formulate mechanically robust, functional DNA loaded dissolvable microneedles. Additionally, complexation of DNA to a cationic delivery peptide, RALA, prior to incorporation into the dissolvable matrix was explored as a means to improve transfection efficacies following release from the polymer matrix. Our data demonstrates that DNA is degraded following incorporation into PVP, but not PVA matrices. The complexation of DNA to RALA prior to incorporation into polymers resulted in higher recovery from dissolvable matrices, and increased transfection efficiencies in vitro. Additionally, RALA/DNA nanoparticles released from dissolvable PVA matrices demonstrated up to 10-fold higher transfection efficiencies than the corresponding complexes released from PVP matrices, indicating that PVA is a superior polymer for this microneedle application. PMID:27846370

Pathological mechanisms underlying single large‐scale mitochondrial DNA deletions

PubMed Central

Rocha, Mariana C.; Rosa, Hannah S.; Grady, John P.; Blakely, Emma L.; He, Langping; Romain, Nadine; Haller, Ronald G.; Newman, Jane; McFarland, Robert; Ng, Yi Shiau; Gorman, Grainne S.; Schaefer, Andrew M.; Tuppen, Helen A.; Taylor, Robert W.

2018-01-01

Objective Single, large‐scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large‐scale mtDNA deletions in skeletal muscle. Methods We investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large‐scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction. Results We have defined 3 “classes” of single, large‐scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class. Interpretation Combining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex‐specific protein‐encoding genes. Furthermore, removal of mt‐tRNA genes impacts specific complexes only at high deletion levels, when complex‐specific protein‐encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115–130 PMID:29283441

Recognition of platinum-DNA adducts by HMGB1a.

PubMed

Ramachandran, Srinivas; Temple, Brenda; Alexandrova, Anastassia N; Chaney, Stephen G; Dokholyan, Nikolay V

2012-09-25

Cisplatin (CP) and oxaliplatin (OX), platinum-based drugs used widely in chemotherapy, form adducts on intrastrand guanines (5'GG) in genomic DNA. DNA damage recognition proteins, transcription factors, mismatch repair proteins, and DNA polymerases discriminate between CP- and OX-GG DNA adducts, which could partly account for differences in the efficacy, toxicity, and mutagenicity of CP and OX. In addition, differential recognition of CP- and OX-GG adducts is highly dependent on the sequence context of the Pt-GG adduct. In particular, DNA binding protein domain HMGB1a binds to CP-GG DNA adducts with up to 53-fold greater affinity than to OX-GG adducts in the TGGA sequence context but shows much smaller differences in binding in the AGGC or TGGT sequence contexts. Here, simulations of the HMGB1a-Pt-DNA complex in the three sequence contexts revealed a higher number of interface contacts for the CP-DNA complex in the TGGA sequence context than in the OX-DNA complex. However, the number of interface contacts was similar in the TGGT and AGGC sequence contexts. The higher number of interface contacts in the CP-TGGA sequence context corresponded to a larger roll of the Pt-GG base pair step. Furthermore, geometric analysis of stacking of phenylalanine 37 in HMGB1a (Phe37) with the platinated guanines revealed more favorable stacking modes correlated with a larger roll of the Pt-GG base pair step in the TGGA sequence context. These data are consistent with our previous molecular dynamics simulations showing that the CP-TGGA complex was able to sample larger roll angles than the OX-TGGA complex or either CP- or OX-DNA complexes in the AGGC or TGGT sequences. We infer that the high binding affinity of HMGB1a for CP-TGGA is due to the greater flexibility of CP-TGGA compared to OX-TGGA and other Pt-DNA adducts. This increased flexibility is reflected in the ability of CP-TGGA to sample larger roll angles, which allows for a higher number of interface contacts between the Pt-DNA adduct and HMGB1a.

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

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

Pröpper, Kevin; Instituto de Biologia Molecular de Barcelona; Meindl, Kathrin

2014-06-01

The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite themore » fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.« less

Fluorescence correlation spectroscopy study of the complexation of DNA hybrids, IgG antibody, and a chimeric protein of IgG-binding ZZ domains fused with a carbohydrate binding module.

PubMed

Rosa, A M M; Prazeres, D M F; Paulo, P M R

2017-06-28

Fluorescence correlation spectroscopy (FCS) was used to characterize the molecular interactions between the four components of a DNA recognition system. A fluorescent DNA probe was used to assess: (i) the hybridization with a complementary biotin-labeled target, (ii) the complexation of the resulting hybrid and an anti-biotin antibody, and (iii) the binding of the latter complex to a ZZ-CBM fusion protein that combines small synthetic IgG Fc-binding Z domains with a carbohydrate binding module (CBM). These binding interactions were monitored by exposing the fluorescent DNA probe to different amounts and combinations of the other molecules in solution. Through the analysis of FCS autocorrelation curves, an association constant (K a ) of 2.9 × 10 7 M -1 was estimated for DNA·DNA hybridization, and the presence of (non-) complementary target DNA in solution could be discriminated. The specific capture of biotinylated DNA hybrids by anti-biotin IgG was verified, with an apparent K a of 2.5 × 10 6 M -1 . The increment in the diffusion time measured when the DNA·DNA:antibody complexes were in contact with the ZZ-CBM fusion protein suggested that the binding occurs at a stoichiometric ratio of DNA/antibody complex to fusion larger than 1 : 1. The FCS-derived information obtained is useful to gain insight into molecular interactions involved in diagnostic assays.

Accelerated Photobleaching of a Cyanine Dye in the Presence of a Ternary Target DNA, PNA Probe, Dye Catalytic Complex: A Molecular Diagnostic

PubMed Central

Wang, M.; Holmes-Davis, R.; Rafinski, Z.; Jedrzejewska, B.; Choi, K. Y.; Zwick, M.; Bupp, C.; Izmailov, A.; Paczkowski, J.; Warner, B.; Koshinsky, H.

2009-01-01

In many settings, molecular testing is needed but unavailable due to complexity and cost. Simple, rapid, and specific DNA detection technologies would provide important alternatives to existing detection methods. Here we report a novel, rapid nucleic acid detection method based on the accelerated photobleaching of the light-sensitive cyanine dye, 3,3′-diethylthiacarbocyanine iodide (DiSC2(3) I−), in the presence of a target genomic DNA and a complementary peptide nucleic acid (PNA) probe. On the basis of the UV–vis, circular dichroism, and fluorescence spectra of DiSC2(3) with PNA–DNA oligomer duplexes and on characterization of a product of photolysis of DiSC2(3) I−, a possible reaction mechanism is proposed. We propose that (1) a novel complex forms between dye, PNA, and DNA, (2) this complex functions as a photosensitizer producing 1O2, and (3) the 1O2 produced promotes photobleaching of dye molecules in the mixture. Similar cyanine dyes (DiSC3(3), DiSC4(3), DiSC5(3), and DiSCpy(3)) interact with preformed PNA–DNA oligomer duplexes but do not demonstrate an equivalent accelerated photobleaching effect in the presence of PNA and target genomic DNA. The feasibility of developing molecular diagnostic assays based on the accelerated photobleaching (the smartDNA assay) that results from the novel complex formed between DiSC2(3) and PNA–DNA is under way. PMID:19231844

EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele.

PubMed

Rein, Katrin; Yanez, Diana A; Terré, Berta; Palenzuela, Lluís; Aivio, Suvi; Wei, Kaichun; Edelmann, Winfried; Stark, Jeremy M; Stracker, Travis H

2015-09-03

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5'-3' exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Evaluation of DNA, BSA binding, and antimicrobial activity of new synthesized neodymium complex containing 29-dimethyl 110-phenanthroline.

PubMed

Moradi, Zohreh; Khorasani-Motlagh, Mozhgan; Rezvani, Ali Reza; Noroozifar, Meissam

2018-02-01

In order to evaluate biological potential of a novel synthesized complex [Nd(dmp) 2 Cl 3 .OH 2 ] where dmp is 29-dimethyl 110-phenanthroline, the DNA-binding, cleavage, BSA binding, and antimicrobial activity properties of the complex are investigated by multispectroscopic techniques study in physiological buffer (pH 7.2).The intrinsic binding constant (K b ) for interaction of Nd(III) complex and FS-DNA is calculated by UV-Vis (K b  = 2.7 ± 0.07 × 10 5 ) and fluorescence spectroscopy (K b  = 1.13 ± 0.03 × 10 5 ). The Stern-Volmer constant (K SV ), thermodynamic parameters including free energy change (ΔG°), enthalpy change (∆H°), and entropy change (∆S°), are calculated by fluorescent data and Vant' Hoff equation. The experimental results show that the complex can bind to FS-DNA and the major binding mode is groove binding. Meanwhile, the interaction of Nd(III) complex with protein, bovine serum albumin (BSA), has also been studied by using absorption and emission spectroscopic tools. The experimental results show that the complex exhibits good binding propensity to BSA. The positive ΔH° and ∆S° values indicate that the hydrophobic interaction is main force in the binding of the Nd(III) complex to BSA, and the complex can quench the intrinsic fluorescence of BSA remarkably through a static quenching process. Also, DNA cleavage was investigated by agarose gel electrophoresis that according to the results cleavage of DNA increased with increasing of concentration of the complex. Antimicrobial screening test gives good results in the presence of Nd(III) complex system.

Silicon supported lipid-DNA thin film structures at varying temperature studied by energy dispersive X-ray diffraction and neutron reflectivity.

PubMed

Domenici, F; Castellano, C; Dell'Unto, F; Albinati, A; Congiu, A

2011-11-01

Non-viral gene transfection by means of lipid-based nanosystems, such as solid supported lipid assemblies, is often limited due to their lack of stability and the consequent loss of efficiency. Therefore not only a detailed thermo-lyotropic study of these DNA-lipid complexes is necessary to understand their interaction mechanisms, but it can also be considered as a first step in conceiving and developing new transfection biosystems. The aim of our study is a structural characterization of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-dimethyl-dioctadecyl-ammonium bromide (DDAB)-DNA complex at varying temperature using the energy dispersive X-ray diffraction (EDXD) and neutron reflectivity (NR) techniques. We have shown the formation of a novel thermo-lyotropic structure of DOPC/DDAB thin film self-organized in multi-lamellar planes on (100)-oriented silicon support by spin coating, thus enlightening its ability to include DNA strands. Our NR measurements indicate that the DOPC/DDAB/DNA complex forms temperature-dependent structures. At 65°C and relative humidity of 100% DNA fragments are buried between single lamellar leaflets constituting the hydrocarbon core of the lipid bilayers. This finding supports the consistency of the hydrophobic interaction model, which implies that the coupling between lipid tails and hypo-hydrated DNA single strands could be the driving force of DNA-lipid complexation. Upon cooling to 25°C, EDXD analysis points out that full-hydrated DOPC-DDAB-DNA can switch in a different metastable complex supposed to be driven by lipid heads-DNA electrostatic interaction. Thermotropic response analysis also clarifies that DOPC has a pivotal role in promoting the formation of our observed thermophylic silicon supported lipids-DNA assembly. Copyright © 2011 Elsevier B.V. All rights reserved.

RNF168 forms a functional complex with RAD6 during the DNA damage response

PubMed Central

Liu, Chao; Wang, Degui; Wu, Jiaxue; Keller, Jennifer; Ma, Teng; Yu, Xiaochun

2013-01-01

Summary Protein ubiquitination plays an important role in initiating the DNA damage response. Following DNA damage, E2 ubiquitin conjugating enzymes are crucial for catalyzing substrate ubiquitination that recruits downstream DNA repair factors to DNA lesions. To identify novel E2 conjugating enzymes important for initiating the DNA-damage-induced ubiquitination cascade, we screened most of the known E2 enzymes and found that RAD6A and RAD6B function together with RNF168 in the ionizing radiation (IR)-induced DNA damage response. Similarly to RNF168-deficient cells, RAD6A- or RAD6B-deficient cells exhibit a reduction in DNA-damage-induced protein ubiquitination. Correspondingly, DNA-damage-induced foci formation of DNA damage repair proteins, such as BRCA1 and 53BP1, is impaired in the absence of RAD6A or RAD6B. Moreover, the RNF168–RAD6 complex targeted histone H1.2 for ubiquitination in vitro and regulated DNA-damage-induced histone H1.2 ubiquitination in vivo. Collectively, these data demonstrate that RNF168, in complex with RAD6A or RAD6B, is activated in the DNA-damage-induced protein ubiquitination cascade. PMID:23525009

Determining the folding and binding free energy of DNA-based nanodevices and nanoswitches using urea titration curves

PubMed Central

Idili, Andrea

2017-01-01

Abstract DNA nanotechnology takes advantage of the predictability of DNA interactions to build complex DNA-based functional nanoscale structures. However, when DNA functional and responsive units that are based on non-canonical DNA interactions are employed it becomes quite challenging to predict, understand and control their thermodynamics. In response to this limitation, here we demonstrate the use of isothermal urea titration experiments to estimate the free energy involved in a set of DNA-based systems ranging from unimolecular DNA-based nanoswitches to more complex DNA folds (e.g. aptamers) and nanodevices. We propose here a set of fitting equations that allow to analyze the urea titration curves of these DNA responsive units based on Watson–Crick and non-canonical interactions (stem-loop, G-quadruplex, triplex structures) and to correctly estimate their relative folding and binding free energy values under different experimental conditions. The results described herein will pave the way toward the use of urea titration experiments in the field of DNA nanotechnology to achieve easier and more reliable thermodynamic characterization of DNA-based functional responsive units. More generally, our results will be of general utility to characterize other complex supramolecular systems based on different biopolymers. PMID:28605461

Experimental studies on the nature of bonding of DNA/bipyridyl-(ethylenediamine)platinum(II) and DNA/netropsin complexes in solution and oriented wet-spun films

NASA Astrophysics Data System (ADS)

Marlowe, R. L.; Szabo, A.; Lee, S. A.; Rupprecht, A.

2002-03-01

The stability of complexes of NaDNA with bipyridyl-(ethylenediamine)platinum(II) (abbreviated [(bipy)Pt(en)]) and with netropsin has been studied using two techniques: (i) ultraviolet melting experiments were done on NaDNA/[(bipy)Pt(en)], showing that the [(bipy)Pt(en)] ligand stabilizes the DNA double helix structure; and (ii) swelling measurements (via optical microscopy) as a function of relative humidity were done on wet-spun oriented films of NaDNA/[(bipy)Pt(en)] and of NaDNA/netropsin. The swelling data shows that an irreversible transition of the films occurs at high relative humidity, first for the NaDNA/netropsin, then for pure NaDNA, and lastly for the NaDNA/[(bipy)Pt(en)]. These results are indicative that the [(bipy)Pt(en)] complex stabilizes the intermolecular bonds which mediate the film swelling characteristics. A model is suggested for the binding of [(bipy)Pt(en)] to DNA to explain why the swelling experiments show this ligand as increasing the intermolecular bond strength between the DNA double helices, while netropsin decreases this degree of stabilization.

The Human Ligase IIIα-XRCC1 Protein Complex Performs DNA Nick Repair after Transient Unwrapping of Nucleosomal DNA*

PubMed Central

Rashid, Ishtiaque; Tomkinson, Alan E.; Pederson, David S.

2017-01-01

Reactive oxygen species generate potentially cytotoxic and mutagenic lesions in DNA, both between and within the nucleosomes that package DNA in chromatin. The vast majority of these lesions are subject to base excision repair (BER). Enzymes that catalyze the first three steps in BER can act at many sites in nucleosomes without the aid of chromatin-remodeling agents and without irreversibly disrupting the host nucleosome. Here we show that the same is true for a protein complex comprising DNA ligase IIIα and the scaffolding protein X-ray repair cross-complementing protein 1 (XRCC1), which completes the fourth and final step in (short-patch) BER. Using in vitro assembled nucleosomes containing discretely positioned DNA nicks, our evidence indicates that the ligase IIIα-XRCC1 complex binds to DNA nicks in nucleosomes only when they are exposed by periodic, spontaneous partial unwrapping of DNA from the histone octamer; that the scaffolding protein XRCC1 enhances the ligation; that the ligation occurs within a complex that ligase IIIα-XRCC1 forms with the host nucleosome; and that the ligase IIIα-XRCC1-nucleosome complex decays when ligation is complete, allowing the host nucleosome to return to its native configuration. Taken together, our results illustrate ways in which dynamic properties intrinsic to nucleosomes may contribute to the discovery and efficient repair of base damage in chromatin. PMID:28184006

Characterization of Ofloxacin Interaction with Mutated (A91V) Quinolone Resistance Determining Region of DNA Gyrase in Mycobacterium Leprae through Computational Simulation.

PubMed

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.

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

PubMed

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

2017-01-01

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

Structure-affinity relationships for the binding of actinomycin D to DNA

NASA Astrophysics Data System (ADS)

Gallego, José; Ortiz, Angel R.; de Pascual-Teresa, Beatriz; Gago, Federico

1997-03-01

Molecular models of the complexes between actinomycin D and 14 different DNA hexamers were built based on the X-ray crystal structure of the actinomycin-d(GAAGCTTC)2 complex. The DNA sequences included the canonical GpC binding step flanked by different base pairs, nonclassical binding sites such as GpG and GpT, and sites containing 2,6-diamino- purine. A good correlation was found between the intermolecular interaction energies calculated for the refined complexes and the relative preferences of actinomycin binding to standard and modified DNA. A detailed energy decomposition into van der Waals and electrostatic components for the interactions between the DNA base pairs and either the chromophore or the peptidic part of the antibiotic was performed for each complex. The resulting energy matrix was then subjected to principal component analysis, which showed that actinomycin D discriminates among different DNA sequences by an interplay of hydrogen bonding and stacking interactions. The structure-affinity relationships for this important antitumor drug are thus rationalized and may be used to advantage in the design of novel sequence-specific DNA-binding agents.

Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation.

PubMed

Glyde, Robert; Ye, Fuzhou; Darbari, Vidya Chandran; Zhang, Nan; Buck, Martin; Zhang, Xiaodong

2017-07-06

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ 54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ 54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ 54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Histone Hl-DNA interaction. Influence of phosphorylation on the interaction of histone Hl with linear fragmented DNA.

PubMed Central

Glotov, B O; Nikolaev, L G; Kurochkin, S N; Severin, E S

1977-01-01

By measuring the fluorescence polarization of fluorescent histone H1 derivatives complexed with DNA, binding of the histone to DNA was studied as a function of ionic strength in the solution prior to and after the H1 phosphorylation on Ser-37 residue. Fluorescent labels were covalently linked either specifically to Tyr-72 residues or unspecifically to lysine residues in the H1 polypeptide chain. The values of the corresponding rotational relaxation times showed that at low ionic strength all the segments of the H1 molecule were immobilized on binding to DNA. The gradual increasing NaC1 concentration in the solution of H1-DNA complex was accompanied at first by additional retardation of the histone mobility in the complex, and then by progressive release of histone H1 from from the complex which was completed at 0.5-0.6 M NaC1 irrespective of phosphorylation. tat the same time the phosphorylation of histone H1 led to removal of the central and, presumably, N-terminal regions of H1 from DNA. PMID:194228

Novel FeII and CoII Complexes of Natural Product Tryptanthrin: Synthesis and Binding with G-Quadruplex DNA

PubMed Central

Zhong, Yi-ning; Zhang, Yan; Gu, Yun-qiong; Wu, Shi-yun; Shen, Wen-ying

2016-01-01

Tryptanthrin is one of the most important members of indoloquinoline alkaloids. We obtained this alkaloid from Isatis. Two novel FeII and CoII complexes of tryptanthrin were first synthesized. Single-crystal X-ray diffraction analyses show that these complexes display distorted four-coordinated tetrahedron geometry via two heterocyclic nitrogen and oxygen atoms from tryptanthrin ligand. Binding with G-quadruplex DNA properties revealed that both complexes were found to exhibit significant interaction with G-quadruplex DNA. This study may potentially serve as the basis of future rational design of metal-based drugs from natural products that target the G-quadruplex DNA. PMID:27698647

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

NASA Astrophysics Data System (ADS)

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G.; Woodbury, Neal W.; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

PubMed

Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G; Woodbury, Neal W; Yan, Hao

2014-07-01

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

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

PubMed

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

2003-07-15

Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1-326 (hRPA70(1-326)), and a fragment of the human XPA protein, residues 98-219 (XPA-MBD). Intensity changes were observed for amide resonances in the (1)H-(15)N correlation spectrum of uniformly (15)N-labeled hRPA70(1-326) after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA70(1-326) fragment. The hRPA70(1-326) residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA70(1-326) suggests that a competitive binding mechanism mediates the formation of the RPA-XPA complex. To determine whether a ternary complex could form between hRPA70(1-326), XPA-MBD and ssDNA, a (1)H-(15)N correlation spectrum was acquired for uniformly (15)N-labeled hRPA70(1-326) after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA70(1-326) in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA70(1-326) suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA70(1-326) may be modulated by ssDNA.

