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Sample records for dna binding specificity

  1. Sequence-Specific DNA Binding by a Short Peptide Dimer

    Science.gov (United States)

    Talanian, Robert V.; McKnight, C. James; Kim, Peter S.

    1990-08-01

    A recently described class of DNA binding proteins is characterized by the "bZIP" motif, which consists of a basic region that contacts DNA and an adjacent "leucine zipper" that mediates protein dimerization. A peptide model for the basic region of the yeast transcriptional activator GCN4 has been developed in which the leucine zipper has been replaced by a disulfide bond. The 34-residue peptide dimer, but not the reduced monomer, binds DNA with nanomolar affinity at 4^circC. DNA binding is sequence-specific as judged by deoxyribonuclease I footprinting. Circular dichroism spectroscopy suggests that the peptide adopts a helical structure when bound to DNA. These results demonstrate directly that the GCN4 basic region is sufficient for sequence-specific DNA binding and suggest that a major function of the GCN4 leucine zipper is simply to mediate protein dimerization. Our approach provides a strategy for the design of short sequence-specific DNA binding peptides.

  2. Quantitative modeling of transcription factor binding specificities using DNA shape.

    Science.gov (United States)

    Zhou, Tianyin; Shen, Ning; Yang, Lin; Abe, Namiko; Horton, John; Mann, Richard S; Bussemaker, Harmen J; Gordân, Raluca; Rohs, Remo

    2015-04-14

    DNA binding specificities of transcription factors (TFs) are a key component of gene regulatory processes. Underlying mechanisms that explain the highly specific binding of TFs to their genomic target sites are poorly understood. A better understanding of TF-DNA binding requires the ability to quantitatively model TF binding to accessible DNA as its basic step, before additional in vivo components can be considered. Traditionally, these models were built based on nucleotide sequence. Here, we integrated 3D DNA shape information derived with a high-throughput approach into the modeling of TF binding specificities. Using support vector regression, we trained quantitative models of TF binding specificity based on protein binding microarray (PBM) data for 68 mammalian TFs. The evaluation of our models included cross-validation on specific PBM array designs, testing across different PBM array designs, and using PBM-trained models to predict relative binding affinities derived from in vitro selection combined with deep sequencing (SELEX-seq). Our results showed that shape-augmented models compared favorably to sequence-based models. Although both k-mer and DNA shape features can encode interdependencies between nucleotide positions of the binding site, using DNA shape features reduced the dimensionality of the feature space. In addition, analyzing the feature weights of DNA shape-augmented models uncovered TF family-specific structural readout mechanisms that were not revealed by the DNA sequence. As such, this work combines knowledge from structural biology and genomics, and suggests a new path toward understanding TF binding and genome function.

  3. Design of sequence-specific DNA-binding molecules.

    Science.gov (United States)

    Dervan, P B

    1986-04-25

    Base sequence information can be stored in the local structure of right-handed double-helical DNA (B-DNA). The question arises as to whether a set of rules for the three-dimensional readout of the B-DNA helix can be developed. This would allow the design of synthetic molecules that bind DNA of any specific sequence and site size. There are four stages of development for each new synthetic sequence-specific DNA-binding molecule: design, synthesis, testing for sequence specificity, and reevaluation of the design. This approach has produced bis(distamycin)fumaramide, a synthetic, crescent-shaped oligopeptide that binds nine contiguous adenine-thymine base pairs in the minor groove of double-helical DNA.

  4. Damage-specific DNA-binding proteins from human cells

    Energy Technology Data Exchange (ETDEWEB)

    Kanjilal, S.

    1992-01-01

    The primary objective of the study was to detect and characterize factors from human cells that bind DNA damaged by ultraviolet radiation. An application of the gel-shift assay was devised in which a DNA probe was UV-irradiated and compared with non-irradiated probe DNA for the ability to bind to such factors in cell extracts. UV-dose dependent binding proteins were identified. Formation of the DNA-protein complexes was independent of the specific sequence, form or source of the DNA. There was a marked preference for lesions on double stranded DNA over those on single stranded DNA. DNA irradiated with gamma rays did not compete with UV-irradiated DNA for the binding activities. Cell lines from patients with genetic diseases associated with disorders of the DNA repair system were screened for the presence of damaged-DNA-binding activities. Simultaneous occurrence of the clinical symptoms of some of these diseases had been previously documented and possible links between the syndromes proposed. However, supporting biochemical or molecular evidence for such associations were lacking. The data from the present investigations indicate that some cases of Xeroderma Pigmentosum group A, Cockayne's Syndrome, Bloom's Syndrome and Ataxia Telangiectasia, all of which exhibit sensitivity to UV or gamma radiation, share an aberrant damaged-DNA-binding factor. These findings support the hypothesis that some of the repair disorder diseases are closely related and may have arisen from a common defect. Partial purification of the binding activities from HeLa cells was achieved. Size-exclusion chromatography resolved the activities into various peaks, one of which was less damage-specific than the others as determined by competition studies using native or UV-irradiated DNA. Some of the activities were further separated by ion-exchange chromatography. On using affinity chromatography methods, the major damage-binding factor could be eluted in the presence of 2 M KCl and 1

  5. Prediction of DNA-binding specificity in zinc finger proteins

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key ... [Roy S, Dutta S, Khanna K, Singla S and Sundar D 2012 Prediction of DNA-binding specificity in zinc finger proteins. J. Biosci. .... well as protection from HIV infection (Reynolds, et al. 2003).

  6. NAD+ Modulates p53 DNA Binding Specificity and Function

    Science.gov (United States)

    McLure, Kevin G.; Takagi, Masatoshi; Kastan, Michael B.

    2004-01-01

    DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53. PMID:15509798

  7. Thermodynamics of sequence-specific binding of PNA to DNA

    DEFF Research Database (Denmark)

    Ratilainen, T; Holmén, A; Tuite, E

    2000-01-01

    For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...... relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kJ mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only...

