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Sample records for relate complex dna

  1. Accurate measurement of the relative abundance of different DNA species in complex DNA mixtures.

    PubMed

    Jeong, Sangkyun; Yu, Hyunjoo; Pfeifer, Karl

    2012-06-01

    A molecular tool that can compare the abundances of different DNA sequences is necessary for comparing intergenic or interspecific gene expression. We devised and verified such a tool using a quantitative competitive polymerase chain reaction approach. For this approach, we adapted a competitor array, an artificially made plasmid DNA in which all the competitor templates for the target DNAs are arranged with a defined ratio, and melting analysis for allele quantitation for accurate quantitation of the fractional ratios of competitively amplified DNAs. Assays on two sets of DNA mixtures with explicitly known compositional structures of the test sequences were performed. The resultant average relative errors of 0.059 and 0.021 emphasize the highly accurate nature of this method. Furthermore, the method's capability of obtaining biological data is demonstrated by the fact that it can illustrate the tissue-specific quantitative expression signatures of the three housekeeping genes G6pdx, Ubc, and Rps27 by using the forms of the relative abundances of their transcripts, and the differential preferences of Igf2 enhancers for each of the multiple Igf2 promoters for the transcription.

  2. Accurate Measurement of the Relative Abundance of Different DNA Species in Complex DNA Mixtures

    PubMed Central

    Jeong, Sangkyun; Yu, Hyunjoo; Pfeifer, Karl

    2012-01-01

    A molecular tool that can compare the abundances of different DNA sequences is necessary for comparing intergenic or interspecific gene expression. We devised and verified such a tool using a quantitative competitive polymerase chain reaction approach. For this approach, we adapted a competitor array, an artificially made plasmid DNA in which all the competitor templates for the target DNAs are arranged with a defined ratio, and melting analysis for allele quantitation for accurate quantitation of the fractional ratios of competitively amplified DNAs. Assays on two sets of DNA mixtures with explicitly known compositional structures of the test sequences were performed. The resultant average relative errors of 0.059 and 0.021 emphasize the highly accurate nature of this method. Furthermore, the method's capability of obtaining biological data is demonstrated by the fact that it can illustrate the tissue-specific quantitative expression signatures of the three housekeeping genes G6pdx, Ubc, and Rps27 by using the forms of the relative abundances of their transcripts, and the differential preferences of Igf2 enhancers for each of the multiple Igf2 promoters for the transcription. PMID:22334570

  3. How to Relate Complex DNA Repair Genotypes to Pathway Function and, Ultimately, Health Risk

    SciTech Connect

    Jones, IM

    2002-01-09

    of this pilot project was to obtain preliminary data on genetic variation in DNA repair function in human cells that might encourage our efforts to establish a research program to relate DNA repair function to complex DNA repair genotype and ultimately to cancer risk of radiation exposure.

  4. Using epigenome-wide association scans of DNA methylation in age-related complex human traits.

    PubMed

    Tsai, Pei-Chien; Spector, Tim D; Bell, Jordana T

    2012-10-01

    With rapid technological advancements emerging epigenetic studies of complex traits have shifted from candidate gene analyses towards epigenome-wide association studies (EWAS). EWAS aim to systematically identify epigenetic variants across the genome that associate with complex phenotypes. Recent EWAS using case-control and disease-discordant identical twin designs have identified phenotype-associated differentially methylated regions for several traits. However, EWAS still face many challenges related to methodology, design and interpretation, owing to the dynamic nature of epigenetic variants over time. This article reviews analytical considerations in conducting EWAS and recent applications of this approach to human aging and age-related complex traits. PMID:23130833

  5. Distinguishing between donors and their relatives in complex DNA mixtures with binary models.

    PubMed

    Slooten, K

    2016-03-01

    While likelihood ratio calculations were until the recent past limited to the evaluation of mixtures in which all alleles of all donors are present in the DNA mixture profile, more recent methods are able to deal with allelic dropout and drop-in. This opens up the possibility to obtain likelihood ratios for mixtures where this was not previously possible, but it also means that a full match between the alleged contributor and the crime stain is no longer necessary. We investigate in this article what the consequences are for relatives of the actual donors, because they typically share more alleles with the true donor than an unrelated individual. We do this with a semi-continuous binary approach, where the likelihood ratios are based on the observed alleles and the dropout probabilities for each donor, but not on the peak heights themselves. These models are widespread in the forensic community. Since in many cases a simple model is used where a uniform dropout probability is assumed for all (or for all unknown) contributors, we explore the extent to which this alters the false positive probabilities for relatives of donors, compared to what would have been obtained with the correct probabilities of dropout for each donor. PMID:26745184

  6. Dlx5 Homeodomain:DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome.

    PubMed

    Proudfoot, Andrew; Axelrod, Herbert L; Geralt, Michael; Fletterick, Robert J; Yumoto, Fumiaki; Deacon, Ashley M; Elsliger, Marc-André; Wilson, Ian A; Wüthrich, Kurt; Serrano, Pedro

    2016-03-27

    The Dlx5 homeodomain is a transcription factor related to the Drosophila distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including split hand and foot malformation 1 in humans. Our characterization of a Dlx5 homeodomain:(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A Q186H mutation linked to split hand and foot malformation 1 likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and they likely destabilize the formation of functional complexes. PMID:26829219

  7. Dlx5 Homeodomain: DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome

    DOE PAGESBeta

    Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael; Fletterick, Robert J.; Yumoto, Fumiaki; Deacon, Ashley M.; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt; Serrano, Pedro

    2016-01-29

    The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked to SHFM-1;more » this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.« less

  8. A chiroptical photoswitchable DNA complex.

    PubMed

    Mammana, Angela; Carroll, Gregory T; Areephong, Jetsuda; Feringa, Ben L

    2011-10-13

    The interesting structural, electronic, and optical properties of DNA provide fascinating opportunities for developing nanoscale smart materials by integrating DNA with opto-electronic components. In this article we demonstrate the electrostatic binding of an amine-terminated dithienylethene (DET) molecular switch to double-stranded synthetic polynucleotides. The DET switch can undergo photochemical ring-closure and opening reactions. Circular dichroism (CD) and UV-vis spectroscopy show that both the open, 1o, and the closed, 1c, forms of the switch bind to DNA. Upon addition of DNA to a solution of 1o or 1c, the UV-vis spectrum displays a hypochromic effect, indicative of an interaction between the switch and the DNA. The chirality of the DNA double-helix is transmitted to the switching unit which displays a well-defined CD signal upon supramolecular complexation to the DNA. Additionally, the CD signal of the DNA attenuates, demonstrating that both components of the complex mutually influence each other's structure; the DNA induces chirality in the switch, and the switch modifies the structure of the DNA. Modulation of the chiroptical properties of the complex is achieved by photochemically switching the DET between its ring open and closed isomers. A pH dependence study of the binding shows that when the pH is increased the switches lose their binding ability, indicating that electrostatic interactions between protonated amines and the negatively charged phosphate backbone are the dominant driving force for binding to the DNA. A comparison of poly(deoxyguanylic-deoxycytidylic) acid [poly(dGdC)(2)] polynucleotides with poly(deoxyadenylic-deoxythymidylic) acid [poly(dAdT)(2)] shows distinct differences in the CD spectra of the complexes. PMID:21879715

  9. Complex DNA structures and structures of DNA complexes

    SciTech Connect

    Chazin, W.J.; Carlstroem, G.; Shiow-Meei Chen; Miick, S.; Gomez-Paloma, L.; Smith, J.; Rydzewski, J.

    1994-12-01

    Complex DNA structures (for example, triplexes, quadruplexes, junctions) and DNA-ligand complexes are more difficult to study by NMR than standard DNA duplexes are because they have high molecular weights, show nonstandard or distorted local conformations, and exhibit large resonance linewidths and severe {sup 1}H spectral overlap. These systems also tend to have limited solubility and may require specialized solution conditions to maintain favorable spectral characteristics, which adds to the spectroscopic difficulties. Furthermore, with more atoms in the system, both assignment and structure calculation become more challenging. In this article, we focus on demonstrating the current status of NMR studies of such systems and the limitations to further progress; we also indicate in what ways isotopic enrichment can be useful.

  10. Complexity and Relations

    ERIC Educational Resources Information Center

    Lancaster, Jeanette Elizabeth

    2013-01-01

    A central feature of complexity is that it is based on non-linear, recursive relations. However, in most current accounts of complexity such relations, while non-linear, are based on the reductive relations of a Newtonian onto-epistemological framework. This means that the systems that are emergent from the workings of such relations are a…

  11. Structural complexity of DNA sequence.

    PubMed

    Liou, Cheng-Yuan; Tseng, Shen-Han; Cheng, Wei-Chen; Tsai, Huai-Ying

    2013-01-01

    In modern bioinformatics, finding an efficient way to allocate sequence fragments with biological functions is an important issue. This paper presents a structural approach based on context-free grammars extracted from original DNA or protein sequences. This approach is radically different from all those statistical methods. Furthermore, this approach is compared with a topological entropy-based method for consistency and difference of the complexity results. PMID:23662161

  12. Structural Complexity of DNA Sequence

    PubMed Central

    Liou, Cheng-Yuan; Cheng, Wei-Chen; Tsai, Huai-Ying

    2013-01-01

    In modern bioinformatics, finding an efficient way to allocate sequence fragments with biological functions is an important issue. This paper presents a structural approach based on context-free grammars extracted from original DNA or protein sequences. This approach is radically different from all those statistical methods. Furthermore, this approach is compared with a topological entropy-based method for consistency and difference of the complexity results. PMID:23662161

  13. Geant4-DNA simulations using complex DNA geometries generated by the DnaFabric tool

    NASA Astrophysics Data System (ADS)

    Meylan, S.; Vimont, U.; Incerti, S.; Clairand, I.; Villagrasa, C.

    2016-07-01

    Several DNA representations are used to study radio-induced complex DNA damages depending on the approach and the required level of granularity. Among all approaches, the mechanistic one requires the most resolved DNA models that can go down to atomistic DNA descriptions. The complexity of such DNA models make them hard to modify and adapt in order to take into account different biological conditions. The DnaFabric project was started to provide a tool to generate, visualise and modify such complex DNA models. In the current version of DnaFabric, the models can be exported to the Geant4 code to be used as targets in the Monte Carlo simulation. In this work, the project was used to generate two DNA fibre models corresponding to two DNA compaction levels representing the hetero and the euchromatin. The fibres were imported in a Geant4 application where computations were performed to estimate the influence of the DNA compaction on the amount of calculated DNA damage. The relative difference of the DNA damage computed in the two fibres for the same number of projectiles was found to be constant and equal to 1.3 for the considered primary particles (protons from 300 keV to 50 MeV). However, if only the tracks hitting the DNA target are taken into account, then the relative difference is more important for low energies and decreases to reach zero around 10 MeV. The computations were performed with models that contain up to 18,000 DNA nucleotide pairs. Nevertheless, DnaFabric will be extended to manipulate multi-scale models that go from the molecular to the cellular levels.

  14. Drug-induced DNA repair: X-ray structure of a DNA-ditercalinium complex.

    PubMed Central

    Gao, Q; Williams, L D; Egli, M; Rabinovich, D; Chen, S L; Quigley, G J; Rich, A

    1991-01-01

    Ditercalinium is a synthetic anticancer drug that binds to DNA by bis-intercalation and activates DNA repair processes. In prokaryotes, noncovalent DNA-ditercalinium complexes are incorrectly recognized by the uvrABC repair system as covalent lesions on DNA. In eukaryotes, mitochondrial DNA is degraded by excess and futile DNA repair. Using x-ray crystallography, we have determined, to 1.7 A resolution, the three-dimensional structure of a complex of ditercalinium bound to the double-stranded DNA fragment [d(CGCG)]2. The DNA in the complex with ditercalinium is kinked (by 15 degrees) and severely unwound (by 36 degrees) with exceptionally wide major and minor grooves. Recognition of the DNA-ditercalinium complex by uvrABC in prokaryotes, and by mitochondrial DNA repair systems in eukaryotes, might be related to drug-induced distortion of the DNA helix. Images PMID:2006181

  15. Imaging of DNA and Protein–DNA Complexes with Atomic Force Microscopy

    PubMed Central

    Lyubchenko, Yuri L.; Shlyakhtenko, Luda S.

    2016-01-01

    This article reviews atomic force microscopy (AFM) studies of DNA structure and dynamics and protein–DNA complexes, including recent advances in the visualization of protein–DNA complexes with the use of cutting-edge, high-speed AFM. Special emphasis is given to direct nanoscale visualization of dynamics of protein–DNA complexes. In the area of DNA structure and dynamics, structural studies of local non-B conformations of DNA and the interplay of local and global DNA conformations are reviewed. The application of time-lapse AFM nanoscale imaging of DNA dynamics is illustrated by studies of Holliday junction branch migration. Structure and dynamics of protein–DNA interactions include problems related to site-specific DNA recombination, DNA replication, and DNA mismatch repair. Studies involving the structure and dynamics of chromatin are also described. PMID:27278886

  16. Complex kinetics of DNA condensation revealed through DNA twist tracing.

    PubMed

    Li, Wei; Wong, Wei Juan; Lim, Ci Ji; Ju, Hai-Peng; Li, Ming; Yan, Jie; Wang, Peng-Ye

    2015-08-01

    Toroid formation is an important mechanism for DNA condensation in cells. The length change during DNA condensation was investigated in previous single-molecule experiments. However, DNA twist is key to understanding the topological kinetics of DNA condensation. In this study, DNA twist as well as DNA length was traced during the DNA condensation by the freely orbiting magnetic tweezers and the tilted magnetic tweezers combined with Brownian dynamics simulations. The experimental results disclose the complex relationship between DNA extension and backbone rotation. Brownian dynamics simulations show that the toroid formation follows a wiggling pathway which leads to the complex DNA backbone rotation as revealed in our experiments. These findings provide the complete description of multivalent cation-dependent DNA toroid formation under tension.

  17. Runt-related Transcription Factor 1 (RUNX1) Stimulates Tumor Suppressor p53 Protein in Response to DNA Damage through Complex Formation and Acetylation*

    PubMed Central

    Wu, Dan; Ozaki, Toshinori; Yoshihara, Yukari; Kubo, Natsumi; Nakagawara, Akira

    2013-01-01

    Representative tumor suppressor p53 plays a critical role in the regulation of proper DNA damage response. In this study, we have found for the first time that Runt-related transcription factor 1 (RUNX1) contributes to p53-dependent DNA damage response. Upon adriamycin (ADR) exposure, p53 as well as RUNX1 were strongly induced in p53-proficient HCT116 and U2OS cells, which were closely associated with significant transactivation of p53 target genes, such as p21WAF1, BAX, NOXA, and PUMA. RUNX1 was exclusively expressed in the cell nucleus and formed a complex with p53 in response to ADR. Chromatin immunoprecipitation assay demonstrated that p53 together with RUNX1 are efficiently recruited onto p53 target gene promoters following ADR exposure, indicating that RUNX1 is involved in p53-mediated transcriptional regulation. Indeed, forced expression of RUNX1 stimulated the transcriptional activity of p53 in response to ADR. Consistent with these observations, knockdown of RUNX1 attenuated ADR-mediated induction of p53 target genes and suppressed ADR-dependent apoptosis. Furthermore, RUNX1 was associated with p300 histone acetyltransferase, and ADR-dependent acetylation of p53 at Lys-373/382 was markedly inhibited in RUNX1 knockdown cells. In addition, knockdown of RUNX1 resulted in a significant decrease in the amount of p53-p300 complex following ADR exposure. Taken together, our present results strongly suggest that RUNX1 is required for the stimulation of p53 in response to DNA damage and also provide novel insight into understanding the molecular mechanisms behind p53-dependent DNA damage response. PMID:23148227

  18. Interaction of DNA and DNA-anti-DNA complexes to fibronectin

    SciTech Connect

    Gupta, R.C.; Simpson, W.A.; Raghow, R.; Hasty, K.

    1986-03-01

    Fibronectin (Fn) is a large multidomain glycoprotein found in the basement membrane, on cell surface and in plasma. The interactions of Fn with DNA may be significant in glomerular deposition of DNA-anti-DNA complexes in patients with systemic lupus erythematosus (SLE). The authors examined the binding of DNA and DNA-anti-DNA complexes to Fn by a solid phase assay in which Fn was coated to microtiter plates and reacted with (/sup 3/H)DNA or DNA complexes with a monoclonal anti-DNA antibody. The optimal interaction of DNA with Fn occurs at <0.1M NaCl suggesting that the binding is charge dependent; the specificity of this binding was shown by competitive inhibition and locking experiments using anti-Fn. The binding was maximum at pH 6.5 and in the absence of Ca/sup 2 +/. The addition of Clq enhanced the binding of DNA and DNA-anti-DNA complexes to Fn, whereas heparan sulfate inhibited such binding. The monomeric or aggregated IgC did not bind Fn but aggregated IgG bound to Fn in the presence of Clq. Furthermore, DNA-anti-DNA complexes in sera from active SLE patients bound Fn which was enhanced in the presence of Clq; DNase abolished this binding indicating that the interaction of these complexes was mediated by DNA. These observations may partially explain the molecular mechanism(s) of the deposition of DNA-anti-DNA complexes in basement membrane.

  19. The kinetic complexity of Acetabularia chloroplast DNA.

    PubMed

    Padmanabhan, U; Green, B R

    1978-11-21

    The kinetic complexity of Acetabularia cliftonii chloroplast DNA is 1.52 +/- 0.26 . 10(9) daltons, compared to 0.2 .10(9) daltons for Chlamydomonas chloroplast DNA. There is an average of three genomes per chloroplast. The unusually large size of the Acetabularia genome may reflect the ancient evolutionary history of this organism.

  20. Electrostatics of DNA complexes with cationic lipids

    NASA Astrophysics Data System (ADS)

    Cherstvy, Andrey

    2007-03-01

    We present the exact solutions of the linear Poisson-Boltzmann theory for several problems relevant to electrostatics of DNA complexes with cationic lipids. We calculate the electrostatic potential and energy for lamellar and inverted hexagonal phases, concentrating on the effects of water-membrane dielectric boundaries. Our results for the complex energy agree qualitatively well with the known numerical solutions of the nonlinear Poisson-Boltzmann equation. Using the solution for the lamellar phase, we calculate its compressibility modulus and compare our findings with experimental data available suggesting a new scaling dependence on DNA-DNA separations in the complex. Also, we treat analytically charge-charge electrostatic interactions across, along, and in between two low-dielectric membranes. We obtain an estimate for the strength of electrostatic interactions of 1D DNA smectic layers across a lipid membrane. We discuss also some aspects of 2D DNA condensation and DNA-DNA attraction in DNA-lipid lamellar phase in the presence of di- and tri-valent cations and analyze the equilibrium intermolecular separations using the recently developed theory of electrostatic interactions of DNA helical charge motifs.

  1. Rapamycin reverses age-related increases in mitochondrial ROS production at complex I, oxidative stress, accumulation of mtDNA fragments inside nuclear DNA, and lipofuscin level, and increases autophagy, in the liver of middle-aged mice.

    PubMed

    Martínez-Cisuelo, V; Gómez, J; García-Junceda, I; Naudí, A; Cabré, R; Mota-Martorell, N; López-Torres, M; González-Sánchez, M; Pamplona, R; Barja, G

    2016-10-01

    Rapamycin consistently increases longevity in mice although the mechanism of action of this drug is unknown. In the present investigation we studied the effect of rapamycin on mitochondrial oxidative stress at the same dose that is known to increase longevity in mice (14mgofrapamycin/kg of diet). Middle aged mice (16months old) showed significant age-related increases in mitochondrial ROS production at complex I, accumulation of mtDNA fragments inside nuclear DNA, mitochondrial protein lipoxidation, and lipofuscin accumulation compared to young animals (4months old) in the liver. After 7weeks of dietary treatment all those increases were totally or partially (lipofuscin) abolished by rapamycin, middle aged rapamycin-treated animals showing similar levels in those parameters to young animals. The decrease in mitochondrial ROS production was due to qualitative instead of quantitative changes in complex I. The decrease in mitochondrial protein lipoxidation was not due to decreases in the amount of highly oxidizable unsaturated fatty acids. Rapamycin also decreased the amount of RAPTOR (of mTOR complex) and increased the amounts of the PGC1-α and ATG13 proteins. The results are consistent with the possibility that rapamycin increases longevity in mice at least in part by lowering mitochondrial ROS production and increasing autophagy, decreasing the derived final forms of damage accumulated with age which are responsible for increased longevity. The decrease in lipofuscin accumulation induced by rapamycin adds to previous information suggesting that the increase in longevity induced by this drug can be due to a decrease in the rate of aging. PMID:27498120

  2. Rapamycin reverses age-related increases in mitochondrial ROS production at complex I, oxidative stress, accumulation of mtDNA fragments inside nuclear DNA, and lipofuscin level, and increases autophagy, in the liver of middle-aged mice.

    PubMed

    Martínez-Cisuelo, V; Gómez, J; García-Junceda, I; Naudí, A; Cabré, R; Mota-Martorell, N; López-Torres, M; González-Sánchez, M; Pamplona, R; Barja, G

    2016-10-01

    Rapamycin consistently increases longevity in mice although the mechanism of action of this drug is unknown. In the present investigation we studied the effect of rapamycin on mitochondrial oxidative stress at the same dose that is known to increase longevity in mice (14mgofrapamycin/kg of diet). Middle aged mice (16months old) showed significant age-related increases in mitochondrial ROS production at complex I, accumulation of mtDNA fragments inside nuclear DNA, mitochondrial protein lipoxidation, and lipofuscin accumulation compared to young animals (4months old) in the liver. After 7weeks of dietary treatment all those increases were totally or partially (lipofuscin) abolished by rapamycin, middle aged rapamycin-treated animals showing similar levels in those parameters to young animals. The decrease in mitochondrial ROS production was due to qualitative instead of quantitative changes in complex I. The decrease in mitochondrial protein lipoxidation was not due to decreases in the amount of highly oxidizable unsaturated fatty acids. Rapamycin also decreased the amount of RAPTOR (of mTOR complex) and increased the amounts of the PGC1-α and ATG13 proteins. The results are consistent with the possibility that rapamycin increases longevity in mice at least in part by lowering mitochondrial ROS production and increasing autophagy, decreasing the derived final forms of damage accumulated with age which are responsible for increased longevity. The decrease in lipofuscin accumulation induced by rapamycin adds to previous information suggesting that the increase in longevity induced by this drug can be due to a decrease in the rate of aging.

  3. Sperm DNA damage and its relation with leukocyte DNA damage.

    PubMed

    Babazadeh, Zahra; Razavi, Shahnaz; Tavalaee, Marziyeh; Deemeh, Mohammad Reza; Shahidi, Maryam; Nasr-Esfahani, Mohammad Hossein

    2010-01-01

    DNA fragmentation in human sperm has been related to endogenous and exogenous factors. Exogenous factors can also affect leukocyte DNA integrity. This study evaluated the relation between sperm DNA damage and leukocyte DNA integrity, as a predictor of exogenous factors. DNA damage in the sperm and leukocytes of 41 individuals undergoing ICSI were measured by Comet assay. In addition, sperm chromatin dispersion (SCD) was carried out on semen samples. A positive correlation was observed between the DNA integrity of sperm with leukocytes. When patients were divided into low and high DNA exposure groups, sperm DNA fragmentation was significantly different between the two groups. Cleavage rate and embryo quality showed significant correlation with leukocyte DNA integrity. The results showed that leukocyte DNA integrity could be used to identify individuals at high risk in order to reduce the extent of DNA damage in patients before ICSI in order to improve the subsequent outcome of this procedure.

  4. 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.

  5. Human DNA polymerase α in binary complex with a DNA:DNA template-primer

    PubMed Central

    Coloma, Javier; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2016-01-01

    The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer – with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes. PMID:27032819

  6. Intercalation processes of copper complexes in DNA

    PubMed Central

    Galindo-Murillo, Rodrigo; García-Ramos, Juan Carlos; Ruiz-Azuara, Lena; Cheatham, Thomas E.; Cortés-Guzmán, Fernando

    2015-01-01

    The family of anticancer complexes that include the transition metal copper known as Casiopeínas® shows promising results. Two of these complexes are currently in clinical trials. The interaction of these compounds with DNA has been observed experimentally and several hypotheses regarding the mechanism of action have been developed, and these include the generation of reactive oxygen species, phosphate hydrolysis and/or base-pair intercalation. To advance in the understanding on how these ligands interact with DNA, we present a molecular dynamics study of 21 Casiopeínas with a DNA dodecamer using 10 μs of simulation time for each compound. All the complexes were manually inserted into the minor groove as the starting point of the simulations. The binding energy of each complex and the observed representative type of interaction between the ligand and the DNA is reported. With this extended sampling time, we found that four of the compounds spontaneously flipped open a base pair and moved inside the resulting cavity and four compounds formed stacking interactions with the terminal base pairs. The complexes that formed the intercalation pocket led to more stable interactions. PMID:25958394

  7. Anionic solid lipid nanoparticles supported on protamine/DNA complexes

    NASA Astrophysics Data System (ADS)

    Ye, Jiesheng; Wang, Aihua; Liu, Chunxi; Chen, Zhijin; Zhang, Na

    2008-07-01

    The objective of this study was to design novel anionic ternary nanoparticles for gene delivery. These ternary nanoparticles were equipped with protamine/DNA binary complexes (150-200 nm) as the support, and the anionic formation was achieved by absorption of anionic solid lipid nanoparticles (<=20 nm) onto the surface of the binary complexes. The small solid lipid nanoparticles (SLNs) were prepared by a modified film dispersion-ultrasonication method, and adsorption of the anionic SLNs onto the binary complexes was typically carried out in water via electrostatic interaction. The formulated ternary nanoparticles were found to be relatively uniform in size (257.7 ± 10.6 nm) with a 'bumpy' surface, and the surface charge inversion from 19.28 ± 1.14 mV to -17.16 ± 1.92 mV could be considered as evidence of the formation of the ternary nanoparticles. The fluorescence intensity measurements from three batches of the ternary nanoparticles gave a mean adsorption efficiency of 96.75 ± 1.13%. Circular dichroism spectra analysis showed that the protamine/DNA complexes had been coated by small SLNs, and that the anionic ternary nanoparticles formed did not disturb the construction of the binary complexes. SYBR Green I analysis suggested that the ternary nanoparticles could protect the DNA from nuclease degradation, and cell viability assay results showed that they exhibit lower cytotoxicity to A549 cells compared with the binary complexes and lipofectamine. The transfection efficiency of the ternary nanoparticles was better than that of naked DNA and the binary complexes, and almost equal to that of lipofectamine/DNA complexes, as revealed by inversion fluorescence microscope observation. These results indicated that the anionic ternary nanoparticles could facilitate gene transfer in cultured cells, and might alleviate the drawbacks of the conventional cationic vector/DNA complexes for gene delivery in vivo.

  8. Intrinsic Dynamics of DNA-Polymer Complexes: a Mechanism for DNA Release

    PubMed Central

    Prevette, Lisa E.; Nikolova, Evgenia N.; Al-Hashimi, Hashim M.; Holl, Mark M. Banaszak

    2012-01-01

    The transfer of genetic material into cells using non-viral vectors offers unique potential for therapeutics; however, the efficacy of delivery depends upon a poorly understood, multistep pathway, limiting the prospects for successful gene delivery. Mechanistic insight into DNA association and release has been hampered by a lack of atomic resolution structural and dynamic information for DNA-polymer complexes (polyplexes). Here, we report a dendrimer-based polyplex system containing poly(ethyleneglycol) (PEG) arms that is suitable for atomic-level characterization by solution NMR spectroscopy. NMR chemical shift, linewidth, and proton transverse relaxation rate measurements reveal that free and dendrimer-bound polyplex DNA exchange rapidly relative to the NMR timescale (< millisecond). The dendrimers retain a high degree of mobility in the polyplex, whereas the DNA shows restrained mobility, suggesting that the polyplex is a highly dynamic complex with a rapidly exchanging dendrimer atmosphere around a more rigid DNA framework. PMID:22823140

  9. Condensation of nonstochiometric DNA/polycation complexes by divalent cations.

    PubMed

    Budker, Vladimir; Trubetskoy, Vladimir; Wolff, Jon A

    2006-12-15

    This study found that divalent cations induced the further condensation of partially condensed DNA within nonstochiometric polycation complexes. The addition of a few mmol of a divalent cation such as calcium reduced by half the inflection point at which DNA became fully condensed by poly-L-lysine (PLL) and a variety of other polycations. The effect on DNA condensation was initially observed using a new method, which is based on the concentration-dependent self-quenching of fluorescent moieties (e.g., rhodamine) covalently linked to the DNA backbone at relatively high densities. Additional analyses, which employed ultracentrifugation, dynamic light scattering, agarose gel electrophoresis, and atomic force microscopy, confirmed the effect of divalent cations. These results provide an additional accounting of the process by which divalent cations induce greater chromatin compaction that is based on the representation of chromatin fibers as a nonstoichiometric polyelectrolyte complex. They also offer a new approach to assemble nonviral vectors for gene therapy.

  10. 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

    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.

  11. DNA based computing for understanding complex shapes.

    PubMed

    Ullah, A M M Sharif; D'Addona, Doriana; Arai, Nobuyuki

    2014-03-01

    This study deals with a computing method called DNA based computing (DBC) that takes inspiration from the Central Dogma of Molecular Biology. The proposed DBC uses a set of user-defined rules to create a DNA-like sequence from a given piece of problem-relevant information (e.g., image data) in a dry-media (i.e., in an ordinary computer). It then uses another set of user-defined rules to create an mRNA-like sequence from the DNA. Finally, it uses the genetic code to translate the mRNA (or directly the DNA) to a protein-like sequence (a sequence of amino acids). The informational characteristics of the protein (entropy, absence, presence, abundance of some selected amino acids, and relationships among their likelihoods) can be used to solve problems (e.g., to understand complex shapes from their image data). Two case studies ((1) fractal geometry generated shape of a fern-leaf and (2) machining experiment generated shape of the worn-zones of a cutting tool) are presented elucidating the shape understanding ability of the proposed DBC in the presence of a great deal of variability in the image data of the respective shapes. The implication of the proposed DBC from the context of Internet-aided manufacturing system is also described. Further study can be carried out in solving other complex computational problems by using the proposed DBC and its derivatives. PMID:24447435

  12. Sensitive determination of DNA based on the interaction between prulifloxacin-terbium(III) complex and DNA.

    PubMed

    Wu, Ting; Fang, Biyun; Chang, Lin; Liu, Min; Chen, Fang

    2013-01-01

    A simple spectrofluorimetric method is described for the determination of DNA, based on its enhancement of the fluorescence intensity of prulifloxacin (PUFX)-Tb(3+). The luminescence intensity of the PUFX-Tb(3+) complex increased up to 10-fold after adding DNA. The excitation and emission wavelengths were 345 and 545 nm, respectively. Under optimum conditions, variations in the fluorescence intensity showed a good linear relationship with the concentration of hsDNA in the range of 3.0 × 10(-9) to 1.0 × 10(-6) g/mL, with a correlation coefficient (R) of 0.997, and the detection limit was 2.1 × 10(-9) g/mL. The method was successfully applied to the determination of DNA in synthetic samples, and recoveries were in the range 97.3-102.0%. The mechanism of fluorescence enhancement of the PUFX-Tb(3+) complex by DNA is also discussed. The mechanism may involve formation of a ternary complex mainly by intercalation binding together with weak electrostatic interaction, which will increase the energy transition from ligand to Tb(3+), increasing the rigidity of the complex, and decreasing the radiationless energy loss through O-H vibration of the H2O molecule in the PUFX-Tb(3+) complex. Compared with the previous DNA probes, the proposed method is not only more robust and friendly to the environment, but also of relatively higher sensitivity.

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

    DOE PAGESBeta

    Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; Johnson, Reid C.; Leng, Fenfei

    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

  14. DNA Sequence Determinants Controlling Affinity, Stability and Shape of DNA Complexes Bound by the Nucleoid Protein Fis

    PubMed Central

    Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; Johnson, Reid C.

    2016-01-01

    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 sequences 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. The affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions. PMID:26959646

  15. Evaluation of DNA extraction methods from complex phototrophic biofilms.

    PubMed

    Ferrera, Isabel; Massana, Ramon; Balagué, Vanessa; Pedrós-Alió, Carles; Sánchez, Olga; Mas, Jordi

    2010-04-01

    Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 +/- 12 to 1893 +/- 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol-chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol-chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms.

  16. Radiation damage to DNA-protein complexes

    NASA Astrophysics Data System (ADS)

    Spotheim-Maurizot, M.; Davídková, M.

    2011-01-01

    We review here the advances in understanding the effects of ionizing radiations on DNA, proteins and their complexes, resulting from the collaboration of the authors' teams. It concerns the preponderant indirect effect of low LET ionizing radiations, thus the attack of the macromolecules in aqueous solution by the most aggressive product of water radiolysis, the hydroxyl radical. A model of simulation of the reaction of these radicals with the macromolecules (called RADACK) was developed and was used for calculating the probabilities of damage of each constituent of DNA or proteins (nucleotide or amino-acid). The calculations allowed to draw conclusions from electrophoresis, mutagenesis, spectroscopic (fluorescence, circular dichroïsm) and mass spectrometry experiments. Thus we have shown that the extent and location of the lesions are strongly dependent on the 3D structure of the macromolecules, which in turns is modulated by their sequence and by the binding of some ligands. Molecular dynamics simulation completed our studies in showing the consequences of each lesion on the stability and structure of the proteins and their complexes with DNA.

  17. Response of base excision repair enzymes to complex DNA lesions.

    PubMed

    Weinfeld, M; Rasouli-Nia, A; Chaudhry, M A; Britten, R A

    2001-11-01

    There is now increasing evidence that ionizing radiation generates complex DNA damage, i.e. two or more lesions--single-strand breaks or modified nucleosides--located within one to two helical turns on the same strand or on opposite strands. Double-strand breaks are the most readily recognizable clustered lesions, but they may constitute a relatively minor fraction of the total. It is anticipated that clustered lesions may play a significant role in cellular response to ionizing radiation since they may present a major challenge to the DNA repair machinery. The degree of lesion complexity increases with increasing LET. This has potential implications for space travel because of exposure to high-LET cosmic radiation. It is therefore critical that we begin to understand the consequences of such damaged sites, including their influence on DNA repair enzymes. This paper presents a short review of our current knowledge of the action of purified DNA repair enzymes belonging to the base excision repair pathway, including DNA glycosylases and apurinic/apyrimidinic endonucleases, on model complex lesions.

  18. Multilamellar structures of DNA complexes with cationic liposomes.

    PubMed

    Dan, N

    1997-10-01

    Studies of DNA complexes with cationic liposomes are prompted by the search for nonviral DNA carriers for gene therapy. Recent experiments have identified a stable multilamellar phase in which ordered smectic layers of DNA alternate with cationic bilayers. In this paper we identify the forces governing DNA adsorption on cationic lamellae, including a membrane-induced attraction between the adsorbed DNA. Calculating the DNA interhelical spacing as a function of system composition, the model successfully explains recent surprising observations.

  19. MHF complex senses branched DNA via binding a pair of crossover DNA duplexes

    PubMed Central

    Zhao, Qi; Saro, Dorina; Sachpatzidis, Aristidis; Singh, Thiyam Ramsing; Schlingman, Daniel; Zheng, Xiao-Feng; Mack, Andrew; Tsai, Miaw-Sheue; Mochrie, Simon; Regan, Lynne; Meetei, Amom Ruhikanta; Sung, Patrick; Xiong, Yong

    2014-01-01

    The conserved MHF1-MHF2 (MHF) complex functions in the activation of the Fanconi anemia (FA) pathway of DNA damage response, in regulating homologous recombination, and in DNA replication fork maintenance. MHF facilitates the processing of multiple types of branched DNAs by the FA DNA translocase FANCM. Here we report the crystal structure of a human MHF-DNA complex that reveals the DNA binding mode of MHF. The structure suggests an MHF preference for branched DNA over double stranded DNA through engaging two duplex arms, which is supported by single molecule studies. Biochemical analyses verify that MHF preferentially engage DNA forks or various four-way junctions independent of the junction-site structure. Genetic experiments provide evidence that the observed DNA-binding interface of MHF is important for cellular resistance to DNA damage. These results provide insights into how the MHF complex recognizes branched DNA and stimulates FANCM activity at such a structure to promote genome maintenance. PMID:24390579

  20. Immunodetection of human topoisomerase I-DNA covalent complexes.

    PubMed

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

    2016-04-01

    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.

  1. Electrophoretic Mobility Shift Assays for Protein–DNA Complexes Involved in DNA Repair*

    PubMed Central

    Tsai, Chun; Smider, Vaughn; Hwang, Byung Joon; Chu, Gilbert

    2014-01-01

    The electrophoretic mobility shift assay (EMSA) can be used to study proteins that bind to DNA structures created by DNA-damaging agents. UV-damaged DNA-binding protein (UV-DDB), which is involved in nucleotide excision repair, binds to DNA damaged by ultraviolet radiation or the anticancer drug cisplatin. Ku, XRCC4/Ligase IV, and DNA–PKcs, which are involved in the repair of DNA double-strand breaks by nonhomologous end joining, assemble in complexes at DNA ends. This chapter will describe several EMSA protocols for detecting different DNA repair protein–DNA complexes. To obtain additional information, one can apply variations of the EMSA, which include the reverse EMSA to detect binding of 35S-labeled protein to damaged DNA, and the antibody supershift assay to detect the presence of a specific protein in the protein–DNA complex. PMID:22941596

  2. Measuring complexity, nonextensivity and chaos in the DNA sequence of the Major Histocompatibility Complex

    NASA Astrophysics Data System (ADS)

    Pavlos, G. P.; Karakatsanis, L. P.; Iliopoulos, A. C.; Pavlos, E. G.; Xenakis, M. N.; Clark, Peter; Duke, Jamie; Monos, D. S.

    2015-11-01

    We analyze 4 Mb sequences of the Major Histocompatibility Complex (MHC), which is a DNA segment on chromosome 6 with high gene density, controlling many immunological functions and associated with many diseases. The analysis is based on modern theoretical and mathematical tools of complexity theory, such as nonlinear time series analysis and Tsallis non-extensive statistics. The results revealed that the DNA complexity and self-organization can be related to fractional dynamical nonlinear processes with low-dimensional deterministic chaotic and non-extensive statistical character, which generate the DNA sequences under the extremization of Tsallis q-entropy principle. While it still remains an open question as to whether the DNA walk is a fractional Brownian motion (FBM), a static anomalous diffusion process or a non-Gaussian dynamical fractional anomalous diffusion process, the results of this study testify for the latter, providing also a possible explanation for the previously observed long-range power law correlations of nucleotides, as well as the long-range correlation properties of coding and non-coding sequences present in DNA sequences.

  3. Orientation of DNA Minicircles Balances Density and Topological Complexity in Kinetoplast DNA.

    PubMed

    Diao, Yuanan; Rodriguez, Victor; Klingbeil, Michele; Arsuaga, Javier

    2015-01-01

    Kinetoplast DNA (kDNA), a unique mitochondrial structure common to trypanosomatid parasites, contains thousands of DNA minicircles that are densely packed and can be topologically linked into a chain mail-like network. Experimental data indicate that every minicircle in the network is, on average, singly linked to three other minicircles (i.e., has mean valence 3) before replication and to six minicircles in the late stages of replication. The biophysical factors that determine the topology of the network and its changes during the cell cycle remain unknown. Using a mathematical modeling approach, we previously showed that volume confinement alone can drive the formation of the network and that it induces a linear relationship between mean valence and minicircle density. Our modeling also predicted a minicircle valence two orders of magnitude greater than that observed in kDNA. To determine the factors that contribute to this discrepancy we systematically analyzed the relationship between the topological properties of the network (i.e., minicircle density and mean valence) and its biophysical properties such as DNA bending, electrostatic repulsion, and minicircle relative position and orientation. Significantly, our results showed that most of the discrepancy between the theoretical and experimental observations can be accounted for by the orientation of the minicircles with volume exclusion due to electrostatic interactions and DNA bending playing smaller roles. Our results are in agreement with the three dimensional kDNA organization model, initially proposed by Delain and Riou, in which minicircles are oriented almost perpendicular to the horizontal plane of the kDNA disk. We suggest that while minicircle confinement drives the formation of kDNA networks, it is minicircle orientation that regulates the topological complexity of the network.

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

    PubMed

    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

  5. 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

  6. DNA delivery systems based on complexes of DNA with synthetic polycations and their copolymers.

    PubMed

    Oupický, D; Konák, C; Ulbrich, K; Wolfert, M A; Seymour, L W

    2000-03-01

    Block and graft copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-(trimethylammonio)ethyl methacrylate were synthesised and used for preparation of polyelectrolyte complexes with calf thymus DNA intended for targeted delivery of genes in vivo. In this study the effects of the speed of component mixing, total concentration of polymers, ionic strength of solvents, copolymer structure and content of HPMA in the copolymers on parameters of the polyelectrolyte complexes was investigated. Static and dynamic light scattering methods were used as a main tool for characterising these complexes. The presence of HPMA units in the polycation had no significant effect on its ability to form complexes with DNA, but did affect molecular parameters and aggregation (precipitation) of the complexes. The size of the complexes increases whereas their molecular weight decreases with increasing content of HPMA units. The density of the complexes decreases with increasing HPMA content independently of the copolymer structure. In order to prepare stable DNA complexes containing single DNA molecule, the following rules should be observed: (1) copolymers should have a content of HPMA units higher than 40%; (2) the DNA concentrations in solutions should be kept below 4 x 10(-5) g/ml and (3) both components should be mixed together in deionized water. The stability of the complexes against precipitation in 0.15 M NaCl and the resistance of the complexed DNA to the action of nucleases was also studied. Whereas DNA complexes of all copolymers showed very good nuclease stability, the presence of a sufficiently high content of HPMA is necessary for their good stability in 0.15 M NaCl. The investigation of the stability and the interaction of DNA complexes in aqueous solutions of serum albumin and dilute human blood serum revealed adsorption of biomacromolecules on DNA complexes accompanied by significant changes in the zeta-potential which finally resulted in formation of a "protein

  7. Chiral heterobimetallic complexes targeting human DNA-topoisomerase Iα.

    PubMed

    Tabassum, Sartaj; Asim, Ahmad; Khan, Rais Ahmad; Hussain, Zahid; Srivastav, Saurabh; Srikrishna, Saripella; Arjmand, Farukh

    2013-12-28

    The chiral monometallic Cu(II) (1) and Zn(II) (2) and heterobimetallic Cu(II)-Sn(IV) and Zn(II)-Sn(IV) complexes with tridentate chiral Schiff base -ONO-ligand in the presence of nitrogen donor heterocyclic ligand imidazole; were prepared and characterized by various physico-chemical and spectroscopic methods. Preliminary complex-DNA interaction studies employing optical methods revealed that 3 displayed a higher propensity towards the drug target DNA double helix and recommended predominantly an electrostatic mode of interaction as well as a groove binding affinity of the complex with CT-DNA. This was quantified by Kb and KSV values of complexes 1-4, which demonstrated a multifold increase in complex 3 binding to CT DNA and clearly demonstrates its potency to act as a chemotherapeutic agent. Furthermore, the gel electrophoretic patterns of supercoiled pBR322 DNA with varying concentrations of complex 3 exhibits the ability to cleave DNA and follow a freely diffusible radical mechanism. The antiproliferative effects of complex 3 on human hepatoma cancer cells (Huh7) was investigated. Human Topo I inhibition assay by complex 3 was performed and results confirmed significantly good activity at lower concentrations than some of the classical Topo I inhibitors. Additionally, complex 3 was investigated for the expression of MMP-2 and TGF-β by real time PCR. The cellular uptake of complex 3 by HeLa cells was studied by confocal microscopy. PMID:24077532

  8. Cryo-EM Imaging of DNA-PK DNA Damage Repair Complexes

    SciTech Connect

    Phoebe L. Stewart

    2005-06-27

    Exposure to low levels of ionizing radiation causes DNA double-strand breaks (DSBs) that must be repaired for cell survival. Higher eukaryotes respond to DSBs by arresting the cell cycle, presumably to repair the DNA lesions before cell division. In mammalian cells, the nonhomologous end-joining DSB repair pathway is mediated by the 470 kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs) together with the DNA-binding factors Ku70 and Ku80. Mouse knock-out models of these three proteins are all exquisitely sensitive to low doses of ionizing radiation. In the presence of DNA ends, Ku binds to the DNA and then recruits DNA-PKcs. After formation of the complex, the kinase activity associated with DNA-PKcs becomes activated. This kinase activity has been shown to be essential for repairing DNA DSBs in vivo since expression of a kinase-dead form of DNA-PKcs in a mammalian cell line that lacks DNA-PKcs fails to complement the radiosensitive phenotype. The immense size of DNA-PKcs suggests that it may also serve as a docking site for other DNA repair proteins. Since the assembly of the DNA-PK complex onto DNA is a prerequisite for DSB repair, it is critical to obtain structural information on the complex. Cryo-electron microscopy (cryo-EM) and single particle reconstruction methods provide a powerful way to image large macromolecular assemblies at near atomic (10-15 ?) resolution. We have already used cryo-EM methods to examine the structure of the isolated DNA-PKcs protein. This structure reveals numerous cavities throughout the protein that may allow passage of single or double-stranded DNA. Pseudo two-fold symmetry was found for the monomeric protein, suggesting that DNA-PKcs may interact with two DNA ends or two Ku heterodimers simultaneously. Here we propose to study the structure of the cross-linked DNA-PKcs/Ku/DNA complex. Difference imaging with our published DNA-PKcs structure will enable us to elucidate the architecture of the complex. A second

  9. ISWI chromatin remodeling complexes in the DNA damage response

    PubMed Central

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA. PMID:25486562

  10. ISWI chromatin remodeling complexes in the DNA damage response.

    PubMed

    Aydin, Özge Z; Vermeulen, Wim; Lans, Hannes

    2014-01-01

    Regulation of chromatin structure is an essential component of the DNA damage response (DDR), which effectively preserves the integrity of DNA by a network of multiple DNA repair and associated signaling pathways. Within the DDR, chromatin is modified and remodeled to facilitate efficient DNA access, to control the activity of repair proteins and to mediate signaling. The mammalian ISWI family has recently emerged as one of the major ATP-dependent chromatin remodeling complex families that function in the DDR, as it is implicated in at least 3 major DNA repair pathways: homologous recombination, non-homologous end-joining and nucleotide excision repair. In this review, we discuss the various manners through which different ISWI complexes regulate DNA repair and how they are targeted to chromatin containing damaged DNA.

  11. Complex relation between triazine-susceptible phenotype and genotype in the weed Senecio vulgaris may be caused by chloroplast DNA polymorphism.

    PubMed

    Frey, J E; Müller-Schärer, H; Frey, B; Frei, D

    1999-08-01

    The weed Senecio vulgaris acquired high levels of resistance to triazine herbicides soon after the latter's introduction. As in most weeds, triazine resistance is conferred by a point mutation in the chloroplast psbA gene that negatively affects the fitness of its carrier. To assess levels of triazine resistance in S. vulgaris field populations, we adopted a PCR-RFLP-based molecular diagnostic test recently developed for the triazine resistance-conferring region of the psbA gene of other weeds, including Brassica napus, Chenopodium spp. and Amaranthus spp., and compared these molecular results to the phenotypic response after triazine application. A highly significant linear correlation was found between phytotoxic symptoms and biomass reduction. Variability in phenotypic response was not only found between populations or inbred lines of S. vulgaris but also within replicates of the same inbred line. No clear relationship, however, was found between the DNA restriction pattern and the phenotypic response to triazine application, thereby throwing doubt on the use of such molecular diagnostic tests to track triazine resistance in S. vulgaris. Our results indicate that the chloroplast genome of S. vulgaris is polymorphic and that the level of polymorphism may be variable within single leaves of individual plants. We discuss the possible genetic basis of this polymorphism and its consequence for the acquisition and inheritance of chloroplast-based traits. PMID:22665192

  12. The role of a microscopic colloidally stabilized phase in solubilizing oligoamine-condensed DNA complexes.

    PubMed

    Trubetskoy, Vladimir S; Wolff, Jon A; Budker, Vladimir G

    2003-02-01

    DNA complexes of spermine and spermidine become resolubilized at very high concentrations of the oligoamine. It has been postulated that high oligoamine concentrations shift the DNA from the globule back to the coil phase. The present study indicates that DNA resolubilization at high concentrations of spermine and spermidine is explained by formation of small particles of condensed DNA that cannot be precipitated by centrifugation. The fact that DNA stays condensed during resolubilization was confirmed using a relatively new condensation assay and three independent microscopic techniques. A considerable portion of DNA was found to be in particles with diameter <100 nm. Formation of such small particles is likely to be caused by colloidal forces. The ability to form small, condensed DNA particles in solutions that contain high concentrations of oligocation should aid in the design of synthetic DNA vectors for gene transfer and gene therapy and in the handling of DNA for diagnostic studies.

  13. The Role of a Microscopic Colloidally Stabilized Phase in Solubilizing Oligoamine-Condensed DNA Complexes

    PubMed Central

    Trubetskoy, Vladimir S.; Wolff, Jon A.; Budker, Vladimir G.

    2003-01-01

    DNA complexes of spermine and spermidine become resolubilized at very high concentrations of the oligoamine. It has been postulated that high oligoamine concentrations shift the DNA from the globule back to the coil phase. The present study indicates that DNA resolubilization at high concentrations of spermine and spermidine is explained by formation of small particles of condensed DNA that cannot be precipitated by centrifugation. The fact that DNA stays condensed during resolubilization was confirmed using a relatively new condensation assay and three independent microscopic techniques. A considerable portion of DNA was found to be in particles with diameter <100 nm. Formation of such small particles is likely to be caused by colloidal forces. The ability to form small, condensed DNA particles in solutions that contain high concentrations of oligocation should aid in the design of synthetic DNA vectors for gene transfer and gene therapy and in the handling of DNA for diagnostic studies. PMID:12547793

  14. A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis1[OPEN

    PubMed Central

    Rutowicz, Kinga; Puzio, Marcin; Halibart-Puzio, Joanna; Lirski, Maciej; Kotliński, Maciej; Kroteń, Magdalena A.; Knizewski, Lukasz; Lange, Bartosz; Muszewska, Anna; Śniegowska-Świerk, Katarzyna; Kościelniak, Janusz; Iwanicka-Nowicka, Roksana; Buza, Krisztián; Janowiak, Franciszek; Żmuda, Katarzyna; Jõesaar, Indrek; Laskowska-Kaszub, Katarzyna; Fogtman, Anna; Kollist, Hannes; Zielenkiewicz, Piotr; Tiuryn, Jerzy; Siedlecki, Paweł; Swiezewski, Szymon; Ginalski, Krzysztof; Koblowska, Marta; Archacki, Rafał; Wilczynski, Bartek; Rapacz, Marcin; Jerzmanowski, Andrzej

    2015-01-01

    Linker (H1) histones play critical roles in chromatin compaction in higher eukaryotes. They are also the most variable of the histones, with numerous nonallelic variants cooccurring in the same cell. Plants contain a distinct subclass of minor H1 variants that are induced by drought and abscisic acid and have been implicated in mediating adaptive responses to stress. However, how these variants facilitate adaptation remains poorly understood. Here, we show that the single Arabidopsis (Arabidopsis thaliana) stress-inducible variant H1.3 occurs in plants in two separate and most likely autonomous pools: a constitutive guard cell-specific pool and a facultative environmentally controlled pool localized in other tissues. Physiological and transcriptomic analyses of h1.3 null mutants demonstrate that H1.3 is required for both proper stomatal functioning under normal growth conditions and adaptive developmental responses to combined light and water deficiency. Using fluorescence recovery after photobleaching analysis, we show that H1.3 has superfast chromatin dynamics, and in contrast to the main Arabidopsis H1 variants H1.1 and H1.2, it has no stable bound fraction. The results of global occupancy studies demonstrate that, while H1.3 has the same overall binding properties as the main H1 variants, including predominant heterochromatin localization, it differs from them in its preferences for chromatin regions with epigenetic signatures of active and repressed transcription. We also show that H1.3 is required for a substantial part of DNA methylation associated with environmental stress, suggesting that the likely mechanism underlying H1.3 function may be the facilitation of chromatin accessibility by direct competition with the main H1 variants. PMID:26351307

  15. Threading of Binuclear Ruthenium Complex Through DNA Bases

    NASA Astrophysics Data System (ADS)

    Paramanathan, Thayaparan; Westerlund, Fredrik; McCauley, Micah; Lincoln, Per; Rouzina, Ioulia; Williams, Mark

    2009-03-01

    Due to steric constraints the dumb-bell shaped binuclear ruthenium complex can only intercalate DNA by threading, which requires local melting of the DNA to occur. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to threading compared to bulk experiments. Stretching single DNA molecules with different drug concentrations and holding a constant force allows the binding to reach equilibrium. We can obtain the equilibrium fractional ligand binding and length of DNA at saturation. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics. In addition, we obtain the minimum binding site size, which may be determined by either electrostatic repulsion or steric constraints.

  16. Effective delivery of DNA into tumor cells and tissues by electroporation of polymer-DNA complex.

    PubMed

    Kang, Jeong-Hun; Toita, Riki; Niidome, Takuro; Katayama, Yoshiki

    2008-07-01

    Electroporation is a useful means for non-viral gene delivery. Here, we investigated the use of electroporation to deliver polymer-DNA complexes into living cells using a protein kinase C (PKC)alpha-responsive polymer. The polymer was complexed with a luciferase-encoding DNA and electroporated into B16 melanoma cells. Gene expression from polymer-DNA complexes was 3- to 5-fold higher than from naked DNA. Moreover, after introduction of the polymer-DNA complex into tissues, luciferase levels were >2-fold higher in B16 melanoma tumors than in normal skin tissue. These results suggest that the combination of our polymer and electroporation is useful for the effective delivery of DNA into tumors. PMID:18375054

  17. 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

    2014-05-01

    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-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. PMID:24497635

  18. 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

    2014-05-01

    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-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.

  19. DNA binding, antioxidant activity, and DNA damage protection of chiral macrocyclic Mn(III) salen complexes.

    PubMed

    Pandya, Nirali; Khan, Noor-ul H; Prathap, K Jeya; Kureshy, Rukhsana I; Abdi, Sayed H R; Mishra, Sandhya; Bajaj, Hari C

    2012-12-01

    We are reporting the synthesis, characterization, and calf thymus DNA binding studies of novel chiral macrocyclic Mn(III) salen complexes S-1, R-1, S-2, and R-2. These chiral complexes showed ability to bind with DNA, where complex S-1 exhibits the highest DNA binding constant 1.20 × 10(6) M(-1). All the compounds were screened for superoxide and hydroxyl radical scavenging activities; among them, complex S-1 exhibited significant activity with IC(50) 1.36 and 2.37 μM, respectively. Further, comet assay was used to evaluate the DNA damage protection in white blood cells against the reactive oxygen species wherein complex S-1 was found effective in protecting the hydroxyl radicals mediated plasmid and white blood cells DNA damage.

  20. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    PubMed

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  1. 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.

  2. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division

    PubMed Central

    Oliva, María A.

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex. PMID:27668216

  3. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division

    PubMed Central

    Oliva, María A.

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex.

  4. Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division.

    PubMed

    Oliva, María A

    2016-01-01

    Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex. PMID:27668216

  5. Complexation Between Cationic Diblock Copolymers and Plasmid DNA

    NASA Astrophysics Data System (ADS)

    Jung, Seyoung; Reineke, Theresa; Lodge, Timothy

    Deoxyribonucleic acids (DNA), as polyanions, can spontaneously bind with polycations to form polyelectrolyte complexes. When the polycation is a diblock copolymer with one cationic block and one uncharged hydrophilic block, the polyelectrolyte complexes formed with plasmid DNA (pDNA) are often colloidally stable, and show great promise in the field of polymeric gene therapy. While the resulting properties (size, stability, and toxicity to biological systems) of the complexes have been studied for numerous cationic diblocks, the fundamentals of the pDNA-diblock binding process have not been extensively investigated. Herein, we report how the cationic block content of a diblock influences the pDNA-diblock interactions. pDNA with 7164 base pairs and poly(2-deoxy-2-methacrylamido glucopyranose)-block-poly(N-(2-aminoethyl) methacrylamide) (PMAG-b-PAEMA) are used as the model pDNA and cationic diblock, respectively. To vary the cationic block content, two PMAG-b-PAEMA copolymers with similar PMAG block lengths but distinct PAEMA block lengths and a PAEMA homopolymer are utilized. We show that the enthalpy change from pDNA-diblock interactions is dependent on the cationic diblock composition, and is closely associated with both the binding strength and the pDNA tertiary structure.