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

PubMed Central

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

2003-01-01

Replication protein A (RPA) is a heterotrimeric single-stranded DNA- (ssDNA) binding protein that can form a complex with the xeroderma pigmentosum group A protein (XPA). This complex can preferentially recognize UV-damaged DNA over undamaged DNA and has been implicated in the stabilization of open complex formation during nucleotide excision repair. In this report, nuclear magnetic resonance (NMR) spectroscopy was used to investigate the interaction between a fragment of the 70 kDa subunit of human RPA, residues 1–326 (hRPA701–326), and a fragment of the human XPA protein, residues 98–219 (XPA-MBD). Intensity changes were observed for amide resonances in the 1H–15N correlation spectrum of uniformly 15N-labeled hRPA701–326 after the addition of unlabeled XPA-MBD. The intensity changes observed were restricted to an ssDNA-binding domain that is between residues 183 and 296 of the hRPA701–326 fragment. The hRPA701–326 residues with the largest resonance intensity reductions were mapped onto the structure of the ssDNA-binding domain to identify the binding surface with XPA-MBD. The XPA-MBD-binding surface showed significant overlap with an ssDNA-binding surface that was previously identified using NMR spectroscopy and X-ray crystallography. Overlapping XPA-MBD- and ssDNA-binding sites on hRPA701–326 suggests that a competitive binding mechanism mediates the formation of the RPA–XPA complex. To determine whether a ternary complex could form between hRPA701–326, XPA-MBD and ssDNA, a 1H–15N correlation spectrum was acquired for uniformly 15N-labeled hRPA701–326 after the simultaneous addition of unlabeled XPA-MBD and ssDNA. In this experiment, the same chemical shift perturbations were observed for hRPA701–326 in the presence of XPA-MBD and ssDNA as was previously observed in the presence of ssDNA alone. The ability of ssDNA to compete with XPA-MBD for an overlapping binding site on hRPA701–326 suggests that any complex formation between RPA and XPA that involves the interaction between XPA-MBD and hRPA701–326 may be modulated by ssDNA. PMID:12853635

Differences in DNA Binding Specificity of Floral Homeotic Protein Complexes Predict Organ-Specific Target Genes.

PubMed

Smaczniak, Cezary; Muiño, Jose M; Chen, Dijun; Angenent, Gerco C; Kaufmann, Kerstin

2017-08-01

Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation. © 2017 American Society of Plant Biologists. All rights reserved.

Effect of Fe3+ on Curcumin-DNA Complex Studied by FT-Raman Spectroscopy

NASA Astrophysics Data System (ADS)

Senthil, K.; Sarojini, R.

2008-11-01

Curcumin is a non toxic and natural antioxidant. The interaction of curcumin with DNA is widely used in clinical treatment of a variety of different forms of cancer, but its antioxidant property turns into proxidant in the presence of high Fe3+ concentrations. However, the relevance of the pro-oxidant nature of curcumin at molecular level is not clearly determined. Thus, in the present study the interaction of Fe(III) on Curcumin DNA complexes were investigated at physiological pH with Fe(III)/drug/DNA (Phosphate) molar ratios (r) 1:10:50, 1:5:25, 1:2:10 and 1:1:5. FT-Raman Spectroscopy was used to establish correlation between spectral changes and drug binding mode, sequence selectivity, DNA Conformation and Structural properties of Fe(III)/drug/DNA complexes in aqueous solution. Spectroscopic results showed that the major spectral changes were observed at 688 cm-1(G), 835 cm-1 (O-P-O), 1092 cm-1 (PO2-), 1485 cm-1 (G,A,T) and 1683 cm-1 (T), 1723 cm-1 (G) in Fe(III)/drug/DNA complex indicating affinity of Fe(III)/drug with the phosphate and DNA base pairs. The present result showed that the combination of Fe-curcumin induced significant DNA damage in a concentration dependent manner.

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

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

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

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 andmore » 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.« less

DNA Binding and Antitumor Activity of α-Diimineplatinum(II) and Palladium(II) Dithiocarbamate Complexes

PubMed Central

Mansouri-Torshizi, Hassan; Saeidifar, Maryam; Khosravi, Fatemeh; Divsalar, Adeleh; Saboury, Ali Akbar; Hassani, Fatemeh

2011-01-01

The two water-soluble designed platinum(II) complex, [Pt(Oct-dtc)(bpy)]NO3 (Oct-dtc = Octyldithiocarbamate and bpy = 2,2′ -bipyridine) and palladium(II) complex, [Pd(Oct-dtc)(bpy)]NO3, have been synthesized and characterized by elemental analyses, molar conductivity measurements, IR, 1H NMR, and electronic spectra studies. Studies of antitumor activity of these complexes against human cell tumor lines (K562) have been carried out. They show Ic50 values lower than that of cisplatin. The complexes have been investigated for their interaction with calf thymus DNA (CT-DNA) by utilizing the electronic absorption spectroscopy, fluorescence spectra, and ethidium bromide displacement and gel filtration techniques. Both of these water-soluble complexes bound cooperatively and intercalatively to the CT-DNA at very low concentrations. Several binding and thermodynamic parameters are also described. PMID:22110410

Comprehensive Characterization of Minichromosome Maintenance Complex (MCM) Protein Interactions Using Affinity and Proximity Purifications Coupled to Mass Spectrometry.

PubMed

Dubois, Marie-Line; Bastin, Charlotte; Lévesque, Dominique; Boisvert, François-Michel

2016-09-02

The extensive identification of protein-protein interactions under different conditions is an important challenge to understand the cellular functions of proteins. Here we use and compare different approaches including affinity purification and purification by proximity coupled to mass spectrometry to identify protein complexes. We explore the complete interactome of the minichromosome maintenance (MCM) complex by using both approaches for all of the different MCM proteins. Overall, our analysis identified unique and shared interaction partners and proteins enriched for distinct biological processes including DNA replication, DNA repair, and cell cycle regulation. Furthermore, we mapped the changes in protein interactions of the MCM complex in response to DNA damage, identifying a new role for this complex in DNA repair. In summary, we demonstrate the complementarity of these approaches for the characterization of protein interactions within the MCM complex.

Molecular docking studies of curcumin natural derivatives with DNA topoisomerase I and II-DNA complexes.

PubMed

Kumar, Anil; Bora, Utpal

2014-12-01

DNA topoisomerase I (topo I) and II (topo II) are essential enzymes that solve the topological problems of DNA by allowing DNA strands or double helices to pass through each other during cellular processes such as replication, transcription, recombination, and chromatin remodeling. Their critical roles make topoisomerases an attractive drug target against cancer. The present molecular docking study provides insights into the inhibition of topo I and II by curcumin natural derivatives. The binding modes suggested that curcumin natural derivatives docked at the site of DNA cleavage parallel to the axis of DNA base pairing. Cyclocurcumin and curcumin sulphate were predicted to be the most potent inhibitors amongst all the curcumin natural derivatives docked. The binding modes of cyclocurcumin and curcumin sulphate were similar to known inhibitors of topo I and II. Residues like Arg364, Asn722 and base A113 (when docked to topo I-DNA complex) and residues Asp479, Gln778 and base T9 (when docked to topo II-DNA complex) seem to play important role in the binding of curcumin natural derivatives at the site of DNA cleavage.

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

PubMed Central

Maréchal, Alexandre; Zou, Lee

2015-01-01

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

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

PubMed

Maréchal, Alexandre; Zou, Lee

2015-01-01

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

Histone Core Phosphorylation Regulates DNA Accessibility*

PubMed Central

Brehove, Matthew; Wang, Tao; North, Justin; Luo, Yi; Dreher, Sarah J.; Shimko, John C.; Ottesen, Jennifer J.; Luger, Karolin; Poirier, Michael G.

2015-01-01

Nucleosome unwrapping dynamics provide transient access to the complexes involved in DNA transcription, repair, and replication, whereas regulation of nucleosome unwrapping modulates occupancy of these complexes. Histone H3 is phosphorylated at tyrosine 41 (H3Y41ph) and threonine 45 (H3T45ph). H3Y41ph is implicated in regulating transcription, whereas H3T45ph is involved in DNA replication and apoptosis. These modifications are located in the DNA-histone interface near where the DNA exits the nucleosome, and are thus poised to disrupt DNA-histone interactions. However, the impact of histone phosphorylation on nucleosome unwrapping and accessibility is unknown. We find that the phosphorylation mimics H3Y41E and H3T45E, and the chemically correct modification, H3Y41ph, significantly increase nucleosome unwrapping. This enhances DNA accessibility to protein binding by 3-fold. H3K56 acetylation (H3K56ac) is also located in the same DNA-histone interface and increases DNA unwrapping. H3K56ac is implicated in transcription regulation, suggesting that H3Y41ph and H3K56ac could function together. We find that the combination of H3Y41ph with H3K56ac increases DNA accessibility by over an order of magnitude. These results suggest that phosphorylation within the nucleosome DNA entry-exit region increases access to DNA binding complexes and that the combination of phosphorylation with acetylation has the potential to significantly influence DNA accessibility to transcription regulatory complexes. PMID:26175159

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

PubMed

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.

A chromatin remodelling complex that loads cohesin onto human chromosomes

NASA Astrophysics Data System (ADS)

Hakimi, Mohamed-Ali; Bochar, Daniel A.; Schmiesing, John A.; Dong, Yuanshu; Barak, Orr G.; Speicher, David W.; Yokomori, Kyoko; Shiekhattar, Ramin

2002-08-01

Nucleosomal DNA is arranged in a higher-order structure that presents a barrier to most cellular processes involving protein DNA interactions. The cellular machinery involved in sister chromatid cohesion, the cohesin complex, also requires access to the nucleosomal DNA to perform its function in chromosome segregation. The machineries that provide this accessibility are termed chromatin remodelling factors. Here, we report the isolation of a human ISWI (SNF2h)-containing chromatin remodelling complex that encompasses components of the cohesin and NuRD complexes. We show that the hRAD21 subunit of the cohesin complex directly interacts with the ATPase subunit SNF2h. Mapping of hRAD21, SNF2h and Mi2 binding sites by chromatin immunoprecipitation experiments reveals the specific association of these three proteins with human DNA elements containing Alu sequences. We find a correlation between modification of histone tails and association of the SNF2h/cohesin complex with chromatin. Moreover, we show that the association of the cohesin complex with chromatin can be regulated by the state of DNA methylation. Finally, we present evidence pointing to a role for the ATPase activity of SNF2h in the loading of hRAD21 on chromatin.

Complexes containing cationic and anionic pH-sensitive liposomes: comparative study of factors influencing plasmid DNA gene delivery to tumors.

PubMed

Chen, Yan; Sun, Ji; Lu, Ying; Tao, Chun; Huang, Jingbin; Zhang, He; Yu, Yuan; Zou, Hao; Gao, Jing; Zhong, Yanqiang

2013-01-01

pH-sensitive liposomes represent an effective gene vector in cancer therapy. However, their use is greatly hampered by their relatively low transfection efficiency. To improve the transfection efficiency of pH-sensitive liposomes, we prepared complexes containing 3β-[N-(N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidyl ethanolamine (DOPE) liposomes and pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and DOPE, and evaluated the influence of various factors on plasmid DNA (pDNA) transfection efficiency. All DC-Chol/DOPE liposome/pDNA and pH-sensitive liposome complexes showed similarly potent pH sensitivity. In the presence of serum-containing medium, two optimized complexes of DC-Chol/DOPE liposomes/pDNA and pH-sensitive PEGylated liposomes showed high transfection efficiency of 22.94% and 20.07%, respectively. Notably, DC-Chol/DOPE (2:3) liposomes/pH-sensitive PEGylated (1%) liposome complexes with a charge ratio of 1:1 (m/m [+/-]) showed enhanced accumulation in tumors in vivo. Our results show the influence of various factors on pDNA transfection efficiency in complexes of DC-Chol/DOPE liposomes and pH-sensitive PEGylated liposomes. Understanding of such mechanisms will lead to better design of complexes of DC-Chol/DOPE liposomes and pH-sensitive liposomes for gene therapy.

Complexes containing cationic and anionic pH-sensitive liposomes: comparative study of factors influencing plasmid DNA gene delivery to tumors

PubMed Central

Chen, Yan; Sun, Ji; Lu, Ying; Tao, Chun; Huang, Jingbin; Zhang, He; Yu, Yuan; Zou, Hao; Gao, Jing; Zhong, Yanqiang

2013-01-01

pH-sensitive liposomes represent an effective gene vector in cancer therapy. However, their use is greatly hampered by their relatively low transfection efficiency. To improve the transfection efficiency of pH-sensitive liposomes, we prepared complexes containing 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidyl ethanolamine (DOPE) liposomes and pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and DOPE, and evaluated the influence of various factors on plasmid DNA (pDNA) transfection efficiency. All DC-Chol/DOPE liposome/pDNA and pH-sensitive liposome complexes showed similarly potent pH sensitivity. In the presence of serum-containing medium, two optimized complexes of DC-Chol/DOPE liposomes/pDNA and pH-sensitive PEGylated liposomes showed high transfection efficiency of 22.94% and 20.07%, respectively. Notably, DC-Chol/DOPE (2:3) liposomes/pH-sensitive PEGylated (1%) liposome complexes with a charge ratio of 1:1 (m/m [+/−]) showed enhanced accumulation in tumors in vivo. Our results show the influence of various factors on pDNA transfection efficiency in complexes of DC-Chol/DOPE liposomes and pH-sensitive PEGylated liposomes. Understanding of such mechanisms will lead to better design of complexes of DC-Chol/DOPE liposomes and pH-sensitive liposomes for gene therapy. PMID:23637529

Synthesis, Structural, DNA Binding and Cleavage Studies of Cu(II) Complexes Containing Benzothiazole Cored Schiff Bases.

PubMed

Tejaswi, Somapangu; Kumar, Marri Pradeep; Rambabu, Aveli; Vamsikrishna, Narendrula; Shivaraj

2016-11-01

Novel benzothiazole Schiff bases L 1 [1-((4,6-difluorobenzo[d]thiazol-2-ylimino)methyl) naphthalen-2-ol], L 2 [3-((4,6-difluorobenzo[d]thiazol-2-ylimino) methyl)benzene-1,2-diol], L 3 [2-((4,6-difluorobenzo[d]thiazol-2-ylimino)methyl)-5-methoxyphenol], L 4 [2-((4,6-difluorobenzo[d]thiazol-2-ylimino)methyl)-4-chlorophenol] and their binary Cu(II) complexes were synthesized. The structures of all the compounds have been discussed on the basis of elemental analysis, FT-IR, NMR, UV-Visible, ESI-Mass, TGA, ESR, SEM, powder XRD and magnetic moments. Based on the analytical and spectral data a square planar geometry has been assigned to all complexes in which the Schiff bases act as monobasic bidentate ligands, coordinating through the azomethine nitrogen and phenolic oxygen atom. DNA binding ability of these complexes was studied on CT-DNA by using UV-Vis absorption, fluorescence and viscometry. DNA cleavage ability of the complexes was examined on pBR322 DNA by using gel electrophoresis method. All the DNA binding studies reveal that they are good intercalators. The bioefficacy of the ligands and their complexes was examined against the growth of bacteria and fungi in vitro to evaluate their antimicrobial potential. The screening data revealed that the complexes showed more antimicrobial activity than the corresponding free ligands.

Electrochemical studies of DNA interaction and antimicrobial activities of MnII, FeIII, CoII and NiII Schiff base tetraazamacrocyclic complexes

NASA Astrophysics Data System (ADS)

Kumar, Anuj; Vashistha, Vinod Kumar; Tevatia, Prashant; Singh, Randhir

2017-04-01

Tetraazamacrocyclic complexes of MnII, FeIII, CoII and NiII have been synthesized by template method. These tetraazamacrocycles have been analyzed with various techniques like molar conductance, IR, UV-vis, mass spectral and cyclic voltammetric studies. On the basis of all these studies, octahedral geometry has been assigned to these tetraazamacrocyclic complexes. The DNA binding properties of these macrocyclic complexes have been investigated by electronic absorption spectra, fluorescence spectra, cyclic voltammetric and differential pulse voltammetric studies. The cyclic voltammetric data showed that ipc and ipa were effectively decreased in the presence of calf thymus DNA, which is a strong evidence for the interaction of these macrocyclic complexes with the calf thymus DNA (ct-DNA). The heterogeneous electron transfer rate constant found in the order: KCoII > KNiII > KMnII which indicates that CoII macrocyclic complex has formed a strong intercalated intermediate. The Stern-Volmer quenching constant (KSV) and voltammetric binding constant were found in the order KSV(CoII) > KSV(NiII) > KSV(MnII) and K+(CoII) > K+(NiII) > K+(MnII) which shows that CoII macrocyclic complex exhibits the high interaction affinity towards ct-DNA by the intercalation binding. Biological studies of the macrocyclic complexes compared with the standard drug like Gentamycin, have shown antibacterial activities against E. coli, P. aeruginosa, B. cereus, S. aureus and antifungal activity against C. albicans.

Protein dynamics during presynaptic complex assembly on individual ssDNA molecules

PubMed Central

Gibb, Bryan; Ye, Ling F.; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick; Greene, Eric C.

2014-01-01

Homologous recombination is a conserved pathway for repairing double–stranded breaks, which are processed to yield single–stranded DNA overhangs that serve as platforms for presynaptic complex assembly. Here we use single–molecule imaging to reveal the interplay between Saccharomyce cerevisiae RPA, Rad52, and Rad51 during presynaptic complex assembly. We show that Rad52 binds RPA–ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 binding extends the ssDNA, and Rad52–RPA clusters remain interspersed along the presynaptic complex. These clusters promote additional binding of RPA and Rad52. Together, our work illustrates the spatial and temporal progression of RPA and Rad52 association with the presynaptic complex, and reveals a novel RPA–Rad52–Rad51–ssDNA intermediate, which has implications for understanding how the activities of Rad52 and RPA are coordinated with Rad51 during the later stages recombination. PMID:25195049

The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex.

PubMed

Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

2013-07-01

A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy=2,2-bipyridine, Hbcpip=2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5±1.4)×10(5) M(-1) in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived. Copyright © 2013 Elsevier B.V. All rights reserved.

The Interaction between Checkpoint Kinase 1 (Chk1) and the Minichromosome Maintenance (MCM) Complex Is Required for DNA Damage-induced Chk1 Phosphorylation*

PubMed Central

Han, Xiangzi; Aslanian, Aaron; Fu, Kang; Tsuji, Toshiya; Zhang, Youwei

2014-01-01

Chk1 is an essential mediator of the DNA damage response and cell cycle checkpoint. However, how exactly Chk1 transduces the checkpoint signaling is not fully understood. Here we report the identification of the heterohexamic minichromosome maintenance (MCM) complex that interacts with Chk1 by mass spectrometry. The interaction between Chk1 and the MCM complex was reduced by DNA damage treatment. We show that the MCM complex, at least partially, contributes to the chromatin association of Chk1, allowing for immediate phosphorylation of Chk1 by ataxia telangiectasia mutated and Rad3-related (ATR) in the presence of DNA damage. Further, phosphorylation of Chk1 at ATR sites reduces the interaction between Chk1 and the MCM complex, facilitating chromatin release of phosphorylated Chk1, a critical step in the initiation and amplification of cell cycle checkpoint. Together, these data provide novel insights into the activation of Chk1 in response to DNA damage. PMID:25049228

GINS complex protein Sld5 recruits SIK1 to activate MCM helicase during DNA replication.

PubMed

Joshi, Kiranmai; Shah, Varun Jayeshkumar; Maddika, Subbareddy

2016-12-01

In eukaryotes, proper loading and activation of MCM helicase at chromosomal origins plays a central role in DNA replication. Activation of MCM helicase requires its association with CDC45-GINS complex, but the mechanism of how this complex activates MCM helicase is poorly understood. Here we identified SIK1 (salt-inducible kinase 1), an AMPK related protein kinase, as a molecular link that connects GINS complex with MCM helicase activity. We demonstrated that Sld5 a component of GINS complex interacts with SIK1 and recruits it to the sites of DNA replication at the onset of S phase. Depletion of SIK1 leads to defective DNA replication. Further, we showed that SIK1 phosphorylates MCM2 at five conserved residues at its N-terminus, which is essential for the activation of MCM helicase. Collectively, our results suggest SIK1 as a novel integral component of CMG replicative helicase during eukaryotic DNA replication. Copyright © 2016 Elsevier Inc. All rights reserved.

Target Capture during Mos1 Transposition*

PubMed Central

Pflieger, Aude; Jaillet, Jerôme; Petit, Agnès; Augé-Gouillou, Corinne; Renault, Sylvaine

2014-01-01

DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide. PMID:24269942

Target capture during Mos1 transposition.

PubMed

Pflieger, Aude; Jaillet, Jerôme; Petit, Agnès; Augé-Gouillou, Corinne; Renault, Sylvaine

2014-01-03

DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide.

Addition of DNA to CrVI and Cytochrome b5 Containing Proteoliposomes Leads to Generation of DNA Strand Breaks and CrIII Complexes

PubMed Central

Borthiry, Griselda R.; Antholine, William E.; Myers, Judith M.; Myers, Charles R.

2009-01-01

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b5 and NADPH :P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of CrVI (hexavalent chromium, as CrO42− ), and the generation of CrV, superoxide (O2·−) , and hydroxyl radical (HO˙). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O2. CrV is formed under both conditions, so the breaks are not mediated directly by CrV. The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO˙ from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO42− , and plasmid DNA, intense EPR signals at g = 5.7and g = 5.0 were observed. These signals are attributed to specific CrIII complexes with large zero field splitting (ZFS). Without DNA, the signals in the g = 5 region were weak. The large ZFS signals were not seen, when CrIIICl3 was incubated with DNA, suggesting that the CrIII–DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g = 2 is attributed to CrIII complexes with a small ZFS. This g = 2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific CrIII complexes that depend on the presence of plasmid DNA and the manner in which the CrIII is formed. PMID:18729091

Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock.