  8. An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro.

    Science.gov (United States)

    Wang, Liangyan; Lu, Huizhi; Wang, Yunguang; Yang, Su; Xu, Hong; Cheng, Kaiying; Zhao, Ye; Tian, Bing; Hua, Yuejin

    2017-03-01

    Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro. This technique is mainly dependent on antibody-antigen immunity, simple and convenient, while radioactive isotope labeling and optimization of partial degradation by deoxyribonuclease (DNase) are avoided. As an example, the specific binding sequence of a target promoter by DdrO (a DNA damage response protein from Deinococcus radiodurans) in vitro was determined by the developed method. The central sequence of the binding site could be easily located using this technique.

  9. Characterization and DNA-binding specificities of Ralstonia TAL-like effectors

    KAUST Repository

    Li, Lixin

    2013-07-01

    Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp. © 2013 The Author.

  10. Enhancement of damage-specific DNA binding of XPA by interaction with the ERCC1 DNA repair protein

    NARCIS (Netherlands)

    A. Nagai; M. Saijo (Masafumi); I. Kuraoka; T. Matsuda (Toshiro); N. Kodo (Naohiko); Y. Nakatsu (Yoshimichi); T. Mimaki; M. Mino; M. Biggerstaff (Maureen); R.D. Wood (Richard); A.M. Sijbers (Anneke); J.H.J. Hoeijmakers (Jan); K. Tanaka (Kiyoji)

    1995-01-01

    textabstractThe human XPA and ERCC1 proteins, which are involved in early steps of nucleotide excision repair of DNA, specifically interacted in an in vitro binding assay and a yeast two-hybrid assay. A stretch of consecutive glutamic acid residues in XPA was needed for binding to ERCC1. Binding of

  11. DNA binding specificity and cleavage activity of Pacmmar transposase.

    Science.gov (United States)

    Delaurière, Laurence; Chénais, Benoît; Pradier, Elisabeth; Hardivillier, Yann; Renault, Sylvaine; Casse, Nathalie

    2009-08-04

    Mariner-like elements (MLEs) are members of the Tc1/mariner superfamily of transposable elements which transpose by a "cut and paste" mechanism. Most of the MLEs characterized to date are transpositionally inactive due to the accumulation of mutations in their transposase gene. Here, we report the biochemical study of two copies of the Pacmmar element (Pacmmar1.1 and Pacmmar1.2), isolated from the coastal crab Pachygrapsus marmoratus. These two copies present an open reading frame encoding a putative active transposase. Using an in vitro transposition assay, we show that Pacmmar transposases are unable to perform by themselves the transposition reaction. However, we demonstrate by an electrophoretic mobility shift assay that both transposases bind specifically to the inverted terminal repeat of the Pacmmar element. Moreover, an in vitro cleavage assay showed that both transposases have the capacity to cleave the transposon. The in vitro cleavage activity of Pacmmar transposases appears imprecise, suggesting the requirement of specific host factors or the presence of mutations which have modified the cleavage specificity of the enzyme.

  12. DNA binding by the plant-specific NAC transcription factors in crystal and solution

    DEFF Research Database (Denmark)

    Welner, Ditte Hededam; Lindemose, Søren; Grossmann, J. Günter

    2012-01-01

    angle X-ray scattering on complexes with oligonucleotides, mutagenesis and (DNase I and uranyl photo-) footprinting, is combined to form a structural view of DNA-binding, and for the first time provide experimental evidence for the speculated relationship between plant-specific NAC proteins, WRKY....... The structure of the DNA-binding NAC domain of ANAC019 has previously been determined by X-ray crystallography, revealing a dimeric and predominantly ß-fold structure, but the mode of binding to cognate DNA has remained elusive. In the present study, information from low resolution X-ray structures and small...... transcription factors and the mammalian GCM (Glial cell missing) transcription factors, which all use a ß-strand motif for DNA-binding. The structure shows that the NAC domain inserts the edge of its core ß-sheet into the major groove, while leaving the DNA largely undistorted. The structure of the NAC-DNA...

  13. Specific binding of eukaryotic ORC to DNA replication origins depends on highly conserved basic residues.

    Science.gov (United States)

    Kawakami, Hironori; Ohashi, Eiji; Kanamoto, Shota; Tsurimoto, Toshiki; Katayama, Tsutomu

    2015-10-12

    In eukaryotes, the origin recognition complex (ORC) heterohexamer preferentially binds replication origins to trigger initiation of DNA replication. Crystallographic studies using eubacterial and archaeal ORC orthologs suggested that eukaryotic ORC may bind to origin DNA via putative winged-helix DNA-binding domains and AAA+ ATPase domains. However, the mechanisms how eukaryotic ORC recognizes origin DNA remain elusive. Here, we show in budding yeast that Lys-362 and Arg-367 residues of the largest subunit (Orc1), both outside the aforementioned domains, are crucial for specific binding of ORC to origin DNA. These basic residues, which reside in a putative disordered domain, were dispensable for interaction with ATP and non-specific DNA sequences, suggesting a specific role in recognition. Consistent with this, both residues were required for origin binding of Orc1 in vivo. A truncated Orc1 polypeptide containing these residues solely recognizes ARS sequence with low affinity and Arg-367 residue stimulates sequence specific binding mode of the polypeptide. Lys-362 and Arg-367 residues of Orc1 are highly conserved among eukaryotic ORCs, but not in eubacterial and archaeal orthologs, suggesting a eukaryote-specific mechanism underlying recognition of replication origins by ORC.

  14. Physical organization of DNA by multiple non-specific DNA-binding modes of integration host factor (IHF.

    Directory of Open Access Journals (Sweden)

    Jie Lin

    Full Text Available The integration host factor (IHF is an abundant nucleoid-associated protein and an essential co-factor for phage λ site-specific recombination and gene regulation in E. coli. Introduction of a sharp DNA kink at specific cognate sites is critical for these functions. Interestingly, the intracellular concentration of IHF is much higher than the concentration needed for site-specific interactions, suggesting that non-specific binding of IHF to DNA plays a role in the physical organization of bacterial chromatin. However, it is unclear how non-specific DNA association contributes to DNA organization. By using a combination of single DNA manipulation and atomic force microscopy imaging methods, we show here that distinct modes of non-specific DNA binding of IHF result in complex global DNA conformations. Changes in KCl and IHF concentrations, as well as tension applied to DNA, dramatically influence the degree of DNA-bending. In addition, IHF can crosslink DNA into a highly compact DNA meshwork that is observed in the presence of magnesium at low concentration of monovalent ions and high IHF-DNA stoichiometries. Our findings provide important insights into how IHF contributes to bacterial chromatin organization, gene regulation, and biofilm formation.

