Science.gov

Sample records for relate complex dna

  1. Force-extension relation of DNA-histone complexes

    NASA Astrophysics Data System (ADS)

    Levine, A. J.; Henle, Mark L.; Chou, Tom

    2007-03-01

    In eukaryotic cells, DNA is packaged inside the nucleus in the form of chromatin, a structure whose basic repeat unit, known as the nucleosome, consists of DNA wrapped around a cylindrical complex of histone proteins. In order for the cell to function properly, these nucleosome complexes must be stable at equilibrium. At the same time, the cell must be able to gain access to the genomic information contained within the DNA, which it can achieve by exerting forces on the nucleosomes that cause the DNA to unwrap from the histones. Single molecule mechanical manipulation techniques, in which DNA/histone complexes are disrupted by an external force, can provide information not only about the equilibrium structure of these complexes, but also about the forces and displacements required to access the DNA in the nucleosome. In this talk, we derive the force-extension relation for these complexes. We allow for the DNA to unwrap from the histones in both a continuous and discontinuous fashion; that is, we allow the histones to ``pop'' off of the DNA, releasing a large amount of DNA in the process. We also include the conformational fluctuations of the unwrapped portions of the DNA.

  2. 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.

  3. Stereodefined phosphorothioate analogues of DNA: relative thermodynamic stability of the model PS-DNA/DNA and PS-DNA/RNA complexes.

    PubMed

    Boczkowska, Małgorzata; Guga, Piotr; Stec, Wojciech J

    2002-10-15

    Thermodynamic data regarding the influence of P-chirality on stability of duplexes formed between phosphorothioate DNA oligonucleotides (of either stereo-defined all-R(P) or all-S(P) or random configuration at the P atoms) and complementary DNA or RNA strands are presented. Measured melting temperatures and calculated DeltaG(37)(o) values showed that duplexes formed by PS-DNA oligomers with DNA strands are less stable than their unmodified counterparts. However, relative stability of the duplexes ([all-R(P)]-PS-DNA/DNA vs [all-S(P)]-PS-DNA/DNA) depends on their sequential composition rather than on the absolute configuration of PS-oligos, contrary to the results of theoretical considerations and molecular modeling reported in the literature. On the other hand, for all six analyzed pairs of diastereomers, the [all-R(P)]-PS isomers form more stable duplexes with RNA templates, but the origin of stereodifferentiation depends on the sequence with more favorable entropy and enthalpy factors which correlated with dT-rich and dA/dG-rich PS-oligomers, respectively.

  4. Relative biological effectiveness for photons: implication of complex DNA double-strand breaks as critical lesions.

    PubMed

    Liang, Ying; Fu, Qibin; Wang, Xudong; Liu, Feng; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2017-03-21

    Current knowledge in radiobiology ascribes the adverse biological effects of ionizing radiation primarily to the induction of DNA double-strand breaks (DSBs), which is supposed to be potentially lethal and may be converted to lethal damage due to misrepair. Soft and ultrasoft x-rays have been found to bear elevated biological effectiveness for cell killing compared with conventional x-rays or (60)Co γ-rays. This phenomenon is qualitatively interpreted as the increased level of DSB induction for low energy photons, however, a thorough quantitative reasoning is lacking. Here, we systematically compared the relative biological effectiveness (RBE) with relative DSB induction for photons from several hundreds of eV up to MeV. Although there is an approximate two-fold increase in the yields of DSB for low energy photons found in our calculation and a large number of experimental measurements, it is far from enough to account for the three- to four-fold increase in RBE. Further theoretical investigations show that DSB complexity (additional single-strand breaks and base damage within 10 base pairs) increases notably for low energy photons, which largely reconciles the discrepancy between RBE and DSB induction. Our theoretical results are in line with accumulating experimental evidence that complex DSBs are refractory to repair machinery and may contribute predominantly to the formation of lethal damage.

  5. Relative biological effectiveness for photons: implication of complex DNA double-strand breaks as critical lesions

    NASA Astrophysics Data System (ADS)

    Liang, Ying; Fu, Qibin; Wang, Xudong; Liu, Feng; Yang, Gen; Luo, Chunxiong; Ouyang, Qi; Wang, Yugang

    2017-03-01

    Current knowledge in radiobiology ascribes the adverse biological effects of ionizing radiation primarily to the induction of DNA double-strand breaks (DSBs), which is supposed to be potentially lethal and may be converted to lethal damage due to misrepair. Soft and ultrasoft x-rays have been found to bear elevated biological effectiveness for cell killing compared with conventional x-rays or 60Co γ-rays. This phenomenon is qualitatively interpreted as the increased level of DSB induction for low energy photons, however, a thorough quantitative reasoning is lacking. Here, we systematically compared the relative biological effectiveness (RBE) with relative DSB induction for photons from several hundreds of eV up to MeV. Although there is an approximate two-fold increase in the yields of DSB for low energy photons found in our calculation and a large number of experimental measurements, it is far from enough to account for the three- to four-fold increase in RBE. Further theoretical investigations show that DSB complexity (additional single-strand breaks and base damage within 10 base pairs) increases notably for low energy photons, which largely reconciles the discrepancy between RBE and DSB induction. Our theoretical results are in line with accumulating experimental evidence that complex DSBs are refractory to repair machinery and may contribute predominantly to the formation of lethal damage.

  6. DNA complexes: Durable binders

    NASA Astrophysics Data System (ADS)

    Urbach, Adam R.

    2011-11-01

    A tetra-intercalator compound that threads through a DNA double-helix to form a remarkably stable complex exhibits an unusual combination of sequence specificity and rapid association yet slow dissociation.

  7. DNA/chitosan electrostatic complex.

    PubMed

    Bravo-Anaya, Lourdes Mónica; Soltero, J F Armando; Rinaudo, Marguerite

    2016-07-01

    Up to now, chitosan and DNA have been investigated for gene delivery due to chitosan advantages. It is recognized that chitosan is a biocompatible and biodegradable non-viral vector that does not produce immunological reactions, contrary to viral vectors. Chitosan has also been used and studied for its ability to protect DNA against nuclease degradation and to transfect DNA into several kinds of cells. In this work, high molecular weight DNA is compacted with chitosan. DNA-chitosan complex stoichiometry, net charge, dimensions, conformation and thermal stability are determined and discussed. The influence of external salt and chitosan molecular weight on the stoichiometry is also discussed. The isoelectric point of the complexes was found to be directly related to the protonation degree of chitosan. It is clearly demonstrated that the net charge of DNA-chitosan complex can be expressed in terms of the ratio [NH3(+)]/[P(-)], showing that the electrostatic interactions between DNA and chitosan are the main phenomena taking place in the solution. Compaction of DNA long chain complexed with low molar mass chitosan gives nanoparticles with an average radius around 150nm. Stable nanoparticles are obtained for a partial neutralization of phosphate ionic sites (i.e.: [NH3(+)]/[P(-)] fraction between 0.35 and 0.80).

  8. Effectiveness of the DNA barcoding approach for closely related conifers discrimination: A case study of the Pinus mugo complex.

    PubMed

    Celiński, Konrad; Kijak, Hanna; Wojnicka-Półtorak, Aleksandra; Buczkowska-Chmielewska, Katarzyna; Sokołowska, Joanna; Chudzińska, Ewa

    DNA barcoding is a standard and efficient method, frequently used for identification, discrimination and discovery of new species. Although this approach is very useful for classifying the world's biodiversity, little is known about its usefulness in barcoding at lower taxonomic level and its discrimination rate for closely related species, like conifers. In this study, we compared the genetic variation of eight chloroplast DNA barcode regions (matK, rbcL, trnH-psbA, trnL-trnF, rpl20-rps18, trnV, ycf1, ycf2) in 17 conifers - three closely related pines from Pinus mugo complex and 14 more distant conifers representing two genera and four sections of the Pinaceae family. The discrimination rate for a single and for multiple DNA barcode regions analyzed in this study was estimated using the Tree-Building and PWG-Distance methods. The usefulness of the DNA barcoding approach for analyzing and resolving taxonomic inconsistency among closely related and more phylogenetically distant conifers was evaluated and discussed. Copyright © 2017 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

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

    SciTech Connect

    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; 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.

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

    DOE PAGES

    Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael; ...

    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

  11. 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.

  12. Supramolecular Complexes of DNA

    NASA Astrophysics Data System (ADS)

    Zuber, G.; Scherman, D.

    Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the

  13. Fluoroquinolone-Gyrase-DNA Complexes

    PubMed Central

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

    2014-01-01

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

  14. A ternary AppA-PpsR-DNA complex mediates light regulation of photosynthesis-related gene expression.

    PubMed

    Winkler, Andreas; Heintz, Udo; Lindner, Robert; Reinstein, Jochen; Shoeman, Robert L; Schlichting, Ilme

    2013-07-01

    The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources, depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the bacterium for using light energy. Illumination, however, enhances repression under semiaerobic conditions. Here, we describe molecular details of two proteins mediating oxygen and light control of photosynthesis-gene expression: the light-sensing antirepressor AppA and the transcriptional repressor PpsR. Our crystal structures of both proteins and their complex and hydrogen/deuterium-exchange data show that light activation of AppA-PpsR2 affects the PpsR effector region within the complex. DNA binding studies demonstrate the formation of a light-sensitive ternary AppA-PpsR-DNA complex. We discuss implications of these results for regulation by light and oxygen, highlighting new insights into blue light-mediated signal transduction.

  15. Indirect DNA Readout by an H-NS Related Protein: Structure of the DNA Complex of the C-Terminal Domain of Ler

    PubMed Central

    Cordeiro, Tiago N.; Schmidt, Holger; Madrid, Cristina; Juárez, Antonio; Bernadó, Pau; Griesinger, Christian; García, Jesús; Pons, Miquel

    2011-01-01

    Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family. PMID:22114557

  16. 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.

  17. The Relation between the Physical Properties of Self-Assembling Cationic Lipid:DNA Complexes and Gene Delivery

    NASA Astrophysics Data System (ADS)

    Ahmad, A.; Slack, N. L.; Evans, Heather M.; Lin, Alison; Martin, A.; Safinya, C. R.

    2000-03-01

    The use of cationic lipids (CL) as carriers of genes (DNA sequences) for delivery in cells is a promising alternative to viral-carriers. Previous work on CL:DNA complexes has focused on binary mixtures of lipids and has shown that the optimal gene delivery vehicle may be mediated by physical properties of the lipid self-assembly(1). Using x-ray diffraction and biological assays, we show that membrane charge density and geometric shape may be universal parameters for successful gene delivery by binary CL mixtures in vitro. Preliminary results from complexes containing novel ternary CL mixtures further elucidate key parameters for gene delivery. Funded by NIH R01-GM59288-01 and R37-AI12520-24, UCBiotechnology Research and Education Program (97-02), NSF-DMR-9972246. 1. J. Raedler et al, Science 275, 810 (1997), Koltover et al Science 281, 78-81 (1998), Koltover et al, Biophysical Journal 77, 95 (1999), A. J. Lin, N. L. Slack, A. Ahmad, I. Koltover, C. X. George, C. E. Samuel, C. R. Safinya, Journal of Drug Targeting (to appear)

  18. Complex reconfiguration of DNA nanostructures.

    PubMed

    Wei, Bryan; Ong, Luvena L; Chen, Jeffrey; Jaffe, Alexander S; Yin, Peng

    2014-07-14

    Nucleic acids have been used to create diverse synthetic structural and dynamic systems. Toehold-mediated strand displacement has enabled the construction of sophisticated circuits, motors, and molecular computers. Yet it remains challenging to demonstrate complex structural reconfiguration in which a structure changes from a starting shape to another arbitrarily prescribed shape. To address this challenge, we have developed a general structural-reconfiguration method that utilizes the modularly interconnected architecture of single-stranded DNA tile and brick structures. The removal of one component strand reveals a newly exposed toehold on a neighboring strand, thus enabling us to remove regions of connected component strands without the need to modify the strands with predesigned external toeholds. By using this method, we reconfigured a two-dimensional rectangular DNA canvas into diverse prescribed shapes. We also used this method to reconfigure a three-dimensional DNA cuboid. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. 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…

  20. 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…

  1. The Effects of Extending of Co-planarity in a Series of Structurally Relative Polypyridyl Palladium(II) Complexes on DNA-binding and Cytotoxicity Properties

    PubMed Central

    Shahraki, Somaye; Mansouri-Torshizi, Hassan; Sori Nezami, Ziba; Ghahghaei, Arezou; Yaghoubi, Fatemeh; Divsalar, Adeleh; Saboury, Ali-Akbar; H. Shirazi, Farshad

    2014-01-01

    In depth interaction studies between calf thymus deoxyribonucleic acid (CT-DNA) and a series of four structurally relative palladium(II) complexes [Pd(en)(HB)](NO3)2 (a-d), where en is ethylenediamine and heterocyclic base (HB) is 2,2'-bipyridine (bpy, a); 1,10-phenanthroline (phen, b); dipyridoquinoxaline (dpq, c) and dipyridophenazine (dppz, d) (Figure 1), were performed. These studies have been investigated by utilizing the electronic absorption spectroscopy, fluorescence spectra and ethidium bromide (EBr) displacement and gel filtration techniques. a-d complexes cooperatively bind and denature the DNA at low concentrations. Their concentration at midpoint of transition, L1/2, follows the order a >> b > c > d. Also the g, the number of binding sites per 1000 nucleotides, follows the order a >> b ~ c > d. EBr and Scatchard experiments for a-d complexes suggest efficient intercalative binding affinity to CT-DNA giving the order: d > c > b > a. Several binding and thermodynamic parameters are also described. The biological activity of these cationic and water soluble palladium complexes were tested against chronic myelogenous leukemia cell line, K562. b, c and d complexes show cytotoxic concentration (Cc50) values much lower than cisplatin. PMID:25587317

  2. 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

  3. 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.

  4. Conformation of DNA in chromatin protein-DNA complexes studied by infrared spectroscopy.

    PubMed Central

    Liquier, J; Gadenne, M C; Taillandier, E; Defer, N; Favatier, F; Kruh, J

    1979-01-01

    The following observations concerning the DNA secondary structures in various nucleohistone complexes were made by infrared spectroscopy: 1/ in chromatin, chromatin extracted by 0.6 M NaCl, nucleosomes, and histone-DNA reconstituted complexes, the DNA remains in a B type conformation at low relative hygrometry; 2/ in chromatin extracted by tRNA and in non histone protein-DNA reconstituted complexes, the DNA can adopt an A type conformation. Infrared linear dichroism data show that in NHP-DNA complexes the low relative hygrometry conformation of DNA may be modified and that the infrared parameter -1090 is close to that measured for RNA's or DNA-RNA hybrids. It is concluded that the histones block the DNA in a B form and that some of the NHP could be involved in the control of the secondary structure of DNA in chromatin. Images PMID:450704

  5. Imaging of DNA and Protein-DNA Complexes with Atomic Force Microscopy.

    PubMed

    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.

  6. 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

  7. DNA clustering and genome complexity.

    PubMed

    Dios, Francisco; Barturen, Guillermo; Lebrón, Ricardo; Rueda, Antonio; Hackenberg, Michael; Oliver, José L

    2014-12-01

    Early global measures of genome complexity (power spectra, the analysis of fluctuations in DNA walks or compositional segmentation) uncovered a high degree of complexity in eukaryotic genome sequences. The main evolutionary mechanisms leading to increases in genome complexity (i.e. gene duplication and transposon proliferation) can all potentially produce increases in DNA clustering. To quantify such clustering and provide a genome-wide description of the formed clusters, we developed GenomeCluster, an algorithm able to detect clusters of whatever genome element identified by chromosome coordinates. We obtained a detailed description of clusters for ten categories of human genome elements, including functional (genes, exons, introns), regulatory (CpG islands, TFBSs, enhancers), variant (SNPs) and repeat (Alus, LINE1) elements, as well as DNase hypersensitivity sites. For each category, we located their clusters in the human genome, then quantifying cluster length and composition, and estimated the clustering level as the proportion of clustered genome elements. In average, we found a 27% of elements in clusters, although a considerable variation occurs among different categories. Genes form the lowest number of clusters, but these are the longest ones, both in bp and the average number of components, while the shortest clusters are formed by SNPs. Functional and regulatory elements (genes, CpG islands, TFBSs, enhancers) show the highest clustering level, as compared to DNase sites, repeats (Alus, LINE1) or SNPs. Many of the genome elements we analyzed are known to be composed of clusters of low-level entities. In addition, we found here that the clusters generated by GenomeCluster can be in turn clustered into high-level super-clusters. The observation of 'clusters-within-clusters' parallels the 'domains within domains' phenomenon previously detected through global statistical methods in eukaryotic sequences, and reveals a complex human genome landscape dominated

  8. Dlx5 homedomain/DNA complex; Structure, binding and effect of mutations related to split-hand and foot malformation syndrome

    PubMed Central

    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

    2016-01-01

    SUMMARY 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; 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. PMID:26829219

  9. Molecular phylogenetics of the Espeletia complex (Asteraceae): evidence from nrDNA ITS sequences on the closest relatives of an Andean adaptive radiation.

    PubMed

    Rauscher, Jason T

    2002-07-01

    The subtribe Espeletiinae (Asteraceae, Heliantheae) comprises morphologically and ecologically diverse plants endemic to the tropical montane paramos of the Andes of Venezuela, Colombia, and Ecuador. Though the ecophysiology and ecology of this adaptive radiation have been well studied, relationships among taxa in the subtribe and between the subtribe and other taxa in the Heliantheae are poorly known. In this study, sequences from the internal transcribed spacer (ITS) region of nuclear ribosomal DNA are used to test previous hypotheses about the phylogenetic position of the Espeletiinae within the Heliantheae and to determine which taxa are the subtribe's closest relatives. Gene phylogenies based on maximum parsimony analyses reveal that the Espeletiinae clade is nested well within the subtribe Melampodiinae and thus should be considered a monophyletic complex of species, not a separate subtribe. The most parsimonious gene trees suggest that the genus Ichthyothere may be the sister taxon to the Espeletia complex and that the genus Smallanthus and a species of Rumfordia are likely among the complex's other closest living relatives. These data offer preliminary insights into the origins of this adaptive radiation and the broader phylogenetic context in which it occurred.

  10. 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.

  11. Molecular differentiation of three closely related members of the mosquito species complex, Anopheles moucheti, by mitochondrial and ribosomal DNA polymorphism.

    PubMed

    Kengne, P; Antonio-Nkondjio, C; Awono-Ambene, H P; Simard, F; Awolola, T S; Fontenille, D

    2007-06-01

    Distinction between members of the equatorial Africa malaria vector Anopheles moucheti (Evans) s.l. (Diptera: Culicidae) has been based mainly on doubtful morphological features. To determine the level of genetic differentiation between the three morphological forms of this complex, we investigated molecular polymorphism in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2). The three genomic regions revealed sequence differences between the three morphological forms similar in degree to the differences shown previously for members of other anopheline species groups or complexes (genetic distance d = 0.047-0.05 for CytB, 0.084-0.166 for ITS1 and 0.03-0.05 for ITS2). Using sequence variation in the ITS1 region, we set up a diagnostic polymerase chain reaction (PCR) for rapid and reliable identification of each subspecies within the An. moucheti complex. Specimens of An. moucheti s.l. collected in Cameroon, the Democratic Republic of Congo (DRC), Uganda and Nigeria were successfully identified, demonstrating the general applicability of this technique.

  12. Hydrodynamic properties of DNA and DNA-lipid complex in an elongational flow field.

    PubMed

    Sasaki, Naoki; Ashitaka, Hidetomo; Ohtomo, Kenji; Fukui, Akimasa

    2007-03-10

    The aim of this study was to determine the difference between hydrodynamic properties of DNA-cetyltrimethylammonium (CTA) complex and those of DNA, which may be related to the difference in fibre-forming ability of DNA-CTA from that of DNA. Responses of DNA and DNA-CTA complex to an elongational flow field were investigated. In both solution systems, results suggesting a coil-stretch transition were obtained. From a critical strain rate value, the radius of gyration of DNA-CTA molecules in ethanol-glycerol solution was revealed to be 0.3-0.5 times of that of DNA in aqueous NaCl solution. Shear viscosity of DNA-CTA solution was much smaller than that of DNA solution, also suggesting a smaller size of DNA-CTA in ethanol-glycerol solution than that of DNA in aqueous NaCl solution. The plateau birefringence value of the DNA-CTA system, a parameter that indicates the local molecular conformation and the molecular arrangement, was only about 1/10 of that of the DNA system. There is an empirically determined molecular model of DNA-CTA complex in which a DNA molecule is sheathed by a cylindrical crust made of CTA chains. This structure reduces the DNA molecular density in a pure elongational flow field region but cannot explain the observed reduction of birefringence intensity. The small plateau birefringence value of DNA-CTA compared with that of DNA was attributed to the reduced molecular polarizability by the particular conformation of DNA molecules and CTA chains in the DNA-CTA system such as that expected by the conformational models.

  13. Ultrafast fluorescence dynamics of Sybr Green I/DNA complexes

    NASA Astrophysics Data System (ADS)

    Trantakis, Ioannis A.; Fakis, Mihalis; Tragoulias, Sotirios S.; Christopoulos, Theodore K.; Persephonis, Peter; Giannetas, Vassilis; Ioannou, Penelope

    2010-01-01

    The ultrafast dynamics of the DNA fluorescent dye Sybr Green I (SG) has been studied in buffer, single-stranded (ssDNA), double-stranded (dsDNA) and triple-stranded DNA (tsDNA). The fluorescence quantum yield of SG increases dramatically when bound to DNA (including tsDNA). The fluorescence dynamics of the free SG has shown two decay components with ˜0.15-0.4 ps and ˜1.3-2.1 ps time constants, depending on the fluorescence wavelength. Upon binding to DNA, the dynamics becomes slower exhibiting four decay components. This is mainly due to the restriction of the internal motions of the dye caused by the relatively rigid environment of the dye complexed with DNA.

  14. 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.

  15. Multivalent Lipid--DNA Complexes: Distinct DNA Compaction Regimes

    NASA Astrophysics Data System (ADS)

    Evans, Heather M.; Ahmad, A.; Ewert, K.; Safinya, C. R.

    2004-03-01

    Cationic liposomes (CL), while intrinsically advantageous in comparison to viruses, still have limited success for gene therapy and require more study. CL spontaneously self-assemble with DNA via counterion release, forming small particles approximately 200nm in diameter. X-ray diffraction reveals CL-DNA structures that are typically a multilamellar organization of lipids with DNA intercalated between the layers. We explore the structural properties of CL-DNA complexes formed with new multivalent lipids (Ewert et al, J. Med. Chem. 2002; 45:5023) that range from 2+ to 16+. Contrary to a simple prediction for the DNA interaxial spacing d_DNA based on a geometrical space-filling model, these lipids show dramatic DNA compaction, down to d_DNA ˜ 25 ÅVariations in the membrane charge density, σ _M, lead to distinct spacing regimes. We propose that this DNA condensation is controlled by a unique locking mechanism between the DNA double helix and the large, multivalent lipid head groups. Funded by NSF DMR-0203755 and NIH GM-59288.

  16. Large branched self-assembled DNA complexes

    NASA Astrophysics Data System (ADS)

    Tosch, Paul; Wälti, Christoph; Middelberg, Anton P. J.; Davies, A. Giles

    2007-04-01

    Many biological molecules have been demonstrated to self-assemble into complex structures and networks by using their very efficient and selective molecular recognition processes. The use of biological molecules as scaffolds for the construction of functional devices by self-assembling nanoscale complexes onto the scaffolds has recently attracted significant attention and many different applications in this field have emerged. In particular DNA, owing to its inherent sophisticated self-organization and molecular recognition properties, has served widely as a scaffold for various nanotechnological self-assembly applications, with metallic and semiconducting nanoparticles, proteins, macromolecular complexes, inter alia, being assembled onto designed DNA scaffolds. Such scaffolds may typically contain multiple branch-points and comprise a number of DNA molecules selfassembled into the desired configuration. Previously, several studies have used synthetic methods to produce the constituent DNA of the scaffolds, but this typically constrains the size of the complexes. For applications that require larger self-assembling DNA complexes, several tens of nanometers or more, other techniques need to be employed. In this article, we discuss a generic technique to generate large branched DNA macromolecular complexes.

  17. Structure of DNA-liposome complexes

    SciTech Connect

    Lasic, D.D.; Strey, H.; Podgornik, R.; Stuart, M.C.A.; Frederik, P.M.

    1997-01-29

    Despite numerous studies and commericially available liposome kits, however, the structure of DNA-cationic liposome complexes is still not yet well understood. We have investigated the structure of these complexes using high-resolution cryo electron microscopy (EM) and small angle X-ray scattering (SAXS). 14 refs., 3 figs.

  18. Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture.

    PubMed

    Mayanagi, Kouta; Kiyonari, Shinichi; Saito, Mihoko; Shirai, Tsuyoshi; Ishino, Yoshizumi; Morikawa, Kosuke

    2009-03-24

    The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

  19. A chromatin remodelling complex involved in transcription and DNA processing.

    PubMed

    Shen, X; Mizuguchi, G; Hamiche, A; Wu, C

    2000-08-03

    The packaging of the eukaryotic genome in chromatin presents barriers that restrict the access of enzymes that process DNA. To overcome these barriers, cells possess a number of multi-protein, ATP-dependent chromatin remodelling complexes, each containing an ATPase subunit from the SNF2/SWI2 superfamily. Chromatin remodelling complexes function by increasing nucleosome mobility and are clearly implicated in transcription. Here we have analysed SNF2/SWI2- and ISWI-related proteins to identify remodelling complexes that potentially assist other DNA transactions. We purified a complex from Saccharomyces cerevisiae that contains the Ino80 ATPase. The INO80 complex contains about 12 polypeptides including two proteins related to the bacterial RuvB DNA helicase, which catalyses branch migration of Holliday junctions. The purified complex remodels chromatin, facilitates transcription in vitro and displays 3' to 5' DNA helicase activity. Mutants of ino80 show hypersensitivity to agents that cause DNA damage, in addition to defects in transcription. These results indicate that chromatin remodelling driven by the Ino80 ATPase may be connected to transcription as well as DNA damage repair.

  20. 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.

  1. Structural polymorphism of DNA-dendrimer complexes

    NASA Astrophysics Data System (ADS)

    Evans, Heather M.; Ahmad, A.; Ewert, K.; Pfohl, T.; Martin-Herranz, A.; Bruinsma, R. F.; Safinya, C. R.

    2003-08-01

    DNA condensation in vivo relies on electrostatic complexation with small cations or large histones. We report a synchrotron x-ray study of the phase behavior of DNA complexed with synthetic cationic dendrimers of intermediate size and charge. We encounter unexpected structural transitions between columnar mesophases with in-plane square and hexagonal symmetries, as well as liquidlike disorder. The isoelectric point is a locus of structural instability. A simple model is proposed based on competing long-range electrostatic interactions and short-range entropic adhesion by counterion release.

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

    PubMed

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

    2016-04-01

    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.

  3. Intercalation processes of copper complexes in DNA.

    PubMed

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

    2015-06-23

    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.

  4. 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

  5. DNA binding, DNA cleavage, and cytotoxicity studies of two new copper (II) complexes.

    PubMed

    Kashanian, Soheila; Khodaei, Mohammad Mehdi; Roshanfekr, Hamideh; Shahabadi, Nahid; Rezvani, Alireza; Mansouri, Ghobad

    2011-05-01

    The DNA binding behavior of [Cu(phen)(phen-dione)Cl]Cl (1) and [Cu(bpy)(phen-dione)Cl]Cl (2) was studied with a series of techniques including UV-vis absorption, circular dichroism spectroscopy, and viscometric methods. Cytotoxicity effect and DNA unwinding properties were also investigated. The results indicate that the Cu(II) complexes interact with calf-thymus DNA by both partially intercalative and hydrogen binding. These findings have been further substantiated by the determination of intrinsic binding constants spectrophotometrically, 12.5 × 10(5) and 5 × 10(5) for 1 and 2, respectively. Our findings suggest that the type of ligands and structure of complexes have marked effect on the binding affinity of complexes involving CT-DNA. Circular dichroism results show that complex 1 causes considerable increase in base stacking of DNA, whereas 2 decreases the base stacking, which is related to more extended aromatic area of 1,10-phenanthroline in 1 rather than bipyridine in 2. Slow decrease in DNA viscosity indicates partially intercalative binding in addition to hydrogen binding on the surface of DNA. The second binding mode was also confirmed by additional tests: interaction in denaturation condition and acidic pH. Also, these new complexes induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) cells.

  6. Design space for complex DNA structures.

    PubMed

    Wei, Bryan; Dai, Mingjie; Myhrvold, Cameron; Ke, Yonggang; Jungmann, Ralf; Yin, Peng

    2013-12-04

    Nucleic acids have emerged as effective materials for assembling complex nanoscale structures. To tailor the structures to function optimally for particular applications, a broad structural design space is desired. Despite the many discrete and extended structures demonstrated in the past few decades, the design space remains to be fully explored. In particular, the complex finite-sized structures produced to date have been typically based on a small number of structural motifs. Here, we perform a comprehensive study of the design space for complex DNA structures, using more than 30 distinct motifs derived from single-stranded tiles. These motifs self-assemble to form structures with diverse strand weaving patterns and specific geometric properties, such as curvature and twist. We performed a systematic study to control and characterize the curvature of the structures, and constructed a flat structure with a corrugated strand pattern. The work here reveals the broadness of the design space for complex DNA nanostructures.

  7. Photoresponsive Supramolecular Complexes as Efficient DNA Regulator

    PubMed Central

    Cheng, Hong-Bo; Zhang, Ying-Ming; Xu, Chao; Liu, Yu

    2014-01-01

    Two supramolecular complexes of trans-1⊂CB[8] and trans-2⊂CB[8] were successfully achieved by the controlled selective complexation process of cucurbit[8]uril (CB[8]) with hetero-guest pair containing azobenzene and bispyridinium moieties in aqueous solution, exhibiting the reversibly light-driven movements of CB[8] upon the photocontrollable isomerization of azophenyl axle components. Significantly, the obtained bistable supramolecular complexes and their corresponding [2]pseudorotaxanes could act as a promising concentrator and cleavage agent to regulate the binding behaviors with DNA molecules. PMID:24572680

  8. 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.

  9. Luminescence sensitization of Tb(3+)-DNA complexes by Ag().

    PubMed

    Xu, Lijun; Zhou, Lu; Chen, Xing; Shen, Xiaoqiang; Wang, Jine; Zhang, Jianye; Pei, Renjun

    2017-03-03

    Terbium ions (Tb(3+)) with unique photophysical properties have been utilized to develop biosensors with low background and high sensitivity. In this study, the Ag(+)-sensitized luminescence of Tb(3+)-DNA complexes was uncovered. The luminescence of Tb(3+)-DNA complexes could be enhanced by more than 30 times in the presence of Ag(+), when Tb(3+) was bound with poly(G) and poly(T) whereas not with other homopolymers. This research confirmed that the sensitization resulted from the interaction of Ag(+) with certain bases involved in DNA, not just with the reported certain G-quadruplex sequence. The coordination of Ag(+) to guanine and thymine bases was expected to increase their rigidities, form Tb(3+)-DNA-Ag(+) ternary structures, and thus enhance energy transfer from guanine and thymine to Tb(3+). These findings benefit the development of sensitive luminescence probes for various nucleic acids-related targets.

  10. Luminescence sensitization of Tb3 +-DNA complexes by Ag+

    NASA Astrophysics Data System (ADS)

    Xu, Lijun; Zhou, Lu; Chen, Xing; Shen, Xiaoqiang; Wang, Jine; Zhang, Jianye; Pei, Renjun

    2017-06-01

    Terbium ions (Tb3 +) with unique photophysical properties have been utilized to develop biosensors with low background and high sensitivity. In this study, the Ag+-sensitized luminescence of Tb3 +-DNA complexes was uncovered. The luminescence of Tb3 +-DNA complexes could be enhanced by more than 30 times in the presence of Ag+, when Tb3 + was bound with poly(G) and poly(T) whereas not with other homopolymers. This research confirmed that the sensitization resulted from the interaction of Ag+ with certain bases involved in DNA, not just with the reported certain G-quadruplex sequence. The coordination of Ag+ to guanine and thymine bases was expected to increase their rigidities, form Tb3 +-DNA-Ag+ ternary structures, and thus enhance energy transfer from guanine and thymine to Tb3 +. These findings benefit the development of sensitive luminescence probes for various nucleic acids-related targets.

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

    PubMed

    Ristic, Dejan; Sanchez, Humberto; Wyman, Claire

    2011-01-01

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

  12. 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. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  13. 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.

  14. Thermally forced transitions of DNA-CTMA complex microstructure

    NASA Astrophysics Data System (ADS)

    Nizioł, Jacek; Ekiert, Robert; Śniechowski, Maciej; Słomiany, Magdalena; Marzec, Mateusz M.

    2016-06-01

    DNA complexed with amphiphilic cationic surfactants is a new class of optical material. In this work DNA and its complex with cetyltrimetyl ammonium chloride were thermally annealed. X-ray diffractometry revealed irreversible changes of DNA-CTMA microstructure. The new microstucture that appeared in result of the first heating course was stable, despite the further thermal annealing. Agarose gel electrophoresis indicated fundamental differences between thermally treated native DNA and DNA-CTMA complex.

  15. DNA interactions of new antitumor aminophosphine platinum(II) complexes.

    PubMed

    Neplechová, K; Kaspárková, J; Vrána, O; Nováková, O; Habtemariam, A; Watchman, B; Sadler, P J; Brabec, V

    1999-07-01

    Mechanistic studies are presented of a novel class of aminophosphine platinum(II) complexes as potential anticancer agents. These new agents, which have demonstrated activity against murine and human tumor cells including those resistant to cisplatin are cis-[PtCl2(Me2N(CH2)3PPh2-P)2] (Com1) and cis-[PtCl(C6H11NH(CH2)2PPh2-N,P)(C6H11NH(CH2) 2PPh2-P)] (Com2). We studied modifications of natural and synthetic DNAs in cell-free media by Com1 and Com2 by various biomedical and biophysical methods and compared the results with those obtained when DNA was modified by cisplatin. The results indicated that Com1 and Com2 coordinated to DNA faster than cisplatin. Bifunctional Com1 formed DNA adducts coordinating to single adenine or guanine residues or by forming cross-links between these residues. In comparison with cisplatin, Com1 formed the adducts more frequently at adenine residues and also formed fewer bidentate lesions. The monofunctional Com2 only formed DNA monodentate adducts at guanine residues. In addition, Com1 terminated DNA synthesis in vitro more efficiently than cisplatin whereas Com2 blocked DNA synthesis only slightly. DNA unwinding studies, measurements of circular dichroism spectra, immunochemical analysis, and studies of the B-Z transition in DNA revealed conformational alterations induced by the adducts of Com1, which were distinctly different from those induced by cisplatin. Com2 had little influence on DNA conformation. It is suggested that the activity profile of aminophosphine platinum(II) complexes, which is different from that of cisplatin and related analogs, might be associated with the specific DNA binding properties of this new class of platinum(II) compounds.

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

    DOE PAGES

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

    2016-03-09

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

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

    SciTech Connect

    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 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. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.

  18. Heteroduplex mobility assay-guided sequence discovery: elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools.

    PubMed

    Oldach, D W; Delwiche, C F; Jakobsen, K S; Tengs, T; Brown, E G; Kempton, J W; Schaefer, E F; Bowers, H A; Glasgow, H B; Burkholder, J M; Steidinger, K A; Rublee, P A

    2000-04-11

    The newly described heterotrophic estuarine dinoflagellate Pfiesteria piscicida has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in Pfiesteria research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect Pfiesteria piscicida 18S rDNA "signature" sequences in complex pools of DNA and used those data as the basis for determination of the complete P. piscicida 18S rDNA sequence. Specific PCR assays for P. piscicida and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.

  19. 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.

  20. Nanoscale structure of protamine/DNA complexes for gene delivery

    NASA Astrophysics Data System (ADS)

    Motta, Simona; Brocca, Paola; Del Favero, Elena; Rondelli, Valeria; Cantù, Laura; Amici, Augusto; Pozzi, Daniela; Caracciolo, Giulio

    2013-02-01

    Understanding the internal packing of gene carriers is a key-factor to realize both gene protection during transport and de-complexation at the delivery site. Here, we investigate the structure of complexes formed by DNA fragments and protamine, applied in gene delivery. We found that complexes are charge- and size-tunable aggregates, depending on the protamine/DNA ratio, hundred nanometers in size. Their compactness and fractal structure depend on the length of the DNA fragments. Accordingly, on the local scale, the sites of protamine/DNA complexation assume different morphologies, seemingly displaying clumping ability for the DNA network only for shorter DNA fragments.

  1. Evolution of DNA replication protein complexes in eukaryotes and Archaea.

    PubMed

    Chia, Nicholas; Cann, Isaac; Olsen, Gary J

    2010-06-02

    The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.

  2. [Liberation of DNA from particles of CD phage of DNA-protein complex: Properties of DNA in the complex and the effect of formaldehyde on the complex structure].

    PubMed

    Dobrov, E N; Ogareva, N A; Manykin, A A; Tikhonenko, T I

    1976-01-01

    Properties of DNA in a complex with protein, which was liberated after destruction of CD phage by heat treatment in solutions with low ionic strength, were studied. DNA in the complex did not differ from free DNA under the same conditions as shown by spectra of circular dichroism and by the type of melting during the thermic denaturation. As demonstrated by viscosimetry and gradient centrifugation in cesium sulfate, 1.5% formaldehyde inhibited the dissociation of the complex studied in a medium containing 0.15 M NaCl. In the medium with 1.5% of HCHO T degrees ml of DNA in the complex was distinctly higher than T degrees ml of free DNA under the same conditions. Electron microscopy showed that the complex studied comprised DNA ;molecules, associated with protein membrane in one or several internal sites.

  3. Analysis of DNA-protein complexes induced by chemical carcinogens

    SciTech Connect

    Costa, M. )

    1990-11-01

    DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.

  4. The Mre11 complex: at the crossroads of dna repair and checkpoint signalling.

    PubMed

    D'Amours, Damien; Jackson, Stephen P

    2002-05-01

    The Mre11 complex is a multisubunit nuclease that is composed of Mre11, Rad50 and Nbs1/Xrs2. Mutations in the genes that encode components of this complex result in DNA- damage sensitivity, genomic instability, telomere shortening and aberrant meiosis. The molecular defect that underlies these phenotypes has long been thought to be related to a DNA repair deficiency. However, recent studies have uncovered functions for the Mre11 complex in checkpoint signalling and DNA replication.

  5. Evolution of DNA Replication Protein Complexes in Eukaryotes and Archaea

    PubMed Central

    Chia, Nicholas; Cann, Isaac; Olsen, Gary J.

    2010-01-01

    Background The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. Methodology/Principal Findings While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex—all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. Conclusion/Significance This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota. PMID:20532250

  6. Release of DNA binary complexes from the ternary complexes by carboxymethyl poly(L-histidine).

    PubMed

    Asayama, Shoichiro; Sudo, Miyuki; Kawakami, Hiroyoshi

    2009-01-01

    The DNA ternary complexes with carboxymethyl poly(L-histidine) (CM-PLH) and poly(ethylenimine) (PEI) have released the DNA binary complexes with PEI by the protonation of CM-PLH at endosomal/lysosomal pH. The dissociation of the CM-PLH from the CM-PLH/PEI/DNA ternary complexes is proved by the fluorescence resonance energy transfer (FRET) analysis between the CM-PLH and PEI. The resulting PEI/DNA binary complexes easily released DNA, as compared with the CM-PLH/PEI/DNA ternary complexes, which was examined by competitive exchange with dextran sulfate. The release of the DNA binary complexes from the ternary complexes is promising mechanism for higher transfection activity by the CM-PLH/PEI/DNA ternary complexes.