PubMed

Hazra, Joyita; Mukherjee, Pooja; Ali, Asif; Poddar, Soumita; Pal, Mahadeb

2017-01-01

An involvement of components of DNA-break repair (DBR) complex including DNA-dependent protein kinase (DNA-PK) and poly-ADP-ribose polymerase 1 (PARP-1) in transcription regulation in response to distinct cellular signalling has been revealed by different laboratories. Here, we explored the involvement of DNA-PK and PARP-1 in the heat shock induced transcription of Hsp70A1A. We find that inhibition of both the catalytic subunit of DNA-PK (DNA-PKc), and Ku70, a regulatory subunit of DNA-PK holo-enzyme compromises transcription of Hsp70A1A under heat shock treatment. In immunoprecipitation based experiments we find that Ku70 or DNA-PK holoenzyme associates with NFκB. This NFκB associated complex also carries PARP-1. Downregulation of both NFκB and PARP-1 compromises Hsp70A1A transcription induced by heat shock treatment. Alteration of three bases by site directed mutagenesis within the consensus κB sequence motif identified on the promoter affected inducibility of Hsp70A1A transcription by heat shock treatment. These results suggest that NFκB engaged with the κB motif on the promoter cooperates in Hsp70A1A activation under heat shock in human cells as part of a DBR complex including DNA-PK and PARP-1.

Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing.

PubMed

Kim, Jin S; Nanfara, Michael T; Chodavarapu, Sundari; Jin, Kyeong S; Babu, Vignesh M P; Ghazy, Mohamed A; Chung, Scisung; Kaguni, Jon M; Sutton, Mark D; Cho, Yunje

2017-04-20

Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda-sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda-β clamp complex. This complex contains two pairs of Hda dimers sandwiched between two β clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the β clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda-β clamp complex indicate that the interaction of the β clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda-β clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing

PubMed Central

Kim, Jin S.; Nanfara, Michael T.; Chodavarapu, Sundari; Jin, Kyeong S.; Babu, Vignesh M. P.; Ghazy, Mohamed A.; Chung, Scisung

2017-01-01

Abstract Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda–sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda–β clamp complex. This complex contains two pairs of Hda dimers sandwiched between two β clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the β clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda–β clamp complex indicate that the interaction of the β clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda–β clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function. PMID:28168278

A unified view of base excision repair: lesion-dependent protein complexes regulated by post-translational modification

PubMed Central

Almeida, Karen H.; Sobol, Robert W.

2007-01-01

Base excision repair (BER) proteins act upon a significantly broad spectrum of DNA lesions that result from endogenous and exogenous sources. Multiple sub-pathways of BER (short-path or long-patch) and newly designated DNA repair pathways (e.g., SSBR and NIR) that utilize BER proteins complicate any comprehensive understanding of BER and its role in genome maintenance, chemotherapeutic response, neurodegeneration, cancer or aging. Herein, we propose a unified model of BER, comprised of three functional processes: Lesion Recognition/Strand Scission, Gap Tailoring and DNA Synthesis/Ligation, each represented by one or more multiprotein complexes and coordinated via the XRCC1/DNA Ligase III and PARP1 scaffold proteins. BER therefore may be represented by a series of repair complexes that assemble at the site of the DNA lesion and mediates repair in a coordinated fashion involving protein-protein interactions that dictate subsequent steps or sub-pathway choice. Complex formation is influenced by post-translational protein modifications that arise from the cellular state or the DNA damage response, providing an increase in specificity and efficiency to the BER pathway. In this review, we have summarized the reported BER protein-protein interactions and protein post-translational modifications and discuss the impact on DNA repair capacity and complex formation. PMID:17337257

Efficient interrupting skills of amino acid metallointercalators with DNA at physiological pH: Evaluation of biological assays

NASA Astrophysics Data System (ADS)

Raman, Natarajan; Selvaganapathy, Muthusamy; Radhakrishnan, Srinivasan

2014-06-01

The 4-aminoantipyrine derivatives (sbnd NO2, sbnd OCH3) and their mixed-ligand complexes with amino acids have been synthesized and investigated for their binding with CT DNA using UV-visible spectroscopy, cyclic voltammetry, and viscosity measurements under physiological conditions of pH (stomach 4.7; blood 7.4). The results from all techniques i.e. binding constant (Kb), and free energy change (ΔG) were in good agreement and inferred spontaneous compound-DNA complexes formation via intercalation. Among all the compounds 1 and 4 showed comparatively greater binding at pH 7.4 as evident from its greater Kb values. All the complexes exhibit oxidative cleavage of supercoiled (SC) pBR322 plasmid DNA in the presence of H2O2 as an activator. It is remarkable that at 25 μM concentration 1 and 4 completely degrade SC DNA into undetectable minor fragments and thus they act as efficient chemical nucleases. Among the new complexes, complexes 1 and 4 have highest potential against all the microorganisms tested. The results of the above biological experiments also reveal that the choice of different metal ions has little influence on the DNA binding, DNA cleavage and antimicrobial assay.

Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR-DNA complexes.

PubMed

Fisher, Susan H; Wray, Lewis V

2008-01-22

The Bacillus subtilis GlnR repressor controls gene expression in response to nitrogen availability. Because all GlnR-regulated genes are expressed constitutively in mutants lacking glutamine synthetase (GS), GS is required for repression by GlnR. Feedback-inhibited GS (FBI-GS) was shown to activate GlnR DNA binding with an in vitro electophoretic mobility shift assay (EMSA). The activation of GlnR DNA binding by GS in these experiments depended on the feedback inhibitor glutamine and did not occur with mutant GS proteins defective in regulating GlnR activity in vivo. Although stable GS-GlnR-DNA ternary complexes were not observed in the EMSA experiments, cross-linking experiments showed that a protein-protein interaction occurs between GlnR and FBI-GS. This interaction was reduced in the absence of the feedback inhibitor glutamine and with mutant GS proteins. Because FBI-GS significantly reduced the dissociation rate of the GlnR-DNA complexes, the stability of these complexes is enhanced by FBI-GS. These results argue that FBI-GS acts as a chaperone that activates GlnR DNA binding through a transient protein-protein interaction that stabilizes GlnR-DNA complexes. GS was shown to control the activity of the B. subtilis nitrogen transcription factor TnrA by forming a stable complex between FBI-GS and TnrA that inhibits TnrA DNA binding. Thus, B. subtilis GS is an enzyme with dual catalytic and regulatory functions that uses distinct mechanisms to control the activity of two different transcription factors.

DNA purification by triplex-affinity capture and affinity capture electrophoresis

DOEpatents

Cantor, C.R.; Ito, Takashi; Smith, C.L.

1996-01-09

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel. 6 figs.

DNA damage and repair after high LET radiation

NASA Astrophysics Data System (ADS)

O'Neill, Peter; Cucinotta, Francis; Anderson, Jennifer

Predictions from biophysical models of interactions of radiation tracks with cellular DNA indicate that clustered DNA damage sites, defined as two or more lesions formed within one or two helical turns of the DNA by passage of a single radiation track, are formed in mammalian cells. These complex DNA damage sites are regarded as a signature of ionizing radiation exposure particularly as the likelihood of clustered damage sites arising endogenously is low. For instance, it was predicted from biophysical modelling that 30-40% of low LET-induced double strand breaks (DSB), a form of clustered damage, are complex with the yield increasing to >90% for high LET radiation, consistent with the reduced reparability of DSB with increasing ionization density of the radiation. The question arises whether the increased biological effects such as mutagenesis, carcinogenesis and lethality is in part related to DNA damage complexity and/or spatial distribution of the damage sites, which may lead to small DNA fragments. With particle radiation it is also important to consider not only delta-rays which may cause clustered damaged sites and may be highly mutagenic but the non-random spatial distribution of DSB which may lead to deletions. In this overview I will concentrate on the molecular aspects of the variation of the complexity of DNA damage on radiation quality and the challenges this complexity presents the DNA damage repair pathways. I will draw on data from micro-irradiations which indicate that the repair of DSBs by non-homologous end joining is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB. In summary the aim is to emphasis the link between the spatial distribution of energy deposition events related to the track, the molecular products formed and the consequence of damage complexity contributing to biological effects and to present some of the outstanding molecular challenges with particle radiation.

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis.

PubMed Central

Arakawa, H; Neault, J F; Tajmir-Riahi, H A

2001-01-01

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases. PMID:11509371

Mononuclear zinc(II) complexes of 2-((2-(piperazin-1-yl)ethylimino)methyl)-4-substituted phenols: Synthesis, structural characterization, DNA binding and cheminuclease activities

NASA Astrophysics Data System (ADS)

Ravichandran, J.; Gurumoorthy, P.; Karthick, C.; Kalilur Rahiman, A.

2014-03-01

Four new zinc(II) complexes [Zn(HL1-4)Cl2] (1-4), where HL1-4 = 2-((2-(piperazin-1-yl)ethylimino)methyl)-4-substituted phenols, have been isolated and fully characterized using various spectro-analytical techniques. The X-ray crystal structure of complex 4 shows the distorted trigonal-bipyramidal coordination geometry around zinc(II) ion. The crystal packing is stabilized by intermolecular NH⋯O hydrogen bonding interaction. The complexes display no d-d electronic band in the visible region due to d10 electronic configuration of zinc(II) ion. The electrochemical properties of the synthesized ligands and their complexes exhibit similar voltammogram at reduction potential due to electrochemically innocent Zn(II) ion, which evidenced that the electron transfer is due to the nature of the ligand. Binding interaction of complexes with calf thymus DNA was studied by UV-Vis absorption titration, viscometric titration and cyclic voltammetry. All complexes bind with CT DNA by intercalation, giving the binding affinity in the order of 2 > 1 ≫ 3 > 4. The prominent cheminuclease activity of complexes on plasmid DNA (pBR322 DNA) was observed in the absence and presence of H2O2. Oxidative pathway reveals that the underlying mechanism involves hydroxyl radical.

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides.

PubMed

Ghosh, Supratim; Mallick, Sumana; Das, Upasana; Verma, Ajay; Pal, Uttam; Chatterjee, Sabyasachi; Nandy, Abhishek; Saha, Krishna D; Maiti, Nakul Chandra; Baishya, Bikash; Suresh Kumar, G; Gmeiner, William H

2018-03-01

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV-Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two ɣ H of curcumin heptadiene chain are closely positioned to the A 16 -H8 and A 17 -H8, while G 12 -H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

The long story of mitochondrial DNA and respiratory complex I.

PubMed

Degli Esposti, Mauro

2017-01-01

This article examines the long story of the relationship between mitochondrial DNA (mtDNA) and respiratory complex I, NADH:Ubiquinone Oxidoreductase, from its beginning  in the genome of the bacterial endosymbiont which then evolved into the mitochondria of our cells. The story begins with the evolution of ancient forms of bacterial complex I into the Nuo14 complex I that was present in the alpha proteobacterial ancestor of mitochondria. The story then becomes complicated in the diversity of eukaryotic organisms that are currently recognized. Therefore, it does not have a clear end, because currently available information shows different situations of metabolic adaptation and gene loss, indicating cases of de-evolution of the original protonmotive complex into a system that may fundamentally assist [FeFe]-hydrogenases in re-oxidising metabolically produced NADH under anaerobic conditions. The history of complex I is thus a never ending story of molecular and physiological evolution producing new perspectives for studying the enzyme complex that occupies the largest proportion of mitochondrial DNA.

A novel nitric oxide-based anticancer therapeutics by macrophage-targeted poly(l-arginine)-based nanoparticles.

PubMed

Kudo, Shinpei; Nagasaki, Yukio

2015-11-10

In the immune system, macrophages in tumor tissue generate nitric oxide (NO), producing versatile effects including apoptosis of tumor cells, because inducible NO synthase (iNOS) in the cytoplasm of a macrophage produces NO using l-arginine as a substrate. Here, we propose novel NO-triggered immune therapeutics based on our newly designed nanoparticle system. We designed a poly(ethylene glycol)-block-poly(l-arginine) (i.e., PEG-b-P(l-Arg)) block copolymer and prepared polyion complex micelles (PEG-b-P(l-Arg)/m) composed of PEG-b-P(l-Arg) and chondroitin sulfate for systemic anticancer immunotherapy. iNOS treatment of PEG-b-P(l-Arg) did not generate NO, but NO molecules were detected after trypsin pretreatment, indicating that hydrolysis of P(l-Arg) to monomeric arginine was taking place in vitro. RAW264.7 macrophages abundantly generated NO from the PEG-b-P(l-Arg)/m in comparison with control micelles; this finding is indicative of robustness of the proposed method. It is interesting to note that systemic administration of PEG-b-P(l-Arg)/m had no noticeable adverse effects and suppressed the tumor growth rate in C26 tumor-bearing mice in a dose-dependent manner. Our newly designed nanoparticle-assisted arginine delivery system seems to hold promise as an NO-mediated anticancer immunotherapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Ionization of short polymethacrylic acid: titration, DLS, and model calculations.

PubMed

Pohlmeier, A; Haber-Pohlmeier, S

2004-05-15

In this work the charging of polymethacrylic acid in excess electrolyte solution is investigated experimentally by titration and dynamic light scattering. The results are analyzed by a penetrable sphere model, which employs the Poisson-Boltzmann equation for the description of electrostatic interactions and takes into account specific binding of H+ and Na+. The evaluation of the DLS data yields two relaxation modes. The slow mode is present only at finite degrees of charging and is therefore caused by collective diffusion. The fast mode, which corresponds to diffusion coefficients in the range from (1.1 to 1.5) x 10(-10) m2 s(-1), is present over the whole pH range. This reflects the diffusional dynamics of the polyion itself and allows the calculation of hydrodynamic radii for equivalent spheres (RH). These increase from 1.5 nm at pH 2.14 up to 1.8 nm for a degree of deprotonation alpha=0.47 at pH 5.86. With a further increase of pH the radii slightly decrease to 1.6 nm. Setting the radius of the penetrable sphere equal to RH, we can successfully model the overall charging curve with logK0H=4.85 and logK0Na=-0.6. This means that weak complexes of the type COO---Na are formed, which reduce the effective charge inside the polyelectrolyte coil.

Metal complexes as DNA intercalators.

PubMed

Liu, Hong-Ke; Sadler, Peter J

2011-05-17

DNA has a strong affinity for many heterocyclic aromatic dyes, such as acridine and its derivatives. Lerman in 1961 first proposed intercalation as the source of this affinity, and this mode of DNA binding has since attracted considerable research scrutiny. Organic intercalators can inhibit nucleic acid synthesis in vivo, and they are now common anticancer drugs in clinical therapy. The covalent attachment of organic intercalators to transition metal coordination complexes, yielding metallointercalators, can lead to novel DNA interactions that influence biological activity. Metal complexes with σ-bonded aromatic side arms can act as dual-function complexes: they bind to DNA both by metal coordination and through intercalation of the attached aromatic ligand. These aromatic side arms introduce new modes of DNA binding, involving mutual interactions of functional groups held in close proximity. The biological activity of both cis- and trans-diamine Pt(II) complexes is dramatically enhanced by the addition of σ-bonded intercalators. We have explored a new class of organometallic "piano-stool" Ru(II) and Os(II) arene anticancer complexes of the type [(η(6)-arene)Ru/Os(XY)Cl](+). Here XY is, for example, ethylenediamine (en), and the arene ligand can take many forms, including tetrahydroanthracene, biphenyl, or p-cymene. Arene-nucleobase stacking interactions can have a significant influence on both the kinetics and thermodynamics of DNA binding. In particular, the cytotoxic activity, conformational distortions, recognition by DNA-binding proteins, and repair mechanisms are dependent on the arene. A major difficulty in developing anticancer drugs is cross-resistance, a phenomenon whereby a cell that is resistant to one drug is also resistant to another drug in the same class. These new complexes are non-cross-resistant with cisplatin towards cancer cells: they constitute a new class of anticancer agents, with a mechanism of action that differs from the anticancer drug cisplatin and its analogs. The Ru-arene complexes with dual functions are more potent towards cancer cells than their nonintercalating analogs. In this Account, we focus on recent studies of dual-function organometallic Ru(II)- and Os(II)-arene complexes and the methods used to detect arene-DNA intercalation. We relate these interactions to the mechanism of anticancer activity and to structure-activity relationships. The interactions between these complexes and DNA show close similarities to those of covalent polycyclic aromatic carcinogens, especially to N7-alkylating intercalation compounds. However, Ru-arene complexes exhibit some new features. Classical intercalation and base extrusion next to the metallated base is observed for {(η(6)-biphenyl)Ru(ethylenediamine)}(2+) adducts of a 14-mer duplex, while penetrating arene intercalation occurs for adducts of the nonaromatic bulky intercalator {(η(6)-tetrahydroanthracene)Ru(ethylenediamine)}(2+) with a 6-mer duplex. The introduction of dual-function Ru-arene complexes introduces new mechanisms of antitumor activity, novel mechanisms for attack on DNA, and new concepts for developing structure- activity relationships. We hope this discussion will stimulate thoughtful and focused research on the design of anticancer chemotherapeutic agents using these unique approaches.

Synthesis and crystal structure elucidation of new copper(II)-based chemotherapeutic agent coupled with 1,2-DACH and orthovanillin: Validated by in vitro DNA/HSA binding profile and pBR322 cleavage pathway.

PubMed

Zaki, Mehvash; Afzal, Mohd; Ahmad, Musheer; Tabassum, Sartaj

2016-08-01

New copper(II)-based complex (1) was synthesized and characterized by analytical, spectroscopic and single crystal X-ray diffraction. The in vitro binding studies of complex 1 with CT DNA and HSA have been investigated by employing biophysical techniques to examine the binding propensity of 1 towards DNA and HSA. The results showed that 1 avidly binds to CT DNA via electrostatic mode along with the hydrogen bonding interaction of NH2 and CN groups of Schiff base ligand with the base pairs of DNA helix, leads to partial unwinding and destabilization of the DNA double helix. Moreover, the CD spectral studies revealed that complex 1 binds through groove binding interaction that stabilizes the right-handed B-form of DNA. Complex 1 showed an impressive photoinduced nuclease activity generating single-strand breaks in comparison with the DNA cleavage activity in presence of visible light. The mechanistic investigation revealed the efficiency of 1 to cleave DNA strands by involving the generation of reactive oxygen species. Furthermore, the time dependent DNA cleavage activity showed that there was gradual increase in the amount of NC DNA on increasing the photoexposure time. However, the interaction of 1 and HSA showed that the change of intrinsic fluorescence intensity of HSA was induced by the microenvironment of Trp residue. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

PubMed Central

Kirouac, Kevin N.; Basu, Ashis K.; Ling, Hong

2013-01-01

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. PMID:23876706

Structural mechanism of replication stalling on a bulky amino-polycyclic aromatic hydrocarbon DNA adduct by a y family DNA polymerase.

PubMed

Kirouac, Kevin N; Basu, Ashis K; Ling, Hong

2013-11-15

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. © 2013.

Binding characteristics and protective capacity of cyanidin-3-glucoside and its aglycon to calf thymus DNA.

PubMed

Zhang, Chao; Guo, Xiaofei; Cai, Wenqian; Ma, Yue; Zhao, Xiaoyan

2015-04-01

The binding characteristics and protective capacity of cyanidin (Cy) and cyanidin-3-glucoside (C3G) to calf thymus DNA were explored for the first time. The Cy and C3G gave a bathochromic shift to the ultraviolet-visible spectra of the DNA, indicating the formation of the DNA-Cy and DNA-C3G complexes. The complexes were formed by an intercalative binding mode based on the results of the fluorescence spectra and competitive binding analysis. Meanwhile, the Cy and C3G protected the DNA from the damage induced by the hydroxyl radical. The binding capacity and protective capacity of the C3G were stronger than that of the Cy. Furthermore, the formation of the DNA-anthocyanin complexes was spontaneous when the hydrogen bond and hydrophobic force played a key role. Hence, the Cy and C3G could protect the DNA automatically from the damage induced by the hydroxyl radical. © 2015 Institute of Food Technologists®

Transcription initiation complex structures elucidate DNA opening.

PubMed

Plaschka, C; Hantsche, M; Dienemann, C; Burzinski, C; Plitzko, J; Cramer, P

2016-05-19

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity

USDA-ARS?s Scientific Manuscript database

The extensive use of DNA barcoding technology in a large inventory of Macrolepidoptera and their parasitoids is documented. The methodology used and its practical applications are summarized, and numerous examples of how DNA barcoding has untangled complexes of cryptic species of butterflies, moths...