  15. Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

    KAUST Repository

    Lee, Seung-Joo

    2010-03-28

    DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5\\'-TGGTC-3\\') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.

  16. Halenaquinone, a chemical compound that specifically inhibits the secondary DNA binding of RAD51.

    Science.gov (United States)

    Takaku, Motoki; Kainuma, Takashi; Ishida-Takaku, Takako; Ishigami, Shintaro; Suzuki, Hidekazu; Tashiro, Satoshi; van Soest, Rob W M; Nakao, Yoichi; Kurumizaka, Hitoshi

    2011-04-01

    Mutations and single-nucleotide polymorphisms affecting RAD51 gene function have been identified in several tumors, suggesting that the inappropriate expression of RAD51 activity may cause tumorigenesis. RAD51 is an essential enzyme for the homologous recombinational repair (HRR) of DNA double-strand breaks. In the HRR pathway, RAD51 catalyzes the homologous pairing between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is the central step of the HRR pathway. To identify a chemical compound that regulates the homologous-pairing activity of RAD51, in the present study, we screened crude extract fractions from marine sponges by the RAD51-mediated homologous-pairing assay. Halenaquinone was identified as an inhibitor of the RAD51 homologous-pairing activity. A surface plasmon resonance analysis indicated that halenaquinone directly bound to RAD51. Intriguingly, halenaquinone specifically inhibited dsDNA binding by RAD51 alone or the RAD51-ssDNA complex, but only weakly affected the RAD51-ssDNA binding. In vivo, halenaquinone significantly inhibited the retention of RAD51 at double-strand break sites. Therefore, halenaquinone is a novel type of RAD51 inhibitor that specifically inhibits the RAD51-dsDNA binding. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  17. DNA recognition by F factor TraI36: highly sequence-specific binding of single-stranded DNA.

    Science.gov (United States)

    Stern, J C; Schildbach, J F

    2001-09-25

    The TraI protein has two essential roles in transfer of conjugative plasmid F Factor. As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid origin of transfer (oriT), cutting the DNA strand that is transferred to the recipient cell. TraI also acts as a helicase, presumably unwinding the plasmid strands prior to transfer. As an essential feature of its nicking activity, TraI is capable of binding and cleaving single-stranded DNA oligonucleotides containing an oriT sequence. The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-terminal domain of TraI that retains the sequence-specific nucleolytic activity. TraI36 recognition is highly sequence-specific for an 11-base region of oriT, with single base changes reducing affinity by as much as 8000-fold. The binding data correlate with plasmid mobilization efficiencies: plasmids containing sequences bound with lower affinities by TraI36 are transferred between cells at reduced frequencies. In addition to the requirement for high affinity binding to oriT, efficient in vitro nicking and in vivo plasmid mobilization requires a pyrimidine immediately 5' of the nick site. The high sequence specificity of TraI single-stranded DNA recognition suggests that despite its recognition of single-stranded DNA, TraI is capable of playing a major regulatory role in initiation and/or termination of plasmid transfer.

  18. Conformational and physicochemical DNA features specific for transcription factor binding sites.

    Science.gov (United States)

    Ponomarenko, J V; Ponomarenko, M P; Frolov, A S; Vorobyev, D G; Overton, G C; Kolchanov, N A

    1999-01-01

    A reliable recognition of transcription factor binding sites is essential for analysis of regulatory genomic sequences. The experimental data make evident an important role of DNA conformational features for site functioning. However, Internet-available tools for revealing conformational and physicochemical DNA features significant for the site functioning and subsequent use of these features for site recognition have not been developed up to now. We suggest an approach for revealing significant conformational and physicochemical properties of functional sites implemented in the database B-DNA-VIDEO. This database is designed to study the sets of various transcription factor binding sites, providing evidence that transcription factor binding sites are characterized by specific sets of significant conformational and physicochemical DNA properties. For a fixed site, by using the B-DNA features selected for this site recognition, the C-program recognizing this site may be generated, control tested and stored in the database B-DNA-VIDEO. Each B-DNA-VIDEO entry links to the Web-applet recognizing the site, whose significant B-DNA features are stored in this entry as the 'site recognition programs'. The pairwise linked entry-applet pairs are compiled within the B-DNA-VIDEO system, which is simultaneously the database and the program tools package applicable immediately for recognizing the sites stored in the database. Indeed, this is the novelty. Hence, B-DNA-VIDEO is the Web resource of both 'searching for static data' and 'active computation' type, that is why it was called an 'activated database'. B-DNA-VIDEO is available at http://wwwmgs.bionet.nsc.ru/systems/BDNAVideo/ and the mirror site at http://www.cbil.upenn.edu/mgs/systems/c onsfreq/.

  19. Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

    Science.gov (United States)

    Brand, Luise H.; Fischer, Nina M.; Harter, Klaus; Kohlbacher, Oliver; Wanke, Dierk

    2013-01-01

    WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function. PMID:23975197

  20. In vitro selection of zinc fingers with altered DNA-binding specificity.

    Science.gov (United States)