  7. Streching of (DNA/functional molecules) complex between electrodes towards DNA molecular wire

    NASA Astrophysics Data System (ADS)

    Kobayashi, Norihisa; Nishizawa, Makoto; Inoue, Shintarou; Nakamura, Kazuki

    2009-08-01

    DNA/functional molecules such as (Ru(bpy)32+ complex, conducting polymer etc.) complex was prepared to study molecular structure and I-V characteristics towards DNA molecular wire. For example, Ru(bpy)32+ was associated with duplex of DNA by not only electrostatic interaction but also intercalation in the aqueous solution. Singlemolecular structure of DNA/Ru(bpy)32+ complex was analyzed with AFM. We found a network structure of DNA/Ru(bpy)32+ complex on the mica substrate, which is similar to native DNA. The height of DNA/Ru(bpy)32+ complex on the mica substrate was ranging from 0.8 to 1.6 nm, which was higher than the naked DNA (0.5-1.0 nm). This indicates that single-molecular DNA/Ru(bpy)32+ complex also connects to each other to form network structure on a mica substrate. In order to stretch DNA complex between electrodes, we employed high frequency and high electric field stretching method proposed by Washizu et al. We stretched and immobilized DNA single molecules between a pair of electrodes and its structures were analyzed with AFM technique. The I-V characteristics of DNA single molecules between electrodes were improved by the association of functional molecules with DNA. The molecular structure and I-V characteristics of DNA complex were discussed.

  8. 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

  9. 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

    2016-04-07

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Immunodetection of human topoisomerase I-DNA covalent complexes

    PubMed Central

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

    2016-01-01

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

  11. Hydrolytic cleavage of DNA by quercetin manganese(II) complexes.

    PubMed

    Jun, Tan; Bochu, Wang; Liancai, Zhu

    2007-04-01

    Quercetin manganese(II) complexes were investigated focusing on its DNA hydrolytic activity. The complexes successfully promote the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid DNA to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complexes with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.2 in the presence of 100 microM of the complexes is found to be 1.32 x 10(-4) s(-1). The hydrolytic cleavage of DNA by the complexes was supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay and T4 ligase ligation.

  12. Evidence of DNA Bending in Transcription Complexes Imaged by Scanning Force Microscopy

    NASA Astrophysics Data System (ADS)

    Rees, William A.; Keller, Rebecca W.; Vesenka, James P.; Yang, Guoliang; Bustamante, Carlos

    1993-06-01

    Complexes of Escherichia coli RNA polymerase with DNA containing the λ P_L promoter have been deposited on mica and imaged in air with a scanning force microscope. The topographic images reveal the gross spatial relations of the polymerase relative to the DNA template. The DNA appears bent in open promoter complexes containing RNA polymerase bound to the promoter and appears more severely bent in elongation complexes in which RNA polymerase has synthesized a 15-nucleotide transcript. This difference could be related to the conformational changes that accompany the maturation of open promoter complexes into elongation complexes and suggests that formation of the elongation complex involves a considerable modification of the spatial relations between the polymerase and the DNA template.

  13. ESR study of the direct radiolysis of DNA, DNA-histones and DNA-intercalators complexes

    NASA Astrophysics Data System (ADS)

    Faucitano, A.; Buttafava, A.; Martinotti, F.; Pedraly-Noy, G.

    The nature of the radicals contributing to the room temperature spectrum of irradiated "dry" DNA, with special reference to the central structure, is discussed, and the thesis of their ionic origin tested by irradiation experiments with intercalators. The mechanism of spin transfer protein→DNA has been investigated through a comparative ESR study on the DNA-histones complex, the structureless random molecular mixture of the DNA-histones and the neat components. The yield of spin transfer is enhanced in the random mixture, presumably because of the greater efficiency of molecular contacts. Evidence of the scavenging of electrons by the thymine and cytosine bases, as a key mechanism for the spin transfer, has been obtained.

  14. Generic technique to generate large branched DNA complexes.

    PubMed

    Tosch, Paul; Wälti, Christoph; Middelberg, Anton P J; Davies, A Giles

    2006-03-01

    The inherent self-recognition properties of DNA have led to its use as a scaffold for various nanotechnology self-assembly applications, with macromolecular complexes, metallic and semiconducting nanoparticles, proteins, inter alia, being assembled onto a designed DNA scaffold. Such structures may typically comprise a number of DNA molecules organized into macromolecules. Many studies have used synthetic methods to produce the constituent DNA molecules, but this typically constrains the molecules to be no longer than around 100 base pairs (30 nm). However, applications that require larger self-assembling DNA complexes, several tens of nanometers or more, need to be generated by other techniques. Here, we present a generic technique to generate large linear, branched, and/or circular DNA macromolecular complexes. The effectiveness of this technique is demonstrated here by the use of Lambda Bacteriophage DNA as a template to generate single- and double-branched DNA structures approximately 120 nm in size.

  15. 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.

  16. Antifungal activity of DNA-lipid complexes and DNA-lipid films against Candida species.

    PubMed

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

    2006-01-01

    In this study amphiphilic lipids, DNA-lipid complexes, and DNA-lipid films were prepared, and their antifungal activity against Candida species was examined. The amphiphilic lipids were synthesized from a reaction of glycine or L-alanine with n-alkyl alcohol in the presence of p-toluene sulfonic acid. DNA-lipid complexes, which were prepared by the simple mixing of DNA and amphiphilic lipids, were insoluble in water. Self-standing, water-insoluble DNA-lipid films were prepared by casting the DNA-lipid complexes from a chloroform/ethanol solution. The antifungal activities of the lipids and DNA-lipid complexes against the Candida species were evaluated by minimum inhibitory concentrations (MICs); those of DNA-lipid films were evaluated by the disk diffusion method. The seven kinds of lipids, DNA-lipid complexes, and DNA-lipid films showed antifungal activity, and no differences were seen in the antifungal activities between glycine and L-alanine derivatives. The lipids, DNA-lipid complexes, and DNA-lipid films, which have shorter alkyl chain length in lipids, showed antifungal activity against all Candida species. However, the effect of antifungal activity against Candida species decreased with increased alkyl chain length in lipids. In this study, it was found that lipids, DNA-lipid complexes, and films with a decyl or dodecyl group exhibit more favorable antifungal activity. (c) 2005 Wiley Periodicals, Inc

  17. Macrocyclic Metal Complex-DNA Conjugates for Electrochemical Sensing of Single Nucleobase Changes in DNA.

    PubMed

    Duprey, Jean-Louis H A; Carr-Smith, James; Horswell, Sarah L; Kowalski, Jarosław; Tucker, James H R

    2016-01-27

    The direct incorporation of macrocyclic cyclidene complexes into DNA via automated synthesis results in a new family of metal-functionalized DNA derivatives that readily demonstrate their utility through the ability of one redox-active copper(II)-containing strand to distinguish electrochemically between all four canonical DNA nucleobases at a single site within a target sequence of DNA.

  18. Complex DNA nanostructures from oligonucleotide ensembles.

    PubMed

    Mathur, Divita; Henderson, Eric R

    2013-04-19

    The first synthetic DNA nanostructures were created by self-assembly of a small number of oligonucleotides. Introduction of the DNA origami method provided a new paradigm for designing and creating two- and three-dimensional DNA nanostructures by folding a large single-stranded DNA and 'stapling' it together with a library of oligonucleotides. Despite its power and wide-ranging implementation, the DNA origami technique suffers from some limitations. Foremost among these is the limited number of useful single-stranded scaffolds of biological origin. This report describes a new approach to creating large DNA nanostructures exclusively from synthetic oligonucleotides. The essence of this approach is to replace the single-stranded scaffold in DNA origami with a library of oligonucleotides termed "scaples" (scaffold staples). Scaples eliminate the need for scaffolds of biological origin and create new opportunities for producing larger and more diverse DNA nanostructures as well as simultaneous assembly of distinct structures in a "single-pot" reaction.

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

    PubMed Central

    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. PMID:26110537

  20. 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

  1. Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV.

    PubMed

    Grob, Patricia; Zhang, Teri T; Hannah, Ryan; Yang, Hui; Hefferin, Melissa L; Tomkinson, Alan E; Nogales, Eva

    2012-01-02

    The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.

  2. Hydrolytic cleavage of DNA by quercetin zinc(II) complex.

    PubMed

    Jun, Tan; Bochu, Wang; Liancai, Zhu

    2007-03-01

    Quercetin zinc(II) complex was investigated focusing on its hydrolytic activity toward DNA. The complex successfully promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The rate of conversion of SC to NC is 1.68x10(-4) s(-1) at pH 7.2 in the presence of 100 microM of the complex. The hydrolytic cleavage of DNA by the complex is supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay, and T4 ligase ligation.

  3. Free Energy Analysis of Protein-DNA Binding: The EcoRI Endonuclease-DNA Complex

    NASA Astrophysics Data System (ADS)

    Jayaram, B.; McConnell, K. J.; Dixit, Surjit B.; Beveridge, D. L.

    1999-05-01

    A detailed theoretical analysis of the thermodynamics and functional energetics of protein-DNA binding in the EcoRI endonuclease-DNA complex is presented. The standard free energy of complexation is considered in terms of a thermodynamic cycle of seven distinct steps decomposed into a total of 24 well-defined components. The model we employ involves explicit all-atom accounts of the energetics of structural adaptation of the protein and the DNA upon complex formation; the van der Waals and electrostatic interactions between the protein and the DNA; and the electrostatic polarization and screening effects, van der Waals components, and cavitation effects of solvation. The ion atmosphere of the DNA is described in terms of a counterion condensation model combined with a Debye-Huckel treatment of added salt effects. Estimates of entropy loss due to decreased translational and rotational degrees of freedom in the complex relative to the unbound species based on classical statistical mechanics are included, as well as corresponding changes in the vibrational and configurational entropy. The magnitudes and signs of the various components are estimated from the AMBER parm94 force field, generalized Born theory, solvent accessibility measures, and empirical estimates of quantities related to ion release. The calculated standard free energy of formation, -11.5 kcal/mol, agrees with experiment to within 5 kcal/mol. This net binding free energy is discerned to be the resultant of a balance of several competing contributions associated with chemical forces as conventionally defined, with 10 out of 24 terms favoring complexation. Contributions to binding compounded from subsets of the 24 components provide a basis for advancing a molecular perspective of binding in terms of structural adaptation, electrostatics, van der Waals interactions, hydrophobic effects, and small ion reorganization and release upon complexation. The van der Waals interactions and water release favor

  4. Disruption of a Topoisomerase-DNA Cleavage Complex by a DNA Helicase

    NASA Astrophysics Data System (ADS)

    Howard, Michael T.; Neece, Sue H.; Matson, Steven W.; Kreuzer, Kenneth N.

    1994-12-01

    The type II DNA topoisomerases are targets for a variety of chemotherapeutic agents, including the antibacterial quinolones and several families of antitumor drugs. These agents stabilize an enzyme-DNA cleavage complex that consists of the topoisomerase covalently linked to the 5' phosphates of a double-stranded DNA break. Although the drug-stabilized cleavage complex is readily reversible, it can result in cell death by a mechanism that remains uncertain. Here we demonstrate that the action of a DNA helicase can convert the cleavage complex into a nonreversible DNA break by displacing DNA strands from the complex. Formation of a nonreversible DNA break, induced by a DNA helicase, could explain the cytotoxicity of these topoisomerase poisons.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

    We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits. PMID:28036377

  7. DNA photo-oxidative damage hazard in transfection complexes.

    PubMed

    Rudiuk, Sergii; Franceschi-Messant, Sophie; Chouini-Lalanne, Nadia; Perez, Emile; Rico-Lattes, Isabelle

    2011-01-01

    Complexes of DNA with various cationic vectors have been largely used for nonviral transfection, and yet the photochemical stability of DNA in such complexes has never been considered. We studied, for the first time, the influence of DNA complexation by a cationic lipid and polymers on the amount of damage induced by benzophenone photosensitization. The localization of benzophenone inside the hydrophobic domains formed by a cationic lipid, DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride), and close to DNA, locally increases the photoinduced cleavage by the reactive oxygen species generated. The same effect was found in the case of DNA complexation with an amphiphilic polymer (polynorbornenemethyleneammonium chloride). However, a decrease in DNA damage was observed in the case of complexation with a hydrophilic polymer (polyethylenimine). The DNA protection in this case was because of the absence of benzophenone hydrophobic incorporation into the complex, and to DNA compaction which decreased the probability of radical attack. These results underline the importance of the chemical structure of the nonviral transfection vector in limiting the risks of photo-oxidative damage of the complexed DNA. © 2010 The Authors. Photochemistry and Photobiology © 2010 The American Society of Photobiology.

  8. Metal Complexes for DNA-Mediated Charge Transport

    PubMed Central

    Barton, Jacqueline K.; Olmon, Eric D.; Sontz, Pamela A.

    2010-01-01

    In all organisms, oxidation threatens the integrity of the genome. DNA-mediated charge transport (CT) may play an important role in the generation and repair of this oxidative damage. In studies involving long-range CT from intercalating Ru and Rh complexes to 5′-GG-3′ sites, we have examined the efficiency of CT as a function of distance, temperature, and the electronic coupling of metal oxidants bound to the base stack. Most striking is the shallow distance dependence and the sensitivity of DNA CT to how the metal complexes are stacked in the helix. Experiments with cyclopropylamine-modified bases have revealed that charge occupation occurs at all sites along the bridge. Using Ir complexes, we have seen that the process of DNA-mediated reduction is very similar to that of DNA-mediated oxidation. Studies involving metalloproteins have, furthermore, shown that their redox activity is DNA-dependent and can be DNA-mediated. Long range DNA-mediated CT can facilitate the oxidation of DNA-bound base excision repair proteins to initiate a redox-active search for DNA lesions. DNA CT can also activate the transcription factor SoxR, triggering a cellular response to oxidative stress. Indeed, these studies show that within the cell, redox-active proteins may utilize the same chemistry as that of synthetic metal complexes in vitro, and these proteins may harness DNA-mediated CT to reduce damage to the genome and regulate cellular processes. PMID:21643528

  9. DNA-DNA kissing complexes as a new tool for the assembly of DNA nanostructures.

    PubMed

    Barth, Anna; Kobbe, Daniela; Focke, Manfred

    2016-02-29

    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. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Evolution of a complex minisatellite DNA sequence.

    PubMed

    Barros, Paula; Blanco, Miguel G; Boán, Francisco; Gómez-Márquez, Jaime

    2008-11-01

    Minisatellites are tandem repeats of short DNA units widely distributed in genomes. However, the information on their dynamics in a phylogenetic context is very limited. Here we have studied the organization of the MsH43 locus in several species of primates and from these data we have reconstructed the evolutionary history of this complex minisatellite. Overall, with the exception of gibbon, MsH43 has an organization that is asymmetric, since the distribution of repeats is distinct between the 5' and 3' halves, and heterogeneous since there are many different repeats, some of them characteristic of each species. Inspection of the MsH43 arrays showed the existence of many duplications and deletions, suggesting the implication of slippage processes in the generation of polymorphism. Concerning the evolutionary history of this minisatellite, we propose that the birth of MsH43 may be situated before the divergence of Old World Monkeys since we found the existence of some MsH43 repeat motifs in prosimians and New World Monkeys. The analysis of MsH43 in apes revealed the existence of an evolutionary breakpoint in the pathway that originated African great apes and humans. Remarkably, human MsH43 is more homologous to orang-utan than to the corresponding sequence in gorilla and chimpanzee. This finding does not comply with the evolutionary paradigm that continuous alterations occur during the course of genome evolution. To adjust our results to the standard phylogeny of primates, we propose the existence of a wandering allele that was maintained almost unaltered during the period that extends between orang-utan and humans.

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

    PubMed Central

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

    1998-01-01

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

  12. The orientation of DNA in an archaeal transcription initiation complex.

    PubMed

    Bartlett, M S; Thomm, M; Geiduschek, E P

    2000-09-01

    RNA polymerase from the hyperthermophile archaeon Pyrococcus furiosus (Pfu) forms specific and transcriptionally active complexes with its conjugate transcription factors TBP (the archaeal TATA binding protein homolog) and TFB (the archaeal homolog of eukaryotic RNA polymerase II and III transcription factors TFIIB and Brf) at the Pfu glutamate dehydrogenase promoter. A photochemical crosslinking method was used to map the vicinity of the catalytic subunits of Pfu RNA polymerase to DNA locations distributed along the polymerase-promoter interface. The largest component of this archaeal polymerase is split into two subunits, A' and A", whose relatively sharp boundary of DNA crosslinking (probed on the transcribed strand) is centered five to six base pairs downstream of the transcriptional start site. A strong argument based on this information, on the well-defined homology between the core bacterial, archaeal and eukaryotic RNA polymerase subunits, and on the recently determined structure of a bacterial RNA polymerase specifies the directionality of DNA in the archaeal transcription complex and its trajectory downstream of the transcriptional start site.

  13. Optimization of DNA delivery by three classes of hybrid nanoparticle/DNA complexes

    PubMed Central

    2010-01-01

    Plasmid DNA encoding a luciferase reporter gene was complexed with each of six different hybrid nanoparticles (NPs) synthesized from mixtures of poly (D, L-lactide-co-glycolide acid) (PLGA 50:50) and the cationic lipids DOTAP (1, 2-Dioleoyl-3-Trimethyammonium-Propane) or DC-Chol {3β-[N-(N', N'-Dimethylaminoethane)-carbamyl] Cholesterol}. Particles were 100-400 nm in diameter and the resulting complexes had DNA adsorbed on the surface (out), encapsulated (in), or DNA adsorbed and encapsulated (both). A luciferase reporter assay was used to quantify DNA expression in 293 cells for the uptake of six different NP/DNA complexes. Optimal DNA delivery occurred for 105 cells over a range of 500 ng - 10 μg of NPs containing 20-30 μg DNA per 1 mg of NPs. Uptake of DNA from NP/DNA complexes was found to be 500-600 times as efficient as unbound DNA. Regression analysis was performed and lines were drawn for DNA uptake over a four week interval. NP/DNA complexes with adsorbed NPs (out) showed a large initial uptake followed by a steep slope of DNA decline and large angle of declination; lines from uptake of adsorbed and encapsulated NPs (both) also exhibited a large initial uptake but was followed by a gradual slope of DNA decline and small angle of declination, indicating longer times of luciferase expression in 293 cells. NPs with encapsulated DNA only (in), gave an intermediate activity. The latter two effects were best seen with DOTAP-NPs while the former was best seen with DC-Chol-NPs. These results provide optimal conditions for using different hybrid NP/DNA complexes in vitro and in the future, will be tested in vivo. PMID:20181278

  14. 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.

  15. 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

  16. Fluorescence anisotropy of DNA/DAPI complex: torsional dynamics and geometry of the complex.

    PubMed Central

    Barcellona, M L; Gratton, E

    1996-01-01

    Fluorescence depolarization of synthetic polydeoxynucleotide/4'-6-diamidino-2-phenylindole dihydrochloride complexes has been investigated as a function of dye/polymer coverage. At low coverage, fluorescence depolarization is due to local torsional motions of the DNA segment where the dye resides. At relatively high coverage, fluorescence depolarization is dominated by energy transfer to other dye molecules along the DNA. The extent of the observed depolarization due to torsional motion depends on the angle the dye molecule forms with the DNA helical axis. A large torsional motion and a small angle produce the same depolarization as a small torsional motion and a large projection angle. Furthermore, the extent of transfer critically depends on the relative orientation of dye molecules along the DNA. The effect of multiple transfer is examined using a Monte Carlo approach. The measurement of depolarization with transfer, at high coverage, allows determination of the dye orientation about the DNA helical axis. The value of the torsional spring constant is then determined, at very low coverage, for few selected polydeoxynucleotides. Images FIGURE 3 PMID:9172758

  17. Spectroscopic studies on the interaction of morin-Eu(III) complex with calf thymus DNA

    NASA Astrophysics Data System (ADS)

    Zhang, Guowen; Guo, Jinbao; Pan, Junhui; Chen, Xiuxia; Wang, Junjie

    2009-04-01

    The interaction between morin-Eu(III) complex and calf thymus DNA in physiological buffer (pH 7.4) was investigated using UV-vis spectrophotometry, fluorescence spectroscopy, viscosity measurements and DNA melting techniques. Hypochromicity and red shift of the absorption spectra of morin-Eu(III) complex were observed in the presence of DNA, and the fluorescence intensity of morin-Eu(III) complex was greatly enhanced with the addition of DNA. Moreover, fluorescence quenching and blue shift of the emission peak were seen in the DNA-ethidium bromide (EB) system when morin-Eu(III) complex was added. The relative viscosity of DNA increased with the addition of morin-Eu(III) complex, whereas the value of melting temperature of DNA-EB system decreased in the presence of morin-Eu(III) complex. All these results indicated that morin-Eu(III) complex can bind to DNA and the major binding mode is intercalative binding. The 3:1 morin:Eu(III) complex (estimated binding constant = 2.36 × 10 6 L mol -1) is stabilized by intercalation into the DNA. The calculated binding constants of morin-Eu(III) complex with DNA at 292, 301 and 310 K were 7.47 × 10 4, 8.89 × 10 4 and 1.13 × 10 5 L mol -1, respectively. The thermodynamic parameters were also obtained: Δ H θ was 20.14 kJ mol -1 > 0 and Δ S θ was 161.70 J mol -1 K -1 > 0, suggesting that hydrophobic force plays a major role in the binding of morin-Eu(III) complex to DNA.

  18. 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.

  19. DNA complexes with Ni nanoparticles: structural and functional properties

    NASA Astrophysics Data System (ADS)

    Tatarinova, Olga N.; Smirnov, Igor P.; Safenkova, Irina V.; Varizhuk, Anna M.; Pozmogova, Galina E.

    2012-10-01

    Supramolecular complexes of biopolymers based on magnetic nanoparticles play an important role in creation of biosensors, implementation of theragnostic and gene therapeutic methods and biosafety evaluation. We investigated the impact of DNA interactions with nanoparticles of nickel (nNi) on the integrity and functionality of DNA. Data obtained by mass spectrometry, electrophoresis, TEM and AFM microscopy techniques, bacterial transformation, and real-time PCR provide evidence that ssDNA and plasmid DNA (pDNA) efficiently form complexes with nNi. AFM data suggest that the complexes are necklace-type structures, in which nanoparticles are randomly distributed along the DNA chains, rather than highly entangled clot-type structures. After desorption, observed DNA characteristics in bioanalytical and biological systems remain unchanged. Only supercoiled pDNA was nicked, but remained, as well as a plasmid-nNi complex, active in expression vector assays. These results are very important for creation of new methods of DNA immobilization and controlled manipulation.

  20. Biological consequences of formation and repair of complex DNA damage.

    PubMed

    Magnander, Karin; Elmroth, Kecke

    2012-12-31

    Endogenous processes or genotoxic agents can induce many types of single DNA damage (single-strand breaks, oxidized bases and abasic sites). In addition, ionizing radiation induces complex lesions such as double-strand breaks and clustered damage. To preserve the genomic stability and prevent carcinogenesis, distinct repair pathways have evolved. Despite this, complex DNA damage can cause severe problems and is believed to contribute to the biological consequences observed in cells exposed to genotoxic stress. In this review, the current knowledge of formation and repair of complex DNA damage is summarized and the risks and biological consequences associated with their repair are discussed. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2014-05-02

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

  2. 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

  3. Direct Observation of Translocation in Individual DNA Polymerase Complexes*

    PubMed Central

    Dahl, Joseph M.; Mai, Ai H.; Cherf, Gerald M.; Jetha, Nahid N.; Garalde, Daniel R.; Marziali, Andre; Akeson, Mark; Wang, Hongyun; Lieberman, Kate R.

    2012-01-01

    Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state. PMID:22378784

  4. Crystallization and initial crystallographic analysis of covalent DNA-cleavage complexes of Staphyloccocus aureus DNA gyrase with QPT-1, moxifloxacin and etoposide.

    PubMed

    Srikannathasan, Velupillai; Wohlkonig, Alexandre; Shillings, Anthony; Singh, Onkar; Chan, Pan F; Huang, Jianzhong; Gwynn, Michael N; Fosberry, Andrew P; Homes, Paul; Hibbs, Martin; Theobald, Andrew J; Spitzfaden, Claus; Bax, Benjamin D

    2015-10-01

    Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15 Å resolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.

  5. Structural analysis of DNA complexation with cationic lipids

    PubMed Central

    Marty, Regis; N'soukpoé-Kossi, Christophe N.; Charbonneau, David; Weinert, Carl Maximilian; Kreplak, Laurent; Tajmir-Riahi, Heidar-Ali

    2009-01-01

    Complexes of cationic liposomes with DNA are promising tools to deliver genetic information into cells for gene therapy and vaccines. Electrostatic interaction is thought to be the major force in lipid–DNA interaction, while lipid-base binding and the stability of cationic lipid–DNA complexes have been the subject of more debate in recent years. The aim of this study was to examine the complexation of calf-thymus DNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant DNA concentration and various lipid contents. Fourier transform infrared (FTIR), UV-visible, circular dichroism spectroscopic methods and atomic force microscopy were used to analyse lipid-binding site, the binding constant and the effects of lipid interaction on DNA stability and conformation. Structural analysis showed a strong lipid–DNA interaction via major and minor grooves and the backbone phosphate group with overall binding constants of KChol = 1.4 (±0.5) × 104 M−1, KDDAB = 2.4 (±0.80) × 104 M−1, KDOTAP = 3.1 (±0.90) × 104 M−1 and KDOPE = 1.45 (± 0.60) × 104 M−1. The order of stability of lipid–DNA complexation is DOTAP>DDAB>DOPE>Chol. Hydrophobic interactions between lipid aliphatic tails and DNA were observed. Chol and DOPE induced a partial B to A-DNA conformational transition, while a partial B to C-DNA alteration occurred for DDAB and DOTAP at high lipid concentrations. DNA aggregation was observed at high lipid content. PMID:19103664

  6. The RuvAB branch migration complex can displace topoisomerase IV.quinolone.DNA ternary complexes.

    PubMed

    Shea, Molly E; Hiasa, Hiroshi

    2003-11-28

    Quinolone antimicrobial drugs target both DNA gyrase and topoisomerase IV (Topo IV) and convert these essential enzymes into cellular poisons. Topoisomerase poisoning results in the inhibition of DNA replication and the generation of double-strand breaks. Double-strand breaks are repaired by homologous recombination. Here, we have investigated the interaction between the RuvAB branch migration complex and the Topo IV.quinolone.DNA ternary complex. A strand-displacement assay is employed to assess the helicase activity of the RuvAB complex in vitro. RuvAB-catalyzed strand displacement requires both RuvA and RuvB proteins, and it is stimulated by a 3'-non-hybridized tail. Interestingly, Topo IV.quinolone.DNA ternary complexes do not inhibit the translocation of the RuvAB complex. In fact, Topo IV.quinolone.DNA ternary complexes are reversed and displaced from the DNA upon their collisions with the RuvAB complex. These results suggest that the RuvAB branch migration complex can actively remove quinolone-induced covalent topoisomerase.DNA complexes from DNA and complete the homologous recombination process in vivo.

  7. 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.

  8. 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.

  9. 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.

  10. Polyamide platinum anticancer complexes designed to target specific DNA sequences.

    PubMed

    Jaramillo, David; Wheate, Nial J; Ralph, Stephen F; Howard, Warren A; Tor, Yitzhak; Aldrich-Wright, Janice R

    2006-07-24

    Two new platinum complexes, trans-chlorodiammine[N-(2-aminoethyl)-4-[4-(N-methylimidazole-2-carboxamido)-N-methylpyrrole-2-carboxamido]-N-methylpyrrole-2-carboxamide]platinum(II) chloride (DJ1953-2) and trans-chlorodiammine[N-(6-aminohexyl)-4-[4-(N-methylimidazole-2-carboxamido)-N-methylpyrrole-2-carboxamido]-N-methylpyrrole-2-carboxamide]platinum(II) chloride (DJ1953-6) have been synthesized as proof-of-concept molecules in the design of agents that can specifically target genes in DNA. Coordinate covalent binding to DNA was demonstrated with electrospray ionization mass spectrometry. Using circular dichroism, these complexes were found to show greater DNA binding affinity to the target sequence: d(CATTGTCAGAC)(2), than toward either d(GTCTGTCAATG)(2,) which contains different flanking sequences, or d(CATTGAGAGAC)(2), which contains a double base pair mismatch sequence. DJ1953-2 unwinds the DNA helix by around 13 degrees , but neither metal complex significantly affects the DNA melting temperature. Unlike simple DNA minor groove binders, DJ1953-2 is able to inhibit, in vitro, RNA synthesis. The cytotoxicity of both metal complexes in the L1210 murine leukaemia cell line was also determined, with DJ1953-6 (34 microM) more active than DJ1953-2 (>50 microM). These results demonstrate the potential of polyamide platinum complexes and provide the structural basis for designer agents that are able to recognize biologically relevant sequences and prevent DNA transcription and replication.

  11. A reliable method for detecting complexed DNA in vitro.

    PubMed

    Holladay, C; Keeney, M; Newland, B; Mathew, A; Wang, W; Pandit, A

    2010-12-01

    Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique hypothesized to allow quantification of both bound and unbound DNA. A comparison of the two methods was performed by quantifying the elution of six different varieties of DNA complexes from a model biomaterial (collagen) scaffold. After seven days of elution, the PicoGreen® assay only allowed detection of three types of complexes (those formed using Lipofectin™ and two synthesised copolymers). However, the Cy5 fluorescent labeling technique enabled detection of all six varieties including those formed via common transfection agents poly(ethylene imine), poly-L-lysine and SuperFect™. This allowed reliable quantification of the elution of all these complexes from the collagen scaffold. Thus, while intercalating dyes may be effective and reliable for detecting double-stranded, unbound DNA, the technique described in this work allowed reliable quantification of DNA independent of complexation state.

  12. RBE of α-particles from (211)At for complex DNA damage and cell survival in relation to cell cycle position.

    PubMed

    Claesson, Kristina; Magnander, Karin; Kahu, Helena; Lindegren, Sture; Hultborn, Ragnar; Elmroth, Kecke

    2011-04-01

    To investigate cell cycle effects and relative biological effectiveness (RBE) of α-particles from the clinically relevant radionuclide Astatine-211 ((211)At), using X-rays as reference radiation. Double-strand breaks (DSB), non-DSB clusters containing oxidised purines and clonogenic survival were investigated. Asynchronous V79-379A fibroblasts or cells synchronised with mimosine in G1, early, mid and late S phase or in mitosis were irradiated with X-rays (100 kV(p)) or (211)At (mean linear energy transfer (LET) 110 keV/μm). Induction of DSB and clusters was determined using pulsed-field gel electrophoresis with fragment analysis. Cell survival was obtained with the clonogenic assay. In asynchronous cells RBE for DSB- and cluster-induction was 3.5 and 0.59, respectively. RBE for 37% cell survival was 8.6. In different cell cycle phases RBE varied from 1.8-3.9 for DSB and 3.1-7.9 for 37% survival (survival at 2 Gy was 6.9-38 times lower after α-irradiation). (211)At induced 6 times more DSB and X-rays induced 11 times more DSB in mitotic cells with highly compacted chromatin relative G1. The radio-response is cell cycle dependent and differs between proliferating and non-cycling cells for both low- and high-LET radiation, resulting in a variation in RBE of α-particles between 1.8 and 8.6.

  13. Mixed DNA/Oligo(ethylene glycol) Functionalized Gold Surface Improve DNA Hybridization in Complex Media

    SciTech Connect

    Lee,C.; Gamble, L.; Grainger, D.; Castner, D.

    2006-01-01

    Reliable, direct 'sample-to-answer' capture of nucleic acid targets from complex media would greatly improve existing capabilities of DNA microarrays and biosensors. This goal has proven elusive for many current nucleic acid detection technologies attempting to produce assay results directly from complex real-world samples, including food, tissue, and environmental materials. In this study, we have investigated mixed self-assembled thiolated single-strand DNA (ssDNA) monolayers containing a short thiolated oligo(ethylene glycol) (OEG) surface diluent on gold surfaces to improve the specific capture of DNA targets from complex media. Both surface composition and orientation of these mixed DNA monolayers were characterized with x-ray photoelectron spectroscopy (XPS) and near-edge x-ray absorption fine structure (NEXAFS). XPS results from sequentially adsorbed ssDNA/OEG monolayers on gold indicate that thiolated OEG diluent molecules first incorporate into the thiolated ssDNA monolayer and, upon longer OEG exposures, competitively displace adsorbed ssDNA molecules from the gold surface. NEXAFS polarization dependence results (followed by monitoring the N 1s{yields}{pi}* transition) indicate that adsorbed thiolated ssDNA nucleotide base-ring structures in the mixed ssDNA monolayers are oriented more parallel to the gold surface compared to DNA bases in pure ssDNA monolayers. This supports ssDNA oligomer reorientation towards a more upright position upon OEG mixed adlayer incorporation. DNA target hybridization on mixed ssDNA probe/OEG monolayers was monitored by surface plasmon resonance (SPR). Improvements in specific target capture for these ssDNA probe surfaces due to incorporation of the OEG diluent were demonstrated using two model biosensing assays, DNA target capture from complete bovine serum and from salmon genomic DNA mixtures. SPR results demonstrate that OEG incorporation into the ssDNA adlayer improves surface resistance to both nonspecific DNA and protein

  14. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

    PubMed Central

    Smale, S T; Tjian, R

    1986-01-01

    We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication. Images PMID:3025630

  15. Solution structure of the luzopeptin-DNA complex

    SciTech Connect

    Zhang, Xiaolu; Patel, D.J. )

    1991-04-23

    The luzopeptin-d(C-A-T-G) complex (1 drug/duplex) has been generated in aqueous solution and its structure characterized by a combined application of two-dimensional NMR experiments and molecular dynamics calculations. Once equivalent of luzopeptin binds to the self-complementary tetranucleotide duplex with the 2-fold symmetry of the antitumor agent and the DNA oligomer retained on complex formation. The authors have assigned the exchangeable and nonexchangeable proton resonances of luzopeptin and the d(C-A-T-G) duplex in the complex and identified the intermolecular proton-proton NOEs that define the alignment of the antitumor agent at its binding site in duplex DNA. The analysis was greatly aided by a large number of intermolecular NOEs involving exchangeable protons on both the luzopeptin and the DNA in the complex. The formation of cis peptide bonds for luzopeptin in the complex results in an increased separation of the long sides of the rectangular cyclic depsipeptide backbone and reorients in the glycine amide proton so that it can form an intermolecular hydrogen bond with the 2-carbonyl of T3 in the complex. This observation explains, in part, the requirement for Watson-Crick A{center dot}T pairs to be sandwiched between the quinolines at the bisintercalation site in the luzopeptin-DNA complex. The NMR studies on the luzopeptin-d(C-A-T-G) complex unequivocally establish that antitumor agents can undergo conformational transitions on complex formation with DNA, and it is the conformation of the drug in the complex that should serve as the starting point for drug design studies. The above structural details on the solution structure of the luzopeptin-DNA complex also explain the sequence selectivity of luzopeptin for bisintercalation at d(C-A){center dot}d(T-G) steps in the d(C-A-T-G) duplex in solution.

  16. 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

  17. Relational Complexity and Theory-of-Mind.

    ERIC Educational Resources Information Center

    Halford, Graeme S.; Andrews, Glenda; Bowden, Darryl

    The concept of relational complexity is applied to explain the persistent difficulties of young children with theory of mind. Relational complexity has been found useful as a general cognitive complexity metric. Children must understand that the relation between an object and a person's percept is conditional on a third variable, such as a filter…

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

    PubMed

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

    2017-05-30

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

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

    PubMed Central

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

    2017-01-01

    A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase–DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development. PMID:28507124

  20. Mystery of DNA repair: the role of the MRN complex and ATM kinase in DNA damage repair.

    PubMed

    Czornak, Kamila; Chughtai, Sanaullah; Chrzanowska, Krystyna H

    2008-01-01

    Genomes are subject to a number of exogenous or endogenous DNA-damaging agents that cause DNA double-strand breaks (DSBs). These critical DNA lesions can result in cell death or a wide variety of genetic alterations, including deletions, translocations, loss of heterozygosity, chromosome loss, or chromosome fusions, which enhance genome instability and can trigger carcinogenesis. The cells have developed an efficient mechanism to cope with DNA damages by evolving the DNA repair machinery. There are 2 major DSB repair mechanisms: nonhomologous end joining (NHEJ) and homologous recombination (HR). One element of the repair machinery is the MRN complex, consisting of MRE11, RAD50 and NBN (previously described as NBS1), which is involved in DNA replication, DNA repair, and signaling to the cell cycle checkpoints. A number of kinases, like ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad-3-related), and DNA PKcs (DNA protein kinase catalytic subunit), phosphorylate various protein targets in order to repair the damage. If the damage cannot be repaired, they direct the cell to apoptosis. The MRN complex as well as repair kinases are also involved in telomere maintenance and genome stability. The dysfunction of particular elements involved in the repair mechanisms leads to genome instability disorders, like ataxia telangiectasia (A-T), A-T-like disorder (ATLD) and Nijmegen breakage syndrome (NBS). The mutated genes responsible for these disorders code for proteins that play key roles in the process of DNA repair. Here we present a detailed review of current knowledge on the MRN complex, kinases engaged in DNA repair, and genome instability disorders.

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

    PubMed

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

    2016-11-01

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

  2. Iridium Complexes as a Roadblock for DNA Polymerase during Amplification.

    PubMed

    Chandra, Falguni; Kumar, Prashant; Tripathi, Suman Kumar; Patra, Srikanta; Koner, Apurba L

    2016-07-05

    Iridium-based metal complexes containing polypyridyl-pyrazine ligands show properties of DNA intercalation. They serve as roadblocks to DNA polymerase activity, thereby inhibiting the polymerization process. Upon the addition of increasing concentrations of these iridium complexes, a rapid polymerase chain reaction (PCR)-based assay reveals the selective inhibition of the DNA polymerization process. This label-free approach to study the inhibition of fundamental cellular processes via physical roadblock can offer an alternative route toward cancer therapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. DNA tile based self-assembly: building complex nanoarchitectures.

    PubMed

    Lin, Chenxiang; Liu, Yan; Rinker, Sherri; Yan, Hao

    2006-08-11

    DNA tile based self-assembly provides an attractive route to create nanoarchitectures of programmable patterns. It also offers excellent scaffolds for directed self-assembly of nanometer-scale materials, ranging from nanoparticles to proteins, with potential applications in constructing nanoelectronic/nanophotonic devices and protein/ligand nanoarrays. This Review first summarizes the currently available DNA tile toolboxes and further emphasizes recent developments toward self-assembling DNA nanostructures with increasing complexity. Exciting progress using DNA tiles for directed self-assembly of other nanometer scale components is also discussed.

  4. Diversity and Complexity in DNA Recognition by Transcription Factors**

    PubMed Central

    Badis, Gwenael; Berger, Michael F.; Philippakis, Anthony A.; Talukder, Shaheynoor; Gehrke, Andrew R.; Jaeger, Savina A.; Chan, Esther T.; Metzler, Genita; Vedenko, Anastasia; Chen, Xiaoyu; Kuznetsov, Hanna; Wang, Chi-Fong; Coburn, David; Newburger, Daniel E.; Morris, Quaid; Hughes, Timothy R.; Bulyk, Martha L.

    2010-01-01

    Sequence preferences of DNA-binding proteins are a primary mechanism by which cells interpret the genome. Despite these proteins’ central importance in physiology, development, and evolution, comprehensive DNA-binding specificities have been determined experimentally for few proteins. Here, we used microarrays containing all 10-base-pair sequences to examine the binding specificities of 104 distinct mouse DNA-binding proteins representing 22 structural classes. Our results reveal a complex landscape of binding, with virtually every protein analyzed possessing unique preferences. Roughly half of the proteins each recognized multiple distinctly different sequence motifs, challenging our molecular understanding of how proteins interact with their DNA binding sites. This complexity in DNA recognition may be important in gene regulation and in evolution of transcriptional regulatory networks. PMID:19443739

  5. 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.