  6. Computational and analytical modeling of cationic lipid-DNA complexes.

    PubMed

    Farago, Oded; Grønbech-Jensen, Niels

    2007-05-01

    We present a theoretical study of the physical properties of cationic lipid-DNA (CL-DNA) complexes--a promising synthetically based nonviral carrier of DNA for gene therapy. The study is based on a coarse-grained molecular model, which is used in Monte Carlo simulations of mesoscopically large systems over timescales long enough to address experimental reality. In the present work, we focus on the statistical-mechanical behavior of lamellar complexes, which in Monte Carlo simulations self-assemble spontaneously from a disordered random initial state. We measure the DNA-interaxial spacing, d(DNA), and the local cationic area charge density, sigma(M), for a wide range of values of the parameter (c) representing the fraction of cationic lipids. For weakly charged complexes (low values of (c)), we find that d(DNA) has a linear dependence on (c)(-1), which is in excellent agreement with x-ray diffraction experimental data. We also observe, in qualitative agreement with previous Poisson-Boltzmann calculations of the system, large fluctuations in the local area charge density with a pronounced minimum of sigma(M) halfway between adjacent DNA molecules. For highly-charged complexes (large (c)), we find moderate charge density fluctuations and observe deviations from linear dependence of d(DNA) on (c)(-1). This last result, together with other findings such as the decrease in the effective stretching modulus of the complex and the increased rate at which pores are formed in the complex membranes, are indicative of the gradual loss of mechanical stability of the complex, which occurs when (c) becomes large. We suggest that this may be the origin of the recently observed enhanced transfection efficiency of lamellar CL-DNA complexes at high charge densities, because the completion of the transfection process requires the disassembly of the complex and the release of the DNA into the cytoplasm. Some of the structural properties of the system are also predicted by a continuum

  7. Assembly of Slx4 signaling complexes behind DNA replication forks.

    PubMed

    Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

    2015-08-13

    Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.

  8. Assembly of Slx4 signaling complexes behind DNA replication forks

    PubMed Central

    Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

    2015-01-01

    Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. PMID:26113155

  9. Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection.

    PubMed

    Xu, Y; Szoka, F C

    1996-05-01

    To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1)-1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.

  10. Statistical mechanics of topologically constrained DNA and nucleoprotein complexes

    NASA Astrophysics Data System (ADS)

    Giovan, Stefan Michael

    A complex connection exists between the 3 dimensional topological state of DNA in living organisms and biological processes including gene expression, DNA replication, recombination and repair. A significant limitation in developing a detailed, quantitative understanding of this connection is due to a lack of rigorous methods to calculate statistical mechanical properties of DNA molecules with complex topologies, including supercoiling, looping and knotting. This dissertation's main focus is on developing such methods and applying them to realistic DNA and nucleoprotein models. In chapter 2, a method is presented to calculate free energies and J factors of protein mediated DNA loops by normal mode analysis (NMA). This method is similar to calculations performed previously but with several significant advances. We apply the method to the specific case of DNA looping mediated by Cre recombinase protein. J factors calculated by our method are compared to experimental measurements to extract geometric and elastic properties of the Cre-DNA synaptic complex. In particular, the results suggest the existence of a synaptic complex that is more flexible than previously expected and may be explained by a stable intermediate in the reaction pathway that deviates significantly from the planar crystal structure. Calculating free energies of DNA looping is difficult in general, especially when considering intermediate length scales such as plasmid sized DNA which may readily adopt multiple topological states. In chapter 3, a novel method is presented to obtain free energies of semiflexible biopolymers with fixed topologies and arbitrary ratios of contour length L to persistence length P. High accuracy is demonstrated by calculating free energies of specific DNA knots with L/P = 20 and L/P = 40, corresponding to DNA lengths of 3000 and 6000 base pairs, respectively. We then apply the method to study the free-energy landscape for a model of a synaptic nucleoprotein complex

  11. Programmed self-assembly of complex DNA nanostructures

    NASA Astrophysics Data System (ADS)

    Tian, Cheng

    DNA has served as an excellent building block to self-assemble into a wide range of one-dimensional (1D), two-dimensional (2D) and three-dimensional (3D) structures with the bottom-up method. Due to the specificity of base pairing, the DNA assembly system is predictable and robust. These DNA structures with higher diversity and complexity have potential applications as templates to organize guest molecules or nanoparticles for the nanofabrication, as biosensors for the genetic diagnosis and environmental detection, and as nanocarriers to deliver and release drugs for the therapy. My major researches focus on designing a novel building block and assembly strategies to self-assemble DNA into complex nanostructures to increase the diversity and complexity. A novel building block was first constructed, which is a parallel, left-handed DNA helix containing multiple domains of half-turn-long standard B-DNA. Such a structure can be used to introduce left-handed crossings in order to increase the diversity and complexity of DNA nanostructures, and can be taken into consideration when predicting the secondary structure of DNA/RNA molecules in cells. In addition, a tile-based directed self-assembly strategy was developed to construct DNA nanocages. In this strategy, directing building blocks were employed to control the self-assembly process of assembly building blocks. This strategy greatly expands the scope of accessible DNA nanostructures and would facilitate technological applications such as nano-guest encapsulation, drug delivery, and nanoparticle organization. As the complexity of DNA nanostructures increases, more errors might be involved in the assembly process. Therefore, a simplified design system based on T-junction was designed to build DNA arrays and minimize the assembly errors. In such system, due to the sequence symmetry, only one DNA single strand is employed and assembled into predesigned 1D and 2D arrays. This design system can be applied to assemble a

  12. Mesoscale Computer Modeling of Lipid-DNA Complexes for Gene Therapy

    NASA Astrophysics Data System (ADS)

    Farago, Oded; Grønbech-Jensen, Niels; Pincus, Philip

    2006-01-01

    We report on a molecular simulation method, which captures the self-assembly of cationic lipid-DNA (CL-DNA) gene delivery complexes. Computational efficiency required for large length- and time-scale simulations is achieved through a coarse-grained representation of the intramolecular details and via intermolecular potentials, which effectively mimic the hydrophobic effect without an explicit solvent. The broad utility of the model is illustrated by demonstrating excellent agreement with x-ray diffraction experimental data for the dependence of the spacing between DNA chains on the concentration of CLs. At high concentrations, the large electrostatic pressure induces the formation of pores in the membranes through which the DNA molecules may escape the complex. We relate this observation to the origin of recently observed enhanced transfection efficiency of lamellar CL-DNA complexes at high charge densities.

  13. Dynamics of DNA bending/unbending in complex with DNA-bending protein IHF

    NASA Astrophysics Data System (ADS)

    Ansari, Anjum; Vivas, Paula; Kuznetsov, Serguei

    2007-03-01

    Kinetics of conformational changes in proteins and DNA that lead to precise recognition of specific DNA binding sites are difficult to observe with the limited time-resolution of stop-flow and single-molecule techniques. Here we use a ˜10 ns laser T-jump apparatus to probe the kinetics of a ˜35-bp DNA substrate bound to E. coli Integration Host Factor (IHF) and end-labeled with a FRET pair. These T-jump measurements, in combination with stop-flow, provide the first direct observation of the DNA bending/unbending kinetics in a protein-DNA complex (Sugimura and Crothers, PNAS, in press; Kuznetsov et al., PNAS, in press). The rates and activation energy of DNA bending are similar to that of a single A:T base pair opening inside uncomplexed DNA, suggesting that spontaneous thermal disruption in base-pairing nucleated at an A:T site may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA. An unusual salt dependence of the binding affinity observed previously for IHF/DNA complex, and explained in terms of DNA binding coupled with disruption of a network of salt bridges within the protein (Holbrook et al., 2001, JMB, 310, 379), is reflected in the salt dependence of the observed bending rates. These results suggest that salt-dependent protein conformational changes may be playing a role in the DNA bending process.

  14. Structure of Cationic Liposome DNA Complexes Incorporating PEG Lipids

    NASA Astrophysics Data System (ADS)

    Winkler, Kathrin; Subramanian, G.; Safinya, C. R.

    1998-03-01

    DNA cationic liposome complexes with PEG lipids are promising candidates for efficient transfection of DNA in vivo. The presence of PEG confers (a) colloidal stability and (b) increased circulation time of the complexes in the blood stream leading to long term transfection activity. However, there has been no elucidation of the structure of these complexes which is crucial for correlating the structure and transfection efficiency. We have carried out a systematic structural investigation of complexes incorporating PEG lipids using X-ray scattering. We have studied the structure of complexes made from DOTAP (a cationic lipid), DOPC (a neutral lipid), PEG-DMPE and λ-DNA both as a function of the PEG-DMPE and neutral lipid concentrations. We have produced stable complexes which form a multilamellar structure with alternating lipid bilayers and DNA molecules. The DNA molecules are ordered in a 2-D smectic array whose spacing is controlled by the concentrations of PEG-DMPE and the neutral lipid. Supported by NSF-DMR-9624091, PRF-31352-AC7, and Los Alamos-STB/UC:96-108.

  15. DNA release from cationic liposome/DNA complexes by anionic lipids

    NASA Astrophysics Data System (ADS)

    Caracciolo, Giulio; Pozzi, Daniela; Caminiti, Ruggero; Marchini, Cristina; Montani, Maura; Amici, Augusto; Amenitsch, Heinz

    2006-12-01

    The authors found that recently developed multicomponent cationic liposome DNA complexes (lipoplexes) exhibit higher transfection efficiency with respect to usually employed binary lipoplexes in NIH 3T3 and A17 cell lines. Interaction of lipoplexes with anionic liposomes (model of cellular membranes) was investigated by synchrotron small angle x-ray diffraction. The authors used one-dimensional DNA packing density to estimate the molar fraction of DNA released from lipoplexes by anionic lipids.

  16. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action.

  17. DNA twisting flexibility and the formation of sharply looped protein-DNA complexes

    NASA Astrophysics Data System (ADS)

    Cloutier, T. E.; Widom, J.

    2005-03-01

    Gene-regulatory complexes often require that pairs of DNA-bound proteins interact by looping-out short (often 100-bp) stretches of DNA. The loops can vary in detailed length and sequence and, thus, in total helical twist, which radically alters their geometry. How this variability is accommodated structurally is not known. Here we show that the inherent twistability of 89- to 105-bp DNA circles exceeds theoretical expectation by up to 400-fold. These results can be explained only by greatly enhanced DNA flexibility, not by permanent bends. They invalidate the use of classic theories of flexibility for understanding sharp DNA looping but support predictions of two recent theories. Our findings imply an active role for DNA flexibility in loop formation and suggest that variability in the detailed helical twist of regulatory loops is accommodated naturally by the inherent twistability of the DNA. activation | gene regulation | repression

  18. DNA packing in stable lipid complexes designed for gene transfer imitates DNA compaction in bacteriophage

    PubMed Central

    Schmutz, M.; Durand, D.; Debin, A.; Palvadeau, Y.; Etienne, A.; Thierry, A. R.

    1999-01-01

    The structure of complexes made from DNA and suitable lipids (lipoplex, Lx) was examined by cryo-electron microscopy (cryoEM). We observed a distinct concentric ring-like pattern with striated shells when using plasmid DNA. These spherical multilamellar particles have a mean diameter of 254 nm with repetitive spacing of 7.5 nm with striation of 5.3 nm width. Small angle x-ray scattering revealed repetitive ordering of 6.9 nm, suggesting a lamellar structure containing at least 12 layers. This concentric and lamellar structure with different packing regimes also was observed by cryoEM when using linear double-stranded DNA, single-stranded DNA, and oligodeoxynucleotides. DNA chains could be visualized in DNA/lipid complexes. Such specific supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of DNA in a liquid crystalline phase. CryoEM examination of T4 phage DNA packed either in T4 capsides or in lipidic particles showed similar patterns. Small angle x-ray scattering suggested an hexagonal phase in Lx-T4 DNA. Our results indicate that both lamellar and hexagonal phases may coexist in the same Lx preparation or particle and that transition between both phases may depend on equilibrium influenced by type and length of the DNA used. PMID:10535915

  19. Molecular Models of STAT5A Tetramers Complexed to DNA Predict Relative Genome-Wide Frequencies of the Spacing between the Two Dimer Binding Motifs of the Tetramer Binding Sites

    PubMed Central

    Sathyanarayana, Bangalore K.; Li, Peng; Lin, Jian-Xin; Leonard, Warren J.

    2016-01-01

    STAT proteins bind DNA as dimers and tetramers to control cellular development, differentiation, survival, and expansion. The tetramer binding sites are comprised of two dimer-binding sites repeated in tandem. The genome-wide distribution of the spacings between the dimer binding sites shows a distinctive, non-random pattern. Here, we report on estimating the feasibility of building possible molecular models of STAT5A tetramers bound to a DNA double helix with all possible spacings between the dimer binding sites. We found that the calculated feasibility estimates correlated well with the experimentally measured frequency of tetramer-binding sites. This suggests that the feasibility of forming the tetramer complex was a major factor in the evolution of this DNA sequence variation. PMID:27537504

  20. Molecular Models of STAT5A Tetramers Complexed to DNA Predict Relative Genome-Wide Frequencies of the Spacing between the Two Dimer Binding Motifs of the Tetramer Binding Sites.

    PubMed

    Sathyanarayana, Bangalore K; Li, Peng; Lin, Jian-Xin; Leonard, Warren J; Lee, Byungkook

    2016-01-01

    STAT proteins bind DNA as dimers and tetramers to control cellular development, differentiation, survival, and expansion. The tetramer binding sites are comprised of two dimer-binding sites repeated in tandem. The genome-wide distribution of the spacings between the dimer binding sites shows a distinctive, non-random pattern. Here, we report on estimating the feasibility of building possible molecular models of STAT5A tetramers bound to a DNA double helix with all possible spacings between the dimer binding sites. We found that the calculated feasibility estimates correlated well with the experimentally measured frequency of tetramer-binding sites. This suggests that the feasibility of forming the tetramer complex was a major factor in the evolution of this DNA sequence variation. PMID:27537504

  1. Comparison of DNA adducts from exposure to complex mixtures in various human tissues and experimental systems

    PubMed Central

    Lewtas, Joellen; Mumford, Judy; Everson, Richard B.; Hulka, Barbara; Wilcosky, Tim; Kozumbo, Walter; Thompson, Claudia; George, Michael; Dobiáš, Lubomir; Šrám, Radim; Li, Xueming; Gallagher, Jane

    1993-01-01

    DNA adducts derived from complex mixtures of polycyclic aromatic compounds emitted from tobacco smoke are compared to industrial pollution sources (e.g., coke ovens and aluminum smelters), smoky coal burning, and urban air pollution. Exposures to coke oven emissions and smoky coal, both potent rodent skin tumor initiators and lung carcinogens in humans, result in high levels of DNA adducts compared to tobacco smoke in the in vitro calf thymus DNA model system, in cultured lymphocytes, and in the mouse skin assay. Using tobacco smoke as a model in human studies, we have compared relative DNA adduct levels detected in blood lymphocytes, placental tissue, bronchoalveolar lung lavage cells, sperm, and autopsy tissues of smokers and nonsmokers. Adduct levels in DNA isolated from smokers were highest in human heart and lung tissue with smaller but detectable differences in placental tissue and lung lavage cells. Comparison of the DNA adduct levels resulting from human exposure to different complex mixtures shows that emissions from coke ovens, aluminum smelters, and smoky coal result in higher DNA adduct levels than tobacco smoke exposure. These studies suggest that humans exposed to complex combustion mixtures will have higher DNA adduct levels in target cells (e.g., lung) as compared to nontarget cells (e.g., lymphocytes) and that the adduct levels will be dependent on the genotoxic and DNA adduct-forming potency of the mixture. ImagesFIGURE 1.FIGURE 1.FIGURE 2.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 4. PMID:8319665

  2. Direct observation of DNA bending/unbending kinetics in complex with DNA-bending protein IHF

    PubMed Central

    Kuznetsov, Serguei V.; Sugimura, Sawako; Vivas, Paula; Crothers, Donald M.; Ansari, Anjum

    2006-01-01

    Regulation of gene expression involves formation of specific protein–DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNA-binding sites. Previous kinetics measurements on several DNA-bending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending/unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from ≈2 ms−1 at ≈37°C to ≈40 ms−1 at ≈10°C and correspond to an activation energy of ≈14 ± 3 kcal/mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend/kink DNA before forming a tight complex with IHF. PMID:17124171

  3. Folding complex DNA nanostructures from limited sets of reusable sequences

    PubMed Central

    Niekamp, Stefan; Blumer, Katy; Nafisi, Parsa M.; Tsui, Kathy; Garbutt, John; Douglas, Shawn M.

    2016-01-01

    Scalable production of DNA nanostructures remains a substantial obstacle to realizing new applications of DNA nanotechnology. Typical DNA nanostructures comprise hundreds of DNA oligonucleotide strands, where each unique strand requires a separate synthesis step. New design methods that reduce the strand count for a given shape while maintaining overall size and complexity would be highly beneficial for efficiently producing DNA nanostructures. Here, we report a method for folding a custom template strand by binding individual staple sequences to multiple locations on the template. We built several nanostructures for well-controlled testing of various design rules, and demonstrate folding of a 6-kb template by as few as 10 unique strand sequences binding to 10 ± 2 locations on the template strand. PMID:27036861

  4. DNA-Cationic Lipid Complexes: Lamellar and Inverted Hexagonal Phases

    NASA Astrophysics Data System (ADS)

    Koltover, I.; Salditt, T.; Raedler, J.; Safinya, C.

    1998-03-01

    Cationic lipid-DNA (CL-DNA) complexes can be efficient non-viral vectors for gene therapy. However, it is not known why transfection rates vary widely for complexes with different lipid compositions. We have discovered a transition between two distinct liquid crystalline (LC) structures of the complex by varying the lipid composition: a lamellar structure ( J. Raedler, I. Koltover, T. Salditt, C. Safinya, Science 275, 810 (1997)) and a novel LC phase with DNA double-strands surrounded by lipid monolayers arranged on a regular hexagonal lattice. The CL-DNA complexes with the two structures interact differently with giant negatively charged liposomes, which represent the simplest model of cellular membranes. We demonstrate the generality of the lamellar-hexagonal transformation by observing it in complexes of cationic lipid with two other negatively charged biopolymers - polyglutamic acid (PGA), a model polypeptide and poly-thymine (polyT), a model single-stranded oligo-nucleotide. We identify the interactions leading to the transformations between the two complex phases for the three different polyelectrolytes. Supported by NSF DMR-9624091 and a Los Alamos CULAR grant No.STB/UC:95-146.

  5. An osmium-DNA interstrand complex: application to facile DNA methylation analysis.

    PubMed

    Tanaka, Kazuo; Tainaka, Kazuki; Umemoto, Tadashi; Nomura, Akiko; Okamoto, Akimitsu

    2007-11-21

    Nucleic acids often acquire new functions by forming a variety of complexes with metal ions. Osmium, in an oxidized state, also reacts with C5-methylated pyrimidines. However, control of the sequence specificity of osmium complexation with DNA is still immature, and the value of the resulting complexes is unknown. We have designed a bipyridine-attached adenine derivative for sequence-specific osmium complexation. Sequence-specific osmium complexation was achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and resulted in the formation of a cross-linked structure. The interstrand cross-link clearly distinguished methylated cytosines from unmethylated cytosines and was used to quantify the degree of methylation at a specific cytosine in the genome.

  6. LL37-DNA complexes and auto-immune diseases

    NASA Astrophysics Data System (ADS)

    Jin, Fan; Sanders, Lori K.; Xian, Wujing; Gilliet, Michel; Wong, Gerard C. L.; Department of Immunology, University of Texas, Houston Collaboration

    2011-03-01

    LL37 is an alpha-helical host defense peptide in humans. Recent work has shown that Toll-like receptor-9 (TLR9), an intracellular receptor in plasmacytoid dendritic cells (pDCs) of the immune system that normally responds to pathogen nucleic acids, can be pathologically triggered by self DNA in the form of DNA-LL37 complexes. Synchrotron small-angle x-ray scattering (SAXS) measurements reveal an unanticipated form of self-assembly between DNA and this positively charged macroion. We examine the generality of this with other macroions, and propose a new geometric criterion for immune cell activation.

  7. Both Complexity and Location of DNA Damage Contribute to Cellular Senescence Induced by Ionizing Radiation

    PubMed Central

    Zhang, Xurui; Ye, Caiyong; Sun, Fang; Wei, Wenjun; Hu, Burong; Wang, Jufang

    2016-01-01

    Persistent DNA damage is considered as a main cause of cellular senescence induced by ionizing radiation. However, the molecular bases of the DNA damage and their contribution to cellular senescence are not completely clear. In this study, we found that both heavy ions and X-rays induced senescence in human uveal melanoma 92–1 cells. By measuring senescence associated-β-galactosidase and cell proliferation, we identified that heavy ions were more effective at inducing senescence than X-rays. We observed less efficient repair when DNA damage was induced by heavy ions compared with X-rays and most of the irreparable damage was complex of single strand breaks and double strand breaks, while DNA damage induced by X-rays was mostly repaired in 24 hours and the remained damage was preferentially associated with telomeric DNA. Our results suggest that DNA damage induced by heavy ion is often complex and difficult to repair, thus presents as persistent DNA damage and pushes the cell into senescence. In contrast, persistent DNA damage induced by X-rays is preferentially associated with telomeric DNA and the telomere-favored persistent DNA damage contributes to X-rays induced cellular senescence. These findings provide new insight into the understanding of high relative biological effectiveness of heavy ions relevant to cancer therapy and space radiation research. PMID:27187621

  8. Physical properties of inner histone-DNA complexes.

    PubMed Central

    Bryan, P N; Wright, E B; Hsie, M H; Olins, A L; Olins, D E

    1978-01-01

    Chicken-erythrocyte inner histone tetramer has been complexed with several natural and synthetic DNA duplexes by salt-gradient dialysis at various protein/DNA ratios. The resulting complexes, in low-ionic-strength buffer, have been examined by electron microscopy, circular dichroism, and thermal denaturation. Electron microscopy reveals nucleosomes (nu bodies) randomly arranged along DNA fibers, including poly(dA-dT)-poly(dA-dT), poly(dI-dC)-poly(dI-dC), but not poly(dA)-poly(dT). Circular dichroism studies showed prominent histone alpha-helix and "suppression" of nucleic acid ellipticity (lambda less than 240 nm). Thermal denaturation experiments revealed Tm behavior comparable to that of H1- (or H5-) depleted chromatin. Tm III and Tm IV increased linearly with G + C%(natural DNAs), but were virtually independent of the histone/DNA ratio; therefore, the melting of nucleosomes along a DNA chain is insensitive to adjacent "spacer" DNA lengths. This suggests that Tm III and Tm IV arise from the melting of different domains of DNA associated with the core nu body. Images PMID:214760

  9. Photoinduced intercalation and coordination of a dirhodium complex to DNA: dual DNA binding.

    PubMed

    Palmer, Alycia M; Burya, Scott J; Gallucci, Judith C; Turro, Claudia

    2014-06-01

    Two new complexes, cis-H,H-[Rh2 (OCCH3 NH)2 (LL)(CH3 CN)2 ](2+) , where LL=bpy (2, bpy=2,2'-bipyridine) and dppz (3, dppz=dipyrido[3,2-a:2',3'-c]phenazine), were prepared from the reaction of cis-H,H-[Rh2 (OCCH3 NH)2 (CH3 CN)6 ](2+) (1) with the corresponding bidentate ligand. The bpy and dppz ligands chelate to the same rhodium atom and are positioned trans to the amidato N atoms, as determined by the single crystal X-ray structure of 2. Irradiation of 2 and 3 with visible light in water results in the exchange of one CH3 CNeq ligand for an H2 O molecule with quantum yields, Φ400 , of 0.040 and 0.044, respectively (λirr =400 nm). The identities of the photoproducts of 2 and 3 were determined to be cis-H,H-[Rh2 (OCCH3 NH)2 (L)(H2 O)(CH3 CN)](2+) , where L is bpy (4) and dppz (5), respectively. Mobility shift assays show that 4 crosslinks double-stranded DNA, and ESI-MS experiments indicate that both 4 and 5 form covalent adducts with single-stranded DNA. In addition, relative viscosity and 2D NMR experiments show that the dppz ligand of 5 also intercalates into DNA upon irradiation, making 3 a dual-binding agent that both intercalates and covalently binds to DNA upon the absorption of visible light.

  10. Application of Anomalous Diffraction Methods to the Study of DNA and DNA-Complexes

    SciTech Connect

    Watkins, Derrick; Moulaei, Tinoush; Komeda, Seiji; Williams, Loren Dean

    2012-01-24

    Anomalous scattering is commonly used to solve X-ray structures. As discussed here, anomalous scattering is also useful for characterizing complex systems with mixed and partial occupancies, where true electron density is represented by unresolvable ensemble averages. The solvent environment surrounding nucleic acids is an example of such a system, as are some DNA-ligand systems. The atomic number and wavelength dependencies of anomalous scattering allow one to filter out the electron densities of C, N, and O, and to cleanly visualize the electron densities of heavier atoms. Therefore, anomalous scattering can make beacons of selected atoms. In addition, anomalous scattering provides a model-independent method for determining atomic identities. Here, we describe applications of anomalous scattering to the structure determination of DNA-platinum complexes and in cation associations of free DNA, of DNA-anthracycline complexes, of chemically modified DNA, and of DNA-protein complexes. The utility of Rb{sup +} and Tl{sup +} as K{sup +} substitutes is supported by similarities in Rb{sup +} and Tl{sup +} association with DNA.

  11. Visualization of DNA and protein-DNA complexes with atomic force microscopy.

    PubMed

    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 purification of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein-DNA complexes.

  12. 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

  13. Design and Fabrication of Nanostructures Based on DNA Ring-Protein Complex

    NASA Astrophysics Data System (ADS)

    Furukawa, Hideki; Endo, Tatsuro; Yanagida, Yasuko; Hatsuzawa, Takeshi

    2008-06-01

    In this report, we describe the design and fabrication of DNA nanostructures (“DNA glasses”, “DNA serial rings”, and “DNA chains”) using DNA ring-protein complexes. An experiment was performed to fabricate DNA ring-conjugated biotin using two types of DNA (“vector DNA” and “insert DNA”). The vector DNA was obtained by cutting plasmid DNA with restriction enzymes. The insert DNA (DNA conjugated with biotin) was obtained using a DNA synthesizer. After ligation and the introduction of streptavidin-modified gold nanoparticles, DNA structures were obtained. Subsequently, the DNA structures were observed by atomic force microscopy (AFM).

  14. Complexation of heterocyclic ligands with DNA in aqueous solution

    NASA Astrophysics Data System (ADS)

    Baranovskii, S. F.; Bolotin, P. A.; Evstigneev, M. P.; Chernyshev, D. N.

    2008-03-01

    We have used spectrophotometry to study self-association and complexation with DNA by organic heterocyclic compounds in the acridine and phenothiazine series: proflavin, thionine, and methylene blue. Based on the experimental concentration dependences of the molar absorption coefficient of the molecules in an aqueous buffer solution (0.01 M NaCl, 0.01 M Na2EDTA, 0.01 M Tris, pH 7.4, T = 298 K), we have determined the equilibrium dimerization constants for the dyes and the DNA complexation parameters using the Scatchard and McGhee-von Hippel models. The observed increase in the cooperativity parameters as the dimerization constants of the ligands increase allowed us to hypothesize that the same interactions occur between dye molecules adsorbed on DNA as in their self-association. The equilibrium DNA-binding constants for the ligands, obtained using the McGhee-von Hippel cooperative model, are (20.9 ± 2.7)·103 M-1 for proflavin and (33.8 ± 4.1)·103 M-1 for thionine. Using the Scatchard model, taking into account intercalation and “external” binding of ligands with DNA, we determined the DNA complexation constants for methylene blue: (26.4 ± 4.6)·103 and (96 ± 17)·103 M-1 respectively. Based on analysis of the data obtained, we hypothesized that the predominant type of binding with DNA is intercalation binding in the case of proflavin and thionine, and “external” binding with the DNA surface in the case of methylene blue.

  15. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  16. Complexes of nitracrine with DNA. Stoichiometry of binding.

    PubMed

    Szmigiero, L; Gniazdowski, M

    1981-01-01

    In the presence of sulfhydryl compounds, an anticancer drug 1-nitro-9-(3-N,N-dimethylaminopropylamino) acridine (nitracrine, Ledakrin) forms irreversible complexes of decreased template activity with DNA. Stoichiometry of the complexes was estimated using the drug labelled with 14C in the acridine ring or in the propyl chain and with 3H in the acridine ring. Up to 50 irreversibly bound 14C-nitracrine molecules were found per 10(3) nucleotides of calf thymus DNA in the presence of dithiothreitol (DTT). Considerably lower binding observed using 3H-labelled drug, particularly when the complexes were formed in the presence of mercaptoethanol (ME) indicates that substitution of tritium atoms occurred during the reaction. Relationship between stoichiometry and template activity in RNA synthesis in vitro system of the complexes was estimated in the paper. PMID:7198467

  17. Hydrodynamic size of DNA/cationic gemini surfactant complex as a function of surfactant structure.

    PubMed

    Devínsky, Ferdinand; Pisárcik, Martin; Lacko, Ivan

    2009-06-01

    The present study deals with the determination of hydrodynamic size of DNA/cationic gemini surfactant complex in sodium bromide solution using the dynamic light scattering method. Cationic gemini surfactants with polymethylene spacer of variable length were used for the interaction with DNA. The scattering experiments were performed at constant DNA and sodium bromide concentrations and variable surfactant concentration in the premicellar and micellar regions as a function of surfactant spacer length. It was found that the DNA conformation strongly depends on the polymethylene spacer length as well as on the surfactant concentration relative to the surfactant critical micelle concentration. Gemini surfactant molecules with 4 methylene groups in the spacer were found to be the least efficient DNA compacting agent in the region above the surfactant cmc. Gemini molecules with the shortest spacer length (2 methylene groups) and the longest spacer length (8 methylene groups) investigated showed the most efficient DNA compaction ability. PMID:19592712

  18. DNA interaction and cytotoxic activities of square planar platinum(II) complexes with N, S-donor ligands

    NASA Astrophysics Data System (ADS)

    Patel, Mohan N.; Patel, Chintan R.; Joshi, Hardik N.; Thakor, Khyati P.

    2014-06-01

    The platinum(II) complexes with N, S-donor ligands have been synthesized and characterized by physicochemical methods viz. elemental, electronic, FT-IR, 1H NMR and LC-MS spectra. The binding mode and potency of the complexes with HS DNA (Herring Sperm) have been examined by absorption titration and viscosity measurement studies. The results revealed that complexes bind to HS DNA via covalent mode with the intrinsic binding constant (Kb) in the range 1.37-7.76 × 105 M-1. Decrease in the relative viscosity of HS DNA also supports the covalent mode of binding. The DNA cleavage activity of synthesized complexes has been carried out by gel electrophoresis experiment using supercoiled form of pUC19 DNA; showing the unwinding of the negatively charged supercoiled DNA. Brine shrimp (Artemia Cysts) lethality bioassay technique has been applied for the determination of toxic property of synthesized complexes in terms of μM.

  19. 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.

  20. Fluorescence of tetrols, tetrols complexed with DNA, and benzo[a]pyrene-DNA adducts in methanol/water solutions

    SciTech Connect

    Steinbach, Paul B.; Hurtubise, Robert J.

    2000-02-01

    Several solution fluorescence parameters were acquired for the four tetrol hydrolysis products of benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts, tetrols complexed with DNA, and BPDE-DNA adducts in several methanol/water solvents. The relative polarity of the environment for the tetrols and BPDE-DNA adducts was determined by using a modified definition of the R value that is commonly employed for pyrene. The R values for the tetrols and BPDE-DNA adducts were calculated by obtaining the ratios of the intensities of the two major fluorescence emission bands at 380 and 400 nm (I{sub 380}/I{sub 400}). The positions of the hydroxyl groups on the hydroaromatic ring of the tetrols were compared in reference to the R values and the changes in the R values as a function of methanol/water composition. This approach resulted in a method for determining whether the hydroxyl groups in the 9 and 10 positions were on the same side or opposite sides of a hydroaromatic ring. The tetrols intercalated between the DNA bases showed quite different fluorescence spectra compared to tetrols not complexed with DNA. Also, the quasi-intercalated BPDE-DNA adducts gave significant changes in the R values with an increase in methanol in the solvent, and excitation spectra showed large shifts and changes in shape with an increase in methanol. The approaches developed provide unique structural and polarity information on tetrols and BPDE-DNA adducts. (c) 2000 Society for Applied Spectroscopy.

  1. Crystal structure of DnaT84–153-dT10 ssDNA complex reveals a novel single-stranded DNA binding mode

    PubMed Central

    Liu, Zheng; Chen, Peng; Wang, Xuejuan; Cai, Gang; Niu, Liwen; Teng, Maikun; Li, Xu

    2014-01-01

    DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriA-dependent primosome assembly is still a black box. Here, we report the 2.83 Å DnaT84–153-dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA. PMID:25053836

  2. Complex formation between phage phi 29 single-stranded DNA binding protein and DNA.

    PubMed

    Soengas, M S; Esteban, J A; Salas, M; Gutiérrez, C

    1994-06-01

    Bacteriophage phi 29 gene 5 encodes a single-stranded DNA (ssDNA) binding protein (SSB) which stimulates viral DNA replication. In the present study, a structural characterization of the complex between ssDNA and the phi 29 SSB was carried out using electron microscopy, band-shift assays and nuclease digestion as well as by monitoring changes in the intrinsic fluorescence of phi 29 SSB upon binding. Phage phi 29 SSB behaves as a monomer in solution and forms complexes with ssDNA which have a homogeneous structure, as if they consist of a continuous array of protein bound to DNA. Interaction of phi 29 SSB with ssDNA leads to a quenching of its tyrosine-dependent intrinsic fluorescence. This fluorescence quenching was directly proportional to the amount of phi 29 SSB bound to the ssDNA and the maximal quenching upon binding was very high (Qmax = 94.6 +/- 3.5%). Direct titration experiments have allowed us to estimate that the stoichiometry (n) of binding to ssDNA was 3.4(+/- 0.3) nucleotides per phi 29 SSB monomer. Both Qmax and n are independent of the salt concentration, suggesting the existence of only one major binding mode. At low salt concentrations, the effective binding constant (Keff = K omega) to poly(dT) was 2.2 x 10(5) M-1, the intrinsic binding constant (K) and the cooperativity parameter (omega) being 4.3 x 10(3) M-1 and 51, respectively. At increasing salt concentrations, the Keff exhibited a small, but significant, decrease. The possible functional significance of the binding parameters of phi 29 SSB during viral DNA replication is discussed.

  3. Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA-protein complexes.

    PubMed

    McAndrew, Christopher P; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L; Pegg, Ian L; Sarkar, Abhijit

    2016-01-01

    We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA-protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether's extension: ~0.05 pN in force and <10 nm change in extension. We have also verified that we can manipulate DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule's force response. Using a calibration technique based on Stokes' drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level.

  4. Lipoplex-Mediated Deintercalation of Doxorubicin from Calf Thymus DNA-Doxorubicin Complex.

    PubMed

    Das, Anupam; Adhikari, Chandan; Chakraborty, Anjan

    2016-09-01

    In this paper, we report the lipoplex-mediated deintercalation of anticancer drug doxorubicin (DOX) from the DOX-DNA complex under controlled experimental conditions. We used three zwitterionic liposomes, namely, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), which are widely different in their phase transition temperatures to form a lipoplex with calf thymus DNA in the presence of Ca(2+) ions. The study revealed that DPPC being in sol-gel phase was more effective in releasing the drug from the DOX-DNA complex compared with liposomes that remain in liquid crystalline phase (DMPC and POPC). The higher extent of drug release in the case of DPPC liposomes was attributed to the stronger lipoplex formation with DNA as compared with that of other liposomes. Owing to the relatively smaller head group area, the DPPC liposomes in their sol-gel phase can absorb a larger number of Ca(2+) ions and hence offer a strong electrostatic interaction with DNA. This interaction was confirmed by time-resolved anisotropy and circular dichroism spectroscopy. Apart from the electrostatic interaction, the possible hydrophobic interaction between the liposomes and DNA was also taken into account for the observed deintercalation. The successful uptake of drug molecules by liposomes from the drug-DNA complex in the post-release period was also confirmed using confocal laser scanning microscopy (CLSM). PMID:27465781

  5. Bell Curve for Transfection by Lamellar Cationic Lipid--DNA Complexes

    NASA Astrophysics Data System (ADS)

    Ahmad, A.; Evans, Heather M.; Ewert, K.; George, C. X.; Samuel, C. E.; Safinya, C. R.

    2004-03-01

    Cationic liposomes (CL) present a viable alternative to viral delivery of therapeutic DNA to cells. We combine CL with DNA in order to form complexes that can deliver foreign DNA (genes) to cells. In trying to improve the transfection efficiency (TE) of lamellar CL-DNA complexes, we have identified universal trends depending on the headgroup size and charge of the cationic lipid. By using new multivalent lipids ranging from 2+ to 16+ (e.g. Ewert et al, J. Med. Chem. 2002; 45: 5023) we are able to access a wide range of membrane charge density values, or σ _M. TE plots vs. σ M for multivalent lipids merge onto a universal curve with a Gaussian shape. The optimal σ M depends on the overall CL/DNA charge. The universal TE curve shows three regimes related to cellular obstacles: at low σ _M, TE is limited by endosomal escape of CL-DNA, while at high σ M TE is limited by complex dissociation and DNA release into the cytoplasm. Funded by NIH GM-59288 and NSF DMR-0203755.

  6. DNA-poly(diallyldimethylammonium chloride) complexation and transfection efficiency.

    PubMed

    Alatorre-Meda, Manuel; Taboada, Pablo; Krajewska, Barbara; Willemeit, Markus; Deml, Alexander; Klösel, Roland; Rodríguez, Julio R

    2010-07-29

    The present work assesses the influence of the cationic charge density (CD) and the cationic valence of poly(diallyldimethylammonium chloride) (pDADMAC) on the DNA compaction and subsequent transfection. Four homopolymers (CD = 1, with different valences) and one copolymer, poly(acrylamide-co-diallyldimethylammonium chloride) (coDADMAC) (CD < 1, equivalent in valence to one of the homopolymers), were studied. The characterization of the DNA-pDADMAC complexes (polyplexes) as a function of the polycation nitrogen to DNA phosphate molar ratios, N/P, was done by means of conductometry, electrophoretic mobility (zeta-potential), dynamic light scattering (DLS), isothermal titration calorimetry (ITC), atomic force microscopy (AFM), and beta-galactosidase (ONPG) and luciferase expression assays at 25 degrees C and physiological pH. In general, all polyplexes rendered compact and stable structures (R(H) approximately 100 nm) with positive surface charges ( approximately 11 mV) but low transfection efficiencies. As revealed by ITC, the DNA-pDADMAC complexation was characterized by a high binding affinity, the process being entropically driven. In particular, two characteristic ratios ((N/P)c and (N/P)*) were detected. Conductometry and ITC data demonstrated that the DNA compaction ratio, (N/P)c, was mainly governed by CD. Meanwhile the ratio from which the polyplex size remained constant, (N/P)*, was found to be valence-dependent as revealed by DLS. On the other hand, the low transfer rate of the polyplexes appeared to be correlated with the high binding affinity observed throughout the complexation process and with a core-shell structure the complexes presumably adopt.

  7. 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

  8. DNA gyrase-mediated wrapping of the DNA strand is required for the replication fork arrest by the DNA gyrase-quinolone-DNA ternary complex.

    PubMed

    Hiasa, H; Shea, M E

    2000-11-01

    The ability of DNA gyrase (Gyr) to wrap the DNA strand around itself allows Gyr to introduce negative supercoils into DNA molecules. It has been demonstrated that the deletion of the C-terminal DNA-binding domain of the GyrA subunit abolishes the ability of Gyr to wrap the DNA strand and catalyze the supercoiling reaction (Kampranis, S. C., and Maxwell, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14416-14421). By using this mutant Gyr, Gyr (A59), we have studied effects of Gyr-mediated wrapping of the DNA strand on its replicative function and its interaction with the quinolone antibacterial drugs. We find that Gyr (A59) can support oriC DNA replication in vitro. However, Gyr (A59)-catalyzed decatenation activity is not efficient enough to complete the decatenation of replicating daughter DNA molecules. As is the case with topoisomerase IV, the active cleavage and reunion activity of Gyr is required for the formation of the ternary complex that can arrest replication fork progression in vitro. Although the quinolone drugs stimulate the covalent Gyr (A59)-DNA complex formation, the Gyr (A59)-quinolone-DNA ternary complexes do not arrest the progression of replication forks. Thus, the quinolone-induced covalent topoisomerase-DNA complex formation is necessary but not sufficient to cause the inhibition of DNA replication. We also assess the stability of ternary complexes formed with Gyr (A59), the wild type Gyr, or topoisomerase IV. The ternary complexes formed with Gyr (A59) are more sensitive to salt than those formed with either the wild type Gyr or topoisomerase IV. Furthermore, a competition experiment demonstrates that the ternary complexes formed with Gyr (A59) readily disassociate from the DNA, whereas the ternary complexes formed with either the wild type Gyr or topoisomerase IV remain stably bound. Thus, Gyr-mediated wrapping of the DNA strand is required for the formation of the stable Gyr-quinolone-DNA ternary complex that can arrest replication fork

  9. DNA-osmium complexes: recent developments in the operative chemical analysis of DNA epigenetic modifications.

    PubMed

    Okamoto, Akimitsu

    2014-09-01

    The development of a reaction for the detection of one epigenetic modification in a long DNA strand is a chemically and biologically challenging research subject. Herein, we report and discuss the formation of 5-methylcytosine-osmium complexes that are used as the basis for a bisulfite-free chemical assay for DNA methylation analysis. Osmium in the oxidized state reacts with C5-methylated pyrimidines in the presence of a bipyridine ligand to give a stable ternary complex. On the basis of this reaction, an adenine derivative with a tethered bipyridine moiety has been designed for sequence-specific osmium complex formation. Osmium complexation is then achieved by hybridization of a short DNA molecule containing this functional nucleotide to a target DNA sequence and results in the formation of a cross-linked structure. This novel concept of methylation-specific reaction, based on a straightforward chemical process, expands the range of methods available for the analysis of epigenetic modifications. Advantages of the described method include amplification-insensitive detection, 5-hydroxymethylcytosine complexation, and visualization through methylation-specific in situ hybridization.

  10. Structures of human exonuclease I DNA complexes suggest a unified mechanism for nuclease family

    PubMed Central

    Orans, Jillian; McSweeney, Elizabeth A.; Iyer, Ravi R.; Hast, Michael A.; Hellinga, Homme W.; Modrich, Paul; Beese, Lorena S.

    2011-01-01

    Summary Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5′ structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5′ ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exo-nucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing. PMID:21496642

  11. Assessment of methods to recover DNA from bacteria, fungi and archaea in complex environmental samples.

    PubMed

    Guillén-Navarro, Karina; Herrera-López, David; López-Chávez, Mariana Y; Cancino-Gómez, Máximo; Reyes-Reyes, Ana L

    2015-11-01

    DNA extraction from environmental samples is a critical step for metagenomic analysis to study microbial communities, including those considered uncultivable. Nevertheless, obtaining good quality DNA in sufficient quantities for downstream methodologies is not always possible, and it depends on the complexity and stability of each ecosystem, which could be more problematic for samples from tropical regions because those ecosystems are less stable and more complex. Three laboratory methods for the extraction of nucleic acids from samples representing unstable (decaying coffee pulp and mangrove sediments) and relatively stable (compost and soil) environments were tested. The results were compared with those obtained using two commercial DNA extraction kits. The quality of the extracted DNA was evaluated by PCR amplification to verify the recovery of bacterial, archaeal, and fungal genetic material. The laboratory method that gave the best results used a lysis procedure combining physical, chemical, and enzymatic steps.

  12. Assessment of methods to recover DNA from bacteria, fungi and archaea in complex environmental samples.

    PubMed

    Guillén-Navarro, Karina; Herrera-López, David; López-Chávez, Mariana Y; Cancino-Gómez, Máximo; Reyes-Reyes, Ana L

    2015-11-01

    DNA extraction from environmental samples is a critical step for metagenomic analysis to study microbial communities, including those considered uncultivable. Nevertheless, obtaining good quality DNA in sufficient quantities for downstream methodologies is not always possible, and it depends on the complexity and stability of each ecosystem, which could be more problematic for samples from tropical regions because those ecosystems are less stable and more complex. Three laboratory methods for the extraction of nucleic acids from samples representing unstable (decaying coffee pulp and mangrove sediments) and relatively stable (compost and soil) environments were tested. The results were compared with those obtained using two commercial DNA extraction kits. The quality of the extracted DNA was evaluated by PCR amplification to verify the recovery of bacterial, archaeal, and fungal genetic material. The laboratory method that gave the best results used a lysis procedure combining physical, chemical, and enzymatic steps. PMID:26014885

  13. DNA-protein noncovalent cross-linking: ruthenium dipyridophenazine biotin complex for the assembly of proteins and gold nanoparticles on DNA templates.

    PubMed

    Slim, Mohamed; Durisic, Nela; Grutter, Peter; Sleiman, Hanadi F

    2007-05-01

    We report the first example of a small molecule that can noncovalently cross-link DNA with streptavidin and streptavidin-labeled materials. Molecule 1 possesses a ruthenium dipyridophenazine DNA-intercalating moiety and a biotin unit; these two units are adequately separated to ensure efficient cross-linking of DNA with protein. Complex 1 is essentially nonemissive in aqueous solution and when bound to streptavidin, however, its luminescence is turned "on" when it binds to DNA. We have used these properties to establish that this complex can simultaneously bind to DNA and streptavidin, and can thus bring these two biomolecules together. We also synthesized a related molecule, 3, in which the biotin and DNA-intercalating moieties are covalently bound. While complex 3 can intercalate into DNA through a threading mechanism, luminescence experiments show that it cannot simultaneously bind DNA and streptavidin, most likely due to the proximity of its two molecular-recognition units. The cross-linking ability of molecule 1 was used to template the assembly of streptavidin molecules on circular plasmid DNA, as visualized by atomic force microscopy. In addition, using 1, we show the organization of discrete groups of gold nanoparticles labeled with streptavidin on a linear DNA template of finite size, with transmission electron microscopy. In these experiments the DNA template acted as a "molecular ruler" that dictated the number of particles in the assembly.

  14. Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

    SciTech Connect

    Pallan, Pradeep S.; Egli, Martin

    2009-06-17

    Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme's interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg{sup 2+} at the active site. A subset of amino acids engaged in contacts to RNA 2{prime}-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme's interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.

  15. DNA Binding Properties of the Actin-Related Protein Arp8 and Its Role in DNA Repair

    PubMed Central

    Murakami, Hirokazu; Otawa, Kenji; Tachiwana, Hiroaki; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-ich; Kurumizaka, Hitoshi; Harata, Masahiko

    2014-01-01

    Actin and actin-related proteins (Arps), which are members of the actin family, are essential components of many of these remodeling complexes. Actin, Arp4, Arp5, and Arp8 are found to be evolutionarily conserved components of the INO80 chromatin remodeling complex, which is involved in transcriptional regulation, DNA replication, and DNA repair. A recent report showed that Arp8 forms a module in the INO80 complex and this module can directly capture a nucleosome. In the present study, we showed that recombinant human Arp8 binds to DNAs, and preferentially binds to single-stranded DNA. Analysis of the binding of adenine nucleotides to Arp8 mutants suggested that the ATP-binding pocket, located in the evolutionarily conserved actin fold, plays a regulatory role in the binding of Arp8 to DNA. To determine the cellular function of Arp8, we derived tetracycline-inducible Arp8 knockout cells from a cultured human cell line. Analysis of results obtained after treating these cells with aphidicolin and camptothecin revealed that Arp8 is involved in DNA repair. Together with the previous observation that Arp8, but not γ-H2AX, is indispensable for recruiting INO80 complex to DSB in human, results of our study suggest an individual role for Arp8 in DNA repair. PMID:25299602

  16. Orientation of non-blue cupric complexes on DNA fibers.

    PubMed

    Chikira, M; Sato, T; Antholine, W E; Petering, D H

    1991-02-15

    Three different orientations of non-blue, type 2 cupric complexes on DNA fibers are obtained from EPR data. The cupric complex of bleomycin, CuBlm, binds as described previously (Shields, H., McGlumphy,C., and Hamrick, P., J., Jr. (1982) Biochim. Biophys. Acta 697, 113-120), except possibly with more restricted motion. The square plane of CuBlm makes an angle of about 65 degrees with the fiber axis. The tridentate complex 2-formylpyridine monothiosemicarbazonato Cu2+ binds with its planar structure perpendicular to the fiber axis. In contrast, other tridentate cupric complexes of tripeptides, CuGHK and CuGHG, bind with the square plane parallel to the fiber axis. The bound forms of Cu(GHK) and Cu(GHG) are determined mostly by the GH moiety in the complex; the contribution of lysine in defining the orientation of the copper moiety is minimal. Thus, the structure of the ligand determines the orientation of these complexes on DNA. PMID:1704368

  17. Complex mitochondrial DNA rearrangements in individual cells from patients with sporadic inclusion body myositis

    PubMed Central

    Rygiel, Karolina A.; Tuppen, Helen A.; Grady, John P.; Vincent, Amy; Blakely, Emma L.; Reeve, Amy K.; Taylor, Robert W.; Picard, Martin; Miller, James; Turnbull, Doug M.

    2016-01-01

    Mitochondrial DNA (mtDNA) rearrangements are an important cause of mitochondrial disease and age related mitochondrial dysfunction in tissues including brain and skeletal muscle. It is known that different mtDNA deletions accumulate in single cells, but the detailed nature of these rearrangements is still unknown. To evaluate this we used a complementary set of sensitive assays to explore the mtDNA rearrangements in individual cells from patients with sporadic inclusion body myositis, a late-onset inflammatory myopathy with prominent mitochondrial changes. We identified large-scale mtDNA deletions in individual muscle fibres with 20% of cytochrome c oxidase-deficient myofibres accumulating two or more mtDNA deletions. The majority of deletions removed only the major arc but ∼10% of all deletions extended into the minor arc removing the origin of light strand replication (OL) and a variable number of genes. Some mtDNA molecules contained two deletion sites. Additionally, we found evidence of mitochondrial genome duplications allowing replication and clonal expansion of these complex rearranged molecules. The extended spectrum of mtDNA rearrangements in single cells provides insight into the process of clonal expansion which is fundamental to our understanding of the role of mtDNA mutations in ageing and disease. PMID:27131788

  18. Ag-DNA Emitter: Metal Nanorod or Supramolecular Complex?

    PubMed

    Ramazanov, Ruslan R; Sych, Tomash S; Reveguk, Zakhar V; Maksimov, Dmitriy A; Vdovichev, Artem A; Kononov, Alexei I

    2016-09-15

    Ligand-stabilized luminescent metal clusters, in particular, DNA-based Ag clusters, are now employed in a host of applications such as sensing and bioimaging. Despite their utility, the nature of their excited states as well as detailed structures of the luminescent metal-ligand complexes remain poorly understood. We apply a new joint experimental and theoretical approach based on QM/MM-MD simulations of the fluorescence excitation spectra for three Ag clusters synthesized on a 12-mer DNA. Contrary to a previously proposed "rod-like" model, our results show that (1) three to four Ag atoms suffice to form a partially oxidized nanocluster emitting in visible range; (2) charge transfer from Ag cluster to DNA contributes to the excited states of the complexes; and (3) excitation spectra of the clusters are strongly affected by the bonding of Ag atoms to DNA bases. The presented approach can also provide a practical way to determine the structure and properties of other luminescent metal clusters. PMID:27564452

  19. Tunable DFB lasers based on DNA-surfactant-dye complexes

    NASA Astrophysics Data System (ADS)

    Chida, Toshifumi; Kawabe, Yutaka

    2012-10-01

    We succeeded to observe fluorescence enhancement and tunable laser emission from spin coated films of DNAcetyltrimetylammonium (CTMA) complex doped with weakly fluorescent cyanine dyes, DiQC2(1) and DiQC2(3) which were optically excited by interfering two beams forming a induced dynamic grating. Wavelength tuning of the laser emission was achieved by varying the angle between the pumping beams determining the grating period on the film. Degradation processes of the dye-doped films of DNA complex and PMMA were investigated and compared by monitoring absorption spectra after continuous excitation with a pulsed laser. Roles of DNA for fluorescence enhancement and improved durability were confirmed for these cyanine dyes.

  20. [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.

  1. Crystal Structure of a Bacterial Topoisomerase IB in Complex with DNA Reveals a Secondary DNA Binding Site

    SciTech Connect

    Patel, Asmita; Yakovleva, Lyudmila; Shuman, Stewart; Mondragón, Alfonso

    2010-10-22

    Type IB DNA topoisomerases (TopIB) are monomeric enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a {approx}21 DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and form protein-DNA synaptic filaments. Here we report a structure of Deinococcus radiodurans TopIB in complex with a 12 bp duplex DNA that demonstrates a secondary DNA binding site located on the surface of the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. Mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.

  2. Molecular Species Delimitation in the Racomitrium canescens Complex (Grimmiaceae) and Implications for DNA Barcoding of Species Complexes in Mosses

    PubMed Central

    Stech, Michael; Veldman, Sarina; Larraín, Juan; Muñoz, Jesús; Quandt, Dietmar; Hassel, Kristian; Kruijer, Hans

    2013-01-01

    In bryophytes a morphological species concept is still most commonly employed, but delimitation of closely related species based on morphological characters is often difficult. Here we test morphological species circumscriptions in a species complex of the moss genus Racomitrium, the R. canescens complex, based on variable DNA sequence markers from the plastid (rps4-trnT-trnL region) and nuclear (nrITS) genomes. The extensive morphological variability within the complex has led to different opinions about the number of species and intraspecific taxa to be distinguished. Molecular phylogenetic reconstructions allowed to clearly distinguish all eight currently recognised species of the complex plus a ninth species that was inferred to belong to the complex in earlier molecular analyses. The taxonomic significance of intraspecific sequence variation is discussed. The present molecular data do not support the division of the R. canescens complex into two groups of species (subsections or sections). Most morphological characters, albeit being in part difficult to apply, are reliable for species identification in the R. canescens complex. However, misidentification of collections that were morphologically intermediate between species questioned the suitability of leaf shape as diagnostic character. Four partitions of the molecular markers (rps4-trnT, trnT-trnL, ITS1, ITS2) that could potentially be used for molecular species identification (DNA barcoding) performed almost equally well concerning amplification and sequencing success. Of these, ITS1 provided the highest species discrimination capacity and should be considered as a DNA barcoding marker for mosses, especially in complexes of closely related species. Molecular species identification should be complemented by redefining morphological characters, to develop a set of easy-to-use molecular and non-molecular identification tools for improving biodiversity assessments and ecological research including mosses. PMID

  3. Molecular species delimitation in the Racomitrium canescens complex (Grimmiaceae) and implications for DNA barcoding of species complexes in mosses.

    PubMed

    Stech, Michael; Veldman, Sarina; Larraín, Juan; Muñoz, Jesús; Quandt, Dietmar; Hassel, Kristian; Kruijer, Hans

    2013-01-01

    In bryophytes a morphological species concept is still most commonly employed, but delimitation of closely related species based on morphological characters is often difficult. Here we test morphological species circumscriptions in a species complex of the moss genus Racomitrium, the R. canescens complex, based on variable DNA sequence markers from the plastid (rps4-trnT-trnL region) and nuclear (nrITS) genomes. The extensive morphological variability within the complex has led to different opinions about the number of species and intraspecific taxa to be distinguished. Molecular phylogenetic reconstructions allowed to clearly distinguish all eight currently recognised species of the complex plus a ninth species that was inferred to belong to the complex in earlier molecular analyses. The taxonomic significance of intraspecific sequence variation is discussed. The present molecular data do not support the division of the R. canescens complex into two groups of species (subsections or sections). Most morphological characters, albeit being in part difficult to apply, are reliable for species identification in the R. canescens complex. However, misidentification of collections that were morphologically intermediate between species questioned the suitability of leaf shape as diagnostic character. Four partitions of the molecular markers (rps4-trnT, trnT-trnL, ITS1, ITS2) that could potentially be used for molecular species identification (DNA barcoding) performed almost equally well concerning amplification and sequencing success. Of these, ITS1 provided the highest species discrimination capacity and should be considered as a DNA barcoding marker for mosses, especially in complexes of closely related species. Molecular species identification should be complemented by redefining morphological characters, to develop a set of easy-to-use molecular and non-molecular identification tools for improving biodiversity assessments and ecological research including mosses.