    Jamieson, A C; Kim, S H; Wells, J A

    1994-05-17

    We have used random mutagenesis and phage display to alter the DNA-binding specificity of Zif268, a transcription factor that contains three zinc finger domains. Four residues in the helix of finger 1 of Zif268 that potentially mediate DNA binding were identified from an X-ray structure of the Zif268-DNA complex. A library was constructed in which these residues were randomly mutated and the Zif268 variants were fused to a truncated version of the gene III coat protein on the surface of M13 filamentous phage particles. The phage displayed the mutant proteins in a monovalent fashion and were sorted by repeated binding and elution from affinity matrices containing different DNA sequences. When the matrix contained the natural nine base pair operator sequence 5'-GCG-TGG-GCG-3', native-like zinc fingers were isolated. New finger 1 variants were found by sorting with two different operators in which the singly modified triplets, GTG and TCG, replaced the native finger 1 triplet, GCG. Overall, the selected finger 1 variants contained a preponderance of polar residues at the four sites. Interestingly, the net charge of the four residues in any selected finger never derived more that one unit from neutrality despite the fact that about half the variants contained three or four charged residues over the four sites. Measurements of the dissociation constants for two of these purified finger 1 variants by gel-shift assay showed their specificities to vary over a 10-fold range, with the greatest affinity being for the DNA binding site for which they were sorted.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

    Science.gov (United States)

    Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

    2016-03-01

    DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

  2. DNA-binding specificities of plant transcription factors and their potential to define target genes.

    Science.gov (United States)

    Franco-Zorrilla, José M; López-Vidriero, Irene; Carrasco, José L; Godoy, Marta; Vera, Pablo; Solano, Roberto

    2014-02-11

    Transcription factors (TFs) regulate gene expression through binding to cis-regulatory specific sequences in the promoters of their target genes. In contrast to the genetic code, the transcriptional regulatory code is far from being deciphered and is determined by sequence specificity of TFs, combinatorial cooperation between TFs and chromatin competence. Here we addressed one of these determinants by characterizing the target sequence specificity of 63 plant TFs representing 25 families, using protein-binding microarrays. Remarkably, almost half of these TFs recognized secondary motifs, which in some cases were completely unrelated to the primary element. Analyses of coregulated genes and transcriptomic data from TFs mutants showed the functional significance of over 80% of all identified sequences and of at least one target sequence per TF. Moreover, combining the target sequence information with coexpression analysis we could predict the function of a TF as activator or repressor through a particular DNA sequence. Our data support the correlation between cis-regulatory elements and the sequence determined in vitro using the protein-binding microarray and provides a framework to explore regulatory networks in plants.

  3. Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes

    DEFF Research Database (Denmark)

    Peng, Xu; Brügger, Kim; Shen, Biao

    2003-01-01

    that are identical in sequence to one of the repeat variants in the S. solfataricus chromosome. Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from S. solfataricus. A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced. The protein is N...... terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function. It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure. It also...... with a helix-turn-helix DNA-binding motif. Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the Sulfolobus genus and in the closely related Acidianus genus. We infer that the genus-specific protein induces an opening of the structure...

  4. DNA-binding specificity and molecular functions of NAC transcription factors

    DEFF Research Database (Denmark)

    Olsen, Addie Nina; Ernst, Heidi Asschenfeldt; Lo Leggio, Leila

    2005-01-01

    . The ability of NAC proteins to dimerize and to bind DNAwas analysed by structure-based mutagenesis. This identified two salt bridge-forming residues essential for NAC protein dimerization. Alteration of basic residues in a loop region containing several highly conserved residues abolished DNA binding. Thus...

  5. Specific interaction of IP6 with human Ku70/80, the DNA-binding subunit of DNA-PK.

    Science.gov (United States)

    Hanakahi, Les A; West, Stephen C

    2002-04-15

    In eukaryotic cells, DNA double-strand breaks can be repaired by non-homologous end-joining, a process dependent upon Ku70/80, XRCC4 and DNA ligase IV. In mammals, this process also requires DNA-PK(cs), the catalytic subunit of the DNA-dependent protein kinase DNA-PK. Previously, inositol hexakisphosphate (IP6) was shown to be bound by DNA-PK and to stimulate DNA-PK-dependent end-joining in vitro. Here, we localize IP6 binding to the Ku70/80 subunits of DNA- PK, and show that DNA-PK(cs) alone exhibits no detectable affinity for IP6. Moreover, proteolysis mapping of Ku70/80 in the presence and absence of IP6 indicates that binding alters the conformation of the Ku70/80 heterodimer. The yeast homologue of Ku70/80, yKu70/80, fails to bind IP6, indicating that the function of IP6 in non-homologous end-joining, like that of DNA-PK(cs), is unique to the mammalian end-joining process.

  6. Eubacterial SpoVG homologs constitute a new family of site-specific DNA-binding proteins.

    Directory of Open Access Journals (Sweden)

    Brandon L Jutras

    Full Text Available A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins.

  7. Structure and DNA-binding of meiosis-specific protein Hop2

    Science.gov (United States)

    Zhou, Donghua; Moktan, Hem; Pezza, Roberto

    2014-03-01

    Here we report structure elucidation of the DNA binding domain of homologous pairing protein 2 (Hop2), which is important to gene diversity when sperms and eggs are produced. Together with another protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. However, the structural and biochemical bases for the function of Hop2 and Mnd1 have not been well understood. As a first step toward such understanding, we recently solved the structure for the N-terminus of Hop2 (1-84) using solution NMR. This fragment shows a typical winged-head conformation with recognized DNA binding activity. DNA interacting sites were then investigated by chemical shift perturbations in a titration experiment. Information of these sites was used to guide protein-DNA docking with MD simulation, revealing that helix 3 is stably lodged in the DNA major groove and that wing 1 (connecting strands 2 and 3) transiently comes in contact with the minor groove in nanosecond time scale. Mutagenesis analysis further confirmed the DNA binding sites in this fragment of the protein.

  8. Molecular and Functional Characterization of ssDNA Aptamers that Specifically Bind Leishmania infantum PABP

    Science.gov (United States)

    Guerra-Pérez, Natalia; Ramos, Edurne; García-Hernández, Marta; Pinto, Celia; Soto, Manuel; Martín, M. Elena; González, Víctor M.