KDM5A demethylase: Erasing histone modifications to promote repair of DNA breaks

PubMed Central

2017-01-01

Repairing DNA breaks within the complexity of the cell chromatin is challenging. In this issue, Gong et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201611135) identify the histone demethylase KDM5A as a critical editor of the cells’ “histone code” that is required to recruit DNA repair complexes to DNA breaks. PMID:28572116

Structure analysis of FAAP24 reveals single-stranded DNA-binding activity and domain functions in DNA damage response

PubMed Central

Wang, Yucai; Han, Xiao; Wu, Fangming; Leung, Justin W; Lowery, Megan G; Do, Huong; Chen, Junjie; Shi, Chaowei; Tian, Changlin; Li, Lei; Gong, Weimin

2013-01-01

The FANCM/FAAP24 heterodimer has distinct functions in protecting cells from complex DNA lesions such as interstrand crosslinks. These functions rely on the biochemical activity of FANCM/FAAP24 to recognize and bind to damaged DNA or stalled replication forks. However, the DNA-binding activity of this complex was not clearly defined. We investigated how FAAP24 contributes to the DNA-interacting functions of the FANCM/FAAP24 complex by acquiring the N-terminal and C-terminal solution structures of human FAAP24. Modeling of the FAAP24 structure indicates that FAAP24 may possess a high affinity toward single-stranded DNA (ssDNA). Testing of various FAAP24 mutations in vitro and in vivo validated this prediction derived from structural analyses. We found that the DNA-binding and FANCM-interacting functions of FAAP24, although both require the C-terminal (HhH)2 domain, can be distinguished by segregation-of-function mutations. These results demonstrate dual roles of FAAP24 in DNA damage response against crosslinking lesions, one through the formation of FANCM/FAAP24 heterodimer and the other via its ssDNA-binding activity required in optimized checkpoint activation. PMID:23999858

Capture of unstable protein complex on the streptavidin-coated single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Liu, Zunfeng; Voskamp, Patrick; Zhang, Yue; Chu, Fuqiang; Abrahams, Jan Pieter

2013-04-01

Purification of unstable protein complexes is a bottleneck for investigation of their 3D structure and in protein-protein interaction studies. In this paper, we demonstrate that streptavidin-coated single-walled carbon nanotubes (Strep•SWNT) can be used to capture the biotinylated DNA- EcoRI complexes on a 2D surface and in solution using atomic force microscopy and electrophoresis analysis, respectively. The restriction enzyme EcoRI forms unstable complexes with DNA in the absence of Mg2+. Capturing the EcoRI-DNA complexes on the Strep•SWNT succeeded in the absence of Mg2+, demonstrating that the Strep•SWNT can be used for purifying unstable protein complexes.

Ultrahigh-resolution crystal structures of Z-DNA in complex with Mn(2+) and Zn(2+) ions.

PubMed

Drozdzal, Pawel; Gilski, Miroslaw; Kierzek, Ryszard; Lomozik, Lechoslaw; Jaskolski, Mariusz

2013-06-01

X-ray crystal structures of the spermine(4+) form of the Z-DNA duplex with the self-complementary d(CG)3 sequence in complexes with Mn(2+) and Zn(2+) cations have been determined at the ultrahigh resolutions of 0.75 and 0.85 Å, respectively. Stereochemical restraints were only used for the sperminium cation (in both structures) and for nucleotides with dual conformation in the Zn(2+) complex. The Mn(2+) and Zn(2+) cations at the major site, designated M(2+)(1), bind at the N7 position of G6 by direct coordination. The coordination geometry of this site was octahedral, with complete hydration shells. An additional Zn(2+)(2) cation was bis-coordinated in a tetrahedral fashion by the N7 atoms of G10 and G12 from a symmetry-related molecule. The coordination distances of Zn(2+)(1) and Zn(2+)(2) to the O6 atom of the guanine residues were 3.613 (6) and 3.258 (5) Å, respectively. Moreover, a chloride ion was also identified in the coordination sphere of Zn(2+)(2). Alternate conformations were observed in the Z-DNA-Zn(2+) structure not only at internucleotide linkages but also at the terminal C3'-OH group of G12. The conformation of the sperminium chain in the Z-DNA-Mn(2+) complex is similar to the spermine(4+) conformation in analogous Z-DNA-Mg(2+) structures. In the Z-DNA-Zn(2+) complex the sperminium cation is disordered and partially invisible in electron-density maps. In the Z-DNA-Zn(2+) complex the sperminium cation only interacts with the phosphate groups of the Z-DNA molecules, while in the Z-DNA-Mn(2+) structure it forms hydrogen bonds to both the phosphate groups and DNA bases.

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

PubMed Central

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-01-01

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

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

PubMed

Mekler, Vladimir; Minakhin, Leonid; Severinov, Konstantin

2017-05-23

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

Following DNA chain extension and protein conformational changes in crystals of a Y-family DNA polymerase via Raman crystallography.

PubMed

Espinoza-Herrera, Shirly J; Gaur, Vineet; Suo, Zucai; Carey, Paul R

2013-07-23

Y-Family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. (13)C- and (15)N-labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg(2+) and Mn(2+), nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of *C modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following a single d*CTP addition. Adding these two bases caused visible damage to the crystal that was possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn(2+) or Mg(2+), the primer extension reaction did not occur; instead, a ternary protein·template·d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features caused by ring modes from the template/primer bases being perturbed and from the DNA backbone appear, as well as features from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary complex than in the binary complex. Using standardized Raman intensities followed as a function of time, the C(*C)TP population in the crystal was maximal at ∼20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred.

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription

PubMed Central

Stefanovsky, Victor Y.; Tremblay, Michel G.; Lindsay, Helen; Robinson, Mark D.

2017-01-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes. PMID:28715449

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

PubMed

Herdman, Chelsea; Mars, Jean-Clement; Stefanovsky, Victor Y; Tremblay, Michel G; Sabourin-Felix, Marianne; Lindsay, Helen; Robinson, Mark D; Moss, Tom

2017-07-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

DNA as a Target for Anticancer Phen-Imidazole Pd(II) Complexes.

PubMed

Heydari, Maryam; Moghadam, Mahboube Eslami; Tarlani, AliAkbar; Farhangian, Hossein

2017-05-01

Imidazole ring is a known structure in many natural or synthetic drug molecules and its metal complexes can interact with DNA and do the cleavage. Hence, to study the influence of the structure and size of the ligand on biological behavior of metal complexes, two water-soluble Pd(II) complexes of phen and FIP ligands (where phen is 1,10-phenanthroline and FIP is 2-(Furan-2-yl)-1H-Imidazo[4,5-f][1, 10]phenanthroline) with the formula of [Pd(phen)(FIP)](NO 3 ) 2 and [Pd(FIP) 2 ]Cl 2 , that were activated against chronic myelogenous leukemia cell line, K562, were selected. Also, the interaction of these anticancer Pd(II) complexes with highly polymerized calf thymus DNA was extensively studied by means of electronic absorption, fluorescence, and circular dichroism in Tris-buffer. The results showed that the binding was positive cooperation and [Pd(phen)(FIP)](NO 3 ) 2 (K f  = 127 M -1 G = 1.2) exhibited higher binding constant and number of binding sites than [Pd(FIP) 2 ]Cl 2 (K f  = 13 M -1 G = 1.03) upon binding to DNA. The fluorescence data indicates that quenching effect for [Pd(phen)(FIP)](NO 3 ) 2 (K SV  = 58 mM -1 ) was higher than [Pd(FIP) 2 ]Cl 2 (K SV  = 12 mM -1 ). Also, [Pd(FIP) 2 ]Cl 2 interacts with ethidium bromide-DNA, as non-competitive inhibition, and can bind to DNA via groove binding and [Pd(phen)(FIP)](NO 3 ) 2 can intercalate in DNA. These results were confirmed by circular dichroism spectra. Docking data revealed that longer complexes have higher interaction energy and bind to DNA via groove binding. Graphical Abstract Two anticancer Pd(II) complexes of imidazole derivative have been synthesized and interacted with calf thymus DNA. Modes of binding have been studied by electronic absorption, fluorescence, and CD measurements. [Pd(FIP) 2 ]Cl 2 can bind to DNA via groove binding while intercalation mode of binding is observed for [Pd(phen)(FIP)](NO 3 ) 2 .

Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome replication.

PubMed

Grimwade, Julia E; Rozgaja, Tania A; Gupta, Rajat; Dyson, Kyle; Rao, Prassanna; Leonard, Alan C

2018-05-25

In all cells, initiation of chromosome replication depends on the activity of AAA+ initiator proteins that form complexes with replication origin DNA. In bacteria, the conserved, adenosine triphosphate (ATP)-regulated initiator protein, DnaA, forms a complex with the origin, oriC, that mediates DNA strand separation and recruitment of replication machinery. Complex assembly and origin activation requires DnaA-ATP, which differs from DnaA-ADP in its ability to cooperatively bind specific low affinity sites and also to oligomerize into helical filaments. The degree to which each of these activities contributes to the DnaA-ATP requirement for initiation is not known. In this study, we compared the DnaA-ATP dependence of initiation from wild-type Escherichia coli oriC and a synthetic origin (oriCallADP), whose multiple low affinity DnaA sites bind DnaA-ATP and DnaA-ADP similarly. OriCallADP was fully occupied and unwound by DnaA-ADP in vitro, and, in vivo, oriCallADP suppressed lethality of DnaA mutants defective in ATP binding and ATP-specific oligomerization. However, loss of preferential DnaA-ATP binding caused over-initiation and increased sensitivity to replicative stress. The findings indicate both DnaA-ATP and DnaA-ADP can perform most of the mechanical functions needed for origin activation, and suggest that a key reason for ATP-regulation of DnaA is to control replication initiation frequency.

Tyrosyl-DNA Phosphodiesterase I a critical survival factor for neuronal development and homeostasis

PubMed Central

van Waardenburg, Robert C.A.M.

2016-01-01

Tyrosyl-DNA phosphodiesterase I (TDP1), like most DNA repair associated proteins, is not essential for cell viability. However, dysfunctioning TDP1 or ATM (ataxia telangiectasia mutated) results in autosomal recessive neuropathology with similar phenotypes, including cerebellar atrophy. Dual inactivation of TDP1 and ATM causes synthetic lethality. A TDP1H493R catalytic mutant is associated with spinocerebellar ataxia with axonal neuropathy (SCAN1), and stabilizes the TDP1 catalytic obligatory enzyme-DNA covalent complex. The ATM kinase activates proteins early on in response to DNA damage. Tdp1−/− and Atm−/− mice exhibit accumulation of DNA topoisomerase I-DNA covalent complexes (TOPO1-cc) explicitly in neuronal tissue during development. TDP1 resolves 3’- and 5’-DNA adducts including trapped TOPO1-cc and TOPO1 protease resistant peptide-DNA complex. ATM appears to regulate the response to TOPO1-cc via a noncanonical function by regulating SUMO/ubiquitin-mediated TOPO1 degradation. In conclusion, TDP1 and ATM are critical factors for neuronal cell viability via two independent but cooperative pathways. PMID:27747316

Tyrosyl-DNA Phosphodiesterase I a critical survival factor for neuronal development and homeostasis.

PubMed

van Waardenburg, Robert C A M

2016-01-01

Tyrosyl-DNA phosphodiesterase I (TDP1), like most DNA repair associated proteins, is not essential for cell viability. However, dysfunctioning TDP1 or ATM (ataxia telangiectasia mutated) results in autosomal recessive neuropathology with similar phenotypes, including cerebellar atrophy. Dual inactivation of TDP1 and ATM causes synthetic lethality. A TDP1H 493 R catalytic mutant is associated with spinocerebellar ataxia with axonal neuropathy (SCAN1), and stabilizes the TDP1 catalytic obligatory enzyme-DNA covalent complex. The ATM kinase activates proteins early on in response to DNA damage. Tdp1-/- and Atm-/- mice exhibit accumulation of DNA topoisomerase I-DNA covalent complexes (TOPO1-cc) explicitly in neuronal tissue during development. TDP1 resolves 3'- and 5'-DNA adducts including trapped TOPO1-cc and TOPO1 protease resistant peptide-DNA complex. ATM appears to regulate the response to TOPO1-cc via a noncanonical function by regulating SUMO/ubiquitin-mediated TOPO1 degradation. In conclusion, TDP1 and ATM are critical factors for neuronal cell viability via two independent but cooperative pathways.

Specificity of the weak binding between the phage SPO1 transcription-inhibitory protein, TF1, and SPO1 DNA.

PubMed

Johnson, G G; Geiduschek, E P

1977-04-05

The interaction of the phage SPO1 protein transcription factor 1 (TF1), with DNA has been analyzed by membrane filter binding and by sedimentation methods. Substantially specific binding of TF1 to helical SPO1 DNA can be demonstrated by nitrocellulose filter-binding assays at relatively low ionic strength (0.08). However, TF1-DNA complexes dissociate and reequilibrate relatively rapidly and this makes filter-binding assays unsuitable for quantitative measurements of binding equilibra. Accordingly, the sedimentation properties of TF1-DNA complexes have been explored and a short-column centrifugation assay has been elaborated for quantitative measurements. Preferential binding of TF1 to the hydroxymethyluracil-containing SPO1 DNA has also been demonstrated by short-column centrifugation. TF1 binds relatively weakly and somewhat cooperatively to SPO1 DNA at many sites; TF1-DNA complexes dissociate and reequilibrate rapidly. At 20 degrees C in 0.01 M phosphate, pH 7.5, 0.15 KC1, one molecule of TF1 can bind to approximately every 60 nucleotide pairs of SPO1 DNA.

Complex DNA Brick Assembly.

PubMed

Ong, Luvena L; Ke, Yonggang

2017-01-01

DNA nanostructures are a useful technology for precisely organizing and manipulating nanomaterials. The DNA bricks method is a modular and versatile platform for applications requiring discrete or periodic structures with complex three-dimensional features. Here, we describe how structures are designed from the fundamental strand architecture through assembly and characterization of the formed structures.

Walking of antitumor bifunctional trinuclear PtII complex on double-helical DNA

PubMed Central

Malina, Jaroslav; Kasparkova, Jana; Farrell, Nicholas P.; Brabec, Viktor

2011-01-01

The trinuclear BBR3464 ([{trans-PtCl(NH3)2}2µ-(trans-Pt(NH3)2(H2N(CH2)6NH2)2)]4+) belongs to the polynuclear class of platinum-based anticancer agents. DNA adducts of this complex differ significantly in structure and type from those of clinically used mononuclear platinum complexes, especially, long-range (Pt, Pt) intrastrand and interstrand cross-links are formed in both 5′–5′ and 3′–3′ orientations. We show employing short oligonucleotide duplexes containing single, site-specific cross-links of BBR3464 and gel electrophoresis that in contrast to major DNA adducts of clinically used platinum complexes, under physiological conditions the coordination bonds between platinum and N7 of G residues involved in the cross-links of BBR3464 can be cleaved. This cleavage may lead to the linkage isomerization reactions between this metallodrug and double-helical DNA. Differential scanning calorimetry of duplexes containing single, site-specific cross-links of BBR3464 reveals that one of the driving forces that leads to the lability of DNA cross-links of this metallodrug is a difference between the thermodynamic destabilization induced by the cross-link and by the adduct into which it could isomerize. The rearrangements may proceed in the way that cross-links originally formed in one strand of DNA can spontaneously translocate from one DNA strand to its complementary counterpart, which may evoke walking of the platinum complex on DNA molecule. PMID:20833634

Hybridization behavior of mixed DNA/alkylthiol monolayers on gold: characterization by surface plasmon resonance and 32P radiometric assay.

PubMed

Gong, Ping; Lee, Chi-Ying; Gamble, Lara J; Castner, David G; Grainger, David W

2006-05-15

Nucleic acid assay from a complex biological milieu is attractive but currently difficult and far from routine. In this study, DNA hybridization from serum dilutions into mixed DNA/mercaptoundecanol (MCU) adlayers on gold was monitored by surface plasmon resonance (SPR). Immobilized DNA probe and hybridized target densities on these surfaces were quantified using 32P-radiometric assays as a function of MCU diluent exposure. SPR surface capture results correlated with radiometric analysis for hybridization performance, demonstrating a maximum DNA hybridization on DNA/MCU mixed adlayers. The maximum target surface capture produced by MCU addition to the DNA probe layer correlates with structural and conformational data on identical mixed DNA/MCU adlayers on gold derived from XPS, NEXAFS, and fluorescence intensity measurements reported in a related study (Lee, C.-Y.; Gong, P.; Harbers, G. M.; Grainger, D. W.; Castner, D. G.; Gamble, L. J. Anal. Chem. 2006, 78, 3316-3325.). MCU addition into the DNA adlayer on gold also improved surface resistance to both nonspecific DNA and serum protein adsorption. Target DNA hybridization from serum dilutions was monitored with SPR on the optimally mixed DNA/MCU adlayers. Both hybridization kinetics and efficiency were strongly affected by nonspecific protein adsorption from a complex milieu even at a minimal serum concentration (e.g., 1%). No target hybridization was detected in SPR assays from serum concentrations above 30%, indicating nonspecific protein adsorption interference of DNA capture and hybridization from complex milieu. Removal of nonsignal proteins from nucleic acid targets prior to assay represents a significant issue for direct sample-to-assay nucleic acid diagnostics from food, blood, tissue, PCR mixtures, and many other biologically complex sample formats.

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system.

PubMed

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

2015-11-19

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database in which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. This database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.

Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

PubMed

Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

2018-05-22

Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

Database of amino acid-nucleotide contacts in contacts in DNA-homeodomain protein

NASA Astrophysics Data System (ADS)

Grokhlina, T. I.; Zrelov, P. V.; Ivanov, V. V.; Polozov, R. V.; Chirgadze, Yu. N.; Sivozhelezov, V. S.

2013-09-01

The analysis of amino acid-nucleotide contacts in interfaces of the protein-DNA complexes, intended to find consistencies in the protein-DNA recognition, is a complex problem that requires an analysis of the physicochemical characteristics of these contacts and the positions of the participating amino acids and nucleotides in the chains of the protein and the DNA, respectively, as well as conservatism of these contacts. Thus, those heterogeneous data should be systematized. For this purpose we have developed a database of amino acid-nucleotide contacts ANTPC (Amino acid Nucleotide Type Position Conservation) following the archetypal example of the proteins in the homeodomain family. We show that it can be used to compare and classify the interfaces of the protein-DNA complexes.

New modulated design and synthesis of chiral CuII/SnIV bimetallic potential anticancer drug entity: In vitro DNA binding and pBR322 DNA cleavage activity

NASA Astrophysics Data System (ADS)

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

2012-05-01

A new chiral ligand scaffold L derived from (R)-2-amino-2-phenyl ethanol and diethyl oxalate was isolated and thoroughly characterized by various spectroscopic methods. The ligand L was allowed to react with CuCl2·2H2O and NiCl2·6H2O to achieve monometallic complexes 1 and 2, respectively. Subsequently modulation of 1 and 2 was carried out in the presence of SnCl4·5H2O to obtain heterobimetallic potential drug candidates 3 and 4 possessing (CuII/SnIV and NiII/SnIV) metallic cores, respectively and characterized by elemental analysis and spectroscopic data including 1H, 13C and 119Sn NMR in case of 3 and 4. In vitro DNA binding studies revealed that complex 3 avidly binds to DNA as quantified by Kb and Ksv values. Complex 3 exhibits a remarkable DNA cleavage activity (concentration dependent) with pBR322 DNA and the cleavage activity of 3 was significantly enhanced in the presence of activators and follows the order H2O2 > Asc > MPA > GSH. Complex 3 cleave pBR322 DNA via hydrolytic pathway and accessible to major groove of DNA.

The Drosophila telomere-capping protein Verrocchio binds single-stranded DNA and protects telomeres from DNA damage response

PubMed Central

Cicconi, Alessandro; Micheli, Emanuela; Vernì, Fiammetta; Jackson, Alison; Gradilla, Ana Citlali; Cipressa, Francesca; Raimondo, Domenico; Bosso, Giuseppe; Wakefield, James G.; Ciapponi, Laura; Cenci, Giovanni; Gatti, Maurizio

2017-01-01

Abstract Drosophila telomeres are sequence-independent structures maintained by transposition to chromosome ends of three specialized retroelements rather than by telomerase activity. Fly telomeres are protected by the terminin complex that includes the HOAP, HipHop, Moi and Ver proteins. These are fast evolving, non-conserved proteins that localize and function exclusively at telomeres, protecting them from fusion events. We have previously suggested that terminin is the functional analogue of shelterin, the multi-protein complex that protects human telomeres. Here, we use electrophoretic mobility shift assay (EMSA) and atomic force microscopy (AFM) to show that Ver preferentially binds single-stranded DNA (ssDNA) with no sequence specificity. We also show that Moi and Ver form a complex in vivo. Although these two proteins are mutually dependent for their localization at telomeres, Moi neither binds ssDNA nor facilitates Ver binding to ssDNA. Consistent with these results, we found that Ver-depleted telomeres form RPA and γH2AX foci, like the human telomeres lacking the ssDNA-binding POT1 protein. Collectively, our findings suggest that Drosophila telomeres possess a ssDNA overhang like the other eukaryotes, and that the terminin complex is architecturally and functionally similar to shelterin. PMID:27940556

Structure of the Mecl Repressor from Staphylococcus aureus in Complex with the Cognate DNA Operator of mec

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

Safo,M.; Ko, T.; Musayev, F.

The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of {beta}-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Angstroms resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA, and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtualmore » DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI-mec complex, but unlike the MecI-bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.« less

Study binding of Al-curcumin complex to ds-DNA, monitoring by multispectroscopic and voltammetric techniques

NASA Astrophysics Data System (ADS)

Ahmadi, F.; Alizadeh, A. A.; Shahabadi, N.; Rahimi-Nasrabadi, M.

2011-09-01

In this work a complex of Al 3+ with curcumin ([Al(curcumin) (EtOH) 2](NO 3) 2) was synthesized and characterized by UV-vis, FT-IR, elemental analysis and spectrophotometric titration techniques. The mole ratio plot revealed a 1:1 complex between Al 3+ and curcumin in solution. For binding studies of this complex to calf thymus-DNA various methods such as: UV-vis, fluorescence, circular dichroism (CD), FT-IR spectroscopy and cyclic voltammetry were used. The intrinsic binding constant of ACC with DNA at 25 °C was calculated by UV-vis and cyclic voltammetry as 2.1 × 10 4 and 2.6 × 10 4, respectively. The thermodynamic studies showed that the reaction is enthalpy and entropy favored. The CD results showed that only the Δ-ACC interacts with DNA and the Δ-ACC form has not any tendency to interact with DNA, also the pure curcumin has not any stereoselective interaction with CT-DNA. Fluorimetric studies showed that fluorescence enhancement was initiated by a static process in the ground state. The cyclic voltammetry showed that ACC interact with DNA with a binding site size of 2. From the FT-IR we concluded that the Δ-ACC interacts with DNA via partial electrostatic and minor groove binding. In comparison with previous works it was concluded that curcumin significantly reduced the affinity of Al 3+ to the DNA.