  4. Oxidative DNA damage in relation to nutrition.

    PubMed

    Krajcovicová-Kudlácková, M; Dusinská, M

    2004-01-01

    Oxidative DNA damage in humans could arise also from incorrect nutritional habit and life style. DNA strand breaks with apurinic/apyrimidinic sites, oxidized purines and oxidized pyrimidines were assessed in 24 subjectively healthy vegetarians (plant food, dairy products, eggs) and compared with 24 non-vegetarians (traditional diet, general population). DNA strand breaks + oxidized purines are significantly reduced in vegetarians (p<0.05), DNA strand breaks are nonsignificantly decreased. The sufficient antioxidative status (overthreshold values of natural essential antioxidants, which mean a reduced risk of free radical disease) is crucial in free radical defense. Intake of protective food commodities (fruit, vegetables, dark grain products, grain sprouts, oil seeds) is significantly higher in vegetarians. Alternative nutrition subjects have a significantly increased plasma levels of vitamin C, vitamin E, beta-carotene with high incidence of overthreshold values (92% vs. 42% - vitamin C, 67% vs. 33% - vitamin E, 67% vs. 17% - beta-carotene). There is recorded a significant inverse linear correlation between values of DNA strand breaks + oxidized purines and vitamin C or beta-carotene levels (p<0.01, p<0.05). Vegetarian diet is significantly more rich source of antioxidants. The results of reduced endogenous DNA damage and higher antioxidative status in vegetarians document that a correct vegetarian nutrition might represent an effective cancer prevention.

  5. Interaction of dinuclear cadmium(II) 5-Cl-salicylaldehyde complexes with calf-thymus DNA.

    PubMed

    Ristovic, Maja Sumar; Zianna, Ariadni; Psomas, George; Hatzidimitriou, Antonios G; Coutouli-Argyropoulou, Evdoxia; Lalia-Kantouri, Maria

    2016-04-01

    Five dinuclear Cd(II) complexes with the anion of 5-Cl-salicylaldehyde (5-Cl-saloH) were synthesized in the absence or presence of the α-diimines: 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen), 2,9-dimethyl-1,10-phenanthroline (neoc) or 2,2'-dipyridylamine (dpamH) and characterized as [Cd(5-Cl-salo)2(CH3OH)]2 (1), [Cd(5-Cl-salo)2(bipy)]2 (2), [Cd(5-Cl-salo)2(phen)]2 (3), [Cd(5-Cl-salo)(neoc)(ONO2)]2 (4) and [Cd(5-Cl-salo)(dpamΗ)(ONO2)]2 (5). The complexes were characterized by spectroscopic techniques (IR, UV-vis, (1)H-NMR and (13)C-NMR), elemental analysis and molar conductivity measurements. The structures of four complexes (1-3 and 5) were determined by X-ray crystallography, providing all three possible coordination modes of the ligand 5-Cl-salicylaldehyde, i.e. bidentate or tridentate chelating and/or bridging mode. The complexes bind to calf-thymus (CT) DNA mainly by intercalation, as concluded by the viscosity measurements and present relatively high DNA-binding constants. The complexes exhibit significant ability to displace ethidium bromide (EB) from the EB-DNA complex, thus indirectly proving the intercalation as the most possible binding mode to CT DNA.

  6. Crystal structures of an archaeal class II DNA photolyase and its complex with UV-damaged duplex DNA

    PubMed Central

    Kiontke, Stephan; Geisselbrecht, Yann; Pokorny, Richard; Carell, Thomas; Batschauer, Alfred; Essen, Lars-Oliver

    2011-01-01

    Class II photolyases ubiquitously occur in plants, animals, prokaryotes and some viruses. Like the distantly related microbial class I photolyases, these enzymes repair UV-induced cyclobutane pyrimidine dimer (CPD) lesions within duplex DNA using blue/near-UV light. Methanosarcina mazei Mm0852 is a class II photolyase of the archaeal order of Methanosarcinales, and is closely related to plant and metazoan counterparts. Mm0852 catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA. We solved crystal structures of Mm0852, the first one for a class II photolyase, alone and in complex with CPD lesion-containing duplex DNA. The lesion-binding mode differs from other photolyases by a larger DNA-binding site, and an unrepaired CPD lesion is found flipped into the active site and recognized by a cluster of five water molecules next to the bound 3′-thymine base. Different from other members of the photolyase-cryptochrome family, class II photolyases appear to utilize an unusual, conserved tryptophane dyad as electron transfer pathway to the catalytic FAD cofactor. PMID:21892138

  7. Crystal structures of an archaeal class II DNA photolyase and its complex with UV-damaged duplex DNA.

    PubMed

    Kiontke, Stephan; Geisselbrecht, Yann; Pokorny, Richard; Carell, Thomas; Batschauer, Alfred; Essen, Lars-Oliver

    2011-11-01

    Class II photolyases ubiquitously occur in plants, animals, prokaryotes and some viruses. Like the distantly related microbial class I photolyases, these enzymes repair UV-induced cyclobutane pyrimidine dimer (CPD) lesions within duplex DNA using blue/near-UV light. Methanosarcina mazei Mm0852 is a class II photolyase of the archaeal order of Methanosarcinales, and is closely related to plant and metazoan counterparts. Mm0852 catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA. We solved crystal structures of Mm0852, the first one for a class II photolyase, alone and in complex with CPD lesion-containing duplex DNA. The lesion-binding mode differs from other photolyases by a larger DNA-binding site, and an unrepaired CPD lesion is found flipped into the active site and recognized by a cluster of five water molecules next to the bound 3'-thymine base. Different from other members of the photolyase-cryptochrome family, class II photolyases appear to utilize an unusual, conserved tryptophane dyad as electron transfer pathway to the catalytic FAD cofactor. PMID:21892138

  8. Synthesis, characterization, DNA binding, DNA cleavage, protein binding and cytotoxic activities of Ru(II) complexes.

    PubMed

    Thota, Sreekanth; Vallala, Srujana; Yerra, Rajeshwar; Rodrigues, Daniel Alencar; Raghavendra, Nulgumnalli Manjunathaiah; Barreiro, Eliezer J

    2016-01-01

    We report on the synthesis of novel Ru(II) compounds (Ru-1 to Ru-8) bearing R-pdc, 4-Cl-pbinh ligands (where R=4-CF3, 4-F, 4-OH pdc=3-phenyl-5-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide, pbinh=phenoxybenzylidene isonicotinyl hydrazides) and their in vitro antitumor activity toward the cell lines murine leukemia L1210, human lymphocyte CEM, human epithelial cervical carcinoma HeLa, BEL-7402 and Molt4/C8. Some of the complexes exhibited more potent antiproliferative activity against cell lines than the standard drug cisplatin. Ruthenium complex Ru-2 displayed potent cytotoxicity with than that of cisplatin. DNA-binding, DNA cleavage and protein binding properties of ruthenium complexes with these ligands are reported. Interactions of these ruthenium complexes with DNA revealed an intercalative mode of binding between them. Synchronous fluorescence spectra proved that the interaction of ruthenium complexes with bovine serum albumin (BSA) resulted in a conformational change of the latter.

  9. Relating equivalence relations to equivalence relations: A relational framing model of complex human functioning

    PubMed Central

    Barnes, Dermot; Hegarty, Neil; Smeets, Paul M.

    1997-01-01

    The current study aimed to develop a behavior-analytic model of analogical reasoning. In Experiments 1 and 2 subjects (adults and children) were trained and tested for the formation of four, three-member equivalence relations using a delayed matching-to-sample procedure. All subjects (Experiments 1 and 2) were exposed to tests that examined relations between equivalence and non-equivalence relations. For example, on an equivalence-equivalence relation test, the complex sample B1/C1 and the two complex comparisons B3/C3 and B3/C4 were used, and on a nonequivalence-nonequivalence relation test the complex sample B1/C2 was presented with the same two comparisons. All subjects consistently related equivalence relations to equivalence relations and nonequivalence relations to nonequivalence relations (e.g., picked B3/C3 in the presence of B1/C1 and picked B3/C4 in the presence of B1/C2). In Experiment 3, the equivalence responding, the equivalence-equivalence responding, and the nonequivalence-nonequivalence responding was successfully brought under contextual control. Finally, it was shown that the contextual cues could function successfully as comparisons, and the complex samples and comparisons could function successfully as contextual cues and samples, respectively. These data extend the equivalence paradigm and contribute to a behaviour-analytic interpretation of analogical reasoning and complex human functioning, in general. PMID:22477120

  10. Interaction of Iron II Complexes with B-DNA. Insights from Molecular Modeling, Spectroscopy and Cellular Biology.

    NASA Astrophysics Data System (ADS)

    Gattuso, Hugo; Duchanois, Thibaut; Besancenot, Vanessa; Barbieux, Claire; Assfeld, Xavier; Becuwe, Philippe; Gros, Philippe; Grandemange, Stephanie; Monari, Antonio

    2015-12-01

    We report the characterization of the interaction between B-DNA and three terpyridin iron II complexes. Relatively long time-scale molecular dynamics is used in order to characterize the stable interaction modes. By means of molecular modeling and UV-vis spectroscopy, we prove that they may lead to stable interactions with the DNA duplex. Furthermore, the presence of larger π-conjugated moieties also leads to the appearance of intercalation binding mode. Non-covalent stabilizing interactions between the iron complexes and the DNA are also characterized and evidenced by the analysis of the gradient of the electronic density. Finally, the structural deformations induced on the DNA in the different binding modes are also evidenced. The synthesis and chemical characterization of the three complexes is reported, as well as their absorption spectra in presence of DNA duplexes to prove the interaction with DNA. Finally, their effects on human cell cultures have also been evidenced to further enlighten their biological effects.

  11. The complex relation between morality and empathy.

    PubMed

    Decety, Jean; Cowell, Jason M

    2014-07-01

    Morality and empathy are fundamental components of human nature across cultures. However, the wealth of empirical findings from developmental, behavioral, and social neuroscience demonstrates a complex relation between morality and empathy. At times, empathy guides moral judgment, yet other times empathy can interfere with it. To better understand such relations, we propose abandoning the catchall term of empathy in favor of more precise concepts, such as emotional sharing, empathic concern, and affective perspective-taking.

  12. A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase alpha on double-stranded DNA.

    PubMed

    Zhang, S; Grosse, F

    1992-11-01

    A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB), also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase alpha to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200-600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase alpha-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.

  13. DNA-METAFECTENE PRO complexation: a physical chemistry study.

    PubMed

    Alatorre-Meda, Manuel; González-Pérez, Alfredo; Rodríguez, Julio R

    2010-07-21

    Complexes formed between cationic liposomes and DNA (also known as lipoplexes or genosomes) have proven, for years now, to be a suitable option for gene delivery to cells, transfection, however, some aspects regarding the liposome-DNA interaction mechanism and complex stability remain still unclear. This work aims to improve the understanding of the poorly defined mechanisms and structural conformation associated with the interaction of METAFECTENE PRO (MEP), a commercial liposomal transfection reagent, with poly-anion DNA at mass ratios around the mass ratio recommended for transfection (L/D congruent with 700). A physical chemistry characterization was conducted at a pH of 6.5 and at a temperature of 25 degrees C by means of dynamic light scattering (DLS), electrophoretic mobility (zeta-potential), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Five parameters important for transfection were determined for the lipoplexes: (i) the hydrodynamic radius, R(H), (ii) the stability with time, (iii) the mass ratio of at which both moieties start to interact, (L/D)(i), (iv) the overall charge, and (v) the morphology. Results in ensemble point to a "beads on a string" conformation, with the lipoplex formation occurring well below isoneutrality from (L/D)(i) congruent with 600. The lipoplexes were found to be stable within at least seven days presenting an average R(H) of 135 nm.

  14. Revisit an old problem -- Complexation between DNA and PEI

    NASA Astrophysics Data System (ADS)

    Wu, Chi

    2009-03-01

    After revisiting the captioned problem by using a combination of chemical synthesis and physical methods, we studied the dynamics of the complexation between branched polyethyleneimine (bPEI) and plasmid DNA (pDNA) and characterized the structure, size and surface charge of the resultant DNA/PEI complexes (polyplexes). As expected, in order to reach a high efficiency in gene transfection into cells it is necessary to use a higher N:P ratio and make the polyplexes positively charged. Our results reveal that it is those uncomplexed bPEI chains free in the solution mixture that plays a vitally important role in enhancing the transfection efficiency, inspiring new thinking of how to correlate in vitro and in vivo studies so that we can improve the in vivo transfection efficiency. Increasing the N:P ratio normally results in a higher cytotoxicity, which is a catch-22 problem. Recently, we found that a proper modification of bPEI can greatly reduce its cytotoxicity without any suffering in the transfection efficiency. In this lecture, we will show that our properly modified bPEI is even much more effective and less cytotoxic in the gene transfection than those commercially available lipoflexes. Our recent breakthrough leads to a complete new direction in the development of non-viral vectors for molecular medicines, including gene transfection.

  15. Complex genomic organization of Indian muntjac centromeric DNA.

    PubMed

    Cheng, Ya-Ming; Li, Tzai-Shiuan; Hsieh, Lie-Jiau; Hsu, Pei-Ching; Li, Yueh-Chun; Lin, Chyi-Chyang

    2009-01-01

    A 69-kb Indian muntjac bacterial artificial chromosome (BAC) clone that screened positive for Cervid satellites I and IV was selected for complete sequence analysis and further characterization. The sequences of this BAC clone were found in the centromeres and in some interstitial sites of Indian muntjac chromosomes. Sequence analyses showed that the BAC clone contained a 14.5 kb Cervid satellite I-like DNA element and a 9 kb Cervid satellite IV-like DNA element. In addition, it contained 51 regions each organized in a complex fashion, with sequences homology to intersperse repetitive sequences such as LINEs, SINEs, LTRs, other published DNA elements, and unassigned sequences. The FISH patterns of seven non-satellite sequence elements generated from the BAC clone showed mainly specific to centromeres of the Indian muntjac representing novel centromeric DNAs of the species. Furthermore, FISH signals and Southern blot patterns of these elements suggest the existence of a not yet identified repetitive sequence with giant repeated monomers. Positive FISH signals of these elements were also detected in the centromeric regions of Formosan muntjac. This suggests that these newly identified non-Cervid satellite DNA sequences have been conserved in the centromere of the Formosan muntjac.

  16. 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.

  17. 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. PMID:24859813

  18. Spatiotemporal Dynamics of Early DNA Damage Response Proteins on Complex DNA Lesions

    PubMed Central

    Tobias, Frank; Löb, Daniel; Lengert, Nicor; Durante, Marco; Drossel, Barbara; Taucher-Scholz, Gisela; Jakob, Burkhard

    2013-01-01

    The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the

  19. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule

    SciTech Connect

    Changela, A.; Digate, R.J.; Mondragon, A.

    2010-03-05

    A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes - DNA topoisomerases - alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 {angstrom} resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.

  20. Testing robustness of relative complexity measure method constructing robust phylogenetic trees for Galanthus L. Using the relative complexity measure

    PubMed Central

    2013-01-01

    Background Most phylogeny analysis methods based on molecular sequences use multiple alignment where the quality of the alignment, which is dependent on the alignment parameters, determines the accuracy of the resulting trees. Different parameter combinations chosen for the multiple alignment may result in different phylogenies. A new non-alignment based approach, Relative Complexity Measure (RCM), has been introduced to tackle this problem and proven to work in fungi and mitochondrial DNA. Result In this work, we present an application of the RCM method to reconstruct robust phylogenetic trees using sequence data for genus Galanthus obtained from different regions in Turkey. Phylogenies have been analyzed using nuclear and chloroplast DNA sequences. Results showed that, the tree obtained from nuclear ribosomal RNA gene sequences was more robust, while the tree obtained from the chloroplast DNA showed a higher degree of variation. Conclusions Phylogenies generated by Relative Complexity Measure were found to be robust and results of RCM were more reliable than the compared techniques. Particularly, to overcome MSA-based problems, RCM seems to be a reasonable way and a good alternative to MSA-based phylogenetic analysis. We believe our method will become a mainstream phylogeny construction method especially for the highly variable sequence families where the accuracy of the MSA heavily depends on the alignment parameters. PMID:23323678

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

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

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

  2. Non-DNA-binding cofactors enhance DNA-binding specificity of a transcriptional regulatory complex.

    PubMed

    Siggers, Trevor; Duyzend, Michael H; Reddy, Jessica; Khan, Sidra; Bulyk, Martha L

    2011-12-06

    Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to >1300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel 'recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4-Met28-Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNA-binding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity.

  3. Mutant Cockayne syndrome group B protein inhibits repair of DNA topoisomerase I-DNA covalent complex.

    PubMed

    Horibata, Katsuyoshi; Saijo, Masafumi; Bay, Mui N; Lan, Li; Kuraoka, Isao; Brooks, Philip J; Honma, Masamitsu; Nohmi, Takehiko; Yasui, Akira; Tanaka, Kiyoji

    2011-01-01

    Two UV-sensitive syndrome patients who have mild photosensitivity without detectable somatic abnormalities lack detectable Cockayne syndrome group B (CSB) protein because of a homozygous null mutation in the CSB gene. In contrast, mutant CSB proteins are produced in CS-B patients with the severe somatic abnormalities of Cockayne syndrome and photosensitivity. It is known that the piggyBac transposable element derived 3 is integrated within the CSB intron 5, and that CSB-piggyBac transposable element derived 3 fusion (CPFP) mRNA is produced by alternative splicing. We found that CPFP or truncated CSB protein derived from CPFP mRNA was stably produced in CS-B patients, and that wild-type CSB, CPFP, and truncated CSB protein interacted with DNA topoisomerase I. We also found that CPFP inhibited repair of a camptothecin-induced topoisomerase I-DNA covalent complex. The inhibition was suppressed by the presence of wild-type CSB, consistent with the autosomal recessive inheritance of Cockayne syndrome. These results suggested that reduced repair of a DNA topoisomerase I-DNA covalent complex because of truncated CSB proteins is involved in the pathogenesis of CS-B. PMID:21143350

  4. DNA–DNA kissing complexes as a new tool for the assembly of DNA nanostructures

    PubMed Central

    Barth, Anna; Kobbe, Daniela; Focke, Manfred

    2016-01-01

    Kissing-loop annealing of nucleic acids occurs in nature in several viruses and in prokaryotic replication, among other circumstances. Nucleobases of two nucleic acid strands (loops) interact with each other, although the two strands cannot wrap around each other completely because of the adjacent double-stranded regions (stems). In this study, we exploited DNA kissing-loop interaction for nanotechnological application. We functionalized the vertices of DNA tetrahedrons with DNA stem-loop sequences. The complementary loop sequence design allowed the hybridization of different tetrahedrons via kissing-loop interaction, which might be further exploited for nanotechnology applications like cargo transport and logical elements. Importantly, we were able to manipulate the stability of those kissing-loop complexes based on the choice and concentration of cations, the temperature and the number of complementary loops per tetrahedron either at the same or at different vertices. Moreover, variations in loop sequences allowed the characterization of necessary sequences within the loop as well as additional stability control of the kissing complexes. Therefore, the properties of the presented nanostructures make them an important tool for DNA nanotechnology. PMID:26773051

  5. Sae2 promotes DNA damage resistance by removing the Mre11–Rad50–Xrs2 complex from DNA and attenuating Rad53 signaling

    PubMed Central

    Chen, Huan; Donnianni, Roberto A.; Handa, Naofumi; Deng, Sarah K.; Oh, Julyun; Timashev, Leonid A.; Kowalczykowski, Stephen C.; Symington, Lorraine S.

    2015-01-01

    The Mre11–Rad50–Xrs2/NBS1 (MRX/N) nuclease/ATPase complex plays structural and catalytic roles in the repair of DNA double-strand breaks (DSBs) and is the DNA damage sensor for Tel1/ATM kinase activation. Saccharomyces cerevisiae Sae2 can function with MRX to initiate 5′-3′ end resection and also plays an important role in attenuation of DNA damage signaling. Here we describe a class of mre11 alleles that suppresses the DNA damage sensitivity of sae2Δ cells by accelerating turnover of Mre11 at DNA ends, shutting off the DNA damage checkpoint and allowing cell cycle progression. The mre11 alleles do not suppress the end resection or hairpin-opening defects of the sae2Δ mutant, indicating that these functions of Sae2 are not responsible for DNA damage resistance. The purified MP110LRX complex shows reduced binding to single- and double-stranded DNA in vitro relative to wild-type MRX, consistent with the increased turnover of Mre11 from damaged sites in vivo. Furthermore, overproduction of Mre11 causes DNA damage sensitivity only in the absence of Sae2. Together, these data suggest that it is the failure to remove Mre11 from DNA ends and attenuate Rad53 kinase signaling that causes hypersensitivity of sae2Δ cells to clastogens. PMID:25831494

  6. Anticancer activity and DNA-binding properties of novel cationic Pt(II) complexes.

    PubMed

    Jamshidi, Mehrnaz; Yousefi, Reza; Nabavizadeh, Seyed Masoud; Rashidi, Mehdi; Haghighi, Mohsen Golbon; Niazi, Ali; Moosavi-Movahedi, Ali-Akbar

    2014-05-01

    In this study, three structurally related cationic Pt complexes, [Pt(ppy)(dppe)]CF3CO2: C1, [Pt(bhq)(dppe)]CF3CO2: C2, and [Pt(bhq)(dppf)]CF3CO2: C3, in which ppy=deprotonated 2-phenylpyridine, bhq=deprotonated benzo[h]quinoline, dppe=bis(diphenylphosphino)ethane and dppf=1,1'-bis(diphenylphosphino)ferrocene, were used for the assessment of their anticancer activities against Jurkat and MCF-7 cancer cell lines. The Pt complexes (C1-C3) demonstrated significant level of anticancer properties, as measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Moreover, the changes in nuclear morphology with Acridine Orange (AO) staining reveal that these complexes are capable to induce apoptosis, and only C1 stimulates activity of Caspase-3 in Jurkat cancer cells. To get a better insight into the nature of binding between these cationic Pt complexes and DNA, different spectroscopic techniques and gel electrophoresis were applied. On the basis of the results of UV/vis absorption spectroscopy, CD experiment and fluorescence quenching of ethidium bromide (EB)-DNA, the interaction between DNA and the Pt complexes is likely to occur through a mixed-binding mode. Overall, the present work suggests that a controlled modification could result in new potentially antitumor complexes which can survive the repair mechanism and induce facile apoptosis. PMID:24530367

  7. A DNA topoisomerase VI-like complex initiates meiotic recombination.

    PubMed

    Vrielynck, Nathalie; Chambon, Aurélie; Vezon, Daniel; Pereira, Lucie; Chelysheva, Liudmila; De Muyt, Arnaud; Mézard, Christine; Mayer, Claudine; Grelon, Mathilde

    2016-02-26

    The SPO11 protein catalyzes the formation of meiotic DNA double strand breaks (DSBs) and is homologous to the A subunit of an archaeal topoisomerase (topo VI). Topo VI are heterotetrameric enzymes comprising two A and two B subunits; however, no topo VIB involved in meiotic recombination had been identified. We characterized a structural homolog of the archaeal topo VIB subunit [meiotic topoisomerase VIB-like (MTOPVIB)], which is essential for meiotic DSB formation. It forms a complex with the two Arabidopsis thaliana SPO11 orthologs required for meiotic DSB formation (SPO11-1 and SPO11-2) and is absolutely required for the formation of the SPO11-1/SPO11-2 heterodimer. These findings suggest that the catalytic core complex responsible for meiotic DSB formation in eukaryotes adopts a topo VI-like structure.

  8. Repair synthesis step involving ERCC1-XPF participates in DNA repair of the Top1-DNA damage complex.

    PubMed

    Takahata, Chiaki; Masuda, Yuji; Takedachi, Arato; Tanaka, Kiyoji; Iwai, Shigenori; Kuraoka, Isao

    2015-08-01

    Topoisomerase 1 (Top1) is the intercellular target of camptothecins (CPTs). CPT blocks DNA religation in the Top1-DNA complex and induces Top1-attached nick DNA lesions. In this study, we demonstrate that excision repair cross complementing 1 protein-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease and replication protein A (RPA) participate in the repair of Top1-attached nick DNA lesions together with other nucleotide excision repair (NER) factors. ERCC1-XPF shows nuclease activity in the presence of RPA on a 3'-phosphotyrosyl bond nick-containing DNA (Tyr-nick DNA) substrate, which mimics a Top1-attached nick DNA lesion. In addition, ERCC1-XPF and RPA form a DNA/protein complex on the nick DNA substrate in vitro, and co-localize in CPT-treated cells in vivo. Moreover, the DNA repair synthesis of Tyr-nick DNA lesions occurred in the presence of NER factors, including ERCC1-XPF, RPA, DNA polymerase delta, flap endonuclease 1 and DNA ligase 1. Therefore, some of the NER repair machinery might be an alternative repair pathway for Top1-attached nick DNA lesions. Clinically, these data provide insights into the potential of ERCC1 as a biomarker during CPT regimens.

  9. The Energy Landscape for the Self-Assembly of a Two-Dimensional DNA Origami Complex.

    PubMed

    Fern, Joshua; Lu, Jennifer; Schulman, Rebecca

    2016-02-23

    While the self-assembly of different types of DNA origami into well-defined complexes could produce nanostructures on which thousands of locations can be independently functionalized with nanometer-scale precision, current assembly processes have low yields. Biomolecular complex formation requires relatively strong interactions and reversible assembly pathways that prevent kinetic trapping. To characterize how these issues control origami complex yields, the equilibrium constants for each possible reaction for the assembly of a heterotetrameric ring, the unit cell of a rectangular lattice, were measured using fluorescence colocalization microscopy. We found that origami interface structure controlled reaction free energies. Cooperativity, measured for the first time for a DNA nanostructure assembly reaction, was weak. Simulations of assembly kinetics suggest assembly occurs via parallel pathways with the primary mechanism of assembly being hierarchical: two dimers form that then bind to one another to complete the ring.

  10. Structure of an 'open' clamp type II topoisomerase-DNA complex provides a mechanism for DNA capture and transport.

    PubMed

    Laponogov, Ivan; Veselkov, Dennis A; Crevel, Isabelle M-T; Pan, Xiao-Su; Fisher, L Mark; Sanderson, Mark R

    2013-11-01

    Type II topoisomerases regulate DNA supercoiling and chromosome segregation. They act as ATP-operated clamps that capture a DNA duplex and pass it through a transient DNA break in a second DNA segment via the sequential opening and closure of ATPase-, G-DNA- and C-gates. Here, we present the first 'open clamp' structures of a 3-gate topoisomerase II-DNA complex, the seminal complex engaged in DNA recognition and capture. A high-resolution structure was solved for a (full-length ParE-ParC55)2 dimer of Streptococcus pneumoniae topoisomerase IV bound to two DNA molecules: a closed DNA gate in a B-A-B form double-helical conformation and a second B-form duplex associated with closed C-gate helices at a novel site neighbouring the catalytically important β-pinwheel DNA-binding domain. The protein N gate is present in an 'arms-wide-open' state with the undimerized N-terminal ParE ATPase domains connected to TOPRIM domains via a flexible joint and folded back allowing ready access both for gate and transported DNA segments and cleavage-stabilizing antibacterial drugs. The structure shows the molecular conformations of all three gates at 3.7 Å, the highest resolution achieved for the full complex to date, and illuminates the mechanism of DNA capture and transport by a type II topoisomerase.

  11. Structural Evolution of Environmentally Responsive Cationic Liposome–DNA Complexes with a Reducible Lipid Linker

    PubMed Central

    Shirazi, Rahau S.; Ewert, Kai K.; Silva, Bruno F. B.; Leal, Cecilia; Li, Youli; Safinya, Cyrus R.

    2012-01-01

    Environmentally responsive materials, i.e., materials that respond to changes in their environment with a change in their properties or structure, are attracting an increasing amount of interest. We recently designed and synthesized a series of cleavable multivalent lipids (CMVLn, with n = 2 to 5 the number of positive headgroup charges at full protonation) with a disulfide bond in the linker between cationic headgroup and hydrophobic tails. The self-assembled complexes of the CMVLs and DNA are a prototypical environmentally responsive material, undergoing extensive structural rearrangement when exposed to reducing agents. We investigated the structural evolution of CMVL–DNA complexes at varied complex composition, temperature and incubation time using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS). A related lipid with a stable linker, TMVL4, was used as a control. In a nonreducing environment CMVL–DNA complexes form the lamellar (LαC) phase, with DNA rods sandwiched between lipid bilayers. However, new self-assembled phases form when the disulfide linker is cleaved by dithiothreitol or the biologically relevant reducing agent glutathione. The released DNA and cleaved CMVL headgroups form a “loosely organized” phase, giving rise to a characteristic broad SAXS correlation profile. CMVLs of high headgroup charge also form condensed DNA bundles. Intriguingly, the cleaved hydrophobic tails of the CMVLs reassemble into tilted chain-ordered Lβ′ phases upon incubation at physiological temperature (37 °C), as indicated by characteristic WAXS peaks. X-ray scattering further reveals that two of the three phases (LβF, LβL, and LβI) comprised by the Lβ′ phase coexist in these samples. The described system may have applications in lipid-based nanotechnologies. PMID:22616637

  12. Polyoma Viral DNA Replicated as a Nucleoprotein Complex in Close Association with the Host Cell Chromatin

    PubMed Central

    Seebeck, Thomas; Weil, Roger

    1974-01-01

    Polyoma viral DNA is shown to be replicated in close association with the mouse cell chromatin. Two virus-specific nucleoprotein complexes, designated complex A and B, can be dissociated from the isolated chromatin by gentle homogenization in 0.5 M NaCl. Complex A contains only replicating polyoma (Py) DNA whereas complex B contains only mature Py DNA I. The results show, furthermore, that complex A, containing viral DNA in different stages of replication, and complex B are both nucleoproteins with the same buoyant density. The data presently available suggest that newly synthesized stretches of Py DNA are immediately complexed with mouse cell histones and that complex B becomes the “core” of progeny Py virions. These results suggested that Py-induced replication of the mouse cell chromatin may be necessary to provide replicating Py DNA with histones. PMID:4362862

  13. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin.

    PubMed

    Tsurimoto, T; Melendy, T; Stillman, B

    1990-08-01

    Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented.

  14. Mixed ligand copper(II) dicarboxylate complexes: the role of co-ligand hydrophobicity in DNA binding, double-strand DNA cleavage, protein binding and cytotoxicity.

    PubMed

    Loganathan, Rangasamy; Ramakrishnan, Sethu; Ganeshpandian, Mani; Bhuvanesh, Nattamai S P; Palaniandavar, Mallayan; Riyasdeen, Anvarbatcha; Akbarsha, Mohamad Abdulkadhar

    2015-06-14

    A few water soluble mixed ligand copper(ii) complexes of the type [Cu(bimda)(diimine)] , where bimda is N-benzyliminodiacetic acid and diimine is 2,2'-bipyridine (bpy, ) or 1,10-phenanthroline (phen, ) or 5,6-dimethyl-1,10-phenanthroline (5,6-dmp, ) or 3,4,7,8-tetramethyl-1,10-phenanthroline (3,4,7,8-tmp, ) and dipyrido[3,2-d: 2',3'-f]quinoxaline (dpq, ), have been successfully isolated and characterized by elemental analysis and other spectral techniques. The coordination geometry around copper(ii) in is described as distorted square based pyramidal while that in is described as square pyramidal. Absorption spectral titrations and competitive DNA binding studies reveal that the intrinsic DNA binding affinity of the complexes depends upon the diimine co-ligand, dpq () > 3,4,7,8-tmp () > 5,6-dmp () > phen () > bpy (). The phen and dpq co-ligands are involved in the π-stacking interaction with DNA base pairs while the 3,4,7,8-tmp/5,6-dmp and bpy co-ligands are involved in respectively hydrophobic and surface mode of binding with DNA. The small enhancement in the relative viscosity of DNA upon binding to supports the DNA binding modes proposed. Interestingly, and are selective in exhibiting a positive induced CD band (ICD) upon binding to DNA suggesting that they induce B to A conformational change. In contrast, and show CD responses which reveal their involvement in strong DNA binding. The complexes are unique in displaying prominent double-strand DNA cleavage while effects only single-strand DNA cleavage, and their ability to cleave DNA in the absence of an activator varies as > > > > . Also, all the complexes exhibit oxidative double-strand DNA cleavage activity in the presence of ascorbic acid, which varies as > > > > . The ability of the complexes to bind and cleave the protein BSA varies in the order > > > > . Interestingly, and cleave the protein non-specifically in the presence of H2O2 as an activator suggesting that they can act also as chemical proteases

  15. Mixed ligand copper(II) dicarboxylate complexes: the role of co-ligand hydrophobicity in DNA binding, double-strand DNA cleavage, protein binding and cytotoxicity.

    PubMed

    Loganathan, Rangasamy; Ramakrishnan, Sethu; Ganeshpandian, Mani; Bhuvanesh, Nattamai S P; Palaniandavar, Mallayan; Riyasdeen, Anvarbatcha; Akbarsha, Mohamad Abdulkadhar

    2015-06-14

    A few water soluble mixed ligand copper(ii) complexes of the type [Cu(bimda)(diimine)] , where bimda is N-benzyliminodiacetic acid and diimine is 2,2'-bipyridine (bpy, ) or 1,10-phenanthroline (phen, ) or 5,6-dimethyl-1,10-phenanthroline (5,6-dmp, ) or 3,4,7,8-tetramethyl-1,10-phenanthroline (3,4,7,8-tmp, ) and dipyrido[3,2-d: 2',3'-f]quinoxaline (dpq, ), have been successfully isolated and characterized by elemental analysis and other spectral techniques. The coordination geometry around copper(ii) in is described as distorted square based pyramidal while that in is described as square pyramidal. Absorption spectral titrations and competitive DNA binding studies reveal that the intrinsic DNA binding affinity of the complexes depends upon the diimine co-ligand, dpq () > 3,4,7,8-tmp () > 5,6-dmp () > phen () > bpy (). The phen and dpq co-ligands are involved in the π-stacking interaction with DNA base pairs while the 3,4,7,8-tmp/5,6-dmp and bpy co-ligands are involved in respectively hydrophobic and surface mode of binding with DNA. The small enhancement in the relative viscosity of DNA upon binding to supports the DNA binding modes proposed. Interestingly, and are selective in exhibiting a positive induced CD band (ICD) upon binding to DNA suggesting that they induce B to A conformational change. In contrast, and show CD responses which reveal their involvement in strong DNA binding. The complexes are unique in displaying prominent double-strand DNA cleavage while effects only single-strand DNA cleavage, and their ability to cleave DNA in the absence of an activator varies as > > > > . Also, all the complexes exhibit oxidative double-strand DNA cleavage activity in the presence of ascorbic acid, which varies as > > > > . The ability of the complexes to bind and cleave the protein BSA varies in the order > > > > . Interestingly, and cleave the protein non-specifically in the presence of H2O2 as an activator suggesting that they can act also as chemical proteases

  16. Visualization of complex DNA double-strand breaks in a tumor treated with carbon ion radiotherapy

    PubMed Central

    Oike, Takahiro; Niimi, Atsuko; Okonogi, Noriyuki; Murata, Kazutoshi; Matsumura, Akihiko; Noda, Shin-Ei; Kobayashi, Daijiro; Iwanaga, Mototaro; Tsuchida, Keisuke; Kanai, Tatsuaki; Ohno, Tatsuya; Shibata, Atsushi; Nakano, Takashi

    2016-01-01

    Carbon ion radiotherapy shows great potential as a cure for X-ray-resistant tumors. Basic research suggests that the strong cell-killing effect induced by carbon ions is based on their ability to cause complex DNA double-strand breaks (DSBs). However, evidence supporting the formation of complex DSBs in actual patients is lacking. Here, we used advanced high-resolution microscopy with deconvolution to show that complex DSBs are formed in a human tumor clinically treated with carbon ion radiotherapy, but not in a tumor treated with X-ray radiotherapy. Furthermore, analysis using a physics model suggested that the complexity of radiotherapy-induced DSBs is related to linear energy transfer, which is much higher for carbon ion beams than for X-rays. Visualization of complex DSBs in clinical specimens will help us to understand the anti-tumor effects of carbon ion radiotherapy. PMID:26925533

  17. DNA binding, photo-induced DNA cleavage and cytotoxicity studies of lomefloxacin and its transition metal complexes

    NASA Astrophysics Data System (ADS)

    Ragheb, Mohamed A.; Eldesouki, Mohamed A.; Mohamed, Mervat S.

    2015-03-01

    This work was focused on a study of the DNA binding and cleavage properties of lomefloxacin (LMF) and its ternary transition metal complexes with glycine. The nature of the binding interactions between compounds and calf thymus DNA (CT-DNA) was studied by electronic absorption spectra, fluorescence spectra and thermal denaturation experiments. The obtained results revealed that LMF and its complexes could interact with CT-DNA via partial/moderate intercalative mode. Furthermore, the DNA cleavage activities of the compounds were investigated by gel electrophoresis. Mechanistic studies of DNA cleavage suggest that singlet oxygen (1O2) is likely to be the cleaving agent via an oxidative pathway, except for Cu(II) complex which proceeds via both oxidative and hydrolytic pathways. Antimicrobial and antitumor activities of the compounds were also studied against some kinds of bacteria, fungi and human cell lines.

  18. Characterization of DNA binding and pairing activities associated with the native SFPQ•NONO DNA repair protein complex

    PubMed Central

    Udayakumar, Durga; Dynan, William S.

    2015-01-01

    Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA double-strand breaks. We have previously shown that a complex of SFPQ (PSF) and NONO (p54nrb) cooperates with Ku protein at an early step of NHEJ, forming a committed preligation complex and stimulating end-joining activity by 10-fold or more. SFPQ and NONO show no resemblance to other repair factors, and their mechanism of action is uncertain. Here, we use an optimized microwell-based assay to characterize the in vitro DNA binding behavior of the native SFPQ•NONO complex purified from human (HeLa) cells. SFPQ•NONO and Ku protein bind independently to DNA, with little evidence of cooperativity and only slight mutual interference at high concentration. Whereas Ku protein requires free DNA ends for binding, SFPQ•NONO does not. Both Ku and SFPQ•NONO have pairing activity, as measured by the ability of DNA-bound protein to capture a second DNA fragment in a microwell-based assay. Additionally, SFPQ•NONO stimulates DNA-dependent protein kinase autophosphorylation, consistent with the ability to promote formation of a synaptic complex formation without occluding the DNA termini proper. These findings suggest that SFPQ•NONO promotes end joining by binding to internal DNA sequences and cooperating with other repair proteins to stabilize a synaptic pre-ligation complex. PMID:25998385

  19. Template synthesis of novel carboxamide dinuclear copper (II) complex: spectral characterization and reactivity towards calf-thymus DNA.

    PubMed

    Mathur, Suvigya; Tabassum, Sartaj

    2008-06-01

    Dinuclear complexes Bis [aqua 1,8-(1,2-dicarboxamido benzene) 3,6-diazaoctane copper (II)/nickel (II)] tetrachloride (1 and 2) were synthesized by a two component one-pot metal template condensation between phthalic anhydride and 1,8-diamino 3,6-diazaoctane. Elemental analysis, molar conductance measurements, electronic absorption, infra-red, electron paramagnetic resonance, nuclear magnetic resonance, atomic absorption, and electron spray mass spectral studies have been performed to probe the nature and structure of the complexes. The interaction of copper (II) complex with calf thymus (CT-DNA) has been studied by using absorption, emission and circular dichoric spectral methods, viscometry, and cyclic voltammetry. A strong hyperchromism along with a red shift in UV bands and hypochromism in the ligand field band of the complex 1 on interaction with CT-DNA imply a covalent mode of DNA binding. This is further confirmed by studying the reactivity of complex 1 using circular dichroism and viscosity measurements. The variation in relative emission intensity of DNA-bound ethidium bromide observed upon treatment with the complex 1 parallel the trend of DNA binding studies. Cyclic voltammetry studies reveal that the complex 1 prefers to bind to DNA in Cu(II) rather than Cu(I) oxidation state.

  20. The DnaB.DnaC complex: a structure based on dimers assembled around an occluded channel.

    PubMed

    Bárcena, M; Ruiz, T; Donate, L E; Brown, S E; Dixon, N E; Radermacher, M; Carazo, J M

    2001-03-15

    Replicative helicases are motor proteins that unwind DNA at replication forks. Escherichia coli DnaB is the best characterized member of this family of enzymes. We present the 26 A resolution three-dimensional structure of the DnaB hexamer in complex with its loading partner, DnaC, obtained from cryo-electron microscopy. Analysis of the volume brings insight into the elaborate way the two proteins interact, and provides a structural basis for control of the symmetry state and inactivation of the helicase by DnaC. The complex is arranged on the basis of interactions among DnaC and DnaB dimers. DnaC monomers are observed for the first time to arrange as three dumb-bell-shaped dimers that interlock into one of the faces of the helicase. This could be responsible for the freezing of DnaB in a C(3) architecture by its loading partner. The central channel of the helicase is almost occluded near the end opposite to DnaC, such that even single-stranded DNA could not pass through. We propose that the DnaB N-terminal domain is located at this face.

  1. Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD–DNA complexes

    PubMed Central

    Carter, Ashley R.; Seaberg, Maasa H.; Fan, Hsiu-Fang; Sun, Gang; Wilds, Christopher J.; Li, Hung-Wen; Perkins, Thomas T.

    2016-01-01

    RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2–4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD–DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5′ strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket. PMID:27220465

  2. 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

  3. The RSC and INO80 chromatin-remodeling complexes in DNA double-strand break repair.

    PubMed

    Chambers, Anna L; Downs, Jessica A

    2012-01-01

    In eukaryotes, DNA is packaged into chromatin and is therefore relatively inaccessible to DNA repair enzymes. In order to perform efficient DNA repair, ATP-dependent chromatin-remodeling enzymes are required to alter the chromatin structure near the site of damage to facilitate processing and allow access to repair enzymes. Two of the best-studied remodeling complexes involved in repair are RSC (Remodels the Structure of Chromatin) and INO80 from Saccharomyces cerevisiae, which are both conserved in higher eukaryotes. RSC is very rapidly recruited to breaks and mobilizes nucleosomes to promote phosphorylation of H2A S129 and resection. INO80 enrichment at a break occurs later and is dependent on phospho-S129 H2A. INO80 activity at the break site also facilitates resection. Consequently, both homologous recombination and nonhomologous end-joining are defective in rsc mutants, while subsets of these repair pathways are affected in ino80 mutants.

  4. Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA

    SciTech Connect

    Sharma A.; Heroux A.; Jenkins K. R.; Bowman G. D.

    2011-12-09

    Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves.

  5. 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

  6. 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

    2016-01-01

    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

  7. Mica functionalization for imaging of DNA and protein-DNA complexes with atomic force microscopy.

    PubMed

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

    2013-01-01

    Surface preparation is a key step for reliable and reproducible imaging of DNA and protein-DNA complexes with atomic force microscopy (AFM). This article describes the approaches for chemical functionalization of the mica surface. One approach utilizes 3-aminopropyl-trietoxy silane (APTES), enabling one to obtain a smooth surface termed AP-mica. 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. Another method utilizes aminopropyl silatrane (APS) to yield an APS-mica surface. The advantage of APS-mica compared with AP-mica is the ability to obtain reliable and reproducible time-lapse images in aqueous solutions. The chapter describes the methodologies for the preparation of AP-mica and APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The applications are illustrated with a number of examples.

  8. Understanding the interaction of an antitumoral platinum(II) 7-azaindolate complex with proteins and DNA.

    PubMed

    Samper, Katia G; Rodríguez, Venancio; Ortega-Carrasco, Elisabeth; Atrian, Sílvia; Maréchal, Jean Didier; Cutillas, Natalia; Zamora, Ana; de Haro, Concepción; Capdevila, Mercè; Ruiz, José; Palacios, Òscar

    2014-12-01

    The reactivity of the [Pt(dmba)(aza-N1)(dmso)] complex 1, (a potential antitumoral drug with lower IC50 than cisplatin in several tumoral cell lines) with different proteins and oligonucleotides is investigated by means of mass spectrometry (ESI-TOF MS). The results obtained show a particular binding behaviour of this platinum(II) complex. The interaction of 1 with the assayed proteins apparently takes place by Pt-binding to the most accessible coordinating amino acids, presumably at the surface of the protein -this avoiding protein denaturation or degradation- with the subsequent release of one or two ligands of 1. The specific reactivity of 1 with distinct proteins allows to conclude that the substituted initial ligand (dmso or azaindolate) is indicative of the nature of the protein donor atom finally bound to the platinum(II) centre, i.e. N- or S-donor amino acid. Molecular modeling calculations suggest that the release of the azaindolate ligand is promoted by a proton transfer to the non-coordinating N present in the azaindolate ring, while the release of the dmso ligand is mainly favoured by the binding of a deprotonated Cys. The interaction of complex 1 with DNA takes always place through the release of the azaindolate ligand. Interestingly, the interaction of 1 with DNA only proceeds when the oligonucleotides are annealed forming a double strand. Complex 1 is also capable to displace ethidium bromide from DNA and it also weakly binds to DNA at the minor groove, as shown by Hoechst 33258 displacement experiments. Furthermore, complex 1 is also a good inhibitor of cathepsin B (an enzyme implicated in a number of cancer related events). Therefore, although compound 1 is definitely able to bind proteins that can hamper its arrival to the nuclear target, it should be taken into consideration as a putative anticancer drug due to its strong interaction with oligonucleotides and its effective inhibition of cat B. PMID:25106460

  9. 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.

  10. DNA binding and nuclease activity of a one-dimensional heterometallic nitrosyl complex.

    PubMed

    Selim, Md; Chowdhury, Sujoy Roy; Mukherjea, Kalyan K

    2007-12-01

    The interaction of a structurally characterized Sr-Fe nitrosyl complex with DNA has been studied by UV-vis and fluorescence spectroscopy, viscometric, and gel electrophoresis techniques. From the absorption titration studies the intrinsic binding constant of the complex with DNA was calculated to be 1.6x10(4)M(-1). Fluorimetric studies indicate that the complex compete with EB in binding to DNA. The complex shows nuclease activity on pUC19 supercoiled DNA in presence of H(2)O(2). PMID:17825903

  11. ATP-dependent DNA binding, unwinding, and resection by the Mre11/Rad50 complex.

    PubMed

    Liu, Yaqi; Sung, Sihyun; Kim, Youngran; Li, Fuyang; Gwon, Gwanghyun; Jo, Aera; Kim, Ae-Kyoung; Kim, Taeyoon; Song, Ok-Kyu; Lee, Sang Eun; Cho, Yunje

    2016-04-01

    ATP-dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here,Methanococcus jannaschii MR-ATPγS-DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS-bound Rad50 nucleotide-binding domains. Duplex DNA cannot access the Mre11 active site in the ATP-free full-length MR complex. ATP hydrolysis drives rotation of the nucleotide-binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis-driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity. PMID:26717941

  12. ATP-dependent DNA binding, unwinding, and resection by the Mre11/Rad50 complex.

    PubMed

    Liu, Yaqi; Sung, Sihyun; Kim, Youngran; Li, Fuyang; Gwon, Gwanghyun; Jo, Aera; Kim, Ae-Kyoung; Kim, Taeyoon; Song, Ok-Kyu; Lee, Sang Eun; Cho, Yunje

    2016-04-01

    ATP-dependent DNA end recognition and nucleolytic processing are central functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair. However, it is still unclear how ATP binding and hydrolysis primes the MR function and regulates repair pathway choice in cells. Here,Methanococcus jannaschii MR-ATPγS-DNA structure reveals that the partly deformed DNA runs symmetrically across central groove between two ATPγS-bound Rad50 nucleotide-binding domains. Duplex DNA cannot access the Mre11 active site in the ATP-free full-length MR complex. ATP hydrolysis drives rotation of the nucleotide-binding domain and induces the DNA melting so that the substrate DNA can access Mre11. Our findings suggest that the ATP hydrolysis-driven conformational changes in both DNA and the MR complex coordinate the melting and endonuclease activity.

  13. Relationships between rDNA, Nop1 and Sir complex in biotechnologically relevant distillery yeasts.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Potocki, Leszek; Kuna, Ewelina; Kaplan, Jakub; Pabian, Sylwia; Kwiatkowska, Aleksandra; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    Distillery yeasts are poorly characterized physiological group among the Saccharomyces sensu stricto complex. As industrial yeasts are under constant environmental stress during fermentation processes and the nucleolus is a stress sensor, in the present study, nucleolus-related parameters were evaluated in 22 commercially available distillery yeast strains. Distillery yeasts were found to be a heterogeneous group with a variable content and length of rDNA and degree of nucleolus fragmentation. The levels of rDNA were negatively correlated with Nop1 (r = -0.59, p = 0.0038). Moreover, the protein levels of Sir transcriptional silencing complex and longevity regulators, namely Sir1, Sir2, Sir3 and Fob1, were studied and negative correlations between Sir2 and Nop1 (r = -0.45, p = 0.0332), and between Sir2 and Fob1 (r = -0.49, p = 0.0211) were revealed. In general, S. paradoxus group of distillery yeasts with higher rDNA pools and Sir2 level than S. bayanus group was found to be more tolerant to fermentation-associated stress stimuli, namely mild cold/heat stresses and KCl treatment. We postulate that rDNA state may be considered as a novel factor that may modulate a biotechnological process. PMID:27329282

  14. Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase

    PubMed Central

    James, Tamara D.; Cardozo, Timothy; Abell, Lauren E.; Hsieh, Meng-Lun; Jenkins, Lisa M. Miller; Jha, Saheli S.; Hinton, Deborah M.

    2016-01-01

    The ability of RNA polymerase (RNAP) to select the right promoter sequence at the right time is fundamental to the control of gene expression in all organisms. However, there is only one crystallized structure of a complete activator/RNAP/DNA complex. In a process called σ appropriation, bacteriophage T4 activates a class of phage promoters using an activator (MotA) and a co-activator (AsiA), which function through interactions with the σ70 subunit of RNAP. We have developed a holistic, structure-based model for σ appropriation using multiple experimentally determined 3D structures (Escherichia coli RNAP, the Thermus aquaticus RNAP/DNA complex, AsiA /σ70 Region 4, the N-terminal domain of MotA [MotANTD], and the C-terminal domain of MotA [MotACTD]), molecular modeling, and extensive biochemical observations indicating the position of the proteins relative to each other and to the DNA. Our results visualize how AsiA/MotA redirects σ, and therefore RNAP activity, to T4 promoter DNA, and demonstrate at a molecular level how the tactful interaction of transcriptional factors with even small segments of RNAP can alter promoter specificity. Furthermore, our model provides a rational basis for understanding how a mutation within the β subunit of RNAP (G1249D), which is far removed from AsiA or MotA, impairs σ appropriation. PMID:27458207

  15. Visualizing the phage T4 activated transcription complex of DNA and E. coli RNA polymerase.

    PubMed

    James, Tamara D; Cardozo, Timothy; Abell, Lauren E; Hsieh, Meng-Lun; Jenkins, Lisa M Miller; Jha, Saheli S; Hinton, Deborah M

    2016-09-19

    The ability of RNA polymerase (RNAP) to select the right promoter sequence at the right time is fundamental to the control of gene expression in all organisms. However, there is only one crystallized structure of a complete activator/RNAP/DNA complex. In a process called σ appropriation, bacteriophage T4 activates a class of phage promoters using an activator (MotA) and a co-activator (AsiA), which function through interactions with the σ(70) subunit of RNAP. We have developed a holistic, structure-based model for σ appropriation using multiple experimentally determined 3D structures (Escherichia coli RNAP, the Thermus aquaticus RNAP/DNA complex, AsiA /σ(70) Region 4, the N-terminal domain of MotA [MotA(NTD)], and the C-terminal domain of MotA [MotA(CTD)]), molecular modeling, and extensive biochemical observations indicating the position of the proteins relative to each other and to the DNA. Our results visualize how AsiA/MotA redirects σ, and therefore RNAP activity, to T4 promoter DNA, and demonstrate at a molecular level how the tactful interaction of transcriptional factors with even small segments of RNAP can alter promoter specificity. Furthermore, our model provides a rational basis for understanding how a mutation within the β subunit of RNAP (G1249D), which is far removed from AsiA or MotA, impairs σ appropriation. PMID:27458207

  16. Relationships between rDNA, Nop1 and Sir complex in biotechnologically relevant distillery yeasts.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Potocki, Leszek; Kuna, Ewelina; Kaplan, Jakub; Pabian, Sylwia; Kwiatkowska, Aleksandra; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    Distillery yeasts are poorly characterized physiological group among the Saccharomyces sensu stricto complex. As industrial yeasts are under constant environmental stress during fermentation processes and the nucleolus is a stress sensor, in the present study, nucleolus-related parameters were evaluated in 22 commercially available distillery yeast strains. Distillery yeasts were found to be a heterogeneous group with a variable content and length of rDNA and degree of nucleolus fragmentation. The levels of rDNA were negatively correlated with Nop1 (r = -0.59, p = 0.0038). Moreover, the protein levels of Sir transcriptional silencing complex and longevity regulators, namely Sir1, Sir2, Sir3 and Fob1, were studied and negative correlations between Sir2 and Nop1 (r = -0.45, p = 0.0332), and between Sir2 and Fob1 (r = -0.49, p = 0.0211) were revealed. In general, S. paradoxus group of distillery yeasts with higher rDNA pools and Sir2 level than S. bayanus group was found to be more tolerant to fermentation-associated stress stimuli, namely mild cold/heat stresses and KCl treatment. We postulate that rDNA state may be considered as a novel factor that may modulate a biotechnological process.

  17. Complexes of poly(ethylene glycol)-based cationic random copolymer and calf thymus DNA: a complete biophysical characterization.

    PubMed

    Nisha, C K; Manorama, Sunkara V; Ganguli, Munia; Maiti, Souvik; Kizhakkedathu, Jayachandran N

    2004-03-16

    Complete biophysical characterization of complexes (polyplexes) of cationic polymers and DNA is needed to understand the mechanism underlying nonviral therapeutic gene transfer. In this article, we propose a new series of synthesized random cationic polymers (RCPs) from methoxy poly(ethylene glycol) monomethacrylate (MePEGMA) and (3-(methacryloylamino)propyl)trimethylammonium chloride with different mole ratios (32:68, 11:89, and 6:94) which could be used as a model system to address and answer the basic questions relating to the mechanism of the interaction of calf thymus DNA (CT-DNA) and cationic polymers. The solubility of the complexes of CT-DNA and RCP was followed by turbidity measurements. It has been observed that complexes of RCP with 68 mol % MePEGMA precipitate near the charge neutralization point, whereas complexes of the other two polymers are water-soluble and stable at all compositions. Dnase 1 digestion experiments show that DNA is inaccessible when it forms complexes with RCP. Ethidium bromide exclusion and gel electrophoretic mobility show that both polymers are capable of binding with CT-DNA. Atomic force microscopy images in conjunction with light scattering experiments showed that the complexes are spherical in nature and 75-100 nm in diameter. Circular dichroism spectroscopy studies indicated that the secondary structure of DNA in the complexes is not perturbed due to the presence of poly(ethylene glycol) segments in the polymer. Furthermore, we used a combination of spectroscopic and calorimetric techniques to determine complete thermodynamic profiles accompanying the helix-coil transition of CT-DNA in the complexes. UV and differential scanning calorimetry melting experiments revealed that DNA in the complexes is more stable than in the free state and the extent of stability depends on the polymer composition. Isothermal titration calorimetry experiments showed that the binding of these RCPs to CT-DNA is associated with small exothermic

  18. [Cancer-vitamins-minerals: Complex relation].