  6. 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

  7. A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase.

    PubMed

    Jost, J P; Schwarz, S; Hess, D; Angliker, H; Fuller-Pace, F V; Stahl, H; Thiry, S; Siegmann, M

    1999-08-15

    We have shown previously that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA. Amino acid sequences of nine peptides derived from a highly purified 5-MeC-DNA glycosylase complex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase. Antibodies directed against human p68 helicase cross-reacted with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase activity. A 2690 bp cDNA coding for the chicken homologue of mammalian p68 was isolated and sequenced. Its derived amino acid sequence is almost identical to the human p68 DEAD box protein up to amino acid position 473 (from a total of 595). This sequence contains all the essential conserved motifs from the DEAD box proteins which are the ATPase, RNA unwinding and RNA binding motifs. The rest of the 122 amino acids in the C-terminal region rather diverge from the human p68 RNA helicase sequence. The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 antibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex. The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helicase activity. However, in the presence or absence of RNA the recombinant protein had no 5-MeC-DNA glycosylase activity. In situ hybridisation of 5 day-old chicken embryos with antisense probes of the chicken DEAD box protein shows a high abundance of its transcripts in differentiating embryonic tissues.

  8. 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

  9. Structures of DNA Polymerase Mispaired DNA Termini Transitioning to Pre-catalytic Complexes Support an Induced-Fit Fidelity Mechanism.

    PubMed

    Batra, Vinod K; Beard, William A; Pedersen, Lars C; Wilson, Samuel H

    2016-11-01

    High-fidelity DNA synthesis requires that polymerases display a strong preference for right nucleotide insertion. When the wrong nucleotide is inserted, the polymerase deters extension from the mismatched DNA terminus. Twenty-three crystallographic structures of DNA polymerase β with terminal template-primer mismatches were determined as binary DNA and ternary pre-catalytic substrate complexes. These structures indicate that the mismatched termini adopt various distorted conformations that attempt to satisfy stacking and hydrogen-bonding interactions. The binary complex structures indicate an induced strain in the mismatched template nucleotide. Addition of a non-hydrolyzable incoming nucleotide stabilizes the templating nucleotide with concomitant strain in the primer terminus. Several dead-end ternary complex structures suggest that DNA synthesis might occur as the enzyme transitions from an open to a closed complex. The structures are consistent with an induced-fit mechanism where a mismatched terminus is misaligned relative to the correct incoming nucleotide to deter or delay further DNA synthesis.

  10. The Complex of Ethidium Bromide with Genomic DNA: Structure Analysis by Polarized Raman Spectroscopy

    PubMed Central

    Tsuboi, Masamichi; Benevides, James M.; Thomas, George J.

    2007-01-01

    Structural properties of the complex formed between genomic DNA and the intercalating drug ethidium bromide (EtBr) have been determined by use of a Raman microscope equipped with near-infrared laser excitation. The polarized spectra, which were obtained from oriented fibers of the EtBr:DNA complex, are interpreted in terms of the relative orientations of the phenanthridinium ring of EtBr and bases of DNA. Quantification of structure parameters of EtBr and DNA in the complex were assessed using Raman tensors obtained from polarized Raman analyses of oriented specimens of EtBr (single crystal) and DNA (hydrated fiber). We find that the phenanthridinium plane is tilted by 35 ± 5° from the plane perpendicular to the fiber (DNA helix) axis. Assuming coplanarity of the phenanthridinium ring and its immediate base neighbors at the intercalation site, such bases would have a tilt angle closer to that of A-DNA (20°) than to that of B-DNA (6°). The average base tilt in stretches of DNA between intercalation sites remains that of B-DNA. PMID:17098786

  11. O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro.

    PubMed

    Liu, Y; Egyhazi, S; Hansson, J; Bhide, S V; Kulkarni, P S; Grafström, R C

    1997-10-01

    Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.

  12. Energy-Complexity Relations by Structural Complexity Methods

    NASA Astrophysics Data System (ADS)

    Ricca, Renzo L.

    2011-09-01

    In this paper we shall review some of the most recent developments and results on work on energy-complexity relations and, if time will allow it, we shall provide an analytical proof of eq. (3) below, a fundamental relation between energy and complexity established by numerical experiments.

  13. Respiratory chain complex I deficiency caused by mitochondrial DNA mutations

    PubMed Central

    Swalwell, Helen; Kirby, Denise M; Blakely, Emma L; Mitchell, Anna; Salemi, Renato; Sugiana, Canny; Compton, Alison G; Tucker, Elena J; Ke, Bi-Xia; Lamont, Phillipa J; Turnbull, Douglass M; McFarland, Robert; Taylor, Robert W; Thorburn, David R

    2011-01-01

    Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality. PMID:21364701

  14. High temperature stabilization of DNA in complexes with cationic lipids.

    PubMed Central

    Tarahovsky, Yury S; Rakhmanova, Vera A; Epand, Richard M; MacDonald, Robert C

    2002-01-01

    The influence on the melting of calf thymus and plasmid DNA of cationic lipids of the type used in gene therapy was studied by ultraviolet spectrophotometry and differential scanning calorimetry. It was found that various membrane-forming cationic lipids are able to protect calf thymus DNA against denaturation at 100 degrees C. After interaction with cationic lipids, the differential scanning calorimetry melting profile of both calf thymus and plasmid DNA revealed two major components, one corresponding to a thermolabile complex with transition temperature, T(m(labile)), close to that of free DNA and a second corresponding to a thermostable complex with a transition temperature, T(m(stable)), at 105 to 115 degrees C. The parameter T(m(stable)) did not depend on the charge ratio, R(+/-). Instead, the amount of thermostable DNA and the enthalpy ratio Delta H((stable))/Delta H((labile)) depended upon R(+/-) and conditions of complex formation. In the case of O-ethyldioleoylphosphatidylcholine, the cationic lipid that was the main subject of the investigation, the maximal stabilization of DNA exceeded 90% between R(+/-) = 1.5 and 3.0. Several other lipids gave at least 75% protection in the range R(+/-) = 1.5 to 2.0. Centrifugal separation of the thermostable and thermolabile fractions revealed that almost all the transfection activity was present at the thermostable fraction. Electron microscopy of the thermostable complex demonstrated the presence of multilamellar membranes with a periodicity 6.0 to 6.5 nm. This periodic multilamellar structure was retained at temperatures as high as 130 degrees C. It is concluded that constraint of the DNA molecules between oppositely charged membrane surfaces in the multilamellar complex is responsible for DNA stabilization. PMID:11751314

  15. Conformational flexibility facilitates self-assembly of complex DNA nanostructures.

    PubMed

    Zhang, Chuan; Su, Min; He, Yu; Zhao, Xin; Fang, Ping-an; Ribbe, Alexander E; Jiang, Wen; Mao, Chengde

    2008-08-05

    Molecular self-assembly is a promising approach to the preparation of nanostructures. DNA, in particular, shows great potential to be a superb molecular system. Synthetic DNA molecules have been programmed to assemble into a wide range of nanostructures. It is generally believed that rigidities of DNA nanomotifs (tiles) are essential for programmable self-assembly of well defined nanostructures. Recently, we have shown that adequate conformational flexibility could be exploited for assembling 3D objects, including tetrahedra, dodecahedra, and buckyballs, out of DNA three-point star motifs. In the current study, we have integrated tensegrity principle into this concept to assemble well defined, complex nanostructures in both 2D and 3D. A symmetric five-point-star motif (tile) has been designed to assemble into icosahedra or large nanocages depending on the concentration and flexibility of the DNA tiles. In both cases, the DNA tiles exhibit significant flexibilities and undergo substantial conformational changes, either symmetrically bending out of the plane or asymmetrically bending in the plane. In contrast to the complicated natures of the assembled structures, the approach presented here is simple and only requires three different component DNA strands. These results demonstrate that conformational flexibility could be explored to generate complex DNA nanostructures. The basic concept might be further extended to other biomacromolecular systems, such as RNA and proteins.

  16. Cooperative dynamics of a DNA polymerase replicating complex.

    PubMed

    Moors, Samuel L C; Herdewijn, Piet; Robben, Johan; Ceulemans, Arnout

    2013-12-01

    Engineered DNA polymerases continue to be the workhorses of many applications in biotechnology, medicine and nanotechnology. However, the dynamic interplay between the enzyme and the DNA remains unclear. In this study, we performed an extensive replica exchange with flexible tempering (REFT) molecular dynamics simulation of the ternary replicating complex of the archaeal family B DNA polymerase from the thermophile Thermococcus gorgonarius, right before the chemical step. The convoluted dynamics of the enzyme are reducible to rigid-body motions of six subdomains. Upon binding to the enzyme, the DNA double helix conformation changes from a twisted state to a partially untwisted state. The twisted state displays strong bending motion, whereby the DNA oscillates between a straight and a bent conformation. The dynamics of double-stranded DNA are strongly correlated with rotations of the thumb toward the palm, which suggests an assisting role of the enzyme during DNA translocation. In the complex, the primer-template duplex displays increased preference for the B-DNA conformation at the n-2 and n-3 dinucleotide steps. Interactions at the primer 3' end indicate that Thr541 and Asp540 are the acceptors of the first proton transfer in the chemical step, whereas in the translocation step both residues hold the primer 3' terminus in the vicinity of the priming site, which is crucial for high processivity.

  17. Gene 5 protein-DNA complex: modeling binding interactions.

    PubMed

    Hutchinson, D L; Barnett, B L; Bobst, A M

    1990-08-01

    A helical (not toroidal) complex consisting of eight gene 5 protein dimers per turn is proposed for the extension of DNA from dimer to dimer using known bond length constraints, postulated protein-nucleic acid interactions (determined from NMR and chemical modification studies), other physical properties of the complex, and data from electron micrographs. The binding channel has been dictated by these known parameters and the relative ease of geometrically fitting these constituents. This channel is different from that previously reported by other modelers. The channel lies underneath the long arm "claw-like" extension of the monomer, so that it rests inside the outer surface of the protein complex. An explanation is proposed for the two binding modes, n = 4 (the predominate mode) and n = 3, based on the weak binding interaction of Tyrosine 34. Also, the site of the less mobile nucleic acid base as reported from ESR studies (S.-C. Kao, E.V. Bobst, G.T. Pauly and A.M. Bobst, J. Biom. Struc. Dyn. 3,261 (1985)) is postulated as involving the fourth nucleotide, and this particular base is stacked between Tyrosine 34 and Phenylalanine 73'.

  18. Structure of a complex between E. coli DNA topoisomerase I and single-stranded DNA.

    PubMed

    Perry, Kay; Mondragón, Alfonso

    2003-11-01

    In order to gain insights into the mechaism of ssDNA binding and recognition by Escherichia coli DNA topoisomerase I, the structure of the 67 kDa N-terminal fragment of topoisomerase I was solved in complex with ssDNA. The structure reveals a new conformational stage in the multistep catalytic cycle of type IA topoisomerases. In the structure, the ssDNA binding groove leading to the active site is occupied, but the active site is not fully formed. Large conformational changes are not seen; instead, a single helix parallel to the ssDNA binding groove shifts to clamp the ssDNA. The structure helps clarify the temporal sequence of conformational events, starting from an initial empty enzyme and proceeding to a ssDNA-occupied and catalytically competent active site.

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

    PubMed

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

    2016-12-17

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

  20. Adsorption behaviors of DNA/cation complexes on amino and silica chip surfaces: a dual polarization interferometry study.

    PubMed

    Huang, Fujian; Liang, Haojun

    2013-06-12

    The adsorption of DNA/Ca(2+), DNA/Cu(2+), and DNA/Co(NH3)6(3+) complexes on amino and silica chip surfaces were investigated using dual polarization interferometry. A more compact DNA/cation complex layer formed on the amino chip surface compared with that on the silica chip surface at the same cation condition. The real-time mass, thickness, and density changes were monitored during the adsorption process. The overall results show that the approaching complexes can cause the conformation rearrangement of the preadsorbed complexes and the preadsorbed complexes affect the deposition pattern of the approaching complexes during the adsorption of DNA/Ca(2+) and DNA/Cu(2+) complexes on both chip surfaces. The relatively strong electrostatic repulsion between the approaching and adsorbed complexes results in multiple mass loading rate changes and loose attachment of the approaching complexes. The weak repulsion between the DNA/Co(NH3)6(3+) complexes cannot induce this kind of conformation rearrangement. Thus, no multiple mass loading rate changes were observed. Meanwhile, the preadsorbed DNA/Co(NH3)6(3+) complex can also affect the deposition pattern of the approaching complex because of the geometric resistance. Therefore, this study will help better understand the conformation change and deposition pattern of complexes with different charge conditions during the adsorption process on the solid-liquid interface.

  1. Optimization of hydrogen bonds for combined DNA/collagen complex.

    PubMed

    Pidaparti, Ramana M; Svintradze, David V; Shan, Yingfeng; Yokota, Hiroki

    2009-01-21

    Many natural and biological systems including collagen and DNA polymers are formed by a process of molecular self-assembly. In this paper, we developed two novel structural models and built heterogeneous DNA/collagen complexes through a preferable arrangement of multiple hydrogen bonds (H-bonds) between DNA and collagen molecules. The simulation results based on three sets of criteria indicate that one of the models with five collagen molecules, which are positioned around each strand of DNA molecules emerged to form a suitable polymer complex with the maximum number of H-bonds. Our predictions quantitatively validated and agreed with the molecular structure reported by Mrevlishvili and Svintradze [2005. Int. J. Biol. Macromol. 36, 324-326].

  2. A reliable method for detecting complexed DNA in vitro

    NASA Astrophysics Data System (ADS)

    Holladay, C.; Keeney, M.; Newland, B.; Mathew, A.; Wang, W.; Pandit, A.

    2010-12-01

    Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique hypothesized to allow quantification of both bound and unbound DNA. A comparison of the two methods was performed by quantifying the elution of six different varieties of DNA complexes from a model biomaterial (collagen) scaffold. After seven days of elution, the PicoGreen® assay only allowed detection of three types of complexes (those formed using Lipofectin™ and two synthesised copolymers). However, the Cy5 fluorescent labeling technique enabled detection of all six varieties including those formed via common transfection agents poly(ethylene imine), poly-l-lysine and SuperFect™. This allowed reliable quantification of the elution of all these complexes from the collagen scaffold. Thus, while intercalating dyes may be effective and reliable for detecting double-stranded, unbound DNA, the technique described in this work allowed reliable quantification of DNA independent of complexation state.Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique

  3. 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.

  4. Dynamics of DNA/intercalator complexes

    NASA Astrophysics Data System (ADS)

    Schurr, J. M.; Wu, Pengguang; Fujimoto, Bryant S.

    1990-05-01

    Complexes of linear and supercoiled DNAs with different intercalating dyes are studied by time-resolved fluorescence polarization anisotropy using intercalated ethidium as the probe. Existing theory is generalized to take account of excitation transfer between intercalated ethidiums, and Forster theory is shown to be valid in this context. The effects of intercalated ethidium, 9-aminoacridine, and proflavine on the torsional rigidity of linear and supercoiled DNAs are studied up to rather high binding ratios. Evidence is presented that metastable secondary structure persists in dye-relaxed supercoiled DNAs, which contradicts the standard model of supercoiled DNAs.

  5. Calculation of complex DNA damage induced by ions

    NASA Astrophysics Data System (ADS)

    Surdutovich, Eugene; Gallagher, David C.; Solov'yov, Andrey V.

    2011-11-01

    This paper is devoted to the analysis of the complex damage of DNA irradiated by ions. The assessment of complex damage is important because cells in which it occurs are less likely to survive because the DNA repair mechanisms may not be sufficiently effective. We study the flux of secondary electrons through the surface of nucleosomes and calculate the radial dose and the distribution of clustered damage around the ion's path. The calculated radial dose distribution is compared to simulations. The radial distribution of the complex damage is found to be different from that of the dose. A comparison with experiments may solve the question of what is more lethal for the cell, damage complexity or absorbed energy. We suggest a way to calculate the probability of cell death based on the complexity of the damage. This work is done within the framework of the phenomenon-based multiscale approach to radiation damage by ions.

  6. Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity.

    PubMed

    Pelletier, H; Sawaya, M R; Wolfle, W; Wilson, S H; Kraut, J

    1996-10-01

    Mammalian DNA polymerase beta (pol beta) is a small (39 kDa) DNA gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a carboxy-terminal 31-kDa domain. In the work reported here, crystal structures of human pol beta complexed with blunt-ended segments of DNA show that, although the crystals belong to a different space group, the DNA is nevertheless bound in the pol beta binding channel in the same way as the DNA in previously reported structures of rat pol beta complexed with a template-primer and ddCTP [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., & Kraut, J. (1994) Science 264, 1891-1903]. The 8-kDa domain is in one of three previously observed positions relative to the 31-kDa domain, suggesting that the 8-kDa domain may assume only a small number of stable conformations. The thumb subdomain is in a more open position in the human pol beta-DNA binary complex than it is in the rat pol beta-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide binding could represent the rate-limiting conformational change that has been observed in pre-steady-state kinetic studies. Intermolecular contacts between the DNA and the 8-kDa domain of a symmetry-related pol beta molecule reveal a plausible binding site on the 8-kDa domain for the downstream oligonucleotide of a gapped-DNA substrate; in addition to a lysine-rich binding pocket that accommodates a 5'-PO4 end group, the 8-kDa domain also contains a newly discovered helix-hairpin-helix (HhH) motif that binds to DNA in the same way as does a structurally and sequentially homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs is facilitated by a metal ion. In that HhH motifs have been identified in other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions observed in pol beta may be applicable to a broad range of DNA binding proteins. The sequence similarity between the HhH motif of endonuclease III from Escherichia coli and the HhH motif of the 8-kDa domain of

  7. 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.

  8. Unusual DNA binding modes for metal anticancer complexes

    PubMed Central

    Pizarro, Ana M.; Sadler, Peter J.

    2010-01-01

    DNA is believed to be the primary target for many metal-based drugs. For example, platinum-based anticancer drugs can form specific lesions on DNA that induce apoptosis. New platinum drugs can be designed that have novel modes of interaction with DNA, such as the trinuclear platinum complex BBR3464. Also it is possible to design inert platinum(IV) pro-drugs which are non-toxic in the dark, but lethal when irradiated with certain wavelengths of light. This gives rise to novel DNA lesions which are not as readily repaired as those induced by cisplatin, and provides the basis for a new type of photoactivated chemotherapy. Finally, newly emerging ruthenium(II) organometallic complexes not only bind to DNA coordinatively, but also by H-bonding and hydrophibic interactions triggered by the introduction of extended arene rings into their versatile structures. Intriguingly osmium (the heavier congener of ruthenium) reacts differently with DNA but can also give rise to highly cytotoxic organometallic complexes. PMID:19344743

  9. 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.

  10. Structural Phase Diagrams for Dendrimer:DNA Complexes

    NASA Astrophysics Data System (ADS)

    Evans, Heather M.; Ahmad, A.; Pfohl, T.; Martin, A.; Safinya, C. R.

    2002-03-01

    Polypropylene imine (PPI) dendrimers become positively charged in aqueous solution and are consequently a useful tool for packaging DNA in gene delivery applications. Studies of PPI and DNA help to improve the efficiency of DNA delivery as well as model more complex biological systems such as histones. PPI is synthesized in a stepwise manner, and at each step the product is referred to as a generation (G1 through G5). We show structural phase diagrams based on synchrotron x-ray diffraction as a function of PPI/DNA charge ratio (P/D). At lower generations PPI bundling with DNA is similar to DNA condensation caused by small multivalent salts such as spermidine. Remarkably, for G4 and G5 distinct structural transitions are seen with increasing P/D. In particular, G4:DNA complexes show a salt-like bundling at low P/D, followed by both square and hexagonal lattices with addition of PPI. Optical microscopy and supporting size and charge measurements will also be shown. Funding provided by NIH GM59288 and NSF DMR-9972246.

  11. 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

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

    PubMed

    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.

  13. Contrasting enantioselective DNA preference: chiral helical macrocyclic lanthanide complex binding to DNA.

    PubMed

    Zhao, Chuanqi; Ren, Jinsong; Gregoliński, Janusz; Lisowski, Jerzy; Qu, Xiaogang

    2012-09-01

    There is great interest in design and synthesis of small molecules which selectively target specific genes to inhibit biological functions in which particular DNA structures participate. Among these studies, chiral recognition has been received much attention because more evidences have shown that conversions of the chirality and diverse conformations of DNA are involved in a series of important life events. Here, we report that a pair of chiral helical macrocyclic lanthanide (III) complexes, (M)-Yb[L(SSSSSS)](3+) and (P)-Yb[L(RRRRRR)](3+), can enantioselectively bind to B-form DNA and show remarkably contrasting effects on GC-rich and AT-rich DNA. Neither of them can influence non-B-form DNA, nor quadruplex DNA stability. Our results clearly show that P-enantiomer stabilizes both poly(dG-dC)(2) and poly(dA-dT)(2) while M-enantiomer stabilizes poly(dA-dT)(2), however, destabilizes poly(dG-dC)(2). To our knowledge, this is the best example of chiral metal compounds with such contrasting preference on GC- and AT-DNA. Ligand selectively stabilizing or destabilizing DNA can interfere with protein-DNA interactions and potentially affect many crucial biological processes, such as DNA replication, transcription and repair. As such, bearing these unique capabilities, the chiral compounds reported here may shed light on the design of novel enantiomers targeting specific DNA with both sequence and conformation preference.

  14. The phase behavior of cationic lipid-DNA complexes.

    PubMed Central

    May, S; Harries, D; Ben-Shaul, A

    2000-01-01

    We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram. PMID:10733951

  15. Antitumor drug nogalamycin binds DNA in both grooves simultaneously: molecular structure of nogalamycin-DNA complex.

    PubMed

    Liaw, Y C; Gao, Y G; Robinson, H; van der Marel, G A; van Boom, J H; Wang, A H

    1989-12-26

    The three-dimensional molecular structures of the complexes between an interesting antitumor drug, nogalamycin, and two DNA hexamers, d[CGT(pS)ACG] and d[m5CGT(pS)Am5CG], were determined at high resolution by X-ray diffraction analyses. Two nogalamycins bind to the DNA double helix in a 2:1 ratio with the aglycon chromophore intercalated between the CpG steps at both ends of the helix. The nogalose and aminoglucose sugars lie in the minor and major grooves, respectively, of the distorted B-DNA double helix. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. Specific hydrogen bonds are found in the complex between the drug and guanine bases. We suggest that nogalamycin may prefer GC sequences embedded in a stretch of AT sequences.

  16. Catalytic Hairpin Assembly-Programmed Porphyrin-DNA Complex as Photoelectrochemical Initiator for DNA Biosensing.

    PubMed

    Zang, Yang; Lei, Jianping; Ling, Pinghua; Ju, Huangxian

    2015-01-01

    A catalytic hairpin assembly (CHA)-programmed porphyrin-DNA complex was designed to trigger the chemiluminescence as photoelectrochemical initiator for DNA sensing. First, the programmed double strand DNA (dsDNA) was formed using two hairpin DNAs as assembly components via target-assisted CHA reaction, and then immobilized on a capture DNA/CdS quantum dots modified electrode. The porphyrin (FeTMPyP) was conveniently assembled on a dsDNA scaffold via the groove interaction. The FeTMPyP@dsDNA complex possessed high catalytic activity toward luminol oxidation to generate the desirable chemiluminescence with high stability under various temperature and alkaline conditions. By integrating the signal amplification capacity of CHA and in situ FeTMPyP-mediated chemiluminescence as excitation light, an amplified photoelectrochemical sensing strategy is proposed for DNA detection. Under optimized conditions, the biosensor shows a wide linear range from 5 to 10000 fM with a detection limit of 2.2 fM. Moreover, the developed photoelectrochemical device exhibits excellent selectivity, high stability, and acceptable fabrication reproducibility. The CHA-programmed porphyrin-DNA strategy not only extends the applications of photoelectrochemistry, but also presents a novel methodology in bioanalysis.

  17. DNA replication origins, ORC/DNA interaction, and assembly of pre-replication complex in eukaryotes.

    PubMed

    Sun, Jingya; Kong, Daochun

    2010-07-01

    Chromosomal DNA replication in eukaryotic cells is highly complicated and sophisticatedly regulated. Owing to its large size, a typical eukaryotic genome contains hundreds to tens of thousands of initiation sites called DNA replication origins where DNA synthesis takes place. Multiple initiation sites remove the constraint of a genome size because only a certain amount of DNA can be replicated from a single origin in a limited time. The activation of these multiple origins must be coordinated so that each segment of chromosomal DNA is precisely duplicated only once per cell cycle. Although DNA replication is a vital process for cell growth and its mechanism is highly conserved, recent studies also reveal significant diversity in origin structure, assembly of pre-replication complex (pre-RC) and regulation of replication initiation along evolutionary lines. The DNA replication origins in the fission yeast Schizosaccharomyces pombe are found to contain a second essential element that is bound by Sap1 protein besides the essential origin recognition complex-binding site. Sap1 is recently demonstrated to be a novel replication initiation protein that plays an essential role in loading the initiation protein Cdc18 to origins and thus directly participates in pre-RC formation. In this review, we summarize the recent advance in understanding how DNA replication origins are organized, how pre-RC is assembled and how DNA replication is initiated and regulated in yeast and metazoans.

  18. 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

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

    PubMed Central

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

    2009-01-01

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

  20. Binding of copper(II) polypyridyl complexes to DNA and consequences for DNA-based asymmetric catalysis.

    PubMed

    Draksharapu, Apparao; Boersma, Arnold J; Leising, Miriam; Meetsma, Auke; Browne, Wesley R; Roelfes, Gerard

    2015-02-28

    The interaction between salmon testes DNA (st-DNA) and a series of Cu(II) polypyridyl complexes, i.e. [Cu(dmbpy)(NO3)2] (1) (dmbpy = 4,4'-dimethyl-2,2'-bipyridine), [Cu(bpy)(NO3)2] (2) (bpy = 2,2'-bipyridine), [Cu(phen)(NO3)2] (3) (phen = phenanthroline), [Cu(terpy)(NO3)2]·H2O (4) (terpy = 2,2':6',2″-terpyridine), [Cu(dpq)(NO3)2] (5) (dpq = dipyrido-[3,2-d:2',3'-f]-quinoxaline) and [Cu(dppz)(NO3)2] (6) (dppz = dipyrido[3,2-a:2',3'-c]phenazine) was studied by UV/Vis absorption, Circular Dichroism, Linear Dichroism, EPR, Raman and (UV and vis) resonance Raman spectroscopies and viscometry. These complexes catalyse enantioselective C-C bond forming reactions in water with DNA as the source of chirality. Complex 1 crystallizes as an inorganic polymer with nitrate ligands bridging the copper ions, which adopt essentially a distorted square pyramidal structure with a fifth bridging nitrate ligand at the axial position. Raman spectroscopy indicates that in solution the nitrate ligands in 1, 2, 3 and 4 are displaced by solvent (H2O). For complex 1, multiple supramolecular species are observed in the presence of st-DNA in contrast to the other complexes, which appear to interact relatively uniformly as a single species predominantly, when st-DNA is present. Overall the data suggest that complexes 1 and 2 engage primarily through groove binding with st-DNA while 5 and 6 undergo intercalation. For complexes 3 and 4 the data indicates that both groove binding and intercalation takes place, albeit primarily intercalation. Although it is tempting to conclude that the groove binders give highest ee and rate acceleration, it is proposed that the flexibility and dynamics in binding of Cu(II) complexes to DNA are key parameters that determine the outcome of the reaction. These findings provide insight into the complex supramolecular structure of these DNA-based catalysts.

  1. Time-resolved fluorescence spectroscopic investigation of cationic polymer/DNA complex formation

    NASA Astrophysics Data System (ADS)

    D'Andrea, Cosimo; Bassi, Andrea; Taroni, Paola; Pezzoli, Daniele; Volonterio, Alessandro; Candiani, Gabriele

    2011-07-01

    Since DNA is not internalized efficiently by cells, the success of gene therapy depends on the availability of carriers to efficiently deliver genetic material into target cells. Gene delivery vectors can be broadly categorized into viral and non-viral ones. Non-viral gene delivery systems are represented by cationic lipids and polymers rely on the basics of supramolecular chemistry termed "self-assembling": at physiological pH, they are cations and spontaneously form lipoplexes (for lipids) and polyplexes (for polymers) complexing nucleic acids. In this scenario, cationic polymers are commonly used as non-viral vehicles. Their effectiveness is strongly related to key parameters including DNA binding ability and stability in different environments. Time-resolved fluorescence spectroscopy of SYBR Green I (DNA dye) was carried out to characterize cationic polymer/DNA complex (polyplex) formation dispersed in aqueous solution. Both fluorescence amplitude and lifetime proved to be very sensitive to the polymer/DNA ratio (N/P ratio, +/-).

  2. Structural basis of human transcription factor Sry-related box 17 binding to DNA.

    PubMed

    Gao, Nana; Jiang, Wei; Gao, Hai; Cheng, Zhong; Qian, Huolian; Si, Shuyi; Xie, Yong

    2013-04-01

    Sry-related box (Sox) transcription factors share a conserved high-mobility-group box domain (HMG-domain) that binds DNA in the minor groove and bends DNA for further assembly of transcriptional machineries. During organogenesis, each member of the Sox family triggers a specific cell lineage differentiation, indicating that their interactions with DNA are different from each other. Therefore, investigating structural rearrangement of each Sox transcription factor HMG-domain upon binding to DNA would help to elucidate the distinctive molecular mechanism by which they interact with DNA. Previous studies have determined the crystal structures of Sox2 HMG-domain/DNA, Sox4 HMGdomain/ DNA, Sox9 HMG-domain/DNA and Sox17 HMG-domain/DNA complexes. However, major gaps remain in the structural information on the Sox transcription factor HMG-domains. Here, we report the crystal structure of the human Sox17 HMG-domain alone at 2.4 A resolution. Comparing this structure and the structure of the mouse Sox17 HMGdomain/ DNA complex provides structural understanding of the mechanism of Sox17 binding to DNA. Specifically, after electrostatic interactions attract Sox17 to DNA, Asn73, Ser99, and Trp106 form hydrogen bonds with DNA, Arg70, Lys80, Arg83, His94, and Asn95 on Sox17 undergo conformational changes and form hydrogen bonds with DNA, contributing to the electrostatic interaction between Sox17 and DNA.

  3. Core nucleosomes by digestion of reconstructed histone-DNA complexes.

    PubMed Central

    Bryan, P N; Wright, E B; Olins, D E

    1979-01-01

    Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (nu 1). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(A-dT.poly(dA-dT) have been examined by circular dichroism, thermaldetenaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260--280 nm and a prominent alpha-helix signal at 222 nm. All particles show biphasic melting except nu 1 (dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of nu 1 (dA-dT) produces a ladder of DNA fragments fiffering in lengthy by one base residue. nu 1 (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4-10.5 bases per turn. There appear to be two regions of different DNA pitch wihtin nu 1 (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles. Images PMID:450703

  4. 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.

  5. 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.

  6. Structural studies on Pax-8 Prd domain/DNA complex.

    PubMed

    Campagnolo, M; Pesaresi, A; Zelezetsky, I; Geremia, S; Randaccio, L; Bisca, A; Tell, G

    2007-04-01

    Pax-8 is a member of the Pax family of transcription factors and is essential in the development of thyroid follicular cells. Pax-8 has two DNA-binding domains: the paired domain and the homeo domain. In this study, a preliminary X-ray diffraction analysis of the mammalian Pax-8 paired domain in complex with the C-site of the thyroglobulin promoter was achieved. The Pax-8 paired domain was crystallized by the hanging-drop vapor-diffusion method in complex with both a blunt-ended 26 bp DNA fragment and with a sticky-ended 24 bp DNA fragment with two additional overhanging bases. Crystallization experiments make clear that the growth of transparent crystals with large dimensions and regular shape is particularly influenced by ionic strength. The crystals of Pax-8 complex with blunt-ended and sticky-ended DNA, diffracted synchrotron radiation to 6.0 and 8.0 A resolution and belongs both to the C centered monoclinic system with cell dimensions: a = 89.88 A, b = 80.05 A, c = 67.73 A, and beta = 124.3 degrees and a = 256.56, b = 69.07, c = 99.32 A, and beta = 98.1 degrees , respectively. Fluorescence experiments suggest that the crystalline disorder, deduced by the poor diffraction, can be attributed to the low homogeneity of the protein-DNA sample. The theoretical comparative model of the Pax-8 paired domain complexed with the C-site of the thyroglobulin promoter shows the probable presence of some specific protein-DNA interactions already observed in other Pax proteins and the important role of the cysteine residues of PAI subdomain in the redox control of the DNA recognition.

  7. Stabilities of Irradiated DNA Complexes from Sarcoma 45 Tumors with Mitoxantrone at Small Fillings

    NASA Astrophysics Data System (ADS)

    Hakobyan, Sergey N.; Shahinyan, Mariam A.; Babayan, Yuri S.

    In the present work, the thermostabilities of mitoxantrone (MTX) complexes with DNA from sarcoma 45 and healthy rat liver were studied. DNAs from both sources were irradiated by resonant (64.5 GHz and 50.3 GHz) and nonresonant (48.3 GHz) frequencies of water. The obtained data showed that DNA solution irradiation by resonant frequencies of water induces a dehydration of nucleotides and Na+ ions in the solution. It is shown that at relatively low concentrations of MTX, when one MTX molecule binds to almost 100 pairs of bases of DNA, the thermostabilities of complexes decrease. Moreover, this change is more pronounced (˜0.8∘C) at the complex formation with DNA released from sarcoma 45 tumor.

  8. Lac repressor: Crystallization of intact tetramer and its complexes with inducer and operator DNA

    SciTech Connect

    Pace, H.C.; Lu, P. ); Lewis, M. Smith Kline and French Labs., King of Prussia, PA )

    1990-03-01

    The intact lac repressor tetramer, which regulates expression of the lac operon in Escherichia coli, has been crystallized in the native form, with an inducer, and in a ternary complex with operator DNA and an anti-inducer. The crystals without DNA diffract to better than 3.5 {angstrom}. They belong to the monoclinic space group C2 and have cell dimensions a = 164.7 {angstrom}, b = 75.6 {angstrom}, and c = 161.2 {angstrom}, with {alpha} = {gamma} = 90{degree} and {beta} = 125.5{degree}. Cocrystals have been obtained with a number of different lac operator-related DNA fragments. The complex with a blunt-ended 16-base-pair strand yielded tetragonal bipyramids that diffract to 6.5 {angstrom}. These protein-DNA cocrystals crack upon exposure to the gratuitous inducer isopropyl {beta}-D-thiogalactoside, suggesting a conformational change in the repressor-operator complex.

  9. Complexity reduction in context-dependent DNA substitution models.

    PubMed

    Majoros, William H; Ohler, Uwe

    2009-01-15

    The modeling of conservation patterns in genomic DNA has become increasingly popular for a number of bioinformatic applications. While several systems developed to date incorporate context-dependence in their substitution models, the impact on computational complexity and generalization ability of the resulting higher order models invites the question of whether simpler approaches to context modeling might permit appreciable reductions in model complexity and computational cost, without sacrificing prediction accuracy. We formulate several alternative methods for context modeling based on windowed Bayesian networks, and compare their effects on both accuracy and computational complexity for the task of discriminating functionally distinct segments in vertebrate DNA. Our results show that substantial reductions in the complexity of both the model and the associated inference algorithm can be achieved without reducing predictive accuracy.

  10. Trajectory of DNA in the RNA polymerase II transcription preinitiation complex

    PubMed Central

    Kim, Tae-Kyung; Lagrange, Thierry; Wang, Yuh-Hwa; Griffith, Jack D.; Reinberg, Danny; Ebright, Richard H.

    1997-01-01

    By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, ≈240 Å channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of ≈50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA. PMID:9356438

  11. TOPOLOGIES OF COMPLEXES CONTAINING O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE AND DNA

    PubMed Central

    Adams, Claire A.; Melikishvili, Manana; Rodgers, David W.; Rasimas, Joseph J.; Pegg, Anthony E.; Fried, Michael G.

    2009-01-01

    SUMMARY The mutagenic and cytotoxic effects of many alkylating agents are reduced by O6-alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we describe and test models for cooperative multi-protein complexes of AGT with single-stranded and duplex DNAs that are based on in vitro binding data and the crystal structure of a 1:1 AGT-DNA complex. These models predict that cooperative assemblies contain a 3-start helical array of proteins with dominant protein-protein interactions between the amino-terminal face of protein n and the carboxy-terminal face of protein n + 3, and they predict that binding duplex DNA does not require large changes in B-form DNA geometry. Experimental tests using protein crosslinking analyzed by mass spectrometry, electrophoretic and analytical ultracentrifugation binding assays and topological analyses with closed circular DNA show that the properties of multiprotein AGT-DNA complexes are consistent with these predictions. PMID:19358853

  12. Fast and continuous-flow detection and separation of DNA complexes and DNA in nanofluidic chip format.

    PubMed

    Viefhues, Martina; Regtmeier, Jan; Anselmetti, Dario

    2015-01-01

    Fast separation of DNA and detection of protein/DNA complexes are important in many state-of-the-art molecular medicine technologies, like the production of gene vaccines or medical diagnostics. Here, we describe a nanofluidic chip-based technique for fast, efficient, and virtually label-free detection and separation of protein/DNA and drug/DNA complexes and topological DNA variants. The mechanism is based on a continuous-flow dielectrophoresis at a nanoslit and allows efficient separation of small DNA fragments (<7,000 base pairs) and fast detection of DNA complexes within 1 min.

  13. Revealing the hidden complexities of mtDNA inheritance.

    PubMed

    White, Daniel James; Wolff, Jonci Nikolai; Pierson, Melanie; Gemmell, Neil John

    2008-12-01

    Mitochondrial DNA (mtDNA) is a pivotal tool in molecular ecology, evolutionary and population genetics. The power of mtDNA analyses derives from a relatively high mutation rate and the apparent simplicity of mitochondrial inheritance (maternal, without recombination), which has simplified modelling population history compared to the analysis of nuclear DNA. However, in biology things are seldom simple, and advances in DNA sequencing and polymorphism detection technology have documented a growing list of exceptions to the central tenets of mitochondrial inheritance, with paternal leakage, heteroplasmy and recombination now all documented in multiple systems. The presence of paternal leakage, recombination and heteroplasmy can have substantial impact on analyses based on mtDNA, affecting phylogenetic and population genetic analyses, estimates of the coalescent and the myriad of other parameters that are dependent on such estimates. Here, we review our understanding of mtDNA inheritance, discuss how recent findings mean that established ideas may need to be re-evaluated, and we assess the implications of these new-found complications for molecular ecologists who have relied for decades on the assumption of a simpler mode of inheritance. We show how it is possible to account for recombination and heteroplasmy in evolutionary and population analyses, but that accurate estimates of the frequencies of biparental inheritance and recombination are needed. We also suggest how nonclonal inheritance of mtDNA could be exploited, to increase the ways in which mtDNA can be used in analyses.

  14. 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.

  15. 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.

  16. Linear Dichroism Characteristics of Ethidium and Proflavine Supercoiled DNA Complexes

    DTIC Science & Technology

    1990-01-01

    of Ethidium-and Proflavine -Supercoi led DNA Complexes CHARLES E. SWENBERG, 1 SUSAN E. CARBERRV, 2 * and NICHOLAS E. GEACINTOV’ SRadliationl Biochenitr...drug were stained with EB, photographed under uv light, molecules, EB and proflavine (PF) (Figure 1), and the bands were quantitated by densitometry

  17. [Contribution of enthalpy to the energetics of complex formation of aromatic ligands with DNA].

    PubMed

    Kostiukov, V V; Khomutova, N M; Evstigneev, M P

    2011-01-01

    The energy contributions of electrostatic, van der Waals interactions, hydrogen bonds, and interactions of charge transfer type to the enthalpy of complex formation of the double-stand DNA with the antitumor antibiotics daunomycin, nogalamycin, and novantron, as well as the mutagens ethidium bromide and proflavine have been calculated. According to the calculations, the van der Waals component (except for nogalamycin) is energetically favorable during complex formation of the antibiotics with DNA, and the contributions of H bonds and electrostatic interactions are unfavorable, with the probability of charge transfer in the complexes being low. It has been shown that the relatively low value of the experimental enthalpy of binding is the sum of components greater in absolute value and different in the sign, which is the cause of large errors in estimating the total enthalpy of complex formation of aromatic ligands with DNA.