    2015-01-01

    Summary A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study. Results We have selected a ssDNA aptamer population against a recombinant 6xHIS–LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes. Conclusion Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be

  9. Crystal structure of an archaeal specific DNA-binding protein (Ape10b2) from Aeropyrum pernix K1.

    Science.gov (United States)

    Kumarevel, Thirumananseri; Sakamoto, Keiko; Gopinath, Subash C B; Shinkai, Akeo; Kumar, Penmetcha K R; Yokoyama, Shigeyuki

    2008-05-15

    DNA binding proteins are essential in all organisms, and they play important roles in both compacting and regulating the genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of Alba or Sso10b, which is a small, abundant, basic protein that binds DNA. Here, we present the crystal structure of Ape10b2 from Aeropyrum pernix K1 at 1.70 A. Although the overall structure resembles the known Alba protein fold, a significant conformational change was observed in the loop regions. Specifically, the L5 loop is slightly longer, as compared to those of other known proteins, and the flexibility of this loop may facilitate the interaction with double stranded DNA. In addition, we showed that Ape10b2 binds to 16 and 39 bp duplex DNAs with high affinity. On the basis of our analyses, we have created a putative protein-DNA complex model. 2007 Wiley-Liss, Inc.

  10. Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans--A Nucleic Acid Binding Protein with Broad Substrate Specificity.

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    Marcin Olszewski

    Full Text Available SSB (single-stranded DNA-binding proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein. This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity. The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m is 100.2 °C as shown by differential scanning calorimetry (DSC analysis.NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

  11. The Arabidopsis SUPERMAN protein is able to specifically bind DNA through its single Cys2-His2 zinc finger motif.

    Science.gov (United States)

    Dathan, Nina; Zaccaro, Laura; Esposito, Sabrina; Isernia, Carla; Omichinski, James G; Riccio, Andrea; Pedone, Carlo; Di Blasio, Benedetto; Fattorusso, Roberto; Pedone, Paolo V

    2002-11-15

    The Arabidopsis SUPERMAN (SUP) gene has been shown to be important in maintaining the boundary between stamens and carpels, and is presumed to act by regulating cell proliferation. In this work, we show that the SUP protein, which contains a single Cys2-His2 zinc finger domain including the QALGGH sequence, highly conserved in the plant zinc finger proteins, binds DNA. Using a series of deletion mutants, it was determined that the minimal domain required for specific DNA binding (residues 15-78) includes the single zinc finger and two basic regions located on either side of this motif. Furthermore, amino acid substitutions in the zinc finger or in the basic regions, including a mutation that knocks out the function of the SUP protein in vivo (glycine 63 to aspartate), have been found to abolish the activity of the SUP DNA-binding domain. These results strongly suggest that the SUP protein functions in vivo by acting as a DNA-binding protein, likely involved in transcriptional regulation. The association of both an N-terminal and a C-terminal basic region with a single Cys2-His2 zinc finger represents a novel DNA-binding motif suggesting that the mechanism of DNA recognition adopted by the SUP protein is different from that described so far in other zinc finger proteins.

  12. Structure elucidation and DNA binding specificity of natural compounds from Cassia siamea leaves: A biophysical approach.

    Science.gov (United States)

    Parveen, Mehtab; Ahmad, Faheem; Malla, Ali Mohammed; Khan, Mohd Sohrab; Rehman, Sayeed Ur; Tabish, Mohammad; Silva, Manuela Ramos; Silva, P S Pereira

    2016-06-01

    A novel isoflavone, 5,6,7-trimethoxy-3-(3',4',5'-trimethoxyphenyl)-4H-chromen-4-one (1) along with a known pyranocoumarin, Seselin (2) have been isolated from the ethanolic extract of the leaves of Cassia siamea (Family: Fabaceae). Compound 1 has been reported for the first time from any natural source and has not been synthesized so far. Their structures were elucidated on the basis of chemical and physical evidences viz. elemental analysis, UV, FT-IR, (1)H-NMR, (13)C-NMR and mass spectral analysis. Structure of compound (1) was further authenticated by single-crystal X-ray analysis and density functional theory (DFT) calculations. A multi-technique approach employing UV-Visible spectroscopy, fluorescence, KI quenching studies, competitive displacement assay, circular dichroism and viscosity studies have been utilized to probe the extent of interaction and possible binding modes of isolated compounds (1-2) with calf thymus DNA (CT-DNA). Both the compounds were found to interact with DNA via non-intercalative binding mode with moderate proficiencies. Groove binding was the major interaction mode in the case of compound 2 while compound 1 probably interacts with DNA through electrostatic interactions. These studies provide deeper insight in understanding of DNA-drug (natural products) interaction which could be helpful to improve their bioavailability for therapeutic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

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    Kareem Mohideen-Abdul

    2017-06-01

    Full Text Available Most nuclear receptors (NRs bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR belongs to the steroid hormone nuclear receptor (SHR family and shares strong similarity in its DNA-binding domain (DBD with that of the estrogen receptor (ER. In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3, but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS, non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3, such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3, where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half

  14. Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

    Science.gov (United States)

    Mohideen-Abdul, Kareem; Tazibt, Karima; Bourguet, Maxime; Hazemann, Isabelle; Lebars, Isabelle; Takacs, Maria; Cianférani, Sarah; Klaholz, Bruno P.; Moras, Dino; Billas, Isabelle M. L.

    2017-01-01

    Most nuclear receptors (NRs) bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs) involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR) belongs to the steroid hormone nuclear receptor (SHR) family and shares strong similarity in its DNA-binding domain (DBD) with that of the estrogen receptor (ER). In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3), but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs)], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS), non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3), such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3), where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half-site is

  15. Conformational changes in DNA-binding proteins: relationships with precomplex features and contributions to specificity and stability.

    Science.gov (United States)

    Andrabi, Munazah; Mizuguchi, Kenji; Ahmad, Shandar

    2014-05-01

    Both Proteins and DNA undergo conformational changes in order to form functional complexes and also to facilitate interactions with other molecules. These changes have direct implications for the stability and specificity of the complex, as well as the cooperativity of interactions between multiple entities. In this work, we have extensively analyzed conformational changes in DNA-binding proteins by superimposing DNA-bound and unbound pairs of protein structures in a curated database of 90 proteins. We manually examined each of these pairs, unified the authors' annotations, and summarized our observations by classifying conformational changes into six structural categories. We explored a relationship between conformational changes and functional classes, binding motifs, target specificity, biophysical features of unbound proteins, and stability of the complex. In addition, we have also investigated the degree to which the intrinsic flexibility can explain conformational changes in a subset of 52 proteins with high quality coordinate data. Our results indicate that conformational changes in DNA-binding proteins contribute significantly to both the stability of the complex and the specificity of targets recognized by them. We also conclude that most conformational changes occur in proteins interacting with specific DNA targets, even though unbound protein structures may have sufficient information to interact with DNA in a nonspecific manner. Copyright © 2013 Wiley Periodicals, Inc.