Structure of the MecI repressor from Staphylococcus aureus in complex with the cognate DNA operator of mec

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

Safo, Martin K., E-mail: msafo@vcu.edu; Ko, Tzu-Ping; Musayev, Faik N.

The up-and-down binding of dimeric MecI to mecA dyad DNA may account for the cooperative effect of the repressor. The dimeric repressor MecI regulates the mecA gene that encodes the penicillin-binding protein PBP-2a in methicillin-resistant Staphylococcus aureus (MRSA). MecI is similar to BlaI, the repressor for the blaZ gene of β-lactamase. MecI and BlaI can bind to both operator DNA sequences. The crystal structure of MecI in complex with the 32 base-pair cognate DNA of mec was determined to 3.8 Å resolution. MecI is a homodimer and each monomer consists of a compact N-terminal winged-helix domain, which binds to DNA,more » and a loosely packed C-terminal helical domain, which intertwines with its counter-monomer. The crystal contains horizontal layers of virtual DNA double helices extending in three directions, which are separated by perpendicular DNA segments. Each DNA segment is bound to two MecI dimers. Similar to the BlaI–mec complex, but unlike the MecI–bla complex, the MecI repressors bind to both sides of the mec DNA dyad that contains four conserved sequences of TACA/TGTA. The results confirm the up-and-down binding to the mec operator, which may account for cooperative effect of the repressor.« less

The N-terminal Region of the DNA-dependent Protein Kinase Catalytic Subunit Is Required for Its DNA Double-stranded Break-mediated Activation*

PubMed Central

Davis, Anthony J.; Lee, Kyung-Jong; Chen, David J.

2013-01-01

DNA-dependent protein kinase (DNA-PK) plays an essential role in the repair of DNA double-stranded breaks (DSBs) mediated by the nonhomologous end-joining pathway. DNA-PK is a holoenzyme consisting of a DNA-binding (Ku70/Ku80) and catalytic (DNA-PKcs) subunit. DNA-PKcs is a serine/threonine protein kinase that is recruited to DSBs via Ku70/80 and is activated once the kinase is bound to the DSB ends. In this study, two large, distinct fragments of DNA-PKcs, consisting of the N terminus (amino acids 1–2713), termed N-PKcs, and the C terminus (amino acids 2714–4128), termed C-PKcs, were produced to determine the role of each terminal region in regulating the activity of DNA-PKcs. N-PKcs but not C-PKcs interacts with the Ku-DNA complex and is required for the ability of DNA-PKcs to localize to DSBs. C-PKcs has increased basal kinase activity compared with DNA-PKcs, suggesting that the N-terminal region of DNA-PKcs keeps basal activity low. The kinase activity of C-PKcs is not stimulated by Ku70/80 and DNA, further supporting that the N-terminal region is required for binding to the Ku-DNA complex and full activation of kinase activity. Collectively, the results show the N-terminal region mediates the interaction between DNA-PKcs and the Ku-DNA complex and is required for its DSB-induced enzymatic activity. PMID:23322783

Synthesis of New Five Coordinated Copper(II) and Nickel(II) Complexes of L-Valine and Kinetic Study of Copper(II) with Calf Thymus DNA

PubMed Central

Tak, Aijaz Ahmad; Arjmand, Farukh

2002-01-01

Five coordinated novel complexes of Cu II and Ni II have been synthesized from benzil and 1,3- diaminopropane- Cu II / Ni II complex and characterized by elemental analysis, i.r., n.m.r., e.p.r, molar conductance and u.v-vis, spectroscopy. The complexes are ionic in nature and exhibit pentaeoordinated geometry around the metal ion. The reaction kinetics of C 25 H 36 N 5 O 2 CuCl with calf thymus DNA was studied by u.v-vis, spectroscopy in aqueous medium. The complex after interaction with calf thymus DNA shows shift in the absorption spectrum and hypochromicity indicating an intercalative binding mode. The K obs values have been calculated under pseudo-first order conditions. The redox behaviour of complex C 25 H 36 N 5 O 2 CuCl in the presence and in the absence of calf thymus DNA in the aqueous solution has been investigated by cyclic voltammetry. The cyclic voitammogram exhibits one quasi-reversible redox wave corresponding to Cu II / Cu I redox couple with E 1 / 2 values of -0.377 and -0.237 V respectively at a scan rate of 0.1V s - 1 .On interaction with calf thymus DNA, the complex C 25 H 36 N 5 O 2 CuCl exhibits shifts in both E p as well as in E 1 / 2 values, indicating strong binding of the complex to the calf thymus DNA. PMID:18475428

Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity.

PubMed

Jacobs, Y; Schnabel, C A; Cleary, M L

1999-07-01

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.

Construction of a fuzzy and Boolean logic gates based on DNA.

PubMed

Zadegan, Reza M; Jepsen, Mette D E; Hildebrandt, Lasse L; Birkedal, Victoria; Kjems, Jørgen

2015-04-17

Logic gates are devices that can perform logical operations by transforming a set of inputs into a predictable single detectable output. The hybridization properties, structure, and function of nucleic acids can be used to make DNA-based logic gates. These devices are important modules in molecular computing and biosensing. The ideal logic gate system should provide a wide selection of logical operations, and be integrable in multiple copies into more complex structures. Here we show the successful construction of a small DNA-based logic gate complex that produces fluorescent outputs corresponding to the operation of the six Boolean logic gates AND, NAND, OR, NOR, XOR, and XNOR. The logic gate complex is shown to work also when implemented in a three-dimensional DNA origami box structure, where it controlled the position of the lid in a closed or open position. Implementation of multiple microRNA sensitive DNA locks on one DNA origami box structure enabled fuzzy logical operation that allows biosensing of complex molecular signals. Integrating logic gates with DNA origami systems opens a vast avenue to applications in the fields of nanomedicine for diagnostics and therapeutics. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

PubMed Central

North, Justin A.; Šimon, Marek; Ferdinand, Michelle B.; Shoffner, Matthew A.; Picking, Jonathan W.; Howard, Cecil J.; Mooney, Alex M.; van Noort, John; Poirier, Michael G.; Ottesen, Jennifer J.

2014-01-01

Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA–histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure. PMID:24561803

Hysteresis in DNA compaction by Dps is described by an Ising model

PubMed Central

Vtyurina, Natalia N.; Dulin, David; Docter, Margreet W.; Meyer, Anne S.; Dekker, Nynke H.; Abbondanzieri, Elio A.

2016-01-01

In all organisms, DNA molecules are tightly compacted into a dynamic 3D nucleoprotein complex. In bacteria, this compaction is governed by the family of nucleoid-associated proteins (NAPs). Under conditions of stress and starvation, an NAP called Dps (DNA-binding protein from starved cells) becomes highly up-regulated and can massively reorganize the bacterial chromosome. Although static structures of Dps–DNA complexes have been documented, little is known about the dynamics of their assembly. Here, we use fluorescence microscopy and magnetic-tweezers measurements to resolve the process of DNA compaction by Dps. Real-time in vitro studies demonstrated a highly cooperative process of Dps binding characterized by an abrupt collapse of the DNA extension, even under applied tension. Surprisingly, we also discovered a reproducible hysteresis in the process of compaction and decompaction of the Dps–DNA complex. This hysteresis is extremely stable over hour-long timescales despite the rapid binding and dissociation rates of Dps. A modified Ising model is successfully applied to fit these kinetic features. We find that long-lived hysteresis arises naturally as a consequence of protein cooperativity in large complexes and provides a useful mechanism for cells to adopt unique epigenetic states. PMID:27091987

Synergistic effect of ultrasound and PEI on DNA transfection in vitro

PubMed Central

Deshpande, Mangesh C.; Prausnitz, Mark R.

2007-01-01

Ultrasound and poly(ethylenimine) (PEI) have each separately been shown to increase DNA transfection efficiency. This study tested the hypothesis that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. This in vitro study assessed transfection efficiency of two different DNA plasmids encoding green fluorescent protein and firefly luciferase in two different cells types, a primary culture of human aortic smooth muscle cells and an immortal line of human prostrate cancer cells. We found that ultrasound sonication increased transfection up to 18-fold, DNA complexation with PEI increased transfection up to 90-fold, and the combination of ultrasound and PEI synergistically increased transfection up to 200-fold, which resulted in reporter gene expression by 34% of cells. Kinetic measurements found that the effects of ultrasound alone acted quickly, whereas increased transfection by PEI either alone or in combination with ultrasound strongly benefited from a 4-h incubation with the DNA plasmid after sonication. Although serum reduced absolute expression levels, it did not affect the relative increase in transfection when ultrasound was added to PEI enhancement. Flow cytometry measurements showed that sonication increased intracellular uptake of labelled DNA complexed to PEI by 55% relative to PEI complexation alone. Electrophoresis assay showed no damage to DNA or PEI-DNA complexes after sonication. Overall, these results suggest that the combination of ultrasound and PEI can have a synergistic effect to increase DNA transfection. PMID:17258835

CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases

PubMed Central

Toliusis, Paulius; Silanskas, Arunas; Szczelkun, Mark D.

2017-01-01

Abstract The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5′-GCCGC-3′ site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes. PMID:28854738

Differential impact of ionic and coordinate covalent chromium (Cr)-DNA binding on DNA replication.

PubMed

Fornsaglio, Jamie L; O'Brien, Travis J; Patierno, Steven R

2005-11-01

The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA. These Cr(III)-DNA interactions consist of Cr-DNA monoadducts, Cr-DNA ternary adducts, and Cr-DNA interstrand cross-links (Cr-ICLs), the latter of which are DNA polymerase arresting lesions (PALs). We sought to determine the impact of Cr-DNA interactions on the formation of replication blocking lesions in S. cerevisiae using a PCR-based method. We found that target sequence (TS) amplification using DNA isolated from Cr(VI)-treated yeast actually increased as a function of Cr(VI) concentration. Moreover, the enhanced TS amplification was reproduced in vitro using Cr(III)-treated DNA. In contrast, PCR amplification of TS from DNA isolated from yeast exposed to equitoxic doses of the inorganic DNA cross-linking agent cisplatin (CDDP), was decreased in a concentration-dependent manner. This paradox suggested that a specific Cr-DNA interaction, such as an ionic Cr-DNA complex, was responsible for the enhanced TS amplification, thereby masking the replication-blocking effect of certain ternary Cr-DNA adducts (i.e. interstrand cross-links). To test this possibility, we removed ionically associated Cr from the DNA using salt extraction prior to PCR analysis. This procedure obviated the increased amplification and revealed a dose-dependent decrease in TS amplification and an increase in Cr-PALs. These data from DNA analyzed ex vivo after treatment of intact cells indicate that ionic interactions of Cr with DNA result in increased DNA amplification whereas coordinate-covalent Cr-DNA complexes lead to formation of Cr-PALs. Thus, these results suggest that treatment of living cells with Cr(VI) leads to two modes of Cr-binding, which may have conflicting effects on DNA replication.

DNA bending and a flip-out mechanism for base excision by the helix–hairpin–helix DNA glycosylase, Escherichia coli AlkA

PubMed Central

Hollis, Thomas; Ichikawa, Yoshitaka; Ellenberger, Tom

2000-01-01

The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 Å crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1–azaribose abasic nucleotide out of DNA and induces a 66° bend in the DNA with a marked widening of the minor groove. The position of the 1–azaribose in the enzyme active site suggests an SN1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA–DNA complex offers the first glimpse of a helix–hairpin–helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner. PMID:10675345

Influence of amino acids Shiff bases on irradiated DNA stability in vivo.

PubMed

Karapetyan, N H; Malakyan, M H; Bajinyan, S A; Torosyan, A L; Grigoryan, I E; Haroutiunian, S G

2013-01-01

To reveal protective role of the new Mn(II) complexes with Nicotinyl-L-Tyrosinate and Nicotinyl-L-Tryptophanate Schiff Bases against ionizing radiation. The DNA of the rats liver was isolated on 7, 14, and 30 days after X-ray irradiation. The differences between the DNA of irradiated rats and rats pre-treated with Mn(II) complexes were studied using the melting, microcalorimetry, and electrophoresis methods. The melting parameters and the melting enthalpy of rats livers DNA were changed after the X-ray irradiation: melting temperature and melting enthalpy were decreased and melting interval was increased. These results can be explained by destruction of DNA molecules. It was shown that pre-treatment of rats with Mn(II) complexes approximates the melting parameters to norm. Agarose gel electrophoresis data confirmed the results of melting studies. The separate DNA fragments were revealed in DNA samples isolated from irradiated animals. The DNA isolated from animals pre-treated with the Mn(II) chelates had better electrophoretic characteristics, which correspond to healthy DNA. Pre-treatment of the irradiated rats with Mn(II)(Nicotinil-L-Tyrosinate) and Mn(II)(Nicotinil-L-Tryptophanate)2 improves the DNA characteristics.

Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks*

PubMed Central

Pohjoismäki, Jaakko L. O.; Goffart, Steffi; Tyynismaa, Henna; Willcox, Smaranda; Ide, Tomomi; Kang, Dongchon; Suomalainen, Anu; Karhunen, Pekka J.; Griffith, Jack D.; Holt, Ian J.; Jacobs, Howard T.

2009-01-01

Analysis of human heart mitochondrial DNA (mtDNA) by electron microscopy and agarose gel electrophoresis revealed a complete absence of the θ-type replication intermediates seen abundantly in mtDNA from all other tissues. Instead only Y- and X-junctional forms were detected after restriction digestion. Uncut heart mtDNA was organized in tangled complexes of up to 20 or more genome equivalents, which could be resolved to genomic monomers, dimers, and linear fragments by treatment with the decatenating enzyme topoisomerase IV plus the cruciform-cutting T7 endonuclease I. Human and mouse brain also contained a population of such mtDNA forms, which were absent, however, from mouse, rabbit, or pig heart. Overexpression in transgenic mice of two proteins involved in mtDNA replication, namely human mitochondrial transcription factor A or the mouse Twinkle DNA helicase, generated abundant four-way junctions in mtDNA of heart, brain, and skeletal muscle. The organization of mtDNA of human heart as well as of mouse and human brain in complex junctional networks replicating via a presumed non-θ mechanism is unprecedented in mammals. PMID:19525233

Investigation of the complex structure, comparative DNA-binding and DNA cleavage of two water-soluble mono-nuclear lanthanum(III) complexes and cytotoxic activity of chitosan-coated magnetic nanoparticles as drug delivery for the complexes

NASA Astrophysics Data System (ADS)

Asadi, Zahra; Nasrollahi, Neda; Karbalaei-Heidari, Hamidreza; Eigner, Vaclav; Dusek, Michal; Mobaraki, Nabiallah; Pournejati, Roya

2017-05-01

Two water-soluble mono-nuclear macrocyclic lanthanum(III) complexes of 2,6-diformyl-4-methylphenol with 1,3-diamino-2-propanol (C1) or 1,3-propylenediamine (C2) were synthesized and characterized by UV-Vis, FT-IR, 13C and 1H NMR spectroscopy and elemental analysis. C1 complex was structurally characterized by single-crystal X-ray diffraction, which revealed that the complex was mononuclear and ten-coordinated. The coordination sites around lanthanum(III) were occupied with a five-dentate ligand, two bidentate nitrates, and one water molecule. The interaction of complexes with DNA was studied in buffered aqueous solution at pH 7.4. UV-Vis absorption spectroscopy, emission spectroscopy, circular dichroism (CD) and viscometric measurements provided clear evidence of the intercalation mechanism of binding. The obtained intrinsic binding constants (Kb) 9.3 × 103 and 1.2 × 103 M- 1 for C1 and C2, respectively confirmed that C1 is better intercalator than C2. The DNA docking studies suggested that the complexes bind with DNA in a groove binding mode with the binding affinity of C1 > C2. Moreover, agarose gel electrophoresis study of the DNA-complex for both compounds revealed that the C1 intercalation cause ethidium bromide replacement in a competitive manner which confirms the suggested mechanism of binding. Finally, the anticancer experiments for the treated cancerous cell lines with both synthesized compounds show that these hydrophilic molecules need a suitable carrier to pass through the hydrophobic nature of cell membrane efficiently.

Synthesis, characterization, DNA-binding and cleavage studies of polypyridyl copper(II) complexes

NASA Astrophysics Data System (ADS)

Gubendran, Ammavasi; Rajesh, Jegathalaprathaban; Anitha, Kandasamy; Athappan, Periyakaruppan

2014-10-01

Six new mixed-ligand copper(II) complexes were synthesized namely [Cu(phen)2OAc]ClO4ṡH2O(1), [Cu(bpy)2OAc]ClO4ṡH2O(2), [Cu(o-ampacac)(phen)]ClO4(3), [Cu(o-ampbzac)(phen)]ClO4(4), [Cu(o-ampacac)(bpy)]ClO4(5), and [Cu(o-ampbzac)(bpy)]ClO4(6) (phen = 1,10-phenanthroline, bpy = 2, 2‧-bipyridine, o-ampacac = (Z)-4-(2-hydroxylamino)pent-3-ene-2-one,o-ampbzac = (Z)-4-(2-hydroxylamino)-4-phenylbut-3-ene-2-one)and characterized by UV-Vis, IR, EPR and cyclic voltammetry. Ligands were characterized by NMR spectra. Single crystal X-ray studies of the complex 1 shows Cu(II) ions are located in a highly distorted octahedral environment. Absorption spectral studies reveal that the complexes 1-6 exhibit hypochromicity during the interaction with DNA and binding constant values derived from spectral and electrochemical studies indicate that complexes 1, 2 and 3 bind strongly with DNA possibly by an intercalative mode. Electrochemical studies reveal that the complexes 1-4 prefer to bind with DNA in Cu(I) rather than Cu(II) form. The shift in the formal potentials E1/2 and CD spectral studies suggest groove or electrostatic binding mode for the complexes 4-6. Complex 1 can cleave supercoiled (SC) pUC18 DNA efficiently into nicked form II under photolytic conditions and into an open circular form (form II) and linear form (form III) in the presence of H2O2 at pH 8.0 and 37 °C, while the complex 2 does not cleave DNA under similar conditions.

Photochemical and DFT studies on DNA-binding ability and antibacterial activity of lanthanum(III)-phenanthroline complex

NASA Astrophysics Data System (ADS)

Niroomand, Sona; Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam; Jahani, Shohreh; Moodi, Asieh

2017-02-01

The binding of the lanthanum(III) complex containing 1,10-phenanthroline (phen), [La(phen)3Cl3·OH2], to DNA is investigated by absorption and emission methods. This complex shows absorption decreasing in a charge transfer band, and fluorescence decrement when it binds to DNA. Electronic absorption spectroscopy (UV-Vis), fluorescence spectra, iodide quenching experiments, salt effect and viscosity measurements, ethidium bromide (EB) competition test, circular dichroism (CD) spectra as well as variable temperature experiments indicate that the La(III) complex binds to fish salmon (FS) DNA, presumably via groove binding mode. The binding constants (Kb) of the La(III) complex with DNA is (2.55 ± 0.02) × 106 M-1. Furthermore, the binding site size, n, the Stern-Volmer constant KSV and thermodynamic parameters; enthalpy change (ΔH0) and entropy change (ΔS0) and Gibb's free energy (ΔG0), are calculated according to relevant fluorescent data and the Van't Hoff equation. The La(III) complex has been screened for its antibacterial activities by the disc diffusion method. Also, in order to supplement the experimental findings, DFT computation and NBO analysis are carried out.

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

NASA Astrophysics Data System (ADS)

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

2010-05-01

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. 119Sn 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 Kb = 8.42 × 104 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.

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

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

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

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

One-step analysis of DNA/chitosan complexes by field-flow fractionation reveals particle size and free chitosan content.

PubMed

Ma, Pei Lian; Buschmann, Michael D; Winnik, Françoise M

2010-03-08

The composition of samples obtained upon complexation of DNA with chitosan was analyzed by asymmetrical flow field flow fractionation (AF4) with online UV-visible, multiangle light scattering (MALS), and dynamic light scattering (DLS) detectors. A chitosan labeled with rhodamine B to facilitate UV-vis detection of the polycation was complexed with DNA under conditions commonly used for transfection (chitosan glucosamine to DNA phosphate molar ratio of 5). AF4 analysis revealed that 73% of the chitosan-rhodamine remained free in the dispersion and that the DNA/chitosan complexes had a broad size distribution ranging from 20 to 160 nm in hydrodynamic radius. The accuracy of the data was assessed by comparison with data from batch-mode DLS and scanning electron microscopy. The AF4 combined with DLS allowed the characterization of small particles that were not detected by conventional batch-mode DLS. The AF4 analysis will prove to be an important tool in the field of gene therapy because it readily provides, in a single measurement, three important physicochemical parameters of the complexes: the amount of unbound polycation, the hydrodynamic size of the complexes, and their size distribution.

Investigation on the correlation between energy deposition and clustered DNA damage induced by low-energy electrons.