  18. Cooperative Stabilization of Zn2+:DNA Complexes Through Netropsin Binding in the Minor Groove of FdU-Substituted DNA

    PubMed Central

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

    2013-01-01

    The simultaneous binding of netropsin in the minor groove and Zn2+ in the major groove of a DNA hairpin that includes 10 consecutive FdU nucleotides at the 3′-terminus (3’FdU) was demonstrated based upon NMR spectroscopy, circular dichroism (CD), and computational modeling studies. The resulting Zn2+ /netropsin:3’FdU complex had very high thermal stability with aspects of the complex intact at 85 °C, conditions that result in complete dissociation of Mg2+ complexes. CD and 19F NMR spectroscopy were consistent with Zn2+ binding in the major groove of the DNA duplex and utilizing F5 and O4 of consecutive FdU nucleotides as ligands with FdU nucleotides hemi-deprotonated in the complex. Netropsin is bound in the minor groove of the DNA duplex based upon 2D NOESY data demonstrating contacts between AH2 1H and netropsin 1H resonances. The Zn2+/netropsin:3’FdU complex displayed increased cytotoxicity towards PC3 prostate cancer (PCa) cells relative to the constituent components or separate complexes (e.g. Zn2+:3’FdU) indicating that this new structural motif may be therapeutically useful for PCa treatment. PMID:23153072

  19. Structure of the Rad50 DNA double-strand break repair protein in complex with DNA.

    PubMed

    Rojowska, Anna; Lammens, Katja; Seifert, Florian U; Direnberger, Carolin; Feldmann, Heidi; Hopfner, Karl-Peter

    2014-12-01

    The Mre11-Rad50 nuclease-ATPase is an evolutionarily conserved multifunctional DNA double-strand break (DSB) repair factor. Mre11-Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50(NBD) (nucleotide-binding domain) in complex with Mre11(HLH) (helix-loop-helix domain), AMPPNP, and double-stranded DNA. DNA binds between both coiled-coil domains of the Rad50 dimer with main interactions to a strand-loop-helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double-strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP-bound state.

  20. Fetal programming: link between early nutrition, DNA methylation, and complex diseases.

    PubMed

    Chmurzynska, Agata

    2010-02-01

    Complex traits, including those involved in diet-related diseases, are determined by multiple genes and environmental influences. Factors influencing the development of complex traits should be expanded to include epigenetic factors, such as DNA methylation, which occurs in utero. Epigenetic factors regulate gene expression and thereby cell differentiation and organogenesis. The process of epigenotype establishment is sensitive to environmental conditions, with nutrition being one of the most important related factors. For example, DNA methylation depends on the availability of several nutrients including methionine and vitamins B(6), B(12), and folate. Epidemiological studies show that undernutrition during fetal life is associated with increased susceptibility to complex diseases. Numerous studies have been conducted on prenatal caloric and protein undernutrition. A reduction in the number of cells and changes in the structure and functioning of organs, as well as permanent changes in DNA methylation and gene expression, have been considered the molecular mechanisms responsible for metabolism programming.

  1. Mechanosensing of DNA bending in a single specific protein-DNA complex

    NASA Astrophysics Data System (ADS)

    Le, Shimin; Chen, Hu; Cong, Peiwen; Lin, Jie; Dröge, Peter; Yan, Jie

    2013-12-01

    Many crucial biological processes are regulated by mechanical stimuli. Here, we report new findings that pico-Newton forces can drastically affect the stability of the site-specific DNA binding of a single transcription factor, the E. coli integration host factor (IHF), by stretching a short ~150 nm DNA containing a single IHF binding site. Dynamic binding and unbinding of single IHF were recorded and analyzed for the force-dependent stability of the IHF-DNA complex. Our results demonstrate that the IHF-DNA interaction is fine tuned by force in different salt concentration and temperature over physiological ranges, indicating that, besides other physiological factors, force may play equally important role in transcription regulation. These findings have broad implications with regard to general mechanosensitivity of site-specific DNA bending proteins.

  2. DNA Sequence-Dependent Deformability Deduced from Protein-DNA Crystal Complexes

    NASA Astrophysics Data System (ADS)

    Olson, Wilma K.; Gorin, Andrey A.; Lu, Xiang-Jun; Hock, Lynette M.; Zhurkin, Victor B.

    1998-09-01

    The deformability of double helical DNA is critical for its packaging in the cell, recognition by other molecules, and transient opening during biochemically important processes. Here, a complete set of sequence-dependent empirical energy functions suitable for describing such behavior is extracted from the fluctuations and correlations of structural parameters in DNA-protein crystal complexes. These elastic functions provide useful stereochemical measures of the local base step movements operative in sequence-specific recognition and protein-induced deformations. In particular, the pyrimidine-purine dimers stand out as the most variable steps in the DNA-protein complexes, apparently acting as flexible ``hinges'' fitting the duplex to the protein surface. In addition to the angular parameters widely used to describe DNA deformations (i.e., the bend and twist angles), the translational parameters describing the displacements of base pairs along and across the helical axis are analyzed. The observed correlations of base pair bending and shearing motions are important for nonplanar folding of DNA in nucleosomes and other nucleoprotein complexes. The knowledge-based energies also offer realistic three-dimensional models for the study of long DNA polymers at the global level, incorporating structural features beyond the scope of conventional elastic rod treatments and adding a new dimension to literal analyses of genomic sequences.

  3. Fidelity of a human cell DNA replication complex.

    PubMed Central

    Roberts, J D; Kunkel, T A

    1988-01-01

    We have measured the fidelity of bidirectional, semiconservative DNA synthesis by a human DNA replication complex in vitro. Replication was performed by extracts of HeLa cells in the presence of simian virus 40 (SV40) large tumor antigen by using a double-stranded phage M13mp2 DNA template containing the SV40 origin of replication and either of two different target sequences for scoring mutations in the lacZ alpha-complementation gene, which encodes the alpha region (specifying the amino-terminal portion) of beta-galactosidase. Replicative synthesis was substantially more accurate than synthesis by the human DNA polymerase alpha-DNA primase complex purified from HeLa cell extracts by immunoaffinity chromatography, suggesting that additional factors or activities in the extract may increase fidelity during bidirectional replication. However, by using a sensitive opal codon reversion assay, single-base substitution errors were readily detected in the replication products at frequencies significantly higher than estimated spontaneous mutation rates in vivo. These data suggest that additional fidelity factors may be present during chromosomal replication in vivo and/or that the fidelity of replication alone does not account for the low spontaneous mutation rates in eukaryotes. Images PMID:3174620

  4. 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.

  5. Unwrapping of DNA-protein complexes under external stretching.

    PubMed

    Sakaue, Takahiro; Löwen, Hartmut

    2004-08-01

    A DNA-protein complex modeled by a semiflexible chain and an attractive spherical core is studied in the situation when an external stretching force is acting on one end monomer of the chain while the other end monomer is kept fixed in space. Without a stretching force, the chain is wrapped around the core. By applying an external stretching force, unwrapping of the complex is induced. We study the statics and dynamics of the unwrapping process by computer simulations and simple phenomenological theory. We find two different scenarios depending on the chain stiffness: For a flexible chain, the extension of the complex scales linearly with the external force applied. The sphere-chain complex is disordered; i.e., there is no clear winding of the chain around the sphere. For a stiff chain, on the other hand, the complex structure is ordered, which is reminiscent of nucleosome. There is a clear winding number, and the unwrapping process under external stretching is discontinuous with jumps of the distance-force curve. This is associated with discrete unwinding processes of the complex. Our predictions are of relevance for experiments, which measure force-extension curves of DNA-protein complexes, such as nucleosome, using optical tweezers.

  6. Biomedical Relation Extraction: From Binary to Complex

    PubMed Central

    Zhong, Dayou

    2014-01-01

    Biomedical relation extraction aims to uncover high-quality relations from life science literature with high accuracy and efficiency. Early biomedical relation extraction tasks focused on capturing binary relations, such as protein-protein interactions, which are crucial for virtually every process in a living cell. Information about these interactions provides the foundations for new therapeutic approaches. In recent years, more interests have been shifted to the extraction of complex relations such as biomolecular events. While complex relations go beyond binary relations and involve more than two arguments, they might also take another relation as an argument. In the paper, we conduct a thorough survey on the research in biomedical relation extraction. We first present a general framework for biomedical relation extraction and then discuss the approaches proposed for binary and complex relation extraction with focus on the latter since it is a much more difficult task compared to binary relation extraction. Finally, we discuss challenges that we are facing with complex relation extraction and outline possible solutions and future directions. PMID:25214883

  7. DNA-surfactant complexes: self-assembly properties and applications.

    PubMed

    Liu, Kai; Zheng, Lifei; Ma, Chao; Göstl, Robert; Herrmann, Andreas

    2017-08-14

    Over the last few years, DNA-surfactant complexes have gained traction as unique and powerful materials for potential applications ranging from optoelectronics to biomedicine because they self-assemble with outstanding flexibility spanning packing modes from ordered lamellar, hexagonal and cubic structures to disordered isotropic phases. These materials consist of a DNA backbone from which the surfactants protrude as non-covalently bound side chains. Their formation is electrostatically driven and they form bulk films, lyotropic as well as thermotropic liquid crystals and hydrogels. This structural versatility and their easy-to-tune properties render them ideal candidates for assembly in bulk films, for example granting directional conductivity along the DNA backbone, for dye dispersion minimizing fluorescence quenching allowing applications in lasing and nonlinear optics or as electron blocking and hole transporting layers, such as in LEDs or photovoltaic cells, owing to their extraordinary dielectric properties. However, they do not only act as host materials but also function as a chromophore itself. They can be employed within electrochromic DNA-surfactant liquid crystal displays exhibiting remarkable absorptivity in the visible range whose volatility can be controlled by the external temperature. Concomitantly, applications in the biological field based on DNA-surfactant bulk films, liquid crystals and hydrogels are rendered possible by their excellent gene and drug delivery capabilities. Beyond the mere exploitation of their material properties, DNA-surfactant complexes proved outstandingly useful for synthetic chemistry purposes when employed as scaffolds for DNA-templated reactions, nucleic acid modifications or polymerizations. These promising examples are by far not exhaustive but foreshadow their potential applications in yet unexplored fields. Here, we will give an insight into the peculiarities and perspectives of each material and are confident to

  8. 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.

  9. Relocalization of DNA lesions to the nuclear pore complex

    PubMed Central

    Freudenreich, Catherine H.; Su, Xiaofeng A.

    2016-01-01

    Early screens in yeast for mutations exhibiting sensitivity to DNA damage identified nuclear pore components, but their role in DNA repair was not well understood. Over the last decade, studies have revealed that several types of persistent DNA lesions relocate to either the nuclear pore complex (NPC) or nuclear envelope (NE). Of these two sites, the nuclear pore appears to be crucial for DNA repair of persistent double-strand breaks, eroded telomeres and sites of fork collapse at expanded CAG repeats. Using a combination of cell biological imaging techniques and yeast genetic assays for DNA repair, researchers have begun to understand both the how and why of lesion relocation to the NPC. Here we review the types of lesions that relocate to the NPC, mediators of relocation and the functional consequences of relocation understood to date. The emerging theme is that relocation to the NPC regulates recombination to influence repair pathway choice and provide a rescue mechanism for lesions or DNA structures that are resistant to repair. PMID:27799300

  10. 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.

  11. DNA adenine methyltransferase facilitated diffusion is enhanced by protein-DNA "roadblock" complexes that induce DNA looping.

    PubMed

    Pollak, Adam J; Reich, Norbert O

    2015-04-07

    The genomes of all cells are intimately associated with proteins, which are important for compaction, scaffolding, and gene regulation. Here we show that pre-existing protein-DNA complexes (roadblocks) diminish and-interestingly-enhance the ability of particular sequence-specific proteins to move along DNA to locate their binding sites. We challenge the bacterial DNA adenine methyltransferase (Dam, recognizes 5'-GATC-3') with tightly bound EcoRV ENase-DNA complexes, which bend DNA. A single EcoRV roadblock does not alter processive (multiple modifications) methylation by Dam. This result disfavors a reliance on heavily touted mechanisms involving sliding or short hops for Dam. Specific conformations of two EcoRV roadblocks cause an increase in processivity. The histone-like leucine-responsive regulatory protein (Lrp) binds DNA nonspecifically as an octamer, and also increases Dam's processivity. These results can be explained by our prior demonstration that Dam moves over large regions (>300 bp) within a single DNA molecule using an "intersegmental hopping" mechanism. This mechanism involves the protein hopping between looped DNA segments. Both roadblock systems can cause the DNA to loop and therefore facilitate intersegmental hopping. For Lrp, this only occurs when the Dam sites are separated (by >134bp) such that they can be looped around the protein. Intersegmental hopping may well be a general mechanism for proteins that navigate long distances along compacted DNA. Unlike Dam, EcoRI ENase (recognizes 5'-GAATTC-3') relies extensively on a sliding mechanism, and as expected, Lrp decreases its processivity. Our systematic use of protein roadblocks provides a powerful strategy to differentiate between site location mechanisms.

  12. DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

    PubMed Central

    Sugiyama, Tomohiko; New, James H.; Kowalczykowski, Stephen C.

    1998-01-01

    Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast. PMID:9600915

  13. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    PubMed

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-02-17

    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.

  14. 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

  15. 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.

  16. 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.

  17. Human RAD50 makes a functional DNA-binding complex.

    PubMed

    Kinoshita, Eri; van Rossum-Fikkert, Sari; Sanchez, Humberto; Kertokalio, Aryandi; Wyman, Claire

    2015-06-01

    The MRE11-RAD50-NBS1 (MRN) complex has several distinct functions in DNA repair including important roles in both non-homologous end-joining (NHEJ) and homologous recombination (HR). The biochemical activities of MR(N) have been well characterized implying specific functional roles for the components. The arrangement of proteins in the complex implies interdependence of their biochemical activities making it difficult to separate specific functions. We obtained purified human RAD50 and observed that it binds ATP, undergoes ATP-dependent conformational changes as well as having ATPase activity. Scanning force microscopy analysis clearly showed that RAD50 binds DNA although not as oligomers. RAD50 alone was not functional in tethering DNA molecules. ATP increased formation of RAD50 multimers which were however globular lacking extended coiled coils, in contrast to the MR complex where ATP induced oligomers have obvious coiled coils protruding from a central domain. These results suggest that MRE11 is important in maintaining the structural arrangement of RAD50 in the protein complex and perhaps has a role in reinforcing proper alignment of the coiled coils in the ATP-bound state. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  18. [Effect of supporting substrates on the structure of DNA and DNA-trivaline complexes studied by atomic force microscopy].

    PubMed

    Klinov, D V; Martynkina, L P; Iurchenko, V Iu; Demin, V V; Strel'tsov, S A; Gerasimov, Iu A; Vengerov, Iu Iu

    2003-01-01

    Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA-TV complexes onto this surface. On mica, both purified DNA and DNA-TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA-TP displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.

  19. Systematical investigation of binding interaction between novel ruthenium(II) arene complex with curcumin analogs and ctDNA.

    PubMed

    Huang, Shan; Liang, Yu; Huang, Chusheng; Su, Wei; Lei, Xiaolin; Liu, Yi; Xiao, Qi

    2016-11-01

    In this study, the interaction between a novel ruthenium(II) arene complex with curcumin analogs and calf thymus DNA (ctDNA) was investigated systematically by viscosity measurement, the DNA melting approach, multispectroscopic techniques and electrochemical methods. The absorption spectra of the ctDNA-drug complex showed a slight red shift and a weak hypochromic effect. The relative viscosity and melting temperature of ctDNA increased on addition of the drug. The evidence obtained from fluorescence competitive experiments indicated that the binding mode of the drug with ctDNA was intercalative. Using acridine orange (AO) as a fluorescence probe, the drug statically quenched the fluorescence of the ctDNA-AO complex, and hydrogen bonding and van der Waals interactions played vital roles in the binding interaction between the drug and ctDNA. The influences of ionic strength, chemical denaturants and pH on the binding interaction were also investigated. Circular dichroism and Fourier transform infrared spectra suggested that this drug might bond with the G-C base pairs of ctDNA and the right-handed B-form helicity of ctDNA remained after drug binding. The intercalative binding between the drug and ctDNA was further investigated using electrochemical techniques. All these results suggested that the biological activity of ctDNA was affected by ruthenium(II) arene complex with curcumin analogs. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  20. Studies on the Interaction Mechanism of 1,10-Phenanthroline Cobalt(II) Complex with DNA and Preparation of Electrochemical DNA Biosensor

    PubMed Central

    Niu, Shuyan; Li, Feng; Zhang, Shusheng; Wang, Long; Li, Xuemei; Wang, Shiying

    2006-01-01

    Fluorescence spectroscopy and ultraviolet (UV) spectroscopy techniques coupled with cyclic voltammetry (CV) were used to study the interaction between salmon sperm DNA and 1,10-Phenanthroline cobalt(II) complex, [Co(phen)2(Cl)(H2O)]Cl·H2O, where phen = 1,10-phenanthroline. The interaction between [Co(phen)2(Cl)(H2O)]+ and double-strand DNA (dsDNA) was identified to be intercalative mode. An electrochemical DNA biosensor was developed by covalent immobilization of probe single-strand DNA (ssDNA) related to human immunodeficiency virus (HIV) on the activated glassy carbon electrode (GCE). With [Co(phen)2(Cl)(H2O)]+ being the novel electrochemical hybridization indicator, the selectivity of ssDNA-modified electrode was investigated and selective detection of complementary ssDNA was achieved using differential pulse voltammetry (DPV).

  1. Complex Archimedean tiling self-assembled from DNA nanostructures.

    PubMed

    Zhang, Fei; Liu, Yan; Yan, Hao

    2013-05-22

    Archimedean tilings are periodic polygonal tessellations that are created by placing regular polygons edge-to-edge around a vertex to fill the plane. Here we show that three- and four-arm DNA junction tiles with specifically designed arm lengths and intertile sticky-end interactions can be used to form sophisticated two-dimensional (2D) and three-dimensional (3D) tessellation patterns. We demonstrate two different complex Archimedean patterns, (3(3).4(2)) and (3(2).4.3.4), and the formation of 2D lattices, 3D tubes, and sealed polygon-shaped pockets from the tessellations. The successful growth of hybrid DNA tile motif arrays suggests that it maybe possible to generate 2D quasi-crystals from DNA building blocks.

  2. Polycomb repressive complex 2 contributes to DNA double-strand break repair

    PubMed Central

    Campbell, Stuart; Ismail, Ismail Hassan; Young, Leah C; Poirier, Guy G; Hendzel, Michael J

    2013-01-01

    Polycomb protein histone methyltransferase, enhancer of Zeste homolog 2 (EZH2), is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether EZH2 has a role in modulating the DNA damage response. Here, we show that polycomb repressive complex 2 (PRC2) is recruited to sites of DNA damage. This recruitment is independent of histone 2A variant X (H2AX) and the PI-3-related kinases ATM and DNA-PKcs. We establish that PARP activity is required for retaining PRC2 at sites of DNA damage. Furthermore, depletion of EZH2 in cells decreases the efficiency of DSB repair and increases sensitivity of cells to gamma-irradiation. These data unravel a crucial role of PRC2 in determining cancer cellular sensitivity following DNA damage and suggest that therapeutic targeting of EZH2 activity might serve as a strategy for improving conventional chemotherapy in a given malignancy. PMID:23907130

  3. DNA and RNA folds in transcription complex as evidenced by iodine-125 radioprobing.

    PubMed

    Karamychev, V N; Panyutin, I G; Neumann, R D; Zhurkin, V B

    2000-01-01

    Abstract Folding of the DNA and RNA strands in an arrested T7 RNA polymerase (RNAP) transcription complex was studied by radioprobing, a novel method based on an analysis of the strand breaks produced by decay of the iodine-125 incorporated at the C5 position of cytosine. (125)I-labeled cytosines were incorporated into transcripts at different positions relative to the site of the arrest. The intensities of the DNA breaks inversely correlate with the distances from the (125)I decay site, so the radioprobing data provide information about the spatial RNA/DNA folding during transcription. We found that the yield of DNA strand breaks is significantly higher in the template than the non-template strand. This is consistent with local opening of the DNA duplex and formation of a hybrid between RNA and the template DNA strand. Our data demonstrate that the RNA-DNA hybrid has a nonuni form A-like structure. When the (125)I is incorporatedseven nucleotides from the active center of RNAP, the yield of strand breaks is substantially lower than if (125)I is positioned at the ends of the hybrid. Consequently, the DNA and RNA strands are located closer to each other at the ends of the hybrid and somehow separated in the middle. Surprisingly, the (125)I-induced breaks were detected in both DNA strands upstream from the transcription "bubble" indicating that DNA and RNA are closely associated outside the RNAP cleft. Thus, radioprobing data imply that the RNA/DNA fold in the complex with T7 RNAP is more complicated than had been anticipated by the existing models. Based on the present data, we suggest a sterically feasible model explaining how formation of the long RNA-DNA hybrid can result in the initiation-to-elongation switch in the T7 transcription complex. According to this model, the topological linkage between the DNA and RNA strands provides the necessary stability for the elongation complex, while permitting movement of the polymerase along the DNA duplex.

  4. PBX and MEIS as Non-DNA-Binding Partners in Trimeric Complexes with HOX Proteins

    PubMed Central

    Shanmugam, Kandavel; Green, Nancy C.; Rambaldi, Isabel; Saragovi, H. Uri; Featherstone, Mark S.

    1999-01-01

    HOX, PBX, and MEIS transcription factors bind DNA through a homeodomain. PBX proteins bind DNA cooperatively as heterodimers with MEIS family members and also with HOX proteins from paralog groups 1 to 10. MEIS proteins cooperatively bind DNA with ABD-B class HOX proteins of groups 9 and 10. Here, we examine aspects of dimeric and higher-order interactions between these three homeodomain classes. The most significant results can be summarized as follows. (i) Most of PBX N terminal to the homeodomain is required for efficient cooperative binding with HOXD4 and HOXD9. (ii) MEIS and PBX proteins form higher-order complexes on a heterodimeric binding site. (iii) Although MEIS does not cooperatively bind DNA with ANTP class HOX proteins, it does form a trimer as a non-DNA-binding partner with DNA-bound PBX-HOXD4. (iv) The N terminus of HOXD4 negatively regulates trimer formation. (v) MEIS forms a similar trimer with DNA-bound PBX-HOXD9. (vi) A related trimer (where MEIS is a non-DNA-binding partner) is formed on a transcriptional promoter within the cell. (vii) We observe an additional trimer class involving non-DNA-bound PBX and DNA-bound MEIS-HOXD9 or MEIS-HOXD10 heterodimers that is enhanced by mutation of the PBX homeodomain. (viii) In this latter trimer, PBX is likely to contact both MEIS and HOXD9/D10. (ix) The stability of DNA binding by all trimers is enhanced relative to the heterodimers. These findings suggest novel functions for PBX and MEIS in modulating the function of DNA-bound MEIS-HOX and PBX-HOX heterodimers, respectively. PMID:10523646

  5. PBX and MEIS as non-DNA-binding partners in trimeric complexes with HOX proteins.

    PubMed

    Shanmugam, K; Green, N C; Rambaldi, I; Saragovi, H U; Featherstone, M S

    1999-11-01

    HOX, PBX, and MEIS transcription factors bind DNA through a homeodomain. PBX proteins bind DNA cooperatively as heterodimers with MEIS family members and also with HOX proteins from paralog groups 1 to 10. MEIS proteins cooperatively bind DNA with ABD-B class HOX proteins of groups 9 and 10. Here, we examine aspects of dimeric and higher-order interactions between these three homeodomain classes. The most significant results can be summarized as follows. (i) Most of PBX N terminal to the homeodomain is required for efficient cooperative binding with HOXD4 and HOXD9. (ii) MEIS and PBX proteins form higher-order complexes on a heterodimeric binding site. (iii) Although MEIS does not cooperatively bind DNA with ANTP class HOX proteins, it does form a trimer as a non-DNA-binding partner with DNA-bound PBX-HOXD4. (iv) The N terminus of HOXD4 negatively regulates trimer formation. (v) MEIS forms a similar trimer with DNA-bound PBX-HOXD9. (vi) A related trimer (where MEIS is a non-DNA-binding partner) is formed on a transcriptional promoter within the cell. (vii) We observe an additional trimer class involving non-DNA-bound PBX and DNA-bound MEIS-HOXD9 or MEIS-HOXD10 heterodimers that is enhanced by mutation of the PBX homeodomain. (viii) In this latter trimer, PBX is likely to contact both MEIS and HOXD9/D10. (ix) The stability of DNA binding by all trimers is enhanced relative to the heterodimers. These findings suggest novel functions for PBX and MEIS in modulating the function of DNA-bound MEIS-HOX and PBX-HOX heterodimers, respectively.

  6. The relative flexibility of B-DNA and A-RNA duplexes: database analysis

    PubMed Central

    Pérez, Alberto; Noy, Agnes; Lankas, Filip; Luque, F. Javier; Orozco, Modesto

    2004-01-01

    An extensive analysis of structural databases is carried out to investigate the relative flexibility of B-DNA and A-RNA duplexes in crystal form. Our results show that the general anisotropic concept of flexibility is not very useful to compare the deformability of B-DNA and A-RNA duplexes, since the flexibility patterns of B-DNA and A-RNA are quite different. In other words, ‘flexibility’ is a dangerous word for describing macromolecules, unless it is clearly defined. A few soft essential movements explain most of the natural flexibility of A-RNA, whereas many are necessary for B-DNA. Essential movements occurring in naked B-DNAs are identical to those necessary to deform DNA in DNA–protein complexes, which suggest that evolution has designed DNA–protein complexes so that B-DNA is deformed according to its natural tendency. DNA is generally more flexible, but for some distortions A-RNA is easier to deform. Local stiffness constants obtained for naked B-DNAs and DNA complexes are very close, demonstrating that global distortions in DNA necessary for binding to proteins are the result of the addition of small concerted deformations at the base-pair level. Finally, it is worth noting that in general the picture of the relative deformability of A-RNA and DNA derived from database analysis agrees very well with that derived from state of the art molecular dynamics (MD) simulations. PMID:15562006

  7. Superspace de Rham complex and relative cohomology

    NASA Astrophysics Data System (ADS)

    Linch, William D.; Randall, Stephen

    2015-09-01

    We investigate the super-de Rham complex of five-dimensional superforms with N = 1 supersymmetry. By introducing a free supercommutative algebra of auxiliary variables, we show that this complex is equivalent to the Chevalley-Eilenberg complex of the translation supergroup with values in superfields. Each cocycle of this complex is defined by a Lorentz- and iso-spin-irreducible superfield subject to a set of constraints. Restricting to constant coefficients results in a subcomplex in which components of the cocycles are coboundaries while the constraints on the defining superfields span the cohomology. This reduces the computation of all of the superspace Bianchi identities to a single linear algebra problem the solution of which implies new features not present in the standard four-dimensional, N = 1 complex. These include splitting/joining in the complex and the existence of cocycles that do not correspond to irreducible supermultiplets of closed differential forms. Interpreting the five-dimensional de Rham complex as arising from dimensional reduction from the six-dimensional complex, we find a second five-dimensional complex associated to the relative de Rham complex of the embedding of the latter in the former. This gives rise to a second source of closed differential forms previously attributed to the phenomenon called "Weyl triviality".

  8. Direct observation of DNA threading in flap endonuclease complexes

    PubMed Central

    AlMalki, Faizah A; Flemming, Claudia S; Zhang, Jing; Feng, Min; Sedelnikova, Svetlana E; Ceska, Tom; Rafferty, John B; Sayers, Jon R; Artymiuk, Peter J

    2016-01-01

    Maintenance of genome integrity requires that branched nucleic acid molecules are accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates, and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate’s single-stranded branch approaches, threads through, and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual “fly-casting, thread, bend and barb” mechanism. PMID:27273516

  9. Islands of Complex DNA Are Widespread in Drosophila Centric Heterochromatin

    PubMed Central

    Le, M. H.; Duricka, D.; Karpen, G. H.

    1995-01-01

    Heterochromatin is a ubiquitous yet poorly understood component of multicellular eukaryotic genomes. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. We have investigated the molecular structure of the 1 Mb of centric heterochromatin in the Drosophila minichromosome Dp1187. A genetic screen of irradiated minichromosomes yielded rearranged derivatives of Dp1187 whose structures were determined by pulsed-field Southern analysis and PCR. Three Dp1187 deletion derivatives and an inversion had one breakpoint in the euchromatin and one in the heterochromatin, providing direct molecular access to previously inaccessible parts of the heterochromatin. End-probed pulsed-field restriction mapping revealed the presence of at least three ``islands'' of complex DNA, Tahiti, Moorea, and Bora Bora, constituting approximately one half of the Dp1187 heterochromatin. Pulsed-field Southern analysis demonstrated that Drosophila heterochromatin in general is composed of alternating blocks of complex DNA and simple satellite DNA. Cloning and sequencing of a small part of one island, Tahiti, demonstrated the presence of a retroposon. The implications of these findings to heterochromatin structure and function are discussed. PMID:8536977

  10. Strong DNA deformation required for extremely slow DNA threading intercalation by a binuclear ruthenium complex

    PubMed Central

    Almaqwashi, Ali A.; Paramanathan, Thayaparan; Lincoln, Per; Rouzina, Ioulia; Westerlund, Fredrik; Williams, Mark C.

    2014-01-01

    DNA intercalation by threading is expected to yield high affinity and slow dissociation, properties desirable for DNA-targeted therapeutics. To measure these properties, we utilize single molecule DNA stretching to quantify both the binding affinity and the force-dependent threading intercalation kinetics of the binuclear ruthenium complex Δ,Δ-[μ‐bidppz‐(phen)4Ru2]4+ (Δ,Δ-P). We measure the DNA elongation at a range of constant stretching forces using optical tweezers, allowing direct characterization of the intercalation kinetics as well as the amount intercalated at equilibrium. Higher forces exponentially facilitate the intercalative binding, leading to a profound decrease in the binding site size that results in one ligand intercalated at almost every DNA base stack. The zero force Δ,Δ-P intercalation Kd is 44 nM, 25-fold stronger than the analogous mono-nuclear ligand (Δ-P). The force-dependent kinetics analysis reveals a mechanism that requires DNA elongation of 0.33 nm for association, relaxation to an equilibrium elongation of 0.19 nm, and an additional elongation of 0.14 nm from the equilibrium state for dissociation. In cells, a molecule with binding properties similar to Δ,Δ-P may rapidly bind DNA destabilized by enzymes during replication or transcription, but upon enzyme dissociation it is predicted to remain intercalated for several hours, thereby interfering with essential biological processes. PMID:25245944

  11. DNA binding properties of the actin-related protein Arp8 and its role in DNA repair.

    PubMed

    Osakabe, Akihisa; Takahashi, Yuichiro; 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.

  12. [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.

  13. Enthused research on DNA-binding and DNA-cleavage aptitude of mixed ligand metal complexes

    NASA Astrophysics Data System (ADS)

    Mahalakshmi, Rajkumar; Raman, Natarajan

    2013-08-01

    Five new Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) mixed ligand complexes have been synthesized using a Schiff base precursor (obtained by the condensation of N-(4-aminophenyl)acetamide and 4-chlorobenzaldehyde) as main ligand and 1,10-phenanthroline as co-ligand. They have been characterized by microanalytical data, IR, UV-Vis, magnetic moment values, conductivity and electrochemical measurements. The spectral data reveal that all the complexes exhibit octahedral geometry. The high electrical conductance of the complexes supports their electrolytic nature. The monomeric nature of the complexes has been assessed from their magnetic susceptibility values. These complexes are better antimicrobial active agents than the free ligands. DNA (CT) binding properties of these complexes have been explored by UV-Vis., viscosity measurements, cyclic voltammetry, and differential pulse voltammetry measurements. The oxidative cleavage activity of the complexes has been studied using supercoiled pUC19 DNA by gel electrophoresis. The experimental results show that the complexes are good intercalators.

  14. Enthused research on DNA-binding and DNA-cleavage aptitude of mixed ligand metal complexes.

    PubMed

    Mahalakshmi, Rajkumar; Raman, Natarajan

    2013-08-01

    Five new Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) mixed ligand complexes have been synthesized using a Schiff base precursor (obtained by the condensation of N-(4-aminophenyl)acetamide and 4-chlorobenzaldehyde) as main ligand and 1,10-phenanthroline as co-ligand. They have been characterized by microanalytical data, IR, UV-Vis, magnetic moment values, conductivity and electrochemical measurements. The spectral data reveal that all the complexes exhibit octahedral geometry. The high electrical conductance of the complexes supports their electrolytic nature. The monomeric nature of the complexes has been assessed from their magnetic susceptibility values. These complexes are better antimicrobial active agents than the free ligands. DNA (CT) binding properties of these complexes have been explored by UV-Vis., viscosity measurements, cyclic voltammetry, and differential pulse voltammetry measurements. The oxidative cleavage activity of the complexes has been studied using supercoiled pUC19 DNA by gel electrophoresis. The experimental results show that the complexes are good intercalators.

  15. 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

  16. 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.

  17. 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.

  18. FANCI-FANCD2 stabilizes the RAD51-DNA complex by binding RAD51 and protects the 5'-DNA end.

    PubMed

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

    2016-12-15

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

  19. Visualization of recombinant DNA and protein complexes using atomic force microscopy.

    PubMed

    Murphy, Patrick J M; Shannon, Morgan; Goertz, John

    2011-07-18

    Atomic force microscopy (AFM) allows for the visualizing of individual proteins, DNA molecules, protein-protein complexes, and DNA-protein complexes. On the end of the microscope's cantilever is a nano-scale probe, which traverses image areas ranging from nanometers to micrometers, measuring the elevation of macromolecules resting on the substrate surface at any given point. Electrostatic forces cause proteins, lipids, and nucleic acids to loosely attach to the substrate in random orientations and permit imaging. The generated data resemble a topographical map, where the macromolecules resolve as three-dimensional particles of discrete sizes (Figure 1). Tapping mode AFM involves the repeated oscillation of the cantilever, which permits imaging of relatively soft biomaterials such as DNA and proteins. One of the notable benefits of AFM over other nanoscale microscopy techniques is its relative adaptability to visualize individual proteins and macromolecular complexes in aqueous buffers, including near-physiologic buffered conditions, in real-time, and without staining or coating the sample to be imaged. The method presented here describes the imaging of DNA and an immunoadsorbed transcription factor (i.e. the glucocorticoid receptor, GR) in buffered solution (Figure 2). Immunoadsorbed proteins and protein complexes can be separated from the immunoadsorbing antibody-bead pellet by competition with the antibody epitope and then imaged (Figure 2A). This allows for biochemical manipulation of the biomolecules of interest prior to imaging. Once purified, DNA and proteins can be mixed and the resultant interacting complex can be imaged as well. Binding of DNA to mica requires a divalent cation, such as Ni(2+) or Mg(2+), which can be added to sample buffers yet maintain protein activity. Using a similar approach, AFM has been utilized to visualize individual enzymes, including RNA polymerase and a repair enzyme, bound to individual DNA strands. These experiments provide

  20. Exploring the Interaction of Ruthenium(II) Polypyridyl Complexes with DNA Using Single-Molecule Techniques†

    PubMed Central

    Mihailovic, Aleksandra; Vladescu, Ioana; McCauley, Micah; Ly, Elaine; Williams, Mark C.; Spain, Eileen M.; Nuñez, Megan E.

    2008-01-01

    Here we explore DNA binding by a family of ruthenium(II) polypyridyl complexes using an atomic force microscope (AFM) and optical tweezers. We demonstrate using AFM that Ru(bpy)2dppz2+ intercalates into DNA (Kb= 1.5 × 105 M−1), as does its close relative Ru(bpy)2dppx2+ (Kb= 1.5 × 105 M−1). However, intercalation by Ru(phen)32+ and other Ru(II) complexes with Kb's lower than Ru(bpy)2dppz2+ are difficult to determine using AFM because of competing aggregation and surface-binding phenomena. At the high Ru(II) concentrations required to evaluate intercalation, most of the DNA strands acquire a twisted, curled conformation that is impossible to measure accurately. The condensation of DNA on mica in the presence of polycations is well known, but it clearly precludes the accurate assessment by AFM of DNA intercalation by most Ru(II) complexes, though not by ethidium bromide and other monovalent intercalators. When stretching individual DNA molecules using optical tweezers the same limitation on high metal concentration does not exist. Using optical tweezers we show that Ru(phen)2dppz2+ intercalates avidly (Kb = 3.2 × 106 M−1) while Ru(bpy)32+ does not intercalate, even at micromolar ruthenium concentrations. Ru(phen)32+ is shown to intercalate weakly, i.e. at micromolar concentrations (Kb= 8.8 × 103 M−1). The distinct differences in DNA stretching behavior between Ru(phen)32+ and Ru(bpy)32+ clearly illustrate that intercalation can be distinguished from groove binding by pulling the DNA with optical tweezers. Our results demonstrate both the benefits and challenges of two single-molecule methods in exploring DNA binding, and help to elucidate the mode of binding of Ru(phen)32+. PMID:16649785

  1. Mre11 Complex and DNA Replication: Linkage to E2F and Sites of DNA Synthesis†

    PubMed Central

    Maser, Richard S.; Mirzoeva, Olga K.; Wells, Julie; Olivares, Heidi; Williams, Bret R.; Zinkel, Robert A.; Farnham, Peggy J.; Petrini, John H. J.

    2001-01-01

    We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus. This association and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is sufficient individually for checkpoint activation. The Nbs1 E2F interaction occurred near the Epstein-Barr virus origin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was restricted to S-phase cells. The Mre11 complex colocalized with PCNA at replication forks throughout S phase, both prior to and coincident with the appearance of nascent DNA. These data suggest that the Mre11 complex suppresses genomic instability through its influence on both the regulation and progression of DNA replication. PMID:11486038

  2. Enantiopure copper(II) complex of natural product rosin derivative: DNA binding, DNA cleavage and cytotoxicity.

    PubMed

    Fei, Bao-Li; Yin, Bin; Li, Dong-Dong; Xu, Wu-Shuang; Lu, Yang

    2016-12-01

    To develop chiral anticancer drug candidates for molecular target DNA, the synthesis and characterization of a novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1) was carried out. The coordination geometry of the copper center is a distorted square-planar arrangement. The interactions of 1 and 2 with salmon sperm DNA were investigated by viscosity measurements, UV, fluorescence and circular dichroism (CD) spectroscopic techniques. All the results reveal that 1 and 2 interacted with DNA through intercalation and 2 exhibited a higher DNA binding ability. Further, 1 and 2 could cleave supercoiled pBR322 DNA by single strand and 2 displayed stronger cleavage ability in the presence of ascorbic acid. In vitro cytotoxicity of 1 and 2 against HeLa, SiHa, HepG-2 and A431 cancer cell lines was studied using CCK-8 assay. The results indicate that 2 had a superior cytotoxicity than 1 and the widely used drug cisplatin under identical conditions. Flow cytometry analysis demonstrates 2 produced death of HeLa cancer cells through an apoptotic pathway. Cell cycle analysis shows that 2 mainly arrested HeLa cells at the S phase. A novel enantiomerically pure copper(II) complex [Cu 1 Cl 2 ] (2) of an optically pure ligand N-(pyridin-2-ylmethylene) dehydroabietylamine (1), based on natural product rosin has been synthesized. 2 has the potential to act as effective anticancer drug.

  3. The FACT complex promotes avian leukosis virus DNA integration.

    PubMed

    Winans, Shelby; Larue, Ross C; Abraham, Carly M; Shkriabai, Nikolozi; Skopp, Amelie; Winkler, Duane; Kvaratskhelia, Mamuka; Beemon, Karen L

    2017-01-25

    All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. Here, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro Likewise, in infected cells, the FACT complex promotes ALV integration activity with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-LTR circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells.