  16. Noc protein binds to specific DNA sequences to coordinate cell division with chromosome segregation.

    Science.gov (United States)

    Wu, Ling Juan; Ishikawa, Shu; Kawai, Yoshikazu; Oshima, Taku; Ogasawara, Naotake; Errington, Jeff

    2009-07-08

    Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis, the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS array recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division, but that its activity is strictly localized by the interaction with NBS sites in vivo. We propose that Noc serves not only as a spatial regulator of cell division to protect the nucleoid, but also as a timing device with an important role in the coordination of chromosome segregation and cell division.

  17. p53 Specifically Binds Triplex DNA In Vitro and in Cells

    NARCIS (Netherlands)

    Brázdová, Marie; Tichý, Vlastimil; Helma, Robert; Bažantová, Pavla; Polášková, Alena; Krejčí, Aneta; Petr, Marek; Navrátilová, Lucie; Tichá, Olga; Nejedlý, Karel; Bennink, Martin L; Subramaniam, Vinod; Bábková, Zuzana; Martínek, Tomáš; Lexa, Matej; Adámik, Matej

    2016-01-01

    Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA

  18. Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein-DNA Complex.

    Science.gov (United States)

    Sinha, Kaustubh; Sangani, Sahil S; Kehr, Andrew D; Rule, Gordon S; Jen-Jacobson, Linda

    2016-11-08

    Metal ion cofactors can alter the energetics and specificity of sequence specific protein-DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein-DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg 2+ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu 3+ or two La 3+ cations bind, and two new crystal structures confirm that Lu 3+ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for 1 H- 15 N amide resonances, for 1 H- 13 C Ile-δ-CH 3 resonances, and for stereospecifically assigned Leu-δ-CH 3 and Val-γ-CH 3 resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex.

  19. Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein–DNA Complex

    Science.gov (United States)

    2016-01-01

    Metal ion cofactors can alter the energetics and specificity of sequence specific protein–DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein–DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg2+ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu3+ or two La3+ cations bind, and two new crystal structures confirm that Lu3+ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for 1H–15N amide resonances, for 1H–13C Ile-δ-CH3 resonances, and for stereospecifically assigned Leu-δ-CH3 and Val-γ-CH3 resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex. PMID:27786446

  20. Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage

    Science.gov (United States)

    Josephs, Eric A.; Kocak, D. Dewran; Fitzgibbon, Christopher J.; McMenemy, Joshua; Gersbach, Charles A.; Marszalek, Piotr E.

    2015-01-01

    CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex. PMID:26384421

  1. Non-specificity and synergy at the binding site of the carboplatin-induced DNA adduct via molecular dynamics simulations of the MutSα-DNA recognition complex

    Science.gov (United States)

    Negureanu, Lacramioara; Salsbury, Freddie R

    2013-01-01

    MutSα is the most abundant mismatch binding factor of human DNA mismatch repair (MMR) proteins. MMR maintain genetic stability by recognizing and repairing DNA defects. Failure to accomplish their function may lead to cancer. In addition, MutSα recognizes at least some types of DNA damage making it a target for anticancer agents. Here, complementing scarce experimental data, we report unique hydrogen bonding motifs associated with the recognition of the carboplatin induced DNA damage by MutSα. These data predict that carboplatin and cisplatin induced damaging DNA adducts are recognized by MutSα in a similar manner. Our simulations also indicate that loss of base pairing at the damage site results in (1) non-specific binding and (2) changes in the atomic flexibility at the lesion site and beyond. To further quantify alterations at MutSα-DNA interface in response to damage recognition non-bonding interactions and salt bridges were investigated. These data indicate (1) possible different packing and (2) disruption of the salt bridges at the MutSα-DNA interface in the damaged complex. These findings (1) underscore the general observation of disruptions at the MutSα-DNA interface and (2) highlight the nature of the anticancer effect of the carboplatin agent. The analysis was carried out from atomistic simulations. PMID:23799640

  2. Biomolecular mirror-image recognition: reciprocal chiral-specific DNA binding of synthetic enantiomers of zinc finger domain from GAGA factor.

    Science.gov (United States)

    Negi, Shigeru; Dhanasekaran, Muthu; Hirata, Tsuyoshi; Urata, Hidehito; Sugiura, Yukio

    2006-05-05

    To experimentally demonstrate the mirror-image recognition in protein and DNA interaction, we have designed a small DNA-binding peptide based on the zinc-finger domain of GAGA transcription factor. Circular dichroism data suggest that the conformations of peptide enantiomers as well as the DNA enantiomers have a mirror-image relationship. The gel mobility shift assay showed that the synthetic enantiomers of the peptide showed reciprocal chiral-specific interactions with the DNA; the natural L-peptide binds specifically with the natural D-DNA substrate, and the unnatural D-peptide binds specifically with the unnatural L-DNA substrate. The present data imply that the folding of the L- and D-enantiomers of the peptide as well as the DNA substrates are exact mirror images of each other in 3-D structure and biological activity, and generalize the chiral-specific nature of biomolecular interaction. 2006 Wiley-Liss, Inc.

  3. A SILAC-based screen for Methyl-CpG binding proteins identifies RBP-J as a DNA methylation and sequence-specific binding protein.