PubMed

Liu, Wei; Tan, Zhenyu; Zhang, Liming; Champion, Christophe

2018-05-01

This study presents the correlation between energy deposition and clustered DNA damage, based on a Monte Carlo simulation of the spectrum of direct DNA damage induced by low-energy electrons including the dissociative electron attachment. Clustered DNA damage is classified as simple and complex in terms of the combination of single-strand breaks (SSBs) or double-strand breaks (DSBs) and adjacent base damage (BD). The results show that the energy depositions associated with about 90% of total clustered DNA damage are below 150 eV. The simple clustered DNA damage, which is constituted of the combination of SSBs and adjacent BD, is dominant, accounting for 90% of all clustered DNA damage, and the spectra of the energy depositions correlating with them are similar for different primary energies. One type of simple clustered DNA damage is the combination of a SSB and 1-5 BD, which is denoted as SSB + BD. The average contribution of SSB + BD to total simple clustered DNA damage reaches up to about 84% for the considered primary energies. In all forms of SSB + BD, the SSB + BD including only one base damage is dominant (above 80%). In addition, for the considered primary energies, there is no obvious difference between the average energy depositions for a fixed complexity of SSB + BD determined by the number of base damage, but average energy depositions increase with the complexity of SSB + BD. In the complex clustered DNA damage constituted by the combination of DSBs and BD around them, a relatively simple type is a DSB combining adjacent BD, marked as DSB + BD, and it is of substantial contribution (on average up to about 82%). The spectrum of DSB + BD is given mainly by the DSB in combination with different numbers of base damage, from 1 to 5. For the considered primary energies, the DSB combined with only one base damage contributes about 83% of total DSB + BD, and the average energy deposition is about 106 eV. However, the energy deposition increases with the complexity of clustered DNA damage, and therefore, the clustered DNA damage with high complexity still needs to be considered in the study of radiation biological effects, in spite of their small contributions to all clustered DNA damage.

Charge-transfer optical absorption mechanism of DNA:Ag-nanocluster complexes

NASA Astrophysics Data System (ADS)

Longuinhos, R.; Lúcio, A. D.; Chacham, H.; Alexandre, S. S.

2016-05-01

Optical properties of DNA:Ag-nanoclusters complexes have been successfully applied experimentally in Chemistry, Physics, and Biology. Nevertheless, the mechanisms behind their optical activity remain unresolved. In this work, we present a time-dependent density functional study of optical absorption in DNA:Ag4. In all 23 different complexes investigated, we obtain new absorption peaks in the visible region that are not found in either the isolated Ag4 or isolated DNA base pairs. Absorption from red to green are predominantly of charge-transfer character, from the Ag4 to the DNA fragment, while absorption in the blue-violet range are mostly associated to electronic transitions of a mixed character, involving either DNA-Ag4 hybrid orbitals or intracluster orbitals. We also investigate the role of exchange-correlation functionals in the calculated optical spectra. Significant differences are observed between the calculations using the PBE functional (without exact exchange) and the CAM-B3LYP functional (which partly includes exact exchange). Specifically, we observe a tendency of charge-transfer excitations to involve purines bases, and the PBE spectra error is more pronounced in the complexes where the Ag cluster is bound to the purines. Finally, our results also highlight the importance of adding both the complementary base pair and the sugar-phosphate backbone in order to properly characterize the absorption spectrum of DNA:Ag complexes.

Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

PubMed Central

Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

2016-01-01

Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

Charge-transfer optical absorption mechanism of DNA:Ag-nanocluster complexes.

PubMed

Longuinhos, R; Lúcio, A D; Chacham, H; Alexandre, S S

2016-05-01

Optical properties of DNA:Ag-nanoclusters complexes have been successfully applied experimentally in Chemistry, Physics, and Biology. Nevertheless, the mechanisms behind their optical activity remain unresolved. In this work, we present a time-dependent density functional study of optical absorption in DNA:Ag_{4}. In all 23 different complexes investigated, we obtain new absorption peaks in the visible region that are not found in either the isolated Ag_{4} or isolated DNA base pairs. Absorption from red to green are predominantly of charge-transfer character, from the Ag_{4} to the DNA fragment, while absorption in the blue-violet range are mostly associated to electronic transitions of a mixed character, involving either DNA-Ag_{4} hybrid orbitals or intracluster orbitals. We also investigate the role of exchange-correlation functionals in the calculated optical spectra. Significant differences are observed between the calculations using the PBE functional (without exact exchange) and the CAM-B3LYP functional (which partly includes exact exchange). Specifically, we observe a tendency of charge-transfer excitations to involve purines bases, and the PBE spectra error is more pronounced in the complexes where the Ag cluster is bound to the purines. Finally, our results also highlight the importance of adding both the complementary base pair and the sugar-phosphate backbone in order to properly characterize the absorption spectrum of DNA:Ag complexes.

The phase behavior of cationic lipid-DNA complexes.

PubMed Central

May, S; Harries, D; Ben-Shaul, A

2000-01-01

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram. PMID:10733951

Development of a slow non-viral DNA release system from PDLLA scaffolds fabricated using a supercritical CO2 technique.

PubMed

Heyde, Mieke; Partridge, Kris A; Howdle, Steven M; Oreffo, Richard O C; Garnett, Martin C; Shakesheff, Kevin M

2007-10-15

Polyamidoamine polymers (PAA) comprising methylene-bisacrylamide/dimethylethylene-diamine monomers were synthesized, complexed with DNA and incorporated into porous P(DL)LA scaffolds by using a supercritical CO(2) (scCO(2)) technique. Scaffolds were made in a dry state consequently there was a need to lyophilize the complexes. A statistically significant reduction of the transfection efficiency was observed in the absence of trehalose when compared to the original complex after freeze-drying. Increasing concentrations (0-10% w/v) of trehalose were added to the complex prior to freeze-drying. Structure dependent differences in DNA binding were evaluated by gel electrophoresis and thermal transition analysis. TEM and PCS showed aggregate formation after freeze-drying without trehalose. Scaffolds were characterized by pore sizes of 173 +/- 73 microm and a porosity of 71%. The transfection potential of the released DNA was investigated by seeding scaffolds with A549 cells and following firefly luciferase as a marker gene after 48 h exposure. Low but continuous levels of transfection were observed for PAA complexes during a 60-day study. Complexes made with Lipofectaminetrade mark gave initially higher levels of DNA release but no further expression was seen after 40 days. Uncomplexed DNA showed background levels of transfection. Culturing cells on 3D scaffolds showed a benefit in retention of transfection activity with time compared to 2D controls. Transfection levels could be increased when cells were grown in OptiMEM. This study demonstrated that PAA/DNA complexes incorporated into a P(DL)LA scaffold made by using scCO(2) processing exhibited a slow release and extended gene expression profile. Copyright 2007 Wiley Periodicals, Inc.

Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein–DNA Complex

PubMed Central

2016-01-01

Metal ion cofactors can alter the energetics and specificity of sequence specific protein–DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein–DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg2+ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu3+ or two La3+ cations bind, and two new crystal structures confirm that Lu3+ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for 1H–15N amide resonances, for 1H–13C Ile-δ-CH3 resonances, and for stereospecifically assigned Leu-δ-CH3 and Val-γ-CH3 resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex. PMID:27786446

Volumetric analysis of formation of the complex of G-quadruplex DNA with hemin using high pressure.

PubMed

Takahashi, Shuntaro; Bhowmik, Sudipta; Sugimoto, Naoki

2017-01-01

DNA guanine-quadruplexes (G-quadruplexes) complexed with the Fe-containing porphyrin, hemin (iron(III)-protoporphyrin IX), can catalyze oxidation reactions. This so-called DNAzyme has been widely used in the field of DNA nanotechnology. To improve DNAzyme properties, we sought to elucidate the interaction mechanism between G-quadruplex DNA and hemin. Here, we performed volumetric analyses of formation of the complex between an oligonucleotide with the sequence of human telomeric DNA (h-telo) and hemin. The G-quadruplex DNA alone and the G-quadruplex DNA-hemin complex were destabilized with increasing pressure in Na + buffer. The pressure required to destabilize the h-telo-hemin complex was less in K + -containing buffer than in buffer with Na + , which indicates that there was a smaller volumetric change upon h-telo formation in K + buffer than in Na + buffer. The calculated change in h-telo-hemin binding volume (∆V b ) in the Na + buffer was 2.5mLmol -1 , whereas it was -41.7 in mLmol -1 the K + buffer. The DNAzyme activity in the K + buffer was higher than that in the Na + buffer at atmospheric pressure. Interestingly, the pressure effect on the destabilization of the h-telo-hemin complex in the presence of poly(ethylene glycol)200 (PEG200) was repressed compared to that in the absence of PEG200. These results suggest that differences in volumetric parameters reflect different mechanisms of interaction between hemin and h-telo due to differences in both the fit of hemin into the h-telo structure and hydration. Thus, the pressure-based thermodynamic analysis provided important information about complex formation and could be a useful index to improve function of DNAzymes. Copyright © 2016 Elsevier Inc. All rights reserved.

Rapid screening for nuclear genes mutations in isolated respiratory chain complex I defects.

PubMed

Pagniez-Mammeri, Hélène; Lombes, Anne; Brivet, Michèle; Ogier-de Baulny, Hélène; Landrieu, Pierre; Legrand, Alain; Slama, Abdelhamid

2009-04-01

Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype-genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.

DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells.

PubMed

Hu, Hao; Gu, Yuanlong; Qian, Yi; Hu, Benshun; Zhu, Congyuan; Wang, Gaohe; Li, Jianping

2014-09-12

Pancreatic cancer is one of the most aggressive human malignancies with extremely poor prognosis. The moderate activity of the current standard gemcitabine and gemcitabine-based regimens was due to pre-existing or acquired chemo-resistance of pancreatic cancer cells. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in gemcitabine resistance, and studied the underlying mechanisms. We found that NU-7026 and NU-7441, two DNA-PKcs inhibitors, enhanced gemcitabine-induced cytotoxicity and apoptosis in PANC-1 pancreatic cancer cells. Meanwhile, PANC-1 cells with siRNA-knockdown of DNA-PKcs were more sensitive to gemcitabine than control PANC-1 cells. Through the co-immunoprecipitation (Co-IP) assay, we found that DNA-PKcs formed a complex with SIN1, the latter is an indispensable component of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). DNA-PKcs-SIN1 complexation was required for Akt activation in PANC-1 cells, while inhibition of this complex by siRNA knockdown of DNA-PKcs/SIN1, or by DNA-PKcs inhibitors, prevented Akt phosphorylation in PANC-1 cells. Further, SIN1 siRNA-knockdown also facilitated gemcitabine-induced apoptosis in PANC-1 cells. Finally, DNA-PKcs and p-Akt expression was significantly higher in human pancreatic cancer tissues than surrounding normal tissues. Together, these results show that DNA-PKcs is important for Akt activation and gemcitabine resistance in PANC-1 pancreatic cancer cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Surfactant-cobalt(III) complexes: The impact of hydrophobicity on interaction with HSA and DNA - insights from experimental and theoretical approach.

PubMed

Veeralakshmi, Selvakumar; Sabapathi, Gopal; Nehru, Selvan; Venuvanalingam, Ponnambalam; Arunachalam, Sankaralingam

2017-05-01

To develop surfactant-based metallodrugs, it is very important to know about their hydrophobicity, micelle forming capacity, their interaction with biomacromolecules such as proteins and nucleic acids, and biological activities. Here, diethylenetriamine (dien) and tetradecylamine ligand (TA) based surfactant-cobalt(III) complexes with single chain domain, [Co(dien)(TA)Cl 2 ]ClO 4 (1) and double chain domain [Co(dien)(TA) 2 Cl](ClO 4 ) 2 (2) were chosen to study the effect of hydrophobicity on the interaction with human serum albumin and calf thymus DNA. The obtained results showed that (i) single chain surfactant-cobalt(III) complex (1) interact with HSA and DNA via electrostatic interaction and groove binding, respectively; (ii) double chain surfactant-cobalt(III) complex (2) interact with HSA and DNA via hydrophobic interaction and partial intercalation, respectively, due to the play of hydrophobicity by single and double chain domains. Further it is noted that, double chain surfactant-cobalt(III) complex interact strongly with HSA and DNA, compared single chain surfactant-cobalt(III) complex due to their more hydrophobicity nature. DFT and molecular docking studies offer insights into the mechanism and mode of binding towards the molecular target CT-DNA and HSA. Hence, the present findings will create new avenue towards the use of hydrophobic metallodrugs for various therapeutic applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

PubMed Central

Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

2016-01-01

CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

Functional Architecture of T7 RNA Polymerase Transcription Complexes

PubMed Central

Nayak, Dhananjaya; Guo, Qing; Sousa, Rui

2007-01-01

Summary T7 RNA polymerase is the best-characterized member of a widespread family of single-subunit RNA polymerases. Crystal structures of T7 RNA polymerase initiation and elongation complexes have provided a wealth of detailed information on RNA polymerase interactions with the promoter and transcription bubble, but the absence of DNA downstream of the melted region of the template in the initiation complex structure, and the absence of DNA upstream of the transcription bubble in the elongation complex structure means that our picture of the functional architecture of T7 RNA polymerase transcription complexes remains incomplete. Here we use the site-specifically tethered chemical nucleases and functional characterization of directed T7 RNAP mutants to both reveal the architecture of the duplex DNA that flanks the transcription bubble in the T7 RNAP initiation and elongation complexes, and to define the function of the interactions made by these duplex elements. We find that downstream duplex interactions made with a cluster of lysines (K711/K713/K714) are present during both elongation and initiation where they contribute to stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is also found to be sharply bent at the upstream edge of the transcription bubble, thereby allowing formation of upstream duplex:polymerase interactions that contribute to elongation complex stability. PMID:17580086

Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

PubMed

Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

2014-10-01

DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

Glycosaminoglycan-resistant and pH-sensitive lipid-coated DNA complexes produced by detergent removal method.

PubMed

Lehtinen, Julia; Hyvönen, Zanna; Subrizi, Astrid; Bunjes, Heike; Urtti, Arto

2008-10-21

Cationic polymers are efficient gene delivery vectors in in vitro conditions, but these carriers can fail in vivo due to interactions with extracellular polyanions, i.e. glycosaminoglycans (GAG). The aim of this study was to develop a stable gene delivery vector that is activated at the acidic endosomal pH. Cationic DNA/PEI complexes were coated by 1,2-dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) (3:2 mol/mol) using two coating methods: detergent removal and mixing with liposomes prepared by ethanol injection. Only detergent removal produced lipid-coated DNA complexes that were stable against GAGs, but were membrane active at low pH towards endosome mimicking liposomes. In relation to the low cellular uptake of the coated complexes, their transfection efficacy was relatively high. PEGylation of the coated complexes increased their cellular uptake but reduced the pH-sensitivity. Detergent removal was thus a superior method for the production of stable, but acid activatable, lipid-coated DNA complexes.

Mononuclear Pd(II) complex as a new therapeutic agent: Synthesis, characterization, biological activity, spectral and DNA binding approaches

NASA Astrophysics Data System (ADS)

Saeidifar, Maryam; Mirzaei, Hamidreza; Ahmadi Nasab, Navid; Mansouri-Torshizi, Hassan

2017-11-01

The binding ability between a new water-soluble palladium(II) complex [Pd(bpy)(bez-dtc)]Cl (where bpy is 2,2‧-bipyridine and bez-dtc is benzyl dithiocarbamate), as an antitumor agent, and calf thymus DNA was evaluated using various physicochemical methods, such as UV-Vis absorption, Competitive fluorescence studies, viscosity measurement, zeta potential and circular dichroism (CD) spectroscopy. The Pd(II) complex was synthesized and characterized using elemental analysis, molar conductivity measurements, FT-IR, 1H NMR, 13C NMR and electronic spectra studies. The anticancer activity against HeLa cell lines demonstrated lower cytotoxicity than cisplatin. The binding constants and the thermodynamic parameters were determined at different temperatures (300 K, 310 K and 320 K) and shown that the complex can bind to DNA via electrostatic forces. Furthermore, this result was confirmed by the viscosity and zeta potential measurements. The CD spectral results demonstrated that the binding of Pd(II) complex to DNA induced conformational changes in DNA. We hope that these results will provide a basis for further studies and practical clinical use of anticancer drugs.

Polyclonal antibodies against a structure mimicking the covalent linkage unit between picornavirus RNA and VPg: an immunochemical study.

PubMed

Ivanova, O A; Venyaminova, A G; Repkova, M N; Drygin, Yu F

2005-09-01

We propose that therapy of patients with anticancer drugs that poison DNA topoisomerases induces formation of covalent complexes of cellular RNAs and DNA topoisomerases. The appearance of these complexes can be detected with antibodies against a synthetic hapten mimicking the covalent linkage unit Tyr-pU(p) of picornavirus RNA and VPg. We synthesized hapten [N(Ac),CO(NH2)]Tyr-(5 P --> O)Up-O-(CH2)6NH2, conjugated it with BSA, and immunized rabbits with the antigen obtained. The raised polyclonal antibodies were purified by successive affinity chromatography on BSA-Sepharose and hapten-Sepharose columns. Target antibodies recognized hapten and encephalomyocarditis virus RNA-VPg complex specifically as found using the dot-immunogold method. We believe that these antibodies might be useful to study mechanism of picorna and similar virus RNA synthesis. The discovery and qualitative determination of the cellular RNA-DNA topoisomerases covalent complexes with these antibodies might be useful to monitor therapy efficacy by drugs "freezing" dead-end complexes of DNA topoisomerases and nucleic acids and to understand the mechanism of DNA topoisomerase poisoning in situ.

21 CFR 314.70 - Supplements and other changes to an approved application.

Code of Federal Regulations, 2014 CFR

2014-04-01

... derived from such studies; (vi) For a natural product, a recombinant DNA-derived protein/polypeptide, or a...) Changes solely affecting a natural protein, a recombinant DNA-derived protein/polypeptide or a complex or..., recombinant DNA-derived protein/polypeptide, complex or conjugate of a drug substance with a monoclonal...

Synthesis, spectroscopic characterization, DNA interaction and antibacterial study of metal complexes of tetraazamacrocyclic Schiff base

NASA Astrophysics Data System (ADS)

Shakir, Mohammad; Khanam, Sadiqa; Firdaus, Farha; Latif, Abdul; Aatif, Mohammad; Al-Resayes, Saud I.

The template condensation reaction between benzil and 3,4-diaminotoulene resulted mononuclear 12-membered tetraimine macrocyclic complexes of the type, [MLCl2] [M = Co(II), Ni(II), Cu(II) and Zn(II)]. The synthesized complexes have been characterized on the basis of the results of elemental analysis, molar conductance, magnetic susceptibility measurements and spectroscopic studies viz. FT-IR, 1H and 13C NMR, FAB mass, UV-vis and EPR. An octahedral geometry has been envisaged for all these complexes, while a distorted octahedral geometry has been noticed for Cu(II) complex. Low conductivity data of all these complexes suggest their non-ionic nature. The interactive studies of these complexes with calf thymus DNA showed that the complexes are avid binders of calf thymus DNA. The in vitro antibacterial studies of these complexes screened against pathogenic bacteria proved them as growth inhibiting agents.

Intrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA.

PubMed

Hyde, Eva I; Callow, Philip; Rajasekar, Karthik V; Timmins, Peter; Patel, Trushar R; Siligardi, Giuliano; Hussain, Rohanah; White, Scott A; Thomas, Christopher M; Scott, David J

2017-08-30

The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder. © 2017 The Author(s).

Detection of damaged DNA bases by DNA glycosylase enzymes.

PubMed

Friedman, Joshua I; Stivers, James T

2010-06-22

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Specific association of mouse MDC1/NFBD1 with NBS1 at sites of DNA-damage.

PubMed

Lee, Alicia C; Fernandez-Capetillo, Oscar; Pisupati, Venkat; Jackson, Stephen P; Nussenzweig, André

2005-01-01

Human MDC1/NFBD1 has been found to interact with key players of the DNA-damage response machinery. Here, we identify and describe a functional homologue of MDC1/ NFBD1 in Mus musculus. The mouse homologue, mMDC1, retains the key motifs identified in the human protein and in response to ionizing radiation forms foci that co-localize with the MRE11-RAD50-NBS1 (MRN) complex and factors such as gammaH2AX and 53BP1. In addition, mMDC1 is associated with DNA damage sites generated during meiotic recombination as well as the X and Y chromosomes during the late stages of meiotic prophase I. Finally, whereas MDC1 shows strong colocalization with the MRN complex in response to DNA damage it does not co-localize with the MRN complex on replicating chromatin. These data suggest that mMDC1 is a marker for both exogenously and endogenously generated DNA double-stranded breaks and that its interaction with the MRN complex is initiated exclusively by DNA damage.

Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA

PubMed Central

Jameson, Katie H; Rostami, Nadia; Fogg, Mark J; Turkenburg, Johan P; Grahl, Anne; Murray, Heath; Wilkinson, Anthony J

2014-01-01

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA-interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N-terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α-helical with predominantly apolar side-chains packing in a hydrophobic interface. Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP–SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor. PMID:25041308

Electrochemical DNA probe for Hg(2+) detection based on a triple-helix DNA and Multistage Signal Amplification Strategy.