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

    PubMed Central

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

    2010-01-01

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

  5. Osteogenic potential for replacing cells in rat cranial defects implanted with a DNA/protamine complex paste.

    PubMed

    Toda, Masako; Ohno, Jun; Shinozaki, Yosuke; Ozaki, Masao; Fukushima, Tadao

    2014-10-01

    Osteoinductive scaffolds are required for bone tissue engineering. The aim of the present study was to assess the osteoinductive capacity of deoxyribonucleic acid (DNA)/protamine complexes in a rat model of critical-size calvarial defects. In addition, we investigated whether cultured mesenchymal-like cells (DP-cells) outgrown from DNA/protamine complex engrafted defects could differentiate to become osteogenic cells in vitro. DNA/protamine complexes were prepared by reactions between DNA and protamine sulfate solutions with stirring. Critical-sized (8mm) calvarial defects were created in the central parietal bones of adult rats. Defects were either left empty or treated with DNA/protamine complex scaffolds. Subsequently, micro-computed tomography (micro-CT), histological, and immunohistochemical analyses were performed. Micro-CT and histological assays showed that DNA/protamine complex engrafted defects had enhanced bone regeneration. DP-cells were expanded from explants of DNA/protamine complex engrafted defects using an explant outgrowth culture system. Osteogenesis-related factors were assessed in DP-cells after treatment with an osteoblast-inducing reagent (OIR). After 3months, nearly complete healing was observed for DNA/protamine complex engrafted calvarial defects. Increased alkaline phosphatase (ALP) activity and Alizarin red staining were found for cultured DP-cells. These cells had high expression levels of osteogenic genes, including those for RUNX-2, ALP, osteopontin, and osteocalcin. These results indicated that DNA/protamine complexes could facilitate bone regeneration in calvarial defects. Moreover, in vitro osteogenic induction experiments showed that DP-cells outgrown from DNA/protamine engrafted defects had an osteogenic potential. Based on these results, we suggest that DNA/protamine complexes may recruit osteocompetent cells in these defects, where they differentiate to osteogenic cells.

  6. 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.

  7. Hepatoma-derived growth factor-related protein 2 promotes DNA repair by homologous recombination

    PubMed Central

    Baude, Annika; Aaes, Tania Løve; Zhai, Beibei; Al-Nakouzi, Nader; Oo, Htoo Zarni; Daugaard, Mads; Rohde, Mikkel; Jäättelä, Marja

    2016-01-01

    We have recently identified lens epithelium-derived growth factor (LEDGF/p75, also known as PSIP1) as a component of the homologous recombination DNA repair machinery. Through its Pro-Trp-Trp-Pro (PWWP) domain, LEDGF/p75 binds to histone marks associated with active transcription and promotes DNA end resection by recruiting DNA endonuclease retinoblastoma-binding protein 8 (RBBP8/CtIP) to broken DNA ends. Here we show that the structurally related PWWP domain-containing protein, hepatoma-derived growth factor-related protein 2 (HDGFRP2), serves a similar function in homologous recombination repair. Its depletion compromises the survival of human U2OS osteosarcoma and HeLa cervix carcinoma cells and impairs the DNA damage-induced phosphorylation of replication protein A2 (RPA2) and the recruitment of DNA endonuclease RBBP8/CtIP to DNA double strand breaks. In contrast to LEDGF/p75, HDGFRP2 binds preferentially to histone marks characteristic for transcriptionally silent chromatin. Accordingly, HDGFRP2 is found in complex with the heterochromatin-binding chromobox homologue 1 (CBX1) and Pogo transposable element with ZNF domain (POGZ). Supporting the functionality of this complex, POGZ-depleted cells show a similar defect in DNA damage-induced RPA2 phosphorylation as HDGFRP2-depleted cells. These data suggest that HDGFRP2, possibly in complex with POGZ, recruits homologous recombination repair machinery to damaged silent genes or to active genes silenced upon DNA damage. PMID:26721387

  8. Architecture of a Serine Recombinase-DNA Regulatory Complex

    SciTech Connect

    Mouw, Kent W.; Rowland, Sally-J.; Gajjar, Mark M.; Boocock, Martin R.; Stark, W. Marshall; Rice, Phoebe A.

    2008-04-29

    An essential feature of many site-specific recombination systems is their ability to regulate the direction and topology of recombination. Resolvases from the serine recombinase family assemble an interwound synaptic complex that harnesses negative supercoiling to drive the forward reaction and promote recombination between properly oriented sites. To better understand the interplay of catalytic and regulatory functions within these synaptic complexes, we have solved the structure of the regulatory site synapse in the Sin resolvase system. It reveals an unexpected synaptic interface between helix-turn-helix DNA-binding domains that is also highlighted in a screen for synapsis mutants. The tetramer defined by this interface provides the foundation for a robust model of the synaptic complex, assembled entirely from available crystal structures, that gives insight into how the catalytic activity of Sin and other serine recombinases may be regulated.

  9. 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.

  10. 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.

  11. Spatiotemporal dynamics of early DNA damage response proteins on complex DNA lesions.

    PubMed

    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

  12. 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.

  13. Design and characterization of a novel lipid-DNA complex that resists serum-induced destabilization.

    PubMed

    Lian, Tianshun; Ho, Rodney J Y

    2003-12-01

    Ineffectiveness of cationic lipids to enhance DNA transfection has been attributed to serum-mediated dissociation and perhaps complement activation of lipid-DNA complexes. To overcome these problems, we have developed a novel lipid-DNA complex that greatly reduces serum-mediated dissociation. The complexes were prepared by mixing cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane and dioleoylphosphatidyl-ethanolamine and DNA in ethanolic (20% v/v ethanol) solution containing 5% sucrose followed by dehydration via rotating evaporation. Upon hydration in H(2)O, the lipid-DNA complexes [ethanol-dried lipid-DNA (EDL) complexes] were formed. The complexes exhibit a low positive zeta potential and enhanced transfection efficiency in contrast to the suppressed efficiency detected with admixed lipid-DNA complexes in the presence of serum across several cell lines. This result may be attributed to the inability of serum to dissociate DNA from lipids in EDL complexes. Using displacement of ethidium bromide intercalation analysis, we found that in serum, only 50% of DNA was exposed in the EDL complexes, compared with 100% in the admixed lipid-DNA complexes. The EDL complexes also showed increased resistance to DNase digestion in the presence of negatively charged lipid, while reducing complement activation in serum. The EDL complexes may improve the transfection activity of lipid-DNA complexes in serum and, perhaps, in vivo.

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

    PubMed

    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.

  15. 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.

  16. Variation of entropic elasticity of DNA-Psoralen complex under UV light

    NASA Astrophysics Data System (ADS)

    Rocha, M. S.; Mesquita, O. N.

    2005-08-01

    We measure the entropic elasticity of a single λ-DNA molecule and of a single DNA-Psoralen complex by doing stretching experiments with an optical tweezers. Psoralen is a photosensitive drug used in the treatment of many skin diseases, by impeding DNA replication. Psolaren intercalates the DNA and can form crosslinks with pyrimidine basis in opposite strands of DNA, when illuminated with UVA light. As crosslinks form the persistence length of the complex increases, indicating an increase in rigidity of the complex. We study the kinetics of DNA-Psoralen crosslink formation via changes in entropic elasticity of the complex.

  17. 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

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

    PubMed Central

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

    2011-01-01

    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. PMID:22146299

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

    PubMed

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

    2016-12-13

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

  20. Noncanonical autophagy is required for type I interferon secretion in response to DNA-immune complexes.

    PubMed

    Henault, Jill; Martinez, Jennifer; Riggs, Jeffrey M; Tian, Jane; Mehta, Payal; Clarke, Lorraine; Sasai, Miwa; Latz, Eicke; Brinkmann, Melanie M; Iwasaki, Akiko; Coyle, Anthony J; Kolbeck, Roland; Green, Douglas R; Sanjuan, Miguel A

    2012-12-14

    Toll-like receptor-9 (TLR9) is largely responsible for discriminating self from pathogenic DNA. However, association of host DNA with autoantibodies activates TLR9, inducing the pathogenic secretion of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Here, we found that in response to DNA-containing immune complexes (DNA-IC), but not to soluble ligands, IFN-α production depended upon the convergence of the phagocytic and autophagic pathways, a process called microtubule-associated protein 1A/1B-light chain 3 (LC3)-associated phagocytosis (LAP). LAP was required for TLR9 trafficking into a specialized interferon signaling compartment by a mechanism that involved autophagy-related proteins, but not the conventional autophagic preinitiation complex, or adaptor protein-3 (AP-3). Our findings unveil a new role for nonconventional autophagy in inflammation and provide one mechanism by which anti-DNA autoantibodies, such as those found in several autoimmune disorders, bypass the controls that normally restrict the apportionment of pathogenic DNA and TLR9 to the interferon signaling compartment.

  1. Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human

    PubMed Central

    Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

    2017-01-01

    DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation. PMID:28212312

  2. BOX DNA: a novel regulatory element related to embryonal carcinoma cell differentiation.

    PubMed Central

    Kihara-Negishi, F; Tsujita, R; Negishi, Y; Ariga, H

    1993-01-01

    BOX DNA was previously isolated from the DNA sequence inserted in the enhancer B domain of mutant polyomavirus (fPyF9) DNA. We also reported that BOX DNA functioned negatively on DNA replication and transcription of another polyomavirus mutant (PyhrN2) in F9-28 cells, a subclone of mouse F9 embryonal carcinoma (EC) cells expressing the polyomavirus large T antigen. In this study, we demonstrate that BOX DNA enhances transcription from the thymidine kinase (TK) promoter in various EC cells. One or three copies of BOX DNA, linked to the bacterial chloramphenicol acetyltransferase gene under the control of the herpes simplex virus TK promoter, activated promoter activity in F9, P19, and ECA2 cells. Band shift assays using BOX DNA as a probe revealed that specific binding proteins were present in all EC cells examined; the patterns of BOX DNA-protein complexes were the same among them. A mutation introduced within BOX DNA abolished enhancer activity as well as the formation of specific DNA-protein complexes. In non-EC cells, including L and BALB/3T3 cells, the enhancer activity of BOX DNA on the TK promoter was not observed, although binding proteins specific to the sequence exist. In band shift assays, the patterns of the DNA-protein complexes of either L or BALB/3T3 cells were different from those of EC cells. Furthermore, the enhancer activity of BOX DNA decreased upon differentiation induction in all EC cells examined, of different origins and distinct differentiation ability. In parallel with the loss of enhancer activity, the binding proteins specific for BOX DNA decreased in these cells. Moreover, we cloned a genomic DNA of F9, termed BOXF1, containing BOX DNA sequence approximately 400 bp upstream from the RNA start site of the gene. BOXF1, containing a TATA-like motif and the binding elements for Sp1 and Oct in addition to BOX DNA, possessed promoter activity deduced by a BOXF1-chloramphenicol acetyltransferase construct. Deletion analyses of the construct

  3. 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

  4. Multifunctional DNA interactions of Ru-Pt mixed metal supramolecular complexes with substituted terpyridine ligands.

    PubMed

    Jain, Avijita; Wang, Jing; Mashack, Emily R; Winkel, Brenda S J; Brewer, Karen J

    2009-10-05

    The coupling of a light absorbing unit to a bioactive site allows for the development of supramolecules with multifunctional interactions with DNA. A series of mixed metal supramolecular complexes that couple a DNA-binding cis-Pt(II)Cl(2) center to a ruthenium chromophore via a polyazine bridging ligand have been prepared, and their DNA interactions have been studied, [(TL)RuCl(dpp)PtCl(2)](PF(6)) (TL = tpy (2,2':6',2''-terpyridine), MePhtpy (4'-(4-methylphenyl)-2,2':6',2''-terpyridine), or (t)Bu(3)tpy (4,4',4''-tri-tert-butyl-2,2':6',2''-terpyridine and dpp = 2,3-bis(2-pyridyl)pyrazine). This series provides for unique tridentate coordinated Ru(II) systems to photocleave DNA with preassociation with the DNA target via coordination of the Pt(II) center. Electronic absorption spectroscopy of the complexes displays intense ligand-based pi-->pi* transitions in the UV region and metal to ligand charge transfer (MLCT) transitions in the visible region. The Ru(dpi)-->dpp(pi*) MLCT transitions occur at 545 nm, red-shifted relative to the 520 nm maxima for the monometallic synthons, [(TL)RuCl(dpp)](PF(6)). The title RuPt complexes display reversible Ru(II/III) oxidative couples at 1.10, 1.10, and 1.01 V vs Ag/AgCl for TL = tpy, MePhtpy, and (t)Bu(3)tpy, respectively. The TL(0/-) reduction occurred at -1.43, -1.44, and -1.59 V vs Ag/AgCl for TL = tpy, MePhtpy, and (t)Bu(3)tpy, respectively. These complexes display a dpp(0/-) couple (-0.50 -0.55, and -0.59 V) significantly shifted to positive potential relative to their monometallic synthons (-1.15, -1.16, and -1.22 V), consistent with the bridging coordination of the dpp ligand. Coupling of (TL)Ru(II)Cl(BL) subunit to a cis-Pt(II)Cl(2) site provides for the application of photochemically inactive Ru(II)(tpy)-based chromophores in DNA photocleavage applications. The [(TL)RuCl(dpp)PtCl(2)](+) complexes display covalent binding to DNA and photocleavage upon irradiation with visible light modulated by TL identity. The redox

  5. A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability.

    PubMed

    Yan, Zhijiang; Delannoy, Mathieu; Ling, Chen; Daee, Danielle; Osman, Fekret; Muniandy, Parameswary A; Shen, Xi; Oostra, Anneke B; Du, Hansen; Steltenpool, Jurgen; Lin, Ti; Schuster, Beatrice; Décaillet, Chantal; Stasiak, Andrzej; Stasiak, Alicja Z; Stone, Stacie; Hoatlin, Maureen E; Schindler, Detlev; Woodcock, Christopher L; Joenje, Hans; Sen, Ranjan; de Winter, Johan P; Li, Lei; Seidman, Michael M; Whitby, Matthew C; Myung, Kyungjae; Constantinou, Angelos; Wang, Weidong

    2010-03-26

    FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

  6. 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.

  7. DNA entropy reveals a significant difference in complexity between housekeeping and tissue specific gene promoters.

    PubMed

    Thomas, David; Finan, Chris; Newport, Melanie J; Jones, Susan

    2015-10-01

    The complexity of DNA can be quantified using estimates of entropy. Variation in DNA complexity is expected between the promoters of genes with different transcriptional mechanisms; namely housekeeping (HK) and tissue specific (TS). The former are transcribed constitutively to maintain general cellular functions, and the latter are transcribed in restricted tissue and cells types for specific molecular events. It is known that promoter features in the human genome are related to tissue specificity, but this has been difficult to quantify on a genomic scale. If entropy effectively quantifies DNA complexity, calculating the entropies of HK and TS gene promoters as profiles may reveal significant differences. Entropy profiles were calculated for a total dataset of 12,003 human gene promoters and for 501 housekeeping (HK) and 587 tissue specific (TS) human gene promoters. The mean profiles show the TS promoters have a significantly lower entropy (p<2.2e-16) than HK gene promoters. The entropy distributions for the 3 datasets show that promoter entropies could be used to identify novel HK genes. Functional features comprise DNA sequence patterns that are non-random and hence they have lower entropies. The lower entropy of TS gene promoters can be explained by a higher density of positive and negative regulatory elements, required for genes with complex spatial and temporary expression. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex.

    PubMed

    Hertzberg, R P; Caranfa, M J; Hecht, S M

    1989-05-30

    Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.

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

    SciTech Connect

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

    1989-03-07

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

  10. Surface-enhanced Raman scattering spectroscopy of topotecan-DNA complexes: Binding to DNA induces topotecan dimerization

    NASA Astrophysics Data System (ADS)

    Mochalov, K. E.; Strel'Tsov, S. A.; Ermishov, M. A.; Grokhovskii, S. L.; Zhuze, A. L.; Ustinova, O. A.; Sukhanova, A. V.; Nabiev, I. R.; Oleinikov, V. A.

    2002-09-01

    The interaction of topotecan (TPT), antitumor inhibitor of human DNA topoisomerase I, with calf thymus DNA was studied by surface-enhanced Raman scattering (SERS) spectroscopy. The SERS spectra of TPT are found to depend on its concentration in solution, which is associated with the dimerization of TPT. The spectral signatures of dimerization are identified. It is shown that binding to DNA induces the formation of TPT dimers. The formation of DNA-TPT-TPT-DNA complexes is considered as one of the possible mechanisms of human DNA topoisomerase I inhibition.

  11. 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.

  12. Whole chromosomal DNA probes for rapid identification of Mycobacterium tuberculosis and Mycobacterium avium complex.

    PubMed Central

    Roberts, M C; McMillan, C; Coyle, M B

    1987-01-01

    Whole chromosomal DNA probes were used to identify clinical isolates of Mycobacterium tuberculosis, Mycobacterium avium complex, and Mycobacterium gordonae. The probe for M. tuberculosis was prepared from Mycobacterium bovis BCG, which has been shown to be closely related to M. tuberculosis. A probe for the M. avium complex was prepared from three strains representing each of the three DNA homology groups in the M. avium complex. The probes were used in dot blot assays to identify clinical isolates of mycobacteria. The dot blot test correctly identified 57 of the 61 (93%) cultures grown on solid media, and 100% of antibiotic-treated broth-grown cells were correctly identified. Identification by dot blot required a maximum of 48 h. When the probes were tested against 63 positive BACTEC (Johnston Laboratories, Inc., Towson, Md.) cultures of clinical specimens, 59% were correctly identified. However, of the 14 BACTEC cultures that had been treated with antibiotics before being lysed, 13 (93%) were correctly identified. PMID:3112180

  13. A tri-copper(II) complex displaying DNA-cleaving properties and antiproliferative activity against cancer cells.

    PubMed

    Suntharalingam, Kogularamanan; Hunt, Douglas J; Duarte, Alexandra A; White, Andrew J P; Mann, David J; Vilar, Ramon

    2012-11-19

    A new disubstituted terpyridine ligand and the corresponding tri-copper(II) complex have been prepared and characterised. The binding affinity and binding mode of this tri-copper complex (as well as the previously reported mono- and di-copper analogues) towards duplex DNA were determined by using UV/Vis spectroscopic titrations and fluorescent indicator displacement (FID) assays. These studies showed the three complexes to bind moderately (in the order of 10(4)  M(-1)) to duplex DNA (ct-DNA and a 26-mer sequence). Furthermore, the number of copper centres and the nature of the substituents were found to play a significant role in defining the binding mode (intercalative or groove binding). The nuclease potential of the three complexes was investigated by using circular plasmid DNA as a substrate and analysing the products by agarose-gel electrophoresis. The cleaving activity was found to be dependent on the number of copper centres present (cleaving potency was in the order: tri-copper>di-copper>mono-copper). Interestingly, the tri-copper complex was able to cleave DNA without the need of external co-reductants. As this complex displayed the most promising nuclease properties, cell-based studies were carried out to establish if there was a direct link between DNA cleavage and cellular toxicity. The tri-copper complex displayed high cytotoxicity against four cancer cell lines. Of particular interest was that it displayed high cytotoxicity against the cisplatin-resistant MOLT-4 leukaemia cell line. Cellular uptake studies showed that the tri-copper complex was able to enter the cell and more importantly localise in the nucleus. Immunoblotting analysis (used to monitor changes in protein levels related to the DNA damage response pathway) and DNA-flow cytometric studies suggested that this tri-copper(II) complex is able to induce cellular DNA damage. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Molecular Recognition in Complexes of TRF Proteins with Telomeric DNA

    PubMed Central

    Wieczór, Miłosz; Tobiszewski, Adrian; Wityk, Paweł; Tomiczek, Bartłomiej; Czub, Jacek

    2014-01-01

    Telomeres are specialized nucleoprotein assemblies that protect the ends of linear chromosomes. In humans and many other species, telomeres consist of tandem TTAGGG repeats bound by a protein complex known as shelterin that remodels telomeric DNA into a protective loop structure and regulates telomere homeostasis. Shelterin recognizes telomeric repeats through its two major components known as Telomere Repeat-Binding Factors, TRF1 and TRF2. These two homologous proteins are therefore essential for the formation and normal function of telomeres. Indeed, TRF1 and TRF2 are implicated in a plethora of different cellular functions and their depletion leads to telomere dysfunction with chromosomal fusions, followed by apoptotic cell death. More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure. Consequently, these proteins are of great interest, not only because of their key role in telomere maintenance and stability, but also as potential drug targets. In the current study, we investigated the molecular basis of telomeric sequence recognition by TRF1 and TRF2 and their DNA binding mechanism. We used molecular dynamics (MD) to calculate the free energy profiles for binding of TRFs to telomeric DNA. We found that the predicted binding free energies were in good agreement with experimental data. Further, different molecular determinants of binding, such as binding enthalpies and entropies, the hydrogen bonding pattern and changes in surface area, were analyzed to decompose and examine the overall binding free energies at the structural level. With this approach, we were able to draw conclusions regarding the consecutive stages of sequence-specific association, and propose a novel aspartate-dependent mechanism of sequence recognition. Finally, our work demonstrates the applicability of computational MD-based methods to studying protein-DNA interactions. PMID:24586793

  15. DNA-binding, spectroscopic and antimicrobial studies of palladium(II) complexes containing 2,2'-bipyridine and 1-phenylpiperazine

    NASA Astrophysics Data System (ADS)

    Shoukry, Azza A.; Mohamed, Mervat S.

    2012-10-01

    With the purpose of evaluating the ability of Pd(II) complex to interact with DNA molecule as the main biological target, two new complexes [Pd(bpy)(OH2)2] (1) and [Pd(Phenpip)(OH2)2] (2), where (bpy = 2,2'-bipyridine; Phenpip = 1-phenylpiperazine), have been synthesized and the binding properties of these complexes with CT-DNA were investigated. The intrinsic binding constants (Kb) calculated from UV-Vis absorption studies were 3.78 × 103 M-1 and 4.14 × 103 M-1 for complexes 1 and 2 respectively. Thermal denaturation has been systematically studied by spectrophotometric method and the calculated ΔTm was nearly 5 °C for each complex. All the results suggest an electrostatic and/or groove binding mode for the interaction between the complexes and CT-DNA. The redox behavior of the two complexes in the absence and in the presence of calf thymus DNA has been investigated by cyclic voltammetry. The cyclic voltammogram exhibits one quasi-reversible redox wave. The change in E1/2, ΔEp and Ipc/Ipa supports that the two complexes exhibit strong binding to calf thymus DNA. Further insight into the binding of complexes with CT-DNA has been made by gel electrophoresis, where the binding of complexes is confirmed through decreasing the intensity of DNA bands. The two complexes have been screened for their antimicrobial activities using the disc diffusion method against some selected Gram-positive and Gram-negative bacteria. The activity data showed that both complexes were more active against Gram-negative than Gram-positive bacteria. It may be concluded that the antimicrobial activity of the compounds is related to cell wall structure of bacteria.

  16. Snf2/Swi2-related ATPase Mot1 drives displacement of TATA-binding protein by gripping DNA

    PubMed Central

    Sprouse, Rebekka O; Brenowitz, Michael; Auble, David T

    2006-01-01

    Mot1 is a conserved Snf2/Swi2-related transcriptional regulator that uses ATP hydrolysis to displace TATA-binding protein (TBP) from DNA. Several models of the enzymatic mechanism have been proposed, including Mot1-catalyzed distortion of TBP structure, competition between Mot1 and DNA for the TBP DNA-binding surface, and ATP-driven translocation of Mot1 along DNA. Here, DNase I footprinting studies provide strong support for a ‘DNA-based' mechanism of Mot1, which we propose involves ATP-driven DNA translocation. Mot1 forms an asymmetric complex with the TBP core domain (TBPc)–DNA complex, contacting DNA both upstream and within the major groove of the TATA Box. Contact with upstream DNA is required for Mot1-mediated displacement of TBPc from DNA. Using the SsoRad54–DNA complex as a model, DNA-binding residues in Mot1 were identified that are critical for Mot1–TBPc–DNA complex formation and catalytic activity, thus placing Mot1 mechanistically within the helicase superfamily. We also report a novel ATP-independent TBPc displacement activity for Mot1 and describe conformational heterogeneity in the Mot1 ATPase, which is likely a general feature of other enzymes in this class. PMID:16541100

  17. Selective nuclei accumulation of ruthenium(II) complex enantiomers that target G-quadruplex DNA.

    PubMed

    Sun, Dongdong; Liu, Yanan; Yu, Qianqian; Liu, Du; Zhou, Yanhui; Liu, Jie

    2015-09-01

    Different enantiomers exhibit large differences in their biological activity and/or toxicity, but they rarely involve the relationship of the agents for molecular and cellular imaging with the chiral structure of ruthenium complexes. Here, we report that an enantiomer of a polypyridyl ruthenium complex can selectively accumulate in the nucleus of HepG2 cells. Confocal laser scanning microscopy studies show that this phenomenon occurs via a non-endocytotic, but temperature-dependent, mechanism of cellular uptake in HepG2 cells. DNA oligonucleotides with repetitive tracts of guanine bases that can form G-quadruplex structures have aroused interest as therapeutic agents and as targets for anticancer drug design. Various biophysical techniques show that the Λ-enantiomer of ruthenium complexes can selectively stabilize human telomeric G-quadruplex DNA and has a strong preference for G-quadruplex over duplex DNA. Judged from the NMR results, we speculate that at higher 4:1 ligand/G-quadruplex stoichiometry, complex Λ-Ru is likely to bind with each groove of the tetraplex in a dimeric form or intercalate with the G-tetrad in the 3' terminal face and coexist with other modes. The molecular modeling analysis is in agreement with the NMR titrations performed in this investigation indicating that ruthenium complexes are actually characterized by a mixed binding mode. The results provide many opportunities for the development of novel agents for living cell-related studies. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories.

    PubMed

    Vago, Riccardo; Leva, Valentina; Biamonti, Giuseppe; Montecucco, Alessandra

    2009-08-15

    DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.

  19. Differentiation of the DnaA-oriC subcomplex for DNA unwinding in a replication initiation complex.

    PubMed

    Ozaki, Shogo; Noguchi, Yasunori; Hayashi, Yasuhisa; Miyazaki, Erika; Katayama, Tsutomu

    2012-10-26

    In Escherichia coli, ATP-DnaA multimers formed on the replication origin oriC promote duplex unwinding, which leads to helicase loading. Based on a detailed functional analysis of the oriC sequence motifs, we previously proposed that the left half of oriC forms an ATP-DnaA subcomplex competent for oriC unwinding, whereas the right half of oriC forms a distinct ATP-DnaA subcomplex that facilitates helicase loading. However, the molecular basis for the functional difference between these ATP-DnaA subcomplexes remains unclear. By analyzing a series of novel DnaA mutants, we found that structurally distinct DnaA multimers form on each half of oriC. DnaA AAA+ domain residues Arg-227 and Leu-290 are specifically required for oriC unwinding. Notably, these residues are required for the ATP-DnaA-specific structure of DnaA multimers in complex with the left half of oriC but not for that with the right half. These results support the idea that the ATP-DnaA multimers formed on oriC are not uniform and that they can adopt different conformations. Based on a structural model, we propose that Arg-227 and Leu-290 play a crucial role in inter-ATP-DnaA interaction and are a prerequisite for the formation of unwinding-competent DnaA subcomplexes on the left half of oriC. These residues are not required for the interaction with DnaB, nucleotide binding, or regulatory DnaA-ATP hydrolysis, which further supports their important role in inter-DnaA interaction. The corresponding residues are evolutionarily conserved and are required for unwinding in the initial complexes of Thermotoga maritima, an ancient hyperthermophile. Therefore, our findings suggest a novel and common mechanism for ATP-DnaA-dependent activation of initial complexes.

  20. Different types of copper complexes with the quinolone antimicrobial drugs ofloxacin and norfloxacin: structure, DNA- and albumin-binding.

    PubMed

    Živec, Petra; Perdih, Franc; Turel, Iztok; Giester, Gerald; Psomas, George

    2012-12-01

    Three novel copper(II) complexes with the second-generation quinolone antibacterial agents norfloxacin (nfH) and ofloxacin (ofloH) have been synthesized resulting in the complexes [Cu(nfH)(phen)Cl]Cl·5H(2)O (1·5H(2)O), [Cu(nfH)(2)]Cl(2)·6H(2)O (2·6H(2)O) and [Cu(II)(ofloH)(2)][(Cu(I)Cl(2))(2)] (3), respectively. The crystal structures of the complexes have been determined by X-ray crystallography revealing that the quinolones act as bidentate ligands coordinated to Cu(II) atom through the pyridone oxygen and a carboxylato oxygen. UV study of the interaction of the quinolones and the complexes with calf-thymus DNA (CT DNA) has shown that they can bind to CT DNA with [Cu(II)(ofloxacin)(2)][(Cu(I)Cl(2))(2)] exhibiting the highest binding constant to CT DNA. The cyclic voltammograms of the complexes in the presence of CT DNA solution have shown that the interaction of the complexes with CT DNA is mainly through electrostatic binding. DNA solution viscosity measurements have shown that the interaction of the compounds with CT DNA by classical intercalation may be ruled out. Competitive studies with ethidium bromide (EB) indicate that the complexes can partially displace the DNA-bound EB suggesting low to moderate competition with EB. Norfloxacin, ofloxacin and their copper complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Simulation of 125I induced DNA strand breaks in a CAP-DNA complex.

    PubMed

    Li, W; Friedland, W; Jacob, P; Paretzke, H G; Panyutin, I; Neumann, R D

    2002-01-01

    The E. coli catabolite gene activator protein (CAP)-DNA complex with 125I located at the position of the H5 atom of the cytosine near the centre was incorporated into the PARTRAC track structure code. DNA strand breaks due to irradiation were calculated by track structure and radical attack simulations; strand breaks due to neutralisation of the highly charged 125Te ion were derived from a semi-empirical distribution. According to the calculations, the neutralisation effect dominates the strand breakage frequency at 2 bases away from the 125I decay site on both strands. The first breakage distribution counted from a 32P labelled end on the strand with 125I agreed well with experimental data, but on the opposite strand, the calculated distribution is more concentrated around the decay site and its yield is about 20% larger than the measured data.

  2. 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.

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

    PubMed

    Udayakumar, Durga; Dynan, William S

    2015-08-07

    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 (p54(nrb)) 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.

  4. Interactions of diastereomeric tripeptides of lysyl-5-fluorotryptophyllysine with DNA. 1. Optical and 19F NMR studies of native DNA complexes.

    PubMed

    Shine, N R; James, T L

    1985-07-30

    Lysyl-5-fluoro-L-tryptophyllysine and lysyl-5-fluoro-D-tryptophyllysine were synthesized, and their interactions with double-stranded DNA were investigated as a model for protein-nucleic acid interactions. The binding to DNA was studied by monitoring various 19F NMR parameters, the fluorescence, and the optical absorbance in thermal denaturation. The 19F resonance of the L-Trp peptide shifts upfield in the presence of DNA, and that of the D-Trp peptide shifts downfield with DNA present. The influence of ionic strength on the binding of each peptide to DNA and the fluorescence quenching titration of each with DNA indicate that electrostatic bonding (approximately 2 per peptide-DNA complex) dominates the binding in each case and accounts for the similar binding constants determined from the fluorescence quenching, i.e., 7.7 X 10(4) M-1 for the L-Trp complex and 6.2 X 10(-1) for the D-Trp complex. The 19F NMR chemical shift, line width, 19F[1H] nuclear Overhauser effect, and spin-lattice relaxation time (T1) changes all indicate that the aromatic moiety of the L-Trp complex, but not that of the D-Trp complex, is stacked between the bases of DNA. The relative increases in DNA melting temperature caused by binding of the tripeptide diastereomers are also consistent with stacking in the case of the L-Trp peptide. The magnitude of the changes and the susceptibility of the 19F NMR chemical shift to altering the solvent isotope (H2O vs. D2O) suggest that the L-Trp ring is not intercalated in the classical sense but is partially inserted between the bases of one strand of the double helix.

  5. A trap potential model investigation of the optical activity induced in dye-DNA intercalation complexes

    NASA Astrophysics Data System (ADS)

    Kamiya, Mamoru

    1988-02-01

    The fundamental features of the optical activity induced in dye-DNA intercalation complexes are studied by application of the trap potential model which is useful to evaluate the induced rotational strength without reference to detailed geometrical information about the intercalation complexes. The specific effect of the potential depth upon the induced optical activity is explained in terms of the relative magnitudes of the wave-phase and helix-phase variations in the path of an electron moving on a restricted helical segment just like an exciton trapped around the dye intercalation site. The parallel and perpendicular components of the induced rotational strength well reflect basic properties of the helicity effects about the longitudinal and tangential axes of the DNA helical cylinder. The trap potential model is applied to optimize the potential parameters so as to reproduce the ionic strength effect upon the optical activity induced to proflavine-DNA intercalation complexes. From relationships between the optimized potential parameters and ionic strengths, it is inferred that increase in the ionic strength contributes to the optical activity induced by the nearest-neighbour interaction between intercalated proflavine and DNA base pairs.

  6. DNA interaction studies of a platinum (II) complex containing an antiviral drug, ribavirin: the effect of metal on DNA binding.

    PubMed

    Shahabadi, Nahid; Mirzaei kalar, Zeinab; Moghadam, Neda Hosseinpour

    2012-10-01

    The water-soluble Pt (II) complex, [PtCl (DMSO)(N(4)N(7)-ribavirin)]· H(2)O (ribavirin is an antiviral drug) has been synthesized and characterized by physico-chemical and spectroscopic methods. The binding interactions of this complex with calf thymus DNA (CT-DNA) were investigated using fluorimetry, spectrophotometry, circular dichroism and viscosimetry. The complex binds to CT-DNA in an intercalative mode. The calculated binding constant, K(b), was 7.2×10(5) M(-1). In fluorimetric studies, the enthalpy (ΔH<0) and entropy (ΔS>0) changes of the reaction between the Pt (II) complex with CT-DNA showed hydrophobic interaction. In addition, CD study showed stabilization of the right-handed B form of CT-DNA. All these results prove that the complex interacts with CT-DNA via intercalative mode of binding. In comparison with the previous study of the DNA interaction with ribavirin, these results show that platinum complex has greater affinity to CT-DNA.

  7. DNA interaction studies of a platinum (II) complex containing an antiviral drug, ribavirin: The effect of metal on DNA binding

    NASA Astrophysics Data System (ADS)

    Shahabadi, Nahid; Mirzaei kalar, Zeinab; Hosseinpour Moghadam, Neda

    2012-10-01

    The water-soluble Pt (II) complex, [PtCl (DMSO)(N4N7-ribavirin)]· H2O (ribavirin is an antiviral drug) has been synthesized and characterized by physico-chemical and spectroscopic methods. The binding interactions of this complex with calf thymus DNA (CT-DNA) were investigated using fluorimetry, spectrophotometry, circular dichroism and viscosimetry. The complex binds to CT-DNA in an intercalative mode. The calculated binding constant, Kb, was 7.2 × 105 M-1. In fluorimetric studies, the enthalpy (ΔH < 0) and entropy (ΔS > 0) changes of the reaction between the Pt (II) complex with CT-DNA showed hydrophobic interaction. In addition, CD study showed stabilization of the right-handed B form of CT-DNA. All these results prove that the complex interacts with CT-DNA via intercalative mode of binding. In comparison with the previous study of the DNA interaction with ribavirin, these results show that platinum complex has greater affinity to CT-DNA.

  8. Characterization of the Structure and DNA Complexity of Mung Bean Mitochondrial Nucleoids

    PubMed Central

    Lo, Yih-Shan; Hsiao, Lin-June; Cheng, Ning; Litvinchuk, Alexandra; Dai, Hwa

    2011-01-01

    Electron microscopic images of mitochondrial nucleoids isolated from mung bean seedlings revealed a relatively homogeneous population of particles, each consisting of a chromatin-like structure associated with a membrane com-ponent. Association of F-actin with mitochondrial nucleoids was also observed. The mitochondrial nucleoid structure identified in situ showed heterogeneous genomic organization. After pulsed-field gel electrophoresis (PFGE), a large proportion of the mitochondrial nucleoid DNA remained in the well, whereas the rest migrated as a 50-200 kb smear zone. This PFGE migration pattern was not af-fected by high salt, topoisomerase I or latrunculin B treatments; however, the mobility of a fraction of the fast-moving DNA decreased conspicuously following an in-gel ethidium-enhanced UV-irradiation treatment, suggesting that molecules with intricately compact structures were present in the 50-200 kb region. Approximately 70% of the mitochondrial nucleoid DNA molecules examined via electron microscopy were open circles, supercoils, complex forms, and linear molecules with interspersed sigma-shaped structures and/or loops. Increased sensitivity of mtDNA to DNase I was found after mitochondrial nucleoids were pretreated with high salt. This result indicates that some loosely bound or peripheral DNA binding proteins protected the mtDNA from DNase I degradation. PMID:21347700

  9. 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

  10. Spectroscopic studies on the interaction of quercetin-terbium(III) complex with calf thymus DNA.

    PubMed

    Dehghan, Gholamreza; Dolatabadi, Jafar Ezzati Nazhad; Jouyban, Abolghasem; Zeynali, Karim Asadpour; Ahmadi, Seyed Mojtaba; Kashanian, Soheila

    2011-03-01

    The interaction of native calf thymus DNA (CT-DNA) with quercetin-terbium(III) [Q-Tb(III)] complex at physiological pH was monitored by UV absorption spectrophotometry, circular dichroism, fluorescence spectroscopy, and viscosimetric techniques. The complex displays binding properties to the CT-DNA and was found to interact with CT-DNA through outside binding, demonstrated by a hypochromic effect of Q-Tb(III) on the UV spectra of CT-DNA and the calculated association constants (K). Also, decrease in the specific viscosity of CT-DNA, decrease in the fluorescence intensity of Q-Tb(III) solutions in the presence of increasing amounts of CT-DNA, and detectable changes in the circular dichroism spectrum of CT-DNA are other evidences to indicate that Q-Tb(III) complex interact with CT-DNA through outside binding.

  11. Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

    PubMed

    Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

    2015-03-31

    Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

  12. Calicheamicin-DNA complexes: warhead alignment and saccharide recognition of the minor groove.

    PubMed

    Ikemoto, N; Kumar, R A; Ling, T T; Ellestad, G A; Danishefsky, S J; Patel, D J

    1995-11-07

    The solution structures of calicheamicin gamma 1I, its cycloaromatized analog (calicheamicin epsilon), and its aryl tetrasaccharide complexed to a common DNA hairpin duplex have been determined by NMR and distance-refined molecular dynamics computations. Sequence specificity is associated with carbohydrate-DNA recognition that places the aryl tetrasaccharide component of all three ligands in similar orientations in the minor groove at the d(T-C-C-T).d(A-G-G-A) segment. The complementary fit of the ligands and the DNA minor groove binding site creates numerous van der Waals contacts as well as hydrogen bonding interactions. Notable are the iodine and sulfur atoms of calicheamicin that hydrogen bond with the exposed amino proton of the 5'- and 3'-guanines, respectively, of the d(A-G-G-A) segment. The sequence-specific carbohydrate binding orients the enediyne aglycone of calicheamicin gamma 1I such that its C3 and C6 proradical centers are adjacent to the cleavage sites. While the enediyne aglycone of calicheamicin gamma 1I is tilted relative to the helix axis and spans the minor groove, the cycloaromatized aglycone is aligned approximately parallel to the helix axis in the respective complexes. Specific localized conformational perturbations in the DNA have been identified from imino proton complexation shifts and changes in specific sugar pucker patterns on complex formation. The helical parameters for the carbohydrate binding site are comparable with corresponding values in B-DNA fibers while a widening of the groove is observed at the adjacent aglycone binding site.

  13. 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.

  14. Isolation and Quantitation of Topoisomerase Complexes Accumulated on Escherichia coli Chromosomal DNA

    PubMed Central

    Aedo, Sandra

    2012-01-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA. PMID:22869559

  15. Isolation and quantitation of topoisomerase complexes accumulated on Escherichia coli chromosomal DNA.

    PubMed

    Aedo, Sandra; Tse-Dinh, Yuk-Ching

    2012-11-01

    DNA topoisomerases are important targets in anticancer and antibacterial therapy because drugs can initiate cell death by stabilizing the transient covalent topoisomerase-DNA complex. In this study, we employed a method that uses CsCl density gradient centrifugation to separate unbound from DNA-bound GyrA/ParC in Escherichia coli cell lysates after quinolone treatment, allowing antibody detection and quantitation of the covalent complexes on slot blots. Using these procedures modified from the in vivo complexes of enzyme (ICE) bioassay, we found a correlation between gyrase-DNA complex formation and DNA replication inhibition at bacteriostatic (1× MIC) norfloxacin concentrations. Quantitation of the number of gyrase-DNA complexes per E. coli cell permitted an association between cell death and chromosomal gyrase-DNA complex accumulation at norfloxacin concentrations greater than 1× MIC. When comparing levels of gyrase-DNA complexes to topoisomerase IV-DNA complexes in the absence of drug, we observed that the gyrase-DNA complex level was higher (∼150-fold) than that of the topoisomerase IV-DNA complex. In addition, levels of gyrase and topoisomerase IV complexes reached a significant increase after 30 min of treatment at 1× and 1.7× MIC, respectively. These results are in agreement with gyrase being the primary target for quinolones in E. coli. We further validated the utility of this method for the study of topoisomerase-drug interactions in bacteria by showing the gyrase covalent complex reversibility after removal of the drug from the medium, and the resistant effect of the Ser83Leu gyrA mutation on accumulation of gyrase covalent complexes on chromosomal DNA.