    PubMed

    Adrianza de Baptista, Gertrudis; Murillo Melo, Carolain

    2014-12-01

    Since nutrition can influence the process of carcinogenesis, this study's objectives are to review the relationship between nutrition and cancer from the point of view of the role of micronutrients in the treatment of cancer patients, and to get to know the deficit relationship and the excess of micronutrients, with the etiology and cancer treatment. At the same time the patient's weight loss relates, among other things, to the type of cancerous tumor, its location, stage thereof, reason for which it may be associated with the deficiency of macro and micronutrients as from psychogenic, anorectics and mal-absorption effects or with mechanical effects as obstruction, among other toxic effects that are common in the treatment of cancer. Hence, the importance that the nutrition expert must have in making an adequate overall nutritional evaluation that allows the nutritional diagnosis, in studying the dietary patterns, to determine the toxic effects of the antineoplastic treatment in order to handle the treatment's timing excellence, symptoms and signs, and thus act effectively optiimizing the patient's life quality, and therewith his/her survival. There are controversies as to which specific dietary factors are related to cancer etiology and the results of studies on metabolic factors, and therefore, the relationship Cancer-Nutrition is quiet complex.

  19. 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.

  20. Creating complex molecular topologies by configuring DNA four-way junctions.

    PubMed

    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. PMID:27657865

  1. Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex

    PubMed Central

    Chand Dakal, Tikam; Giudici, Paolo; Solieri, Lisa

    2016-01-01

    Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5’ end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding. PMID:27501051

  2. The DNA cleavage reaction of DNA gyrase. Comparison of stable ternary complexes formed with enoxacin and CcdB protein.

    PubMed

    Scheirer, K E; Higgins, N P

    1997-10-24

    The potent synthetic fluoroquinolones and the natural CcdB protein encoded by the F plasmid both inhibit bacterial growth by attacking DNA gyrase and by stimulating enzyme-induced breaks in bacterial DNA. The cleavage mechanisms of these structurally diverse compounds were analyzed by purifying and characterizing stable ternary complexes of enoxacin and CcdB protein with gyrase bound to a strong gyrase binding site from bacteriophage Mu. Three differences between enoxacin- and CcdB-derived complexes were discovered. 1) Enoxacin binds to the DNA active site and alters the breakage/reunion activity of the enzyme. CcdB binds gyrase-DNA complexes but does not influence enzymatic activity directly. 2) Complexes that produce DNA cleavage with enoxacin are reversible, whereas similar complexes made with CcdB protein are not. 3) Enoxacin stimulates cleavage of both relaxed and supercoiled forms of DNA in the absence of ATP, whereas CcdB induces cleavage only after many cycles of ATP-dependent breakage and reunion. These differences in mechanisms can be explained by a model in which enoxacin induces formation of a novel "cleavable" complex, whereas CcdB protein traps a very rare "cleaved" conformation of the enzyme.

  3. Further purification and characterization of a multienzyme complex for DNA synthesis in human cells.

    PubMed

    Li, C; Cao, L G; Wang, Y L; Baril, E F

    1993-12-01

    The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract-post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362-6374] was purified by poly (ethylene glycol) precipitation, Q-Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase alpha-primase, a 3',5' exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase alpha,beta and DNA ligase I, showed that polymerase alpha and DNA ligase I are localized to granular-like foci within the nucleus during S-phase. In contrast, DNA polymerase beta, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm. PMID:8300757

  4. Choosing relatives for DNA identification of missing persons.

    PubMed

    Ge, Jianye; Budowle, Bruce; Chakraborty, Ranajit

    2011-01-01

    DNA-based analysis is integral to missing person identification cases. When direct references are not available, indirect relative references can be used to identify missing persons by kinship analysis. Generally, more reference relatives render greater accuracy of identification. However, it is costly to type multiple references. Thus, at times, decisions may need to be made on which relatives to type. In this study, pedigrees for 37 common reference scenarios with 13 CODIS STRs were simulated to rank the information content of different combinations of relatives. The results confirm that first-order relatives (parents and fullsibs) are the most preferred relatives to identify missing persons; fullsibs are also informative. Less genetic dependence between references provides a higher on average likelihood ratio. Distant relatives may not be helpful solely by autosomal markers. But lineage-based Y chromosome and mitochondrial DNA markers can increase the likelihood ratio or serve as filters to exclude putative relationships.

  5. Visually Relating Gene Expression and in vivo DNA Binding Data

    SciTech Connect

    Huang, Min-Yu; Mackey, Lester; Ker?,; nen, Soile V. E.; Weber, Gunther H.; Jordan, Michael I.; Knowles, David W.; Biggin, Mark D.; Hamann, Bernd

    2011-09-20

    Gene expression and in vivo DNA binding data provide important information for understanding gene regulatory networks: in vivo DNA binding data indicate genomic regions where transcription factors are bound, and expression data show the output resulting from this binding. Thus, there must be functional relationships between these two types of data. While visualization and data analysis tools exist for each data type alone, there is a lack of tools that can easily explore the relationship between them. We propose an approach that uses the average expression driven by multiple of ciscontrol regions to visually relate gene expression and in vivo DNA binding data. We demonstrate the utility of this tool with examples from the network controlling early Drosophila development. The results obtained support the idea that the level of occupancy of a transcription factor on DNA strongly determines the degree to which the factor regulates a target gene, and in some cases also controls whether the regulation is positive or negative.

  6. Design and synthesis of DNA-tethered ruthenium complexes that self-assemble into linear arrays.

    PubMed

    Stewart, Kristen M; McLaughlin, Larry W

    2003-12-01

    Ruthenium(II) bis(terpyridine) complexes have been prepared with two triethylene glycol linkers to which DNA sequences have been attached; hybridization at various complex ratios results in linear arrays of varying lengths.

  7. DNA Double-Strand Break Rejoining in Complex Normal Tissues

    SciTech Connect

    Ruebe, Claudia E.; Kuehne, Martin; Fricke, Andreas

    2008-11-15

    Purpose: The clinical radiation responses of different organs vary widely and likely depend on the intrinsic radiosensitivities of their different cell populations. Double-strand breaks (DSBs) are the most deleterious form of DNA damage induced by ionizing radiation, and the cells' capacity to rejoin radiation-induced DSBs is known to affect their intrinsic radiosensitivity. To date, only little is known about the induction and processing of radiation-induced DSBs in complex normal tissues. Using an in vivo model with repair-proficient mice, the highly sensitive {gamma}H2AX immunofluorescence was established to investigate whether differences in DSB rejoining could account for the substantial differences in clinical radiosensitivity observed among normal tissues. Methods and Materials: After whole body irradiation of C57BL/6 mice (0.1, 0.5, 1.0, and 2.0 Gy), the formation and rejoining of DSBs was analyzed by enumerating {gamma}H2AX foci in various organs representative of both early-responding (small intestine) and late-responding (lung, brain, heart, kidney) tissues. Results: The linear dose correlation observed in all analyzed tissues indicated that {gamma}H2AX immunofluorescence allows for the accurate quantification of DSBs in complex organs. Strikingly, the various normal tissues exhibited identical kinetics for {gamma}H2AX foci loss, despite their clearly different clinical radiation responses. Conclusion: The identical kinetics of DSB rejoining measured in different organs suggest that tissue-specific differences in radiation responses are independent of DSB rejoining. This finding emphasizes the fundamental role of DSB repair in maintaining genomic integrity, thereby contributing to cellular viability and functionality and, thus, tissue homeostasis.

  8. Chemistry specificity of DNA-polycation complex salt response: a simulation study of DNA, polylysine and polyethyleneimine.

    PubMed

    Antila, Hanne S; Härkönen, Marc; Sammalkorpi, Maria

    2015-02-21

    In this work, the chemistry specific stability determining factors of DNA-polycation complexes are examined by performing all-atom molecular dynamics simulations. To this end, we conduct a systematic variation of polycation line charge through polyethyleneimine (PEI) protonation and polycation chemistry via comparison with poly-l-lysine (PLL). Our simulations show that increasing line charge of the polycation alone does not lead to more salt tolerant complexes. Instead, the effective charge compensation by the polycation correlates with the increased stability of the complex against additional salt. The salt stability of PEI-DNA complexes also links to the proton sponge property of weak polycations, commonly assumed to be behind the effectivity of PEI as a gene delivery vector. Examination of the complexes reveals the mechanism behind this behaviour; more Cl(-) ions are attracted by the protonated complexes but, in contrast to the common depiction of the proton sponge behaviour, the ion influx does not cause swelling of the complex structure itself. However, PEI protonation leads to release of PEI while DNA remains tightly bound to the complex. Jointly, these findings shed light on the stability determining factors of DNA-polycation complexes, raise charge distribution as an important stability determining contributor, and indicate that the effectivity of PEI in gene delivery is likely to result from the freed PEI facilitating gene transfection.

  9. ct-DNA Binding and Antibacterial Activity of Octahedral Titanium (IV) Heteroleptic (Benzoylacetone and Hydroxamic Acids) Complexes

    PubMed Central

    Kaushal, Raj; Thakur, Sheetal; Nehra, Kiran

    2016-01-01

    Five structurally related titanium (IV) heteroleptic complexes, [TiCl2(bzac)(L1–4)] and [TiCl3(bzac)(HL5)]; bzac = benzoylacetonate; L1–5 = benzohydroximate (L1), salicylhydroximate (L2), acetohydroximate (L3), hydroxyurea (L4), and N-benzoyl-N-phenyl hydroxylamine (L5), were used for the assessment of their antibacterial activities against ten pathogenic bacterial strains. The titanium (IV) complexes (1–5) demonstrated significant level of antibacterial properties as measured using agar well diffusion method. UV-Vis absorption spectroscopic technique was applied, to get a better insight into the nature of binding between titanium (IV) complexes with calf thymus DNA (ct-DNA). On the basis of the results of UV-Vis absorption spectroscopy, the interaction between ct-DNA and the titanium (IV) complexes is likely to occur through the same mode. Results indicated that titanium (IV) complex can bind to calf thymus DNA (ct-DNA) via an intercalative mode. The intrinsic binding constant (Kb) was calculated by absorption spectra by using Benesi-Hildebrand equation. Further, Gibbs free energy was also calculated for all the complexes. PMID:27119022

  10. Initiation of DNA damage responses through XPG-related nucleases

    PubMed Central

    Kuntz, Karen; O'Connell, Matthew J

    2013-01-01

    Lesion-specific enzymes repair different forms of DNA damage, yet all lesions elicit the same checkpoint response. The common intermediate required to mount a checkpoint response is thought to be single-stranded DNA (ssDNA), coated by replication protein A (RPA) and containing a primer-template junction. To identify factors important for initiating the checkpoint response, we screened for genes that, when overexpressed, could amplify a checkpoint signal to a weak allele of chk1 in fission yeast. We identified Ast1, a novel member of the XPG-related family of endo/exonucleases. Ast1 promotes checkpoint activation caused by the absence of the other XPG-related nucleases, Exo1 and Rad2, the homologue of Fen1. Each nuclease is recruited to DSBs, and promotes the formation of ssDNA for checkpoint activation and recombinational repair. For Rad2 and Exo1, this is independent of their S-phase role in Okazaki fragment processing. This XPG-related pathway is distinct from MRN-dependent responses, and each enzyme is critical for damage resistance in MRN mutants. Thus, multiple nucleases collaborate to initiate DNA damage responses, highlighting the importance of these responses to cellular fitness. PMID:23211746

  11. 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.

  12. DNA-binding affinity and nuclease activity of two cytotoxic copper terpyridine complexes.

    PubMed

    Shi, Pengfei; Lin, Miaoxin; Zhu, Jianhui; Zhang, Yangmiao; Jiang, Qin

    2009-01-01

    Two copper(II) terpyridine complexes, [Cu(atpy)(NO(3))(H(2)O)](NO(3)) 3H(2)O (1) and [Cu(ttpy)(NO(3))(2)] (2) (atpy = 4'-p-N9-adeninylmethyl-phenyl-2,2':6,2''-terpyridine; ttpy = 4'-p-tolyl-2,2':6,2''-terpyridine) exhibited high cytotoxicity, with average ten times more potency than cisplatin against the human cervix carcinoma cell line (HeLa), the human liver carcinoma cell line (HepG2), the human galactophore carcinoma cell line (MCF7), and the human prostate carcinoma cell line (PC-3). The cytotoxicity of the complex 1 was lower than that of the complex 2. Both complexes showed more efficient oxidative DNA cleavage activity under irradiation with UV light at 260 nm than in the presence of ascorbic acid. Especially, complex 1 exhibited evident photoinduced double-stranded DNA cleavage activity. The preliminary mechanism experiments revealed that hydrogen peroxide was involved in the oxidative DNA damage induced by both complexes. From the absorption titration data, the DNA-binding affinity of the complexes with surpersoiled plasmid pUC19 DNA, polydAdT, and polydGdC was calculated and complex 2 showed higher binding affinity than complex 1 with all these substrates. The DNA cleavage ability and DNA-binding affinity of both complexes depended on the substituent group on the terpyrdine ligands. PMID:19705364

  13. Interactions of CT-DNA with Hypocrellin A and its Al3+-Hypocrellin A complex

    NASA Astrophysics Data System (ADS)

    Ma, Fei; Ge, Xuefeng; Huang, Heyong; Yang, Chao; Han, Lina; Zhou, Jiahong; Yang, Xiaodi

    2013-05-01

    In this study, the chelation of Hypocrellin A (HA) with Al3+ in water solution has been synthesized, and the interactions of HA and Al3+-HA complex with calf thymus DNA are in detail compared by UV-vis and fluorescence spectroscopic techniques, circular dichroism spectroscopy and viscosity measurement. The experiment results suggest that HA and Al3+-HA complex both could bind to CT DNA by intercalation mode, and double helix of DNA was damaged. Moreover, Al3+-HA complex not only displays higher absorption at therapeutic window but also displays stronger binding affinity to CT DNA than HA.

  14. Immune cell activation from multivalent interactions with liquid-crystalline polycation-DNA complexes

    NASA Astrophysics Data System (ADS)

    Schmidt, Nathan; Jin, Fan; Lande, Roberto; Curk, Tine; Xian, Wujing; Frasca, Loredana; Dobnikar, Jure; Frenkel, Daan; Gilliet, Michel; Wong, Gerard

    2014-03-01

    Microbial DNA can trigger type I interferon (IFN) production in plasmacytoid cells (pDCs) by binding to endosomal toll-like receptor 9 (TLR9). TLR9 in pDCs do not normally respond to self-DNA, but in certain autoimmune diseases self-DNA can complex with the polycationic antimicrobial peptide LL37 into condensed structures which allow DNA to access endosomal compartments and stimulate TLR9 in pDCs. We use x-ray studies and cell measurements of IFN secretion by pDCs to show that a broad range of polycation-DNA complexes stimulate pDCs and elucidate the criterion for high IFN production. Furthermore, we show via experiments and computer simulations that the distinguishing factor for why certain complexes activate pDCs while others do not is the self-assembled structure of the liquid-crystalline polycation-DNA complex.

  15. Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery.

    PubMed

    Trubetskoy, V S; Wong, S C; Subbotin, V; Budker, V G; Loomis, A; Hagstrom, J E; Wolff, J A

    2003-02-01

    The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.

  16. 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.

  17. An 'open' structure of the RecOR complex supports ssDNA binding within the core of the complex.

    PubMed

    Radzimanowski, Jens; Dehez, François; Round, Adam; Bidon-Chanal, Axel; McSweeney, Sean; Timmins, Joanna

    2013-09-01

    Efficient DNA repair is critical for cell survival and the maintenance of genome integrity. The homologous recombination pathway is responsible for the repair of DNA double-strand breaks within cells. Initiation of this pathway in bacteria can be carried out by either the RecBCD or the RecFOR proteins. An important regulatory player within the RecFOR pathway is the RecOR complex that facilitates RecA loading onto DNA. Here we report new data regarding the assembly of Deinococcus radiodurans RecOR and its interaction with DNA, providing novel mechanistic insight into the mode of action of RecOR in homologous recombination. We present a higher resolution crystal structure of RecOR in an 'open' conformation in which the tetrameric RecR ring flanked by two RecO molecules is accessible for DNA binding. We show using small-angle neutron scattering and mutagenesis studies that DNA binding does indeed occur within the RecR ring. Binding of single-stranded DNA occurs without any major conformational changes of the RecOR complex while structural rearrangements are observed on double-stranded DNA binding. Finally, our molecular dynamics simulations, supported by our biochemical data, provide a detailed picture of the DNA binding motif of RecOR and reveal that single-stranded DNA is sandwiched between the two facing oligonucleotide binding domains of RecO within the RecR ring. PMID:23814185

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-08-01

    Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA. PMID:27311715

  20. Ultraselective homogeneous electrochemical biosensor for DNA species related to oral cancer based on nicking endonuclease assisted target recycling amplification.

    PubMed

    Tan, Yue; Wei, Xiaofeng; Zhao, Mengmeng; Qiu, Bin; Guo, Longhua; Lin, Zhenyu; Yang, Huang-Hao

    2015-09-15

    Traditional electrochemical DNA biosensors need DNA immobilization on the electrode surface, which is tedious and time-consuming. In this study, a simple but ultraselective electrochemical DNA biosensor had been designed to determine target DNA species related to oral cancer overexpressed 1 in saliva, which combines the signal amplification of nicking endonuclease assisted target recycling with the immobilization-free electrochemical method. The complementary substrate strand of target DNA species contains a simple asymmetric sequence had been modified with a methylene blue at the 3' terminal first, which cannot diffuse easily to the negative charged ITO electrode surface due to the abundant negative charges. The presence of the target DNA would trigger the formation of double-stranded DNA (dsDNA). Then the nicking endonuclease can recognize the simple asymmetric sequence in the dsDNA and cleave the substrate strand of ds-DNA into two pieces, a long ssDNA and a 2-base ssDNA linked with methylene blue. The short one can diffuse easily to the negative charged ITO electrode surface and results in the enhanced electrochemical response detected. At the same time, the target DNA can dissociate from the dsDNA and trigger the next round of hybridization, cleavage, and releasing, which results in the signal amplification. This homogeneous DNA biosensor can detect as low as 0.35 pM (S/N = 3) target DNA. Compared with the traditional heterogeneous electrochemical DNA biosensors, which are tedious and time-consuming due to the complex DNA immobilization process, the assay not only owns the merits of simple and high efficiency since performed in a homogeneous solution but also exhibits a high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence.

  1. DNA Origami with Complex Curvatures in Three-Dimensional Space

    SciTech Connect

    Han, Dongran; Pal, Suchetan; Nangreave, Jeanette; Deng, Zhengtao; Liu, Yan; Yan, Hao

    2011-04-14

    We present a strategy to design and construct self-assembling DNA nanostructures that define intricate curved surfaces in three-dimensional (3D) space using the DNA origami folding technique. Double-helical DNA is bent to follow the rounded contours of the target object, and potential strand crossovers are subsequently identified. Concentric rings of DNA are used to generate in-plane curvature, constrained to 2D by rationally designed geometries and crossover networks. Out-of-plane curvature is introduced by adjusting the particular position and pattern of crossovers between adjacent DNA double helices, whose conformation often deviates from the natural, B-form twist density. A series of DNA nanostructures with high curvature—such as 2D arrangements of concentric rings and 3D spherical shells, ellipsoidal shells, and a nanoflask—were assembled.

  2. Genetics Home Reference: MPV17-related hepatocerebral mitochondrial DNA depletion syndrome

    MedlinePlus

    ... mitochondrial DNA depletion syndrome MPV17-related hepatocerebral mitochondrial DNA depletion syndrome Enable Javascript to view the expand/ ... All Close All Description MPV17 -related hepatocerebral mitochondrial DNA depletion syndrome is an inherited disorder that can ...

  3. Genetics Home Reference: TK2-related mitochondrial DNA depletion syndrome, myopathic form

    MedlinePlus

    ... DNA depletion syndrome, myopathic form TK2-related mitochondrial DNA depletion syndrome, myopathic form Enable Javascript to view ... Open All Close All Description TK2 -related mitochondrial DNA depletion syndrome, myopathic form ( TK2 -MDS) is an ...

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

    SciTech Connect

    Tang, K.H.; Niebuhr, M.; Aulabaugh, A.; Tsai, M.D.; /Ohio State U. /SLAC, SSRL

    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 kDa 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}.

  5. Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering

    SciTech Connect

    Tang, Kuo-Hsiang; Niebuhr, Marc; Aulabaugh, Ann; Tsai, Ming-Daw

    2008-03-25

    We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase β (Pol β) 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 β-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol β-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 kDa 5'-dRP lyase domain of the second Pol β molecule with the active site of the 1 : 1 Pol β-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'-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'-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol β-DNA complex enhances the function of Pol β.

  6. Luminescent Iridium(III) Complex Labeled DNA for Graphene Oxide-Based Biosensors.

    PubMed

    Zhao, Qingcheng; Zhou, Yuyang; Li, Yingying; Gu, Wei; Zhang, Qi; Liu, Jian

    2016-02-01

    There has been growing interest in utilizing highly photostable iridium(III) complexes as new luminescent probes for biotechnology and life science. Herein, iridium(III) complex with carboxyl group was synthesized and activated with N-hydroxysuccinimide, followed by tagging to the amino terminate of single-stranded DNA (ssDNA). The Ir-ssDNA probe was further combined with graphene oxide (GO) nanosheets to develop a GO-based biosensor for target ssDNA detection. The quenching efficiency of GO, and the photostability of iridium(III) complex and GO-Ir-ssDNA biosensor, were also investigated. On the basis of the high luminescence quenching efficiency of GO toward iridium(III) complex, the GO-Ir-ssDNA biosensor exhibited minimal background signals, while strong emission was observed when Ir-ssDNA desorbed from GO nanosheets and formed a double helix with the specific target, leading to a high signal-to-background ratio. Moreover, it was found that luminescent intensities of iridium(III) complex and GO-Ir-ssDNA biosensor were around 15 and 3 times higher than those of the traditional carboxyl fluorescein (FAM) dye and the GO-FAM-ssDNA biosensor after UV irradiation, respectively. Our study suggested the sensitive and selective Ir-ssDNA probe was suitable for the development of highly photostable GO-based detection platforms, showing promise for application beyond the OLED (organic light emitting diode) area. PMID:26753824

  7. A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

    SciTech Connect

    Evrin, C.; Li, H.; Clarke, P.; Zech, J.; Lurz, R.; Sun, J.; Uhle, S.; Stillman, B.; Speck, C.

    2009-12-01

    During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.

  8. Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex.

    PubMed

    Millet, Armêl; Strauss, François; Delagoutte, Emmanuelle

    2015-01-01

    The enzymatic DNA relaxation requires the DNA to be transiently nicked and rejoined, the covalent topoisomerase-DNA complex being a key intermediate of the nicking-joining reaction. Practically, this reaction is most often characterized by oligonucleotides. However, the incision-religation of an oligonucleotide does not fully recapitulate the incision-religation occuring during relaxation and the preferred substrate for such reaction characterization is supercoiled DNA. We therefore developed a method that used radiolabeled supercoiled DNA mini-circles to characterize the covalent enzyme-DNA complex formed during a relaxation reaction. Resolution of the relaxation products under different conditions permitted to quantify the proportion of covalent complex formed during the relaxation catalyzed by two topoisomerase models, the Escherichia coli topoisomerase I and the calf thymus topoisomerase I. As expected, the covalent complex formed with the calf thymus topoisomerase I was significantly enriched by camptothecin, a widely-used inhibitor of this topoisomerase, and a salt jump permitted the multiple topoisomerases trapped per mini-circle to complete the reaction cycle. The identified positions of the camptothecin-induced incision sites were shown to be independent of the linking number and the substrate circular nature Overall, our results demonstrate that supercoiled mini-circles constitute a powerful and polyvalent substrate to characterize the mechanism of action of novel topoisomerases and inhibitors, including the incision-religation reaction.

  9. Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex

    PubMed Central

    Millet, Armêl; Strauss, François; Delagoutte, Emmanuelle

    2015-01-01

    The enzymatic DNA relaxation requires the DNA to be transiently nicked and rejoined, the covalent topoisomerase-DNA complex being a key intermediate of the nicking-joining reaction. Practically, this reaction is most often characterized by oligonucleotides. However, the incision-religation of an oligonucleotide does not fully recapitulate the incision-religation occuring during relaxation and the preferred substrate for such reaction characterization is supercoiled DNA. We therefore developed a method that used radiolabeled supercoiled DNA mini-circles to characterize the covalent enzyme-DNA complex formed during a relaxation reaction. Resolution of the relaxation products under different conditions permitted to quantify the proportion of covalent complex formed during the relaxation catalyzed by two topoisomerase models, the Escherichia coli topoisomerase I and the calf thymus topoisomerase I. As expected, the covalent complex formed with the calf thymus topoisomerase I was significantly enriched by camptothecin, a widely-used inhibitor of this topoisomerase, and a salt jump permitted the multiple topoisomerases trapped per mini-circle to complete the reaction cycle. The identified positions of the camptothecin-induced incision sites were shown to be independent of the linking number and the substrate circular nature Overall, our results demonstrate that supercoiled mini-circles constitute a powerful and polyvalent substrate to characterize the mechanism of action of novel topoisomerases and inhibitors, including the incision-religation reaction. PMID:26300432

  10. NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1.

    PubMed

    Omichinski, J G; Clore, G M; Schaad, O; Felsenfeld, G; Trainor, C; Appella, E; Stahl, S J; Gronenborn, A M

    1993-07-23

    The three-dimensional solution structure of a complex between the DNA binding domain of the chicken erythroid transcription factor GATA-1 and its cognate DNA site has been determined with multidimensional heteronuclear magnetic resonance spectroscopy. The DNA binding domain consists of a core which contains a zinc coordinated by four cysteines and a carboxyl-terminal tail. The core is composed of two irregular antiparallel beta sheets and an alpha helix, followed by a long loop that leads into the carboxyl-terminal tail. The amino-terminal part of the core, including the helix, is similar in structure, although not in sequence, to the amino-terminal zinc module of the glucocorticoid receptor DNA binding domain. In the other regions, the structures of these two DNA binding domains are entirely different. The DNA target site in contact with the protein spans eight base pairs. The helix and the loop connecting the two antiparallel beta sheets interact with the major groove of the DNA. The carboxyl-terminal tail, which is an essential determinant of specific binding, wraps around into the minor groove. The complex resembles a hand holding a rope with the palm and fingers representing the protein core and the thumb, the carboxyl-terminal tail. The specific interactions between GATA-1 and DNA in the major groove are mainly hydrophobic in nature, which accounts for the preponderance of thymines in the target site. A large number of interactions are observed with the phosphate backbone. PMID:8332909

  11. Cellular uptake, cytotoxicity, apoptosis, DNA-binding, photocleavage and molecular docking studies of ruthenium(II) polypyridyl complexes.

    PubMed

    Srishailam, A; Kumar, Yata Praveen; Venkat Reddy, P; Nambigari, Navaneetha; Vuruputuri, Uma; Singh, Surya S; Satyanarayana, S

    2014-03-01

    Three new mononuclear [Ru (phen)2 ptip](2+) (1), [Ru (bpy)2 ptip](2+) (2) and [Ru (dmb)2 ptip](2+) (3) [ptip=(2-(5-phenylthiophen-2-yl)-1H-imidazo[4, 5-f][1,10 phenanthroline, phen=1, 10 phenanthroline, bpy=2, 2' bipyridine, dmb=4, 4'-dimethyl 2, 2' bipyridine] complexes were synthesized and characterised by elemental analysis, IR, NMR and Mass spectra. The DNA-binding behaviours were investigated by electronic absorption titration, luminescence spectra, viscosity measurements and photo-activated cleavage. The DNA-binding constants Kb of complexes 1, 2 and 3 were determined to be 7.0 (± 0.06)× 10(5), 3.87 (± 0.04) × 10(5), 2.79 (±0.07) × 10(5) respectively. The results showed that these complexes interact with CT-DNA by intercalative mode. Cell viability experiments indicated that the Ru(II) complex showed significant dose-dependent cytotoxicity to HeLa tumour cell lines. Further flow cytometry experiments showed that the cytotoxic Ru(II) complex induced apoptosis of HeLa tumour cell lines. Our data demonstrated that the Ru(II) polypyridyl complex binds to DNA and thereby induces apoptosis in tumor cells, suggesting that anti-tumor activity of the Ru(II) complex could be related to its interaction with DNA. The molecular dynamic simulations and docking methods were used to predict the DNA binding affinity of ruthenium complexes and with good visualisation images supporting with experimental results.

  12. Osmium complexation of mismatched DNA: effect of the bases adjacent to mismatched 5-methylcytosine.

    PubMed

    Nomura, Akiko; Tainaka, Kazuki; Okamoto, Akimitsu

    2009-03-18

    The efficiency of osmium complex formation at 5-methylcytosine in mismatched DNA duplexes is a key point for the design of sequence-specific detection of DNA methylation. Osmium complexation was not observed in fully matched duplexes, whereas the complexation site and efficiency in mismatched duplexes changed depending on the type of 5'-neighboring base of the 5-methylcytosine forming a mismatched base pair. In particular, when the base adjacent to the 5' side of the mismatched base pair was thymine, a unique "side reaction" was observed. However, the nature of the mismatched base pairs in the reaction site did not influence the selectivity of osmium complex formation with methylated DNA.

  13. Human replication protein A binds single-stranded DNA in two distinct complexes.

    PubMed Central

    Blackwell, L J; Borowiec, J A

    1994-01-01

    Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes. Images PMID:8196638

  14. Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation

    PubMed Central

    Marques, Catarina A.; Tiengwe, Calvin; Lemgruber, Leandro; Damasceno, Jeziel D.; Scott, Alan; Paape, Daniel; Marcello, Lucio; McCulloch, Richard

    2016-01-01

    Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying. PMID:26951375

  15. Direct electrochemical stripping detection of cystic-fibrosis-related DNA linked through cadmium sulfide quantum dots

    NASA Astrophysics Data System (ADS)

    Marin, Sergio; Merkoçi, Arben

    2009-02-01

    Electrochemical detection of a cadmium sulfide quantum dots (CdS QDs)-DNA complex connected to paramagnetic microbeads (MB) was performed without the need for chemical dissolving. The method is based on dropping 20 µl of CdS QD-DNA-MB suspension on the surface of a screen-printed electrode. It is followed by magnetic collection on the surface of the working electrode and electrochemical detection using square-wave voltammetry (SWV), giving a well-shaped and sensitive analytical signal. A cystic-fibrosis-related DNA sequence was sandwiched between the two DNA probes. One DNA probe is linked via biotin-streptavidin bonding with MB and the other one via thiol groups with the CdS QD used as tags. Nonspecific signals of DNA were minimized using a blocking agent and the results obtained were successfully employed in a model DNA sensor with an interest in future applications in the clinical field. The developed nanoparticle biosensing system may offer numerous opportunities in other fields where fast, low cost and efficient detection of small volume samples is required.

  16. 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.

  17. DNA damage and repair in telomeres: relation to aging.

    PubMed Central

    Kruk, P A; Rampino, N J; Bohr, V A

    1995-01-01

    We have established a method for the detection of DNA damage and its repair in human telomeres, the natural ends of chromosomes which are necessary for replication and critical for chromosomal stability. We find that ultraviolet light-induced pyrimidine dimers in telomeric DNA are repaired less efficiently than endogenous genes but more efficiently than inactive, noncoding regions. We have also measured telomeric length, telomeric DNA damage, and its repair in relation to the progression of aging. Telomeres are shorter in fibroblasts from an old donor compared to fibroblasts from a young donor, shortest in cells from a patient with the progeroid disorder Werner syndrome, and relatively long in fibroblasts from a patient with Alzheimer disease. Telomeric DNA repair efficiency is lower in cells from an old donor than in cells from a young donor, normal in Alzheimer cells, and slightly lower in Werner cells. It is possible that this decline in telomeric repair with aging is of functional significance to an age-related decline in genomic stability. Images Fig. 1 Fig. 2 PMID:7816828

  18. Sex-reversing mutations affect the architecture of SRY-DNA complexes.

    PubMed Central

    Pontiggia, A; Rimini, R; Harley, V R; Goodfellow, P N; Lovell-Badge, R; Bianchi, M E

    1994-01-01

    The testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination. Images PMID:7813448

  19. A Potential Rhodium Cancer Therapy: Studies of a Cytotoxic Organorhodium (I) Complex that Binds DNA

    PubMed Central

    McConnell, Jeanette R.; Rananaware, Dimple P; Ramsey, Deborah M.; Buys, Kai N.; Cole, Marcus L.; McAlpine, Shelli R.

    2013-01-01

    Described is a novel organorhodium(I) complex that is cytotoxic to the colon cancer cell line HCT116 and alters cell migration, DNA replication, and DNA condensation. Most importantly, the mechanism observed is not seen for the parent organorhodium dimer complex [{RhCl(COD)}2], RhCl3, or the free ligand/proligands (COD and 1-nbutyl-3-methylimidazolium chloride). Thus, the activity of this organorhodium complex is attributable to its unique structure. PMID:23541673

  20. DNA Interactions with Ruthenium(ll) Polypyridine Complexes Containing Asymmetric Ligands

    PubMed Central

    Chao, Hui

    2005-01-01

    In an attempt to probe nucleic acid structures, numerous Ru(II) complexes with different ligands have been synthesized and investigated. In this contribution we focus on the DNA-binding properties of ruthenium(II) complexes containing asymmetric ligands that have attracted little attention in the past decades. The influences of the shape and size of the ligand on the binding modes, affinity, enantioselectivities and photocleavage of the complexes to DNA are described. PMID:18365086

  1. DNA Ends Alter the Molecular Composition and Localization of Ku Multicomponent Complexes*

    PubMed Central

    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

    2012-01-01

    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. PMID:22535209

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

    SciTech Connect

    Joo, Woo; Xu, Guozhou; Persky, Nicole S.; Smogorzewska, Agata; Rudge, Derek G.; Buzovetsky, Olga; Elledge, Stephen J.; Pavletich, Nikola P.

    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 protein 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.

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

    SciTech Connect

    W Joo; G Xu; n Persky; A Smogorzewska; D Rudge; O Buzovetsky; S Elledge; N Pavletich

    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 protein 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26658024

  5. Dynamics and recognition within a protein–DNA complex: a molecular dynamics study of the SKN-1/DNA interaction

    PubMed Central

    Etheve, Loïc; Martin, Juliette; Lavery, Richard

    2016-01-01

    Molecular dynamics simulations of the Caenorhabditis elegans transcription factor SKN-1 bound to its cognate DNA site show that the protein–DNA interface undergoes significant dynamics on the microsecond timescale. A detailed analysis of the simulation shows that movements of two key arginine side chains between the major groove and the backbone of DNA generate distinct conformational sub-states that each recognize only part of the consensus binding sequence of SKN-1, while the experimentally observed binding specificity results from a time-averaged view of the dynamic recognition occurring within this complex. PMID:26721385

  6. UV-damaged DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation

    PubMed Central

    Brand, Marjorie; Moggs, Jonathan G.; Oulad-Abdelghani, Mustapha; Lejeune, Fabrice; Dilworth, F.Jeffrey; Stevenin, James; Almouzni, Geneviève; Tora, Làszlò

    2001-01-01

    Initiation of transcription of protein-encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, a complex comprised of the TATA box-binding protein (TBP) and TBP-associated factors (TAFIIs). In the presence of TBP-free TAFII complex (TFTC), initiation of Pol II transcription can occur in the absence of TFIID. TFTC containing the GCN5 acetyltransferase acetylates histone H3 in a nucleosomal context. We have identified a 130 kDa subunit of TFTC (SAP130) that shares homology with the large subunit of UV-damaged DNA-binding factor. TFTC preferentially binds UV-irradiated DNA, UV-damaged DNA inhibits TFTC-mediated Pol II transcription and TFTC is recruited in parallel with the nucleotide excision repair protein XP-A to UV-damaged DNA. TFTC preferentially acetylates histone H3 in nucleosomes assembled on UV-damaged DNA. In agreement with this, strong histone H3 acetylation occurs in intact cells after UV irradiation. These results suggest that the access of DNA repair machinery to lesions within chromatin may be facilitated by TFTC via covalent modification of chromatin. Thus, our experiments reveal a molecular link between DNA damage recognition and chromatin modification. PMID:11406595

  7. Tetrameric structure of the restriction DNA glycosylase R.PabI in complex with nonspecific double-stranded DNA

    PubMed Central

    Wang, Delong; Miyazono, Ken-ichi; Tanokura, Masaru

    2016-01-01

    R.PabI is a type II restriction enzyme that recognizes the 5′-GTAC-3′ sequence and belongs to the HALFPIPE superfamily. Although most restriction enzymes cleave phosphodiester bonds at specific sites by hydrolysis, R.PabI flips the guanine and adenine bases of the recognition sequence out of the DNA helix and hydrolyzes the N-glycosidic bond of the flipped adenine in a similar manner to DNA glycosylases. In this study, we determined the structure of R.PabI in complex with double-stranded DNA without the R.PabI recognition sequence by X-ray crystallography. The 1.9 Å resolution structure of the complex showed that R.PabI forms a tetrameric structure to sandwich the double-stranded DNA and the tetrameric structure is stabilized by four salt bridges. DNA binding and DNA glycosylase assays of the R.PabI mutants showed that the residues that form the salt bridges (R70 and D71) are essential for R.PabI to find the recognition sequence from the sea of nonspecific sequences. R.PabI is predicted to utilize the tetrameric structure to bind nonspecific double-stranded DNA weakly and slide along it to find the recognition sequence. PMID:27731370

  8. Transitions Between Distinct Compaction Regimes in Complexes of Multivalent Cationic Lipids And DNA

    SciTech Connect

    Farago, O.; Ewert, K.; Ahmad, A.; Evans, H.M.; Gronbech-Jensen, N.; Safinya, C.R.

    2009-05-18

    Cationic lipids (CLs) have found widespread use as nonviral gene carriers (vectors), including applications in clinical trials of gene therapy. However, their observed transfection efficiencies (TEs) are inferior to those of viral vectors, providing a strong incentive for a detailed understanding of CL-DNA complex behavior. In recent systematic studies employing monovalent as well as newly synthesized multivalent lipids (MVLs), the membrane cationic charge density has been identified as a key parameter governing the TE of lamellar CL-DNA complexes. In this work, we use x-ray scattering and molecular simulations to investigate the structural properties of complexes containing MVLs. At low mole fraction of neutral lipids (NLs), {phi}{sub NL}, the complexes show dramatic DNA compaction, down to essentially close-packed DNA arrays with a DNA interaxial spacing d{sub DNA} = 25{angstrom}. A gradual increase in {phi}{sub NL} does not lead to a continuous increase in d{sub DNA} as observed for DNA complexes of monovalent CLs. Instead, distinct spacing regimes exist, with sharp transitions between the regimes. Three packing states have been identified: (1), close packed, (2), condensed, but not close packed, with d{sub DNA} = 27-28{angstrom}, and (3), an expanded state, where d{sub DNA} increases gradually with {phi}{sub NL}. Based on our experimental and computational results, we conclude that the DNA condensation is mediated by the multivalent cationic lipids, which assemble between the negatively charged DNA rods. Quite remarkably, the computational results show that the less tightly packed structure in regime 2 is thermodynamically more stable than the close packed structure in regime 1. Accordingly, the constant DNA spacing observed in regime 2 is attributed to lateral phase coexistence between this stable CL-DNA complex and neutral membranes. This finding may explain the reduced TE measured for such complexes: transfection involves endosomal escape and disassembly of

  9. Interaction of Iron II Complexes with B-DNA. Insights from Molecular Modeling, Spectroscopy, and Cellular Biology

    PubMed Central

    Gattuso, Hugo; Duchanois, Thibaut; Besancenot, Vanessa; Barbieux, Claire; Assfeld, Xavier; Becuwe, Philippe; Gros, Philippe C.; Grandemange, Stephanie; Monari, Antonio

    2015-01-01

    We report the characterization of the interaction between B-DNA and three terpyridin iron II complexes. Relatively long time-scale molecular dynamics (MD) is used in order to characterize the stable interaction modes. By means of molecular modeling and UV-vis spectroscopy, we prove that they may lead to stable interactions with the DNA duplex. Furthermore, the presence of larger π-conjugated moieties also leads to the appearance of intercalation binding mode. Non-covalent stabilizing interactions between the iron complexes and the DNA are also characterized and evidenced by the analysis of the gradient of the electronic density. Finally, the structural deformations induced on the DNA in the different binding modes are also evidenced. The synthesis and chemical characterization of the three complexes is reported, as well as their absorption spectra in presence of DNA duplexes to prove the interaction with DNA. Finally, their effects on human cell cultures have also been evidenced to further enlighten their biological effects. PMID:26734600

  10. Interaction of Iron II Complexes with B-DNA. Insights from Molecular Modeling, Spectroscopy, and Cellular Biology.

    PubMed

    Gattuso, Hugo; Duchanois, Thibaut; Besancenot, Vanessa; Barbieux, Claire; Assfeld, Xavier; Becuwe, Philippe; Gros, Philippe C; Grandemange, Stephanie; Monari, Antonio

    2015-01-01

    We report the characterization of the interaction between B-DNA and three terpyridin iron II complexes. Relatively long time-scale molecular dynamics (MD) is used in order to characterize the stable interaction modes. By means of molecular modeling and UV-vis spectroscopy, we prove that they may lead to stable interactions with the DNA duplex. Furthermore, the presence of larger π-conjugated moieties also leads to the appearance of intercalation binding mode. Non-covalent stabilizing interactions between the iron complexes and the DNA are also characterized and evidenced by the analysis of the gradient of the electronic density. Finally, the structural deformations induced on the DNA in the different binding modes are also evidenced. The synthesis and chemical characterization of the three complexes is reported, as well as their absorption spectra in presence of DNA duplexes to prove the interaction with DNA. Finally, their effects on human cell cultures have also been evidenced to further enlighten their biological effects. PMID:26734600

  11. Contrasting patterns of the 5S and 45S rDNA evolutions in the Byblis liniflora complex (Byblidaceae).

    PubMed

    Fukushima, Kenji; Imamura, Kaori; Nagano, Katsuya; Hoshi, Yoshikazu

    2011-03-01

    To clarify the evolutionary dynamics of ribosomal RNA genes (rDNAs) in the Byblis liniflora complex (Byblidaceae), we investigated the 5S and 45S rDNA genes through (1) chromosomal physical mapping by fluorescence in situ hybridization (FISH) and (2) phylogenetic analyses using the nontranscribed spacer of 5S rDNA (5S-NTS) and the internal transcribed spacer of 45S rDNA (ITS). In addition, we performed phylogenetic analyses based on rbcL and trnK intron. The complex was divided into 2 clades: B. aquatica-B. filifolia and B. guehoi-B. liniflora-B. rorida. Although members of the complex had conservative symmetric karyotypes, they were clearly differentiated on chromosomal rDNA distribution patterns. The sequence data indicated that ITS was almost homogeneous in all taxa in which two or four 45S rDNA arrays were frequently found at distal regions of chromosomes in the somatic karyotype. ITS homogenization could have been prompted by relatively distal 45S rDNA positions. In contrast, 2-12 5S rDNA arrays were mapped onto proximal/interstitial regions of chromosomes, and some paralogous 5S-NTS were found in the genomes harboring 4 or more arrays. 5S-NTS sequence type-specific FISH analysis showed sequence heterogeneity within and between some 5S rDNA arrays. Interlocus homogenization may have been hampered by their proximal location on chromosomes. Chromosomal location may have affected the contrasting evolutionary dynamics of rDNAs in the B. liniflora complex.

  12. Coarse-grained Molecular Simulation Studies of Complexation of Sulfobetaine-Lysine Copolymer and DNA for Gene Delivery

    NASA Astrophysics Data System (ADS)

    Ghobadi, Ahmadreza F.; Jayaraman, Arthi

    2015-03-01

    Gene delivery involves successful transfection of therapeutic DNA by a vector into target cells and protein expression of that genetic material. Viral vectors are effective at gene delivery but elicit harmful immunogenic responses, thus motivating ongoing research on non-viral transfection agents. Cationic polymers are a promising class of non-viral vectors due to their low immugenic responses and low toxicity, and their ability to bind to the polyanionic DNA backbone to form a polycation-DNA complex (polyplex) that is then internalized in the target cell. While past studies have shown many polycations with differing DNA transfection efficacies, there is a need for general design guidelines that can relate the molecular features of the polycation to its DNA transfection efficiency. Using atomistic and coarse-grained molecular dynamics simulations we connect polycation design to polycation-DNA binding and experimentally observed transfection efficiency. Specifically in this presentation we will discuss our recent work looking into the effect of incorporating zwitterions into lysine based polycations on the resulting polyplex structure, shape, surface charge density and stability of DNA-polycation complexes.

  13. Rolling cycle amplification based single-color quantum dots-ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA.

    PubMed

    Su, Chen; Liu, Yufei; Ye, Tai; Xiang, Xia; Ji, Xinghu; He, Zhike

    2015-01-01

    In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots-ruthenium complex (QDs-Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen)2(dppx)](2+) (dppx=7,8-dimethyldipyrido [3,2-a:2',3'-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen)2(dppx)](2+) is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen)2(dppx)](2+) through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover, this strategy applies QDs-Ru assembling dyads to the detection of single-strand DNA (ssDNA) without any functionalization and separation techniques.

  14. Dynamic Expression of DNA Complexation with Self-assembled Biomolecular Clusters.

    PubMed

    Bartolami, Eline; Bessin, Yannick; Gervais, Virginie; Dumy, Pascal; Ulrich, Sébastien

    2015-08-24

    We report herein the implementation of a dynamic covalent chemistry approach to the generation of multivalent clusters for DNA recognition. We show that biomolecular clusters can be expressed in situ by a programmed self-assembly process using chemoselective ligations. The cationic clusters are shown, by fluorescence displacement assay, gel electrophoresis and isothermal titration calorimetry, to effectively complex DNA through multivalent interactions. The reversibility of the ligation was exploited to demonstrate that template effects occur, whereby DNA imposes component selection in order to favor the most active DNA-binding clusters. Furthermore, we show that a chemical effector can be used to trigger DNA release through component exchange reactions. PMID:26177835

  15. Synthesis of dihydromyricetin-manganese (II) complex and interaction with DNA

    NASA Astrophysics Data System (ADS)

    Guo, Qingquan; Yuan, Juan; Zeng, Jinhua; He, Xiangzhu; Li, Daguang

    2012-11-01

    Dihydromyricetin has many physiological functions and its metal complex could have better effects. DNA is very important in biological body, but little attention has been devoted to the relationship between dihydromyricetin-metal complex and the DNA. In this paper, dihydromyricetin-Mn (II) complex has been prepared and characterized using UV-vis absorption spectrophotometry, IR spectroscopy, elemental analysis, and thermal gravimetric analysis (TG-DTA Analysis). The interaction of dihydromyricetin-Mn (II) complex with DNA was investigated using UV-vis spectra, fluorescence measurements and viscosity measurements. The results indicate that the dihydromyricetin-manganese (II) complex can intercalate into the stacked base pairs of DNA with binding constant Kb = 5.64 × 104 M and compete with the strong intercalator ethidium bromide for the intercalative binding sites with Stern-Volmer quenching constant, Ksq = 1.16.

  16. Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage.

    PubMed

    Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson; Weinert, Brian T; Passmore, Lori A; Patel, Ketan J; Olsen, Jesper V; Choudhary, Chunaram; Bekker-Jensen, Simon; Mailand, Niels

    2015-01-01

    We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage.

  17. DNA-binding drugs caught in action: the latest 3D pictures of drug-DNA complexes.

    PubMed

    Boer, D Roeland; Canals, Albert; Coll, Miquel

    2009-01-21

    In this paper, we review recent DNA-binding agents that are expected to influence the field of DNA-targeting. We restrict ourselves to binders for which the three-dimensional structure in complex with DNA or RNA has been determined by X-ray crystallography or NMR. Furthermore, we primarily focus on unprecedented ways of targeting peculiar DNA structures, such as junctions, quadruplexes, and duplex DNAs different from the B-form. Classical binding modes of small molecular weight compounds to DNA, i.e. groove binding, intercalation and covalent addition are discussed in those cases where the structures represent a novelty. In addition, we review 3D structures of triple-stranded DNA, of the so-called Peptide Nucleic Acids (PNAs), which are oligonucleotide bases linked by a polypeptide backbone, and of aptamers, which are DNA or RNA receptors that are designed combinatorially. A discussion on perspectives in the field of DNA-targeting and on sequence recognition is also provided.

  18. 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.

  19. 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.

  20. Complexing of amino acids to DNA by chromate in intact cells.

    PubMed Central

    Voitkun, V; Zhitkovich, A; Costa, M

    1994-01-01

    Using o-pthaldialdehyde (OPT) fluorescence, the amino acids associated with DNA were studied following exposure of intact Chinese hamster ovary cells to chromate. Rigorous extraction with EDTA, acid, or base was required to release the amino acids cross-linked to the DNA isolated from control or chromate-treated cells by standard procedures (i.e., proteinase K, phenol, etc.). Amino acids resisting extraction from DNA were not studied since analysis was limited to those that could be released by these procedures. There was a chromate dose-dependent increase in amino acids complexed with the DNA that could be released by EDTA, acid, and base, and these amino acids were separated by HPLC and identified. Substantial increases in cysteine, glutamine, glutamic acid, histidine, threonine, and tyrosine were found as a function of increasing concentrations of chromate. There was also a time-dependent increase in complexing of these amino acids to the DNA by chromate. The amino acids found complexed to DNA in intact cells by chromate were thought to originate from reactions of free amino acids or small peptides with the DNA rather than being proteolytic products derived from larger proteins that were cross-linked to the DNA. This was supported by a number of experiments: a) free amino acids or bovine serum albumin (BSA) were cross-linked by chromium to DNA in vitro and the DNA was isolated by standard procedures.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7843108

  1. Interaction of Bis-Zn(II) salphen complex with calf thymus-DNA

    NASA Astrophysics Data System (ADS)

    Yussof, Aida Mastura Binti Mohd; Karim, Nurul Huda Abd

    2014-09-01

    Metal salphen family has been extensively studied over the past few years and has been reported to be good DNA stabilizers due to its high binding affinity. Binding studies of metal complex with DNA are useful for understanding the interaction mechanism and to provide an insight about the application and design of a novel effective drug target to DNA. In this study, a bis-zinc (II) salphen metal complex derived from 4-methyl-2,6-diformylphenol and 1,2-diaminobenzene (H2L) via condensation reactions has been synthesised. The zinc(II) macrocyclic complex is characterised using standard spectroscopic and structural techniques such as 1H NMR spectroscopy and FTIR spectroscopy. The binding interaction between the synthesised metal complex with calf thymus-DNA (ct-DNA) has been investigated by preliminary UV/Vis DNA study. From the preliminary UV/Vis DNA study, it shows that Bis-Zn(II) salphen complex has interaction with ct-DNA.

  2. Effective DNA binding and cleaving tendencies of malonic acid coupled transition metal complexes

    NASA Astrophysics Data System (ADS)

    Pravin, Narayanaperumal; Utthra, Ponnukalai Ponya; Kumaravel, Ganesan; Raman, Natarajan

    2016-11-01

    Eight transition metal complexes were designed to achieve maximum biological efficacy. They were characterized by elemental analysis and various other spectroscopic techniques. The monomeric complexes were found to espouse octahedral geometry and non-electrolytic nature. The DNA interaction propensity of the complexes with calf thymus DNA (CT-DNA), studied at physiological pH by spectrophotometric, spectrofluorometric, cyclic voltammetry, and viscometric techniques revealed intercalation as the possible binding mode. Fascinatingly, the complexes were found to exhibit greater binding strength than that of the free ligands. A strong hypochromism and a slight red shift were exhibited by complex 5 among the other complexes. The intrinsic binding constant values of all the complexes compared to cisplatin reveal that they are excellent metallonucleases than that of cisplatin. The complexes were also shown to reveal displacement of the ethidium bromide, a strong intercalator using fluorescence titrations. Gel electrophoresis was used to divulge the competence of the complexes in cleaving the supercoiled pBR322 plasmid DNA. From the results, it is concluded that the complexes, especially 5, are excellent chemical nucleases in the presence of H2O2. Furthermore, the in vitro antimicrobial screening of the complexes exposes that these complexes are excellent antimicrobial agents. Overall the effect of coligands is evident from the results of all the investigations.

  3. Influence of salt bridge interactions on the gas-phase stability of DNA/peptide complexes

    NASA Astrophysics Data System (ADS)

    Alves, Sandra; Woods, Amina; Delvolvé, Alice; Tabet, Jean Claude

    2008-12-01

    Negative ion mode electrospray ionization mass spectrometry was used to study DNA duplexes-peptide interaction. In the present study, we show that peptides that contain two adjacent basic residues interact noncovalently with DNA single strand or duplex. Fragmentation of the complexes between peptides containing basic residues and DNA were studied under collisions and showed unexpected dissociation pathways, as previously reported for peptide-peptide interactions. The binary complexes are dissociated either along fragmentation of the covalent bonds of the peptide backbone and/or along the single DNA strand backbone cleavage without disruption of noncovalent interaction, which demonstrates the strong binding of peptide to the DNA strand. Sequential MS/MS and MSn were further performed on ternary complexes formed between duplexes and peptides to investigate the nature of interaction. The CID spectra showed as major pathway the disruption of the noncovalent interactions and the formation of binary complexes and single-strand ions, directed by the nucleic acid gas-phase acidity. Indeed, a preferential formation of complexes with thymidine containing single strands is observed. An alternative pathway is also detected, in which complexes are dissociated along the covalent bond of the peptide and/or DNA according to the basicity. Our experimental data suggest the presence of strong salt bridge interactions between DNA and peptides containing basic residues.

  4. 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

  5. A 2,2"-bipyridine ligand for incorporation into oligodeoxynucleotides: synthesis, stability and fluorescence properties of ruthenium-DNA complexes.

    PubMed

    Wiederholt, K; McLaughlin, L W

    1999-06-15

    A non-nucleoside linker based upon the ligand 2,2'-bipyridine and ethylene glycol is prepared and placed into the backbone of a number of oligonucleo-tides. The bipyridine ligand is reacted with cis -dichloro bis(2,2'-bipyridyl) Ru(II) to generate the relatively substitutionally inert complex based upon the well-characterized tris -2,2'-bipyridyl Ru(II). The ruthenium-containing DNA complexes exhibited UV and fluorescence characteristics that are consistent with those previously observed for simple tris -2,2'-bipyridyl Ru(II) complexes. Oligonucleotides containing the ruthenium complex will form both DNA duplexes and triplexes with stabilities that are slightly better than those formed from simple tethered oligonucleotide probes in which the two hybridizing sequences are tethered by simple tri(ethylene glycol) or hexa(ethylene glycol) linkers.

  6. DNA bending by retinoid X receptor-containing retinoid and thyroid hormone receptor complexes.

    PubMed Central

    Lu, X P; Eberhardt, N L; Pfahl, M

    1993-01-01

    Retinoid X receptors (RXR) have been identified as common subunits in the regulation of multiple hormonal signaling pathways. Using circular permutation and phasing analysis of specific response elements, we present evidence that RXR-retinoic acid receptor and RXR-thyroid hormone receptor heterodimer or RXR-RXR homodimer complexes induce directed DNA bends when bound to their cognate response elements. The extent of DNA bending induced by the RXR alpha-containing complexes varied and depended on the structure of the DNA-binding sites and the RXR partners. The overall bending orientation for RXR-containing complexes is directed toward the major groove of the DNA helix at the center of hormone response elements. Our observation implicates DNA bending as a possible mechanism underlying transcriptional regulation of distinct retinoid and thyroid hormone responsive genes. Images PMID:8413250

  7. Binding of netropsin to DNA in complexes with polypeptides containing repetitive lysine sequences.

    PubMed

    Votavová, H; Burckhardt, G; Sponar, J; Zimmer, C

    1981-07-27

    The interaction of the antibiotic netropsin with calf thymus DNA, T4 DNA and poly(dA-dT) . poly(dA-dT) in complexes with sequential polypeptides containing repetitive lysine sequences and histone H1 was investigated using circular dichroism spectroscopy and equilibrium dialysis. Both soluble DNA-polypeptide complexes and insoluble complexes showed binding of netropsin. The possibility of displacement of polypeptides from DNA binding sites by competition with netropsin molecules was eliminated by experiments using 14C-labelled polypeptides. From the analysis of CD titration behavior as well as from the results of equilibrium dialysis studies it follows that netropsin does not compete with polypeptides for DNA binding sites, which suggests that these two ligands occupy different sites. Various explanations for minor differences in the CD behavior of the bound netropsin in the saturation region are also discussed.