PubMed

Wang, Huan; Zhang, Yihe; Ma, Hongmin; Ren, Xiang; Wang, Yaoguang; Zhang, Yong; Wei, Qin

2016-12-15

In this work, an ultrasensitive electrochemical sensor was developed for detection of Hg(2+). Gold nanoparticles decorated bovine serum albumin reduction of graphene oxide (AuNP-BSA-rGO) were used as subsurface material for the immobilization of triple-helix DNA. The triple-helix DNA containing a thiol labelled single-stranded DNA (sDNA) and a thymine-rich DNA (T-rich DNA), which could be unwinded in the present of Hg(2+) to form more stable thymine-Hg(2+)-thymine (T-Hg(2+)-T) complex. T-Hg(2+)-T complex was then removed and the sDNA was left on the electrode. At this time, gold nanoparticle carrying thiol labelled cytosine-rich complementary DNA (cDNA-AuNP) could bind with the free sDNA. Meanwhile, the other free cDNA on AuNP could bind with each other in the present of Ag(+) to form the stable cytosine-Ag(+)-cytosine (C-Ag(+)-C) complex and circle amplification. Plenty of C-Ag(+)-C could form silver nanoclusters by electrochemical reduction and the striping signal of Ag could be measured for purpose of the final electrochemical detection of Hg(2+). This sensor could detect Hg(2+) over a wide concentration range from 0.1 to 130nM with a detection limit of 0.03nM. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular dynamics studies on the DNA-binding process of ERG.

PubMed

Beuerle, Matthias G; Dufton, Neil P; Randi, Anna M; Gould, Ian R

2016-11-15

The ETS family of transcription factors regulate gene targets by binding to a core GGAA DNA-sequence. The ETS factor ERG is required for homeostasis and lineage-specific functions in endothelial cells, some subset of haemopoietic cells and chondrocytes; its ectopic expression is linked to oncogenesis in multiple tissues. To date details of the DNA-binding process of ERG including DNA-sequence recognition outside the core GGAA-sequence are largely unknown. We combined available structural and experimental data to perform molecular dynamics simulations to study the DNA-binding process of ERG. In particular we were able to reproduce the ERG DNA-complex with a DNA-binding simulation starting in an unbound configuration with a final root-mean-square-deviation (RMSD) of 2.1 Å to the core ETS domain DNA-complex crystal structure. This allowed us to elucidate the relevance of amino acids involved in the formation of the ERG DNA-complex and to identify Arg385 as a novel key residue in the DNA-binding process. Moreover we were able to show that water-mediated hydrogen bonds are present between ERG and DNA in our simulations and that those interactions have the potential to achieve sequence recognition outside the GGAA core DNA-sequence. The methodology employed in this study shows the promising capabilities of modern molecular dynamics simulations in the field of protein DNA-interactions.

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

PubMed Central

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

2013-01-01

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

Understanding the mechanism of DNA deactivation in ion therapy of cancer cells: hydrogen peroxide action*

NASA Astrophysics Data System (ADS)

Piatnytskyi, Dmytro V.; Zdorevskyi, Oleksiy O.; Perepelytsya, Sergiy M.; Volkov, Sergey N.

2015-11-01

Changes in the medium of biological cells under ion beam irradiation has been considered as a possible cause of cell function disruption in the living body. The interaction of hydrogen peroxide, a long-lived molecular product of water radiolysis, with active sites of DNA macromolecule was studied, and the formation of stable DNA-peroxide complexes was considered. The phosphate groups of the macromolecule backbone were picked out among the atomic groups of DNA double helix as a probable target for interaction with hydrogen peroxide molecules. Complexes consisting of combinations including: the DNA phosphate group, H2O2 and H2O molecules, and Na+ counterion, were considered. The counterions have been taken into consideration insofar as under the natural conditions they neutralise DNA sugar-phosphate backbone. The energy of the complexes have been determined by considering the electrostatic and the Van der Waals interactions within the framework of atom-atom potential functions. As a result, the stability of various configurations of molecular complexes was estimated. It was shown that DNA phosphate groups and counterions can form stable complexes with hydrogen peroxide molecules, which are as stable as the complexes with water molecules. It has been demonstrated that the formation of stable complexes of H2O2-Na+-PO4- may be detected experimentally by observing specific vibrations in the low-frequency Raman spectra. The interaction of H2O2 molecule with phosphate group of the double helix backbone can disrupt DNA biological function and induce the deactivation of the cell genetic apparatus. Thus, the production of hydrogen peroxide molecules in the nucleus of living cells can be considered as an additional mechanism by which high-energy ion beams destroy tumour cells during ion beam therapy. Contribution to the Topical Issue "COST Action Nano-IBCT: Nano-scale Processes Behind Ion-Beam Cancer Therapy", edited by Andrey Solov'yov, Nigel Mason, Gustavo García, Eugene Surdutovich.

Synthesis and structures of a pincer-type rhodium(iii) complex: reactivity toward biomolecules.

PubMed

Milutinović, Milan M; Bogojeski, Jovana V; Klisurić, Olivera; Scheurer, Andreas; Elmroth, Sofi K C; Bugarčić, Živadin D

2016-10-04

A novel rhodium(iii) complex [Rh III (H 2 L tBu )Cl 3 ] (1) (H 2 L tBu = 2,6-bis(5-tert-butyl-1H-pyrazol-3-yl)pyridine) containing a pincer type, tridentate nitrogen-donor chelate system was synthesized. Single crystal X-ray structure analysis revealed that 1 crystallizes in the orthorhombic space group Pbcn with a = 20.7982(6), b = 10.8952(4), c = 10.9832(4) Å, V = 2488.80(15) Å 3 , and eight molecules in the unit cell. The rhodium center in the complex [Rh III (H 2 L tBu )Cl 3 ] (1) is coordinated in a slightly distorted octahedral geometry by the tridentate N,N,N-donor and three chloro ligands, adopting a mer arrangement with an essentially planar ligand skeleton. Due to the tridentate coordination of the N,N,N-donor, the central nitrogen atom N1 is located closer to the Rh III center. The reactivity of the synthesized complex toward small biomolecules (l-methionine (l-Met), guanosine-5'-monophosphate (5'-GMP), l-histidine (l-His) and glutathione (GSH)) and to a series of duplex DNAs and RNA was investigated. The order of reactivity of the studied small biomolecules is: 5'-GMP > GSH > l-Met > l-His. Duplex RNA reacts faster with the [Rh III (H 2 L tBu )Cl 3 ] complex than duplex DNA, while shorter duplex DNA (15mer GG) reacts faster compared with 22mer GG duplex DNA. In addition, a higher reactivity is achieved with a DNA duplex with a centrally located GG-sequence than with a 22GTG duplex DNA, in which the GG-sequence is separated by a T base. Furthermore, the interaction of this metal complex 1 with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) was examined by absorption (UV-Vis) and emission spectral studies (EthBr displacement studies). Overall, the studied complex exhibited good DNA and BSA interaction ability.

Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.

PubMed Central

Slama-Schwok, A; Zakrzewska, K; Léger, G; Leroux, Y; Takahashi, M; Käs, E; Debey, P

2000-01-01

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation. PMID:10777751

Synthesis, molecular docking and DNA binding studies of phthalimide-based copper(II) complex: In vitro antibacterial, hemolytic and antioxidant assessment

NASA Astrophysics Data System (ADS)

Arif, Rizwan; Nayab, Pattan Sirajuddin; Ansari, Istikhar A.; Shahid, M.; Irfan, Mohammad; Alam, Shadab; Abid, Mohammad; Rahisuddin

2018-05-01

In the present research work, we prepared N-substituted phthalimide, 2-(-(2-(2-(2-(1,3-dioxoisoindoline-2-yl-ethylamino)ethylamino)ethyl)isoindoline-1,3-dione (DEEI) and its copper(II) complex. The ligand (DEEI) and its Cu(II) complex were structurally identified using absorption, FTIR, NMR, electron spin resonance, X-ray diffraction spectral studies, thermogravimetric and elemental analyses. The electronic spectrum and magnetic moment value proposed that Cu(II) complex has square planar geometry. The DNA interaction ability of the ligand (DEEI) and Cu(II) complex was studied by means of absorption and fluorescence spectrophotometer, viscosity measurements, cyclic voltammetery, and circular dichroism methods. The extent of DNA binding (Kb) with Calf thymus (Ct-DNA) follows the order of Cu(II) complex (1.11 × 106 M-1) > DEEI (1.0 × 105 M-1), indicating that Cu(II) complex interact with Ct-DNA through groove binding mode and more sturdily than ligand (DEEI). Interestingly, in silico predictions were corroborated with in vitro DNA binding studies. The antibacterial evaluation of these compounds was screened against a panel of bacterial strains Pseudomonas aeruginosa (MTCC 2453), Salmonella enterica (MTCC 3224), Streptococcus pneumoniae (MTCC 655), Enterococcus faecalis (MTCC 439), Klebsiella pneumonia and Escherichia coli (ATCC 25922). The results showed that the copper(II) complex has significant antibacterial potential (IC50 = 0.0019 μg/mL) against Salmonella enteric comparable with ligand (DEEI) and standard drug ciprofloxacin. Growth curve study of Cu(II) complex against only three bacterial strains S. enterica, E. faecalis and S. pneumoniae showed its bactericidal nature. Cu(II) complex showed less than 2% hemolysis on human RBCs indicating its non toxic nature. The results of antioxidant assay demonstrated that scavenging activity of Cu(II) complex is higher as compared to ligand and ascorbic acid as standard.

Quantitative Analysis of HIV-1 Preintegration Complexes

PubMed Central

Engelman, Alan; Oztop, Ilker; Vandegraaff, Nick; Raghavendra, Nidhanapati K.

2009-01-01

Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3′ processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3′ processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3′-OHs to the 5′-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3′ processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3′ processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3′ processing and DNA strand transfer activities. PMID:19233280

Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

PubMed Central

Kautz, Armin R.; Weisshart, Klaus; Schneider, Annerose; Grosse, Frank; Nasheuer, Heinz-Peter

2001-01-01

Although p48 is the most conserved subunit of mammalian DNA polymerase α-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag. PMID:11507202

Environmentally-relevant concentrations of Al(III) and Fe(III) cations induce aggregation of free DNA by complexation with phosphate group.

PubMed

Qin, Chao; Kang, Fuxing; Zhang, Wei; Shou, Weijun; Hu, Xiaojie; Gao, Yanzheng

2017-10-15

Environmental persistence of free DNA is influenced by its complexation with other chemical species and its aggregation mechanisms. However, it is not well-known how naturally-abundant metal ions, e.g., Al(III) and Fe(III), influence DNA aggregation. This study investigated aggregation behaviors of model DNA from salmon testes as influenced by metal cations, and elucidated the predominant mechanism responsible for DNA aggregation. Compared to monovalent (K + and Na + ) and divalent (Ca 2+ and Mg 2+ ) cations, Al(III) and Fe(III) species in aqueous solution caused rapid DNA aggregations. The maximal DNA aggregation occurred at 0.05 mmol/L Al(III) or 0.075 mmol/L Fe(III), respectively. A combination of atomic force microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy revealed that Al(III) and Fe(III) complexed with negatively charged phosphate groups to neutralize DNA charges, resulting in decreased electrostatic repulsion and subsequent DNA aggregation. Zeta potential measurements and molecular computation further support this mechanism. Furthermore, DNA aggregation was enhanced at higher temperature and near neutral pH. Therefore, DNA aggregation is collectively determined by many environmental factors such as ion species, temperature, and solution pH. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystallization and preliminary X-ray analysis of a complex formed between the antibiotic simocyclinone D8 and the DNA breakage–reunion domain of Escherichia coli DNA gyrase

PubMed Central

Edwards, Marcus J.; Flatman, Ruth H.; Mitchenall, Lesley A.; Stevenson, Clare E. M.; Maxwell, Anthony; Lawson, David M.

2009-01-01

Crystals of a complex formed between the 59 kDa N-terminal fragment of the Escherichia coli DNA gyrase A subunit (also known as the breakage–reunion domain) and the antibiotic simocyclinone D8 were grown by vapour diffusion. The complex crystallized with I-centred orthorhombic symmetry and X-ray data were recorded to a resolution of 2.75 Å from a single crystal at the synchrotron. DNA gyrase is an essential bacterial enzyme and thus represents an attractive target for drug development. PMID:19652356

Thermal treatment effects imposed on solid DNA cationic lipid complex with hexadecyltrimethylammonium chloride, observed by variable angle spectroscopic ellipsometry

NASA Astrophysics Data System (ADS)

Nizioł, Jacek

2014-12-01

DNA cationic lipid complexes are materials of properties required for applications in organic electronics and optoelectronics. Often, their thermal stability demonstrated by thermogravimetry is cited in the literature as important issue. However, little is known about processes occurring in heated solid DNA cationic lipid complexes. In frame of this work, thin films of Deoxyribonucleic acid-hexadecyltrimethylammonium chloride (DNA-CTMA) were deposited on silicon wafers. Samples were thermally annealed, and simultaneously, their optical functions were measured by spectroscopic ellipsometry. At lower temperatures, thermal expansion coefficient of solid DNA-CTMA was negative, but at higher temperatures positive. Thermally induced modification of absorption spectrum in UV-vis was observed. It occurred at a range of temperatures higher than this of DNA denaturation in solution. The observed phenomenon was irreversible, at least in time scale of the experiment (one day).

Effect of uv light on formation and synthetic capacity of DNA-membrane complexes from T7-infected cells of E. coli

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

Wintermantel, G.

1974-02-01

The action of uv light on the attachment of T7-DNA to the bacterial cell membrane, and the RNA- and DNA-synthesizing activity of the DNA-membrane complex, were investigated. In E. coli H560 cells infected by uv-irradiated T7 phages, the amount of membrane-associated parental /sup 32/P-labeled T7-DNA was determined. To measure the RNA- and DNA-synthesizing activity, the isolated DNA- membrane complexes were incubated with an equimolar mixture of the corresponding four nucleoside triphosphates. The uv-sensitivities of the functions tested were calculated from the slopes of the dose-effect curves. In relation to the uv- sensitivity for plaque-forming ability of the T7bacteriophages they amountmore » to 0.07 ( plus or minus 0.01); 0.15 ( plus or minus 0.01); and 0.56 ( plus or minus 0.04), respectively. (auth)« less

Structure of a preternary complex involving a prokaryotic NHEJ DNA polymerase.

PubMed

Brissett, Nigel C; Martin, Maria J; Pitcher, Robert S; Bianchi, Julie; Juarez, Raquel; Green, Andrew J; Fox, Gavin C; Blanco, Luis; Doherty, Aidan J

2011-01-21

In many prokaryotes, a specific DNA primase/polymerase (PolDom) is required for nonhomologous end joining (NHEJ) repair of DNA double-strand breaks (DSBs). Here, we report the crystal structure of a catalytically active conformation of Mycobacterium tuberculosis PolDom, consisting of a polymerase bound to a DNA end with a 3' overhang, two metal ions, and an incoming nucleotide but, significantly, lacking a primer strand. This structure represents a polymerase:DNA complex in a preternary intermediate state. This polymerase complex occurs in solution, stabilizing the enzyme on DNA ends and promoting nucleotide extension of short incoming termini. We also demonstrate that the invariant Arg(220), contained in a conserved loop (loop 2), plays an essential role in catalysis by regulating binding of a second metal ion in the active site. We propose that this NHEJ intermediate facilitates extension reactions involving critically short or noncomplementary DNA ends, thus promoting break repair and minimizing sequence loss during DSB repair. Copyright © 2011 Elsevier Inc. All rights reserved.

Spectral investigations on binding of DNA-CTMA complex with tetrameric copper phthalocyanines

NASA Astrophysics Data System (ADS)

Venkat, Narayanan; Haley, Joy E.; Swiger, Rachel; Zhu, Lei; Wei, Xiaoliang; Ouchen, Fahima; Grote, James G.

2013-10-01

The binding of DNA-CTMA (Deoxyribonucleic acid-cetyltrimethylammonium) complex with two tetrameric Copper Phthalocyanine (CuPc) systems, substituted with carboxylic acid (CuPc-COOH) and derivatized further as an imidazolium salt (CuPc-COOR), was investigated in dimethylsulfoxide (DMSO) solutions using UV/Visible Spectroscopy. Absorbance changes at 685 nm (Q band of the CuPc) were monitored as a function of DNA-CTMA added to the dye solution and stock concentrations of DNA-CTMA in DMSO were varied to facilitate observation of the full binding process. Our findings indicated that while binding with DNA-CTMA was more well-defined in the case of CuPc-COOH, the binding profile of the CuPc-COOR showed initial growth followed by decay in its Q-band absorbance which was indicative of a more complex binding mechanism involving the dye and DNA-CTMA. Preliminary findings from photophysical studies involving the CuPc tetramers and DNA-CTMA are also discussed in this paper.

Structural analysis of a eukaryotic sliding DNA clamp-clamp loadercomplex.

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

Bowman, Gregory D.; O'Donnell, Mike; Kuriyan, John

2006-06-17

Sliding clamps are ring-shaped proteins that encircle DNA and confer high processivity on DNA polymerases. Here we report the crystal structure of the five-protein clamp loader complex (replication factor-C, RFC) of the yeast Saccharomyces cerevisiae, bound to the sliding clamp (proliferating cell nuclear antigen, PCNA). Tight interfacial coordination of the ATP analogue ATP-?-S by RFC results in a spiral arrangement of the ATPase domains of the clamp loader above the PCNA ring. Placement of a model for primed DNA within the central hole of PCNA reveals a striking correspondence between the RFC spiral and the grooves of the DNA doublemore » helix. This model, in which the clamp loader complex locks onto primed DNA in a screw-cap-like arrangement, provides a simple explanation for the process by which the engagement of primer-template junctions by the RFC:PCNA complex results in ATP hydrolysis and release of the sliding clamp on DNA.« less

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

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

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Addition of an RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σA dissociation.« less

Structure of a bacterial RNA polymerase holoenzyme open promoter complex

DOE PAGES

Bae, Brian; Feklistov, Andrey; Lass-Napiorkowska, Agnieszka; ...

2015-09-08

Initiation of transcription is a primary means for controlling gene expression. In bacteria, the RNA polymerase (RNAP) holoenzyme binds and unwinds promoter DNA, forming the transcription bubble of the open promoter complex (RPo). We have determined crystal structures, refined to 4.14 Å-resolution, of RPo containing Thermus aquaticus RNAP holoenzyme and promoter DNA that includes the full transcription bubble. The structures, combined with biochemical analyses, reveal key features supporting the formation and maintenance of the double-strand/single-strand DNA junction at the upstream edge of the -10 element where bubble formation initiates. The results also reveal RNAP interactions with duplex DNA just upstreammore » of the -10 element and potential protein/DNA interactions that direct the DNA template strand into the RNAP active site. Additionally a RNA primer to yield a 4 base-pair post-translocated RNA:DNA hybrid mimics an initially transcribing complex at the point where steric clash initiates abortive initiation and σ A dissociation.« less

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA

PubMed Central

Mori, Tetsuya; Saveliev, Sergei V.; Xu, Yao; Stafford, Walter F.; Cox, Michael M.; Inman, Ross B.; Johnson, Carl H.

2002-01-01

KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecA/DnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecA/DnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns. PMID:12477935

Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.

PubMed

Brewer, Laurence R; Friddle, Raymond; Noy, Aleksandr; Baldwin, Enoch; Martin, Shelley S; Corzett, Michele; Balhorn, Rod; Baskin, Ronald J

2003-10-01

Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

Jab1 Mediates Protein Degradation of Rad9/Rad1/Hus1 Checkpoint Complex

PubMed Central

Huang, Jin; Yuan, Honglin; Lu, Chongyuan; Liu, Ximeng; Cao, Xu; Wan, Mei

2009-01-01

Summary The Rad1-Rad9-Hus1 (9-1-1) complex serves a dual role as a DNA-damage sensor in checkpoint signaling and as a mediator in DNA repair pathway. However, the intercellular mechanisms that regulate 9-1-1 complex are poorly understood. Jab1, the fifth component of the COP9 signalosome complex, plays a central role in the degradation of multiple proteins and is emerging as an important regulator in cancer development. Here, we tested the hypothesis that Jab1 controls the protein stability of the 9-1-1 complex via the proteosome pathway. We provide evidence that Jab1 physically associates with the 9-1-1 complex. This association is mediated through direct interaction between Jab1 and Rad1, one of the subunits of 9-1-1 complex. Importantly, Jab1 causes the translocation of the 9-1-1 complex from the nucleus to the cytoplasm, mediating rapid degradation of the 9-1-1 complex via 26S proteasome. Furthermore, Jab1 significantly suppresses checkpoint signaling activation, DNA synthesis recovery from blockage and cell viability after replication stresses such as UV exposure, γ radiation and hydroxyurea treatment. These results suggest that Jab1 is an important regulator for 9-1-1 protein stability control in cells, which may provide novel information on the involvement of Jab1 in checkpoint and DNA repair signaling in response to DNA damage. PMID:17583730

Efficient plasmid DNA cleavage by a mononuclear copper(II) complex.