  16. 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

  17. [Energetics of complex formation of the DNA hairpin structure d(GCGAAGC) with aromatic ligands].

    PubMed

    Kostiukov, V V

    2011-01-01

    The energy contributions of various physical interactions to the total Gibbs energy of complex formation of the biologically important DNA hairpin d(GCGAAGC) with aromatic antitumor antibiotics daunomycin and novantron and the mutagens ethidium and proflavine have been calculated. It has been shown that the relatively small value of the total energy of binding of the ligands to the hairpin is the sum of components great in absolute value and different in sign. The contributions of van der Waals interactions and both intra- and intermolecular hydrogen bonds and bonds with aqueous environment have been studied. According to the calculations, the hydrophobic and van der Waals components are energetically favorable in complex formation of the ligands with the DNA pairpin d(GCGAAGC), whereas the electrostatic (with consideration of hydrogen bonds) and entropic components are unfavorable.

  18. The syntactic complexity of Russian relative clauses

    PubMed Central

    Fedorenko, Evelina; Gibson, Edward

    2012-01-01

    Although syntactic complexity has been investigated across dozens of studies, the available data still greatly underdetermine relevant theories of processing difficulty. Memory-based and expectation-based theories make opposite predictions regarding fine-grained time course of processing difficulty in syntactically constrained contexts, and each class of theory receives support from results on some constructions in some languages. Here we report four self-paced reading experiments on the online comprehension of Russian relative clauses together with related corpus studies, taking advantage of Russian’s flexible word order to disentangle predictions of competing theories. We find support for key predictions of memory-based theories in reading times at RC verbs, and for key predictions of expectation-based theories in processing difficulty at RC-initial accusative noun phrase (NP) objects, which corpus data suggest should be highly unexpected. These results suggest that a complete theory of syntactic complexity must integrate insights from both expectation-based and memory-based theories. PMID:24711687

  19. The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif.

    PubMed

    Huth, J R; Bewley, C A; Nissen, M S; Evans, J N; Reeves, R; Gronenborn, A M; Clore, G M

    1997-08-01

    The solution structure of a complex between a truncated form of HMG-I(Y), consisting of the second and third DNA binding domains (residues 51-90), and a DNA dodecamer containing the PRDII site of the interferon-beta promoter has been solved by multidimensional nuclear magnetic resonance spectroscopy. The stoichiometry of the complex is one molecule of HMG-I(Y) to two molecules of DNA. The structure reveals a new architectural minor groove binding motif which stabilizes B-DNA, thereby facilitating the binding of other transcription factors in the opposing major groove. The interactions involve a central Arg-Gly-Arg motif together with two other modules that participate in extensive hydrophobic and polar contracts. The absence of one of these modules in the third DNA binding domain accounts for its-100 fold reduced affinity relative to the second one.

  20. Comparative Analysis of Interaction of Human and Yeast DNA Damage Recognition Complexes with Damaged DNA in Nucleotide Excision Repair*

    PubMed Central

    Krasikova, Yuliya S.; Rechkunova, Nadejda I.; Maltseva, Ekaterina A.; Pestryakov, Pavel E.; Petruseva, Irina O.; Sugasawa, Kaoru; Chen, Xuejing; Min, Jung-Hyun; Lavrik, Olga I.

    2013-01-01

    The human XPC-RAD23B complex and its yeast ortholog, Rad4-Rad23, are the primary initiators of global genome nucleotide excision repair. The interaction of these proteins with damaged DNA was analyzed using model DNA duplexes containing a single fluorescein-substituted dUMP analog as a lesion. An electrophoretic mobility shift assay revealed similarity between human and yeast proteins in DNA binding. Quantitative analyses of XPC/Rad4 binding to the model DNA structures were performed by fluorescent depolarization measurements. XPC-RAD23B and Rad4-Rad23 proteins demonstrate approximately equal binding affinity to the damaged DNA duplex (KD ∼ (0.5 ± 0.1) and (0.6 ± 0.3) nm, respectively). Using photoreactive DNA containing 5-iodo-dUMP in defined positions, XPC/Rad4 location on damaged DNA was shown. Under conditions of equimolar binding to DNA both proteins exhibited the highest level of cross-links to 5I-dUMP located exactly opposite the damaged nucleotide. The positioning of the XPC and Rad4 proteins on damaged DNA by photocross-linking footprinting is consistent with x-ray analysis of the Rad4-DNA crystal complex. The identity of the XPC and Rad4 location illustrates the common principles of structure organization of DNA damage-scanning proteins from different Eukarya organisms. PMID:23443653

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

    PubMed

    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 K(b), 5.21×10(4)M(-1) that are higher than that obtained for 2 (red-shift, 2 nm; K(b), 1.73×10(4)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 3-10nm 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 E(pc) and E(0)' 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 (HO()) and the singlet oxygen are believed to be the reactive species responsible for the cleavage.

  2. Molecular dynamics simulations of the glucocorticoid receptor DNA-binding domain in complex with DNA and free in solution.

    PubMed Central

    Eriksson, M A; Härd, T; Nilsson, L

    1995-01-01

    Molecular dynamics simulations have been performed on the glucocorticoid receptor DNA binding domain (GR DBD) in aqueous solution as a dimer in complex with DNA and as a free monomer. In the simulated complex, we find a slightly increased bending of the DNA helix axis compared with the crystal structure in the spacer region of DNA between the two half-sites that are recognized by GR DBD. The bend is mainly caused by an increased number of interactions between DNA and the N-terminal extended region of the sequence specifically bound monomer. The recognition helices of GR DBD are pulled further into the DNA major groove leading to a weakening of the intrahelical hydrogen bonds in the middle of the helices. Many ordered water molecules with long residence times are found at the intermolecular interfaces of the complex. The hydrogen-bonding networks (including water bridges) on either side of the DNA major groove involve residues that are highly conserved within the family of nuclear receptors. Very similar hydrogen-bonding networks are found in the estrogen receptor (ER) DBD in complex with DNA, which suggests that this is a common feature for proper positioning of the recognition helix in ER DBD and GR DBD. Images FIGURE 1 FIGURE 6 FIGURE 8 FIGURE 10 FIGURE 11 FIGURE 14 PMID:7696496

  3. 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

  4. 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.

  5. DNA interactions of new cytotoxic tetrafunctional dinuclear platinum complex trans,trans-[{PtCl2(NH3)}2(piperazine)].

    PubMed

    Brabec, Viktor; Christofis, Petros; Slámová, Martina; Kostrhunová, Hana; Nováková, Olga; Najajreh, Yousef; Gibson, Dan; Kaspárková, Jana

    2007-06-15

    A new tetrafunctional dinuclear platinum complex trans,trans-[{PtCl2(NH3)}2(piperazine)] with sterically rigid linking group was designed, synthesized and characterized. In this novel molecule, the DNA-binding features of two classes of the platinum compounds with proven antitumor activity are combined, namely trans oriented bifunctional mononuclear platinum complexes with a heterocyclic ligand and polynuclear platinum complexes. DNA-binding mode of this new complex was analyzed by various methods of molecular biology and biophysics. The complex coordinates DNA in a unique way and interstrand and intrastrand cross-links are the predominant lesions formed in DNA in cell-free media and in absence of proteins. An intriguing aspect of trans,trans-[{PtCl2(NH3)}2(piperazine)] is that, using a semi-rigid linker, interstrand cross-linking is diminished relative to other dinuclear platinum complexes with flexible linking groups and lesions that span several base pairs, such as tri- and tetrafunctional adducts, become unlikely. In addition, in contrast to the inability of trans,trans-[{PtCl2(NH3)}2(piperazine)] to cross-link two DNA duplexes, the results of the present work convincingly demonstrate that this dinuclear platinum complex forms specific DNA lesions which can efficiently cross-link proteins to DNA. The results substantiate the view that trans,trans-[{PtCl2(NH3)}2(piperazine)] or its analogues could be used as a tool for studies of DNA properties and their interactions or as a potential antitumor agent. The latter view is also corroborated by the observation that trans,trans-[{PtCl2(NH3)}2(piperazine)] is a more effective cytotoxic agent than cisplatin against human tumor ovarian cell lines.

  6. 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.

  7. Cobalt complexes of terpyridine ligand: crystal structure and photocleavage of DNA.

    PubMed

    Indumathy, Ramasamy; Radhika, Srinivasan; Kanthimathi, Mookandi; Weyhermuller, T; Unni Nair, Balachandran

    2007-03-01

    Two new cobalt complexes, [Co(pytpy)(2)](ClO(4))(2), 1, and [Co(pytpy)(2)](ClO(4))(3), 2 where pytpy=pyridine terpyridine, have been synthesized and characterized. Single-crystal X-ray structure of both the complexes has been resolved. The structure shows the complexes to be a monomeric cobalt(II) and cobalt(III) species with two pytpy ligands coordinated to the metal ion to give a six coordinate complex. Both cobalt(II) and cobalt(III) complexes crystallize in meridional configuration. The interaction of these complexes with calf thymus DNA has been explored by using absorption, emission spectral, electrochemical studies and viscosity measurements. From the experimental results the DNA binding constants of 1 and 2 are found to be (1.97+/-0.15)x10(4)M(-1) and (2.7+/-0.20)x10(4)M(-1) respectively. The ratio of DNA binding constants of 1 and 2 have been estimated to be 0.82 from electrochemical studies, which is in close agreement with the value of 0.73 obtained from spectral studies. The observed changes in viscosity of DNA in the presence of increasing amount of complexes 1 and 2 suggest intercalating binding of these complexes to DNA. Results of DNA cleaving experiments reveal that complex 2 efficiently cleaves DNA under photolytic conditions while complex 1 does not cleave DNA under similar conditions.

  8. Delineating relative homogeneous G+C domains in DNA sequences.

    PubMed

    Li, W

    2001-10-03

    The concept of homogeneity of G+C content is always relative and subjective. This point is emphasized and quantified in this paper using a simple example of one sequence segmented into two subsequences. Whether the sequence is homogeneous or not can be answered by whether the two-subsequence model describes the DNA sequence better than the one-sequence model. There are at least three equivalent ways of looking at the 1-to-2 segmentation: Jensen-Shannon divergence measure, log likelihood ratio test, and model selection using Bayesian information criterion. Once a criterion is chosen, a DNA sequence can be recursively segmented into multiple domains. We use one subjective criterion called segmentation strength based on the Bayesian information criterion. Whether or not a sequence is homogeneous and how many domains it has depend on this criterion. We compare six different genome sequences (yeast S. cerevisiae chromosome III and IV, bacterium M. pneumoniae, human major histocompatibility complex sequence, longest contigs in human chromosome 21 and 22) by recursive segmentations at different strength criteria. Results by recursive segmentation confirm that yeast chromosome IV is more homogeneous than yeast chromosome III, human chromosome 21 is more homogeneous than human chromosome 22, and bacterial genomes may not be homogeneous due to short segments with distinct base compositions. The recursive segmentation also provides a quantitative criterion for identifying isochores in human sequences. Some features of our recursive segmentation, such as the possibility of delineating domain borders accurately, are superior to those of the moving-window approach commonly used in such analyses.

  9. Mitochondrial DNA Sequence Divergence among Lycopersicon and Related Solanum Species

    PubMed Central

    McClean, Phillip E.; Hanson, Maureen R.

    1986-01-01

    Sequence divergence among the mitochondrial (mt) DNA of nine Lycopersicon and two closely related Solanum species was estimated using the shared fragment method. A portion of each mt genome was highlighted by probing total DNA with a series of plasmid clones containing mt-specific DNA fragments from Lycopersicon pennellii. A total of 660 fragments were compared. As calculated by the shared fragment method, sequence divergence among the mtDNAs ranged from 0.4% for the L. esculentum-L. esculentum var. cerasiforme pair to 2.7% for the Solanum rickii-L. pimpinellifolium and L. cheesmanii-L. chilense pairs. The mtDNA divergence is higher than that reported for Lycopersicon chloroplast (cp) DNA, which indicates that the DNAs of the two plant organelles are evolving at different rates. The percentages of shared fragments were used to construct a phenogram that illustrates the present-day relationships of the mtDNAs. The mtDNA-derived phenogram places L. hirsutum closer to L. esculentum than taxonomic and cpDNA comparisons. Further, the recent assignment of L. pennellii to the genus Lycopersicon is supported by the mtDNA analysis. PMID:17246320

  10. 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

  11. 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.

  12. Application of genomic DNA affinity chromatography identifies multiple interferon-alpha-regulated Stat2 complexes.

    PubMed

    Ghislain, J J; Fish, E N

    1996-05-24

    Interferon-alpha (IFN-alpha)-induced signal transduction is mediated by the phosphorylation-activation of the signal transducer and activator of transcription (STAT) proteins Stat1, Stat2, and Stat3. Previous studies have shown that these activated STATs dimerize to form four distinct STAT complexes which translocate to the nucleus and activates transcription by binding to specific promoter elements. The interferon-stimulated gene factor-3 (ISGF3) consists of Stat2 and Stat1 heterodimers in association with a DNA-binding protein, p48, that binds to the interferon stimulated response element. Homo-and heterodimers of Stat1 and Stat3 bind to the palindromic interferon response element (pIRE). In this report we demonstrate the utility of a biochemical procedure that we have developed, based on genomic DNA affinity chromatography, for the identification of IFN-alpha-induced STAT complexes. Using this approach, we identified ISGF3-independent Stat2-containing STAT complexes. Results from the analysis of Stat2 complexes in the electrophoretic mobility shift assay were consistent with genomic DNA affinity chromatography results and identified a Stat2:1 complex that binds with low affinity to the pIRE of the interferon regulatory factor-1 gene. Immunoprecipitation studies of Stat2 revealed an IFN-alpha dependent co-precipitation of both Stat1 and Stat3. Taken together, our results suggest that IFN-alpha activates, in addition to ISGF3, other Stat2-containing STAT complexes, one of which binds to an element related to the interferon regulatory factor-1 pIRE.

  13. A stable double-stranded DNA-ethidium homodimer complex: Application to picogram fluorescence detection of DNA in agarose gels

    SciTech Connect

    Glazer, A.N.; Mathies, R.A. Lawrence Berkeley Laboratory, CA ); Peck, K. )

    1990-05-01

    The complex between double-stranded DNA and ethidium homodimer (5,5{prime}-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridinium) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the performed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble.

  14. A stable double-stranded DNA-ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels.

    PubMed Central

    Glazer, A N; Peck, K; Mathies, R A

    1990-01-01

    The complex between double-stranded DNA and ethidium homodimer (5,5'-diazadecamethylene)bis(3,8-diamino-6-phenylphenanthridini um) cation, formed at a ratio of 1 homodimer per 4 or 5 base pairs, is stable in agarose gels under the usual conditions for electrophoresis. This unusual stability allows formation of the complex before electrophoresis and then separation and detection in the absence of background stain. Competition experiments between the preformed DNA-ethidium homodimer complex and a 50-fold molar excess of unlabeled DNA show that approximately one-third of the dye is retained within the original complex independent of the duration of the competition. However, dye-extraction experiments show that these are not covalent complexes. After electrophoretic separation, detection of bands containing 25 pg of DNA was readily achieved in 1-mm thick agarose gels with laser excitation at 488 nm and a scanning confocal fluorescence imaging system. The band intensity was linear with the amount of DNA applied from 0.2 to 1.0 ng per lane and with the number of kilobase pairs (kbp) per band within a lane. Analysis of an aliquot of a polymerase-chain-reaction mixture permitted ready detection of 80 pg of a 1.6-kbp amplified fragment. The use of the ethidium homodimer complex together with laser excitation for DNA detection on gels is at least two orders of magnitude more sensitive than conventional fluorescence-based procedures. The homodimer-DNA complex exemplifies a class of fluorescent probes where the intercalation of dye chromophores in DNA forms a stable, highly fluorescent ensemble. Images PMID:2339125

  15. 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.

  16. Transient association of the DNA-ligand complex during gel electrophoresis.

    PubMed

    Protozanova, E; Macgregor, R B

    1999-07-01

    DNA frayed wires are extremely stable multistranded complexes arising from the association of oligonucleotides with long terminal runs of consecutive guanines. Frayed wires originating from d(A15G15) have multiple binding sites for short complementary oligonucleotides such as dT10. We examine unusual band patterns obtained when complexes formed between dT10 and DNA frayed wires are resolved on nondenaturing polyacrylamide gels. Since the lifetime of the dT10-frayed wire complexes is shorter than the time of the gel run, the interaction between the components during the gel electrophoresis affects their band patterns. We have conducted chasing experiments to show that (i) the binding of dT10 to the frayed wires can occur during gel electrophoresis, and (ii) dissociation of the complexes occurs during the gel run. Rapid repetitive dissociation-reassociation of the complexes leads to a constant partitioning of dT10 between their binding sites within frayed wires. Consequently, complexes composed of frayed wires and various numbers of bound ligands appear on the gel as a single well-defined band. The mobilities of these bands decrease continuously with the concentration of the ligand reaching saturation when all the binding sites are occupied. This characteristic pattern is observed only for relatively unstable interactions. Longer ligands, i.e., oligonucleotides with higher affinity towards the binding sites, cease to exhibit the dynamic character of interaction during gel electrophoresis. These ligands form long-lived complexes with the frayed wires that appear on the gel as faint smeared bands reflecting the presence of multiple stable complexes.

  17. Potential-modulated DNA cleavage by (N-salicylideneglycinato)copper(II) complex.

    PubMed

    Yang, Zhou-Sheng; Wang, Yan-Ling; Liu, Yun-Chun; Zhao, Guang-Chao

    2005-11-01

    The interaction of aqua (N-salicylideneglycinato)copper(II) (Cu(salgly)2+) complex with calf thymus DNA has been investigated by cyclic voltammetry. Potential-modulated DNA cleavage in the presence of Cu(salgly)2+ complex was performed at a gold electrode in a thin layer cell. DNA can be efficiently cleaved by electrochemically reducing Cu(salgly)2+ complex to Cu(salgly)+ complex at -0.7 V (vs. Ag/AgCl). When the solution was aerated with a small flow of O2 during electrolysis, the extent of DNA cleavage was dramatically enhanced, and hydroxyl radical scavengers inhibited DNA cleavage. These results suggested that O2 and hydroxyl radical were involved in potential-modulated DNA cleavage reaction. The percentage of DNA cleavage was enhanced as the working potential was shifted to more negative values and the electrolysis time was increased. It was also dependent on the ratio of Cu(salgly)2+ complex to DNA concentration. The cleaved DNA fragments were separated by high performance liquid chromatography (HPLC). The experimental results indicated that the method for potential-modulated DNA cleavage by Cu(salgly)2+ complex was simple and efficient.

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

    PubMed

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

    2012-08-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  20. 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

  1. 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.

  2. Structure of the DNA-SspC Complex: Implications for DNA Packaging, Protection, and Repair in Bacterial Spores

    PubMed Central

    Frenkiel-Krispin, Daphna; Sack, Rinat; Englander, Joseph; Shimoni, Eyal; Eisenstein, Miriam; Bullitt, Esther; Horowitz-Scherer, Rachel; Hayes, Christopher S.; Setlow, Peter; Minsky, Abraham; Wolf, Sharon Grayer

    2004-01-01

    Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed α/β-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the α/β-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the α/β-type SASP. PMID:15150240

  3. 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.

  4. Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore

    NASA Astrophysics Data System (ADS)

    Benner, Seico; Chen, Roger J. A.; Wilson, Noah A.; Abu-Shumays, Robin; Hurt, Nicholas; Lieberman, Kate R.; Deamer, David W.; Dunbar, William B.; Akeson, Mark

    2007-11-01

    Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules. Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates. In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.

  5. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links

    PubMed Central

    Räschle, Markus; Smeenk, Godelieve; Hansen, Rebecca K.; Temu, Tikira; Oka, Yasuyoshi; Hein, Marco Y.; Nagaraj, Nagarjuna; Long, David T.; Walter, Johannes C.; Hofmann, Kay; Storchova, Zuzana; Cox, Jürgen; Bekker-Jensen, Simon; Mailand, Niels; Mann, Matthias

    2017-01-01

    DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells. PMID:25931565

  6. Synthesis, spectral characterization and eukaryotic DNA degradation of thiosemicarbazones and their platinum(IV) complexes.

    PubMed

    Al-Hazmi, G A; El-Metwally, N M; El-Gammal, O A; El-Asmy, A A

    2008-01-01

    The condensation products of acetophenone (or its derivatives), salicylaldehyde and o-hydroxy-p-methoxybenzophenone with thiosemicarbazide and ethyl- or phenyl-thiosemicarbazide are the investigated thiosemicarbazones. Their reactions with H(2)PtCl(6) produced Pt(IV) complexes characterized by elemental, thermal, mass, IR and electronic spectral studies. The coordination modes were found mononegative bidentate in the acetophenone derivatives and binegative tridentate in the salicylaldehyde derivatives. The complexes were analyzed thermogravimetrically and found highly stable. Some ligands and their complexes were screened against Sarcina sp. and E. coli using the cup-diffusion technique. [Pt(oHAT)(OH)Cl] shows higher activity against E. coli than the other compounds. The degradation power of the tested compounds on the calf thymus DNA supports their selectivity against bacteria and not against the human or related eukaryotic organisms.

  7. Synthesis, spectral characterization and eukaryotic DNA degradation of thiosemicarbazones and their platinum(IV) complexes

    NASA Astrophysics Data System (ADS)

    Al-Hazmi, G. A.; El-Metwally, N. M.; El-Gammal, O. A.; El-Asmy, A. A.

    2008-01-01

    The condensation products of acetophenone (or its derivatives), salicylaldehyde and o-hydroxy- p-methoxybenzophenone with thiosemicarbazide and ethyl- or phenyl-thiosemicarbazide are the investigated thiosemicarbazones. Their reactions with H 2PtCl 6 produced Pt(IV) complexes characterized by elemental, thermal, mass, IR and electronic spectral studies. The coordination modes were found mononegative bidentate in the acetophenone derivatives and binegative tridentate in the salicylaldehyde derivatives. The complexes were analyzed thermogravimetrically and found highly stable. Some ligands and their complexes were screened against Sarcina sp. and E. coli using the cup-diffusion technique. [Pt( oHAT)(OH)Cl] shows higher activity against E. coli than the other compounds. The degradation power of the tested compounds on the calf thymus DNA supports their selectivity against bacteria and not against the human or related eukaryotic organisms.

  8. Polymorphism of DNA-anionic liposome complexes reveals hierarchy of ion-mediated interactions.

    PubMed

    Liang, Hongjun; Harries, Daniel; Wong, Gerard C L

    2005-08-09

    Self-assembled DNA delivery systems based on anionic lipids (ALs) complexed with DNA mediated by divalent cations have been recently introduced as an alternative to cationic lipid-DNA complexes because of their low cytotoxicity. We investigate AL-DNA complexes induced by different cations by using synchrotron small angle x-ray scattering and confocal microscopy to show how different ion-mediated interactions are expressed in the self-assembled structures and phase behavior of AL-DNA complexes. The governing interactions in AL-DNA systems are complex: divalent ions can mediate strong attractions between different combinations of the components (such as DNA-DNA and membrane-membrane). Moreover, divalent cations can coordinate non-electrostatically with lipids and modify the resultant membrane structure. We find that at low membrane charge densities AL-DNA complexes organize into a lamellar structure of alternating DNA and membrane layers crosslinked by ions. At high membrane charge densities, a new phase with no analog in cationic lipid-DNA systems is observed: DNA is expelled from the complex, and a lamellar stack of membranes and intercalated ions is formed. For a subset of the ionic species, high ion concentrations generate an inverted hexagonal phase comprised of DNA strands wrapped by ion-coated lipid tubes. A simple theoretical model that takes into account the electrostatic and membrane elastic contributions to the free energy shows that this transition is consistent with an ion-induced change in the membrane spontaneous curvature, c0. Moreover, the crossover between the lamellar and inverted hexagonal phases occurs at a critical c0 that agrees well with experimental values.

  9. Modulation of photo-oxidative DNA damage by cationic surfactant complexation.

    PubMed

    Rudiuk, Sergii; Franceschi-Messant, Sophie; Chouini-Lalanne, Nadia; Perez, Emile; Rico-Lattes, Isabelle

    2008-08-19

    The natural packaging of DNA in the cell by histones provides a particular environment affecting its sensitivity to oxidative damage. In this work, we used the complexation of DNA by cationic surfactants to modulate the conformation, the dynamics, and the environment of the double helix. Photo-oxidative damage initiated by benzophenone as the photosensitizer on a plasmid DNA complexed by dodecyltrimethylammonium chloride (DTAC), tetradecyltrimethylammonium chloride (TTAC), cetyltrimethyammonium chloride (CTAC) and bromide (CTAB) was detected by agarose gel electrophoresis. By fluorescent titration in the presence of ethidium bromide (EB) and agarose gel electrophoresis, we experimentally confirmed the complexation diagrams with a critical aggregation concentration on DNA matrix (CAC DNA) delimiting two regions of complexation, according to the DNA-phosphate concentration. The study of the photo-oxidative damage shows, for the first time, a direct correlation between the DNA complexation by these surfactants and the efficiency of DNA cleavage, with a maximum corresponding to the CAC DNA for DTAC and CTAC, and to DNA neutralization for CTAC and CTAB. The localization of a photosensitizer having low water solubility, such as benzophenone, inside the hydrophobic domains formed by the surfactant aggregated on DNA, locally increases the photoinduced cleavage by the free radical oxygen species generated. The inefficiency of a water-soluble quencher of hydroxyl radicals, such as mannitol, confirmed this phenomenon. The detection of photo-oxidative damage constitutes a new tool for investigating DNA complexation by cationic surfactants. Moreover, highlighting the drastically increased sensitivity of a complexed DNA to photo-oxidative damage is of crucial importance for the biological use of surfactants as nonviral gene delivery systems.

  10. A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide complex of d(CGTACG)2

    PubMed Central

    Adams, Adrienne; Guss, J. Mitchell; Collyer, Charles A.; Denny, William A.; Wakelin, Laurence P. G.

    2000-01-01

    The structures of the complexes formed between 9-amino-[N-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG5BrUACG)2 and d(CGTACG)2 have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 Å. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native). PMID:11058124

  11. Intranasal Administration of Novel Chitosan Nanoparticle/DNA Complexes Induces Antibody Response to Hepatitis B Surface Antigen in Mice.

    PubMed

    Lebre, F; Borchard, G; Faneca, H; Pedroso de Lima, M C; Borges, O

    2016-02-01

    The generation of strong pathogen-specific immune responses at mucosal surfaces where hepatitis B virus (HBV) transmission can occur is still a major challenge. Therefore, new vaccines are urgently needed in order to overcome the limitations of existing parenteral ones. Recent studies show that this may be achieved by intranasal immunization. Chitosan has gained attention as a nonviral gene delivery system; however, its use in vivo is limited due to low transfection efficiency mostly related to strong interaction between the negatively charged DNA and the positively charged chitosan. We hypothesize that the adsorption of negatively charged human serum albumin (HSA) onto the surface of the chitosan particles would facilitate the intracellular release of DNA, enhancing transfection activity. Here, we demonstrate that a robust systemic immune response was induced after vaccination using HSA-loaded chitosan nanoparticle/DNA (HSA-CH NP/DNA) complexes. Furthermore, intranasal immunization with HSA-CH NP/DNA complexes induced HBV specific IgA in nasal and vaginal secretions; no systemic or mucosal responses were detected after immunization with DNA alone. Overall, our results show that chitosan-based DNA complexes elicited both humoral and mucosal immune response, making them an interesting and valuable gene delivery system for nasal vaccination against HBV.

  12. Comparing the interaction of vanadyl-hypocrellin A complex and hypocrellin A with CT DNA

    NASA Astrophysics Data System (ADS)

    Yang, Chao; Ma, Fei; Tang, Jing; Han, Lina; Wei, Shaohua; Zhou, Lin; Zhou, Jiahong; Shen, Jian; Ge, Xuefeng

    2013-03-01

    Interaction studies of the vanadyl-hypocrellin A (VO2+-HA) complex and hypocrellin A (HA) with CT DNA were investigated by UV-Vis spectroscopy, DNA melting, fluorescence spectra and zeta potential measurements. The binding constants and binding mechanisms of HA and VO2+-HA with CT DNA had also been studied by fluorescence quenching experiments. Both spectrophotometric and DNA melting studies indicated that VO2+-HA and HA could bind to CT DNA. Thermodynamic parameters together with zeta potential studies revealed that the binding force between HA or VO2+-HA with CT DNA was hydrophobic and electrostatic force, separately.

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

    PubMed

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

    2016-08-05

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

  14. 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.

  15. Photo-thermal effects in gold nanorods/DNA complexes

    NASA Astrophysics Data System (ADS)

    De Sio, Luciano; Caracciolo, Giulio; Annesi, Ferdinanda; Placido, Tiziana; Pozzi, Daniela; Comparelli, Roberto; Pane, Alfredo; Curri, Maria Lucia; Agostiano, Angela; Bartolino, Roberto

    2015-12-01

    An ingenious combination of plasmonic nanomaterials and one of the most relevant biological systems, deoxyribonucleic acid (DNA) is achieved by bioconjugating gold nanorods (GNRs) with DNA via electrostatic interaction between positively charged GNRs and negatively charged short DNA. The obtained system is investigated as a function of DNA concentration by means of gel electrophoresis, zeta-potential, DNA melting and morphological analysis. It turns out that the obtained bioconjugated systems present both effective electric charge and aggregate size that are particularly amenable for gene therapy and nanomedicine applications. Finally, the effect of the localized (photo-thermal heating) and delocalized temperature variation on the DNA melting by performing both light induced bio-transparent optical heating experiments and a thermographic analysis is investigated, demonstrating that the developed system can be exploited for monitoring nanoscale temperature variation under optical illumination with very high sensitivity.

  16. 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.

  17. Zebularine: A Novel DNA Methylation Inhibitor that Forms a Covalent Complex with DNA Methyltransferases

    PubMed Central

    Zhou, L.; Connolly, B.A.; Dickman, M.J.; Hurd, P.J.

    2009-01-01

    Mechanism-based inhibitors of enzymes, which mimic reactive intermediates in the reaction pathway, have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here, we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.Hha I) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone, an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.Hha I and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine. PMID:12206775

  18. Prediction of DNA-binding proteins from relational features

    PubMed Central

    2012-01-01

    Background The process of protein-DNA binding has an essential role in the biological processing of genetic information. We use relational machine learning to predict DNA-binding propensity of proteins from their structures. Automatically discovered structural features are able to capture some characteristic spatial configurations of amino acids in proteins. Results Prediction based only on structural relational features already achieves competitive results to existing methods based on physicochemical properties on several protein datasets. Predictive performance is further improved when structural features are combined with physicochemical features. Moreover, the structural features provide some insights not revealed by physicochemical features. Our method is able to detect common spatial substructures. We demonstrate this in experiments with zinc finger proteins. Conclusions We introduced a novel approach for DNA-binding propensity prediction using relational machine learning which could potentially be used also for protein function prediction in general. PMID:23146001

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

    PubMed

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

    2016-02-02

    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.

  20. A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex.

    PubMed

    Ishimi, Y

    1997-09-26

    All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain which is conserved from yeast to mammals. Our group purified MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins from HeLa cells by using histone-Sepharose column chromatography (Ishimi, Y., Ichinose, S., Omori, A., Sato K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115-24122). The present study revealed that both ATPase activity and DNA helicase activity that displaces oligonucleotides annealed to single-stranded circular DNA are associated with an MCM protein complex. Both ATPase and DNA helicase activities were co-purified with a 600-kDa protein complex that is consisted of equal amounts of MCM4, -6, and -7 proteins. An immunodepletion of the MCM protein complex from the purified fraction using anti-MCM4 antibody resulted in the severe reduction of the DNA helicase activity. Displacement of DNA fragments by the DNA helicase suggested that it migrated along single-stranded DNA in the 3' to 5' direction, and the DNA helicase activity was detected only in the presence of hydrolyzable ATP or dATP. These results suggest that this helicase may be involved in the initiation of DNA replication as a DNA unwinding enzyme.

  1. Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA

    PubMed Central

    Zabrady, Katerina; Adamus, Marek; Vondrova, Lucie; Liao, Chunyan; Skoupilova, Hana; Novakova, Marketa; Jurcisinova, Lenka; Alt, Aaron; Oliver, Antony W.; Lehmann, Alan R.; Palecek, Jan J.

    2016-01-01

    SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin. PMID:26446992

  2. 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}.

  3. 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.

  4. Polarized fluorescence correlation spectroscopy of DNA-DAPI complexes.

    PubMed

    Barcellona, Maria Luisa; Gammon, Seth; Hazlett, Theodore; Digman, Michelle A; Gratton, Enrico

    2004-11-01

    We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules are present at any instant of time. The measurements of long DNA molecules labeled with the fluorescent probe DAPI show local rotational motions of the polymers in addition to translation motions of the entire polymer. For smaller molecules such as EGFP, the viscosity of the solution must be increased to bring the relaxation due to rotational motion into the measurable range. Overall, our results show that polarized fluorescence correlation spectroscopy can be used to detect fast and slow rotational motion in the time scale from microsecond to second, a range that cannot be easily reached by conventional fluorescence anisotropy decay methods.

  5. Mononuclear metal complexes with ciprofloxacin: Synthesis, characterization and DNA-binding properties.

    PubMed

    Psomas, George

    2008-09-01

    Five novel metal complexes of the quinolone antibacterial agent ciprofloxacin with Mn(2+), Fe(3+), Co(2+), Ni(2+) and MoO(2)(2+) have been prepared and characterized with physicochemical, spectroscopic and electrochemical techniques. In all these complexes, ciprofloxacin acts as a bidentate deprotonated ligand bound to the metal through the pyridone oxygen and one carboxylate oxygen. The central metal in each complex is six-coordinate and a slightly distorted octahedral geometry is proposed. The lowest energy model structures of the Mn(2+), Fe(3+) and MoO(2)(2+) complexes have been determined with molecular modeling calculations. The cyclic voltammograms of the complexes have been recorded in dmso solution and in 1/2 dmso/buffer (containing 150mM NaCl and 15mM trisodium citrate at pH 7.0) solution and the corresponding redox potentials have been estimated. The biological activity of the complexes has been evaluated by examining their ability to bind to calf-thymus DNA (CT DNA) with UV and fluorescence spectroscopies and cyclic voltammetry. UV studies of the interaction of the complexes with DNA have shown that these compounds can bind to CT DNA. The binding constants of the complexes with CT DNA have also been calculated. The cyclic voltammograms of the complexes in the presence of CT DNA have shown that the complexes can bind to CT DNA by both the intercalative and the electrostatic binding mode. Competitive studies with ethidium bromide (EB) have shown that the complexes exhibit the ability to displace the DNA-bound EB indicating that the complexes bind to DNA probably via intercalation in strong competition with EB for the intercalative binding site.

  6. Insights into the Replisome from the Structure of a Ternary Complex of the DNA Polymerase III [alpha]-Subunit

    SciTech Connect

    Wing, R.A.; Bailey, S.; Steitz, T.A.

    2009-03-27

    The crystal structure of the catalytic {alpha}-subunit of the DNA polymerase III (PolIII{alpha}) holoenzyme bound to primer-template DNA and an incoming deoxy-nucleoside 5{prime}-triphosphate has been determined at 4.6-{angstrom} resolution. The polymerase interacts with the sugar-phosphate backbone of the DNA across its minor groove, which is made possible by significant movements of the thumb, finger, and {beta}-binding domains relative to their orientations in the unliganded polymerase structure. Additionally, the DNA and incoming nucleotide are bound to the active site of PolIII{alpha} nearly identically as they are in their complex with DNA polymerase {beta}, thereby proving that the eubacterial replicating polymerase, but not the eukaryotic replicating polymerase, is homologous to DNA polymerase {beta}. Finally, superimposing a recent structure of the clamp bound to DNA on this PolIII{alpha} complex with DNA places a loop of the {beta}-binding domain into the appropriate clamp cleft and supports a mechanism of polymerase switching.

  7. Mode of formation and structural features of DNA-cationic liposome complexes used for transfection.

    PubMed

    Gershon, H; Ghirlando, R; Guttman, S B; Minsky, A

    1993-07-20

    Complexes formed between cationic liposomes and nucleic acids represent a highly efficient vehicle for delivery of DNA and RNA molecules into a large variety of eukaryotic cells. By using fluorescence, gel electrophoresis, and metal-shadowing electron microscopy techniques, the factors that affect the, yet unclear, interactions between DNA and cationic liposomes as well as the structural features of the resulting complexes have been elucidated. A model is suggested according to which cationic liposomes bind initially to DNA molecules to form clusters of aggregated vesicles along the nucleic acids. At a critical liposome density, two processes occur, namely, DNA-induced membrane fusion, indicated by lipid mixing studies, and liposome-induced DNA collapse, pointed out by the marked cooperativity of the encapsulation processes, by their modulations by DNA-condensing agents, and also by their conspicuous independence upon DNA length. The DNA collapse leads to the formation of condensed structures which can be completely encapsulated within the fused lipid bilayers in a fast, highly cooperative process since their exposed surface is substantially smaller than that of extended DNA molecules. The formation of the transfecting DNA-liposome complexes in which the nucleic acids are fully encapsulated within a positively-charged lipid bilayer is proposed, consequently, to be dominated by mutual effects exerted by the DNA and the cationic liposomes, leading to interrelated lipid fusion and DNA collapse.

  8. DNA barcoding of the ichthyofauna of Pánuco-Tamesí complex: evidence for taxonomic conflicts in some groups.

    PubMed

    Mejía, Omar; León-Romero, Yatzil; Soto-Galera, Eduardo

    2012-12-01

    The Pánuco-Tamesí complex in eastern Mexico is globally recognized as an important ecoregion due to its high level of endemism. In this study, DNA barcodes were generated for 152 individuals of 31 species. Additionally, 170 DNA barcodes for the related species available in the Barcode of Life Database (BOLD) system were included to test the ability of barcoding technique to discriminate between the closely related species. DNA barcoding allowed the discrimination of 79.2% of the analyzed species; poor resolution was observed in four genera in which the levels of resolution ranged from 16.6% in the genus Herichthys to 77.7% in the genus Xiphophorus. The results of this study demonstrate that DNA barcoding is a useful exploratory tool but fails to discriminate between closely related species.

  9. Design and synthesis of novel hybrid metal complex-DNA conjugates: key building blocks for multimetallic linear DNA nanoarrays.

    PubMed

    Ghosh, Sumana; Pignot-Paintrand, Isabelle; Dumy, Pascal; Defrancq, Eric

    2009-07-07

    We describe here a short and efficient synthetic route for incorporating terpyridine based metal complexes at the 3'-extremity of both single and bis-oligonucleotide (bis-ODN) stretches to form novel metal complex-ODN conjugates. All single stranded mono and bis-ODN tethered metal complexes and the respective duplex ODNs were characterized by circular dichroism spectroscopy and UV-Vis melting experiments. Duplexes formed by these hybrid metal complex-DNA conjugates showed around 4-5 degrees C stabilization with respect to the unmodified duplexes. Moreover hybridization of two complementary bis-ODN tethered metal complexes at different ratios in solution gave rise to a self-assembled nanometric linear network, which was characterized by non-denaturing gel electrophoresis and TEM studies. Thus, our simple synthetic strategy would contribute to develop multimetallic 2D-DNA nanoarrays where we can place different metal complexes at regular intervals along the ODN stretches.

  10. 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.

  11. [Studies on the interaction of the metal complex of hydrazide of podophyllic acid with DNA].