  8. Specific Photocrosslinking of DNA-Protein Complexes: Identification of Contacts Between Integration Host Factor and Its Target DNA

    NASA Astrophysics Data System (ADS)

    Yang, Shu-Wei; Nash, Howard A.

    1994-12-01

    Azide moieties have been specifically placed in the backbone of DNA by chemical coupling between azidophenacyl bromide and uniquely positioned phosphorothioate residues. The derivatized DNA forms specific complexes with a DNA-binding protein and, following irradiation with 302-nm light, makes specific crosslinks to the protein. Isolation of this covalent complex, followed by tryptic digestion and Edman degradation of the resulting crosslinked peptide, identifies the portion of the protein that is near the derivatized segment of the target DNA. We use this method to probe the interaction between a specific DNA sequence and integration host factor (IHF) protein. A single IHF heterodimer is known to contact >25 bp of DNA and thereby introduce a sharp bend. Two segments of a typical IHF site were derivatized with aryl azide. Although the segments were separated by only 5 bp, they crosslinked to different subunits of IHF. The locations of the crosslinks support our current view for the way IHF protein binds to and bends its specific targets.

  9. RAD50 and NBS1 form a stable complex functional in DNA binding and tethering.

    PubMed

    van der Linden, Eddy; Sanchez, Humberto; Kinoshita, Eri; Kanaar, Roland; Wyman, Claire

    2009-04-01

    The RAD50/MRE11/NBS1 protein complex (RMN) plays an essential role during the early steps of DNA double-strand break (DSB) repair by homologous recombination. Previous data suggest that one important role for RMN in DSB repair is to provide a link between DNA ends. The striking architecture of the complex, a globular domain from which two extended coiled coils protrude, is essential for this function. Due to its DNA-binding activity, ability to form dimers and interact with both RAD50 and NBS1, MRE11 is considered to be crucial for formation and function of RMN. Here, we show the successful expression and purification of a stable complex containing only RAD50 and NBS1 (RN). The characteristic architecture of the complex was not affected by absence of MRE11. Although MRE11 is a DNA-binding protein it was not required for DNA binding per se or DNA-tethering activity of the complex. The stoichiometry of NBS1 in RMN and RN complexes was estimated by SFM-based volume analysis. These data show that in vitro, R, M and N form a variety of stable complexes with variable subunit composition and stoichiometry, which may be physiologically relevant in different aspects of RMN function.

  10. DNA strand exchange stimulated by spontaneous complex formation with cationic comb-type copolymer.

    PubMed

    Kim, Won Jong; Akaike, Toshihiro; Maruyama, Atsushi

    2002-10-30

    Cationic comb-type copolymers (CCCs) composed of a polycation backbone and water-soluble side chains accelerate by 4-5 orders the DNA strand exchange reaction (SER) between double helical DNA and its homologous single-strand DNA. The accelerating effect is considered due to alleviation of counterion association during transitional intermediate formation in sequential displacement pathway. CCCs stabilize not only matured hybrids but also the nucleation complex to accelerate hybridization. PMID:12392411

  11. Exercise and inflammation-related epigenetic modifications: focus on DNA methylation.

    PubMed

    Horsburgh, Steven; Robson-Ansley, Paula; Adams, Rozanne; Smith, Carine

    2015-01-01

    Epigenetics is the study of mitotically or meiotically heritable phenotypes that occur as a result of modifications to DNA, thereby regulating gene expression independently of changes in base sequence due to manipulation of the chromatin structure. These modifications occur through a variety of mechanisms, such as DNA methylation, post-translational histone modifications, and non-coding RNAs, and can cause transcriptional suppression or activation depending on the location within the gene. Environmental stimuli, such as diet and exercise, are thought to be able to regulate these mechanisms, with inflammation as a probable contributory factor. Research into these areas is still in its infancy however. This review will focus on DNA methylation in the context of inflammation (both pro- and anti-inflammatory processes) and exercise. The complexity and relative shortcomings of some existing techniques for studying epigenetics will be highlighted, and recommendations for future study approaches made.

  12. Interaction of drug based copper(II) complexes with Herring Sperm DNA and their biological activities

    NASA Astrophysics Data System (ADS)

    Patel, Mohan N.; Patel, Chintan R.; Joshi, Hardik N.

    2012-11-01

    Square pyramidal Cu(II) complexes with NS donor ligand and ciprofloxacin have been synthesized and characterized using analytical and spectral techniques. The synthesized complexes have been tested for their antimicrobial activity using double dilution technique in terms of minimum inhibitory concentration (MIC) and colony forming unit (CFU). The DNA binding ability of the complexes with Sperm Herring DNA has been performed using absorption titration and viscosity measurement. The nuclease activity of complexes with plasmid DNA (pUC19) has been carried out using agarose gel electrophoresis technique. Synthesized complexes have been tested for their SOD mimic activity using NBT/NADH/PMS system. The cytotoxic properties of metal complexes have been evaluated using brine shrimp lethality bioassay.

  13. Morphology characterization and single-molecule study of DNA-dodecyltrimethylammonium bromide complex.

    PubMed

    Ran, Shi-Yong; Wang, Yan-Wei; Yang, Guang-Can; Zhang, Lin-Xi

    2011-04-28

    DNA compaction induced by dodecyltrimethylammonium bromide (DTAB) is studied using atomic force microscopy (AFM) and magnetic tweezers. The morphology of DNA-DTAB complex is dependent on the DTAB concentration and incubation time. With magnesium ions, the complexes show rod- and network-like structures after approximately 5 min of incubation at low DTAB concentrations. With increasing incubation time, more toroids and globules appeared, resulting in the formation of scattered condensed particles. At high DTAB concentrations, the complexes show swollen globular structures independent of the incubation time. The compaction and unraveling of the DNA-DTAB complex are also analyzed at the single-molecule level using magnetic tweezers. The extension-time curves show a staircase structure with typical sizes of ∼40, 60, 80, and 112 nm, suggesting that the complexes are well organized and more compacted than those induced by multivalent ions. Finally, the high DTAB concentration stabilized the complex and increased the unraveling energy barrier.

  14. Protein Crystal Eco R1 Endonulease-DNA Complex

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Type II restriction enzymes, such as Eco R1 endonulease, present a unique advantage for the study of sequence-specific recognition because they leave a record of where they have been in the form of the cleaved ends of the DNA sites where they were bound. The differential behavior of a sequence -specific protein at sites of differing base sequence is the essence of the sequence-specificity; the core question is how do these proteins discriminate between different DNA sequences especially when the two sequences are very similar. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  15. Probabilistic expert systems for handling artifacts in complex DNA mixtures.

    PubMed

    Cowell, R G; Lauritzen, S L; Mortera, J

    2011-06-01

    This paper presents a coherent probabilistic framework for taking account of allelic dropout, stutter bands and silent alleles when interpreting STR DNA profiles from a mixture sample using peak size information arising from a PCR analysis. This information can be exploited for evaluating the evidential strength for a hypothesis that DNA from a particular person is present in the mixture. It extends an earlier Bayesian network approach that ignored such artifacts. We illustrate the use of the extended network on a published casework example.

  16. Synthesis, characterization, DNA binding, DNA cleavage and antimicrobial studies of Schiff base ligand and its metal complexes.

    PubMed

    Mendu, Padmaja; Kumari, C Gyana; Ragi, Rajesh

    2015-03-01

    A series of Cu(II), Ni(II), Co(II), Mn(II) and Zn(II) complexes have been synthesized from the Schiff base ligand L. The Schiff base ligand 4-chloro-2-((4-oxo-4H-chromen-3yl) methylene amino) benzoic acid (L) has been synthesized by the reaction between chromone-3-carbaldehyde and 4-chloro-2-amino benzoic acid. The nature of bonding and geometry of the transition metal complexes as well as ligand L have been deduced from elemental analysis, FT-IR, UV-vis, (1)H NMR, (13)C NMR, ESR spectral studies, mass, magnetic susceptibility and molar conductance measurements. The complexes are found to have ML2 composition and are neutral in DMSO. Based on elemental, conductance and spectral studies, six-coordinated geometry was assigned for these complexes. The ligand L acts as tridentate and coordinates through nitrogen atom of azomethine group, hydroxyl of the carboxyl group and oxygen atom of keto group of γ-pyrone ring. The interaction of Cu(II) complex with CT-DNA was carried out by UV-vis, fluorescence titrations and viscosity measurements. The complex binds to DNA through intercalative binding mode. The nuclease activity of the above metal complexes shows that Cu(II) and Co(II) complexes cleave DNA through redox chemistry. The biological activity of the ligand and its complexes have been studied on four bacteria E. coli, B. subtilis, pseudomonas and Edwardella and two fungi penicillium and trichoderma by well disc and fusion method and found that the metal complexes are more active than the free Schiff base ligand.

  17. Activity of site-specific endonucleases on complexes of plasmid DNA with multiwalled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Veligura, A. A.; Shulitsky, B. G.; Asayonok, A. A.; Vaskovtsev, E. V.

    2016-08-01

    We have synthesized and investigated structural and functional properties of plasmid DNA complexes with multi-walled carbon nanotubes (MWCNTs) for detection of changes in structural state of the plasmid DNA at its recognition by site-specific endonuclease. It has been also established that the site-specific endonuclease is functionally active on the surface of MWCNTs.

  18. Integration of DNA barcoding into an ongoing inventory of complex tropical biodiversity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  19. Molecular beacon-equilibrium cyclization detection of DNA-protein complexes.

    PubMed

    Vitko, Jason; Rujan, Iulian; Androga, Lagu; Mukerji, Ishita; Bolton, Philip H

    2007-11-01

    Molecular beacon detection of equilibrium cyclization (MBEC) is a novel, high sensitivity technique that can allow DNA-protein complex formation to be studied under diverse conditions in a cost effective and rapid manner that can be adapted to high throughput screening. To demonstrate the ease and utility of applying MBEC to the investigation of the K(D) values of protein-DNA complexes, the sequence-specific Escherichia coli integration host factor (IHF) protein has been used as a test system. Competition between a labeled MBEC DNA construct and unlabeled duplex DNA for IHF binding allows the determination of K(D) values as a function of the DNA duplex sequence. This allows sequence specificity to be monitored while using only a single molecular beacon-labeled DNA. The robustness of MBEC for monitoring protein-DNA complex formation has been further demonstrated by determining the K(D) values as a function of salt concentration to investigate the net number of salt bridges formed in sequence-specific and -nonspecific IHF-DNA complexes. These MBEC results have been compared with those from other approaches.

  20. The Structure of the Dead ringer-DNA complex reveals how AT-rich interaction domains (ARIDs) recognize DNA

    SciTech Connect

    Iwahara, Junji; Iwahara, Mizuho; Daughdrill, Gary W.; Ford, Joe J.; Clubb, Robert T.

    2002-03-01

    The AT-rich interaction domain (ARID) is a DNA-binding module found in many eukaryotic transcription factors. Using NMR Spectroscopy, we have determined the first ever three-dimensional structure of an ARID-DNA complex (mol.wt 25.7 kDa) formed by Dead ringer from Drosophila melanogaster, ARIDs recognize DNA through a novel mechanism involving major groove immobilization of a large loop that connects the helices of a non-canonical helix-turn-helix motif, and through a concomitant structural rearrangement. that produces stabilizing contacts from a B-hairpin. Dead ringer's preference for a AT-rich DNA originates from three positions within the ARID fold that form energetically significant contacts to an adenine thymine base step.

  1. Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid.

    PubMed

    Ito, Tomoko; Iida-Tanaka, Naoko; Koyama, Yoshiyuki

    2008-05-01

    Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells. PMID:18446606

  2. Force-dependent persistence length of DNA-intercalator complexes measured in single molecule stretching experiments.

    PubMed

    Bazoni, R F; Lima, C H M; Ramos, E B; Rocha, M S

    2015-06-01

    By using optical tweezers with an adjustable trap stiffness, we have performed systematic single molecule stretching experiments with two types of DNA-intercalator complexes, in order to investigate the effects of the maximum applied forces on the mechanical response of such complexes. We have explicitly shown that even in the low-force entropic regime the persistence length of the DNA-intercalator complexes is strongly force-dependent, although such behavior is not exhibited by bare DNA molecules. We discuss the possible physicochemical effects that can lead to such results. In particular, we propose that the stretching force can promote partial denaturation on the highly distorted double-helix of the DNA-intercalator complexes, which interfere strongly in the measured values of the persistence length. PMID:25913936

  3. Enoxacin-Tb3+ complex as an environmentally friendly fluorescence probe for DNA and its application.

    PubMed

    Tong, Changlun; Hu, Zhou; Liu, Weiping

    2007-02-15

    The fluorescence intensity of the enoxacin (ENX)-Tb(3+) complex enhanced by DNA was studied. On the basis of this study, an environmentally friendly fluorescence probe of enoxacin-Tb(3+) for the determination of single-stranded and double-stranded DNA was developed. Under the optimal conditions, the enhanced fluorescence intensity was in proportion to the concentration of DNA in the range of 2.0x10(-8) to 2.0x10(-6)g mL(-1) for hsDNA, 1.0x10(-8) to 1.0x10(-6)g mL(-1) for ctDNA and 5.0x10(-9) to 1.0x10(-6)g mL(-1) for thermally denatured ctDNA. The detection limits (S/N=3) were 5.0, 9.0 and 3.0ng mL(-1), respectively. The interaction modes between ENX-Tb(3+) and DNA and the mechanism of the fluorescence enhancement were also discussed in details. The experimental results from UV absorption spectra, fluorescence spectra and the competing combination tests between the ENX-Tb(3+) complex and EB probe indicated that the possible interaction modes between enoxacin-Tb(3+) complex and DNA had at least two different binding modes: the electrostatic binding and the intercalation binding. Additionally, this fluorescence probe was used to study the interaction between heavy metals and DNA.

  4. Structure of the LexA-DNA complex and implications for SOS box measurement

    SciTech Connect

    Zhang, Adrianna P.P.; Pigli, Ying Z; Rice, Phoebe A

    2010-09-08

    The eubacterial SOS system is a paradigm of cellular DNA damage and repair, and its activation can contribute to antibiotic resistance. Under normal conditions, LexA represses the transcription of many DNA repair proteins by binding to SOS 'boxes' in their operators. Under genotoxic stress, accumulating complexes of RecA, ATP and single-stranded DNA (ssDNA) activate LexA for autocleavage. To address how LexA recognizes its binding sites, we determined three crystal structures of Escherichia coli LexA in complex with SOS boxes. Here we report the structure of these LexA-DNA complexes. The DNA-binding domains of the LexA dimer interact with the DNA in the classical fashion of a winged helix-turn-helix motif. However, the wings of these two DNA-binding domains bind to the same minor groove of the DNA. These wing-wing contacts may explain why the spacing between the two half-sites of E. coli SOS boxes is invariant.

  5. Dose effects in electron beam irradiation of DNA-complex thin films

    SciTech Connect

    Li, W.; Jones, R.; Spaeth, H.; Steckl, A. J.

    2010-08-09

    Electron beam irradiation of double-stranded DNA (dsDNA)-surfactant thin films was investigated. Irradiation caused dissociation, leading to increasing thin film solubility in water and degradation of dsDNA. These two effects produced a maximum concentration of dsDNA in aqueous solution at 400 {mu}C/cm{sup 2} dose. These properties resulted in dual-mode resist characteristics of the DNA-surfactant films. At low dose, the DNA films functioned as positive resist while at high dose they worked as negative resist. The transition between the two regimes also occurred at 400 {mu}C/cm{sup 2}. This implies that the cross-linking process (typical for negative resists) first requires the dissociation of the DNA-surfactant complex.

  6. BstYI bound to noncognate DNA reveals a "hemispecific" complex: implications for DNA scanning.

    PubMed

    Townson, Sharon A; Samuelson, James C; Bao, Yongming; Xu, Shuang-Yong; Aggarwal, Aneel K

    2007-04-01

    DNA recognition by proteins is essential for specific expression of genes in a living organism. En route to a target DNA site, a protein will often sample noncognate DNA sites through nonspecific protein-DNA interactions, resulting in a variety of conformationally different binding states. We present here the crystal structure of endonuclease BstYI bound to a noncognate DNA. Surprisingly, the structure reveals the enzyme in a "hemispecific" binding state on the pathway between nonspecific and specific recognition. A single base pair change in the DNA abolishes binding of only one monomer, with the second monomer bound specifically. We show that the enzyme binds essentially as a rigid body, and that one end of the DNA is accommodated loosely in the binding cleft while the other end is held tightly. Another intriguing feature of the structure is Ser172, which has a dual role in establishing nonspecific and specific contacts. Taken together, the structure provides a snapshot of an enzyme in a "paused" intermediate state that may be part of a more general mechanism of scanning DNA. PMID:17437717

  7. BstYI bound to noncognate DNA reveals a "hemispecific" complex: implications for DNA scanning.

    PubMed

    Townson, Sharon A; Samuelson, James C; Bao, Yongming; Xu, Shuang-Yong; Aggarwal, Aneel K

    2007-04-01

    DNA recognition by proteins is essential for specific expression of genes in a living organism. En route to a target DNA site, a protein will often sample noncognate DNA sites through nonspecific protein-DNA interactions, resulting in a variety of conformationally different binding states. We present here the crystal structure of endonuclease BstYI bound to a noncognate DNA. Surprisingly, the structure reveals the enzyme in a "hemispecific" binding state on the pathway between nonspecific and specific recognition. A single base pair change in the DNA abolishes binding of only one monomer, with the second monomer bound specifically. We show that the enzyme binds essentially as a rigid body, and that one end of the DNA is accommodated loosely in the binding cleft while the other end is held tightly. Another intriguing feature of the structure is Ser172, which has a dual role in establishing nonspecific and specific contacts. Taken together, the structure provides a snapshot of an enzyme in a "paused" intermediate state that may be part of a more general mechanism of scanning DNA.

  8. Cationic Liposome-DNA Complexes: From supramolecular assembly toward gene delivery

    NASA Astrophysics Data System (ADS)

    Evans, Heather M.; Ahmad, A.; Ewert, K.; Martin, A.; Safinya, Cr

    2003-03-01

    Cationic liposomes (CL) present a viable alternative to viral delivery of therapeutic DNA and peptides to cells. We complex CL with DNA to deliver foreign DNA (genes) to cells. Typical self-assembly of CL-DNA shown by x-ray diffraction reveals multilamellar lipids with DNA intercalated between the lipid layers, having a DNA interaxial spacing d(DNA)[1]. The length d(DNA) can be tuned at the subnanometer level (from 35 down to 5 angstroms) by control of the membrane charge density and other parameters. Three distinct DNA-DNA interaction regimes were found due to repulsive long-range electrostatic forces, repulsive short-range hydration forces, and a polymer induced attractive depletion force [2-4]. We correlate d(DNA) to transfection in mammalian cells. These compact DNA structures suggest use for high density storage of genetic information, as well as for biological templates. Supported by NSF DMR-0203755, NIH GM59288. 1. J Radler et al, Science 275, 810 (1997). 2. AJ Lin et al, Biophys. J. (in press). 3. K Ewert, A Ahmad, H Evans et al, J. Med. Chem. 45, 5023 (2002). 4. A Martin et al, (submitted).

  9. Conformation of nucleoplasmin and its interaction with DNA-protamine complex as a simple model of fish sperm nuclei.

    PubMed

    Iwata, K; Hozumi, K; Itoh, T; Sakairi, N; Tokura, S; Katagiri, C; Nishi, N

    1997-06-01

    Nucleoplasmin was isolated from Xenopus laevis eggs and purified by an improved method using an open column. Its conformation was investigated spectrophotometrically by UV, CD and fluorescence. It was shown that alpha-helix content of nucleoplasmin was 30-40%, and one of the two tryptophan residues in nucleoplasmin located in the hydrophobic surroundings and the other in the relatively hydrophilic surroundings. The isolated nucleoplasmin was found to decondense sperm nuclei of salmon also, suggesting a possibility of the existence of nucleoplasmin-like protein in fish as well. Collapse of the protamine (salmine)-DNA complex as a simple model for fish sperm nuclei by nucleoplasmin was directly observed by measuring OD320 of aqueous protamine-DNA mixtures. This is a molecular level observation for the removal of protamine from DNA-protamine complex. PMID:9218166

  10. Thermodynamics of cationic lipid-DNA complex formation as studied by isothermal titration calorimetry.

    PubMed Central

    Pozharski, Edwin; MacDonald, Robert C

    2002-01-01

    The detailed analysis of the cationic lipid-DNA complex formation by means of isothermal titration calorimetry is presented. Most experiments were done using 1,2-dioleyl-sn-glycero-3-ethylphosphocholine (EDOPC), but basic titrations were also done using DOTAP, DOTAP:DOPC, and DOTAP:DOPE mixtures. Complex formation was endothermic with less than 1 kcal absorbed per mole of lipid or DNA charge. This enthalpy change was attributed to DNA-DNA mutual repulsion within the lamellar complex. The exception was DOTAP:DOPE-containing lipoplex for which the enthalpy of formation was exothermic, presumably because of DOPE amine group protonation. Experimental conditions, namely, direction and titration increment as well as concentration of titrant, which dictate the structure of resulting lipoplex (whether lamellar complex or DNA-coated vesicle), were found to affect the apparent thermodynamics of complex formation. The structure, in turn, influences the biological properties of the lipoplex. If the titration of lipid into DNA was carried out in large increments, the DeltaH was larger than when the injection increments were smaller, a finding that is consistent with increased vesicle disruption under large increments and which is expected theoretically. Cationic lipid-DNA binding was weak in high ionic strength solutions, however, the effective binding constant is within micromolar range because of macromolecular nature of the interaction. PMID:12080142

  11. Determining the Architecture of a Protein-DNA Complex by Combining FeBABE Cleavage Analyses, 3-D Printed Structures, and the ICM Molsoft Program.

    PubMed

    James, Tamara; Hsieh, Meng-Lun; Knipling, Leslie; Hinton, Deborah

    2015-01-01

    Determining the structure of a protein-DNA complex can be difficult, particularly if the protein does not bind tightly to the DNA, if there are no homologous proteins from which the DNA binding can be inferred, and/or if only portions of the protein can be crystallized. If the protein comprises just a part of a large multi-subunit complex, other complications can arise such as the complex being too large for NMR studies, or it is not possible to obtain the amounts of protein and nucleic acids needed for crystallographic analyses. Here, we describe a technique we used to map the position of an activator protein relative to the DNA within a large transcription complex. We determined the position of the activator on the DNA from data generated using activator proteins that had been conjugated at specific residues with the chemical cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE). These analyses were combined with 3-D models of the available structures of portions of the activator protein and B-form DNA to obtain a 3-D picture of the protein relative to the DNA. Finally, the Molsoft program was used to refine the position, revealing the architecture of the protein-DNA within the transcription complex. PMID:26404142

  12. TDP1 promotes assembly of non-homologous end joining protein complexes on DNA

    PubMed Central

    Heo, Jinho; Li, Jing; Summerlin, Matthew; Hays, Annette; Katyal, Sachin; McKinnon, Peter J.; Nitiss, Karin C.; Nitiss, John L.; Hanakahi, Leslyn A.

    2015-01-01

    The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3′-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ. PMID:25841101

  13. Selective DNA purine base photooxidation by bis-terdentate iridium(III) polypyridyl and cyclometalated complexes.

    PubMed

    Jacques, Alexandre; Kirsch-De Mesmaeker, Andrée; Elias, Benjamin

    2014-02-01

    Two bis-terdentate iridium(III) complexes with polypyridyl and cyclometalated ligands have been prepared and characterized. Their spectroscopic and electrochemical properties have been studied, and a photophysical scheme addressing their properties is proposed. Different types of excited states have been considered to account for the deactivation processes in each complex. Interestingly, in the presence of mono- or polynucleotides, a photoinduced electron-transfer process from a DNA purine base (i.e., guanine or adenine) to the excited complex is shown through luminescence quenching experiments. For the first time, this work reports evidence for selective DNA purine bases oxidation by excited iridium(III) bis-terdentate complexes.

  14. Development of an effective gene delivery system: a study of complexes composed of a peptide-based amphiphilic DNA compaction agent and phospholipid

    PubMed Central

    Murphy, Eric A.; Waring, Alan J.; Murphy, Jason C.; Willson, Richard C.; Longmuir, Kenneth J.

    2001-01-01

    We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidylethanolamine/µg DNA produced the highest luciferase gene expression, exceeding 1 × 109 relative light units/s/mg protein (>3 µg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidylethanolamine are highly effective non-viral gene delivery systems. PMID:11522841

  15. Synthesis, characterization, DNA interactions, DNA cleavage, radical scavenging activity, antibacterial, anti-proliferative and docking studies of new transition metal complexes.

    PubMed

    Chennam, Kishan Prasad; Ravi, Mudavath; Ushaiah, B; Srinu, V; Eslavath, Ravi Kumar; Devi, Ch Sarala

    2016-01-01

    The compound N-(2-hydroxybenzylidene)-1-ethyl-1, 4-dihydro-7-methyl-4-oxo-1, 8 naphthyridine-3-carbohydrazide (LH) and its Cu (II), Co (II) and Zn (II) complexes were synthesized and characterized. The absorption spectral titrations and competitive DNA binding studies depicted those complexes of title compound bind to CT-DNA through intercalation. Interestingly [Cu (II)-(L2)] showed relatively high binding constant value (6.61 x 10(5) M(-1)) compared to [Co (II)-(L2)] (4.378× 10(5) M(-1)) and [Zn (II)-(L2)] (3.1x10(5) M(-1)). Ligand and its complexes were also examined for DNA nuclease activity against pBR-322 plasmid DNA, which showed that [Cu (II)-(L2)] had the best hydrolytic cleavage property displaying prominent double-strand DNA cleavage. In addition, antioxidant activities of the ligand and its metal complexes investigated through scavenging effects for DPPH radical in- vitro, indicated their potentiality as good antioxidants. The in vitro anti-bacterial study inferred the better anti-bacterial activity of [Cu (II)-(L2)] and this was also correlated theoretically by employing docking studies wherein [Cu (II)-(L2)] displayed good Gold score and Chem score. Finally the in vitro anti- proliferative activity of studied compounds was tested against HeLa and MCF-7 cell lines. Interestingly [Cu (II)-(L2)] displayed lower IC50 value and lower percentage of viability in both HeLa and MCF-7 cell lines.

  16. Transfer of DNA into lymphoma cells by DNA-bound to T101-biotinylated-avidin-polylysine antibody complex

    SciTech Connect

    Chakrabarti, M.; Panyutin, I.; Neumann, R.D.; Carrasquillo, J.A.

    1996-05-01

    Monoclonal antibodies (MoAb) can be used to target specific cell types. MoAb have been used to selectively deliver DNA molecules to target cells. Our long term goal is to evaluate the use of rapidly internalizing antibody to selectively deliver radiolabeled DNA to the nucleus. As a model system we used T101, an IgG2a murine MoAb that recognizes CD5 and upon binding to this antigen rapidly internalizes. As a DNA sequence we used the pGL2 plasmid containing the luciferase reported gene. Avidin-polylysine was prepared by periodate oxidation of carbohydrate chains of avidin which were then covalently attached to polylysine (Mol.wt 56 kD) and stabilized by sodium borohydride reduction. This conjugate was bound with biotinylated T101 MoAb (biotinylation in lysine residue of MoAb). The final avidin-polylysine-biotin-T101 was purified by size exclusion HPLC. The luciferase DNA was cut by Xba1 restriction enzyme followed by labeling with I-125-dCTP and purified through a G-50 minicolumn. Specific activity was found to be 1{mu}Ci/{mu}g. The I-125 pGL2 was incubated with the T101-biotin-avidin-polylysine complex and 95% of DNA was complexed as determined by nitrocellulose binding. The immunoreactivity of the resulting I-125 pGL2 complex was {approximately} 73%. The retention of I-125 by CD5 positive cells in culture at 37{degrees}C was longer than that of directly iodinated T101. For DNA transfection study CCRF-CEM cell were incubated with 25{mu}G T101-biotin conjugated with avidin-polylysine (10{mu}g) and pGL2 plasmid DNA(10{mu}g). Luciferase activity was observed in the CCRF-CEM cell line that was 35 times higher than the control experiments with pGL2-avidin-polylysine in absence of T101-biotin. In conclusion, these result suggest that DNA conjugated antibody complex can be used to deliver radioactive DNA to the cell nucleus.

  17. Interpreting DNA mixtures with the presence of relatives.

    PubMed

    Hu, Yue-Qing; Fung, Wing K

    2003-02-01

    The assessment of DNA mixtures with the presence of relatives is discussed in this paper. The kinship coefficients are incorporated into the evaluation of the likelihood ratio and we first derive a unified expression of joint genotypic probabilities. A general formula and seven types of detailed expressions for calculating likelihood ratios are then developed for the case that a relative of the tested suspect is an unknown contributor to the mixed stain. These results can also be applied to the case of a non-tested suspect with one tested relative. Moreover, the formula for calculating the likelihood ratio when there are two related unknown contributors is given. Data for a real situation are given for illustration, and the effect of kinship on the likelihood ratio is shown therein. Some interesting findings are obtained.

  18. PEGylated Cationic Liposome – DNA Complexation in Brine is Pathway-Dependent

    PubMed Central

    Silva, Bruno F.B.; Majzoub, Ramsey N.; Chan, Chia-Ling; Li, Youli; Olsson, Ulf; Safinya, Cyrus R.

    2013-01-01

    Cationic liposome-DNA (CL-DNA) complexes, are regarded as promising materials for safe and efficient delivery of genes for therapeutical applications. In order to be used in vivo, these complexes may be coated with a hydrophilic polymer (e.g. polyethylene-glycol, PEG) that provides steric stabilization towards adhesion of proteins and removal by the immune system. In this work we study the influence of the initial salt concentration (Cs) – which modulates the electrostatic interaction between oppositely charged vesicles and DNA – on the structure and stability of PEGylated CL-DNA particles. Previous small-angle X-ray scattering has shown that if non-PEGylated or PEGylated CL-DNA lamellar complexes are prepared in water, their structure is well defined with a high number of lipid membrane-DNA layers (larger than 20). Here we show that if these complexes are transferred to saline media (150 mM NaCl or DMEM, both near physiological conditions), this structure remains nearly unchanged. Conversely, if PEGylated complexes are prepared in saline media, their lamellar structure is much looser, with fewer number of layers. This pathway dependent behavior of PEGylated complex formation in brine is modulated by the liposome membrane charge density and the mole fraction of PEG 2000 in the membranes, with the average number of layers decreasing with increasing Cs and in going from 5 mol% to 10 mol% PEG-lipid. Each of these structures (high and low number of layers) is stable with time, suggesting that despite complex formation being thermodynamically favored, the complexation process in PEGylated membranes, which determines the number of layers per particle, is kinetically controlled. In the extreme case (when polymer repulsions from 10 mol% PEG-lipid are maximized and electrostatic attraction between PEGylated CLs and DNA are minimized at low membrane charge density) complex formation is suppressed at high Cs=150 mM. PMID:24060564

  19. Transfection of Escherichia coli spheroplasts with a bacteriophage Mu DNA-protein complex.

    PubMed Central

    Chase, C D; Benzinger, R H

    1982-01-01

    We disrupted bacteriophage Mu particles by freeze-thaw treatment and recovered the DNA by CsCl density gradient centrifugation. This CsCl-purified DNA had a buoyant density which was indistinguishable from that of phenol-extracted Mu DNA. It was, however, 10(3) times more infective than phenol-extracted DNA for spheroplasts of exoV endI Escherichia coli. Infectivity was destroyed by proteinase K as well as by pancreatic DNase, indicating that the infective form was a DNA-protein complex. The infective properties of the complex demonstrated that the protein protects. Mu DNA against degradation by exonuclease V and that it serves at least one other function in bacteriophage Mu infection. The infectivity of the CsCl-purified DNA was due to a small class of highly infective molecules which sedimented 1.2. times faster than phenol-extracted Mu DNA on neutral sucrose gradients. This change in sedimentation rate is best explained by the formation of protein-linked circular monomers or linear dimers of Mu DNA. In vitro labeling of the DNA-protein complex, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that the CsCl-purified DNA contained a noncovalently associated 65,000-dalton polypeptide. A 65,000-dalton protein was also found to be a minor component of the bacteriophage Mu particle. No protein was found in phenol-extracted Mu DNA. These results suggest that the 65,000-dalton protein is necessary for successful phage infection and is normally injected into the host cell with the Mu genome. PMID:6211551

  20. Binding of piano-stool Ru(II) complexes to DNA; QM/MM study.

    PubMed

    Futera, Zdeněk; Platts, James A; Burda, Jaroslav V

    2012-10-01

    Ru(II) "piano-stool" complexes belong to group of biologically active metallocomplexes with promising anticancer activity. In this study, we investigate the reaction mechanism of [(η(6)-benzene)Ru(II)(en)(H(2)O)](2+) (en = ethylenediamine) complex binding to DNA by hybrid QM/MM computational techniques. The reaction when the Ru(II) complex is coordinated on N7-guanine from major groove is explored. Two reaction pathways, direct binding to N7 position and two-step mechanism passing through O6 position, are considered. It was found that the reaction is exothermic and the direct binding process is preferred kinetically. In analogy to cisplatin, we also explored the possibility of intrastrand cross-link formation where the Ru(II) complex makes a bridge between two adjacent guanines. Two different pathways were found, leading to a final structure with released benzene ligand. This process is exothermic; however, one pathway is blocked by relatively high initial activation barrier. Geometries, energies, and electronic properties analyzed by atoms in molecules and natural population analysis methods are discussed. PMID:22707416

  1. Stacking of Short DNA Induces the Gyroid Cubic-to-Inverted Hexagonal Phase Transition in Lipid–DNA Complexes

    PubMed Central

    Leal, Cecília; Ewert, Kai K.; Bouxsein, Nathan F.; Shirazi, Rahau S.; Li, Youli; Safinya, Cyrus R.

    2012-01-01

    Lyotropic phases of amphiphiles are a prototypical example of self-assemblies. Their structure is generally determined by amphiphile shape and their phase transitions are primarily governed by composition. In this paper, we demonstrate a new paradigm for membrane shape control where the electrostatic coupling of charged membranes to short DNA (sDNA), with tunable temperature-dependent end-to-end stacking interactions, enables switching between the inverted gyroid cubic structure (QIIG) and the inverted hexagonal phase (HIIC). We investigated the structural shape transitions induced in the QIIG phase upon complexation with a series of sDNAs (5, 11, 24, and 48 bp) with three types of end structure (“sticky” adenine (A)–thymine (T) (dAdT) overhangs, no overhang (blunt), and “nonsticky” dTdT overhangs) using synchrotron small-angle X-ray scattering. Very short 5 bp sDNA with dAdT overhangs and blunt ends induce coexistence of the QIIG and the HIIC phase, with the fraction of QIIG increasing with temperature. Phase coexistence for blunt 5 bp sDNA is observed from 27 °C to about 65 °C, where the HIIC phase disappears and the temperature dependence of the lattice spacing of the QIIG phase indicates that the sDNA duplexes melt into single strands. The only other sDNA for which melting is observed is 5 bp sDNA with dTdT overhangs, which forms the QIIG phase throughout the studied range of temperature (27 °C to 85.2 °C). The longer 11 bp sDNA forms coexisting QIIG and HIIC phases (with the fraction of QIIG again increasing with temperature) only for “nonsticky” dTdT overhangs, while dAdT overhangs and blunt ends exclusively template the HIIC phase. For 24 and 48 bp sDNAs the HIIC phase replaces the QIIG phase at all investigated temperatures, independent of sDNA end structure. Our work demonstrates how the combined effects of sDNA length and end structure (which determine the temperature-dependent stacking length) tune the phase behavior of the complexes

  2. 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

  3. Interactions of Ru(II) polypyridyl complexes with DNA mismatches and abasic sites.

    PubMed

    Nandhini, T; Anju, K R; Manikandamathavan, V M; Vaidyanathan, V G; Nair, B U

    2015-05-21

    Polypyridyl based ruthenium(II) complexes, [Ru(bpy)2(furphen)](PF6)2 (1) and [Ru(bpy)2(imiphen)](PF6)2 (2) {furphen: 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline and imiphen: 2-(1H-imidazol-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline} were synthesized and characterized by ESI-MS, NMR, UV-Visible and fluorescence spectroscopic techniques. The interaction of Ru(II) complexes with calf-thymus DNA (CT DNA) as well as oligonucleotides containing mismatches and abasic sites was studied along with unmodified control DNA. Based on absorption titration studies, binding constants (Kb) for the interaction of complexes 1 and 2 with DNA were found to be 6.7 ± 0.2 × 10(3) and 4.9 ± 0.2 × 10(4) M(-1), respectively. Hydrodynamic studies revealed weak interactions between the two complexes and CT-DNA. Luminescence studies revealed that both the complexes exhibit a five-fold increase in emission upon addition of CT-DNA. The integrated emission intensity of complexes 1 and 2 with CC mismatch oligonucleotides was 1.5 and 1.2 fold higher than that of control GC match oligonucleotides, respectively. Both the complexes did not show any specificity towards abasic or other mismatch sites except for CC mismatch. The results from this study provide an insight into the requirements of ligand shape in recognising DNA mutations such as mismatch and selectivity between DNA mismatches. PMID:25893583

  4. Synthesis, characterization, thermal and DNA-binding properties of new zinc complexes with 2-hydroxyphenones.

    PubMed

    Mrkalić, Emina; Zianna, Ariadni; Psomas, George; Gdaniec, Maria; Czapik, Agnieszka; Coutouli-Argyropoulou, Evdoxia; Lalia-Kantouri, Maria

    2014-05-01

    The neutral mononuclear zinc complexes with 2-hydroxyphenones (ketoH) having the formula [Zn(keto)2(H2O)2] and [Zn(keto)2(enR)], where enR stands for a N,N'-donor heterocyclic ligand such as 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen) or 2,2'-dipyridylamine (dpamH), have been synthesized and characterized by IR, UV and (1)H NMR spectroscopies. The 2-hydroxyphenones are chelated to the metal ion through the phenolate and carbonyl oxygen atoms. The crystal structures of [bis(2-hydroxy-4-methoxy-benzophenone)(2,2'-bipyridine)zinc(II)] dimethanol solvate and [bis(2-hydroxy-benzophenone)(2,2'-bipyridine)zinc(II)] dimethanol solvate have been determined by X-ray crystallography. The thermal stability of the zinc complexes has been investigated by simultaneous TG/DTG-DTA technique. The ability of the complexes to bind to calf-thymus DNA (CT DNA) has been studied by UV-absorption and fluorescence emission spectroscopy as well as viscosity measurements. UV studies of the interaction of the complexes with DNA have shown that they can bind to CT DNA and the corresponding binding constants to DNA have been calculated and evaluated. The complexes most probably bind to CT DNA via intercalation as concluded by studying the viscosity of a DNA solution in the presence of the complexes. Competitive studies with ethidium bromide (EB) have shown that the reported complexes can displace the DNA-bound EB, suggesting strong competition with EB for the intercalation site. PMID:24561277

  5. An ultraviolet melting study of the stability of the DNA double helix in the NaDNA-bipyridyl-(ethylenediamine)platintum(II) complex.

    PubMed

    Szabó, S; Lee, S A

    2008-08-01

    Complexes of NaDNA with the bipyridyl-(ethylenediamine)platintum(II) (abbreviated [(bipy)Pt(en)]2+) molecular ion have been studied in solution via ultraviolet absorption experiments at 260 nm between 50 and 90 degrees C. These measurements, performed as a function of the molar ratio of the [(bipy)Pt(en)]2+ complex to DNA base pairs, show that the stability of the DNA double helix is increased by the formation of the DNA.[(bipy)Pt(en)]2+ complex: at a molar ratio of 0.33, the temperature at which the DNA double helix separates into two single strands is increased by about 15 degrees C.

  6. Stacked Graphene-Al2O3 Nanopore Sensors for Sensitive Detection of DNA and DNA-Protein Complexes

    PubMed Central

    Venkatesan, Bala Murali; Estrada, David; Banerjee, Shouvik; Jin, Xiaozhong; Dorgan, Vincent E.; Bae, Myung-Ho; Aluru, Narayana R.; Pop, Eric; Bashir, Rashid

    2012-01-01

    We report the development of a multilayered graphene-Al2O3 nanopore platform for the sensitive detection of DNA and DNA-protein complexes. Graphene-Al2O3 nanolaminate membranes are formed by sequentially depositing layers of graphene and Al2O3 with nanopores being formed in these membranes using an electron-beam sculpting process. The resulting nanopores are highly robust, exhibit low electrical noise (significantly lower than nanopores in pure graphene), are highly sensitive to electrolyte pH at low KCl concentrations (attributed to the high buffer capacity of Al2O3) and permit the electrical biasing of the embedded graphene electrode, thereby allowing for three terminal nanopore measurements. In proof-of-principle biomolecule sensing experiments, the folded and unfolded transport of single DNA molecules and RecA coated DNA complexes could be discerned with high temporal resolution. The process described here also enables nanopore integration with new graphene based structures, including nanoribbons and nanogaps, for single molecule DNA sequencing and medical diagnostic applications. PMID:22165962

  7. Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

    SciTech Connect

    Cappadocia, Laurent; Maréchal, Alexandre; Parent, Jean-Sébastien; Lepage, Étienne; Sygusch, Jurgen; Brisson, Normand

    2010-09-07

    DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

  8. Mobile DNA Elements: The Seeds of Organic Complexity on Earth.

    PubMed

    Habibi, Laleh; Pedram, Mehrdad; AmirPhirozy, Akbar; Bonyadi, Khadijeh

    2015-10-01

    Mobile DNA or transposable elements (TEs) are genomic sequences capable of moving themselves independently into different parts of the genome. Viral invasion of eukaryotic genomes is assumed to be the main source of TEs. Selfish transposition of these elements could be a serious threat to the host cell, as they can insert themselves into the middle of coding genes and/or induce genomic instability. In response, through millions of years of evolution, cells have come up with various mechanisms such as genomic imprinting, DNA methylation, heterochromatin formation, and RNA interference to deactivate them. Interestingly, these processes have also greatly contributed to important cellular functions involved in cell differentiation, development, and differential gene expression. Propagation of TE copies during the course of evolution have resulted in increasing the genome size and providing proper space and flexibility in shaping the genome by creating new genes and establishing essential cellular structures such as heterochromatin, centromere, and telomeres. Yet, these elements are mostly labeled for playing a role in pathogenesis of human diseases. Here, we attempt to introduce TEs as factors necessary for making us human rather than just selfish sequences or obligatory guests invading our DNA.

  9. Synthesis, Characterization, Molecular Modeling, and DNA Interaction Studies of Copper Complex Containing Food Additive Carmoisine Dye.

    PubMed

    Shahabadi, Nahid; Akbari, Alireza; Jamshidbeigi, Mina; Khodarahmi, Reza

    2016-06-01

    A copper complex of carmoisine dye; [Cu(carmoisine)2(H2O)2]; was synthesized and characterized by using physico-chemical and spectroscopic methods. The binding of this complex with calf thymus (ct) DNA was investigated by circular dichroism, absorption studies, emission spectroscopy, and viscosity measurements. UV-vis results confirmed that the Cu complex interacted with DNA to form a ground-state complex and the observed binding constant (2× 10(4) M(-1)) is more in keeping with the groove bindings with DNA. Furthermore, the viscosity measurement result showed that the addition of complex causes no significant change on DNA viscosity and it indicated that the intercalation mode is ruled out. The thermodynamic parameters are calculated by van't Hoff equation, which demonstrated that hydrogen bonds and van der Waals interactions played major roles in the reaction. The results of circular dichroism (CD) suggested that the complex can change the conformation of DNA from B-like form toward A-like conformation. The cytotoxicity studies of the carmoisine dye and its copper complex indicated that both of them had anticancer effects on HT-29 (colon cancer) cell line and they may be new candidates for treatment of the colon cancer.

  10. Synthesis, Characterization, Molecular Modeling, and DNA Interaction Studies of Copper Complex Containing Food Additive Carmoisine Dye.

    PubMed

    Shahabadi, Nahid; Akbari, Alireza; Jamshidbeigi, Mina; Khodarahmi, Reza

    2016-06-01

    A copper complex of carmoisine dye; [Cu(carmoisine)2(H2O)2]; was synthesized and characterized by using physico-chemical and spectroscopic methods. The binding of this complex with calf thymus (ct) DNA was investigated by circular dichroism, absorption studies, emission spectroscopy, and viscosity measurements. UV-vis results confirmed that the Cu complex interacted with DNA to form a ground-state complex and the observed binding constant (2× 10(4) M(-1)) is more in keeping with the groove bindings with DNA. Furthermore, the viscosity measurement result showed that the addition of complex causes no significant change on DNA viscosity and it indicated that the intercalation mode is ruled out. The thermodynamic parameters are calculated by van't Hoff equation, which demonstrated that hydrogen bonds and van der Waals interactions played major roles in the reaction. The results of circular dichroism (CD) suggested that the complex can change the conformation of DNA from B-like form toward A-like conformation. The cytotoxicity studies of the carmoisine dye and its copper complex indicated that both of them had anticancer effects on HT-29 (colon cancer) cell line and they may be new candidates for treatment of the colon cancer. PMID:27152751

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

    PubMed

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

    2015-01-01

    Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature. PMID:26737172

  12. 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.

  13. Structure of DNMT1-DNA Complex Reveals a Role for Autoinhibition in Maintenance DNA Methylation

    SciTech Connect

    Song, Jikui; Rechkoblit, Olga; Bestor, Timothy H.; Patel, Dinshaw J.

    2011-09-06

    Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.

  14. Structure of DNMT1-DNA Complex Reveals a Role for Autoinhibition in Maintenance DNA Methylation

    SciTech Connect

    J Song; O Rechkoblit; T Bestor; D Patel

    2011-12-31

    Maintenance of genomic methylation patterns is mediated primarily by DNA methyltransferase-1 (DNMT1). We have solved structures of mouse and human DNMT1 composed of CXXC, tandem bromo-adjacent homology (BAH1/2), and methyltransferase domains bound to DNA-containing unmethylated CpG sites. The CXXC specifically binds to unmethylated CpG dinucleotide and positions the CXXC-BAH1 linker between the DNA and the active site of DNMT1, preventing de novo methylation. In addition, a loop projecting from BAH2 interacts with the target recognition domain (TRD) of the methyltransferase, stabilizing the TRD in a retracted position and preventing it from inserting into the DNA major groove. Our studies identify an autoinhibitory mechanism, in which unmethylated CpG dinucleotides are occluded from the active site to ensure that only hemimethylated CpG dinucleotides undergo methylation.

  15. Characterising the atypical 5'-CG DNA sequence specificity of 9-aminoacridine carboxamide Pt complexes.

    PubMed

    Kava, Hieronimus W; Galea, Anne M; Md Jamil, Farhana; Feng, Yue; Murray, Vincent

    2014-08-01

    In this study, the DNA sequence specificity of four DNA-targeted 9-aminoacridine carboxamide Pt complexes was compared with cisplatin, using two specially constructed plasmid templates. One plasmid contained 5'-CG and 5'-GA insert sequences while the other plasmid contained a G-rich transferrin receptor gene promoter insert sequence. The damage profiles of each compound on the different DNA templates were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. With the plasmid that contained 5'-CG and 5'-GA dinucleotides, the four 9-aminoacridine carboxamide Pt complexes produced distinctly different damage profiles as compared with cisplatin. These 9-aminoacridine complexes had greatly increased levels of DNA damage at CG and GA dinucleotides as compared with cisplatin. It was shown that the presence of a CG or GA dinucleotide was sufficient to reveal the altered DNA sequence selectivity of the 9-aminoacridine carboxamide Pt analogues. The DNA sequence specificity of the Pt complexes was also found to be similarly altered utilising the transferrin receptor DNA sequence. PMID:24827388

  16. DNA methylation in complex disease: applications in nursing research, practice, and policy.

    PubMed

    Wright, Michelle L; Ralph, Jody L; Ohm, Joyce E; Anderson, Cindy M

    2013-01-01

    DNA methylation is an epigenomic modification that is essential to normal human development and biological processes. DNA methylation patterns are heritable and dynamic throughout the life span. Environmental exposures can alter DNA methylation patterns, contributing to the development of complex disease. Identification and modulation of environmental factors influencing disease susceptibility through alterations in DNA methylation are amenable to nursing intervention and form the basis for individualized patient care. Here we describe the evidence supporting the translation of DNA methylation analyses as a tool for screening, diagnosis, and treatment of complex disease in nursing research and practice. The ethical, legal, social, and economic considerations of advances in genomics are considered as a model for epigenomic policy. We conclude that contemporary and informed nurse scientists and clinicians are uniquely poised to apply innovations in epigenomic research to clinical populations and develop appropriate policies that guide equitable and ethical use of new strategies to improve patient care. PMID:23849553

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

    PubMed

    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 298K. 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)×10(5)) between complex and protein have been obtained at 298K. 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)×10(6)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.

  18. 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.

  19. 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.

  20. DNA computing, computation complexity and problem of biological evolution rate.

    PubMed

    Melkikh, Alexey V

    2008-12-01

    An analogy between the evolution of organisms and some complex computational problems (cryptosystem cracking, determination of the shortest path in a graph) is considered. It is shown that in the absence of a priori information about possible species of organisms such a problem is complex (is rated in the class NP) and cannot be solved in a polynomial number of steps. This conclusion suggests the need for re-examination of evolution mechanisms. Ideas of a deterministic approach to the evolution are discussed.

  1. Optical tweezers reveal a dynamic mechanical response of cationic peptide-DNA complexes

    NASA Astrophysics Data System (ADS)

    Lee, Amy; Zheng, Tai; Sucayan, Sarah; Chou, Szu-Ting; Tricoli, Lucas; Hustedt, Jason; Kahn, Jason; Mixson, A. James; Seog, Joonil

    2013-03-01

    Nonviral carriers have been developed to deliver nucleic acids by forming nanoscale complexes; however, there has been limited success in achieving high transfection efficiency. Our hypothesis is that a factor affecting gene delivery efficiency is the mechanical response of the condensed complex. To begin to test this hypothesis, we directly measured the mechanical properties of DNA-carrier complexes using optical tweezers. Histidine-lysine (HK) polymer, Asparagine-lysine (NK) polymer and poly-L-lysine were used to form complexes with a single DNA molecule. As carriers were introduced, a sudden decrease in DNA extension occurrs at a force level which is defined as critical force (Fc). Fc is carrier and concentration dependent. Pulling revealed reduction in DNA extension length for HK-DNA complexes. The characteristics of force profiles vary by agent and can be dynamically manipulated by changes in environmental conditions such as ionic strength of the buffer as well as pH. Heparin can remove cationic reagents which are otherwise irreversibly bound to DNA. The implications for optimizing molecular interactions to enhance transfection efficiency will be discussed.

  2. Structures of HIV-1 RT-DNA complexes before and after incorporation of the anti-AIDS drug tenofovir.

    PubMed

    Tuske, Steve; Sarafianos, Stefan G; Clark, Arthur D; Ding, Jianping; Naeger, Lisa K; White, Kirsten L; Miller, Michael D; Gibbs, Craig S; Boyer, Paul L; Clark, Patrick; Wang, Gang; Gaffney, Barbara L; Jones, Roger A; Jerina, Donald M; Hughes, Stephen H; Arnold, Eddy

    2004-05-01

    Tenofovir, also known as PMPA, R-9-(2-(phosphonomethoxypropyl)adenine, is a nucleotide reverse transcriptase (RT) inhibitor. We have determined the crystal structures of two related complexes of HIV-1 RT with template primer and tenofovir: (i) a ternary complex at a resolution of 3.0 A of RT crosslinked to a dideoxy-terminated DNA with tenofovir-diphosphate bound as the incoming substrate; and (ii) a RT-DNA complex at a resolution of 3.1 A with tenofovir at the 3' primer terminus. The tenofovir nucleotide in the tenofovir-terminated structure seems to adopt multiple conformations. Some nucleoside reverse transcriptase inhibitors, including 3TC and AZT, have elements ('handles') that project beyond the corresponding elements on normal dNTPs (the 'substrate envelope'). HIV-1 RT resistance mechanisms to AZT and 3TC take advantage of these handles; tenofovir's structure lacks handles that could protrude through the substrate envelope to cause resistance.

  3. Transient HMGB protein interactions with B-DNA duplexes and complexes

    PubMed Central

    Zimmerman, Jeff; Maher, L. James

    2008-01-01

    HMGB proteins are abundant, non-histone proteins in eukaryotic chromatin. HMGB proteins contain one or two conserved “HMG boxes” and can be sequence specific or nonspecific in their DNA binding. HMGB proteins cause strong DNA bending and bind preferentially to deformed DNAs. We wish to understand how HMGB proteins increase the apparent flexibility of non-distorted B-form DNA. We test the hypothesis that HMGB proteins bind transiently, creating an ensemble of distorted DNAs with rapidly-interconverting conformations. We show that binding of B-form DNA by HMGB proteins is both weak and transient under conditions where DNA cyclization is strongly enhanced. We also detect novel complexes in which HMGB proteins simultaneously bind more than one DNA duplex. PMID:18413230

  4. Biochemical Identification and Characterization of DNA Groups within the Proteus vulgaris Complex

    PubMed Central

    Janda, J. Michael; Abbott, Sharon L.; Khashe, Shideh; Probert, Will

    2001-01-01

    We placed 43 isolates belonging to the Proteus vulgaris complex into proposed DNA groups (genomovars) using five previously recommended tests (tests for esculin hydrolysis, production of acid from salicin, l-rhamnose fermentation, and elaboration of DNase and lipase). On the basis of the results of these five tests, 49% of the isolates fell into DNA groups 5 and 6, 37% fell into DNA group 2, and the remaining 14% fell into DNA groups 3 and 4. Sequencing of the 16S rRNA genes of 11 members of DNA groups 5 and 6 indicated that 10 of these isolates (91%) could be unambiguously assigned to one of these two genomospecies. Overall expression of selected enzymatic and virulence-associated characteristics did not differ significantly among DNA groups. PMID:11283033

  5. Synthesis, characterization, DNA/BSA interactions and anticancer activity of achiral and chiral copper complexes.

    PubMed

    Zhou, Xue-Quan; Sun, Qian; Jiang, Lin; Li, Si-Tong; Gu, Wen; Tian, Jin-Lei; Liu, Xin; Yan, Shi-Ping

    2015-05-28

    Six novel copper(ii) complexes of [CuCl]ClO4 (), [Cu(acac)]PF6 (), [CuCl]2(PF6)2 (), [CuCl]2(PF6)2 (), [Cu(acac)]PF6 () and [Cu(acac)]PF6 (), ( = 1-naphthyl-N,N-[bis(2-pyridyl)methyl]amine, = R/S-1-naphthyl-N,N-[bis(2-pyridyl)methyl]ethanamine, acac = diacetone) were synthesized to serve as artificial nucleases. All complexes were structurally characterized using X-ray crystallography. The crystal structures showed the presence of distorted square-planar CuLCl (, and ) and distorted tetragonal-pyramidal CuL(acac) (, and ) geometry. The interaction of these complexes with calf thymus DNA (CT-DNA) was researched by means of several spectroscopy methods, which indicated that the complexes were bound to CT-DNA by an intercalation binding mode. DNA cleavage experiments revealed that the complexes exhibited remarkable DNA cleavage activities in the presence of H2O2, and single oxygen ((1)O2) or hydroxyl radicals may serve as the major cleavage active species. In particular, the in vitro cytotoxicity of the complexes on four human cancer cell lines (HeLa, MCF-7, Bel-7404 and HepG-2) demonstrated that the six compounds had broad-spectrum anti-cancer activity with low IC50 values. The stronger cytotoxicity and DNA cleavage activity of the chiral enantiomers compared with chiral analogues verified the influence of chirality on the antitumor activity of complexes. Meanwhile, the protein binding ability was revealed by quenching of tryptophan emission with the addition of complexes using BSA as a model protein. The results indicated that the quenching mechanism of BSA by the complexes was a static process.

  6. Photoinduced interactions of supramolecular ruthenium(II) complexes with plasmid DNA: synthesis and spectroscopic, electrochemical, and DNA photocleavage studies.