PubMed

Sissi, Claudia; Mancin, Fabrizio; Gatos, Maddalena; Palumbo, Manlio; Tecilla, Paolo; Tonellato, Umberto

2005-04-04

The Cu(II) complex of the ligand all-cis-2,4,6-triamino-1,3,5-trihydroxycyclohexane (TACI) is a very efficient catalyst of the cleavage of plasmid DNA in the absence of any added cofactor. The maximum rate of degradation of the supercoiled plasmid DNA form, obtained at pH 8.1 and 37 degrees C, in the presence of 48 microM TACI.Cu(II), is 2.3 x 10(-3) s(-1), corresponding to a half-life time of only 5 min for the cleavage of form I (supercoiled) to form II (relaxed circular). The dependence of the rate of plasmid DNA cleavage from the TACI.Cu(II) complex concentration follows an unusual and very narrow bell-like profile, which suggests an high DNA affinity of the complexes but also a great tendency to form unreactive dimers. The reactivity of the TACI.Cu(II) complexes is not affected by the presence of several scavengers for reactive oxygen species or when measured under anaerobic conditions. Moreover, no degradation of the radical reporter Rhodamine B is observed in the presence of such complexes. These results are consistent with the operation of a prevailing hydrolytic pathway under the normal conditions used, although the failure to obtain enzymatic religation of the linearized DNA does not allow one to rule out the occurrence of a nonhydrolytic oxygen-independent cleavage. A concurrent oxidative mechanism becomes competitive upon addition of reductants or in the presence of high levels of molecular oxygen: under such conditions, in fact, a remarkable increase in the rate of DNA cleavage is observed.

Imparting the unique properties of DNA into complex material architectures and functions.

PubMed

Xu, Phyllis F; Noh, Hyunwoo; Lee, Ju Hun; Domaille, Dylan W; Nakatsuka, Matthew A; Goodwin, Andrew P; Cha, Jennifer N

2013-07-01

While the remarkable chemical and biological properties of DNA have been known for decades, these properties have only been imparted into materials with unprecedented function much more recently. The inimitable ability of DNA to form programmable, complex assemblies through stable, specific, and reversible molecular recognition has allowed the creation of new materials through DNA's ability to control a material's architecture and properties. In this review we discuss recent progress in how DNA has brought unmatched function to materials, focusing specifically on new advances in delivery agents, devices, and sensors.

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

PubMed Central

Felgner, P L; Gadek, T R; Holm, M; Roman, R; Chan, H W; Wenz, M; Northrop, J P; Ringold, G M; Danielsen, M

1987-01-01

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique. Images PMID:2823261

Exploring the DNA binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases and their platinum(II) complexes.

PubMed

Neves, Amanda P; Pereira, Michelle X G; Peterson, Erica J; Kipping, Ralph; Vargas, Maria D; Silva, Floriano P; Carneiro, J Walkimar M; Farrell, Nicholas P

2013-02-01

Several chlorido and amino Pt(2+) complexes of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases HL exhibiting moderate to high cytotoxicity against cancer cell lines were studied in order to investigate their modes of DNA binding, in vitro DNA strand breaks, mechanism of topoisomerase (Topo I) inhibition and cellular accumulation. DNA model base studies have shown that complex 1a [Pt(HL1)Cl(2)] was capable of binding covalently to 9-ethylguanine (9-EtG) and 5'-GMP. (1)H NMR and mass spectrometry studies have shown that both chlorides were substituted by 9-EtG ligands, whereas 5'-GMP was able to replace only one chlorido ligand, due to steric hindrance. The chlorido Pt(2+) complexes [Pt(HL)Cl(2)] highly accumulate in prostate (PC-3) and melanoma (MDA-MB-435) cell lines, being able to induce DNA strand breaks in vitro and inhibit Topo I by a catalytic mode. On the other hand, the free 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones HL and the amino Pt(2+) complexes [Pt(L(-))(NH(3))(2)]NO(3) neither cause DNA strand breakage nor exhibit strong DNA interaction, nevertheless the latter were also found to be catalytic inhibitors of Topo I at 100μM. Thus, coordination of the Mannich bases HL to the "PtCl(2)" fragment substantially affects the chemical and biophysical properties of the pro-ligands, leading to an improvement of their DNA binding properties and generating compounds that cleave DNA and catalytically inhibit Topo I. Finally, the high cytotoxicity exhibited by the free (uncomplexed) 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones might be associated with their decomposition in solution, which is not observed for the Pt(2+) complexes. Copyright © 2012 Elsevier Inc. All rights reserved.

The electrostatic role of the Zn-Cys2His2 complex in binding of operator DNA with transcription factors: mouse EGR-1 from the Cys2His2 family.

PubMed

Chirgadze, Y N; Boshkova, E A; Polozov, R V; Sivozhelezov, V S; Dzyabchenko, A V; Kuzminsky, M B; Stepanenko, V A; Ivanov, V V

2018-01-07

The mouse factor Zif268, known also as early growth response protein EGR-1, is a classical representative for the Cys2His2 transcription factor family. It is required for binding the RNA polymerase with operator dsDNA to initialize the transcription process. We have shown that only in this family of total six Zn-finger protein families the Zn complex plays a significant role in the protein-DNA binding. Electrostatic feature of this complex in the binding of factor Zif268 from Mus musculus with operator DNA has been considered. The factor consists of three similar Zn-finger units which bind with triplets of coding DNA. Essential contacts of the factor with the DNA phosphates are formed by three conservative His residues, one in each finger. We describe here the results of calculations of the electrostatic potentials for the Zn-Cys2His2 complex, Zn-finger unit 1, and the whole transcription factor. The potential of Zif268 has a positive area on the factor surface, and it corresponds exactly to the binding sites of each of Zn-finger units. The main part of these areas is determined by conservative His residues, which form contacts with the DNA phosphate groups. Our result shows that the electrostatic positive potential of this histidine residue is enhanced due to the Zn complex. The other contacts of the Zn-finger with DNA are related to nucleotide bases, and they are responsible for the sequence-specific binding with DNA. This result may be extended to all other members of the Cys2His2 transcription factor family.

The Chemical and Biological Effects of cis-Dichlorodiammineplatinum (II), an Antitumor Agent, on DNA

PubMed Central

Munchausen, Linda L.

1974-01-01

cis-Dichlorodiammineplatinum (II) binds irreversibly to the bases in DNA; the amount of platinum complex bound can be determined from changes in the ultraviolet absorption spectrum. As the ratio of platinum to phosphate is increased, an increasing inactivation of bacterial transforming DNA is observed. At a ratio that corresponds to spectrometric saturation, transforming activity is inactivated >105-fold. The trans isomer of the platinum complex, which is not effective against tumors, induces a similar inactivation of transforming DNA but with half the efficiency, indicating a different mode of binding. The sensitivity to inactivation by cis isomer varies slightly with the genetic marker assayed but is not dependent on the excision repair system. Uptake of DNA by competent cells is unaffected by bound platinum complex; however, integration of platinum-bound transforming DNA into the host genome decreases as the mole fraction of platinum increases. This loss of integration parallels the decreased transforming activity of the DNA. Although the drug induces interstrand crosslinks in DNA in vitro, these crosslinks are relatively rare events and cannot account for the observed inactivation. PMID:4548188

DNA AND THE FINE STRUCTURE OF SYNAPTIC CHROMOSOMES IN THE DOMESTIC ROOSTER (GALLUS DOMESTICUS)

PubMed Central

Coleman, James R.; Moses, Montrose J.

1964-01-01

The indium trichloride method of Watson and Aldridge (38) for staining nucleic acids for electron microscopy was employed to study the relationship of DNA to the structure of the synaptinemal complex in meiotic prophase chromosomes of the domestic rooster. The selectivity of the method was demonstrated in untreated and DNase-digested testis material by comparing the distribution of indium staining in the electron microscope to Feulgen staining and ultraviolet absorption in thicker sections seen with the light microscope. Following staining by indium, DNA was found mainly in the microfibril component of the synaptinemal complex. When DNA was known to have been removed from aldehyde-fixed material by digestion with DNase, indium stainability was also lost. However, staining of the digested material with non-selective heavy metal techniques demonstrated the presence of material other than DNA in the microfibrils and showed that little alteration in appearance of the chromosome resulted from DNA removal. The two dense lateral axial elements of the synaptinemal complex, but not the central one to any extent, also contained DNA, together with non-DNA material. PMID:14228519

Two sides of the same coin: TFIIH complexes in transcription and DNA repair.

PubMed

Zhovmer, Alexander; Oksenych, Valentyn; Coin, Frédéric

2010-04-13

TFIIH is organized into a seven-subunit core associated with a three-subunit Cdk-activating kinase (CAK) module. TFIIH has roles in both transcription initiation and DNA repair. During the last 15 years, several studies have been conducted to identify the composition of the TFIIH complex involved in DNA repair. Recently, a new technique combining chromatin immunoprecipitation and western blotting resolved the hidden nature of the TFIIH complex participating in DNA repair. Following the recruitment of TFIIH to the damaged site, the CAK module is released from the core TFIIH, and the core subsequently associates with DNA repair factors. The release of the CAK is specifically driven by the recruitment of the DNA repair factor XPA and is required to promote the incision/excision of the damaged DNA. Once the DNA lesions have been repaired, the CAK module returns to the core TFIIH on the chromatin, together with the release of the repair factors. These data highlight the dynamic composition of a fundamental cellular factor that adapts its subunit composition to the cell needs.

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

PubMed Central

Kosturko, L D; Daub, E; Murialdo, H

1989-01-01

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure. Images PMID:2521383

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

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

Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at

2012-01-20

Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less

Programmable DNA tile self-assembly using a hierarchical sub-tile strategy.

PubMed

Shi, Xiaolong; Lu, Wei; Wang, Zhiyu; Pan, Linqiang; Cui, Guangzhao; Xu, Jin; LaBean, Thomas H

2014-02-21

DNA tile based self-assembly provides a bottom-up approach to construct desired nanostructures. DNA tiles have been directly constructed from ssDNA and readily self-assembled into 2D lattices and 3D superstructures. However, for more complex lattice designs including algorithmic assemblies requiring larger tile sets, a more modular approach could prove useful. This paper reports a new DNA 'sub-tile' strategy to easily create whole families of programmable tiles. Here, we demonstrate the stability and flexibility of our sub-tile structures by constructing 3-, 4- and 6-arm DNA tiles that are subsequently assembled into 2D lattices and 3D nanotubes according to a hierarchical design. Assembly of sub-tiles, tiles, and superstructures was analyzed using polyacrylamide gel electrophoresis and atomic force microscopy. DNA tile self-assembly methods provide a bottom-up approach to create desired nanostructures; the sub-tile strategy adds a useful new layer to this technique. Complex units can be made from simple parts. The sub-tile approach enables the rapid redesign and prototyping of complex DNA tile sets and tiles with asymmetric designs.

Translational Dielectric Friction on a Chain of Charged Spheres

PubMed Central

Boughammoura, Sondès; M'halla, Jalel

2014-01-01

We have proved in details that the dielectric friction remains the principal frictional effect for a stretched polyion modeled as a chain of charged spheres, whereas, in the case of Manning's model (infinite thread with a continuous distribution of charge), this friction effect is nonexistent. According to this chain model, it is therefore possible to detect by conductivity measurements any transition from a coiled configuration (ellipsoidal model) to a stretched configuration during dilution process. We have also underlined the important interdependence between the dielectric friction and the ionic condensation of the counterions, in order to distinguish between the Ostwald regime and the Manning regime for which the degree of condensation is practically constant in a large range of concentrations. PMID:24672333

[Principles for intensive therapy of status epilepticus in children].

PubMed

Bondarenko, E S; Freĭdkov, V I

1979-01-01

A total of 113 children with brain edema of infectious and toxic nature were studied. The main clinical syndrome in these conditions was an epileptical status. Among anticonvulsive drugs used seduxen appeared to be the most effective. The use of GABA, gas narcosis and myorelaxants is recommended as well. The rehydration by plasma, low molecular plasma substitutes and glucose-polyion solutions are discussed. The paper contains some information on contraindications for early dehydration and rehydration. Hypoxia is eliminated by providing adequate lung ventilation, oxygenotherapy or hyperbaric oxygenation, by using oxidative enzymes and drugs increasing the resistance of the brain to hypoxia and hypothermia. It is recommended that hormonal therapy should be used. The techniques of urgent measures and their sequence are given.

Energetic basis for selective recognition of T*G mismatched base pairs in DNA by imidazole-rich polyamides.

PubMed

Lacy, Eilyn R; Nguyen, Binh; Le, Minh; Cox, Kari K; OHare, Caroline; Hartley, John A; Lee, Moses; Wilson, W David

2004-01-01

To complement available structure and binding results and to develop a detailed understanding of the basis for selective molecular recognition of T.G mismatches in DNA by imidazole containing polyamides, a full thermodynamic profile for formation of the T.G-polyamide complex has been determined. The amide-linked heterocycles f-ImImIm and f-PyImIm (where f is formamido group, Im is imidazole and Py is pyrrole) were studied by using biosensor-surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) with a T.G mismatch containing DNA hairpin duplex and a similar DNA with only Watson-Crick base pairs. Large negative binding enthalpies for all of the polyamide-DNA complexes indicate that the interactions are enthalpically driven. SPR results show slower complex formation and stronger binding of f-ImImIm to the T.G than to the match site. The thermodynamic analysis indicates that the enhanced binding to the T.G site is the result of better entropic contributions. Negative heat capacity changes for the complex are correlated with calculated solvent accessible surface area changes and indicate hydrophobic contributions to complex formation. DNase I footprinting analysis in a long DNA sequence provided supporting evidence that f-ImImIm binds selectively to T.G mismatch sites.

Energetic basis for selective recognition of T·G mismatched base pairs in DNA by imidazole-rich polyamides

PubMed Central

Lacy, Eilyn R.; Nguyen, Binh; Le, Minh; Cox, Kari K.; O'Hare, Caroline; Hartley, John A.; Lee, Moses; Wilson, W. David

2004-01-01

To complement available structure and binding results and to develop a detailed understanding of the basis for selective molecular recognition of T·G mismatches in DNA by imidazole containing polyamides, a full thermodynamic profile for formation of the T·G–polyamide complex has been determined. The amide-linked heterocycles f-ImImIm and f-PyImIm (where f is formamido group, Im is imidazole and Py is pyrrole) were studied by using biosensor-surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) with a T·G mismatch containing DNA hairpin duplex and a similar DNA with only Watson–Crick base pairs. Large negative binding enthalpies for all of the polyamide–DNA complexes indicate that the interactions are enthalpically driven. SPR results show slower complex formation and stronger binding of f-ImImIm to the T·G than to the match site. The thermodynamic analysis indicates that the enhanced binding to the T·G site is the result of better entropic contributions. Negative heat capacity changes for the complex are correlated with calculated solvent accessible surface area changes and indicate hydrophobic contributions to complex formation. DNase I footprinting analysis in a long DNA sequence provided supporting evidence that f-ImImIm binds selectively to T·G mismatch sites. PMID:15064359

Retrosynthetic Analysis-Guided Breaking Tile Symmetry for the Assembly of Complex DNA Nanostructures.

PubMed

Wang, Pengfei; Wu, Siyu; Tian, Cheng; Yu, Guimei; Jiang, Wen; Wang, Guansong; Mao, Chengde

2016-10-11

Current tile-based DNA self-assembly produces simple repetitive or highly symmetric structures. In the case of 2D lattices, the unit cell often contains only one basic tile because the tiles often are symmetric (in terms of either the backbone or the sequence). In this work, we have applied retrosynthetic analysis to determine the minimal asymmetric units for complex DNA nanostructures. Such analysis guides us to break the intrinsic structural symmetries of the tiles to achieve high structural complexities. This strategy has led to the construction of several DNA nanostructures that are not accessible from conventional symmetric tile designs. Along with previous studies, herein we have established a set of four fundamental rules regarding tile-based assembly. Such rules could serve as guidelines for the design of DNA nanostructures.

Atomic Simulation of Complex DNA DSBs and the Interactions with the Ku70/80 Heterodimer

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2011-01-01

DNA double strand breaks (DSBs) induced by ionizing radiation (IR) usually contain modified bases such as 8-oxo-7,8-dihydroguanine (8-oxoG) and thymine glycol, apurinic/apyrimidinic (AP) sites, 2-deoxyribonolactone, or single-strand breaks (SSBs). The presence of such lesions in close proximity to the DSB terminus makes the DNA nicks more difficult to repair and rejoin than endogenously induced simple DSBs, and as such a major determinant of the biological effects of high linear energy transfer (LET) radiation as encountered in space travel. In this study we conducted molecular dynamics simulations on a series of DNA duplexes with various complex lesions of 8-oxoG and AP sites, in an effort to investigate the effects of such lesions to the structural integrity and stability of DNA after insulted by IR. We also simulated the interaction of such complex DSBs with the Ku70/80 heterodimer, the first protein in mammalian cells to embark the non-homologous end joining (NHEJ) DNA repair pathway. The results indicate, compared to DNA with simple DSBs, the complex lesions can enhance the hydrogen bonds opening rate at the DNA terminus, and increase the mobility of the whole duplex, thus they present more deleterious effects to the genome integrity if not captured and repaired promptly in cells. Simulations also demonstrate the binding of Ku drastically reduces structural disruption and flexibility caused by the complex lesions, and the interactions of Ku with complex DSBs have a different potential energy landscape from the bound structure with simple DSB. In all complex DSBs systems, the binding of DSB terminus with Ku70 is softened while the binding of the middle duplex with Ku80 is tightened. This energy shift may help the Ku protein to secure at the DSB terminus for a longer time, so that other end processing factors or repair pathways can proceed at the lesions before NHEJ repair process starts. These atomic simulations may provide valuable new insight into the selective action of repair proteins on damaged DNA.

Recognition of thymine in DNA bulges by a Zn(II) macrocyclic complex.

PubMed

del Mundo, Imee Marie A; Fountain, Matthew A; Morrow, Janet R

2011-08-14

A Zn(II) macrocyclic complex with appended quinoline is a bifunctional recognition agent that uses both the Zn(II) center and the pendent aromatic group to bind to thymine in bulges with good selectivity over DNA containing G, C or A bulges. Spectroscopic studies show that the stem containing the bulge stays largely intact in a DNA hairpin with the Zn(II) complex bound to the thymine bulge. This journal is © The Royal Society of Chemistry 2011

Synthesis, characterization, redox behavior, DNA and protein binding and antibacterial activity studies of ruthenium(II) complexes of bidentate schiff bases.

PubMed

Paul, Hena; Sen, Buddhadeb; Mondal, Tapan Kumar; Chattopadhyay, Pabitra

2017-08-03

Two new ruthenium(II) complexes of Schiff base ligands (L) derived from cinnamaldehyde and ethylenediamine formulated as [Ru(L)(bpy) 2 ](ClO 4 ) 2 , where L 1 = N,N'-bis(4-nitrocinnamald-ehyde)ethylenediamine and L 2 = N,N'-bis(2-nitrocinnamaldehyde)-ethylenediamine for complex 1 and 2, respectively, were isolated in pure form. The complexes were characterized by physicochemical and spectroscopic methods. The electrochemical behavior of the complexes showed the Ru(III)/Ru(II) couple at different potentials with quasi-reversible voltammograms. The interaction of the complexes with calf thymus DNA (CT-DNA) using absorption, emission spectral studies and electrochemical techniques have been used to determine the binding constant, K b and the linear Stern-Volmer quenching constant, K SV . The results indicate that the ruthenium(II) complexes interact with CT-DNA strongly in a groove binding mode. The interactions of bovine serum albumin (BSA) with the complexes were also investigated with the help of absorption and fluorescence spectroscopy tools. Absorption spectroscopy proved the formation of a ground state BSA-[Ru(L)(bpy) 2 ](ClO 4 ) 2 complex. The antibacterial study showed that the Ru(II) complexes (1 and 2) have better activity than the standard antibiotics but weak activity than the ligands.

Non-enolisable Knoevenagel condensate appended Schiff bases-metal (II) complexes: Spectral characteristics, DNA-binding and nuclease activities

NASA Astrophysics Data System (ADS)

Gubendran, Ammavasi; Kesavan, Mookkandi Palsamy; Ayyanaar, Srinivasan; Mitu, Liviu; Athappan, Periyakaruppan; Rajesh, Jegathalaprathaban

2017-06-01

New Schiff base complexes [Cu(L1)Cl] (1), [Ni(L1)Cl] (2), [Zn(L1)Cl] (3), and [Fe(L2)H2OCl] (4) {L1 = (4E)-3-(2-hydroxybenzylidene)-4-(2-hydroxyphenylimino)pentan-2-one, L2 = 2,2‧-(1E,1‧E)-(3-(2-hydroxybenzylidene)-pentane-2,4-diylidene)bis(azan-1-yl-1 idene)diphenol} have been synthesized and characterized by elemental analysis, UV-Vis, IR, FAB-mass, EPR, spectral studies and electrochemical studies, the ligands L1 &L2 were characterized by 1H and 13C NMR spectra. Complex 1 show a visible spectral d-d band near 600 nm and display cyclic voltammetric quasireversible response for the Cu(II)/Cu(I) couple vs Ag/AgCl in DMSO. The EPR spectrum of 1 show g‖ > g⊥ suggesting a square planar geometry around copper with dx2 - y2 as the ground state. The mass spectral results have confirmed the proposed structure for complexes 1-4. DNA binding properties of these complexes 1-4 have been investigated by absorption titrations, cyclic voltammetric studies and circular dichroism studies. On titration with DNA, the complexes 1-4 show hypochromism at the MLCT band (13-31%) with a red shift of 1-8 nm in the electronic spectrum and positive shift of voltammetric E1/2 in the CV studies are in favour of intercalative binding. CD spectra of 1 showed an increase in molar ellipticity (θ278) of the positive band with a minor red shift indicating the transition of B-form of DNA to A like form. DNA cleavage studies of complexes 1 and 4 with pUC18 DNA were studied by gel electrophoresis and complex 4 cleaves supercoiled pUC18 DNA in an oxidative manner in the presence of H2O2 and on photo irradiation at 312 nm.