    PubMed

    Wang, Ping-Hong; Zhang, Qi; Wang, Liu-Fang; Song, Yu-Min; Qu, Jian-Qiang; Liu, Ying-Qian

    2006-05-01

    The interaction between the metal complex of hydrazide of podophyllic acid and calf thymus (CT) DNA was studied by using absorption spectra, fluorescence spectra and DNA heat denaturation. It was found that the intensity of the maximal absorption peaks from absorption spectra is weakened in the presence of the metal complex of hydrazide of podophyllic acid compared with that in the absence of the metal complex. All the experimental results show that the intercalation mode was proved to exist between HDPP-Ni complexes and CT DNA.

  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. Mesencephalic complex I deficiency does not correlate with parkinsonism in mitochondrial DNA maintenance disorders.

    PubMed

    Palin, Eino J H; Paetau, Anders; Suomalainen, Anu

    2013-08-01

    Genetic evidence from recessively inherited Parkinson's disease has indicated a clear causative role for mitochondrial dysfunction in Parkinson's disease. This role has long been discussed based on findings that toxic inhibition of mitochondrial respiratory complex I caused parkinsonism and that tissues of patients with Parkinson's disease show complex I deficiency. Disorders of mitochondrial DNA maintenance are a common cause of inherited neurodegenerative disorders, and lead to mitochondrial DNA deletions or depletion and respiratory chain defect, including complex I deficiency. However, parkinsonism associates typically with defects of catalytic domain of mitochondrial DNA polymerase gamma. Surprisingly, however, not all mutations affecting DNA polymerase gamma manifest as parkinsonism, but, for example, spacer region mutations lead to spinocerebellar ataxia and/or severe epilepsy. Furthermore, defective Twinkle helicase, a close functional companion of DNA polymerase gamma in mitochondrial DNA replication, results in infantile-onset spinocerebellar ataxia, epilepsy or adult-onset mitochondrial myopathy, but not typically parkinsonism. Here we sought for clues for this specificity in the neurological manifestations of mitochondrial DNA maintenance disorders by studying mesencephalic neuropathology of patients with DNA polymerase gamma or Twinkle defects, with or without parkinsonism. We show here that all patients with mitochondrial DNA maintenance disorders had neuronopathy in substantia nigra, most severe in DNA polymerase gamma-associated parkinsonism. The oculomotor nucleus was also affected, but less severely. In substantia nigra, all patients had a considerable decrease of respiratory chain complex I, but other respiratory chain enzymes were not affected. Complex I deficiency did not correlate with parkinsonism, age, affected gene or inheritance. We conclude that the cell number in substantia nigra correlated well with parkinsonism in DNA polymerase gamma

  14. Hh signaling inhibitors from Vitex negundo; naturally occurring inhibitors of the GLI1-DNA complex.

    PubMed

    Arai, Midori A; Fujimatsu, Teruhisa; Uchida, Kyoko; Sadhu, Samir K; Ahmed, Firoj; Ishibashi, Masami

    2013-05-01

    The hedgehog (Hh) signaling pathway has crucial roles in embryonic development, cell maintenance and proliferation, and is also known to contribute to cancer cell growth. New naturally occurring Hh inhibitors (1, 7 and 9) were isolated from Vitex negundo using our previously constructed cell-based assay. Bioactivity guided isolation provided 9 natural compounds including a new diterpene, nishindanol (9). Compounds 7 and 9 showed cytotoxicity against cancer cell lines in which Hh signaling was aberrantly activated. Vitetrifolin D (7; GLI1 transcriptional inhibition IC50 = 20.2 μM) showed inhibition of Hh related protein (PTCH and BCL2) production. Interestingly, the constructed electrophoresis mobility shift assay revealed that vitetrifolin D (7) disrupted GLI1 binding on its DNA binding domain. epi-Sclareol (8; inactive), possessing a similar structure to 7, did not show inhibition of GLI1–DNA complex formation. This is the first example of naturally occurring inhibitors of GLI1–DNA complex formation.

  15. 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

  16. Single Molecule Measurements of Protelomerase TelK-DNA Complexes

    NASA Astrophysics Data System (ADS)

    Landry, Markita; Khafizov, Rustem; Huang, Wai Mun; Chemla, Yann

    2008-10-01

    Protein-DNA interactions lie at the heart of many essential cellular processes such as replication, recombination, and repair. Recent advances in optical ``tweezers'' have made it possible to resolve motions on the scale of a single base pair of DNA, 3.4å. High-resolution optical traps have the potential to reveal these interactions at their fundamental length scales and should reveal how certain proteins bind to DNA or recognize target sequences. Telomerases are enzymes that have been actively studied in various organisms because of their fundamental involvement with both cancer and aging^1. Protelomerase TelK is an enzyme responsible for forming closed DNA hairpin ends in linear DNA. TelK is not an ATP dependant enzyme, which is surprising given the degree of DNA distortion accomplished by the enzyme, and the large energy barrier intrinsic in DNA hairpin formation. Therefore, our focus is on TelK mutants lacking their c-terminal domain, and TelK YF mutants lacking their tyrosine active site amino acid. Preliminary data have shown remarkable differences in protein binding and unbinding forces caused by the removal of a single oxygen atom from a 73 kDa protein. Further measurements using high-resolution optical tweezers should provide fundamental insights into the nature and importance of the electrostatic interactions between TelK and its DNA substrate. 1. Shay, J. et al. Rad. Res. 155, 188 (2001) [1] Huang, W. et al. Mol. Cell. 27, 901 (2007).

  17. 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.

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

    PubMed Central

    Witosch, Justine; Wolf, Eva; Mizuno, Naoko

    2014-01-01

    The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork. PMID:25348395

  19. DNA Damage: From Chronic Inflammation to Age-Related Deterioration

    PubMed Central

    Ioannidou, Anna; Goulielmaki, Evi; Garinis, George A.

    2016-01-01

    To lessen the “wear and tear” of existence, cells have evolved mechanisms that continuously sense DNA lesions, repair DNA damage and restore the compromised genome back to its native form. Besides genome maintenance pathways, multicellular organisms may also employ adaptive and innate immune mechanisms to guard themselves against bacteria or viruses. Recent evidence points to reciprocal interactions between DNA repair, DNA damage responses and aspects of immunity; both self-maintenance and defense responses share a battery of common players and signaling pathways aimed at safeguarding our bodily functions over time. In the short-term, this functional interplay would allow injured cells to restore damaged DNA templates or communicate their compromised state to the microenvironment. In the long-term, however, it may result in the (premature) onset of age-related degeneration, including cancer. Here, we discuss the beneficial and unrewarding outcomes of DNA damage-driven inflammation in the context of tissue-specific pathology and disease progression. PMID:27826317

  20. Preparation, characterization, and DNA binding studies of water-soluble quercetin--molybdenum(VI) complex.

    PubMed

    Ahmadi, Seyed Mojtaba; Dehghan, Gholamreza; Hosseinpourfeizi, Muhammad Ali; Dolatabadi, Jafar Ezzati Nazhad; Kashanian, Soheila

    2011-07-01

    DNA binding studies of flavonoids are needed to understand the reaction mechanism and improve drugs that target DNA. Quercetin (Q) is one of the most common flavonoids that can chelate metal ions and interact with double-stranded DNA. In the present work, UV absorption spectrophotometry, viscosimetry, circular dichroism, and fluorescence spectroscopic techniques were employed to study the interaction of water-soluble quercetin--molybdenum(VI) complex [Q-Mo(VI)] with calf thymus DNA. The binding constants (K(b)) for the complex with DNA were estimated to be 2.9 × 10(3) through spectroscopic titrations. Upon addition of the complex, significant decreases were observed in the viscosity of calf thymus DNA. Circular dichroic spectra indicated that there are certain detectable conformational changes in the DNA double helix when complex was added. Further, competitive methylene blue binding studies with fluorescence spectroscopy have shown that the complex can bind to DNA through nonintercalative mode. The experimental results suggest that Q-Mo(VI) binds to DNA via an outside binding mode.

  1. 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

  2. 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

  3. Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer

    PubMed Central

    Lin, Jason L. J.; Wu, Chyuan-Chuan; Yang, Wei-Zen; Yuan, Hanna S.

    2016-01-01

    Endonuclease G (EndoG) is an evolutionarily conserved mitochondrial protein in eukaryotes that digests nucleus chromosomal DNA during apoptosis and paternal mitochondrial DNA during embryogenesis. Under oxidative stress, homodimeric EndoG becomes oxidized and converts to monomers with diminished nuclease activity. However, it remains unclear why EndoG has to function as a homodimer in DNA degradation. Here, we report the crystal structure of the Caenorhabditis elegans EndoG homologue, CPS-6, in complex with single-stranded DNA at a resolution of 2.3 Å. Two separate DNA strands are bound at the ββα-metal motifs in the homodimer with their nucleobases pointing away from the enzyme, explaining why CPS-6 degrades DNA without sequence specificity. Two obligatory monomeric CPS-6 mutants (P207E and K131D/F132N) were constructed, and they degrade DNA with diminished activity due to poorer DNA-binding affinity as compared to wild-type CPS-6. Moreover, the P207E mutant exhibits predominantly 3′-to-5′ exonuclease activity, indicating a possible endonuclease to exonuclease activity change. Thus, the dimer conformation of CPS-6 is essential for maintaining its optimal DNA-binding and endonuclease activity. Compared to other non-specific endonucleases, which are usually monomeric enzymes, EndoG is a unique dimeric endonuclease, whose activity hence can be modulated by oxidation to induce conformational changes. PMID:27738134

  4. Insights into protein -- DNA interactions, stability and allosteric communications: A computational study of MutS-DNA recognition complexes

    NASA Astrophysics Data System (ADS)

    Negureanu, Lacramioara; Salsbury, Freddie

    2012-02-01

    DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. The most abundant MMR mismatch-binding factor in eukaryotes, MutS, recognizes and initiates the repair of base-base mismatches and small insertion/deletions. We performed molecular dynamics simulations on mismatched and damaged MutS-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutS proteins recognize the mismatched and platinum cross-linked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutS binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. A large number of known MMR cancer causing mutations among the residues were found.

  5. 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 ...

  6. High Density Data Storage Systems by DNA Complexes and Nano-Particles from DNA Hybrid Materials

    DTIC Science & Technology

    2006-12-20

    Research 2 In-situ Intercalation of Phtharocyanine dye with DNA 11 Intercalation of DNA by various photochromic dyes into... photochromic dyes which are intercalated into DNA double helical structures. It was reported in 2005 that photochromic dyes such as spiropyran derivatives...enhance photochromic responces in DNA wave guide which had a grating structure by using refractive resonances through the wave guide. The design is

  7. Evaluation of DNA Binding Drugs as Inhibitors of ESX, and ETS Domain Transcription Factor Associated With Breast Cancer: Effects of ESX/DNA Complex Disruption

    DTIC Science & Technology

    2000-08-01

    the major groove simultaneously narrows. This distortion of the helix may contribute to the agent’s inhibition of TF/ DNA complex formation. Another...class of DNA binding agents is the intercalators, which slide the chromophore between the base pairs of DNA and lengthen the helix . This can adversely...7). This distortion of the helix may contribute to the agent’s inhibition of TF/ DNA complex formation (8-10). Another class of DNA binding agents is

  8. Synthesis, characterization, DNA and HSA binding studies of isomeric Pd (II) antitumor complexes using spectrophotometry techniques.

    PubMed

    Zareian-Jahromi, Sareh; Mansouri-Torshizi, Hassan

    2017-05-07

    Two new Palladium(II) isomeric complexes, [Pd (Gly)(Leu)](I) and [Pd (Gly)(Ile)](II), where Gly is glycine, and Leu and Ile are isomeric amino acids (leucine and isoleucine), have been synthesized and characterized by elemental analysis, molar conductivity measurements, FT-IR, (1)H NMR, and UV-Vis. The complexes have been tested for their In vitro cytotoxicity against cancer cell line K562 and their binding properties to calf thymus DNA (CT-DNA) and human serum albumin (HSA) have also been investigated by multispectroscopic techniques. Interactions of these complexes with CT-DNA were monitored using gel electrophoresis. The energy transfer from HSA to these complexes and the binding distance between HSA and the complexes (r) were calculated. The results obtained from these studies indicated that at very low concentrations, both complexes effectively interact with CT-DNA and HSA. Fluorescence studies revealed that the complexes strongly quench DNA bound ethidium bromide as well as the intrinsic fluorescence of HSA through the static quenching procedures. Binding constant (Kb), apparent biomolecular quenching constant (kq), and number of binding sites (n) for CT-DNA and HSA were calculated using Stern-Volmer equation. The calculated thermodynamic parameters indicated that the hydrogen binding and vander Waals forces might play a major role in the interaction of these complexes with HSA and DNA. Thus, we propose that the complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of I > II with DNA- and II > I with HSA-binding.

  9. Quinolone-DNA interaction: sequence-dependent binding to single-stranded DNA reflects the interaction within the gyrase-DNA complex.

    PubMed

    Noble, Christian G; Barnard, Faye M; Maxwell, Anthony

    2003-03-01

    We have investigated the interaction of quinolones with DNA by a number of methods to establish whether a particular binding mode correlates with quinolone potency. The specificities of the quinolone-mediated DNA cleavage reaction of DNA gyrase were compared for a number of quinolones. Two patterns that depended on the potency of the quinolone were identified. Binding to plasmid DNA was examined by measuring the unwinding of pBR322 by quinolones; no correlation with quinolone potency was observed. Quinolone binding to short DNA oligonucleotides was measured by surface plasmon resonance. The quinolones bound to both single- and double-stranded oligonucleotides in an Mg(2+)-dependent manner. Quinolones bound to single-stranded DNA with a higher affinity, and the binding exhibited sequence dependence; binding to double-stranded DNA was sequence independent. The variations in binding in the presence of metal ions showed that Mg(2+) promoted tighter, more specific binding to single-stranded DNA than softer metal ions (Mn(2+) and Cd(2+)). Single-stranded DNA binding by quinolones correlated with the in vitro quinolone potency, indicating that this mode of interaction may reflect the interaction of the quinolone with DNA in the context of the gyrase-DNA complex.

  10. [Studies on the interaction of metalic complex and DNA by the spectral methods].

    PubMed

    Lin, Qiu-Yue; Hu, Rui-Ding; Zheng, Xiao-Hua

    2004-08-01

    The interaction of [Cu(A)2]2+ (A = phen, bpy, en) complex ion and ct-DNA was investigated by using absorption spectra and fluorescence spectra. Based on the measurement of A260 in the different pH mediums, the alkaline denaturation curves of DNA in the absence and in the presence of [Cu(phen)2]2+ complex ion were obtained. The result showed that the alkaline denaturation for [Cu(phen)2]2+ complex ion occured at a higher pH than that for DNA alone. The absorption of EB-DNA system in the presence of [Cu(phen)2]2+ showed that [Cu(phen)2]2+ could partly sustain the place of EB. The result of fluorescence spectra suggested that the [Cu(phen)2]2+ complex ion could make the fluorescence intensity of EB-DNA system to decreased sharply. The effect of [Fe(CN)6]4- on the emission of [Cu(phen)2]2+ and [Cu(phen)2](2+) -DNA system was studied. All the experimental results indicated that the intercalative binding mode was the major mode for the interaction of [Cu(phen)2]2+ complex ion and DNA, and the size of ligands can effect the binding of complexes to DNA.

  11. MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA

    PubMed Central

    Lee, Ka Cheong; Padget, Kay; Curtis, Hannah; Cowell, Ian G.; Moiani, Davide; Sondka, Zbyslaw; Morris, Nicholas J.; Jackson, Graham H.; Cockell, Simon J.; Tainer, John A.; Austin, Caroline A.

    2012-01-01

    Summary Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5′-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay. Recombinant MRE11 from 2 sources removed topoisomerase IIα from genomic DNA in vitro, as did MRE11 immunoprecipitates isolated from A-TLD or K562 cells. Basal topoisomerase II complex levels were very high in A-TLD cells lacking full-length wild type MRE11, suggesting that MRE11 facilitates the processing of topoisomerase complexes that arise as part of normal cellular metabolism. In K562 cells inhibition of MRE11, PARP or replication increased topoisomerase IIα and β complex levels formed in the absence of an anti-topoisomerase II drug. PMID:23213480

  12. 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.

  13. 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.

  14. Structure of the FANCI-FANCD2 complex: insights into the Fanconi anemia DNA repair pathway.

    PubMed

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

    2011-07-15

    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 ~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.

  15. DNA interaction of europium(III) complex containing 2,2'-bipyridine and its antimicrobial activity.

    PubMed

    Jahani, Shohreh; Khorasani-Motlagh, Mozhgan; Noroozifar, Meissam

    2016-01-01

    The interaction of native fish salmon DNA (FS-DNA) with [Eu(bpy)3Cl2(H2O)]Cl, where bpy is 2,2'-bipyridine, is studied at physiological pH in Tris-HCl buffer by spectroscopic methods, viscometric techniques as well as circular dichroism (CD). These experiments reveal that Eu(III) complex has interaction with FS-DNA. Moreover, binding constant and binding site size have been determined. The value of Kb has been defined 2.46 ± .02 × 10(5) M(-1). The thermodynamic parameters are calculated by Van't Hoff equation, the results show that the interaction of the complex with FS-DNA is an entropically driven phenomenon. CD spectroscopy followed by viscosity as well as fluorescence and UV--Vis measurements indicate that the complex interacts with FS-DNA via groove binding mode. Also, the synthesized Eu(III) complex has been screened for antimicrobial activities.

  16. Quantitative determination of anti-dsDNA antibodies and antibody/dsDNA stoichiometries in prepared, soluble complement-fixing antibody/dsDNA immune complexes.

    PubMed

    Taylor, R P; Horgan, C

    1984-10-01

    We have investigated quantitatively the complement-mediated binding of prepared, soluble 125I-7S IgG antibody/3H-dsDNA immune complexes to human red blood cells (RBCs). We have performed these studies by using a detailed modification of the RBC-CF assay [Pedersen et al., J. Immun. Meth. 38, 269-280 (1980)] which now allows for the simultaneous measurement of both 3H-DNA and 125I-binding to the cells. Our results indicate that, in the case of three SLE patients, their anti-dsDNA antibody titers are sufficiently high that a small fraction of their 125I-7S IgG antibodies (ca 0.1-0.2%) can be identified as specifically anti-dsDNA. We have also used an indirect method (with 125I-labelled rabbit anti-human IgG) for the determination of IgG anti-dsDNA antibodies in complement-fixing antibody/dsDNA immune complexes that bind to RBCs, and the results of these measurements are in reasonable agreement with the direct binding experiments. These studies have also allowed us to estimate the antibody/DNA stoichiometries in complement-fixing immune complexes. The results of these experiments may provide a useful standard for the analysis of monoclonal anti-dsDNA antibodies.

  17. DNA-DNA relatedness and phylogenetic positions of Slackia exigua, Slackia heliotrinireducens, Eggerthella lenta, and other related bacteria.

    PubMed

    Nakazawa, F; Hoshino, E

    2004-10-01

    Recently, two asaccharolytic Eubacterium species, Eubacterium exiguum and Eubacterium lentum, and Peptostreptococcus heliotrinreducens have been reclassified as Slackia exigua, Eggerthella lenta and Slackia heliotrinireducens in the novel genera on the basis of 16S rDNA sequence analysis. But DNA-DNA relatedness among these species and other related bacteria have not been reported yet. DNA-DNA relatedness is the standard arbiter and the recommended method for the designation and evaluation of new species, particularly closely related ones. In the present study, DNA-DNA hybridization studies were performed on S. exigua, S. heliotrinireducens and E. lenta together with the other bacterial species in the related genera. The phylogenetic relationships of these species were also investigated by comparison analysis of 16S rDNA sequence data. In the DNA-DNA hybridization studies, S. exigua showed a DNA homology level of 33% to S. heliotrinireducens and 11% to E. lenta. DNA-DNA homology between S. heliotrinireducens and E. lenta was 10%. But these three species showed very low homology (less than 5%) to the related asaccharolytic species such as Eubacterium and Mogibacterium. In conclusion, the DNA-DNA relatedness data together with the evolutionary data in the present paper further support the reclassification of Eubacterium exiguum, Peptostreptococcus heliotrinreducens and Eubacterium lentum as Slackia exigua, Slackia heliotrinireducens and Eggerthella lenta, respectively.

  18. An AP Endonuclease 1–DNA Polymerase β Complex: Theoretical Prediction of Interacting Surfaces

    PubMed Central

    Abyzov, Alexej; Uzun, Alper; Strauss, Phyllis R.; Ilyin, Valentin A.

    2008-01-01

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

  19. How charge fluctuations modulate complexation of DNA around a histone cylinder

    NASA Astrophysics Data System (ADS)

    Kim, Won Kyu; Sung, Wokyung

    2013-10-01

    It is an enigma that in a nucleosome complex, a single DNA winds around a histone protein core about 1.6-1.8 times. We find that the optimal winding can be given by the charge density fluctuations on the DNA contour due to multivalent counterions binding and unbinding in a physiological ionic background. From a model free energy of the DNA wound on the histone cylinder, where the charge fluctuations induce the intra-DNA attraction, we establish the conditions and a phase diagram for stability of the complex as a function of the salt and divalent cations concentrations.

  20. Vanadium(IV) and copper(II) complexes of salicylaldimines and aromatic heterocycles: Cytotoxicity, DNA binding and DNA cleavage properties.

    PubMed

    Correia, Isabel; Roy, Somnath; Matos, Cristina P; Borovic, Sladjana; Butenko, Nataliya; Cavaco, Isabel; Marques, Fernanda; Lorenzo, Julia; Rodríguez, Alejandra; Moreno, Virtudes; Pessoa, João Costa

    2015-06-01

    Five copper(II) complexes, [Cu(sal-Gly)(bipy)](1), [Cu(sal-Gly)(phen)] (2), [Cu(sal-l-Ala)(phen)] (3), [Cu(sal-D-Ala)(phen)] (4), [Cu(sal-l-Phe)(phen)] (5) and five oxidovanadium(IV) complexes, [V(IV)O(sal-Gly)(bipy)] (6), [V(IV)O(sal-Gly)(phen)] (7), [V(IV)O(sal-l-Phe)(H2O)] (8), [V(IV)O(sal-l-Phe)(bipy)] (9), [V(IV)O(sal-l-Phe)(phen)] (10) (sal=salicylaldehyde, bipy=2,2'-bipyridine, phen=1,10-phenanthroline) were synthesized and characterized, and their interaction with DNA was evaluated by different techniques: gel electrophoresis, fluorescence, UV-visible and circular dichroism spectroscopy. The complexes interact with calf-thymus DNA and efficiently cleave plasmid DNA in the absence (only 2 and 5) and/or presence of additives. The cleavage ability is concentration-dependent as well as metal and ligand-dependent. Moreover, DNA binding experiments show that the phen-containing Cu(II) and V(IV)O compounds display stronger DNA interaction ability than the corresponding bipy analogues. The complexes present cytotoxic activity against human ovarian (A2780) and breast (MCF7) carcinoma cells. Cell-growth inhibition (IC50) of compounds 1, 2 and 5 in human promyelocytic leukemia (HL60) and human cervical cancer (HeLa) cells were also determined. The copper complexes show much higher cytotoxic activity than the corresponding vanadium complexes and the reference drug cisplatin (except for the sal-Gly complexes); namely, the phenanthroline copper complexes 2-5 are ca. 10-fold more cytotoxic than cisplatin and more cytotoxic than their bipyridine analogues.

  1. 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

  2. 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

  3. 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

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

    PubMed

    Marin, Sergio; Merkoçi, Arben

    2009-02-04

    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 microl 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

    2015-01-08

    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.

  9. 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.

  10. First-order Judd-Ofelt optical characterization of DNA-Ln3+ complexes

    NASA Astrophysics Data System (ADS)

    Paulson, Bjorn; Sauer, Gregor; Cheon, Seungwuk; Dugasani, Sreekantha Reddy; Oh, Kyunghwan

    2016-09-01

    Complexes formed of deoxyribose nucleic acid (DNA) and trivalent lanthanide ions (Ln3+) promise a combination of high optical gain and low optical loss in an organic polymer host matrix. However, there has been some dispute about the binding mechanism between the DNA helix and the positively-charged lanthanide ions. Here we introduce an attempt to resolve the mechanism for binding through Judd-Ofelt analysis on DNA-Eu3+, DNA-Tb3+, and DNA-Sm3+ to first order. From initial Judd-Ofelt parameters extrapolations can be made to the line strengths, Einstein coefficients, and fluorescence lifetimes.

  11. Cyclometalated Iridium(III) Imidazole Phenanthroline Complexes as Luminescent and Electrochemiluminescent G-Quadruplex DNA Binders.

    PubMed

    Castor, Katherine J; Metera, Kimberly L; Tefashe, Ushula M; Serpell, Christopher J; Mauzeroll, Janine; Sleiman, Hanadi F

    2015-07-20

    Six cyclometalated iridium(III) phenanthroimidazole complexes with different modifications to the imidazole phenanthroline ligand exhibit enhanced luminescence when bound to guanine (G-) quadruplex DNA sequences. The complexes bind with low micromolar affinity to human telomeric and c-myc sequences in a 1:1 complex:quadruplex stoichiometry. Due to the luminescence enhancement upon binding to G-quadruplex DNA, the complexes can be used as selective quadruplex indicators. In addition, the electrogenerated chemiluminescence of all complexes increases in the presence of specific G-quadruplex sequences, demonstrating potential for the development of an ECL-based G-quadruplex assay.

  12. 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

  13. 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.

  14. Structure of the CAP-DNA complex at 2.5 angstroms resolution: a complete picture of the protein-DNA interface.

    PubMed

    Parkinson, G; Wilson, C; Gunasekera, A; Ebright, Y W; Ebright, R H; Ebright, R E; Berman, H M

    1996-07-19

    The crystallographic structure of the CAP-DNA complex at 3.0 A resolution has been reported previously. For technical reasons, the reported structure had been determined using a gapped DNA molecule lacking two phosphates important for CAP-DNA interaction. In this work, we report the crystallographic structure of the CAP-DNA complex at 2.5 A resolution using a DNA molecule having all phosphates important for CAP-DNA interaction. The present resolution permits unambiguous identification of amino acid-base and amino acid-phosphate hydrogen bonded contacts in the CAP-DNA complex. In addition, the present resolution permits accurate definition of the kinked DNA conformation in the CAP-DNA complex.

  15. Eukaryotic Mismatch Repair in Relation to DNA Replication.

    PubMed

    Kunkel, Thomas A; Erie, Dorothy A

    2015-01-01

    Three processes act in series to accurately replicate the eukaryotic nuclear genome. The major replicative DNA polymerases strongly prevent mismatch formation, occasional mismatches that do form are proofread during replication, and rare mismatches that escape proofreading are corrected by mismatch repair (MMR). This review focuses on MMR in light of increasing knowledge about nuclear DNA replication enzymology and the rate and specificity with which mismatches are generated during leading- and lagging-strand replication. We consider differences in MMR efficiency in relation to mismatch recognition, signaling to direct MMR to the nascent strand, mismatch removal, and the timing of MMR. These studies are refining our understanding of relationships between generating and repairing replication errors to achieve accurate replication of both DNA strands of the nuclear genome.

  16. DNA codes and information: formal structures and relational causes.

    PubMed

    Sternberg, Richard V

    2008-09-01

    Recently the terms "codes" and "information" as used in the context of molecular biology have been the subject of much discussion. Here I propose that a variety of structural realism can assist us in rethinking the concepts of DNA codes and information apart from semantic criteria. Using the genetic code as a theoretical backdrop, a necessary distinction is made between codes qua symbolic representations and information qua structure that accords with data. Structural attractors are also shown to be entailed by the mapping relation that any DNA code is a part of (as the domain). In this framework, these attractors are higher-order informational structures that obviate any "DNA-centric" reductionism. In addition to the implications that are discussed, this approach validates the array of coding systems now recognized in molecular biology.

  17. Structure, antimicrobial activity, DNA- and albumin-binding of manganese(II) complexes with the quinolone antimicrobial agents oxolinic acid and enrofloxacin.

    PubMed

    Zampakou, Marianthi; Akrivou, Melpomeni; Andreadou, Eleni G; Raptopoulou, Catherine P; Psycharis, Vassilis; Pantazaki, Anastasia A; Psomas, George

    2013-04-01

    The reaction of MnCl2 with the quinolone antibacterial drug oxolinic acid (Hoxo) results to the formation of [KMn(oxo)3(MeOH)3]. Interaction of MnCl2 with the quinolone Hoxo or enrofloxacin (Herx) and the N,N'-donor heterocyclic ligand 1,10-phenanthroline (phen) results in the formation of metal complexes with the general formula [Mn(quinolonato)2(phen)]. The crystal structures of [KMn(oxo)3(MeOH)3] and [Mn(erx)2(phen)], exhibiting a 1D polymeric and a mononuclear structure, respectively, have been determined by X-ray crystallography. In these complexes, the deprotonated bidentate quinolonato ligands are coordinated to manganese(II) ion through the pyridone oxygen and a carboxylato oxygen. All complexes can act as potential antibacterial agents with [Mn(erx)2(phen)] exhibiting the most pronounced antimicrobial activity against five different microorganisms. Interaction of the complexes with calf-thymus DNA (CT DNA), studied by UV spectroscopy, has shown that they bind to CT DNA. Competitive study with ethidium bromide (EB) has shown that all complexes can displace the DNA-bound EB indicating their binding to DNA in strong competition with EB. Intercalative binding mode is proposed for the interaction of the complexes with CT DNA and has also been verified by DNA solution viscosity measurements and cyclic voltammetry. DNA electrophoretic mobility experiments suggest that [Mn(erx)2(phen)] binds strongly to supercoiled pDNA and to linearized pDNA possibly by an intercalative manner provoking double-stranded cleavage reflecting in a nuclease-like activity. The complexes exhibit good binding propensity to human or bovine serum albumin protein showing relatively high binding constant values. The binding constants of the complexes towards CT DNA and albumins have been compared to their corresponding zinc(II) and nickel(II) complexes.

  18. The NF90/NF45 complex participates in DNA break repair via nonhomologous end joining.

    PubMed

    Shamanna, Raghavendra A; Hoque, Mainul; Lewis-Antes, Anita; Azzam, Edouard I; Lagunoff, David; Pe'ery, Tsafi; Mathews, Michael B

    2011-12-01

    Nuclear factor 90 (NF90), an RNA-binding protein implicated in the regulation of gene expression, exists as a heterodimeric complex with NF45. We previously reported that depletion of the NF90/NF45 complex results in a multinucleated phenotype. Time-lapse microscopy revealed that binucleated cells arise by incomplete abscission of progeny cells followed by fusion. Multinucleate cells arose through aberrant division of binucleated cells and displayed abnormal metaphase plates and anaphase chromatin bridges suggestive of DNA repair defects. NF90 and NF45 are known to interact with the DNA-dependent protein kinase (DNA-PK), which is involved in telomere maintenance and DNA repair by nonhomologous end joining (NHEJ). We hypothesized that NF90 modulates the activity of DNA-PK. In an in vitro NHEJ assay system, DNA end joining was reduced by NF90/NF45 immunodepletion or by RNA digestion to an extent similar to that for catalytic subunit DNA-PKcs immunodepletion. In vivo, NF90/NF45-depleted cells displayed increased γ-histone 2A.X foci, indicative of an accumulation of double-strand DNA breaks (DSBs), and increased sensitivity to ionizing radiation consistent with decreased DSB repair. Further, NF90/NF45 knockdown reduced end-joining activity in vivo. These results identify the NF90/NF45 complex as a regulator of DNA damage repair mediated by DNA-PK and suggest that structured RNA may modulate this process.

  19. 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.

  20. Direct measurement of interaction forces between a platinum dichloride complex and DNA molecules.

    PubMed

    Muramatsu, Hiroshi; Shimada, Shogo; Okada, Tomoko

    2017-06-29

    The interaction forces between a platinum dichloride complex and DNA molecules have been studied using atomic force microscopy (AFM). The platinum dichloride complex, di-dimethylsulfoxide-dichloroplatinum (II) (Pt(DMSO)2Cl2), was immobilized on an AFM probe by coordinating the platinum to two amino groups to form a complex similar to Pt(en)Cl2, which is structurally similar to cisplatin. The retraction forces were measured between the platinum complex and DNA molecules immobilized on mica plates using force curve measurements. The histogram of the retraction force for λ-DNA showed several peaks; the unit retraction force was estimated to be 130 pN for a pulling rate of 60 nm/s. The retraction forces were also measured separately for four single-base DNA oligomers (adenine, guanine, thymine, and cytosine). Retraction forces were frequently observed in the force curves for the DNA oligomers of guanine and adenine. For the guanine DNA oligomer, the most frequent retraction force was slightly lower than but very similar to the retraction force for λ-DNA. A higher retraction force was obtained for the adenine DNA oligomer than for the guanine oligomer. This result is consistent with a higher retraction activation energy of adenine with the Pt complex being than that of guanine because the kinetic rate constant for retraction correlates to exp(FΔx - ΔE) where ΔE is an activation energy, F is an applied force, and Δx is a displacement of distance.

  1. Details of ssDNA annealing revealed by an HSV-1 ICP8–ssDNA binary complex

    PubMed Central

    Tolun, Gökhan; Makhov, Alexander M.; Ludtke, Steven J.; Griffith, Jack D.

    2013-01-01

    Infected cell protein 8 (ICP8) from herpes simplex virus 1 was first identified as a single-strand (ss) DNA-binding protein. It is essential for, and abundant during, viral replication. Studies in vitro have shown that ICP8 stimulates model replication reactions, catalyzes annealing of complementary ssDNAs and, in combination with UL12 exonuclease, will catalyze ssDNA annealing homologous recombination. DNA annealing and strand transfer occurs within large oligomeric filaments of ssDNA-bound ICP8. We present the first 3D reconstruction of a novel ICP8–ssDNA complex, which seems to be the basic unit of the DNA annealing machine. The reconstructed volume consists of two nonameric rings containing ssDNA stacked on top of each other, corresponding to a molecular weight of 2.3 MDa. Fitting of the ICP8 crystal structure suggests a mechanism for the annealing reaction catalyzed by ICP8, which is most likely a general mechanism for protein-driven DNA annealing. PMID:23605044

  2. Comparative Analysis of Satellite DNA in the Drosophila melanogaster Species Complex.

    PubMed

    Jagannathan, Madhav; Warsinger-Pepe, Natalie; Watase, George J; Yamashita, Yukiko M

    2017-02-09

    Satellite DNAs are highly repetitive sequences that account for the majority of constitutive heterochromatin in many eukaryotic genomes. It is widely recognized that sequences and locations of satellite DNAs are highly divergent even in closely related species, contributing to the hypothesis that satellite DNA differences may underlie speciation. However, due to its repetitive nature, the mapping of satellite DNAs has been mostly left out of recent genomics analyses, hampering the use of molecular genetics techniques to better understand their role in speciation and evolution. Satellite DNAs are most extensively and comprehensively mapped in Drosophila melanogaster, a species that is also an excellent model system with which to study speciation. Yet the lack of comprehensive knowledge regarding satellite DNA identity and location in its sibling species (D. simulans, D. mauritiana, and D. sechellia) has prevented the full utilization of D. melanogaster in studying speciation. To overcome this problem, we initiated the mapping of satellite DNAs on the genomes of the D. melanogaster species complex (D. melanogaster, D. simulans, D. mauritiana, and D. sechellia) using multi-color fluorescent in situ hybridization (FISH) probes. Our study confirms a striking divergence of satellite DNAs in the D. melanogaster species complex, even among the closely related species of the D. simulans clade (D. simulans, D. mauritiana, and D. sechellia), and suggests the presence of unidentified satellite sequences in these species.

  3. Comparative Analysis of Satellite DNA in the Drosophila melanogaster Species Complex

    PubMed Central

    Jagannathan, Madhav; Warsinger-Pepe, Natalie; Watase, George J.; Yamashita, Yukiko M.

    2016-01-01

    Satellite DNAs are highly repetitive sequences that account for the majority of constitutive heterochromatin in many eukaryotic genomes. It is widely recognized that sequences and locations of satellite DNAs are highly divergent even in closely related species, contributing to the hypothesis that satellite DNA differences may underlie speciation. However, due to its repetitive nature, the mapping of satellite DNAs has been mostly left out of recent genomics analyses, hampering the use of molecular genetics techniques to better understand their role in speciation and evolution. Satellite DNAs are most extensively and comprehensively mapped in Drosophila melanogaster, a species that is also an excellent model system with which to study speciation. Yet the lack of comprehensive knowledge regarding satellite DNA identity and location in its sibling species (D. simulans, D. mauritiana, and D. sechellia) has prevented the full utilization of D. melanogaster in studying speciation. To overcome this problem, we initiated the mapping of satellite DNAs on the genomes of the D. melanogaster species complex (D. melanogaster, D. simulans, D. mauritiana, and D. sechellia) using multi-color fluorescent in situ hybridization (FISH) probes. Our study confirms a striking divergence of satellite DNAs in the D. melanogaster species complex, even among the closely related species of the D. simulans clade (D. simulans, D. mauritiana, and D. sechellia), and suggests the presence of unidentified satellite sequences in these species. PMID:28007840

  4. 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.

  5. Structure, antimicrobial activity and DNA-binding properties of the cobalt(II)-sparfloxacin complex.

    PubMed

    Efthimiadou, Eleni K; Karaliota, Alexandra; Psomas, George

    2008-07-15

    The neutral mononuclear cobalt(II) complex with sparfloxacin has been prepared and characterized with physicochemical, spectroscopic and electrochemical techniques, and molecular mechanics calculations. The interaction of the complex with calf-thymus DNA has been investigated with UV spectroscopy, cyclic voltammetry, and competitive studies with ethidium bromide. The antimicrobial activity of the complex has been tested against three microorganisms.

  6. Eukaryotic DNA replication: Orchestrated action of multi-subunit protein complexes.

    PubMed

    Kang, Sukhyun; Kang, Mi-Sun; Ryu, Eunjin; Myung, Kyungjae

    2017-05-01

    Genome duplication is an essential process to preserve genetic information between generations. The eukaryotic cell cycle is composed of functionally distinct phases: G1, S, G2, and M. One of the key replicative proteins that participate at every stage of DNA replication is the Mcm2-7 complex, a replicative helicase. In the G1 phase, inactive Mcm2-7 complexes are loaded on the replication origins by replication-initiator proteins, ORC and Cdc6. Two kinases, S-CDK and DDK, convert the inactive origin-loaded Mcm2-7 complex to an active helicase, the CMG complex in the S phase. The activated CMG complex begins DNA unwinding and recruits enzymes essential for DNA synthesis to assemble a replisome at the replication fork. After completion of DNA synthesis, the inactive CMG complex on the replicated DNA is removed from chromatin to terminate DNA replication. In this review, we will discuss the structure, function, and regulation of the molecular machines involved in each step of DNA replication. Copyright © 2017. Published by Elsevier B.V.

  7. Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

    PubMed Central

    Su, Mei-Tzu; Liu, I-Hua; Wu, Chia-Wei; Chang, Shu-Ming; Tsai, Ching-Hwa; Yang, Pei-Wen; Chuang, Yu-Chia; Lee, Chung-Pei

    2014-01-01

    ABSTRACT Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmunoprecipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCE Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme

  8. Synthesis, Spectral Characterization, SEM, Antimicrobial, Antioxidative Activity Evaluation, DNA Binding and DNA Cleavage Investigation of Transition Metal(II) Complexes Derived from a tetradentate Schiff base bearing thiophene moiety.

    PubMed

    Abdel Aziz, Ayman A; Seda, Sabry H

    2017-03-01

    A novel series of Co(II), Ni(II), Cu(II) and Zn(II) mononuclear complexes have been synthesized involving a potentially tetradentate Schiff base ligand, which was obtained by condensation of 2-aminophenol with 2,5-thiophene-dicarboxaldehyde. The complexes were synthesized via reflux reaction of methanolic solution of the appropriate Schiff base ligand with one equivalent of corresponding metal acetate salt. Based on different techniques including micro analysis, FT-IR, NMR, UV-Vis, ESR, ESI-mass and conductivity measurements, four-coordinated geometry was assigned for all complexes. Spectroscopic data have shown that, the reported Schiff base coordinated to metal ions as a dibasic tetradentate ligand through the phenolic oxygen and the azomethine nitrogen. The antimicrobial activities of the parent ligand and its complexes were investigated by using the agar disk diffusion method. Antioxidation properties of the novel complexes were investigated and it was found that all the complexes have good radical scavenging properties. The binding of complexes to calf thymus DNA (CT-DNA) was investigated by absorption, emission and viscosity measurements. Binding studies have shown that all the complexes interacted with CT-DNA via intercalation mode and the binding affinity varies with relative order as Cu(II) complex > Co(II) complex > Zn(II) complex > Ni(II) complex. Furthermore, DNA cleavage properties of the metal complexes were also investigated. The results suggested the possible utilization of novel complexes for pharmaceutical applications.