    PubMed

    Swavey, Shawn; DeBeer, Madeleine; Li, Kaiyu

    2015-04-01

    Two new bridging ligands have been synthesized by combining substituted benzaldehydes with phenanthrolinopyrrole (php), resulting in new polyazine bridging ligands. The ligands have been characterized by (1)H NMR, mass spectroscopy, and elemental analysis. These new ligands display π-π* transitions above 500 nm with modest molar absorptivities. Upon excitation at the ligand-centered charge-transfer transition, weak emission with a maximum wavelength of 612 nm is observed. When coordinated to two ruthenium(II) bis(bipyridyl) groups, the new bimetallic complexes generated give an overall 4+ charge. The electronic transitions of the bimetallic ruthenium(II) complexes display traditional π-π* transitions at 287 nm and metal-to-ligand charge-transfer transitions at 452 nm with molar absorptivities greater than 30000 M(-1) cm(-1). Oxidation of the ruthenium(II) metal centers to ruthenium(III) occurs at potentials above 1.4 V versus the Ag/AgCl reference electrode. Spectroscopic and electrochemical measurements indicate that the ruthenium(II) moieties behave independently. Both complexes are water-soluble and show the ability to photonick plasmid DNA when irradiated with low-energy light above 550 nm. In addition, one of the complexes, [Ru(bpy)2php]2Van(4+), shows the ability to linearize plasmid DNA and gives evidence, by gel electrophoresis, of photoinduced binding to plasmid DNA. PMID:25798576

  7. Structure of an aprataxin-DNA complex with insights into AOA1 neurodegenerative disease

    SciTech Connect

    Tumbale, Percy; Appel, C Denise; Kraehenbuehl, Rolf; Robertson, Patrick D; Williams, Jessica S; Krahn, Joe; Ahel, Ivan; Williams, R Scott

    2012-09-17

    DNA ligases finalize DNA replication and repair through DNA nick-sealing reactions that can abort to generate cytotoxic 5'-adenylation DNA damage. Aprataxin (Aptx) catalyzes direct reversal of 5'-adenylate adducts to protect genome integrity. Here the structure of a Schizosaccharomyces pombe Aptx-DNA-AMP-Zn2+ complex reveals active site and DNA interaction clefts formed by fusing a histidine triad (HIT) nucleotide hydrolase with a DNA minor groove-binding C2HE zinc finger (Znf). An Aptx helical 'wedge' interrogates the base stack for sensing DNA ends or DNA nicks. The HIT-Znf, the wedge and an '[F/Y]PK' pivot motif cooperate to distort terminal DNA base-pairing and direct 5'-adenylate into the active site pocket. Structural and mutational data support a wedge-pivot-cut HIT-Znf catalytic mechanism for 5'-adenylate adduct recognition and removal and suggest that mutations affecting protein folding, the active site pocket and the pivot motif underlie Aptx dysfunction in the neurodegenerative disorder ataxia with oculomotor apraxia 1 (AOA1).

  8. Deictic Relational Complexity and the Development of Deception

    ERIC Educational Resources Information Center

    McHugh, Louise; Barnes-Holmes, Yvonne; Barnes-Holmes, Dermot; Stewart, Ian; Dymond, Simon

    2007-01-01

    An empirical investigation of age-related development of the ability to deceive was conducted from the perspective of Relational Frame Theory, which, unlike the traditional approach, Theory of Mind, has been used to analyze deception in terms of the complexity of the relational responding involved. A derived relational responding-based protocol…

  9. DNA profiling of complex bacterial populations: toxic cyanobacterial blooms.

    PubMed

    Saker, Martin; Moreira, Cristiana; Martins, Joana; Neilan, Brett; Vasconcelos, Vitor Manuel

    2009-11-01

    Cyanobacteria are prokaryotic photosynthetic living organisms that inhabit our planet for over three billion years. With a worldwide distribution, they can be found in all types of environments: fresh, brackish and saltwater as well as terrestrial. Though beneficial in the development of life on earth, they also constitute a serious risk to our ecosystems since they can biologically produce harmful secondary metabolites named cyanotoxins. When studying cyanobacteria and their cyanotoxins, several methodologies have been applied with an increasing relevance to molecular methods. Therefore, the aim of this review is to describe alternative molecular methods that can be used as alternative methods for the identification of cyanobacteria. More traditional chemotaxonomic methods are discussed briefly as are the standard and somewhat dated techniques for assessing genome content for taxonomic classification schemes. The use of DNA amplification technology has been applied to the systematics and phylogeny of many bacterial groups, and the optimisation of methods for rapid identification and classification of cyanobacteria are presented. Together with novel methods developed for these photosynthetic microorganisms, the generated DNA profiles have been utilised to study cyanobacterial bloom population diversity and prediction of strain toxigenicity. Finally, the genotypes found were applied to a variety of phylogenetic analyses; trees were reconstructed and compared to the current morphological system of classification. The ecology and diversity of the cyanobacteria is discussed with respect to the derived molecular phylogenies and systematics.

  10. Synthesis and characterisation of platinum (II) salphen complex and its interaction with calf thymus DNA

    NASA Astrophysics Data System (ADS)

    Sukri, Shahratul Ain Mohd; Heng, Lee Yook; Karim, Nurul Huda Abd

    2014-09-01

    A platinum (II) salphen complex was synthesised by condensation reaction of 2,4-dihydroxylbenzaldehyde and o-phenylenediamine with potassium tetrachloroplatinate to obtain N,N'-Bis-4-(hydroxysalicylidene)-phenylenediamine-platinum (II). The structure of the complex was confirmed by 1H and 13C NMR spectroscopy, FTIR spectroscopy, CHN elemental analyses and ESI-MS spectrometry. The platinum (II) salphen complex with four donor atoms N2O2 from its salphen ligand coordinated to platinum (II) metal centre were determined. The binding mode and interaction of this complex with calf thymus DNA was determined by UV/Vis DNA titration and emission titration. The intercalation between the DNA bases by π-π stacking due to its square planar geometry and aromatic rings structures was proposed.

  11. LL37:DNA complexes provide antimicrobial activity against intracellular bacteria in human macrophages.

    PubMed

    Stephan, Alexander; Batinica, Marina; Steiger, Julia; Hartmann, Pia; Zaucke, Frank; Bloch, Wilhelm; Fabri, Mario

    2016-08-01

    As part of the innate host response neutrophils release neutrophil extracellular traps (NETs), protein:DNA complexes that contain a number of antimicrobial peptides (AMPs), such as cathelicidin. Human cathelicidin in its active form, LL37, has potent antimicrobial activity against bacteria. However, whether LL37 derived from NETs contributes to antimicrobial activity against intracellular pathogens remains unclear. Here, we report that NETs induced by mycobacteria contain cathelicidin. Human macrophages internalized NET-bound cathelicidin, which is transported to lysosomal compartments. Furthermore, using a model of in vitro-generated LL37:DNA complexes we found that LL37 derived from such complexes attacks mycobacteria in macrophage phagolysosomes resulting in antimicrobial activity. Taken together, our results suggest a mechanism by which LL37 in complex with DNA contributes to host defence against intracellular bacteria in human macrophages.

  12. Synthesis and characterisation of platinum (II) salphen complex and its interaction with calf thymus DNA

    SciTech Connect

    Sukri, Shahratul Ain Mohd; Heng, Lee Yook; Karim, Nurul Huda Abd

    2014-09-03

    A platinum (II) salphen complex was synthesised by condensation reaction of 2,4-dihydroxylbenzaldehyde and o-phenylenediamine with potassium tetrachloroplatinate to obtain N,N′-Bis-4-(hydroxysalicylidene)-phenylenediamine-platinum (II). The structure of the complex was confirmed by {sup 1}H and {sup 13}C NMR spectroscopy, FTIR spectroscopy, CHN elemental analyses and ESI-MS spectrometry. The platinum (II) salphen complex with four donor atoms N{sub 2}O{sub 2} from its salphen ligand coordinated to platinum (II) metal centre were determined. The binding mode and interaction of this complex with calf thymus DNA was determined by UV/Vis DNA titration and emission titration. The intercalation between the DNA bases by π-π stacking due to its square planar geometry and aromatic rings structures was proposed.

  13. Intracellular disassembly and localization of a new P123-PEI-R13/DNA complex.

    PubMed

    Zhu, Manman; Liu, Kehai; Zhu, Qing; Chen, Shunsheng; Lv, Hui; Zhao, Wenfang; Mao, Yuan; Hu, Jing

    2014-01-01

    The appropriate location and release of target gene is necessary for gene therapy. In our previous paper, a gene vector named P123-PEI-R13 has been successfully synthesized, and the physical characteristics and cellular trafficking of nanoparticle P123-PEI-R13/DNA has been explored explicitly, but little was known about its disassembly within cells. In order to investigate its intracellular disassembly, P123-PEI-R13/DNA complex was exposed to the different competitors (RNA, DNA, proteins) or different conditions of pH and osmolarity, DNA release was determined by gel electrophoresis. Meanwhile, confocal laser technology was used to locate the complex in cells. The results revealed that DNA, RNA and osmolarity could affect the stability of the complex obviously, especially RNA which exist in nucleus. In addition, the speed of DNA release decreased as the weight ratio of polymer increased. Images got by a confocal fluorescence microscope confirmed that after cell uptake, P123-PEI-R13 could translocate DNA into nucleus.

  14. Human telomeric G-quadruplex DNA interactions of N-phenanthroline glycosylamine copper(II) complexes.

    PubMed

    Duskova, Katerina; Sierra, Sara; Arias-Pérez, María-Selma; Gude, Lourdes

    2016-01-01

    We report in this article the interactions of five N-(1,10-phenanthrolin-5-yl)-β-glycopyranosylamine copper(II) complexes with G-quadruplex DNA. Specifically, the interactions of these compounds with a human telomeric oligonucleotide have been assessed by fluorescence-based assays (FRET melting and G4-FID), circular dichroism and competitive equilibrium dialysis experiments. The metal complexes bind and stabilize G-quadruplex DNA structures with apparent association constants in the order of 10(4)-10(5)M(-1) and the affinity observed is dependent on the ionic conditions utilized and the specific nature of the carbohydrate moiety tethered to the 1,10-phenanthroline system. The compounds showed only a slight preference to bind G-quadruplex DNA over duplex DNA when the quadruplex DNA was folded in sodium ionic conditions. However, the binding affinity and selectivity, although modest, were notably increased when the G-quadruplex DNA was folded in the presence of potassium metal ions. Moreover, the study points towards a significant contribution of groove and/or loop binding in the recognition mode of quadruplex structures by these non-classical quadruplex ligands. The results reported herein highlight the potential and the versatility of carbohydrate bis-phenanthroline metal-complex conjugates to recognize G-quadruplex DNA structures. PMID:26678174

  15. Human telomeric G-quadruplex DNA interactions of N-phenanthroline glycosylamine copper(II) complexes.

    PubMed

    Duskova, Katerina; Sierra, Sara; Arias-Pérez, María-Selma; Gude, Lourdes

    2016-01-01

    We report in this article the interactions of five N-(1,10-phenanthrolin-5-yl)-β-glycopyranosylamine copper(II) complexes with G-quadruplex DNA. Specifically, the interactions of these compounds with a human telomeric oligonucleotide have been assessed by fluorescence-based assays (FRET melting and G4-FID), circular dichroism and competitive equilibrium dialysis experiments. The metal complexes bind and stabilize G-quadruplex DNA structures with apparent association constants in the order of 10(4)-10(5)M(-1) and the affinity observed is dependent on the ionic conditions utilized and the specific nature of the carbohydrate moiety tethered to the 1,10-phenanthroline system. The compounds showed only a slight preference to bind G-quadruplex DNA over duplex DNA when the quadruplex DNA was folded in sodium ionic conditions. However, the binding affinity and selectivity, although modest, were notably increased when the G-quadruplex DNA was folded in the presence of potassium metal ions. Moreover, the study points towards a significant contribution of groove and/or loop binding in the recognition mode of quadruplex structures by these non-classical quadruplex ligands. The results reported herein highlight the potential and the versatility of carbohydrate bis-phenanthroline metal-complex conjugates to recognize G-quadruplex DNA structures.

  16. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    SciTech Connect

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  17. Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

    SciTech Connect

    Pröpper, Kevin; Meindl, Kathrin; Sammito, Massimo; Dittrich, Birger; Sheldrick, George M.; Pohl, Ehmke; Usón, Isabel

    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 the 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.

  18. The FANCD2-FANCI complex is recruited to DNA interstrand crosslinks before monoubiquitination of FANCD2.

    PubMed

    Liang, Chih-Chao; Li, Zhuolun; Lopez-Martinez, David; Nicholson, William V; Vénien-Bryan, Catherine; Cohn, Martin A

    2016-01-01

    The Fanconi anaemia (FA) pathway is important for the repair of DNA interstrand crosslinks (ICL). The FANCD2-FANCI complex is central to the pathway, and localizes to ICLs dependent on its monoubiquitination. It has remained elusive whether the complex is recruited before or after the critical monoubiquitination. Here, we report the first structural insight into the human FANCD2-FANCI complex by obtaining the cryo-EM structure. The complex contains an inner cavity, large enough to accommodate a double-stranded DNA helix, as well as a protruding Tower domain. Disease-causing mutations in the Tower domain are observed in several FA patients. Our work reveals that recruitment of the complex to a stalled replication fork serves as the trigger for the activating monoubiquitination event. Taken together, our results uncover the mechanism of how the FANCD2-FANCI complex activates the FA pathway, and explains the underlying molecular defect in FA patients with mutations in the Tower domain. PMID:27405460

  19. Some relations between two stages DNA splicing languages

    NASA Astrophysics Data System (ADS)

    Mudaber, Mohammad Hassan; Yusof, Yuhani; Mohamad, Mohd Sham

    2014-06-01

    A new symbolization of Yusof-Goode (Y-G) rule, which is associated with Y-G splicing system, was introduced by Yusof in 2012 under the framework of formal language theory. The purpose of this investigation is to present the biological process of DNA splicing in a translucent way. In this study, two stages splicing languages are introduced based on Y-G approach and some relations between stage one and stage two splicing languages are presented, given as theorems. Additionally, the existing relations between two stages splicing languages based on crossings and contexts of restriction enzymes factors with respect to two initial strings (having two cutting sites) and two rules are presented as subset.

  20. 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.

  1. Redox-active and DNA-binding coordination complexes of clotrimazole.

    PubMed

    Betanzos-Lara, Soledad; Chmel, Nikola P; Zimmerman, Matthew T; Barrón-Sosa, Lidia R; Garino, Claudio; Salassa, Luca; Rodger, Alison; Brumaghim, Julia L; Gracia-Mora, Isabel; Barba-Behrens, Norah

    2015-02-28

    DNA interactions of anticancer mononuclear Cu(2+), Co(2+), Zn(2+), and Ni(2+) complexes with the biologically active ligand clotrimazole (clotri) are reported. To fully characterize DNA binding modes for these complexes of the formulae [M(clotri)2Cl2]·nH2O (1-4), [M(clotri)2Br2]·nH2O (5,6), [M(clotri)3NO3]NO3·nH2O (9), and [M(clotri)3(NO3)2] (10), circular dichroism (CD) and linear dichroism (LD) spectroscopy, UV melting experiments, atomic force microscopy (AFM) and ethidium bromide (EtBr) displacement methods were used. Results indicate mixed electrostatic interactions, possibly through groove binding, that result in accretion and coiling of DNA. Electrochemical studies indicate that the Cu(2+) complex 9 readily reduces to the reactive-oxygen-species-generating Cu(+), which oxidatively damages DNA. There is a subtle correlation between log P values, calculated electrostatic potentials, and cytotoxicity of the complexes. The extent of cell-nucleus DNA-metal adduct formation in the HeLa cervix-uterine carcinoma cell line does not necessarily correlate with cytotoxicity, indicating that the nature of DNA lesions may be crucial to activity. PMID:25561277

  2. Electrical properties of nanofibers and structural characterization of DNA-Au(III) complexes.

    PubMed

    Kwon, Young-Wan; Lee, Chang Hoon; Jin, Jung-Il; Hwang, Jong Seung; Hwang, Sung Woo

    2014-05-23

    In order to realize deoxyribonucleic acid (DNA)-based molecular electronics, chemical modifications of DNA are needed to improve electrical conductivity. We developed a novel method utilizing the incorporation of Au(III) ions into DNA bases to alter their electronic properties. When Au(III) ions were incorporated proportionally into DNA bases, conductance increased up to an Au(III) content of 0.42 Au(III) ion/nucleotide. Surprisingly, electron paramagnetic resonance signals of Au(II) ions were detected at g ∼1.98, and the calculated spin number of Au(II) ions ranged from ∼10(13) to ∼10(15). The structural deformation of the DNA helix occurred when complexed with Au(III); simultaneously, the conductance of DNA-Au(III) complexes decreased when the content of Au(III) was higher than 0.42 atom/nucleotide. This observation implies that the maintenance of helical structure in the Au(III) doped state of DNA molecules is very important to the enhancement of the carrier mobility of DNA. PMID:24786616

  3. Salt-Dependent DNA-DNA Spacings in Intact Bacteriophage lambda Reflect Relative Importance of DNA Self-Repulsion and Bending Energies

    SciTech Connect

    X Qiu; D Rau; V Parsegian; L Fang; C Knobler; W Gelbart

    2011-12-31

    Using solution synchrotron x-ray scattering, we measure the variation of DNA-DNA d spacings in bacteriophage {lambda} with mono-, di-, and polyvalent salt concentrations, for wild-type [48.5 x 10{sup 3} base pairs (bp)] and short-genome-mutant (37.8 kbp) strains. From the decrease in d spacings with increasing salt, we deduce the relative contributions of DNA self-repulsion and bending to the energetics of packaged phage genomes. We quantify the DNA-DNA interaction energies within the intact phage by combining the measured d spacings in the capsid with measurements of osmotic pressure in DNA assemblies under the same salt conditions in bulk solution. In the commonly used Tris-Mg buffer, the DNA-DNA interaction energies inside the phage capsids are shown to be about 1 kT/bp, an order of magnitude larger than the bending energies.

  4. DNA/polyethyleneimine/hyaluronic acid small complex particles and tumor suppression in mice.

    PubMed

    Ito, Tomoko; Yoshihara, Chieko; Hamada, Katsuyuki; Koyama, Yoshiyuki

    2010-04-01

    The highest barriers for non-viral vectors to an efficient in vivo gene transfection would be (1) non-specific interaction with biological molecules, and (2) large size of the DNA complex particles. Protective coating of the DNA/polyethyleneimine (PEI) complexes by hyaluronic acid (HA) effectively diminished the adverse interactions with biological molecules. Here we found HA also protected the DNA/PEI complexes against aggregation and inactivation through lyophilization-and-rehydration procedures. It allows us to prepare the concentrated very small DNA complex particles (<70 nm) suspension by preparing the complexes at highly diluted conditions, followed by lyophilized-and-rehydrated to a small volume. In vivo gene expression efficiency of the small complex was examined with mice subcutaneously inoculated with B16 melanoma cells. These formulations showed high reporter-gene expression level in tumor after intravenous injection into tumor-bearing mice. Small complex was then made of the plasmid encoding GM-CSF gene, and injected into the mice bearing subcutaneous solid B16 tumor. After intravenous injection, it induced apparent tumor growth suppression in 50% of the mice. Notably, significant therapeutic effect was detected in the mice that received intratumoral injection, and 75% of the mice were completely cured with disappearance of tumor. PMID:20047759

  5. Stable RNA-DNA-RNA polymerase complexes can accompany formation of a single phosphodiester bond.

    PubMed

    Sylvester, J E; Cashel, M

    1980-03-18

    Incubation of RNA polymerase with poly[d(A-T)n] template results in a binary enzyme-DNA complex. Further addition of the dinucleotide UpA and [alpha-32P]UTP results in catalytic formation of the labeled trinucleotide UpApU until substrate exhaustion. In contrast, incubation of binary enzyme-DNA complexes with ApU and [alpha-32P]ATP results in labeled ApUpA formation to an extent that is stoichiometric with the amount of enzyme present despite an excess of substrates. The occurrence of ApUpA in a stable DNA-enzyme-RNA ternary complex is shown by gel exclusion chromatography, Millipore filtration, and the ability of ternary complexes to support subsequent RNA chain elongation. Radioactivity is not bound to Millipore filters when purified, labeled ApUpA is added to enzyme-DNA binary complexes. Hence, phosphodiester bond formation is required for stable ternary complex formation. The absence of the delta subunit of RNA polymerase or the addition of rifampicin to the reaction before ribonucleotide substrates results in catalytic ApUpA formation instead of stable ternary complexes.

  6. The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage

    PubMed Central

    De Zio, D; Bordi, M; Tino, E; Lanzuolo, C; Ferraro, E; Mora, E; Ciccosanti, F; Fimia, G M; Orlando, V; Cecconi, F

    2011-01-01

    Apaf1 is a key regulator of the mitochondrial intrinsic pathway of apoptosis, as it activates executioner caspases by forming the apoptotic machinery apoptosome. Its genetic regulation and its post-translational modification are crucial under the various conditions where apoptosis occurs. Here we describe Ku70/86, a mediator of non-homologous end-joining pathway of DNA repair, as a novel regulator of Apaf1 transcription. Through analysing different Apaf1 promoter mutants, we identified an element repressing the Apaf1 promoter. We demonstrated that Ku70/86 is a nuclear factor able to bind this repressing element and downregulating Apaf1 transcription. We also found that Ku70/86 interaction with Apaf1 promoter is dynamically modulated upon DNA damage. The effect of this binding is a downregulation of Apaf1 expression immediately following the damage to DNA; conversely, we observed Apaf1 upregulation and apoptosis activation when Ku70/86 unleashes the Apaf1-repressing element. Therefore, besides regulating DNA repair, our results suggest that Ku70/86 binds to the Apaf1 promoter and represses its activity. This may help to inhibit the apoptosome pathway of cell death and contribute to regulate cell survival. PMID:20966962

  7. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

    PubMed Central

    Weitzman, M D; Kyöstiö, S R; Kotin, R M; Owens, R A

    1994-01-01

    AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration. Images PMID:8016070

  8. The elimination of DNA from the Cry toxin-DNA complex is a necessary step in the mode of action of the Cry8 toxin.

    PubMed

    Ai, Bingjie; Li, Jie; Feng, Dongmei; Li, Feng; Guo, Shuyuan

    2013-01-01

    Several crystal (Cry) proteins are known to occur as DNA-protein complexes. However, the role of the DNA associated with the activated toxin in the mechanism of action of the Cry toxin has long been ignored. Here, we focused on the DNA-activated Cry toxin complex. Both forms of the Cry8Ca2 and Cry8Ea1 toxins, i.e., with or without bound DNA, were separately obtained. Size-exclusion chromatography analysis indicated that the Cry8Ca2 toxin-DNA complex has a tight or compact structure. The Cry8Ca2 toxin-DNA complex is more likely to move toward the air/water interface and is more hydrophobic than the toxin without DNA. Competitive binding assays indicated that the Cry8Ca2 and Cry8Ea1 toxins without DNA specifically bind to the midgut of Anomala corpulenta and Holotrichia parallela larvae, respectively. In contrast, the association of DNA with each toxin might result in the nonspecific recognition of the Cry toxin and its target receptor in the insect midgut. The association of the DNA fragment with the Cry8 toxin was shown to protect the Cry protein from digestion by proteases. Based on our results, we propose an additional step in the mechanism of action of the Cry8 toxin and elucidate the function of the associated DNA as well as the importance of the removal of this DNA for the insecticidal activity of the toxin. PMID:24324685

  9. The elimination of DNA from the Cry toxin-DNA complex is a necessary step in the mode of action of the Cry8 toxin.

    PubMed

    Ai, Bingjie; Li, Jie; Feng, Dongmei; Li, Feng; Guo, Shuyuan

    2013-01-01

    Several crystal (Cry) proteins are known to occur as DNA-protein complexes. However, the role of the DNA associated with the activated toxin in the mechanism of action of the Cry toxin has long been ignored. Here, we focused on the DNA-activated Cry toxin complex. Both forms of the Cry8Ca2 and Cry8Ea1 toxins, i.e., with or without bound DNA, were separately obtained. Size-exclusion chromatography analysis indicated that the Cry8Ca2 toxin-DNA complex has a tight or compact structure. The Cry8Ca2 toxin-DNA complex is more likely to move toward the air/water interface and is more hydrophobic than the toxin without DNA. Competitive binding assays indicated that the Cry8Ca2 and Cry8Ea1 toxins without DNA specifically bind to the midgut of Anomala corpulenta and Holotrichia parallela larvae, respectively. In contrast, the association of DNA with each toxin might result in the nonspecific recognition of the Cry toxin and its target receptor in the insect midgut. The association of the DNA fragment with the Cry8 toxin was shown to protect the Cry protein from digestion by proteases. Based on our results, we propose an additional step in the mechanism of action of the Cry8 toxin and elucidate the function of the associated DNA as well as the importance of the removal of this DNA for the insecticidal activity of the toxin.

  10. New Dendritic Lipids for Improved Gene Delivery by Cationic Liposome-DNA Complexes

    NASA Astrophysics Data System (ADS)

    Ewert, Kai

    2005-03-01

    Cationic Liposome-DNA (CL-DNA) complexes are widely used in non-viral gene delivery, including clinical trials, but their efficiency still requires optimization. Membrane charge density is a universal parameter for transfection with lamellar CL-DNA complexes (Lin AJ et al., Biophys. J. 2003; 84: 3307; Ahmad A et al., J. Gene Med., accepted). Newly synthesized lipids with dendritic headgroups, based on an ornithine scaffold, have headgroup charges of +4e to +16e. These lipids form lamellar complexes if the headgroup charge is small or the fraction of dendritic lipid in the membrane (in mixtures with DOPC) is low. Higher contents of highly charged lipids exhibit a novel phase of CL-DNA complexes, whose structure was determined by synchrotron x-ray diffraction. Cylindrical micelles of lipid are arranged on a hexagonal lattice, with DNA rods placed around them in the interstices. Complexes with this structure are highly transfecting, preventing the previously observed drop in transfection efficiency at very high membrane charge densities. Funded by NIH GM-59288.

  11. A unique binding cavity for divalent cations in the DNA-metal-chromomycin A3 complex.

    PubMed

    Itzhaki, L; Weinberger, S; Livnah, N; Berman, E

    1990-02-15

    Binding of chromomycin A3 (CRA) to calf thymus DNA was investigated in the presence of divalent cations using visible absorption and 1H-nmr spectroscopies. An apparent equilibrium binding constant (approximately 10(11) M-1) was obtained from metal competition experiments using EDTA to remove the metal cation from the DNA-M-CRA (M: metal) complex. The large binding constant of the drug to DNA enabled us to obtain essentially complete complexation of CRA to the short homogeneous d(ATGCAT)2 duplex using stoichiometric amounts of the metal cation. Large induced chemical shifts were observed in the 1H-nmr spectrum of the above complex using the paramagnetic Co2+ cation, indicating that the metal occupies a unique binding site. Since no induced 1H-nmr chemical shifts were observed for the drug-Co2+ mixture, it was concluded that no metal-drug complex is formed. In addition, it was found that bound CRA is negatively charged at physiological pH and binding to the DNA could be affected only by using metal cations whose ionic radius size (less than 0.85 A) and charge (2+) were simultaneously satisfied. Stringent metal cation selectivity for the DNA-M-CRA complex may be intimately connected with the antitumor selectivity of CRA, since different types of cells generally possess widely differing molar concentrations of metal cations.

  12. Replitase: a complex integrating dNTP synthesis and DNA replication.

    PubMed

    Reddy, G P; Fager, R S

    1993-01-01

    Replitase is a multienzyme complex of mammalian cells that produces deoxynucleoside triphosphates and delivers them to the DNA polymerase activity, which also resides in the complex. Structural interactions within this complex form the basis of internal controls to keep these key biosynthetic processes efficient and in balance. The active complex is formed in the nuclear region in only the S phase of the cell cycle, when the cell's DNA is being replicated. Replitase is a member of the growing family of structured, multienzyme, biosynthetic complexes for which very similar complexes are found in prokaryotes and eukaryotes. Logically, the most basic of all biosynthetic pathways should show the efficiency and precise controls that even lesser pathways possess and, in fact, this seems to be so. In this article, we have outlined a broad range of evidence supporting the existence of the replitase complex in mammalian cells, a complex for dNTP synthesis and polymerase that exists only in the S phase and only in the nuclear region. This is consistent with localization studies in intact cells and after various forms of cell fractionation and, particularly, with experiments of incorporation of precursors into DNA in isolated complexes and S phase permeabilized cells. A most forceful argument for replitase is the existence of three striking phenomena--channeling, compartmentation, and cross-inhibition. These are very difficult, perhaps impossible, to explain without replitase; with replitase, their explanation is beautifully simple.

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  14. Homodinuclear lanthanide complexes of phenylthiopropionic acid: synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity.

    PubMed

    Shiju, C; Arish, D; Kumaresan, S

    2013-03-15

    Lanthanide complexes of La(III), Pr(III), Nd(III), Sm(III), and Ho(III) with phenylthiopropionic acid were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the lanthanide complexes are homodinuclear in nature. The two lanthanide ions are bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles are consistent with the proposed formulations. Powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H(2)O(2). The result shows that the Pr(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the La(III) and Nd(III) complexes are more active than the corresponding Pr(III), Sm(III), Ho(III) complexes, and the free ligand on both the cancer cells.

  15. Homodinuclear lanthanide complexes of phenylthiopropionic acid: Synthesis, characterization, cytotoxicity, DNA cleavage, and antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Shiju, C.; Arish, D.; Kumaresan, S.

    2013-03-01

    Lanthanide complexes of La(III), Pr(III), Nd(III), Sm(III), and Ho(III) with phenylthiopropionic acid were synthesized and characterized by elemental analysis, mass, IR, electronic spectra, molar conductance, TGA, and powder XRD. The results show that the lanthanide complexes are homodinuclear in nature. The two lanthanide ions are bridged by eight oxygen atoms from four carboxylate groups. Thermal decomposition profiles are consistent with the proposed formulations. Powder XRD studies show that all the complexes are amorphous in nature. Antimicrobial studies indicate that these complexes exhibit more activity than the ligand itself. The DNA cleavage activity of the ligand and its complexes were assayed on Escherichia coli DNA using gel electrophoresis in the presence of H2O2. The result shows that the Pr(III) and Nd(III) complexes have completely cleaved the DNA. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa) and colon cancer cells (HCT116) and it was found that the La(III) and Nd(III) complexes are more active than the corresponding Pr(III), Sm(III), Ho(III) complexes, and the free ligand on both the cancer cells.

  16. The DNA-binding and bioactivity of rare earth metal complexes.

    PubMed

    Yang, Li; Wang, Bochu; Tan, Jun; Zhu, Liancai

    2013-08-01

    Recently more and more attention is paid to the rare earth metal complexes, because the properties of the rare earth metals are similar to those of the transition metals such as the similar atomic and the ionic radius. A large number of rare metal complexes were synthesized, and their bioactivities were also studied. This review highlights recent researches on the interaction of some rare earth metal complexes with DNA, analyzes how the configuration of the complexes influences the binding affinity, and focuses on the pharmacological activities of the complexes, such as anticancer, antibacterial, antioxidant, anti-inflammatory and anti-virus.

  17. Synthesis, DNA binding, photo-induced DNA cleavage, cytotoxicity studies of a family of heavy rare earth complexes.

    PubMed

    Chen, Gong-Jun; Wang, Zhi-Gang; Qiao, Xin; Xu, Jing-Yuan; Tian, Jin-Lei; Yan, Shi-Ping

    2013-10-01

    As a continuing investigation of our previous studies about the influence of the different rare earth metal ions on the bioactivity, a family of heavy rare earth metal complexes, [RE(acac)3(dpq)] (RE=Tb (1), Dy (2), Ho (3), Er (4), Tm (5), Yb (6), Lu (7)) and [RE(acac)3(dppz)]·CH3OH (RE=Tb (8), Dy (9), Ho (10), Er (11), Tm (12), Yb (13), Lu (14) viz. acetylacetonate (acac), dipyrido[3,2-d:20,30-f]quinoxaline (dpq), dipyrido[3,2-a:20,30-c] phenazine (dppz)), has been synthesized and their biological activities were also investigated. On the irradiation with UV-A light of 365nm or ambient light, all complexes exhibit efficient DNA cleavage activity via the mechanistic pathway involving the formation of singlet oxygen and hydroxyl radical as the reactive species. In addition, the in vitro cytotoxicity of these complexes on HeLa cells has been examined by MTT assay, which indicate that these compounds have the potential to act as effective anticancer drugs. The results of the above biological experiments also reveal that the choice of different rare earth metal ions has little influence on the DNA binding, DNA cleavage and cytotoxicity.

  18. DNA Binding Mode Transitions of Escherichia coli HUαβ: Evidence for Formation of a Bent DNA – Protein Complex on Intact, Linear Duplex DNA

    PubMed Central

    Koh, Junseock; Saecker, Ruth M.; Record, M. Thomas

    2008-01-01

    Escherichia coli HUαβ, a major nucleoid associated protein (NAP), organizes the DNA chromosome and facilitates numerous DNA transactions. Using isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) and a series of DNA lengths (8, 15, 34, 38 and 160 base pairs) we establish that HUαβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA mole ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 base pairs), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 (± 0.4) × 106 M−1, and a binding enthalpy of +7.7 (± 0.6) kcal/mol at 15 °C and 0.15 M Na+. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω ~ 20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 (± 0.2) × 105 M−1 for the 10 bp mode, 3.5 (± 1.4) × 104 M−1 for the 6 bp mode) and binding enthalpies (4.2 (± 0.3) kcal/mol for the 10 bp mode, −1.6 (±0.3) kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. FRET data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement, AFM), fluorescence, and electrophoretic gel mobility shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA. PMID:18657548

  19. 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.

  20. Study on the interaction between the inclusion complex of hematoxylin with β-cyclodextrin and DNA.

    PubMed

    Xu, Dongling; Wang, Xingming; Fei, Dan; Ding, Lisheng

    2010-11-01

    Ultraviolet-visible (UV-vis) spectra, fluorescence spectra, electrochemistry, and the thermodynamic method were used to discuss the interaction mode between the inclusion complex of hematoxylin with β-cyclodextrin and herring sperm DNA. On the condition of physiological pH, the result showed that hematoxylin and β-cyclodextrin formed an inclusion complex with binding ratio n(hematoxylin):n(β-cyclodextrin) = 1:1. The interaction mode between β-cyclodextrin-hematoxylin and DNA was a mixed binding, which contained intercalation and electrostatic mode. The binding ratio between β-cyclodextrin-hematoxylin and DNA was n(β-cyclodextrin -hematoxylin):n(DNA) = 2:1, binding constant was K(⊖)(298.15K) = 5.29 × 10⁴ L·mol⁻¹, and entropy worked as driven force in this action.

  1. Crystal Structure of the Lactose Operon Repressor and Its Complexes with DNA and Inducer

    NASA Astrophysics Data System (ADS)

    Lewis, Mitchell; Chang, Geoffrey; Horton, Nancy C.; Kercher, Michele A.; Pace, Helen C.; Schumacher, Maria A.; Brennan, Richard G.; Lu, Ponzy

    1996-03-01

    The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-β-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

  2. Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction

    PubMed Central

    Laxmikanthan, Gurunathan; Xu, Chen; Brilot, Axel F; Warren, David; Steele, Lindsay; Seah, Nicole; Tong, Wenjun; Grigorieff, Nikolaus; Landy, Arthur; Van Duyne, Gregory D

    2016-01-01

    The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation. DOI: http://dx.doi.org/10.7554/eLife.14313.001 PMID:27223329

  3. Counterion effects on nano-confined metal-drug-DNA complexes.

    PubMed

    Biswas, Nupur; Chakraborty, Sreeja; Datta, Alokmay; Sarkar, Munna; Mukhopadhyay, Mrinmay K; Bera, Mrinal K; Seto, Hideki

    2016-01-01

    We have explored morphology of DNA molecules bound with Cu complexes of piroxicam (a non-steroidal anti-inflammatory drug) molecules under one-dimensional confinement of thin films and have studied the effect of counterions present in a buffer. X-ray reflectivity at and away from the Cu K absorption edge and atomic force microscopy studies reveal that confinement segregates the drug molecules preferentially in a top layer of the DNA film, and counterions enhance this segregation. PMID:26925353

  4. DNA content, kinetic complexity, and the ploidy question in Candida albicans.

    PubMed Central

    Riggsby, W S; Torres-Bauza, L J; Wills, J W; Townes, T M

    1982-01-01

    Candida albicans is a dimorphic fungus that is pathogenic for humans. No sexual cycle has been reported for this fungus, and earlier reports have differed on whether typical strains of C. albicans are haploid or diploid. Previous estimates of the DNA content of C. albicans varied by one order of magnitude. We used three independent methods to measure the kinetic complexity of the single-copy DNA from a typical strain of C. albicans (strain H317) to determine the DNA content per haploid genote; we obtained values of 15 and 20 fg per cell by using S1 nuclease and hydroxyapatite assays, respectively. Optical assays for DNA reassociation kinetics, although not definitive in themselves, yielded values in this range. Chemical measurements of the DNA content of several typical strains, including strain H317, yielded values clustered about a mean of 37 fg per cell. We concluded that these strains are diploid. PMID:6765567

  5. Conformation transformation determined by different self-assembled phases in a DNA complex with cationic polyhedral oligomeric silsesquioxane lipid.

    PubMed

    Cui, Li; Chen, Daoyong; Zhu, Lei

    2008-05-01

    In this work, a novel cube-shaped cationic lipid based on the imidazolium salt of polyhedral oligomeric silsesquioxane (POSS) was complexed with double-stranded DNA. Because of the negative spontaneous curvature of the cationic POSS imidazolium lipid, an inverted hexagonal phase resulted above the melting point of POSS crystals. Depending on the competition between the crystallization of POSS molecules and the negative spontaneous curvature of cationic POSS imidazolium lipids, different self-assembled phase morphologies were obtained. A lamellar phase was obtained when the POSS crystallization was relatively slow. When the POSS crystallization was fast, an inverted hexagonal phase was obtained with POSS lamellar crystals grown in the interstitials of DNA cylinders. On the basis of a circular dichroism study, double-stranded DNA adopted the B-form helical conformation in the inverted hexagonal phase, whereas the helical conformation was largely destroyed in the lamellar phase.

  6. The ``complex trick'' in five-dimensional relativity

    NASA Astrophysics Data System (ADS)

    Lessner, G.

    2008-10-01

    The generation method developed by Newman et al. which creates from spherically symmetric solutions of the field equations by a complex coordinate transformation axisymmetric solutions is extended to five-dimensional relativity.

  7. Liquid-crystalline ordering of antimicrobial peptide-DNA complexes controls TLR9 activation

    NASA Astrophysics Data System (ADS)

    Schmidt, Nathan W.; Jin, Fan; Lande, Roberto; Curk, Tine; Xian, Wujing; Lee, Calvin; Frasca, Loredana; Frenkel, Daan; Dobnikar, Jure; Gilliet, Michel; Wong, Gerard C. L.

    2015-07-01

    Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs , , , , ). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically.

  8. Preparation of plasmid DNA in transfection complexes for fluorescence and electron spectroscopic imaging.

    PubMed

    Malecki, M

    1996-01-01

    The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured glioma cells. Preparation of the transfected DNA for imaging was pursued by two methods. In the first method tetramethylrhodamine, nanogold, and ferritin were linked through streptavidin to the biotinylated plasmid DNA. Trafficking of the fluorescent derivatives was studied in living cells with fluorescence microscopy. Then, selected cells were rapidly cryo-immobilized. Ultra-structural distribution of the transfected DNA was imaged with energy filtering transmission electron microscopy. In the second method, the unmodified transfected DNA was detected in cryo-immobilized cells by in situ polymerase chain reaction and in situ hybridization. For laser scanning fluorescence microscopy probes were labeled with tetramethylrhodamine. For ultrastructural analysis by electron spectroscopic imaging, probes containing incorporated digoxigenin were labeled with anti-digoxigenin boronated antibodies. Based upon the developed procedures, it has been demonstrated that the presence of the nuclear localization signal in the transfection complex resulted in rapid nuclear import of the transfected DNA. PMID:9601525

  9. Liquid-crystalline ordering of antimicrobial peptide-DNA complexes controls TLR9 activation.

    PubMed

    Schmidt, Nathan W; Jin, Fan; Lande, Roberto; Curk, Tine; Xian, Wujing; Lee, Calvin; Frasca, Loredana; Frenkel, Daan; Dobnikar, Jure; Gilliet, Michel; Wong, Gerard C L

    2015-07-01

    Double-stranded DNA (dsDNA) can trigger the production of type I interferon (IFN) in plasmacytoid dendritic cells (pDCs) by binding to endosomal Toll-like receptor-9 (TLR9; refs 1-5). It is also known that the formation of DNA-antimicrobial peptide complexes can lead to autoimmune diseases via amplification of pDC activation. Here, by combining X-ray scattering, computer simulations, microscopy and measurements of pDC IFN production, we demonstrate that a broad range of antimicrobial peptides and other cationic molecules cause similar effects, and elucidate the criteria for amplification. TLR9 activation depends on both the inter-DNA spacing and the multiplicity of parallel DNA ligands in the self-assembled liquid-crystalline complex. Complexes with a grill-like arrangement of DNA at the optimum spacing can interlock with multiple TLR9 like a zipper, leading to multivalent electrostatic interactions that drastically amplify binding and thereby the immune response. Our results suggest that TLR9 activation and thus TLR9-mediated immune responses can be modulated deterministically.

  10. Enhanced nasal mucosal delivery and immunogenicity of anti-caries DNA vaccine through incorporation of anionic liposomes in chitosan/DNA complexes.

    PubMed

    Chen, Liulin; Zhu, Junming; Li, Yuhong; Lu, Jie; Gao, Li; Xu, Huibi; Fan, Mingwen; Yang, Xiangliang

    2013-01-01

    The design of optimized nanoparticles offers a promising strategy to enable DNA vaccines to cross various physiological barriers for eliciting a specific and protective mucosal immunity via intranasal administration. Here, we reported a new designed nanoparticle system through incorporating anionic liposomes (AL) into chitosan/DNA (CS/DNA) complexes. With enhanced cellular uptake, the constructed AL/CS/DNA nanoparticles can deliver the anti-caries DNA vaccine pGJA-P/VAX into nasal mucosa. TEM results showed the AL/CS/DNA had a spherical structure. High DNA loading ability and effective DNA protection against nuclease were proved by gel electrophoresis. The surface charge of the AL/CS/DNA depended strongly on pH environment, enabling the intracellular release of loaded DNA via a pH-mediated manner. In comparison to the traditional CS/DNA system, our new design rendered a higher transfection efficiency and longer residence time of the AL/CS/DNA at nasal mucosal surface. These outstanding features enable the AL/CS/DNA to induce a significantly (p<0.01) higher level of secretory IgA (SIgA) than the CS/DNA in animal study, and a longer-term mucosal immunity. On the other hand, the AL/CS/DNA exhibited minimal cytotoxicity. These results suggest that the developed nanoparticles offer a potential platform for DNA vaccine packaging and delivery for more efficient elicitation of mucosal immunity. PMID:23977186

  11. Imparting the unique properties of DNA into complex material architectures and functions

    PubMed Central

    Xu, Phyllis F.; Noh, Hyunwoo; Lee, Ju Hun; Domaille, Dylan W.; Nakatsuka, Matthew A.; Goodwin, Andrew P.; Cha, Jennifer N.

    2014-01-01

    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. PMID:25525408

  12. Self-assembly of DNA-polymer complexes using template polymerization.

    PubMed Central

    Trubetskoy, V S; Budker, V G; Hanson, L J; Slattum, P M; Wolff, J A; Hagstrom, J E

    1998-01-01

    The self-assembly of supramolecular complexes of nucleic acids and polymers is of relevance to several biological processes including viral and chromatin formation as well as gene therapy vector design. We now show that template polymerization facilitates condensation of DNA into particles that are <150 nm in diameter. Inclusion of a poly(ethylene glycol)-containing monomer prevents aggregation of these particles. The DNA within the particles remains biologically active and can express foreign genes in cells. The formation or breakage of covalent bonds has until now not been employed to compact DNA into artificial particles. PMID:9722638

  13. Self-assembly of DNA-polymer complexes using template polymerization.

    PubMed

    Trubetskoy, V S; Budker, V G; Hanson, L J; Slattum, P M; Wolff, J A; Hagstrom, J E

    1998-09-15

    The self-assembly of supramolecular complexes of nucleic acids and polymers is of relevance to several biological processes including viral and chromatin formation as well as gene therapy vector design. We now show that template polymerization facilitates condensation of DNA into particles that are <150 nm in diameter. Inclusion of a poly(ethylene glycol)-containing monomer prevents aggregation of these particles. The DNA within the particles remains biologically active and can express foreign genes in cells. The formation or breakage of covalent bonds has until now not been employed to compact DNA into artificial particles.

  14. 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.

  15. Enhanced efficiency of P-element mediated transgenesis in Drosophila: Microinjection of DNA complexed with nanomaterial.

    PubMed

    Sonane, Madhavi; Goyal, Ritu; Chowdhuri, Debapratim K; Ram, Kristipati Ravi; Gupta, Kailash C

    2013-01-01

    The efficiency of genetic transformation technology to generate stable transgenics depends upon the successful delivery of plasmid DNA in embryonic cells. The available gene vectors facilitate efficient plasmid DNA delivery to the cellular milieu but are exposed to nuclease degradation. Recent in vitro studies suggest encapsulation of plasmid DNA with nanomaterial(s) for better protection against nucleases. Therefore, in this study, we tested if complexing of free plasmid DNA with linear polyethylenimine (LPEI, 25 kDa) based nanoparticle (LPN) enhances the efficiency of transformation (transgenesis) by using Drosophila based germ-line transformation technology. Here, we show that the LPN-DNA complex not only enhances the efficiency of this transgenic technology at a DNA concentration of 0.04 μg/μl but also reduces the DNA quantity required to generate transgenics by ten folds. This approach has potential applications for other types of transgenesis and nucleic acid injection methods in Drosophila as well as other popular genetic model systems.

  16. SERS as an advanced tool for investigating chloroethyl nitrosourea derivatives complexation with DNA.

    PubMed

    Agarwal, Shweta; Ray, Bhumika; Mehrotra, Ranjana

    2015-11-01

    We report surface-enhanced Raman spectroscopic (SERS) studies on free calf thymus DNA and its complexes with anti-tumor chloroethyl nitrosourea derivatives; semustine and nimustine. Since, first incident of SERS in 1974, it has rapidly established into an analytical tool, which can be used for the trace detection and characterization of analytes. Here, we depict yet another application of SERS in the field of drug-DNA interaction and thereby, its promising role in rational designing of new chemotherapeutic agents. Vibrational spectral analysis has been performed in an attempt to delineate the anti-cancer action mechanism of above mentioned nitrosourea derivatives. Strong SERS bands associated with the complexation of DNA with semustine and nimustine have been observed, which reveal binding of nitrosourea derivatives with heterocyclic nitrogenous base pair of DNA duplex. Formation of dG-dC interstrand cross-link in DNA double helices is also suggested by the SERS spectral outcomes of CENUs-DNA adduct. Results, demonstrated here, reflect recent progress in the newly developing field of drug-DNA interaction analysis via SERS.

  17. Quantitative model of R-loop forming structures reveals a novel level of RNA-DNA interactome complexity.

    PubMed

    Wongsurawat, Thidathip; Jenjaroenpun, Piroon; Kwoh, Chee Keong; Kuznetsov, Vladimir

    2012-01-01

    R-loop is the structure co-transcriptionally formed between nascent RNA transcript and DNA template, leaving the non-transcribed DNA strand unpaired. This structure can be involved in the hyper-mutation and dsDNA breaks in mammalian immunoglobulin (Ig) genes, oncogenes and neurodegenerative disease related genes. R-loops have not been studied at the genome scale yet. To identify the R-loops, we developed a computational algorithm and mapped R-loop forming sequences (RLFS) onto 66,803 sequences defined by UCSC as 'known' genes. We found that ∼59% of these transcribed sequences contain at least one RLFS. We created R-loopDB (http://rloop.bii.a-star.edu.sg/), the database that collects all RLFS identified within over half of the human genes and links to the UCSC Genome Browser for information integration and visualisation across a variety of bioinformatics sources. We found that many oncogenes and tumour suppressors (e.g. Tp53, BRCA1, BRCA2, Kras and Ptprd) and neurodegenerative diseases related genes (e.g. ATM, Park2, Ptprd and GLDC) could be prone to significant R-loop formation. Our findings suggest that R-loops provide a novel level of RNA-DNA interactome complexity, playing key roles in gene expression controls, mutagenesis, recombination process, chromosomal rearrangement, alternative splicing, DNA-editing and epigenetic modifications. RLFSs could be used as a novel source of prospective therapeutic targets.

  18. Phylogenetic relationships among the Caribbean members of the Cliona viridis complex (Porifera, Demospongiae, Hadromerida) using nuclear and mitochondrial DNA sequences.

    PubMed

    Escobar, Dairo; Zea, Sven; Sánchez, Juan A

    2012-08-01

    Species complexes - groups of closely related species in which intraspecific and interspecific variability overlap - have generated considerable interest and study. Frequently, members of a species complex do not have complete reproductive isolation; therefore, the complex may go through extensive gene flow. In the Caribbean Sea, some encrusting and excavating sponges of the genus Cliona (Porifera, Hadromerida, Clionaidae) are grouped within the great "Cliona viridis" complex because of their morphological similarities. This study examined the evolutionary relationships of the Caribbean members of this complex (C. caribbaea, C. tenuis, C. aprica and C. varians) and related taxa based on nuclear (ITS1 and ITS2) and mitochondrial (3' end of ND6) DNA sequences. The intragenomic ITS variation and its secondary structures were evaluated using a mixed approach of Denaturing Gradient Gel Electrophoresis (DGGE), DNA sequencing and secondary structure prediction. Considerable intragenomic variation was found in all the species, with apparently functional ITS1 and ITS2 secondary structures. Despite the subtle but clear morphological differentiation in these excavating sponges, the intragenomic copies of C. caribbaea, C. tenuis and C. aprica had a polyphyletic placement in the ITS1 and ITS2 genealogies and very low divergence. Therefore, it is clear that these species constitute a species complex (herein called Ct-complex). Genetic distances within the Ct-complex revealed that an important part of the interspecific variation overlapped with intraspecific variation, suggesting either incomplete lineage sorting or extensive gene flow. In contrast, C. varians and an unidentified "Pione" species emerged as monophyletic clades, being the closest sister groups to the Ct-complex. Additionally, our results support that C. laticavicola and C. delitrix conform a monophyletic group, but absence of reciprocal monophyly in these species suggests they may be life stages or ecophenotypes of

  19. Phylogenetic relationships among the Caribbean members of the Cliona viridis complex (Porifera, Demospongiae, Hadromerida) using nuclear and mitochondrial DNA sequences.

    PubMed

    Escobar, Dairo; Zea, Sven; Sánchez, Juan A

    2012-08-01

    Species complexes - groups of closely related species in which intraspecific and interspecific variability overlap - have generated considerable interest and study. Frequently, members of a species complex do not have complete reproductive isolation; therefore, the complex may go through extensive gene flow. In the Caribbean Sea, some encrusting and excavating sponges of the genus Cliona (Porifera, Hadromerida, Clionaidae) are grouped within the great "Cliona viridis" complex because of their morphological similarities. This study examined the evolutionary relationships of the Caribbean members of this complex (C. caribbaea, C. tenuis, C. aprica and C. varians) and related taxa based on nuclear (ITS1 and ITS2) and mitochondrial (3' end of ND6) DNA sequences. The intragenomic ITS variation and its secondary structures were evaluated using a mixed approach of Denaturing Gradient Gel Electrophoresis (DGGE), DNA sequencing and secondary structure prediction. Considerable intragenomic variation was found in all the species, with apparently functional ITS1 and ITS2 secondary structures. Despite the subtle but clear morphological differentiation in these excavating sponges, the intragenomic copies of C. caribbaea, C. tenuis and C. aprica had a polyphyletic placement in the ITS1 and ITS2 genealogies and very low divergence. Therefore, it is clear that these species constitute a species complex (herein called Ct-complex). Genetic distances within the Ct-complex revealed that an important part of the interspecific variation overlapped with intraspecific variation, suggesting either incomplete lineage sorting or extensive gene flow. In contrast, C. varians and an unidentified "Pione" species emerged as monophyletic clades, being the closest sister groups to the Ct-complex. Additionally, our results support that C. laticavicola and C. delitrix conform a monophyletic group, but absence of reciprocal monophyly in these species suggests they may be life stages or ecophenotypes of

  20. Cationic Lipid-Nucleic Acid Complexes for Gene Delivery And Silencing: Pathways And Mechanisms for Plasmid Dna And Sirna

    SciTech Connect

    Ewert, K.K.; Zidovska, A.; Ahmad, A.; Bouxsein, N.F.; Evans, H.M.; McAllister, C.S.; Samuel, C.E.; Safinya, C.R.; /SLAC

    2012-07-17

    Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

  1. Synthesis and evaluation of gold(III) complexes as efficient DNA binders and cytotoxic agents.

    PubMed

    Patel, Mohan N; Bhatt, Bhupesh S; Dosi, Promise A

    2013-06-01

    In recent years, great interest has been focused on gold(III) complexes as cytotoxic and antitumor drugs. Recent studies demonstrated that simple bidentate or polydentate ligands containing nitrogen donor atoms may offer sufficient redox stabilization to produce viable Au(III) anticancer drug targets under physiologic conditions. So, we have synthesized square planer Au(III) complexes of type [Au(A(n))Clx]·Cly and characterized them using UV-Vis absorption, C, H, N elemental analysis, FT-IR, LC-MS, (1)H and (13)C NMR spectroscopy. These compounds manifested significant cytotoxic properties in vitro for brine shrimp lethality bioassay. The metal complexes were screened for series of DNA binding activity using UV-Vis absorption titration, hydrodynamic measurement and thermal DNA denaturation study. The nucleolytic activity was performed on plasmid pUC19 DNA. The Michaelis-Menten kinetic studies were performed to evaluate rate of enhancement in metal complexes mediated DNA cleavage over the non-catalyzed DNA cleavage.

  2. Synthesis and evaluation of gold(III) complexes as efficient DNA binders and cytotoxic agents

    NASA Astrophysics Data System (ADS)

    Patel, Mohan N.; Bhatt, Bhupesh S.; Dosi, Promise A.

    2013-06-01

    In recent years, great interest has been focused on gold(III) complexes as cytotoxic and antitumor drugs. Recent studies demonstrated that simple bidentate or polydentate ligands containing nitrogen donor atoms may offer sufficient redox stabilization to produce viable Au(III) anticancer drug targets under physiologic conditions. So, we have synthesized square planer Au(III) complexes of type [Au(An)Clx]·Cly and characterized them using UV-Vis absorption, C, H, N elemental analysis, FT-IR, LC-MS, 1H and 13C NMR spectroscopy. These compounds manifested significant cytotoxic properties in vitro for brine shrimp lethality bioassay. The metal complexes were screened for series of DNA binding activity using UV-Vis absorption titration, hydrodynamic measurement and thermal DNA denaturation study. The nucleolytic activity was performed on plasmid pUC19 DNA. The Michaelis-Menten kinetic studies were performed to evaluate rate of enhancement in metal complexes mediated DNA cleavage over the non-catalyzed DNA cleavage.

  3. Chiroptical properties, binding affinity, and photostability of a conjugated zinc porphyrin dimer complexed with left-handed Z-DNA and right-handed B-DNA.