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    Stefanie J J Bartels

    Full Text Available BACKGROUND: DNA methylation is an epigenetic modification that plays a crucial role in a variety of biological processes. Methylated DNA is specifically bound by Methyl-CpG Binding Proteins (MBPs. Three different types of MBPs have been identified so far: the Methyl-CpG Binding Domain (MBD family proteins, three BTB/POZ-Zn-finger proteins, and UHRF1. Most of the known MBPs have been identified via homology with the MBD and Zn-finger domains as present in MeCP2 and Kaiso, respectively. It is conceivable that other proteins are capable of recognizing methylated DNA. METHODOLOGY/PRINCIPAL FINDINGS: For the purpose of identifying novel 'readers' we set up a methyl-CpG pull-down assay combined with stable-isotope labeling by amino acids in cell culture (SILAC. In a methyl-CpG pull-down with U937 nuclear extracts, we recovered several known MBPs and almost all subunits of the MBD2/NuRD complex as methylation specific binders, providing proof-of-principle. Interestingly, RBP-J, the transcription factor downstream of Notch receptors, also bound the DNA in a methylation dependent manner. Follow-up pull-downs and electrophoretic mobility shift assays (EMSAs showed that RBP-J binds methylated DNA in the context of a mutated RBP-J consensus motif. CONCLUSIONS/SIGNIFICANCE: The here described SILAC/methyl-CpG pull-down constitutes a new approach to identify potential novel DNAme readers and will advance unraveling of the complete methyl-DNA interactome.

  4. DNA binding specificity of ATAF2, a NAC domain transcription factor targeted for degradation by Tobacco mosaic virus

    Directory of Open Access Journals (Sweden)

    Wang Xiao

    2012-08-01

    Full Text Available Abstract Background Control of the host transcriptome represents a key battleground in the interaction of plants and pathogens. Specifically, plants have evolved complex defense systems that induce profound transcriptional changes in response to pathogen attack while pathogens have evolved mechanisms to subvert or disable these defenses. Several NAC transcription factors such as ATAF2 have been linked to plant defense responses, including those targeting viruses. The replication protein of Tobacco mosaic virus (TMV has been shown to interact with and target the degradation of ATAF2. These findings suggest that the transcriptional targets of ATAF2 are involved in defense against TMV. Results To detect potential ATAF2 transcriptional targets, a genomic pull-down assay was utilized to identify ATAF2 promoter binding sequences. Subsequent mobility shift and DNA footprinting assays identified a 30-bp ATAF2 binding sequence. An in vivo GUS reporter system confirmed the function of the identified 30-bp binding sequence as an ATAF2 specific transcriptional activator in planta. Gel filtration studies of purified ATAF2 protein and mutagenesis studies of the 30-bp binding sequence indicate ATAF2 functions as a dimer. Computational analysis of interacting promoter sequences identified a corresponding 25-bp A/T-rich consensus sequence with repeating [GC]AAA motifs. Upon ATAF2 induction real-time qRT-PCR studies confirmed the accumulation of select gene transcripts whose promoters contain this consensus sequence. Conclusion We report the identification of a cis-regulatory binding sequence for ATAF2. Different from other known NAC protein binding sequences, the A/T-rich ATAF2 binding motif represents a novel binding sequence for NAC family proteins. Combined this information represents a unique tool for the identification of ATAF2 target genes.

  5. Sequence Discrimination by DNA-binding Domain of ETS Family Transcription Factor PU.1 Is Linked to Specific Hydration of Protein-DNA Interface*

    Science.gov (United States)

    Poon, Gregory M. K.

    2012-01-01

    PU.1 is an essential transcription factor in normal hematopoietic lineage development. It recognizes a large number of promoter sites differing only in bases flanking a core consensus of 5′-GGAA-3′. DNA binding is mediated by its ETS domain, whose sequence selectivity directly corresponds to the transactivational activity and frequency of binding sites for full-length PU.1 in vivo. To better understand the basis of sequence discrimination, we characterized its binding properties to a high affinity and low affinity site. Despite sharing a homologous structural framework as confirmed by DNase I and hydroxyl radical footprinting, the two complexes exhibit striking heterogeneity in terms of hydration properties. High affinity binding is destabilized by osmotic stress, whereas low affinity binding is insensitive. Dimethyl sulfate footprinting showed that the major groove at the core consensus is protected in the high affinity complex but accessible in the low affinity one. Finally, destabilization of low affinity binding by salt is in quantitative agreement with the number of phosphate contacts but is substantially attenuated in high affinity binding. These observations support a mechanism of sequence discrimination wherein specifically bound water molecules couple flanking backbone contacts with base-specific interactions in a sequestered cavity at the core consensus. The implications of this model with respect to other ETS paralogs are discussed. PMID:22474303

  6. DNA end-binding specificity of human Rad50/Mre11 is influenced by ATP

    NARCIS (Netherlands)

    M. de Jager (Martijn); C. Wyman (Claire); D.C. van Gent (Dik); R. Kanaar (Roland)

    2002-01-01

    textabstractThe Rad50, Mre11 and Nbs1 complex is involved in many essential chromosomal organization processes dealing with DNA ends, including two major pathways of DNA double-strand break repair, homologous recombination and non-homologous end joining. Previous data on the

  7. Replicative senescence is associated with nuclear reorganization and with DNA methylation at specific transcription factor binding sites.

    Science.gov (United States)

    Hänzelmann, Sonja; Beier, Fabian; Gusmao, Eduardo G; Koch, Carmen M; Hummel, Sebastian; Charapitsa, Iryna; Joussen, Sylvia; Benes, Vladimir; Brümmendorf, Tim H; Reid, George; Costa, Ivan G; Wagner, Wolfgang

    2015-01-01

    Primary cells enter replicative senescence after a limited number of cell divisions. This process needs to be considered in cell culture experiments, and it is particularly important for regenerative medicine. Replicative senescence is associated with reproducible changes in DNA methylation (DNAm) at specific sites in the genome. The mechanism that drives senescence-associated DNAm changes remains unknown - it may involve stochastic DNAm drift due to imperfect maintenance of epigenetic marks or it is directly regulated at specific sites in the genome. In this study, we analyzed the reorganization of nuclear architecture and DNAm changes during long-term culture of human fibroblasts and mesenchymal stromal cells (MSCs). We demonstrate that telomeres shorten and shift towards the nuclear center at later passages. In addition, DNAm profiles, either analyzed by MethylCap-seq or by 450k IlluminaBeadChip technology, revealed consistent senescence-associated hypermethylation in regions associated with H3K27me3, H3K4me3, and H3K4me1 histone marks, whereas hypomethylation was associated with chromatin containing H3K9me3 and lamina-associated domains (LADs). DNA hypermethylation was significantly enriched in the vicinity of genes that are either up- or downregulated at later passages. Furthermore, specific transcription factor binding motifs (e.g. EGR1, TFAP2A, and ETS1) were significantly enriched in differentially methylated regions and in the promoters of differentially expressed genes. Senescence-associated DNA hypermethylation occurs at specific sites in the genome and reflects functional changes in the course of replicative senescence. These results indicate that tightly regulated epigenetic modifications during long-term culture contribute to changes in nuclear organization and gene expression.