  9. Selenite induces topoisomerase I and II-DNA complexes in K562 leukemia cells.

    PubMed

    López-Lázaro, Miguel; Willmore, Elaine; Elliott, Sarah L; Austin, Caroline A

    2008-11-01

    The essential trace element selenium is one of the most promising cancer chemopreventive agents. Data from preclinical studies have revealed that selenite, an inorganic form of selenium, may also be useful in cancer chemotherapy. DNA topoisomerases (topos) are the target of several useful anticancer drugs. These drugs induce DNA complexes with either topo I or topo II; then cellular processing coverts these topo-DNA complexes into permanent DNA strand breaks that ultimately lead to cell death. Previous reports have revealed that selenite can induce apoptosis in cancer cells selectively and that selenite-induced apoptosis is preceded by the formation of DNA strand breaks. In vitro experiments have shown that selenite induces topo II-DNA complexes, which seem to be involved in selenite-induced apoptosis. Using the cell-based assay TARDIS, here we show that selenite induces topo II-DNA complexes (topo IIalpha and topo IIbeta) in K562 leukemia cells; these complexes appeared in a time-dependent manner and correlated with the induction of apoptosis. Cells lacking topo IIbeta were resistant to selenite-induced cell growth inhibition, suggesting that this isoenzyme is a target for selenite. We report for the first time that selenite induces topo I-DNA complexes in K562 cells; the levels of these complexes were high at short exposure times and seem to appear before the induction of apoptosis. Overall, our results show that selenite induces topo-DNA complexes in cells with both topo I and II, and support previous data that suggest that this agent has potential for the treatment of cancer. (c) 2008 Wiley-Liss, Inc.

  10. 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

  11. New fluorescence reactions in DNA cytochemistry. 2. Microscopic and spectroscopic studies on fluorescent aluminum complexes

    SciTech Connect

    Del Castillo, P.; Llorente, A.R.; Gomez, A.; Gosalvez, J.; Goyanes, V.J.; Stockert, J.C. )

    1990-02-01

    Metal-dye complexes are widely applied in light microscopic techniques for chromatin staining (e.g., hematoxylin and carmine), but fluorescent complexes between phosphate-binding cations and suitable ligands have been little used. Preformed and postformed Al complexes with different anionic dyes induced strong and selective fluorescence reactions in nuclei from chicken blood smears, frozen sections, paraffin-embedded sections and Epon-embedded sections of mouse and rat tissues, mitotic chromosomes, meiotic chromosomes and kinetoplasts of Trypanosoma cruzi epimastigotes. The DNA-dependent fluorescence of these structures showed a very low fading rate. The emission colors were related to the ligand. The most suitable compounds for forming fluorescent Al chelates were 8-hydroxyquinoline, morin, nuclear fast red and purpurin. Staining with diluted carmine solutions and InCl3 mordanting, followed by 8-hydroxyquinoline, also induced chromatin fluorescence. After treating isolated mouse chromosomes with the preformed complex Al-nuclear fast red, x-ray microanalysis indicated a P:Al:dye binding ratio of about 40:15:1. The selectivity, stability and easy formation of these fluorescent Al complexes are obvious advantages for their use as new cytochemical probes in cytologic studies.

  12. Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate-mediated targeting in vitro and prolonged plasma circulation in vivo.

    PubMed

    Ward, Christopher M; Pechar, Michal; Oupicky, David; Ulbrich, Karel; Seymour, Leonard W

    2002-01-01

    Gene delivery vectors based on poly(L-lysine) and DNA (pLL/DNA complexes) have limited use for targeted systemic application in vivo since they bind cells and proteins non-specifically. In this study we have attempted to form folate-targeted vectors with extended systemic circulation by surface modification of pLL/DNA complexes with hydrophilic polymers. pLL/DNA complexes were stabilised by surface modification with a multivalent reactive polymer based on alternating segments of poly(ethylene glycol) and tripeptides bearing reactive ester groups. Folate moieties were incorporated into the vectors either by direct attachment of folate to the polymer or via intermediate poly(ethylene glycol) spacers of 800 and 3400 Da. Polymer-coated complexes show similar morphology to uncoated complexes, their zeta potential is decreased towards zero, serum protein binding is inhibited and aqueous solubility is substantially increased. Intravenous (i.v.) administration to mice of coated complexes produced extended systemic circulation, with up to 2000-fold more DNA measured in the bloodstream after 30 min compared with simple pLL/DNA complexes. In further contrast to simple pLL/DNA complexes, coated complexes do not bind blood cells in vivo. Folate receptor targeting is shown to mediate targeted association with HeLa cells in vitro, leading to increased transgene expression. We demonstrate for the first time that DNA uptake via the folate receptor is dependent on pEG spacer length, with the transgene expression relatively independent of the level of internalised DNA. We show increased systemic circulation, decreased blood cell and protein binding, and folate-targeted transgene expression using pLL/DNA complexes surface-modified with a novel multireactive hydrophilic polymer. This work provides the basis for the development of plasma-circulating targeted vectors for in vivo applications. Copyright 2002 John Wiley & Sons, Ltd.

  13. Anti-cancer palladium complexes: a focus on PdX2L2, palladacycles and related complexes.

    PubMed

    Kapdi, Anant R; Fairlamb, Ian J S

    2014-07-07

    Much success has been achieved with platinum-based chemotherapeutic agents, i.e. through interactions with DNA. The long-term application of Pt complexes is thwarted by issues, leading scientists to examine other metals such as palladium which could exhibit complementary modes of action (given emphasis wherever known). Over the last 10 years several research groups have focused on the application of an eclectic array of palladium complexes (of the type PdX2L2, palladacycles and related structures) as potential anti-cancer agents. This review therefore provides readers with an up to date account of the advances that have taken place over the past several decades.

  14. DNA-binding, cytotoxicity, cellular uptake, apoptosis and photocleavage studies of Ru(II) complexes.

    PubMed

    N Deepika; C Shobha Devi; Y Praveen Kumar; K Laxma Reddy; P Venkat Reddy; D Anil Kumar; Surya S Singh; S Satyanarayana

    2016-07-01

    Two Ru(II) complexes [Ru(phen)2bppp](ClO4)2 (1) and [Ru(phen)27-Br-dppz](ClO4)2 (2) [phen=1,10 phenanthroline, 7-Br-dppz=7-fluorodipyrido[3,2-a:2',3'-c]phenazine, bppp=11-bromo-pyrido[2',3':5,6]pyrazino[2,3-f] [1,10]phenanthroline] have been synthesized and characterized by elemental analysis, ES-MS, (1)H-NMR, (13)C-NMR and IR. The in vitro cytotoxicity of the complexes examined against a panel of cancer cell lines (HeLa, Du145 and A549) by MTT method, both complexes show prominent anticancer activity against various cancer cells. Live cell imaging study and flow cytometric analysis demonstrate that both the complexes 1 and 2 could cross the cell membrane accumulating in the nucleus. Further, flow cytometry experiments showed that the cytotoxic Ru(II) complexes 1 and 2 induced apoptosis of HeLa tumor cell lines. Photo induced DNA cleavage studies have been performed and results indicate that both the complexes efficiently photo cleave pBR322 DNA. The binding properties of two complexes toward CT-DNA were investigated by various optical methods and viscosity measurements. The experimental results suggested that both Ru(II) complexes can intercalate into DNA base pairs. The complexes were docked into DNA-base pairs using the GOLD docking program.

  15. Effects of temperature and relative humidity on DNA methylation.

    PubMed

    Bind, Marie-Abele; Zanobetti, Antonella; Gasparrini, Antonio; Peters, Annette; Coull, Brent; Baccarelli, Andrea; Tarantini, Letizia; Koutrakis, Petros; Vokonas, Pantel; Schwartz, Joel

    2014-07-01

    Previous studies have found relationships between DNA methylation and various environmental contaminant exposures. Associations with weather have not been examined. Because temperature and humidity are related to mortality even on non-extreme days, we hypothesized that temperature and relative humidity may affect methylation. We repeatedly measured methylation on long interspersed nuclear elements (LINE-1), Alu, and 9 candidate genes in blood samples from 777 elderly men participating in the Normative Aging Study (1999-2009). We assessed whether ambient temperature and relative humidity are related to methylation on LINE-1 and Alu, as well as on genes controlling coagulation, inflammation, cortisol, DNA repair, and metabolic pathway. We examined intermediate-term associations of temperature, relative humidity, and their interaction with methylation, using distributed lag models. Temperature or relative humidity levels were associated with methylation on tissue factor (F3), intercellular adhesion molecule 1 (ICAM-1), toll-like receptor 2 (TRL-2), carnitine O-acetyltransferase (CRAT), interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), and glucocorticoid receptor, LINE-1, and Alu. For instance, a 5°C increase in 3-week average temperature in ICAM-1 methylation was associated with a 9% increase (95% confidence interval: 3% to 15%), whereas a 10% increase in 3-week average relative humidity was associated with a 5% decrease (-8% to -1%). The relative humidity association with ICAM-1 methylation was stronger on hot days than mild days. DNA methylation in blood cells may reflect biological effects of temperature and relative humidity. Temperature and relative humidity may also interact to produce stronger effects.

  16. Effects of Temperature and Relative Humidity on DNA Methylation

    PubMed Central

    Bind, Marie-Abele; Zanobetti, Antonella; Gasparrini, Antonio; Peters, Annette; Coull, Brent; Baccarelli, Andrea; Tarantini, Letizia; Koutrakis, Petros; Vokonas, Pantel; Schwartz, Joel

    2014-01-01

    Background Previous studies have found relationships between DNA methylation and various environmental contaminant exposures. Associations with weather have not been examined. Because temperature and humidity are related to mortality even on non-extreme days, we hypothesized that temperature and relative humidity may affect methylation. Methods We repeatedly measured methylation on long interspersed nuclear elements (LINE-1), Alu, and 9 candidate genes in blood samples from 777 elderly men participating in the normative aging Study (1999–2009). We assessed whether ambient temperature and relative humidity are related to methylation on LINE-1 and Alu, as well as on genes controlling coagulation, inflammation, cortisol, DNA repair, and metabolic pathway. We examined intermediate-term associations of temperature, relative humidity, and their interaction with methylation, using distributed lag models. Results Temperature or relative humidity levels were associated with methylation on tissue factor (F3), intercellular adhesion molecule 1 (ICAM-1), toll-like receptor 2 (TRL-2), carnitine O-acetyltransferase (CRAT), interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), and glucocorticoid receptor, LINE-1, and Alu. For instance, a 5°c increase in 3-week average temperature in ICAM-1 methylation was associated with a 9% increase (95% confidence interval: 3% to 15%), whereas a 10% increase in 3-week average relative humidity was associated with a 5% decrease (−8% to −1%). The relative humidity association with ICAM-1 methylation was stronger on hot days than mild days. Conclusions DNA methylation in blood cells may reflect biological effects of temperature and relative humidity. Temperature and relative humidity may also interact to produce stronger effects. PMID:24809956

  17. Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: Insights from DNA complex structures

    PubMed Central

    Pike, Ashley C. W.; Gomathinayagam, Shivasankari; Swuec, Paolo; Berti, Matteo; Schnecke, Christina; Marino, Francesca; von Delft, Frank; Renault, Ludovic; Costa, Alessandro; Vindigni, Alessandro

    2015-01-01

    RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration. PMID:25831490

  18. Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: insights from DNA complex structures.

    PubMed

    Pike, Ashley C W; Gomathinayagam, Shivasankari; Swuec, Paolo; Berti, Matteo; Zhang, Ying; Schnecke, Christina; Marino, Francesca; von Delft, Frank; Renault, Ludovic; Costa, Alessandro; Gileadi, Opher; Vindigni, Alessandro

    2015-04-07

    RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.

  19. Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

    PubMed Central

    Kaufmann, W K; Boyer, J C; Estabrooks, L L; Wilson, S J

    1991-01-01

    Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes. PMID:1646393

  20. Probing physical properties of a DNA-protein complex using nanofluidic channels.

    PubMed

    Frykholm, Karolin; Alizadehheidari, Mohammadreza; Fritzsche, Joachim; Wigenius, Jens; Modesti, Mauro; Persson, Fredrik; Westerlund, Fredrik

    2014-03-12

    A method to investigate physical properties of a DNA-protein complex in solution is demonstrated. By using tapered nanochannels and lipid passivation the persistence length of a RecA filament formed on double-stranded DNA is determined to 1.15 μm, in agreement with the literature, without attaching protein or DNA to any handles or surfaces. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. 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.

  2. 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

  3. 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

  4. Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK.

    PubMed

    Costantini, Silvia; Woodbine, Lisa; Andreoli, Lucia; Jeggo, Penny A; Vindigni, Alessandro

    2007-06-01

    DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.

  5. DNA interactions of cobalt(III) mixed-polypyridyl complexes containing asymmetric ligands.

    PubMed

    Wang, Xiang-Li; Chao, Hui; Li, Hong; Hong, Xian-Lan; Liu, Yun-Jun; Tan, Li-Feng; Ji, Liang-Nian

    2004-06-01

    Three novel asymmetric ligands, 3-(pyridine-2-yl)-5,6-diphenyl-as-triazine (pdtb), 3-(pyridine-2-yl)-as-triazino[5,6-f]acenaphthylene (pdta) and 3-(pyridine-2-yl)-as-triazino[5,6-f]phenanthroline (pdtp) and their cobalt(III) complexes have been synthesized and characterized. Binding of the three complexes with calf thymus DNA (CT-DNA) has been investigated by spectroscopic methods, viscosity, cyclic voltammetry, and electrophoresis measurements. The experimental results indicate that the size and shape of the intercalated ligand have a marked effect on the binding affinity of complexes to CT-DNA. Complexes 2 and 3 have also been found to promote cleavage of plasmid pBR322 DNA from the supercoiled form I to the open circular form II upon irradiation.

  6. 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

    2015-06-07

    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.

  7. Unusual reactivity in a commercial chromium supplement compared to baseline DNA cleavage with synthetic chromium complexes.

    PubMed

    Chaudhary, Shveta; Pinkston, Joel; Rabile, M Mohamed; Van Horn, J David

    2005-03-01

    Commercially available chromium supplements were tested for their DNA cleavage ability compared with synthetic chromium(III) complexes, including chromium(III) tris-picolinate [Cr(pic)3], basic chromium acetate [Cr3O(OAc)6]+, model complexes, and recently patented Cr-complexes for use in supplements or therapy. Four different supplements (P1-P4) were tested for their DNA cleaving activity in the presence and the absence of H2O2, dithiothreitol (DTT) or ascorbate. One supplement, P1, showed nicking of DNA in the absence of oxidant or reductant at 120 microM metal concentration. Different lot numbers of P1 were also tested for DNA cleavage activity with similar results. Commercial supplements containing Cr(pic)3 nicked DNA at 120 microM metal concentrations in the presence of 5 mM ascorbate or with excess hydrogen peroxide, analogous to reactions with synthetic Cr(pic)3 reported elsewhere. Another chromium (non-Cr(pic)3) supplement, P2, behaves in a comparable manner to simple Cr(III) salts in the DNA nicking assay. Chromium(III) malonate [Cr(mal)2] and chromium(III) acetate [Cr(OAc)] can nick DNA in the presence of ascorbate or hydrogen peroxide, respectively, only at higher metal concentrations. The Cr(III) complexes of histidine, succinate or N-acetyl-L-glutamate do not nick DNA to a significant degree.

  8. Cleavage enhancement of specific chemical bonds in DNA-Cisplatin complexes induced by X-rays

    NASA Astrophysics Data System (ADS)

    Zheng, Yi; Yao, Xiaobin; Luo, Xinglan; Fu, Xianzhi

    2014-04-01

    The chemical bond transformation of cisplatin-DNA complexes can be probed efficiently by XPS which provides a concomitant X-ray irradiation source as well. The presence to Pt could considerably increase formation of the SE induced by X-ray and that the further interaction of these LEE with DNA leads to the enhancement of bond cleavages.

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

    USDA-ARS?s Scientific Manuscript database

    The extensive use of DNA barcoding technology in a large inventory of Macrolepidoptera and their parasitoids is documented. The methodology used and its practical applications are summarized, and numerous examples of how DNA barcoding has untangled complexes of cryptic species of butterflies, moths...

  10. Photoinduced formation of stable Ag-nanoparticles from a ternary ligand-DNA-Ag(+) complex.

    PubMed

    Berdnikova, Daria V; Ihmels, Heiko; Schönherr, Holger; Steuber, Marc; Wesner, Daniel

    2015-03-28

    The combination of (i) the light-harvesting nature and excited-state redox reactivity of a cationic DNA intercalator, (ii) a conjugated Ag(+)-binding crown ether, and (iii) the stabilizing effect of DNA on AgNPs in one integral ternary complex enables the mild photoinduced formation of Ag nanoparticles.

  11. Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex.

    PubMed

    Jiang, Xiaohua; Klimovich, Vitaly; Arunkumar, Alphonse I; Hysinger, Erik B; Wang, Yingda; Ott, Robert D; Guler, Gulfem D; Weiner, Brian; Chazin, Walter J; Fanning, Ellen

    2006-11-29

    We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.

  12. 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.

  13. 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.

  14. Characteristic effect of an anticancer dinuclear platinum(II) complex on the higher-order structure of DNA.

    PubMed

    Kida, Naoko; Katsuda, Yousuke; Yoshikawa, Yuko; Komeda, Seiji; Sato, Takaji; Saito, Yoshihiro; Chikuma, Masahiko; Suzuki, Mari; Imanaka, Tadayuki; Yoshikawa, Kenichi

    2010-06-01

    It is known that a 1,2,3-triazolato-bridged dinuclear platinum(II) complex, [{cis-Pt(NH(3))(2)}(2)(micro-OH)(micro-1,2,3-ta-N (1),N (2))](NO(3))(2) (AMTA), shows high in vitro cytotoxicity against several human tumor cell lines and circumvents cross-resistance to cisplatin. In the present study, we examined a dose- and time-dependent effect of AMTA on the higher-order structure of a large DNA, T4 phage DNA (166 kbp), by adapting single-molecule observation with fluorescence microscopy. It was found that AMTA induces the shrinking of DNA into a compact state with a much higher potency than cisplatin. From a quantitative analysis of the Brownian motion of individual DNA molecules in solution, it became clear that the density of a DNA segment in the compact state is about 2,000 times greater than that in the absence of AMTA. Circular dichroism spectra suggested that AMTA causes a transition from the B to the C form in the secondary structure of DNA, which is characterized by fast and slow processes. Electrophoretic measurements indicated that the binding of AMTA to supercoiled DNA induces unwinding of the double helix. Our results indicate that AMTA acts on DNA through both electrostatic interaction and coordination binding; the former causes a fast change in the secondary structure from the B to the C form, whereas the latter promotes shrinking in the higher-order structure as a relatively slow kinetic process. The shrinking effect of AMTA on DNA is attributable to the possible increase in the number of bridges along a DNA molecule. It is concluded that AMTA interacts with DNA in a manner markedly different from that of cisplatin.

  15. Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay.

    PubMed

    Bahjat, Mahnoush; Bloedjes, Timon A; van der Veen, Amélie; de Wilde, Guus; Maas, Chiel; Guikema, Jeroen E J

    2017-06-09

    The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

  16. 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.

  17. Supermolecules of poly(N-isopropylacrylamide) complexating Herring sperm DNA with bio-multiple hydrogen bonding.

    PubMed

    Lee, Ai-Wei; Hsu, Cheng-Chen; Liu, Yi-Zu; Wei, Po-Li; Chen, Jem-Kun

    2016-12-01

    In this study we used the poly(N-isopropylacrylamide) (PNIPAAm) as a medium to blend with an organic DNA, herring sperm DNA (HSD), to generate PNIPAAm-HSD supramolecular complexes. Bio-multiple hydrogen bonding (BMHB) between PNIPAAm and HSD was investigated that changed the temperature responsiveness of PNIPAAm relatively to the HSD concentrations. With blending the HSD into PNIPAAm matrix, the phase separation in solution is completely opposite from that of neat PNIPAAm. Surface property in static water contact angle (SWCA) is also opposite from that of pure PNIPAAm upon increasing HSD content over 60%. In addition, we found that the PNIPAAm and HSD self-assembled a specific triangle-like structure at a PNIPAAm-to-HSD weight ratio of 1:4 at 25°C; while the triangle-like structure disappeared with increasing temperature to 45°C. Furthermore, both PNIPAAm and HSD could be regarded as insulator, but it transformed into a semiconductive matter after blending with the HSD. Incorporation of organic DNA with hydrogel could significantly change their properties, which might facilitate their use as novel materials in bioelectronics. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. 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).

  19. The HIR corepressor complex binds to nucleosomes generating a distinct protein/DNA complex resistant to remodeling by SWI/SNF

    PubMed Central

    Prochasson, Philippe; Florens, Laurence; Swanson, Selene K.; Washburn, Michael P.; Workman, Jerry L.

    2005-01-01

    The histone regulatory (HIR) and histone promoter control (HPC) repressor proteins regulate three of the four histone gene loci during the Saccharomyces cerevisiae cell cycle. Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 proteins form a stable HIR repressor complex. The HIR complex promotes histone deposition onto DNA in vitro and constitutes a novel nucleosome assembly complex. The HIR complex stably binds to DNA and nucleosomes. Furthermore, HIR complex binding to nucleosomes forms a distinct protein/DNA complex resistant to remodeling by SWI/SNF. Thus, the HIR complex is a novel nucleosome assembly complex which functions with SWI/SNF to regulate transcription. PMID:16264190

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

    PubMed

    Pozharski, Edwin; MacDonald, Robert C

    2002-07-01

    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.

  1. 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

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

    PubMed Central

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

    2011-01-01

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

  3. A Method for isolation of DNA-binding proteins based on solubility of DNA-protein complexes.

    PubMed

    Yang, Hua; Li, Huang; Rao, Li-qun; Long, Gui-you; Peng, Guo-ping; Jin, Lu

    2012-10-01

    The study of DNA-binding proteins is crucial in understanding gene regulatory networks. We developed a new method for the enrichment of DNA-binding proteins based on the variability of DNA-protein complexes' solubility in different ionic strength solutions. 0.14M sodium chloride was determined as the most efficient extraction concentration to precipitate DNA-binding proteins. SDS-PAGE analysis revealed that some high-abundance proteins were removed effectively and at the same time DNA-binding proteins were isolated in this simple process. Twenty kinds of proteins were identified in the acquired sample by 1-D gel-LC-MS/MS. Furthermore, computerized analysis of MS data showed that quite a number of unmatched peptides have the classic structure of leucine zipper or zinc finger, which were symbolic elements of transcription factors. These results suggested that this new method can acquire DNA-binding proteins effectively and allow improvement in the isolation of high-quality DNA-binding proteins.

  4. Recognition of thymine in DNA bulges by a Zn(II) macrocyclic complex.

    PubMed

    del Mundo, Imee Marie A; Fountain, Matthew A; Morrow, Janet R

    2011-08-14

    A Zn(II) macrocyclic complex with appended quinoline is a bifunctional recognition agent that uses both the Zn(II) center and the pendent aromatic group to bind to thymine in bulges with good selectivity over DNA containing G, C or A bulges. Spectroscopic studies show that the stem containing the bulge stays largely intact in a DNA hairpin with the Zn(II) complex bound to the thymine bulge. This journal is © The Royal Society of Chemistry 2011

  5. 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.

  6. SbcCD-mediated processing of covalent gyrase-DNA complex in Escherichia coli.

    PubMed

    Aedo, Sandra; Tse-Dinh, Yuk-Ching

    2013-10-01

    Quinolones trap the covalent gyrase-DNA complex in Escherichia coli, leading to cell death. Processing activities for trapped covalent complex have not been characterized. A mutant strain lacking SbcCD nuclease activity was examined for both accumulation of gyrase-DNA complex and viability after quinolone treatment. Higher complex levels were found in ΔsbcCD cells than in wild-type cells after incubation with nalidixic acid and ciprofloxacin. However, SbcCD activity protected cells against the bactericidal action of nalidixic acid but not ciprofloxacin.

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

    PubMed

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

    2014-02-03

    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.

  8. Synthesis, spectroscopic characterization, DNA interaction and antibacterial study of metal complexes of tetraazamacrocyclic Schiff base

    NASA Astrophysics Data System (ADS)

    Shakir, Mohammad; Khanam, Sadiqa; Firdaus, Farha; Latif, Abdul; Aatif, Mohammad; Al-Resayes, Saud I.

    The template condensation reaction between benzil and 3,4-diaminotoulene resulted mononuclear 12-membered tetraimine macrocyclic complexes of the type, [MLCl2] [M = Co(II), Ni(II), Cu(II) and Zn(II)]. The synthesized complexes have been characterized on the basis of the results of elemental analysis, molar conductance, magnetic susceptibility measurements and spectroscopic studies viz. FT-IR, 1H and 13C NMR, FAB mass, UV-vis and EPR. An octahedral geometry has been envisaged for all these complexes, while a distorted octahedral geometry has been noticed for Cu(II) complex. Low conductivity data of all these complexes suggest their non-ionic nature. The interactive studies of these complexes with calf thymus DNA showed that the complexes are avid binders of calf thymus DNA. The in vitro antibacterial studies of these complexes screened against pathogenic bacteria proved them as growth inhibiting agents.

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

    PubMed

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

    2015-06-01

    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.

  10. 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

  11. Highly sensitive surface plasmon resonance biosensor for the detection of HIV-related DNA based on dynamic and structural DNA nanodevices.

    PubMed

    Diao, Wei; Tang, Min; Ding, Shijia; Li, Xinmin; Cheng, Wenbin; Mo, Fei; Yan, Xiaoyu; Ma, Hongmin; Yan, Yurong

    2017-09-09

    Early detection, diagnosis and treatment of human immune deficiency virus (HIV) infection is the key to reduce acquired immunodeficiency syndrome (AIDS) mortality. In our research, an innovative surface plasmon resonance (SPR) biosensing strategy has been developed for highly sensitive detection of HIV-related DNA based on entropy-driven strand displacement reactions (ESDRs) and double-layer DNA tetrahedrons (DDTs). ESDRs as enzyme-free and label-free signal amplification circuit can be specifically triggered by target DNA, leading to the cyclic utilization of target DNA and the formation of plentiful double-stranded DNA (dsDNA) products. Subsequently, the dsDNA products bind to the immobilized hairpin capture probes and further combine with DDTs nanostructures. Due to the high efficiency of ESDRs and large molecular weight of DDTs, the SPR response signal was enhanced dramatically. The proposed SPR biosensor could detect target DNA sensitively and specifically in a linear range from 1pM to 150nM with a detection limit of 48fM. In addition, the whole detecting process can be accomplished in 60min with high accuracy and duplicability. In particular, the developed SPR biosensor was successfully used to analyze target DNA in complex biological sample, indicating that the developed strategy is promising for rapid and early clinical diagnosis of HIV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Is the formation of cationic lipid-DNA complexes a thermodynamically driven phenomenon? Structure and phase behavior of DC-Chol/DNA complexes say not

    NASA Astrophysics Data System (ADS)

    Caracciolo, Giulio; Pozzi, Daniela; Caminiti, Ruggero

    2006-07-01

    The currently accepted mechanism of formation of cationic lipid-DNA complexes (lipoplexes) relies on the basic assumption that equilibrium structure of lipoplexes is regulated by thermodynamics. The main consequence is that neutral lipoplexes are one phase whereas positively (or negatively) charged ones coexist with excess lipid (or excess DNA). The authors report a small angle x-ray diffraction study on the structure of lipoplexes made of the cationic lipid 3β-[N-(N ,N-dimethylaminoethane)-carbamoyl]cholesterol and calf thymus Na-DNA. Here the authors show that positively charged lipoplexes can coexist with unbound DNA and they claim that steric size effects are definitely important to determine the equilibrium structure of lipoplexes.

  13. Phylogeny of "Philoceanus complex" seabird lice (Phthiraptera: Ischnocera) inferred from mitochondrial DNA sequences.

    PubMed

    Page, Roderic D M; Cruickshank, Robert H; Dickens, Megan; Furness, Robert W; Kennedy, Martyn; Palma, Ricardo L; Smith, Vincent S

    2004-03-01

    The Philoceanus complex is a large assemblage of lice that parasitise procellariiform seabirds (petrels, albatrosses, and their relatives). We obtained mitochondrial 12S rRNA and cytochrome oxidase I DNA sequences from 39 species from diverse hosts and localities. Resolution of deeper relationships between genera was limited, however there is evidence for two major clades, one hosted by albatrosses, the other by petrels. Based on our results, the genera hosted by albatrosses are excellent candidates for detailed analysis of cospeciation. Our results also suggest that a previous estimate of a 5-fold difference in the relative rate of sequence evolution in lice and their avian hosts is an artefact of limited taxonomic sampling.

  14. Studies on Photocleavage, DNA Binding, Cytotoxicity, and Docking Studies of Ruthenium(II) Mixed Ligand Complexes.

    PubMed

    Kumar, Yata Praveen; Devi, C Shobha; Srishailam, A; Deepika, N; Kumar, V Ravi; Reddy, P Venkat; Nagasuryaprasad, K; Singh, Surya S; Nagababu, Penumaka; Satyanarayana, S

    2016-11-01

    This article describes the synthesis and characterization of three new Ru(II) polypyridyl complexes including [Ru(phen)2(dpphz)](2+) (1), [Ru(bpy)2(dpphz)](2+) (2) and [Ru(dmb)2(dpphz)](2+) (3) where dpphz = dipyrido[3,2-a:2',3'-c] phenazine-11-hydrazide, phen =1,10-phenanthroline, bpy = 2,2'-bipyridine and dmb = 4,4'-dimethyl2,2'-bipyridine. The binding behaviors of these complexes to calf thymus DNA (CT-DNA) were explored by spectroscopic titrations, viscosity measurements. Results suggest that these complexes can bind to CT-DNA through intercalation. However, their binding strength differs from each other; this may be attributed to difference in the ancillary ligand. The cytotoxicity of 1-3 was evaluated by MTT assay; results indicated that all complexes have significant dose dependent cytotoxicity with HeLa tumor cell line. All complexes exhibited efficient photocleavage of pBR322 DNA upon irradiation. The DNA binding ability of 1-3 was also studied by docking the complexes into B-DNA using docking program.

  15. Comparison of fungi within the Gaeumannomyces-Phialophora complex by analysis of ribosomal DNA sequences.

    PubMed

    Bryan, G T; Daniels, M J; Osbourn, A E

    1995-02-01

    Four ascomycete species of the genus Gaeumannomyces infect roots of monocotyledons. Gaeumannomyces graminis contains four varieties, var. tritici, var. avenae, var. graminis, and var. maydis. G. graminis varieties tritici, avenae, and graminis have Phialophora-like anamorphs and, together with the other Gaeumannomyces and Phialophora species found on cereal roots, constitute the Gaeumannomyces-Phialophora complex. Relatedness of a number of Gaeumannomyces and Phialophora isolates was assessed by comparison of DNA sequences of the 18S rRNA gene, the 5.8S rRNA gene, and the internal transcribed spacers (ITS). G. graminis var. tritici, G. graminis var. avenae, and G. graminis var. graminis isolates can be distinguished from each other by nucleotide sequence differences in the ITS regions. The G. graminis var. tritici isolates can be further subdivided into R and N isolates (correlating with ability [R] or inability [N] to infect rye). Phylogenetic analysis of the ITS regions of several oat-infecting G. graminis var. tritici isolates suggests that these isolates are actually more closely related to G. graminis var. avenae. The isolates of Magnaporthe grisea included in the analysis showed a surprising degree of relatedness to members of the Gaeumannomyces-Phialophora complex. G. graminis variety-specific oligonucleotide primers were used in PCRs to amplify DNA from cereal seedlings infected with G. graminis var. tritici or G. graminis var. avenae, and these should be valuable for sensitive detection of pathogenic isolates and for diagnosis of take-all.

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

    PubMed Central

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

    2016-01-01

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

  17. 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.

  18. Effects of Tris and Hepes buffers on the interaction of palladium-diaminopropane complexes with DNA.

    PubMed

    Akdi, Khalid; Vilaplana, Rosario A; Kamah, Sanae; González-Vílchez, Francisco

    2005-06-01

    The Pd(II) complexes, [PdCl(2)(1,2-pn)] and [PdCl(2)(1,3-pn)] (pn is diaminopropane), were synthesized and characterized by analytical and spectroscopic (FT-IR, (1)H NMR and (13)C NMR) techniques. UV difference spectral study performed on Pd-pn/DNA systems, indicate a pronounced interaction of palladium complexes with DNA in cell-free media; comparison of lambda(max), Abs(max) and %H values observed for the two compounds might be attributed to structural differences of the chelated ligand rings. Results obtained from electrophoretic analysis of Pd complexes in presence of pBR322 plasmid DNA show a clear decreasing of the supercoiled (SC) DNA form mobility, that could be attributed to unwinding of the double helix; a parallel increasing of the open-circular (OC) DNA form mobility is also noted, this fact implying that the binding of complexes either shortens or condenses the DNA helix. Interaction studies of Pd complexes with plasmid DNA in different buffer systems indicate that DNA binding efficiency capable of modifying the tertiary structure of pBR322 decreased from NaClO(4) to Hepes 2, Hepes 1 [Hepes=4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], and Tris [(hydroxymethyl)aminomethane] buffers, in this order. Moreover, the level of DNA modifications produced by palladium complexes in 10 mM NaClO(4) remains unchanged after transferring the samples into the medium required for subsequent biophysical or biochemical analyses.

  19. Energetics, conformation, and recognition of DNA duplexes containing a major adduct of an anticancer azolato-bridged dinuclear Pt(II) complex.

    PubMed

    Mlcouskova, Jarmila; Malina, Jaroslav; Novohradsky, Vojtech; Kasparkova, Jana; Komeda, Seiji; Brabec, Viktor

    2012-10-01

    The design of anticancer metallodrugs is currently focused on platinum complexes which form on DNA major adducts that cannot readily be removed by DNA repair systems. Hence, antitumor azolato-bridged dinuclear Pt(II) complexes, such as [{cis-Pt(NH(3))(2)}(2)(μ-OH)(μ-pyrazolate)](2+) (AMPZ), have been designed and synthesized. These complexes exhibit markedly higher toxic effects in tumor cell lines than mononuclear conventional cisplatin. Biophysical and biochemical aspects of the alterations induced in short DNA duplexes uniquely and site-specifically modified by the major DNA adduct of AMPZ, namely 1,2-GG intrastrand cross-links, were examined. Attention was also paid to conformational distortions induced in DNA by the adducts of AMPZ and cisplatin, associated alterations in the thermodynamic stability of the duplexes, and recognition of these adducts by high-mobility-group (HMG) domain proteins. Chemical probing of DNA conformation, DNA bending studies and translesion synthesis by DNA polymerase across the platinum adduct revealed that the distortion induced in DNA by the major adduct of AMPZ was significantly less pronounced than that induced by similar cross-links from cisplatin. Concomitantly, the cross-link from AMPZ reduced the thermodynamic stability of the modified duplex considerably less. In addition, HMGB1 protein recognizes major DNA adducts of AMPZ markedly less than those of cisplatin. The experimental evidence demonstrates why the major DNA adducts of the new anticancer azolato-bridged dinuclear Pt(II) complexes are poor substrates for DNA repair observed in a previously published report. The relative resistance to DNA repair explains why these platinum complexes show major pharmacological advantages over cisplatin in tumor cells. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2012-10-05

    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. Copyright © 2012 Wiley Periodicals, Inc.

  1. Synthesis, anticancer activities, interaction with DNA and mitochondria of manganese complexes.

    PubMed

    Qiu-Yun, Chen; Dong-Fang, Zhou; Juan, Huang; Wen-Jie, Guo; Jing, Gao

    2010-11-01

    Two new complexes [(Etdpa)MnCl(2)] and [(Adpa)Mn(Cl)(H(2)O)] (Etdpa = ethyl bis(2-pyridylmethyl)amino-2-propionate; Adpa = bis(2-pyridylmethyl)amino-2-propionic acid) were synthesized and characterized by spectral methods. The crystal structure of [(Etdpa)MnCl(2)] shows that the Mn(II) atom is coordinated by three N atoms (N1, N2, N3), one oxygen atom (O1) of the ligand (Etdpa) and two chloride atoms (Cl1, Cl2), forming a distorted octahedral geometry. The binding interaction between ct-DNA and the synthesized complexes was relatively weak, but they can inhibit the induced swelling of Ca(2+)-loaded mitochondria in a dose-dependent manner. The [(Adpa)Mn(Cl)(H(2)O)] can cause the obvious decrease of mitochondria membrane potential. The MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenpyltetra-zolium bromide) assay shows that the two Mn(II) complexes are more active against cancer cells. Especially [(Adpa)Mn(Cl)(H(2)O)] can inhibit the proliferation of glioma cells with IC(50) 9.5 μM. Experimental results indicate that the [(Adpa)Mn(Cl)(H(2)O)] could be a new potential antitumor complex to target the mitochondria.

  2. 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.

  3. 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

  4. RNA-activated DNA cleavage by the Type III-B CRISPR–Cas effector complex

    PubMed Central

    Estrella, Michael A.; Kuo, Fang-Ting; Bailey, Scott

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat) system is an RNA-guided immune system that protects prokaryotes from invading genetic elements. This system represents an inheritable and adaptable immune system that is mediated by multisubunit effector complexes. In the Type III-B system, the Cmr effector complex has been found to cleave ssRNA in vitro. However, in vivo, it has been implicated in transcription-dependent DNA targeting. We show here that the Cmr complex from Thermotoga maritima can cleave an ssRNA target that is complementary to the CRISPR RNA. We also show that binding of a complementary ssRNA target activates an ssDNA-specific nuclease activity in the histidine–aspartate (HD) domain of the Cmr2 subunit of the complex. These data suggest a mechanism for transcription-coupled DNA targeting by the Cmr complex and provide a unifying mechanism for all Type III systems. PMID:26848046

  5. 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

  6. 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.

  7. Biophysical characterization of quaternary pyridinium functionalized polynorbornenes for DNA complexation and their cellular interactions.

    PubMed

    Guler Gokce, Zeliha; Zuhal Birol, Semra; Eren, Tarık; Ercelen Ceylan, Sebnem

    2017-04-01

    Cationic polymers with hydrophobic side chains have gained great interest as DNA carriers since they form a compact complex with negatively charged DNA phosphate groups and interact with the cell membrane. Amphiphilic polyoxanorbornenes with different quaternary alkyl pyridinium side chains with ethyl-p(OPy2) and hexyl units-p(OPy6) bearing 10 kDa MWT were synthesized by living Ring-Opening Metathesis Polymerization method. The physicochemical characteristics: critical micellar concentration, size distribution, surface charge, and condensation of polymer/DNA complex were investigated. Morphology of complexes was monitored by Atomic force microscopy. Cytotoxicity and interaction of these complexes with model lipid vesicles mimicking the cell membrane were examined. These polymers were enabled to form small sized complexes of DNA, which interact with model membrane vesicles. It was found that the nature of hydrophobicity of the homopolymers significantly impacts rates of DNA complexation and the surface charge of the resulting complexes. These results highlight the prospect of the further examinations of these polymers as gene carriers. © 2016 Wiley Periodicals, Inc.

  8. 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

    2016-06-02

    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.

  9. Spectroscopic and molecular modeling studies of caffeine complexes with DNA intercalators.

    PubMed Central

    Larsen, R W; Jasuja, R; Hetzler, R K; Muraoka, P T; Andrada, V G; Jameson, D M

    1996-01-01

    Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation. PMID:8770220

  10. 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.

  11. 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

  12. Stacked graphen