    PubMed

    Choi, Jung Kyu; Reed, Aisha; Balaz, Milan

    2014-01-14

    We have studied the UV-vis absorption and chiroptical properties, binding affinity and photostability of a conjugated positively charged butadiyne-linked Zn(ii) porphyrin dimer bound to DNA sequence poly(dG-dC)2. Right-handed B-DNA, spermine-induced Z-DNA and Co(iii)-induced Z-DNA have been explored. Resonance light scattering (RLS) spectra showed formation of porphyrin aggregates in the presence of all DNA forms with the largest aggregates formed with B-DNA. The porphyrin dimer gave rise to induced bisignate circular dichroism (CD) signals in the presence of the left-handed Z-DNA conformations. On the other hand, the dimer stayed nearly chiroptically silent when complexed with the B-form of poly(dG-dC)2. Our results indicated that the conjugated Zn(ii) porphyrin dimer can be used as a sensor for the chiroptical detection of Z-DNA in the visible (400-500 nm) and near-infrared region of the electromagnetic spectrum (700-800 nm). The helicity of DNA had little effect on the dimer binding affinities. The photostability of the porphyrin dimer complexed with any form of DNA was higher than that of the free molecule. The porphyrin dimer bound to Z-DNA exhibited slower photobleaching than the B-DNA dimer complex.

  4. The infrared spectrum and structure of the type I complex of silver and DNA.

    PubMed Central

    DiRico, D E; Keller, P B; Hartman, K A

    1985-01-01

    Infrared spectroscopy was used to study films of the type I complex of Ag+ and DNA as a function of hydration with the following conclusions. Ag+ binds to guanine residues but not to cytosine or thymine residues. Cytosine becomes protonated as Ag+ binds to guanine. (These conclusions confirm previous models.) The type I complex remains in the B family of structures with slight modifications of the sugar-phosphate geometry. This modified B structure remains stable at lower values of hydration for which pure DNA is in the A form. Binding of Ag+ to PO2-, O-P-O or the deoxyribose oxygen is excluded. PMID:4000921

  5. Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA.

    PubMed

    Iyer, Lakshminarayan M; Zhang, Dapeng; Burroughs, A Maxwell; Aravind, L

    2013-09-01

    Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel 'readers' of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and

  6. Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA

    PubMed Central

    Iyer, Lakshminarayan M.; Zhang, Dapeng; Maxwell Burroughs, A.; Aravind, L.

    2013-01-01

    Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel ‘readers’ of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems

  7. Is a fully wrapped SSB–DNA complex essential for Escherichia coli survival?

    PubMed Central

    Waldman, Vincent M.; Weiland, Elizabeth; Kozlov, Alexander G.; Lohman, Timothy M.

    2016-01-01

    Escherichia coli single-stranded DNA binding protein (SSB) is an essential homotetramer that binds ssDNA and recruits multiple proteins to their sites of action during genomic maintenance. Each SSB subunit contains an N-terminal globular oligonucleotide/oligosaccharide binding fold (OB-fold) and an intrinsically disordered C-terminal domain. SSB binds ssDNA in multiple modes in vitro, including the fully wrapped (SSB)65 and (SSB)56 modes, in which ssDNA contacts all four OB-folds, and the highly cooperative (SSB)35 mode, in which ssDNA contacts an average of only two OB-folds. These modes can both be populated under physiological conditions. While these different modes might be used for different functions, this has been difficult to assess. Here we used a dimeric SSB construct with two covalently linked OB-folds to disable ssDNA binding in two of the four OB-folds thus preventing formation of fully wrapped DNA complexes in vitro, although they retain a wild-type-like, salt-dependent shift in cooperative binding to ssDNA. These variants complement wild-type SSB in vivo indicating that a fully wrapped mode is not essential for function. These results do not preclude a normal function for a fully wrapped mode, but do indicate that E. coli tolerates some flexibility with regards to its SSB binding modes. PMID:27084941

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

    SciTech Connect

    Suh, Myungkoo

    1995-12-06

    Laser-induced fluorescence line narrowing and non-line narrowing spectroscopic methods were applied to conformational studies of stable DNA adducts of the 7{beta}, 8{alpha}-dihydoxy-9{alpha}, l0{alpha}-epoxy-7,8,9, 10-tetrahydrobenzo[{alpha}]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, ({minus})-trans-, (+)-cis- and ({minus})-cis adducts of anti-BPDE bound to exocyclic amino group of the central guanine in an 11-mer oligonucleotide, exist in a mixture of conformations in frozen aqueous buffer matrices. The (+)-trans adduct adopts primarily an external conformation with a smaller fraction ( {approximately} 25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant {pi}-{pi} stacking interactions between the pyrenyl residues and the bases. Conformations of the trans-adduct of (+)-anti -BPDE in 11-mer oligonucleotides were studied as a function of flanking bases. In single stranded form the adduct at G{sub 2} or G{sub 3} (5 ft-flanking, base guanine) adopts a conformation with strong, interaction with the bases. In contrast, the adduct with a 5ft-flanking, thymine exists in a primarily helixexternal conformation. Similar differences were observed in the double stranded oligonucleotides. The nature of the 3ft-flanking base has little influence on the conformational equilibrium of the (+)-trans-anti BPDE-dG adduct. The formation and repair of BPDE-N{sup 2}-dG in DNA isolated from the skin of mice treated topically with benzo[{alpha}]pyrene (BP) was studied. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N-dG, and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N{sup 2}-dG.

  9. Serum Albumin Binding Inhibits Nuclear Uptake of Luminescent Metal-Complex-Based DNA Imaging Probes.

    PubMed

    Wragg, Ashley; Gill, Martin R; McKenzie, Luke; Glover, Caroline; Mowll, Rachel; Weinstein, Julia A; Su, Xiaodi; Smythe, Carl; Thomas, Jim A

    2015-08-10

    The DNA binding and cellular localization properties of a new luminescent heterobimetallic Ir(III) Ru(II) tetrapyridophenazine complex are reported. Surprisingly, in standard cell media, in which its tetracationic, isostructural Ru(II) Ru(II) analogue is localized in the nucleus, the new tricationic complex is poorly taken up by live cells and demonstrates no nuclear staining. Consequent cell-free studies reveal that the Ir(III) Ru(II) complex binds bovine serum albumin, BSA, in Sudlow's Site I with a similar increase in emission and binding affinity to that observed with DNA. Contrastingly, in serum-free conditions the complex is rapidly internalized by live cells, where it localizes in cell nuclei and functions as a DNA imaging agent. The absence of serum proteins also greatly alters the cytotoxicity of the complex, where high levels of oncosis/necrosis are observed due to this enhanced uptake. This suggests that simply increasing the lipophilicity of a DNA imaging probe to enhance cellular uptake can be counterproductive as, due to increased binding to serum albumin protein, this strategy can actually disrupt nuclear targeting.

  10. Interactions of the DNA polymerase X of African swine fever virus with double-stranded DNA. Functional structure of the complex.

    PubMed

    Jezewska, Maria J; Bujalowski, Paul J; Bujalowski, Wlodzimierz

    2007-10-12

    Interactions of the polymerase X of African swine fever virus with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence energy transfer techniques. Studies with unmodified dsDNAs were performed, using competition titration method. ASV pol X binds the dsDNA with a site-size of n=10(+/-2) base-pairs, which is significantly shorter than the total site-size of 16(+/-2) nucleotides of the enzyme-ssDNA complex. The small site size indicates that the enzyme binds the dsDNA exclusively using the proper DNA-binding subsite. Fluorescence energy transfer studies between the tryptophan residue W92 and the acceptor, located at the 5' or 3' end of the dsDNA, suggest strongly that the proper DNA-binding subsite is located on the non-catalytic C-terminal domain. Moreover, intrinsic interactions with the dsDNA 10-mer or 20-mer are accompanied by the same net number of ions released, independent of the length of the DNA, indicating the same length of the DNA engaged in the complex. The dsDNA intrinsic affinity is about two orders of magnitude higher than the ssDNA affinity, indicating that the proper DNA-binding subsite is, in fact, the specific dsDNA-binding site. Surprisingly, ASFV pol X binds the dsDNA with significant positive cooperativity, which results from protein-protein interactions. Cooperative interactions are accompanied by the net ion release, with anions participating in the ion-exchange process. The significance of these results for ASFV pol X activity in the recognition of damaged DNA is discussed.

  11. Complexity of genetic sequences modified by horizontal gene transfer and degraded-DNA uptake

    NASA Astrophysics Data System (ADS)

    Tremberger, George; Dehipawala, S.; Nguyen, A.; Cheung, E.; Sullivan, R.; Holden, T.; Lieberman, D.; Cheung, T.

    2015-09-01

    Horizontal gene transfer has been a major vehicle for efficient transfer of genetic materials among living species and could be one of the sources for noncoding DNA incorporation into a genome. Our previous study of lnc- RNA sequence complexity in terms of fractal dimension and information entropy shows a tight regulation among the studied genes in numerous diseases. The role of sequence complexity in horizontal transferred genes was investigated with Mealybug in symbiotic relation with a 139K genome microbe and Deinococcus radiodurans as examples. The fractal dimension and entropy showed correlation R-sq of 0.82 (N = 6) for the studied Deinococcus radiodurans sequences. For comparison the Deinococcus radiodurans oxidative stress tolerant catalase and superoxide dismutase genes under extracellular dGMP growth condition showed R-sq ~ 0.42 (N = 6); and the studied arsenate reductase horizontal transferred genes for toxicity survival in several microorganisms showed no correlation. Simulation results showed that R-sq < 0.4 would be improbable at less than one percent chance, suggestive of additional selection pressure when compared to the R-sq ~ 0.29 (N = 21) in the studied transferred genes in Mealybug. The mild correlation of R-sq ~ 0.5 for fractal dimension versus transcription level in the studied Deinococcus radiodurans sequences upon extracellular dGMP growth condition would suggest that lower fractal dimension with less electron density fluctuation favors higher transcription level.

  12. Efficient plasmid DNA cleavage by a mononuclear copper(II) complex.

    PubMed

    Sissi, Claudia; Mancin, Fabrizio; Gatos, Maddalena; Palumbo, Manlio; Tecilla, Paolo; Tonellato, Umberto

    2005-04-01

    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. PMID:15792466

  13. A planar Schiff base platinum(II) complex: crystal structure, cytotoxicity and interaction with DNA.

    PubMed

    Peng, Yan; Zhong, Hui; Chen, Zhen-Feng; Liu, Yan-Cheng; Zhang, Guo-Hai; Qin, Qi-Pin; Liang, Hong

    2014-01-01

    A new platinum(II) complex of salphen derivative, namely Schiff base ligand that derived from o-phenylenediamine and 5-chlorosalicylaldehyde was synthesized. The complex possessed a planar mononuclear structure. The in vitro cytotoxicities of the complex were evaluated by microculture tetrozolium (MTT) assay against seven human tumor cell lines with the IC50 values of ca. 11.61 µM. Cell cycle analysis indicated that the complex induced apoptosis and G1-phase arrest in A549 cells. The results of colony formation assay showed that the complex could suppress the proliferation and viability of A549 cells. The binding of the complex to potential target DNA were investigated by fluorescence spectroscopy, viscosity measurements, fluorescence polarization and agarose gel electrophoresis. The results suggest that the most probable binding mode of the complex is intercalation. PMID:24583776

  14. In vitro recognition of DNA base pairs by histones in histone-DNA complexes and reconstituted core particles: an ultraviolet resonance Raman study.

    PubMed Central

    Laigle, A; Chinsky, L; Turpin, P Y; Liquier, J; Taillandier, E

    1982-01-01

    Resonance Raman spectra of complexes between DNA and the four core histones, alone or associated, have been investigated in vitro using excitations at 300 and 257 nm, which give complementary informations about the DNA bases. H2A and H2B fractions recognize the G-C base pairs, while H3 and H4 (arginine rich fractions) recognize the A-T base pairs. The associated fractions form complexes with DNA which yield about the same DNA spectral modifications as the DNA-H4 complexes. This reveals the important role of the arginine rich fractions in the core particle formation and confirms the preferential in vitro assembly of nucleosome cores on A-T rich regions of DNA (25). PMID:7155896

  15. Mitochondrial DNA phylogeography of the Labeobarbus intermedius complex (Pisces, Cyprinidae) from Ethiopia.

    PubMed

    Beshera, K A; Harris, P M

    2014-08-01

    Mitochondrial DNA phylogeography of populations of the Labeobarbus intermedius complex (hexaploid barb) was investigated using 88 complete and 71 partial cytochrome b (cytb) sequences originating from 21 localities in five major drainages in Ethiopia and two localities in northern Kenya. The samples included 14 of the 15 Labeobarbus species described from Lake Tana. Discrete phylogeographic analyses of 159 cytb sequences employing Bayesian Markov Chain Monte Carlo (MCMC) simulations using Bayesian evolutionary analysis by sampling trees (BEAST) supported the monophyly of the L. intermedius complex, including the Lake Tana species. This analysis, in combination with statistical parsimony analysis, identified two mitochondrial DNA lineages within the complex. Divergence dating employing coalescent simulations suggested that the geographic split in the L. intermedius complex that led to the formation of these lineages occurred during the Pleistocene (c. 0.5 M b.p.), consistent with the timing of volcano-tectonic events postulated to have shaped the current landscape of East Africa.

  16. Formation of alkali labile linkages in DNA by hedamycin and use of hedamycin as a probe of protein-DNA complexes.

    PubMed Central

    Bennett, G N

    1982-01-01

    Hedamycin forms a stable complex with DNA and introduces alkali labile linkages in the DNA. These labile linkages are located at deoxyguanosine residues and are cleaved by the treatment used for breakage at bases alkylated by dimethyl sulfate. The reaction of hedamycin with all G residues in the chain is not uniform, and certain positions, particularily those in TG tracts, are especially reactive. The reaction of hedamycin with DNA can be inhibited by ethidium bromide, suggesting that intercalation is important in positioning the reactive group of hedamycin near to the base which is modified. The low amount of hedamycin needed to produce observable breakage, its specificity for reaction with DNA and its ability to react with DNA under mild conditions make it suitable for use as a probe of protein-DNA complexes. This was shown by the ability of lac repressor and RNA polymerase to block reaction of hedamycin with the DNA of the lac regulatory region. Images PMID:7133991

  17. Algorithmic complexity theory and the relative efficiency of financial markets

    NASA Astrophysics Data System (ADS)

    Giglio, R.; Matsushita, R.; Figueiredo, A.; Gleria, I.; Da Silva, S.

    2008-11-01

    Financial economists usually assess market efficiency in absolute terms. This is to be viewed as a shortcoming. One way of dealing with the relative efficiency of markets is to resort to the efficiency interpretation provided by algorithmic complexity theory. We employ such an approach in order to rank 36 stock exchanges and 20 US dollar exchange rates in terms of their relative efficiency.

  18. Structure of the PolIIIα-τc-DNA complex suggests an atomic model of the replisome.

    PubMed

    Liu, Bin; Lin, Jinzhong; Steitz, Thomas A

    2013-04-01

    The C-terminal domain (CTD) of the τ subunit of the clamp loader (τc) binds to both the DnaB helicase and the DNA polymerase III α subunit (PolIIIα), and determines their relative positions and orientations on the leading and lagging strands. Here, we present a 3.2 Å resolution structure of Thermus aquaticus PolIIIα in complex with τc and a DNA substrate. The structure reveals that the CTD of τc interacts with the CTD of PolIIIα through its C-terminal helix and the adjacent loop. Additionally, in this complex PolIIIα displays an open conformation that includes the reorientations of the oligonucleotide-binding fold and the thumb domain, which may be an indirect result of crystal packing due to the presence of the τc. Nevertheless, the position of the τc on PolIIIα allows us to suggest an approximate model for how the PolIIIα is oriented and positioned on the DnaB helicase.

  19. The architecture of ArgR-DNA complexes at the genome-scale in Escherichia coli

    PubMed Central

    Cho, Suhyung; Cho, Yoo-Bok; Kang, Taek Jin; Kim, Sun Chang; Palsson, Bernhard; Cho, Byung-Kwan

    2015-01-01

    DNA-binding motifs that are recognized by transcription factors (TFs) have been well studied; however, challenges remain in determining the in vivo architecture of TF-DNA complexes on a genome-scale. Here, we determined the in vivo architecture of Escherichia coli arginine repressor (ArgR)-DNA complexes using high-throughput sequencing of exonuclease-treated chromatin-immunoprecipitated DNA (ChIP-exo). The ChIP-exo has a unique peak-pair pattern indicating 5′ and 3′ ends of ArgR-binding region. We identified 62 ArgR-binding loci, which were classified into three groups, comprising single, double and triple peak-pairs. Each peak-pair has a unique 93 base pair (bp)-long (±2 bp) ArgR-binding sequence containing two ARG boxes (39 bp) and residual sequences. Moreover, the three ArgR-binding modes defined by the position of the two ARG boxes indicate that DNA bends centered between the pair of ARG boxes facilitate the non-specific contacts between ArgR subunits and the residual sequences. Additionally, our approach may also reveal other fundamental structural features of TF-DNA interactions that have implications for studying genome-scale transcriptional regulatory networks. PMID:25735747

  20. INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

    SciTech Connect

    Ausin, Israel; Greenberg, Maxim V.C.; Simanshu, Dhirendra K.; Hale, Christopher J.; Vashisht, Ajay A.; Simon, Stacey A.; Lee, Tzuu-fen; Feng, Suhua; Española, Sophia D.; Meyers, Blake C.; Wohlschlegel, James A.; Patel, Dinshaw J.; Jacobsen, Steven E.

    2012-10-23

    At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.

  1. Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA–protein complexes

    PubMed Central

    McAndrew, Christopher P.; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L.; Pegg, Ian L.; Sarkar, Abhijit

    2016-01-01

    We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA–protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether’s extension: ~0.05 pN in force and <10 nm change in extension. We have also verified that we can manipulate DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule’s force response. Using a calibration technique based on Stokes’ drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level. PMID:26757808

  2. Atomic-Scale Molecular Dynamics Simulations of DNA-Polycation Complexes: Two Distinct Binding Patterns.

    PubMed

    Kondinskaia, Diana A; Kostritskii, Andrei Yu; Nesterenko, Alexey M; Antipina, Alexandra Yu; Gurtovenko, Andrey A

    2016-07-14

    Synthetic cationic polymers represent a promising class of delivery vectors for gene therapy. Here, we employ atomistic molecular dynamics simulations to gain insight into the structure and properties of complexes of DNA with four linear polycations: polyethylenimine (PEI), poly-l-lysine (PLL), polyvinylamine (PVA), and polyallylamine (PAA). These polycations differ in their polymer geometries, protonation states, and hydrophobicities of their backbone chains. Overall, our results demonstrate for the first time the existence of two distinct patterns of binding of DNA with polycations. For PEI, PLL, and PAA, the complex is stabilized by the electrostatic attraction between protonated amine groups of the polycation and phosphate groups of DNA. In contrast, PVA demonstrates an alternative binding pattern as it gets embedded into the DNA major groove. It is likely that both the polymer topology and affinity of the backbone chain of PVA to the DNA groove are responsible for such behavior. The differences in binding patterns can have important biomedical implications: embedding PVA into a DNA groove makes it less sensitive to changes in the aqueous environment (pH level, ionic strength, etc.) and could therefore hinder the intracellular release of genetic material from a delivery vector, leading to lower transfection activity. PMID:27280954

  3. Diagnosis of skin cancer by correlation and complexity analyses of damaged DNA.

    PubMed

    Namazi, Hamidreza; Kulish, Vladimir V; Delaviz, Fatemeh; Delaviz, Ali

    2015-12-15

    Skin cancer is a common, low-grade cancerous (malignant) growth of the skin. It starts from cells that begin as normal skin cells and transform into those with the potential to reproduce in an out-of-control manner. Cancer develops when DNA, the molecule found in cells that encodes genetic information, becomes damaged and the body cannot repair the damage. A DNA walk of a genome represents how the frequency of each nucleotide of a pairing nucleotide couple changes locally. In this research in order to diagnose the skin cancer, first DNA walk plots of genomes of patients with skin cancer were generated. Then, the data so obtained was checked for complexity by computing the fractal dimension. Furthermore, the Hurst exponent has been employed in order to study the correlation of damaged DNA. By analysing different samples it has been found that the damaged DNA sequences are exhibiting higher degree of complexity and less correlation compared to normal DNA sequences. This investigation confirms that this method can be used for diagnosis of skin cancer. The method discussed in this research is useful not only for diagnosis of skin cancer but can be applied for diagnosis and growth analysis of different types of cancers.

  4. Diagnosis of skin cancer by correlation and complexity analyses of damaged DNA

    PubMed Central

    Namazi, Hamidreza; Kulish, Vladimir V.; Delaviz, Fatemeh; Delaviz, Ali

    2015-01-01

    Skin cancer is a common, low-grade cancerous (malignant) growth of the skin. It starts from cells that begin as normal skin cells and transform into those with the potential to reproduce in an out-of-control manner. Cancer develops when DNA, the molecule found in cells that encodes genetic information, becomes damaged and the body cannot repair the damage. A DNA walk of a genome represents how the frequency of each nucleotide of a pairing nucleotide couple changes locally. In this research in order to diagnose the skin cancer, first DNA walk plots of genomes of patients with skin cancer were generated. Then, the data so obtained was checked for complexity by computing the fractal dimension. Furthermore, the Hurst exponent has been employed in order to study the correlation of damaged DNA. By analysing different samples it has been found that the damaged DNA sequences are exhibiting higher degree of complexity and less correlation compared to normal DNA sequences. This investigation confirms that this method can be used for diagnosis of skin cancer. The method discussed in this research is useful not only for diagnosis of skin cancer but can be applied for diagnosis and growth analysis of different types of cancers. PMID:26497203

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

    PubMed

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

    2014-01-01

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

  6. RNA and DNA binding of inert oligonuclear ruthenium(II) complexes in live eukaryotic cells.

    PubMed

    Li, Xin; Gorle, Anil K; Ainsworth, Tracy D; Heimann, Kirsten; Woodward, Clifford E; Collins, J Grant; Keene, F Richard

    2015-02-28

    Confocal microscopy was used to study the intracellular localisation of a series of inert polypyridylruthenium(II) complexes with three eukaryotic cells lines - baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (Hep-G2). Co-staining experiments with the DNA-selective dye DAPI demonstrated that the di-, tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,12-dodecane bridging ligand ("bb12") showed a high degree of selectivity for the nucleus of the eukaryotic cells. Additional co-localisation experiments with the general nucleic acid stain SYTO 9 indicated that the ruthenium complexes showed a considerable preference for the RNA-rich nucleolus, rather than chromosomal DNA. No significant differences were observed in the intracellular localisation between the ΔΔ and ΛΛ enantiomers of the dinuclear complex. Cytotoxicity assays carried out over 72 hours indicated that the ruthenium complexes, particularly the tri- and tetra-nuclear species, were significantly toxic to the eukaryotic cells. However, when the activity of the least cytotoxic compound (the ΔΔ enantiomer of the dinuclear species) was determined over a 24 hour period, the results indicated that the ruthenium complex was approximately a 100-fold less toxic to liver and kidney cells than to Gram positive bacteria. Circular dichroism (CD) spectroscopy was used to examine the effect of the ΔΔ and ΛΛ enantiomers of the dinuclear complex on the solution conformations of RNA and DNA. The CD experiments indicated that the RNA maintained the A-type conformation, and the DNA the B-type structure, upon binding by the ruthenium complexes.

  7. A Tightly Coupled Particle-Fluid Model for DNA-Laden Flows in Complex Microscale Geometries

    SciTech Connect

    Trebotich, D; Miller, G H; Colella, P; Graves, D T; Martin, D F; Schwartz, P O

    2004-11-18

    We present a stable and convergent method for the computation of flows of DNA-laden fluids in microchannels with complex geometry. The numerical strategy combines a ball-rod model representation for polymers tightly coupled with a projection method for incompressible viscous flow. We use Cartesian grid embedded boundary methods to discretize the fluid equations in the presence of complex domain boundaries. A sample calculation is presented showing flow through a packed array microchannel in 2D.

  8. RNA and DNA binding of inert oligonuclear ruthenium(II) complexes in live eukaryotic cells.

    PubMed

    Li, Xin; Gorle, Anil K; Ainsworth, Tracy D; Heimann, Kirsten; Woodward, Clifford E; Collins, J Grant; Keene, F Richard

    2015-02-28

    Confocal microscopy was used to study the intracellular localisation of a series of inert polypyridylruthenium(II) complexes with three eukaryotic cells lines - baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (Hep-G2). Co-staining experiments with the DNA-selective dye DAPI demonstrated that the di-, tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,12-dodecane bridging ligand ("bb12") showed a high degree of selectivity for the nucleus of the eukaryotic cells. Additional co-localisation experiments with the general nucleic acid stain SYTO 9 indicated that the ruthenium complexes showed a considerable preference for the RNA-rich nucleolus, rather than chromosomal DNA. No significant differences were observed in the intracellular localisation between the ΔΔ and ΛΛ enantiomers of the dinuclear complex. Cytotoxicity assays carried out over 72 hours indicated that the ruthenium complexes, particularly the tri- and tetra-nuclear species, were significantly toxic to the eukaryotic cells. However, when the activity of the least cytotoxic compound (the ΔΔ enantiomer of the dinuclear species) was determined over a 24 hour period, the results indicated that the ruthenium complex was approximately a 100-fold less toxic to liver and kidney cells than to Gram positive bacteria. Circular dichroism (CD) spectroscopy was used to examine the effect of the ΔΔ and ΛΛ enantiomers of the dinuclear complex on the solution conformations of RNA and DNA. The CD experiments indicated that the RNA maintained the A-type conformation, and the DNA the B-type structure, upon binding by the ruthenium complexes. PMID:25333883

  9. Molecular recognition of DNA-protein complexes: a straightforward method combining scanning force and fluorescence microscopy.

    PubMed

    Sanchez, Humberto; Kanaar, Roland; Wyman, Claire

    2010-06-01

    Combining scanning force and fluorescent microscopy allows simultaneous identification of labeled biomolecules and analysis of their nanometer level architectural arrangement. Fluorescent polystyrene nano-spheres were used as reliable objects for alignment of optical and topographic images. This allowed the precise localization of different fluorescence particles within complex molecular assemblies whose structure was mapped in nanometer detail topography. Our experiments reveal the versatility of this method for analysis of proteins and protein-DNA complexes.

  10. DNA binding, DNA cleavage and cytotoxicity studies of a new water soluble copper(II) complex: The effect of ligand shape on the mode of binding

    NASA Astrophysics Data System (ADS)

    Kashanian, Soheila; Khodaei, Mohammad Mehdi; Roshanfekr, Hamideh; Shahabadi, Nahid; Mansouri, Ghobad

    2012-02-01

    The interaction of native calf thymus DNA (CT-DNA) with [Cu(ph 2phen)(phen-dione)Cl]Cl was studied at physiological pH by spectrophotometric, spectrofluorometric, circular dichroism, and viscometric techniques. Considerable hypochromicity and red shift are observed in the UV absorption band of the Cu complex. Binding constants ( Kb) of DNA with the complex were calculated at different temperatures. Thermodynamic parameters, enthalpy and entropy changes were calculated according to Van't Hoff equation, which indicated that reaction is predominantly enthalpically driven. All these results indicate that Cu(II) complex interacts with CT-DNA via intercalative mode. Also, this new complex induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) and human T lymphocyte carcinoma-Jurkat cell lines.

  11. DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes

    NASA Astrophysics Data System (ADS)

    Raman, N.; Sobha, S.; Selvaganapathy, M.; Mahalakshmi, R.

    2012-10-01

    Few transition metal complexes of tetradentate N2O2 donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL1/KHL2 have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL1/KHL2 are found to act as tetradentate ligands using N2O2 donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen (1O2) and superoxide anion radical (O2rad -) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method.

  12. DNA binding mode of novel tetradentate amino acid based 2-hydroxybenzylidene-4-aminoantipyrine complexes.

    PubMed

    Raman, N; Sobha, S; Selvaganapathy, M; Mahalakshmi, R

    2012-10-01

    Few transition metal complexes of tetradentate N(2)O(2) donor Schiff base ligands containing 2-hydroxybenzylidene-4-aminoantipyrine and amino acids (alanine/valine) abbreviated to KHL(1)/KHL(2) have been synthesized. All the metal complexes have been fully characterized with the help of elemental analyses, molecular weights, molar conductance values, magnetic moments and spectroscopic data. The Schiff bases KHL(1)/KHL(2) are found to act as tetradentate ligands using N(2)O(2) donor set of atoms leading to a square-planar geometry for the complexes around the metal ions. The binding behaviors of the complexes to calf thymus DNA have been investigated by absorption spectra, viscosity measurements and cyclic voltammetry. The DNA binding constants reveal that all these complexes interact with DNA through minor groove binding mode. The studies on mechanism of photocleavage reveal that singlet oxygen ((1)O(2)) and superoxide anion radical (O(2)(-)) may play an important role in the photocleavage. The Schiff bases and their metal complexes have been screened for their in vitro antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae and antifungal activities against Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans by MIC method. PMID:22885083

  13. 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

  14. Calf thymus DNA binding studies of the new neodymium-naproxen complex.

    PubMed

    Huo, Ruina; Xu, Guiqing; Jiang, Xiaoying; Ge, Yao; Xue, Zaikun; Cui, Fengling

    2012-05-01

    Fluorescence spectroscopy in combination with UV absorption spectroscopy was carried out to investigate the interaction between the neodymium-naproxen complex (Nd-NAP) and calf thymus DNA (ctDNA). The experimental results showed that Nd-NAP intercalated with the ctDNA base pairs. Analysis of fluorescence quenching data of Nd-NAP by ctDNA at different temperatures using a Stern-Volmer equation revealed that dynamic and static quenching occurred simultaneously. The binding constants and the number of binding sites at 293 and 310 K were obtained as 2.904 × 10(4) L mol(-1), 1.172 and 2.432 × 10(4) L mol(-1), 1.143, respectively. The thermodynamic parameters ΔG, ΔH, and ΔS calculated at different temperatures indicated that hydrogen bonding and van der Waals force were the main binding forces.

  15. Effectiveness, against tuberculosis, of pseudo-ternary complexes: peptide-DNA-cationic liposome.

    PubMed

    Rosada, Rogério Silva; Silva, Célio Lopes; Santana, Maria Helena Andrade; Nakaie, Clóvis Ryuichi; de la Torre, Lucimara Gaziola

    2012-05-01

    We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex. The same cationic liposomes, composed of egg chicken L-α-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane (2:1:1M), were previously evaluated as a gene carrier for tuberculosis immunization protocols with DNAhsp65. The pseudo-ternary complex presented a controlled size (250 nm), spherical-like shape, and various lamellae in liposomes as evaluated by transmission electron microscopy. An assay of fluorescence probe accessibility confirmed insertion of the peptide/DNA into the liposome structure. Peptide addition conferred no cytotoxicity in vitro, and similar therapeutic effects against tuberculosis were seen with four times less DNA compared with naked DNA treatment. Taken together, the results indicate that the pseudo-ternary complex is a promising gene vaccine for tuberculosis treatment. This work contributes to the development of multifunctional nanostructures in the search for strategies for in vivo DNA delivery. PMID:21999959

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

    PubMed Central

    2015-01-01

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

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

    PubMed

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

    2015-09-14

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

  18. Bacterial conjugation protein MobA mediates integration of complex DNA structures into plant cells.

    PubMed

    Bravo-Angel, A M; Gloeckler, V; Hohn, B; Tinland, B

    1999-09-01

    Agrobacterium tumefaciens transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2. Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria. A detailed analysis of MobA-mediated DNA mobilization by Agrobacterium to plants was performed. We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2. MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus. However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies. In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5' end. Isolation and analysis of the most conserved 5' ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another. These complex integration patterns indicate a specific deficiency in MobA. The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins. PMID:10482518

  19. G9a/GLP Complex Maintains Imprinted DNA Methylation in Embryonic Stem Cells

    PubMed Central

    Zhang, Tuo; Termanis, Ausma; Özkan, Burak; Bao, Xun X.; Culley, Jayne; de Lima Alves, Flavia; Rappsilber, Juri; Ramsahoye, Bernard; Stancheva, Irina

    2016-01-01

    Summary DNA methylation at imprinting control regions (ICRs) is established in gametes in a sex-specific manner and has to be stably maintained during development and in somatic cells to ensure the correct monoallelic expression of imprinted genes. In addition to DNA methylation, the ICRs are marked by allele-specific histone modifications. Whether these marks are essential for maintenance of genomic imprinting is largely unclear. Here, we show that the histone H3 lysine 9 methylases G9a and GLP are required for stable maintenance of imprinted DNA methylation in embryonic stem cells; however, their catalytic activity and the G9a/GLP-dependent H3K9me2 mark are completely dispensable for imprinting maintenance despite the genome-wide loss of non-imprinted DNA methylation in H3K9me2-depleted cells. We provide additional evidence that the G9a/GLP complex protects imprinted DNA methylation by recruitment of de novo DNA methyltransferases, which antagonize TET dioxygenass-dependent erosion of DNA methylation at ICRs. PMID:27052169

  20. The histone-fold complex MHF is remodeled by FANCM to recognize branched DNA and protect genome stability

    PubMed Central

    Fox, David; Yan, Zhijiang; Ling, Chen; Zhao, Ye; Lee, Duck-Yeon; Fukagawa, Tatsuo; Yang, Wei; Wang, Weidong

    2014-01-01

    Histone-fold proteins typically assemble in multiprotein complexes to bind duplex DNA. However, one histone-fold complex, MHF, associates with Fanconi anemia (FA) protein FANCM to form a branched DNA remodeling complex that senses and repairs stalled replication forks and activates FA DNA damage response network. How the FANCM-MHF complex recognizes branched DNA is unclear. Here, we solved the crystal structure of MHF and its complex with the MHF-interaction domain (referred to as MID) of FANCM, and performed structure-guided mutagenesis. We found that the MID-MHF complex consists of one histone H3-H4-like MHF heterotetramer wrapped by a single polypeptide of MID. We identified a zinc atom-liganding structure at the central interface between MID and MHF that is critical for stabilization of the complex. Notably, the DNA-binding surface of MHF was altered by MID in both electrostatic charges and allosteric conformation. This leads to a switch in the DNA-binding preference — from duplex DNA by MHF alone, to branched DNA by the MID-MHF complex. Mutations that disrupt either the composite DNA-binding surface or the protein-protein interface of the MID-MHF complex impaired activation of the FA network and genome stability. Our data provide the structural basis of how FANCM and MHF work together to recognize branched DNA, and suggest a novel mechanism by which histone-fold complexes can be remodeled by their partners to bind special DNA structures generated during DNA metabolism. PMID:24699063

  1. Quantitative characterization of protein–protein complexes involved in base excision DNA repair

    PubMed Central

    Moor, Nina A.; Vasil'eva, Inna A.; Anarbaev, Rashid O.; Antson, Alfred A.; Lavrik, Olga I.

    2015-01-01

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process. PMID:26013813

  2. Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

    PubMed

    Moor, Nina A; Vasil'eva, Inna A; Anarbaev, Rashid O; Antson, Alfred A; Lavrik, Olga I

    2015-07-13

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

  3. Metal-polybenzimidazole complexes as a nonviral gene carrier: effects of the DNA affinity on gene delivery.

    PubMed

    Huang, Xueying; Dong, Xiongwei; Li, Xue; Meng, Xianggao; Zhang, Dan; Liu, Changlin

    2013-12-01

    The metal complex-based carriers are emerging likely as a new type of gene-delivery systems prone to systematic structural alteration and chemical tailoring. In our work, the DNA affinity of metal complexes with polybenzimidazoles was found to be one of the determinants that can regulate expression of the transgenes. Here, the correlations between the DNA affinity and transfection efficacy were explored by characterizing gene-delivering properties of a series of Co(2+)- and Ca(2+)-polybenzimidazole complexes. The binding equilibrium constants (Kobs) of the divalent metal complexes to DNA, which is considered as a measure of the DNA affinity of metal complexes, were evaluated by isothermal titration calorimetry (ITC) and UV-visible absorption titration. The properties of DNA condensates formed with the metal complexes including sizes, ζ potential and morphology were observed to be altered with Kobs values. The monodispersed spherical condensates were found only for the Ca(2+) complexes whose DNA affinity is weaker than that of the Co(2+) complexes. However, the cell internalization examination indicated that cell uptake of the DNA condensates is independent of homogeneity in their sizes and morphology. The comparison of transgene expression showed that that the Ca(2+) complex-mediated transfection has higher efficiency than the Co(2+) complexes under the conditions tested, and the transfection efficacy cannot be correlated with the cell uptake of DNA condensates. Moreover, the Ca(2+) complexes and their DNA condensates had lower cytotoxicity than the Co(2+) complexes. Thus, the DNA affinity should be one of the factors to be capable of regulating the gene-delivering property of metal complexes. PMID:24099694

  4. Redefining the roles of mitochondrial DNA-encoded subunits in respiratory Complex I assembly

    PubMed Central

    Vartak, Rasika; Deng, Janice; Fang, Hezhi; Bai, Yidong

    2015-01-01

    Respiratory Complex I deficiency is implicated in numerous degenerative and metabolic diseases. In particular, mutations in several mitochondrial DNA (mtDNA)-encoded Complex I subunits including ND4, ND5 and ND6 have been identified in several neurological diseases. We previously demonstrated that these subunits played essential roles in Complex I assembly which in turn affected mitochondrial function. Here, we carried out a comprehensive study of the Complex I assembly pathway. We identified a new Complex I intermediate containing both membrane and matrix arms at an early assembly stage. We find that lack of the ND6 subunit does not hinder membrane arm formation; instead it recruits ND1 and ND5 enter the intermediate. While ND4 is important for the formation of the newly identified intermediate, the addition of ND5 stabilizes the complex and is required for the critical transition from Complex I to supercomplexes assembly. As a result, the Complex I assembly pathway has been redefined in this study. PMID:25887158

  5. Structural biology of disease-associated repetitive DNA sequences and protein-DNA complexes involved in DNA damage and repair

    SciTech Connect

    Gupta, G.; Santhana Mariappan, S.V.; Chen, X.; Catasti, P.; Silks, L.A. III; Moyzis, R.K.; Bradbury, E.M.; Garcia, A.E.

    1997-07-01

    This project is aimed at formulating the sequence-structure-function correlations of various microsatellites in the human (and other eukaryotic) genomes. Here the authors have been able to develop and apply structure biology tools to understand the following: the molecular mechanism of length polymorphism microsatellites; the molecular mechanism by which the microsatellites in the noncoding regions alter the regulation of the associated gene; and finally, the molecular mechanism by which the expansion of these microsatellites impairs gene expression and causes the disease. Their multidisciplinary structural biology approach is quantitative and can be applied to all coding and noncoding DNA sequences associated with any gene. Both NIH and DOE are interested in developing quantitative tools for understanding the function of various human genes for prevention against diseases caused by genetic and environmental effects.

  6. Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding

    PubMed Central

    Janzen, Daniel H; Hajibabaei, Mehrdad; Burns, John M; Hallwachs, Winnie; Remigio, Ed; Hebert, Paul D.N

    2005-01-01

    By facilitating bioliteracy, DNA barcoding has the potential to improve the way the world relates to wild biodiversity. Here we describe the early stages of the use of cox1 barcoding to supplement and strengthen the taxonomic platform underpinning the inventory of thousands of sympatric species of caterpillars in tropical dry forest, cloud forest and rain forest in northwestern Costa Rica. The results show that barcoding a biologically complex biota unambiguously distinguishes among 97% of more than 1000 species of reared Lepidoptera. Those few species whose barcodes overlap are closely related and not confused with other species. Barcoding also has revealed a substantial number of cryptic species among morphologically defined species, associated sexes, and reinforced identification of species that are difficult to distinguish morphologically. For barcoding to achieve its full potential, (i) ability to rapidly and cheaply barcode older museum specimens is urgent, (ii) museums need to address the opportunity and responsibility for housing large numbers of barcode voucher specimens, (iii) substantial resources need be mustered to support the taxonomic side of the partnership with barcoding, and (iv) hand-held field-friendly barcorder must emerge as a mutualism with the taxasphere and the barcoding initiative, in a manner such that its use generates a resource base for the taxonomic process as well as a tool for the user. PMID:16214742

  7. Non-uniform velocity of homogeneous DNA in a uniform electric field: consequence of electric-field-induced slow dissociation of highly stable DNA-counterion complexes.

    PubMed

    Musheev, Michael U; Kanoatov, Mirzo; Krylov, Sergey N

    2013-05-29

    Identical molecules move with identical velocities when placed in a uniform electric field within a uniform electrolyte. Here we report that homogeneous DNA does not obey this fundamental rule. While most DNA moves with similar velocities, a fraction of DNA moves with velocities that vary within a multiple-fold range. The size of this irregular fraction increases several orders of magnitude when exogenous counterions are added to DNA. The irregular fraction decreases several orders of magnitude when DNA counterions are removed by dialysis against deionized water in the presence of a strong electric field (0.6 kV/cm). Dialysis without the field is ineffective in decreasing the size of irregular fraction. These results suggest that (i) DNA can form very stable complexes with counterions, (ii) these complexes can be dissociated by an electric field, and (iii) the observed non-uniform velocity of DNA is caused by electric-field-induced slow dissociation of these stable complexes. Our findings help to better understand a fundamental property of DNA: its interaction with counterions. In addition, these findings suggest a practical way of making electromigration of DNA more uniform: removal of strongly bound DNA counterions by electro-dialysis against deionized water.

  8. An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase-DNA complexes to surfaces.

    PubMed

    Williams, John G K; Steffens, David L; Anderson, Jon P; Urlacher, Teresa M; Lamb, Donald T; Grone, Daniel L; Egelhoff, Jolene C

    2008-10-01

    Single molecule analysis of individual enzymes can require oriented immobilization of the subject molecules on a detection surface. As part of a technology development project for single molecule DNA sequencing, we faced the multiple challenges of immobilizing both a DNA polymerase and its DNA template together in an active, stable complex capable of highly processive DNA synthesis on a nonstick surface. Here, we report the genetic modification of the archaeal DNA polymerase 9 degrees N in which two biotinylated peptide 'legs' are inserted at positions flanking the DNA-binding cleft. Streptavidin binding on either side of the cleft both traps the DNA template in the polymerase and orients the complex on a biotinylated surface. We present evidence that purified polymerase-DNA-streptavidin complexes are active both in solution and immobilized on a surface. Processivity is improved from <20 nt in the unmodified polymerase to several thousand nucleotides in the engineered complexes. High-molecular weight DNA synthesized by immobilized complexes is observed moving above the surface even as it remains tethered to the polymerase. Pre-formed polymerase-DNA-streptavidin complexes can be stored frozen and subsequently thawed without dissociation or loss of activity, making them convenient for use in single molecule analysis. PMID:18723573

  9. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-01-01

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. PMID:18834537

  10. Genetic variants in multisynthetase complex genes are associated with DNA damage levels in Chinese populations.

    PubMed

    Liu, Jia; Zhu, Meng; Chen, Weihong; Xie, Kaipeng; Shen, Wei; Yuan, Jing; Cheng, Yang; Geng, Liguo; Wang, Yuzhuo; Jin, Guangfu; Dai, Juncheng; Ma, Hongxia; Du, Jiangbo; Wang, Meilin; Zhang, Zhengdong; Hu, Zhibin; Wu, Tangchun; Shen, Hongbing

    2016-04-01

    Aminoacyl-tRNA synthetases (ARSs) and ARS-interacting multi-functional proteins (AIMPs) form a multisynthetase complex (MSC) and play an important role in the process of DNA damage repair. We hypothesized that genetic variants in key ARSs and AIMPs might regulate the DNA damage response. Therefore, we systematically screened 23 potentially functional polymorphisms in MSC genes and evaluated the association between the genetic variants and DNA damage levels in 307 subjects from three cities in southern, central and northern China (Zhuhai, Wuhan and Tianjin, respectively). We examined personal 24-h PM2.5 exposure levels and DNA damage levels in peripheral blood lymphocytes for each subject. We found that the variant allele of rs12199241 in AIMP3 was significantly associated with DNA damage levels (β=0.343, 95%CI: 0.133-0.554, P=0.001). Meanwhile, the results of rs5030754 in EPRS and rs3784929 in KARS indicated their suggestive roles in DNA damage processes (β=0.331, 95%CI: 0.062-0.599, P=0.016 for rs5030754; β=0.192, 95%CI: 0.016-0.368, P=0.033 for rs3784929, respectively). After multiple testing, rs12199241 was still significantly associated with DNA damage levels. Combined analysis of these three polymorphisms showed a significant allele-dosage association between the number of risk alleles and higher DNA damage levels (Ptrend<0.001). These findings indicate that genetic variants in MSC genes may account for PM2.5-modulated DNA damage levels in Chinese populations.

  11. Genetic variants in multisynthetase complex genes are associated with DNA damage levels in Chinese populations.

    PubMed

    Liu, Jia; Zhu, Meng; Chen, Weihong; Xie, Kaipeng; Shen, Wei; Yuan, Jing; Cheng, Yang; Geng, Liguo; Wang, Yuzhuo; Jin, Guangfu; Dai, Juncheng; Ma, Hongxia; Du, Jiangbo; Wang, Meilin; Zhang, Zhengdong; Hu, Zhibin; Wu, Tangchun; Shen, Hongbing

    2016-04-01

    Aminoacyl-tRNA synthetases (ARSs) and ARS-interacting multi-functional proteins (AIMPs) form a multisynthetase complex (MSC) and play an important role in the process of DNA damage repair. We hypothesized that genetic variants in key ARSs and AIMPs might regulate the DNA damage response. Therefore, we systematically screened 23 potentially functional polymorphisms in MSC genes and evaluated the association between the genetic variants and DNA damage levels in 307 subjects from three cities in southern, central and northern China (Zhuhai, Wuhan and Tianjin, respectively). We examined personal 24-h PM2.5 exposure levels and DNA damage levels in peripheral blood lymphocytes for each subject. We found that the variant allele of rs12199241 in AIMP3 was significantly associated with DNA damage levels (β=0.343, 95%CI: 0.133-0.554, P=0.001). Meanwhile, the results of rs5030754 in EPRS and rs3784929 in KARS indicated their suggestive roles in DNA damage processes (β=0.331, 95%CI: 0.062-0.599, P=0.016 for rs5030754; β=0.192, 95%CI: 0.016-0.368, P=0.033 for rs3784929, respectively). After multiple testing, rs12199241 was still significantly associated with DNA damage levels. Combined analysis of these three polymorphisms showed a significant allele-dosage association between the number of risk alleles and higher DNA damage levels (Ptrend<0.001). These findings indicate that genetic variants in MSC genes may account for PM2.5-modulated DNA damage levels in Chinese populations. PMID:26871430

  12. Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates.

    PubMed Central

    Li, Y.; Kong, Y.; Korolev, S.; Waksman, G.

    1998-01-01

    The crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 A resolution. The dNTPs bind adjacent to the O helix of Klentaq1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaq1 structures demonstrate appropriate stacking of the nucleotide base with the 3' end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant. PMID:9605316

  13. Architecture of the human XPC DNA repair and stem cell coactivator complex

    PubMed Central

    He, Yuan; Grob, Patricia; Fong, Yick W.; Nogales, Eva; Tjian, Robert

    2015-01-01

    The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair. PMID:26627236

  14. Complex Oncogenic Translocations with Gene Amplification are Initiated by Specific DNA Breaks in Lymphocytes

    PubMed Central

    Wright, Sarah M.; Woo, Yong H.; Alley, Travis L.; Shirley, Bobbi-Jo; Akeson, Ellen C.; Snow, Kathy J.; Maas, Sarah A.; Elwell, Rachel L.; Foreman, Oded; Mills, Kevin D.

    2009-01-01

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. While chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now demonstrate that specific DNA double strand breaks, occurring within a narrow segment of Igh are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Eμ are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability, and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors. PMID:19435904

  15. Complex oncogenic translocations with gene amplification are initiated by specific DNA breaks in lymphocytes.

    PubMed

    Wright, Sarah M; Woo, Yong H; Alley, Travis L; Shirley, Bobbi-Jo; Akeson, Ellen C; Snow, Kathy J; Maas, Sarah A; Elwell, Rachel L; Foreman, Oded; Mills, Kevin D

    2009-05-15

    Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. Whereas chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double-strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now show that specific DNA double-strand breaks, occurring within a narrow segment of Igh, are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Emu are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors.

  16. Age-Related and Heteroplasmy-Related Variation in Human mtDNA Copy Number

    PubMed Central

    Li, Mingkun; Madea, Burkhard; Stoneking, Mark

    2016-01-01

    The mitochondrial (mt) genome is present in many copies in human cells, and intra-individual variation in mtDNA sequences is known as heteroplasmy. Recent studies found that heteroplasmies are highly tissue-specific, site-specific, and allele-specific, however the functional implications have not been explored. This study investigates variation in mtDNA copy numbers (mtCN) in 12 different tissues obtained at autopsy from 152 individuals (ranging in age from 3 days to 96 years). Three different methods to estimate mtCN were compared: shotgun sequencing (in 4 tissues), capture-enriched sequencing (in 12 tissues) and droplet digital PCR (ddPCR, in 2 tissues). The highest precision in mtCN estimation was achieved using shotgun sequencing data. However, capture-enrichment data provide reliable estimates of relative (albeit not absolute) mtCNs. Comparisons of mtCN from different tissues of the same individual revealed that mtCNs in different tissues are, with few exceptions, uncorrelated. Hence, each tissue of an individual seems to regulate mtCN in a tissue-related rather than an individual-dependent manner. Skeletal muscle (SM) samples showed an age-related decrease in mtCN that was especially pronounced in males, while there was an age-related increase in mtCN for liver (LIV) samples. MtCN in SM samples was significantly negatively correlated with both the total number of heteroplasmic sites and with minor allele frequency (MAF) at two heteroplasmic sites, 408 and 16327. Heteroplasmies at both sites are highly specific for SM, accumulate with aging and are part of functional elements that regulate mtDNA replication. These data support the hypothesis that selection acting on these heteroplasmic sites is reducing mtCN in SM of older individuals. PMID:26978189

  17. DNA Methylation of Lipid-Related Genes Affects Blood Lipid Levels

    PubMed Central

    Pfeiffer, Liliane; Wahl, Simone; Pilling, Luke C.; Reischl, Eva; Sandling, Johanna K.; Kunze, Sonja; Holdt, Lesca M.; Kretschmer, Anja; Schramm, Katharina; Adamski, Jerzy; Klopp, Norman; Illig, Thomas; Hedman, Åsa K.; Roden, Michael; Hernandez, Dena G.; Singleton, Andrew B.; Thasler, Wolfgang E.; Grallert, Harald; Gieger, Christian; Herder, Christian; Teupser, Daniel; Meisinger, Christa; Spector, Timothy D.; Kronenberg, Florian; Prokisch, Holger; Melzer, David; Peters, Annette; Deloukas, Panos; Ferrucci, Luigi; Waldenberger, Melanie

    2016-01-01

    Background Epigenetic mechanisms might be involved in the regulation of interindividual lipid level variability and thus may contribute to the cardiovascular risk profile. The aim of this study was to investigate the association between genome-wide DNA methylation and blood lipid levels high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol. Observed DNA methylation changes were also further analyzed to examine their relationship with previous hospitalized myocardial infarction. Methods and Results Genome-wide DNA methylation patterns were determined in whole blood samples of 1776 subjects of the Cooperative Health Research in the Region of Augsburg F4 cohort using the Infinium HumanMethylation450 BeadChip (Illumina). Ten novel lipid-related CpG sites annotated to various genes including ABCG1, MIR33B/SREBF1, and TNIP1 were identified. CpG cg06500161, located in ABCG1, was associated in opposite directions with both high-density lipoprotein cholesterol (β coefficient=−0.049; P=8.26E-17) and triglyceride levels (β=0.070; P=1.21E-27). Eight associations were confirmed by replication in the Cooperative Health Research in the Region of Augsburg F3 study (n=499) and in the Invecchiare in Chianti, Aging in the Chianti Area study (n=472). Associations between triglyceride levels and SREBF1 and ABCG1 were also found in adipose tissue of the Multiple Tissue Human Expression Resource cohort (n=634). Expression analysis revealed an association between ABCG1 methylation and lipid levels that might be partly mediated by ABCG1 expression. DNA methylation of ABCG1 might also play a role in previous hospitalized myocardial infarction (odds ratio, 1.15; 95% confidence interval=1.06–1.25). Conclusions Epigenetic modifications of the newly identified loci might regulate disturbed blood lipid levels and thus contribute to the development of complex lipid-related diseases. PMID:25583993

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

    NASA Astrophysics Data System (ADS)

    Halid, Nurul Izni Abdullah; Hasbullah, Siti Aishah; Ahmad, Haslina; Heng, Lee Yook; Karim, Nurul Huda Abd; Harun, Siti Norain

    2014-09-01

    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)2(PIP)]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)2(PIP)]2+ was used as electrochemical redox intercalator label to detect DNA hybridization event. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the ruthenium (II) complex was active. The results indicate that the interaction of [Ru(bpy)2(PIP)]2+ with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.

  19. Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution.

    PubMed

    McClarin, J A; Frederick, C A; Wang, B C; Greene, P; Boyer, H W; Grable, J; Rosenberg, J M

    1986-12-19

    The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.

  20. Cyclic Amphipathic Peptide-DNA Complexes Mediate High-Efficiency Transfection of Adherent Mammalian Cells

    NASA Astrophysics Data System (ADS)

    Legendre, Jean-Yves; Szoka, Francis C., Jr.

    1993-02-01

    A DNA transfection protocol has been developed that makes use of the cyclic cationic amphipathic peptide gramicidin S and dioleoyl phosphatidylethanolamine. The DNA complex is formed by mixing gramicidin S with DNA at a 1:1 charge ratio and then adding phosphatidylethanolamine at a lipid/peptide molar ratio of 5:1. The complex mediates rapid association of DNA with cells and leads to transient expression levels of β-galactosidase ranging from 1 to 30% of the transfected cells, with long-term expression being about an order of magnitude lower. The respective roles of peptide and phospholipid are not yet resolved but optimal transfection requires both the cyclic peptide and the hexagonal phase-competent phospholipid PtdEtn. Transfection in CV-1 cells is not affected by lysomotrophic agents, which suggests that DNA entry into the cell is via the plasma membrane. This technique that is simple, economical, and reproducible mediates transfection levels up to 20-fold higher than cationic liposomes in adherent mammalian cells.

  1. Identification and characterization of specific DNA-binding complexes containing members of the Myc/Max/Mad network of transcriptional regulators.

    PubMed

    Sommer, A; Bousset, K; Kremmer, E; Austen, M; Lüscher, B

    1998-03-20

    In the past, eukaryotic cell-derived complexes of the Myc/Max/Mad network of transcriptional regulators have largely been refractory to DNA binding studies. We have developed electrophoretic mobility shift assay conditions to measure specific DNA binding of Myc/Max/Mad network complexes using a COS7 cell-based overexpression system. With the established protocol, we have measured on- and off-rates of c-Myc/Max, Max/Max, and Mad1/Max complexes and determined relative affinities. All three complexes appeared to bind with comparable affinity to a Myc E-box sequence. Furthermore, our data derived from competition experiments suggested that the Mad3/Max and Mad4/Max complexes also possess comparable DNA binding affinities. The conditions established for COS7 cell-overexpressed proteins were then used to identify c-Myc/Max, Max/Max, and Mnt/Max complexes in HL-60 cells. However, no Mad1/Max could be detected, despite the induction of Mad1 expression during differentiation. Whereas the DNA binding activity of c-Myc/Max complexes was down-regulated, Max/Max binding increased, and Mnt/Max binding remained unchanged. In addition, we have also tested for upstream stimulatory factor (USF) binding and observed that, in agreement with published data, USF comprises a major Myc E-box-binding factor that is more abundant than any of the Myc/Max/Mad network complexes. Similar to the Mnt/Max complex, the binding activity of USF remained constant during HL-60 differentiation. Our findings establish conditions for the analysis of DNA binding of Myc/Max/Mad complexes and indicate posttranslational regulation of the Max/Max complex.

  2. Mesomorphic complexes of DNA with the mixtures of a cationic surfactant and a neutral lipid.

    PubMed

    Hsu, Wei-Long; Chen, Hsin-Lung; Liou, Willisa; Lin, Hsien-Kuang; Liu, Wen-Liang

    2005-10-11

    Polyanionic DNA binds to cationic lipids to form electrostatic complexes exhibiting rich self-assembled structures. These types of complexes have been considered as a nonviral carrier in gene therapy and as a template for nanostructure construction. For the latter application where biocompatibility is not the key issue, replacement of cationic lipid by cationic surfactant is advantageous due to the wide availability of surfactant. Here we report the self-assembly behavior of the complexes of DNA with a cationic surfactant, dodecyltrimethylammonium bromide (DTAB), mixed with a neutral lipid, dioleoylphosphatidylethanolamine (DOPE), in fully hydrated state as a function of DTAB-to-DNA base pair molar ratio (x), DOPE-to-DTAB molar ratio (m) and temperature. The binary complexes of DNA with DTAB microphase separated to form hydrophilic and hydrophobic domains without long-range order. Incorporating DOPE into the complexes effectively strengthened the hydrophobic interaction and hence promoted the formations of long-range ordered mesophases, including a condensed multilamellar phase (L(alpha)(c)) at small to intermediate m (m approximately 6). The lyotropic mesophase transition with respect to the change of m was properly predicted by a formula for calculating the packing parameter of amphiphile mixture. In addition to the lyotropic transition, an unusual thermotropic order-order transition (OOT) between L(alpha)(c) and H(II)(c) phases was revealed for the isoelectric complex with m = 3. This OOT was thermally reversible and was postulated to be driven by the reduction of the effective headgroup area due to the release of trapped water molecules.

  3. Synthesis, DNA binding and photocleavage, and cellular uptake of an alkyl chain-linked dinuclear ruthenium(II) complex.

    PubMed

    Liu, Ping; Liu, Jin; Zhang, Yu-Qi; Wu, Bao-Yan; Wang, Ke-Zhi

    2015-02-01

    A dinuclear ruthenium(II) complex [(bpy)2Ru(L(1))Ru(bpy)2]Cl4 {bpy=2,2'-bipyridine, L(1)=1,6-bis(3-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-9H-carbazol-9-yl)hexane} was synthesised and characterized. The calf thymus DNA (ct-DNA) binding properties of the complex were investigated by means of UV-Visible absorption and emission spectrophotometric titrations, ethidium bromide competitive binding, steady-state emission quenching with ferrocyanide, DNA viscosity measurements, and DNA thermal denaturation. The results indicated that the complex avidly binds to ct-DNA most probably through a threading bis-intercalative mode. The complex was also evidenced to act as an efficient DNA photocleaver, and an effective luminescent stain for cytoplasmic of HeLa cells with low cytotoxicity.

  4. Non-metallocene organometallic complexes and related methods and systems

    DOEpatents

    Agapie, Theodor; Golisz, Suzanne Rose; Tofan, Daniel; Bercaw, John E.

    2010-12-07

    A non-metallocene organometallic complex comprising a tridentate ligand and a metal bonded to a tridentate ligand, wherein two substituted aryl groups in the tridentate ligand are connected to a cyclic group at the ortho position via semi-rigid ring-ring linkages, and selected so to provide the resulting non-metallocene organometallic complex with a C.sub.S geometry, a C.sub.1 geometry, a C.sub.2 geometry or a C.sub.2v geometry. Method for performing olefin polymerization with a non-metallocene organometallic complex as a catalyst, related catalytic systems, tridentate ligand and method for providing a non-metallocene organometallic complex.

  5. Complex wireframe DNA origami nanostructures with multi-arm junction vertices.

    PubMed

    Zhang, Fei; Jiang, Shuoxing; Wu, Siyu; Li, Yulin; Mao, Chengde; Liu, Yan; Yan, Hao

    2015-09-01

    Structural DNA nanotechnology and the DNA origami technique, in particular, have provided a range of spatially addressable two- and three-dimensional nanostructures. These structures are, however, typically formed of tightly packed parallel helices. The development of wireframe structures should allow the creation of novel designs with unique functionalities, but engineering complex wireframe architectures with arbitrarily designed connections between selected vertices in three-dimensional space remains a challenge. Here, we report a design strategy for fabricating finite-size wireframe DNA nanostructures with high complexity and programmability. In our approach, the vertices are represented by n × 4 multi-arm junctions (n = 2-10) with controlled angles, and the lines are represented by antiparallel DNA crossover tiles of variable lengths. Scaffold strands are used to integrate the vertices and lines into fully assembled structures displaying intricate architectures. To demonstrate the versatility of the technique, a series of two-dimensional designs including quasi-crystalline patterns and curvilinear arrays or variable curvatures, and three-dimensional designs including a complex snub cube and a reconfigurable Archimedean solid were constructed.

  6. Complex wireframe DNA origami nanostructures with multi-arm junction vertices

    NASA Astrophysics Data System (ADS)

    Zhang, Fei; Jiang, Shuoxing; Wu, Siyu; Li, Yulin; Mao, Chengde; Liu, Yan; Yan, Hao

    2015-09-01

    Structural DNA nanotechnology and the DNA origami technique, in particular, have provided a range of spatially addressable two- and three-dimensional nanostructures. These structures are, however, typically formed of tightly packed parallel helices. The development of wireframe structures should allow the creation of novel designs with unique functionalities, but engineering complex wireframe architectures with arbitrarily designed connections between selected vertices in three-dimensional space remains a challenge. Here, we report a design strategy for fabricating finite-size wireframe DNA nanostructures with high complexity and programmability. In our approach, the vertices are represented by n × 4 multi-arm junctions (n = 2-10) with controlled angles, and the lines are represented by antiparallel DNA crossover tiles of variable lengths. Scaffold strands are used to integrate the vertices and lines into fully assembled structures displaying intricate architectures. To demonstrate the versatility of the technique, a series of two-dimensional designs including quasi-crystalline patterns and curvilinear arrays or variable curvatures, and three-dimensional designs including a complex snub cube and a reconfigurable Archimedean solid were constructed.

  7. The solution structure of a specific GAGA factor-DNA complex reveals a modular binding mode.

    PubMed

    Omichinski, J G; Pedone, P V; Felsenfeld, G; Gronenborn, A M; Clore, G M

    1997-02-01

    The structure of a complex between the DNA binding domain of the GAGA factor (GAGA-DBD) and an oligonucleotide containing its GAGAG consensus binding site has been determined by nuclear magnetic resonance spectroscopy. The GAGA-DBD comprises a single classical Cys2-His2 zinc finger core, and an N-terminal extension containing two highly basic regions, BR1 and BR2. The zinc finger core binds in the major groove and recognizes the first three GAG bases of the consensus in a manner similar to that seen in other classical zinc finger-DNA complexes. Unlike the latter, which require tandem zinc finger repeats with a minimum of two units for high affinity binding, the GAGA-DBD makes use of only a single finger complemented by BR1 and BR2. BR2 forms a helix that interacts in the major groove recognizing the last G of the consensus, while BR1 wraps around the DNA in the minor groove and recognizes the A in the fourth position of the consensus. The implications of the structure of the GAGA-DBD-DNA complex for chromatin remodelling are discussed. PMID:9033593

  8. Directing folding pathways for multi-component DNA origami nanostructures with complex topology

    NASA Astrophysics Data System (ADS)

    Marras, A. E.; Zhou, L.; Kolliopoulos, V.; Su, H.-J.; Castro, C. E.

    2016-05-01

    Molecular self-assembly has become a well-established technique to design complex nanostructures and hierarchical mesoscale assemblies. The typical approach is to design binding complementarity into nucleotide or amino acid sequences to achieve the desired final geometry. However, with an increasing interest in dynamic nanodevices, the need to design structures with motion has necessitated the development of multi-component structures. While this has been achieved through hierarchical assembly of similar structural units, here we focus on the assembly of topologically complex structures, specifically with concentric components, where post-folding assembly is not feasible. We exploit the ability to direct folding pathways to program the sequence of assembly and present a novel approach of designing the strand topology of intermediate folding states to program the topology of the final structure, in this case a DNA origami slider structure that functions much like a piston-cylinder assembly in an engine. The ability to program the sequence and control orientation and topology of multi-component DNA origami nanostructures provides a foundation for a new class of structures with internal and external moving parts and complex scaffold topology. Furthermore, this work provides critical insight to guide the design of intermediate states along a DNA origami folding pathway and to further understand the details of DNA origami self-assembly to more broadly control folding states and landscapes.

  9. Dinuclear Zinc (II) Complexes of Macrocyclic Polyamine Ligands Containing an Imidazolium Bridge: Synthesis, Characterization, and Their Interaction with Plasmid DNA

    PubMed Central

    Huang, Jun; Huang, Qing-Dong; Zhang, Ji; Zhou, Li-Hong; Li, Qiang-Lin; Li, Kun; Jiang, Ning; Lin, Hong-Hui; Wu, Jiang; Yu, Xiao-Qi

    2007-01-01

    Two novel macrocyclic polyamine ligands and their dinuclear zinc (II) complexes were synthesized and characterized. Their interaction with plasmid DNA was studied by gel electrophoresis and fluorescence quenching experiment. The result showed that these complexes could bind DNA efficiently under physiological conditions.

  10. Evaluation of DNA binding, DNA cleavage, protein binding, radical scavenging and in vitro cytotoxic activities of ruthenium(II) complexes containing 2,4-dihydroxy benzylidene ligands.

    PubMed

    Mohanraj, Maruthachalam; Ayyannan, Ganesan; Raja, Gunasekaran; Jayabalakrishnan, Chinnasamy

    2016-12-01

    The new ruthenium(II) complexes with hydrazone ligands, 4-Methyl-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(1)), 4-Methoxy-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(2)), 4-Bromo-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(3)), were synthesized and characterized by various spectro analytical techniques. The molecular structures of the ligands were confirmed by single crystal X-ray diffraction technique. The DNA binding studies of the ligands and complexes were examined by absorption, fluorescence, viscosity and cyclic voltammetry methods. The results indicated that the ligands and complexes could interact with calf thymus DNA (CT-DNA) through intercalation. The DNA cleavage activity of the complexes was evaluated by gel electrophoresis assay, which revealed that the complexes are good DNA cleaving agents. The binding interaction of the ligands and complexes with bovine serum albumin (BSA) was investigated using fluorescence spectroscopic method. Antioxidant studies showed that the complexes have a strong radical scavenging properties. Further, the cytotoxic effect of the complexes examined on cancerous cell lines showed that the complexes exhibit significant anticancer activity. PMID:27612830

  11. Crystal Structure of the Human NKX2.5 Homeodomain in Complex with DNA Target

    SciTech Connect

    Pradhan, Lagnajeet; Genis, Caroli; Scone, Peyton; Weinberg, Ellen O.; Kasahara, Hideko; Nam, Hyun-Joo

    2012-10-16

    NKX2.5 is a homeodomain containing transcription factor regulating cardiac formation and function, and its mutations are linked to congenital heart disease. Here we provide the first report of the crystal structure of the NKX2.5 homeodomain in complex with double-stranded DNA of its endogenous target, locating within the proximal promoter -242 site of the atrial natriuretic factor gene. The crystal structure, determined at 1.8 {angstrom} resolution, demonstrates that NKX2.5 homeodomains occupy both DNA binding sites separated by five nucleotides without physical interaction between themselves. The two homeodomains show identical conformation despite the differences in the DNA sequences they bind, and no significant bending of the DNA was observed. Tyr54, absolutely conserved in NK2 family proteins, mediates sequence-specific interaction with the TAAG motif. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients.

  12. Structure of the Human Rev1-DNA-dNTP Ternary Complex

    SciTech Connect

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

    2009-01-01

    Y-family DNA polymerases have proven to be remarkably diverse in their functions and in strategies for replicating through DNA lesions. The structure of yeast Rev1 ternary complex has revealed the most radical replication strategy, where the polymerase itself dictates the identity of the incoming nucleotide, as well as the identity of the templating base. We show here that many of the key elements of this highly unusual strategy are conserved between yeast and human Rev1, including the eviction of template G from the DNA helix and the pairing of incoming deoxycytidine 5'-triphosphate with a surrogate arginine residue. We also show that the catalytic core of human Rev1 is uniquely augmented by two large inserts, I1 and I2, wherein I1 extends > 20 A away from the active site and may serve as a platform for protein-protein interactions specific for Rev1's role in translesion DNA synthesis in human cells, and I2 acts as a 'flap' on the hydrophobic pocket accommodating template G. We suggest that these novel structural features are important for providing human Rev1 greater latitude in promoting efficient and error-free translesion DNA synthesis through the diverse array of bulky and potentially carcinogenic N2-deoxyguanosine DNA adducts in human cells.

  13. Efficacy of cationic lipid-DNA complexes (CLDC) on hepatitis B virus in transgenic mice.

    PubMed

    Morrey, John D; Motter, Neil E; Taro, Brandon; Lay, Marla; Fairman, Jeffery

    2008-07-01

    Cationic lipid-DNA (non-coding) complexes (CLDC) are activators of the innate immune response that increase survival of rodents with some acute viral infections and cancers. CLDC were evaluated for their ability to impact viral DNA levels in transgenic mice carrying an infectious clone of hepatitis B virus (HBV). Mice used in the studies were diet-restricted as nursing pups from solid food, because the expression of HBV DNA in the liver was increased above background levels in some mice with this restriction. Survival surgery was performed on these mice to obtain liver biopsies from which to select animals with suitable levels of liver HBV DNA for entry into the experimental protocols. Intravenous administration of 5 microg/mouse of CLDC on days 1, 7 and 13 reduced liver HBV DNA to similar low levels achieved with the positive control, adefovir dipivoxil. In a subsequent experiment, the same treatment schedule was used to determine that the minimal effective CLDC dose was between 0.5 and 0.05 microg/mouse. Selective cytokines were increased in the livers of CLDC-treated compared to placebo-treated mice in a dose-responsive manner. CLDC were effective in reducing liver HBV DNA and could be considered for further evaluation in other hepatitis models. PMID:18358544

  14. Structures of complexes comprised of Fischerella transcription factor HetR with Anabaena DNA targets.

    PubMed

    Kim, Youngchang; Ye, Zi; Joachimiak, Grazyna; Videau, Patrick; Young, Jasmine; Hurd, Kathryn; Callahan, Sean M; Gornicki, Piotr; Zhao, Jindong; Haselkorn, Robert; Joachimiak, Andrzej

    2013-05-01

    HetR is an essential regulator of heterocyst development in cyanobacteria. Many mutations in HetR render Anabaena incapable of nitrogen fixation. The protein binds to a DNA palindrome upstream of hetP and other genes. We have determined the crystal structures of HetR complexed with palindromic DNA targets, 21, 23, and 29 bp at 2.50-, 3.00-, and 3.25-Å resolution, respectively. The highest-resolution structure shows fine details of specific protein-DNA interactions. The lower-resolution structures with longer DNA duplexes have similar interaction patterns and show how the flap domains interact with DNA in a sequence nonspecific fashion. Fifteen of 15 protein-DNA contacts predicted on the basis of the structure were confirmed by single amino acid mutations that abolished binding in vitro and complementation in vivo. A striking feature of the structure is the association of glutamate 71 from each subunit of the HetR dimer with three successive cytosines in each arm of the palindromic target, a feature that is conserved among all known heterocyst-forming cyanobacteria sequenced to date. PMID:23610410

  15. Complex interactions between the DNA-damage response and mammalian telomeres

    PubMed Central

    Arnoult, Nausica; Karlseder, Jan

    2016-01-01

    Natural chromosome ends resemble double-stranded DNA breaks, but they do not activate a damage response in healthy cells. Telomeres therefore have evolved to solve the ‘end-protection problem’ by inhibiting multiple DNA damage–response pathways. During the past decade, the view of telomeres has progressed from simple caps that hide chromosome ends to complex machineries that have an active role in organizing the genome. Here we focus on mammalian telomeres and summarize and interpret recent discoveries in detail, focusing on how repair pathways are inhibited, how resection and replication are controlled and how these mechanisms govern cell fate during senescence, crisis and transformation. PMID:26581520

  16. Cooperativity or phase transition? Unfolding transition of DNA cationic surfactant complex

    NASA Astrophysics Data System (ADS)

    Mel'nikov, Sergey M.; Sergeyev, Vladimir G.; Yoshikawa, Kenichi; Takahashi, Hiroshi; Hatta, Ichiro

    1997-11-01

    We recently reported that single duplex DNA, with the size above the order of several tens kilobase pairs, undergoes a large discrete transition from an elongated coil into a collapsed globule with the addition of a cationic surfactant. In the present article, we describe the manner of the unfolding transition of compact long DNA, or globule DNA, complexed with cationic surfactants, cetyltrimethylammonium bromide (CTAB) and distearyldimethylammonium bromide (D18DAB), as is induced by the addition of sodium bromide. The conformational dynamics of individual single duplex T4DNA molecules was directly observed with the use of fluorescence microscopy. We found that on the level of individual DNAs, the salt-induced unfolding transition of the globules is largely discrete, or first-order phase transition for the both complexes with CTAB and D18DAB. On the other hand, for the ensemble average of the DNAs, the transition is discrete with CTAB but is continuous (sigmoidal) with D18DAB. The discreteness for the coil-globule transition in the ensemble of DNAs complexed with CTAB is attributed to the existence of the phase transition in whole over the bulk solution: the sphere-rod transition in surfactant micelles. On the other hand, for D18DAB such phase transition on the micelle structure in the bulk solution seems to be absent. In correspondence to such a large difference on the manner of the transition, x-ray diffraction analysis indicates marked difference on the structure of DNA complexes with CTAB and with D18DAB.

  17. DNA fragmentation, transgene expression and embryo development after intracytoplasmic injection of DNA-liposome complexes in IVF bovine zygotes.

    PubMed

    Vichera, G; Moro, L N; Buemo, C; Salamone, D

    2014-05-01

    Summary This study was designed to evaluate the quality and viability of bovine embryos produced by in vitro fertilization (IVF), after intracytoplasmic injection of pCX-EGFP-liposome complexes or pBCKIP2.8-liposome complexes (plasmids that codify the human insulin gene). Cleavage, blastocysts and expanded blastocysts rates of these both groups were not different from that of controls (IVF or IVF embryos injected with liposomes alone; IVF-L). The percentage of EGFP-positive (EGFP+) blastocysts was 41.8%. In Experiment 2, the blastocysts obtained after injection of pCX-EGFP-liposome complexes that did or did not express the transgene, were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling) assay at days 6, 7 and 8 of culture in vitro(Bd6, Bd7 and Bd8), in order to evaluate DNA fragmentation. The EGFP+ blastocysts showed different proportions of TUNEL-positive cells (T+) at Bd6, Bd7 and Bd8 (91, 73.7 and 99.5%, respectively) while blastocysts without EGFP expression (EGFP-) showed statistically lower numbers of fragmented nuclei (0, 44.6 and 85%, respectively; P < 0.05). There was no evidence of DNA fragmentation in either Bd6 or Bd7 IVF and IVF-L control blastocysts, but T+ nuclei were detected at Bd8 in both groups (66.4 and 85.8% respectively). Finally, IVF blastocysts (n = 21) injected with insulin-liposome complexes, cultured for 6, 7 and 8 days, were transferred to recipient cows. Pregnancy rates of 18.2% (2/11) and 40% (2/5) resulted from the transfer of Bd6 and Bd7 cells, respectively. Two pregnancies developed to term but they were not transgenic for the insulin gene. In conclusion, EGFP expression affects DNA integrity but not embryo development. Moreover, additional transfers are required in order to overcome the drawbacks generated by in vitro culture length and transgene expression.

  18. The structural basis for partitioning of the XRCC1/DNA ligase III-[alpha] BRCT-mediated dimer complexes

    SciTech Connect

    Cuneo, Matthew J.; Gabel, Scott A.; Krahn, Joseph M.; Ricker, Melissa A.; London, Robert E.

    2011-11-17

    The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-{alpha}. For efficient ligation, ligase III-{alpha} is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-{alpha} BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.

  19. The Structural Basis for Partitioning of the XRCC1/DNA Ligase III-alpha BRCT-mediated Dimer Complexes

    SciTech Connect

    M Cuneo; S Gabel; J Krahn; M Ricker; R London

    2011-12-31

    The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-{alpha}. For efficient ligation, ligase III-{alpha} is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-{alpha} BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.

  20. Docking of ethanamine Schiff base imines & metal (II) complexes, cytotoxicity & DNA interaction studies

    NASA Astrophysics Data System (ADS)

    Sujarani, S.; Ramu, A.

    2015-01-01

    The present study deals with a series of biologically and stereo chemically important novel transition metal (II) Schiff base chelates. The Cu (II), Co (II), Mn (II) and Ni (II) ions containing complexes were synthesized by using diphenylethanamine and 2-hydroxy/2, 4-dihydroxy/2-hydroxy-4-methoxybenzaldehydes. The synthesized complexes were characterized using micro analytical, IR, NMR, ESI-Mass, UV-Visible, cyclic voltammetry and the EPR spectroscopic techniques. The spectral data evidenced the action of ligands as a neutral bidentate Schiff bases, coordinating through azomethine nitrogen and oxygen atom of hydroxyl group. The interaction studies revealed the groove binding nature of complexes with CT-DNA. The ligand and synthesized metal complexes showed cytotoxicity against cancerous cells. The strong binding affinity of the imine and metal complexes was also confirmed by molecular docking studies.

  1. The semiotics of control and modeling relations in complex systems.

    PubMed

    Joslyn, C

    2001-01-01

    We provide a conceptual analysis of ideas and principles from the systems theory discourse which underlie Pattee's semantic or semiotic closure, which is itself foundational for a school of theoretical biology derived from systems theory and cybernetics, and is now being related to biological semiotics and explicated in the relational biological school of Rashevsky and Rosen. Atomic control systems and models are described as the canonical forms of semiotic organization, sharing measurement relations, but differing topologically in that control systems are circularly and models linearly related to their environments. Computation in control systems is introduced, motivating hierarchical decomposition, hybrid modeling and control systems, and anticipatory or model-based control. The semiotic relations in complex control systems are described in terms of relational constraints, and rules and laws are distinguished as contingent and necessary functional entailments, respectively. Finally, selection as a meta-level of constraint is introduced as the necessary condition for semantic relations in control systems and models.

  2. Spectroscopic study on the interaction of ct-DNA with manganese Salen complex containing triphenyl phosphonium groups

    NASA Astrophysics Data System (ADS)

    Dehkordi, Maryam Nejat; Bordbar, Abdol-Khalegh; Lincoln, Per; Mirkhani, Valiollah

    2012-05-01

    The DNA binding properties of a bulky and hydrophobic Schiff base complex of manganese(III) [N,N'-bis(5-(triphenyl phosphonium methyl)salicylidene)-1,2-ethylene diamine chloride Mn(III) acetate] was examined by spectroscopic techniques. UV-vis titration data indicate both hypo and hyperchromic effect with addition of DNA to complex. A competitive binding study showed that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by adding Mn Salen complex. This finding indicates that Mn Salen complex displaces EB from its binding site in DNA. Helix melting studies indicate improvement in the helix stability, and an increase in the melting temperature. The analysis of CD spectra represents the structural changes in DNA due to the binding of Mn Salen complex. The binding constant has been calculated using absorbance and fluorescence data. The results also represent that the binding process proceeds by strong electrostatic and hydrophobic interactions.

  3. Fluorescence studies, DNA binding properties and antimicrobial activity of a dysprosium(III) complex containing 1,10-phenanthroline.

    PubMed

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

    2013-10-01

    Luminescence and binding properties of dysprosium(III) complex containing 1,10-phenanthroline (phen), [Dy(phen)2(OH2)3Cl]Cl2⋅H2O with DNA has been studied by electronic absorption, emission spectroscopy and viscosity measurement. The thermodynamic studies suggest that the interaction process to be endothermic and entropically driven, which indicates that the dysprosium(III) complex might interact with DNA by a non intercalation binding mode. Additionally, the competitive fluorescence study with ethidium bromide and also the effect of iodide ion and salt concentration on fluorescence of the complex-DNA system is investigated. Experimental results indicate that the Dy(III) complex strongly binds to DNA, presumably via groove binding mode. Furthermore, the complex shows a potent antibacterial activity and DNA cleavage ability.

  4. Crystallization and preliminary X-ray analysis of a complex of the FOXO1 and Ets1 DNA-binding domains and DNA

    SciTech Connect

    Choy, Wing W.; Datta, Drishadwatti; Geiger, Catherine A.; Birrane, Gabriel; Grant, Marianne A.

    2013-12-24

    The DNA-binding domains of Ets1 and FOXO1 were expressed, purified, and crystallized in complex with DNA containing a composite sequence for a noncanonical forkhead binding site and an ETS site. Diffraction data were collected to a resolution of 2.2 Å.

  5. Mechanistic study of transfection of chitosan/DNA complexes coated by anionic poly(γ-glutamic acid).

    PubMed

    Liao, Zi-Xian; Peng, Shu-Fen; Ho, Yi-Cheng; Mi, Fwu-Long; Maiti, Barnali; Sung, Hsing-Wen

    2012-04-01

    Chitosan (CS) has been investigated as a non-viral carrier for gene delivery, but resulting in a relatively low transfection. To address this concern, we developed a ternary system comprised the core of CS/DNA complex and the outer coating of an anionic polymer, poly(γ-glutamic acid) (γ-PGA). In molecular dynamic (MD) simulations, we found that γ-PGA was entangle tightly with the excess CS emanating from the surface of test complexes, thus making them more compact. With γ-PGA coating, the extent of test complexes internalized and their transfection efficiency were evidently enhanced. Trypsin treatment induced a concentration-dependent decrease in internalization of the γ-PGA-coated complexes, suggesting a specific protein-mediated endocytosis. The endocytosis inhibition study indicates that the γ-glutamyl transpeptidase (GGT) present on cell membranes was responsible for the uptake of test complexes. The amine group in the N-terminal γ-glutamyl unit on γ-PGA played an essential role in the interaction with GGT. When entangled with CS, the free N-terminal γ-glutamyl unit of γ-PGA on test complexes was exposed and might thus be accommodated within the γ-glutamyl binding pocket of the membrane GGT. Above results suggest that the γ-PGA coating on CS/DNA complexes can significantly enhance their cellular uptake via a specific GGT-mediated pathway. Knowledge of the uptake mechanism is crucial for the development of an efficient vector for gene transfection.

  6. Conformational fluctuations of a protein-DNA complex and the structure and ordering of water around it

    NASA Astrophysics Data System (ADS)

    Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy

    2011-12-01

    Protein-DNA binding is an important process responsible for the regulation of genetic activities in living organisms. The most crucial issue in this problem is how the protein recognizes the DNA and identifies its target base sequences. Water molecules present around the protein and DNA are also expected to play an important role in mediating the recognition process and controlling the structure of the complex. We have performed atomistic molecular dynamics simulations of an aqueous solution of the protein-DNA complex formed between the DNA binding domain of human TRF1 protein and a telomeric DNA. The conformational fluctuations of the protein and DNA and the microscopic structure and ordering of water around them in the complex have been explored. In agreement with experimental studies, the calculations reveal conformational immobilization of the terminal segments of the protein on complexation. Importantly, it is discovered that both structural adaptations of the protein and DNA, and the subsequent correlation between them to bind, contribute to the net entropy loss associated with the complex formation. Further, it is found that water molecules around the DNA are more structured with significantly higher density and ordering than that around the protein in the complex.

  7. A mononuclear zinc(II) complex with piroxicam: Crystal structure, DNA- and BSA-binding studies; in vitro cell cytotoxicity and molecular modeling of oxicam complexes

    NASA Astrophysics Data System (ADS)

    Jannesari, Zahra; Hadadzadeh, Hassan; Amirghofran, Zahra; Simpson, Jim; Khayamian, Taghi; Maleki, Batool

    2015-02-01

    A new mononuclear Zn(II) complex, trans-[Zn(Pir)2(DMSO)2], where Pir- is 4-hydroxy-2-methyl-N-2-pyridyl-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide (piroxicam), has been synthesized and characterized. The crystal structure of the complex was obtained by the single crystal X-ray diffraction technique. The interaction of the complex with DNA and BSA was investigated. The complex interacts with FS-DNA by two binding modes, viz., electrostatic and groove binding (major and minor). The microenvironment and the secondary structure of BSA are changed in the presence of the complex. The anticancer effects of the seven complexes of oxicam family were also determined on the human K562 cell lines and the results showed reasonable cytotoxicities. The interactions of the oxicam complexes with BSA and DNA were modeled by molecular docking and molecular dynamic simulation methods.

  8. Structural insights into the functions of the FANCM-FAAP24 complex in DNA repair.

    PubMed

    Yang, Hui; Zhang, Tianlong; Tao, Ye; Wang, Fang; Tong, Liang; Ding, Jianping

    2013-12-01

    Fanconi anemia (FA) is a genetically heterogeneous disorder associated with deficiencies in the FA complementation group network. FA complementation group M (FANCM) and FA-associated protein 24 kDa (FAAP24) form a stable complex to anchor the FA core complex to chromatin in repairing DNA interstrand crosslinks. Here, we report the first crystal structure of the C-terminal segment of FANCM in complex with FAAP24. The C-terminal segment of FANCM and FAAP24 both consist of a nuclease domain at the N-terminus and a tandem helix-hairpin-helix (HhH)2 domain at the C-terminus. The FANCM-FAAP24 complex exhibits a similar architecture as that of ApXPF. However, the variations of several key residues and the electrostatic property at the active-site region render a catalytically inactive nuclease domain of FANCM, accounting for the lack of nuclease activity. We also show that the first HhH motif of FAAP24 is a potential binding site for DNA, which plays a critical role in targeting FANCM-FAAP24 to chromatin. These results reveal the mechanistic insights into the functions of FANCM-FAAP24 in DNA repair. PMID:24003026

  9. Structural Mechanism behind Distinct Efficiency of Oct4/Sox2 Proteins in Differentially Spaced DNA Complexes

    PubMed Central

    Yesudhas, Dhanusha; Anwar, Muhammad Ayaz; Panneerselvam, Suresh; Durai, Prasannavenkatesh; Shah, Masaud; Choi, Sangdun

    2016-01-01

    The octamer-binding transcription factor 4 (Oct4) and sex-determining region Y (SRY)-box 2 (Sox2) proteins induce various transcriptional regulators to maintain cellular pluripotency. Most Oct4/Sox2 complexes have either 0 base pairs (Oct4/Sox20bp) or 3 base pairs (Oct4/Sox23bp) separation between their DNA-binding sites. Results from previous biochemical studies have shown that the complexes separated by 0 base pairs are associated with a higher pluripotency rate than those separated by 3 base pairs. Here, we performed molecular dynamics (MD) simulations and calculations to determine the binding free energy and per-residue free energy for the Oct4/Sox20bp and Oct4/Sox23bp complexes to identify structural differences that contribute to differences in induction rate. Our MD simulation results showed substantial differences in Oct4/Sox2 domain movements, as well as secondary-structure changes in the Oct4 linker region, suggesting a potential reason underlying the distinct efficiencies of these complexes during reprogramming. Moreover, we identified key residues and hydrogen bonds that potentially facilitate protein-protein and protein-DNA interactions, in agreement with previous experimental findings. Consequently, our results confess that differential spacing of the Oct4/Sox2 DNA binding sites can determine the magnitude of transcription of the targeted genes during reprogramming. PMID:26790000

  10. Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome-DNA complexes

    NASA Astrophysics Data System (ADS)

    Torchilin, Vladimir P.; Levchenko, Tatyana S.; Rammohan, Ram; Volodina, Natalia; Papahadjopoulos-Sternberg, Brigitte; D'Souza, Gerard G. M.

    2003-02-01

    Liposomes modified with TAT peptide (TATp-liposomes) showed fast and efficient translocation into the cell cytoplasm with subsequent migration into the perinuclear zone. TATp-liposomes containing a small quantity (10 mol %) of a cationic lipid formed firm noncovalent complexes with DNA. Here, we present results demonstrating both in vitro and in vivo transfection with TATp-liposome-DNA complexes. Mouse NIH/3T3 fibroblasts and rat H9C2 cardiomyocytes were transfected with such complexes in vitro. The transfection with the TATp-liposome-associated pEGFP-N1 plasmid encoding for the green fluorescent protein (GFP) was high, whereas the cytotoxicity was lower than that of commonly used cationic lipid-based gene-delivery systems. Intratumoral injection of TATp-liposome-DNA complexes into the Lewis lung carcinoma tumor of mice also resulted in an expression of GFP in tumor cells. This transfection system should be useful for various protocols of cell treatment in vitro or ex vivo as well as for localized in vivo gene therapy.

  11. The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis

    PubMed Central

    Haye, Joanna E.; Gammie, Alison E.

    2015-01-01

    During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/β to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles. PMID:26684201

  12. Architecture and DNA recognition elements of the Fanconi anemia FANCM-FAAP24 complex.

    PubMed

    Coulthard, Rachel; Deans, Andrew J; Swuec, Paolo; Bowles, Maureen; Costa, Alessandro; West, Stephen C; McDonald, Neil Q

    2013-09-01

    Fanconi anemia (FA) is a disorder associated with a failure in DNA repair. FANCM (defective in FA complementation group M) and its partner FAAP24 target other FA proteins to sites of DNA damage. FANCM-FAAP24 is related to XPF/MUS81 endonucleases but lacks endonucleolytic activity. We report a structure of an FANCM C-terminal fragment (FANCMCTD) bound to FAAP24 and DNA. This S-shaped structure reveals the FANCM (HhH)2 domain is buried, whereas the FAAP24 (HhH)2 domain engages DNA. We identify a second DNA contact and a metal center within the FANCM pseudo-nuclease domain and demonstrate that mutations in either region impair double-stranded DNA binding in vitro and FANCM-FAAP24 function in vivo. We show the FANCM translocase domain lies in proximity to FANCMCTD by electron microscopy and that binding fork DNA structures stimulate its ATPase activity. This suggests a tracking model for FANCM-FAAP24 until an encounter with a stalled replication fork triggers ATPase-mediated fork remodeling. PMID:23932590

  13. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

    PubMed Central

    Gell, D; Jackson, S P

    1999-01-01

    In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80. PMID:10446239

  14. Bacteriophage T4 Dam DNA-[N6-adenine]methyltransferase. Kinetic evidence for a catalytically essential conformational change in the ternary complex.

    PubMed

    Evdokimov, Alexey A; Zinoviev, Victor V; Malygin, Ernst G; Schlagman, Samuel L; Hattman, Stanley

    2002-01-01

    We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N6-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction from S-adenosyl-l-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA(Me) and S-adenosyl-l-homocysteine, AdoHcy) release was AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNA(Me) dissociates relatively rapidly (k(off) = 1.7 x s(-1)) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (k(off) = 0.018 x s(-1)), indicating that its release is the rate-limiting step, consistent with kcat = 0.015 x s(-1). After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is

  15. A reinvestigation of phylogeny and divergence times of the Ablepharus kitaibelii species complex (Sauria, Scincidae) based on mtDNA and nuDNA genes.

    PubMed

    Skourtanioti, Eirini; Kapli, Paschalia; Ilgaz, Çetin; Kumlutaş, Yusuf; Avcı, Aziz; Ahmadzadeh, Faraham; Crnobrnja-Isailović, Jelka; Gherghel, Iulian; Lymberakis, Petros; Poulakakis, Nikos

    2016-10-01

    Morphological and DNA data support that the East Mediterranean snake-eyed skink Ablepharus kitaibelii represents a species complex that includes four species A. kitaibelii, A. budaki, A. chernovi, and A. rueppellii, highlighting the need of its taxonomic reevaluation. Here, we used Bayesian and Maximum Likelihood methods to estimate the phylogenetic relationships of all members of the complex based on two mitochondrial (cyt b, 16S rRNA) and two nuclear markers (MC1R, and NKTR) and using Chalcides, Eumeces, and Eutropis as outgroups. The biogeographic history of the complex was also investigated through the application of several phylogeographic (BEAST) and biogeographic (BBM) analyses. Paleogeographic and paleoclimatic data were used to support the inferred phylogeographic patterns. The A. kitaibelli species complex exhibits high genetic diversity, revealing cases of hidden diversity and cases of non-monophyletic species such as A. kitaibelii and A. budaki. Our results indicate that A. pannonicus branches off first and a group that comprises specimens of A. kitaibelli and A. budaki from Kastelorizo Island group (southeast Greece) and southwest Turkey, respectively is differentiated from the rest A. kitaibelli and A. budaki populations and may represent a new species. The estimated divergence times place the origin of the complex in the Middle Miocene (∼16Mya) and the divergence of most currently recognized species in the Late Miocene. The inferred ancestral distribution suggests that the complex originated in Anatolia, supposing that several vicariance and dispersal events that are related with the formation of the Mid-Aegean Trench, the Anatolian Diagonal and the orogenesis of the mountain chains in southern and eastern Anatolia have led to current distribution pattern of A. kitaibelii species complex in the Balkans and Middle East. PMID:27404043

  16. A reinvestigation of phylogeny and divergence times of the Ablepharus kitaibelii species complex (Sauria, Scincidae) based on mtDNA and nuDNA genes.

    PubMed

    Skourtanioti, Eirini; Kapli, Paschalia; Ilgaz, Çetin; Kumlutaş, Yusuf; Avcı, Aziz; Ahmadzadeh, Faraham; Crnobrnja-Isailović, Jelka; Gherghel, Iulian; Lymberakis, Petros; Poulakakis, Nikos

    2016-10-01

    Morphological and DNA data support that the East Mediterranean snake-eyed skink Ablepharus kitaibelii represents a species complex that includes four species A. kitaibelii, A. budaki, A. chernovi, and A. rueppellii, highlighting the need of its taxonomic reevaluation. Here, we used Bayesian and Maximum Likelihood methods to estimate the phylogenetic relationships of all members of the complex based on two mitochondrial (cyt b, 16S rRNA) and two nuclear markers (MC1R, and NKTR) and using Chalcides, Eumeces, and Eutropis as outgroups. The biogeographic history of the complex was also investigated through the application of several phylogeographic (BEAST) and biogeographic (BBM) analyses. Paleogeographic and paleoclimatic data were used to support the inferred phylogeographic patterns. The A. kitaibelli species complex exhibits high genetic diversity, revealing cases of hidden diversity and cases of non-monophyletic species such as A. kitaibelii and A. budaki. Our results indicate that A. pannonicus branches off first and a group that comprises specimens of A. kitaibelli and A. budaki from Kastelorizo Island group (southeast Greece) and southwest Turkey, respectively is differentiated from the rest A. kitaibelli and A. budaki populations and may represent a new species. The estimated divergence times place the origin of the complex in the Middle Miocene (∼16Mya) and the divergence of most currently recognized species in the Late Miocene. The inferred ancestral distribution suggests that the complex originated in Anatolia, supposing that several vicariance and dispersal events that are related with the formation of the Mid-Aegean Trench, the Anatolian Diagonal and the orogenesis of the mountain chains in southern and eastern Anatolia have led to current distribution pattern of A. kitaibelii species complex in the Balkans and Middle East.

  17. The cis-Diammineplatinum(II) Complex of Curcumin: A Dual Action DNA Crosslinking and Photochemotherapeutic Agent.

    PubMed

    Mitra, Koushambi; Gautam, Srishti; Kondaiah, Paturu; Chakravarty, Akhil R

    2015-11-16

    [Pt(cur)(NH3)2](NO3) (1), a curcumin-bound cis-diammineplatinum(II) complex, nicknamed Platicur, as a novel photoactivated chemotherapeutic agent releases photoactive curcumin and an active platinum(II) species upon irradiation with visible light. The hydrolytic instability of free curcumin reduces upon binding to platinum(II). Interactions of 1 with 5'-GMP and ct-DNA indicated formation of platinum-bound DNA adducts upon exposure to visible light (λ=400-700 nm). It showed apoptotic photocytotoxicity in cancer cells (IC50 ≈ 15 μM), thus forming (⋅)OH, while remaining passive in the darkness (IC50 >200 μM). A comet assay and platinum estimation suggest Pt-DNA crosslink formation. The fluorescence microscopic images showed cytosolic localization of curcumin, thus implying possibility of dual action as a chemo- and phototherapeutic agent.

  18. A naproxen complex of dysprosium intercalates into calf thymus DNA base pairs

    NASA Astrophysics Data System (ADS)

    Yang, Mengsi; Jin, Jianhua; Xu, Guiqing; Cui, Fengling; Luo, Hongxia

    2014-01-01

    The binding mode and mechanism of dysprosium-naproxen complex (Dy-NAP) with calf thymus deoxyribonucleic acid (ctDNA) were studied using UV-vis and fluorescence spectra in physiological buffer (pH 7.4). The results showed that more than one type of quenching process occurred and the binding mode between Dy-NAP with ctDNA might be intercalation. In addition, ionic strength, iodide quenching and fluorescence polarization experiments corroborated the intercalation binding mode between Dy-NAP and ctDNA. The calculated thermodynamic parameters ΔG, ΔH and ΔS at different temperature demonstrated that hydrophobic interaction force played a major role in the binding process.

  19. Release of cationic polymer-DNA complexes from the endosome: A theoretical investigation of the proton sponge hypothesis

    NASA Astrophysics Data System (ADS)

    Yang, Shuang; May, Sylvio

    2008-11-01

    Polyplexes are complexes composed of DNA and cationic polymers; they are promising transport vehicles for nonviral gene delivery. Cationic polymers that contain protonatable groups, such as polyethylenimine, have been suggested to trigger endosomal escape of polyplexes according to the "proton sponge hypothesis." Here, osmotic swelling is induced by a decrease in the endosomal pH value, leading to an accumulation of polymer charge accompanied by the influx of Cl- ions to maintain overall electroneutrality. We study a theoretical model of the proton sponge mechanism. The model is based on the familiar Poisson-Boltzmann approach, modified so as to account for the presence of ionizable polyelectrolytes within self-consistent field theory with assumed ground state dominance. We consider polyplexes, composed of fixed amounts of DNA and cationic polymer, to coexist with uncomplexed cationic polymer in an enclosing vesicle of fixed volume. For such a system, we calculate the increase in osmotic pressure upon moderately decreasing the pH value and relate that pressure to the rupture tension of the enclosing membrane. Our model predicts membrane rupture upon pH decrease only within a certain range of free polymer content in the vesicle. That range narrows with increasing amount of DNA. Consequently, there exists a maximal amount of DNA that can be incorporated into a vesicle while maintaining the ability of content release through the proton sponge mechanism.

  20. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-05-11

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E₂, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional "repair and survive, or die" hypothesis.

  1. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-01-01

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E₂, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional "repair and survive, or die" hypothesis. PMID:27187358

  2. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    PubMed Central

    Mirzayans, Razmik; Andrais, Bonnie; Kumar, Piyush; Murray, David

    2016-01-01

    It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence) in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress) DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E2, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional “repair and survive, or die” hypothesis. PMID:27187358

  3. Pedagogy of complex relationality exploring complexity theory, neuro-phenomenology, and attentiveness for education

    NASA Astrophysics Data System (ADS)

    Khattar, Randa

    What do the new sciences of complex relationality offer education? This work draws on complexity theory, neurological understandings of biology and phenomenology, and attentiveness to study what the new sciences might offer education and the possibilities of a pedagogical understanding of embodied knowing. Complexity theory provides understandings of local-global relationality, self-organization, far-from-equilibrium conditions, and emergent dynamics that are important for describing pedagogical relationality. In itself, however, complexity theory is lacking an attention to issues of embodiment that respond directly to an ethical understanding of relationality. Phenomenology provides important views on the human experience of perception, for example, Merleau-Ponty's, whose research opens up possibilities for embodiment and attentiveness. At the level of pedagogical practice, I will pose, following biologists Humberto Maturana and Francisco Varela's autopoietic self-making understanding of life, that attentiveness perspectives, which have been largely absent from western pedagogical theory and practice, are crucial to promoting embodied knowing for education. Maturana and Varela's autopoietic perspective offers an embodied understanding of living---and therefore education---which opens up necessary attentive spaces to listen to one another in non judgmental awareness in the present moment of experience. I offer insights into a relationally complex conception of education drawing on this biological and autopoietically-grounded framework. These insights are framed in the context of five clusters of relations: (1) emergence, far-from-equilibrium, and local-global relationality; (2) autopoietic autonomy, structural determination and sensory-motor coupling; (3) triggering perturbations, structural plasticity, and autopoietic organization; (4) knowing as adequate action, domains of interaction, and blind spots; and (5) attentiveness and responsive relationality. Four

  4. DNA binding propensity and nuclease efficacy of biosensitive Schiff base complexes containing pyrazolone moiety: Synthesis and characterization

    NASA Astrophysics Data System (ADS)

    Paulpandiyan, Rajakkani; Raman, Natarajan

    2016-12-01

    A series of novel Co(II), Cu(II), Ni(II) and Zn(II) complexes (1-8) were synthesized from pyrazolone precursor Schiff base(s), obtained by the condensation of 4-amino-2,3-dimethyl-1-phenyl-3-pyrazolin-5-one (4-aminoantipyrine) with cinnamaldehyde/benzaldehyde and respective metal(II) chloride. They have been characterized by elemental analysis, magnetic susceptibility, molar conductance measurements, UV-Vis., IR, NMR, ESI mass spectra and EPR studies. These complexes show lower conductance values, supporting their non-electrolytic nature. Spectroscopic and other analytical data of the complexes suggest octahedral geometry. The binding properties of these complexes with DNA have been explored by electronic absorption spectra, cyclic voltammetry and viscosity measurements which reveal that the complexes have the ability to interact with calf thymus DNA (CT DNA) by intercalative mode. The binding constant (Kb) values clearly signify that the complex 1 has more intercalating ability than other complexes. DNA cleavage efficacy of these complexes with pUC18 DNA has been investigated by gel electrophoresis technique. All the complexes have been found to promote cleavage of pUC18 DNA from the super coiled form I to the open circular form II in presence of hydrogen peroxide. The in vitro antibacterial and antifungal assay, investigated by Minimum Inhibitory Concentration (MIC) method indicates that these complexes are good antimicrobial agents against various pathogens.

  5. Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA.

    PubMed

    Boskovic, Jasminka; Bragado-Nilsson, Elisabeth; Saligram Prabhakar, Bhargrav; Yefimenko, Igor; Martínez-Gago, Jaime; Muñoz, Sergio; Méndez, Juan; Montoya, Guillermo

    2016-09-16

    DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replication. After the assembly of a double heterohexameric MCM2-7 complex at replication origins in G1, the 2 heterohexamers separate from each other and associate with Cdc45 and GINS proteins in a CMG complex that is capable of unwinding dsDNA during S phase. Here, we have reconstituted and characterized the purified human MCM2-7 (hMCM2-7) hexameric complex by co-expression of its 6 different subunits in insect cells. The conformational variability of the complex has been analyzed by single particle electron microscopy in the presence of different nucleotide analogs and DNA. The interaction with nucleotide stabilizes the complex while DNA introduces conformational changes in the hexamer inducing a cylindrical shape. Our studies suggest that the assembly of GINS and Cdc45 to the hMCM2-7 hexamer would favor conformational changes on the hexamer bound to ssDNA shifting the cylindrical shape of the complex into a right-handed spiral conformation as observed in the CMG complex bound to DNA.

  6. A look at the effect of sequence complexity on pressure destabilisation of DNA polymers.

    PubMed

    Rayan, Gamal; Macgregor, Robert B

    2015-04-01

    Our previous studies on the helix-coil transition of double-stranded DNA polymers have demonstrated that molar volume change (ΔV) accompanying the thermally-induced transition can be positive or negative depending on the experimental conditions, that the pressure-induced transition is more cooperative than the heat-induced transition [Rayan and Macgregor, J Phys Chem B2005, 109, 15558-15565], and that the pressure-induced transition does not occur in the absence of water [Rayan and Macgregor, Biophys Chem, 2009, 144, 62-66]. Additionally, we have shown that ΔV values obtained by pressure-dependent techniques differ from those obtained by ambient pressure techniques such as PPC [Rayan et al. J Phys Chem B2009, 113, 1738-1742] thus shedding light on the effects of pressure on DNA polymers. Herein, we examine the effect of sequence complexity, and hence cooperativity on pressure destabilisation of DNA polymers. Working with Clostridium perfringes DNA under conditions such that the estimated ΔV of the helix-coil transition corresponds to -1.78 mL/mol (base pair) at atmospheric pressure, we do not observe the pressure-induced helix-coil transition of this DNA polymer, whereas synthetic copolymers poly[d(A-T)] and poly[d(I-C)] undergo cooperative pressure-induced transitions at similar ΔV values. We hypothesise that the reason for the lack of pressure-induced helix-coil transition of C. perfringens DNA under these experimental conditions lies in its sequence complexity.

  7. The role of DNA repair in brain related disease pathology.

    PubMed

    Canugovi, Chandrika; Misiak, Magdalena; Ferrarelli, Leslie K; Croteau, Deborah L; Bohr, Vilhelm A

    2013-08-01

    Oxidative DNA damage is implicated in brain aging, neurodegeneration and neurological diseases. Damage can be created by normal cellular metabolism, which accumulates with age, or by acute cellular stress conditions which create bursts of oxidative damage. Brain cells have a particularly high basal level of metabolic activity and use distinct oxidative damage repair mechanisms to remove oxidative damage from DNA and dNTP pools. Accumulation of this damage in the background of a functional DNA repair response is associated with normal aging, but defective repair in brain cells can contribute to neurological dysfunction. Emerging research strongly associates three common neurodegenerative conditions, Alzheimer's, Parkinson's and stroke, with defects in the ability to repair chronic or acute oxidative damage in neurons. This review explores the current knowledge of the role of oxidative damage repair in preserving brain function and highlights the emerging models and methods being used to advance our knowledge of the pathology of neurodegenerative disease.

  8. Expanding the scope of CE reactor to ssDNA-binding protein-ssDNA complexes as exemplified for a tool for direct measurement of dissociation kinetics of biomolecular complexes.

    PubMed

    Takahashi, Toru; Ohtsuka, Kei-Ichirou; Tomiya, Yoriyuki; Iki, Nobuhiko; Hoshino, Hitoshi

    2009-09-01

    CE reactor (CER), which was developed as a tool for direct measurement of the dissociation kinetics of metal complexes, was successfully applied to the complexes of Escherichia coli ssDNA-binding protein (SSB) with ssDNA. The basic concept of CER is the application of CE separation process as a dissociation kinetic reactor for the complex, and the observation of the on-capillary dissociation reaction profile of the complex as the decrease of the peak height of the complex with increase of the migration time. The peak height of [SSB-ssDNA] decreases as the migration time increases since the degree of the decrease of [SSB-ssDNA] through the on-capillary dissociation reaction is proportional to the degree of the decrease of the peak height of [SSB-ssDNA]. The dissociation degree-time profiles for the complexes are quantitatively described by analyzing a set of electropherograms with different migration times. Dissociation rate constants of [SSB-ssDNA] consisting of 20-mer, 25-mer and 31-mer ssDNA were directly determined to be 3.99x10(-4), 4.82x10(-4) and 1.50x10(-3)/s, respectively. CER is a concise and effective tool for dissociation kinetic analysis of biomolecular complexes.

  9. Role of intercalation and redox potential in DNA photosensitization by ruthenium(II) polypyridyl complexes: assessment using DNA repair protein tests.

    PubMed

    Gicquel, Etienne; Souchard, Jean-Pierre; Magnusson, Fay; Chemaly, Jad; Calsou, Patrick; Vicendo, Patricia

    2013-08-01

    Here we report that the photoreactivity of ruthenium(II) complexes with nucleobases may not only be modulated by their photoredox properties but also by their DNA binding mode. The damage resulting from photolysis of synthetic oligonucleotides and plasmid DNA by [Ru(bpz)3](2+), [Ru(bipy)3](2+) and the two DNA intercalating agents [Ru(bpz)2dppz](2+) and [Ru(bipy)2dppz](2+) has been monitored by polyacrylamide gel electrophoresis and by tests using proteins involved in DNA repair processes (DNA-PKCs, Ku80, Ku70, and PARP-1). The data show that intercalation controls the nature of the DNA damage photo-induced by ruthenium(II) complexes reacting with DNA via an electron transfer process. The intercalating agent [Ru(bpz)2dppz](2+) is a powerful DNA breaker inducing the formation of both single and double (DSBs) strand breaks which are recognized by the PARP-1 and DNA-PKCs proteins respectively. [Ru(bpz)2dppz](2+) is the first ruthenium(II) complex described in the literature that is able to induce DSBs by an electron transfer process. In contrast, its non-intercalating parent compound, [Ru(bpz)3](2+), is mostly an efficient DNA alkylating agent. Photoadducts are recognized by the proteins Ku70 and Ku80 as with cisplatin adducts. This result suggests that photoaddition of [Ru(bpz)2dppz](2+) is strongly affected by its DNA intercalation whereas its photonuclease activity is exalted. The data clearly show that DNA intercalation decreases drastically the photonuclease activity of ruthenium(II) complexes oxidizing guanine via the production of singlet oxygen. Interestingly, the DNA sequencing data revealed that the ligand dipyridophenazine exhibits on single-stranded oligonucleotides a preference for the 5'-TGCGT-3' sequence. Moreover the use of proteins involved in DNA repair processes to detect DNA damage was a powerful tool to examine the photoreactivity of ruthenium(II) complexes with nucleic acids. PMID:23835850

  10. Replication of single-stranded DNA templates by primase-polymerase complexes of the yeast, Saccharomyces cerevisiae.

    PubMed Central

    Biswas, E E; Biswas, S B

    1988-01-01

    A partially purified primase-polymerase complex from the yeast, Saccharomyces cerevisiae, was capable of replicating a single stranded circular phage DNA into a replicative form with high efficiency. The primase-polymerase complex exhibited primase activity and polymerase activity on singly primed circular ssDNA as well as on gapped DNA. In addition, it was able to replicate an unprimed, single-stranded, circular phage DNA through a coupled primase-polymerase action. On Biogel A-O.5m filtration the primase-polymerase activities appeared in the void volume, demonstrating a mass of greater than 500 kilodaltons. Primase and various primase-polymerase complexes synthesized unique primers on single stranded DNA templates and the size distribution of primers was dependent on the structure of the DNA and the nature of the primase-polymerase assembly. Images PMID:3041377

  11. SNP-microarrays can accurately identify the presence of an individual in complex forensic DNA mixtures.

    PubMed

    Voskoboinik, Lev; Ayers, Sheri B; LeFebvre, Aaron K; Darvasi, Ariel

    2015-05-01

    Common forensic and mass disaster scenarios present DNA evidence that comprises a mixture of several contributors. Identifying the presence of an individual in such mixtures has proven difficult. In the current study, we evaluate the practical usefulness of currently available "off-the-shelf" SNP microarrays for such purposes. We found that a set of 3000 SNPs specifically selected for this purpose can accurately identify the presence of an individual in complex DNA mixtures of various compositions. For example, individuals contributing as little as 5% to a complex DNA mixture can be robustly identified even if the starting DNA amount was as little as 5.0ng and had undergone whole-genome amplification (WGA) prior to SNP analysis. The work presented in this study represents proof-of-principle that our previously proposed approach, can work with real "forensic-type" samples. Furthermore, in the absence of a low-density focused forensic SNP microarray, the use of standard, currently available high-density SNP microarrays can be similarly used and even increase statistical power due to the larger amount of available information.

  12. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    PubMed

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications.

  13. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer

    SciTech Connect

    Lewis, M.; Chang, G.; Horton, N.C.

    1996-03-01

    The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor a product of the lacl gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-B-D-1thiogalactoside (IPTG) and the lac repressor complexed with a 21 base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and the repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quarternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites in the genomic DNA. 76 refs., 11 figs., 1 tab.

  14. In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex

    PubMed Central

    Cao, Bo; Zheng, Xiaoqing; Cheng, Qiuxiang; Yao, Fen; Zheng, Tao; Ramesh Babu, I.; Zhou, Huchen; Dedon, Peter; You, Delin

    2015-01-01

    A wide variety of prokaryotes possess DNA modifications consisting of sequence-specific phosphorothioates (PT) inserted by members of a five-gene cluster. Recent genome mapping studies revealed two unusual features of PT modifications: short consensus sequences and partial modification of a specific genomic site in a population of bacteria. To better understand the mechanism of target selection of PT modifications that underlies these features, we characterized the substrate recognition of the PT-modifying enzymes termed DptC, D and E in a cell extract system from Salmonella. The results revealed that double-stranded oligodeoxynucleotides underwent de novo PT modification in vitro, with the same modification pattern as in vivo, i. e., GpsAAC/GpsTTC motif. Unexpectedly, in these in vitro analyses we observed no significant effect on PT modification by sequences flanking GAAC/GTTC motif, while PT also occurred in the GAAC/GTTC motif that could not be modified in vivo. Hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA. The PT-modifying enzymes were then found to function as a large protein complex, with all of three subunits in tetrameric conformations. This study provided the first demonstration of in vitro DNA PT modification by PT-modifying enzymes that fu