  8. Halenaquinone, a chemical compound that specifically inhibits the secondary DNA binding of RAD51

    NARCIS (Netherlands)

    Takaku, M.; Kainuma, T.; Ishida-Takaku, T.; Ishigami, S.; Suzuki, H.; Tashiro, S.; van Soest, R.W.M.; Nakao, Y.; Kurumizaka, H.

    2011-01-01

    Mutations and single-nucleotide polymorphisms affecting RAD51 gene function have been identified in several tumors, suggesting that the inappropriate expression of RAD51 activity may cause tumorigenesis. RAD51 is an essential enzyme for the homologous recombinational repair (HRR) of DNA

  9. Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

    Energy Technology Data Exchange (ETDEWEB)

    Adámik, Matej [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Bažantová, Pavla [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava (Czech Republic); Navrátilová, Lucie; Polášková, Alena [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Pečinka, Petr [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Biology and Ecology, Faculty of Science, University of Ostrava, Chittussiho 10, 701 03 Ostrava (Czech Republic); Holaňová, Lucie [Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 61242 Brno (Czech Republic); Tichý, Vlastimil [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Brázdová, Marie, E-mail: maruska@ibp.cz [Institute of Biophysics, Academy of Science of the Czech Republic, v.v.i., Královopolská 135, 612 65 Brno (Czech Republic); Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 61242 Brno (Czech Republic)

    2015-01-02

    Highlights: • DNA binding of p53 family core domains is inhibited by cadmium, cobalt and nickel. • Binding to DNA protects p53 family core domains from metal induced inhibition. • Cadmium, cobalt and nickel induced inhibition was reverted by EDTA in vitro. - Abstract: Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50 μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA–protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA–p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed.

  10. Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair

    Science.gov (United States)

    Shinohara, Takeshi; Ikawa, Shukuko; Iwasaki, Wakana; Hiraki, Toshiki; Hikima, Takaaki; Mikawa, Tsutomu; Arai, Naoto; Kamiya, Nobuo; Shibata, Takehiko

    2015-01-01

    In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions. PMID:25561575

  11. De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

    KAUST Repository

    Mahfouz, Magdy M.

    2011-01-24

    Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

  12. Delta9-tetrahydrocannabinol increases sequence-specific AP-1 DNA-binding activity and Fos-related antigens in the rat brain.

    Science.gov (United States)

    Porcella, A; Gessa, G L; Pani, L

    1998-05-01

    Delta-9-tetrahydrocannabinol (delta9-THC), the psychoactive principle of marijuana, has been shown to upregulate the mRNA levels of immediate-early genes in the rat brain. Using electrophoretic mobility-shift assay and one-dimensional Western blot, we here report that delta9-THC increases Activator protein-1 (AP-1) DNA-binding and Fos-related antigen activity in discrete areas of the rat brain. One hour after the intraperitoneal administration of delta9-THC at a dose of 10 or 15 mg/kg, AP-1 DNA-binding activity in the nucleus accumbens increased by 33 and 49%, respectively, while Western blot showed an increase in both c-Fos, FosB, Fra-1 (Fos-related antigen) and Fra-2. In the cingulate cortex and caudate-putamen, delta9-THC significantly increased AP-1 DNA-binding activity only at the highest dose used (57 and 71%, respectively). While in the caudate-putamen the increase in AP-1 DNA binding was mainly due to an elevation of the c-Fos and FosB proteins, the same phenomenon depended on the FosB, Fra-1 and Fra-2 peptides in the cingulate cortex. The effect of delta9-THC on the AP-1 DNA binding and the Fos-related antigens in the nucleus accumbens was blocked by the specific cannabinoid antagonist SR141716 A (3 mg/kg i.p.). delta9-THC failed to modify Specificity protein 1 (Sp1) DNA-binding activity. The results indicate that delta9-THC activates gene coding for AP-1 DNA-binding proteins by acting on cannabinoid receptors, and induces a different transcriptional program on the early-immediate gene of the Fos family, in different areas in the rat brain, suggesting that this mechanism might be involved in the central actions of cannabinoids.

  13. The Arabidopsis SUPERMAN protein is able to specifically bind DNA through its single Cys2–His2 zinc finger motif

    Science.gov (United States)

    Dathan, Nina; Zaccaro, Laura; Esposito, Sabrina; Isernia, Carla; Omichinski, James G.; Riccio, Andrea; Pedone, Carlo; Di Blasio, Benedetto; Fattorusso, Roberto; Pedone, Paolo V.

    2002-01-01

    The Arabidopsis SUPERMAN (SUP) gene has been shown to be important in maintaining the boundary between stamens and carpels, and is presumed to act by regulating cell proliferation. In this work, we show that the SUP protein, which contains a single Cys2–His2 zinc finger domain including the QALGGH sequence, highly conserved in the plant zinc finger proteins, binds DNA. Using a series of deletion mutants, it was determined that the minimal domain required for specific DNA binding (residues 15–78) includes the single zinc finger and two basic regions located on either side of this motif. Furthermore, amino acid substitutions in the zinc finger or in the basic regions, including a mutation that knocks out the function of the SUP protein in vivo (glycine 63 to aspartate), have been found to abolish the activity of the SUP DNA-binding domain. These results strongly suggest that the SUP protein functions in vivo by acting as a DNA-binding protein, likely involved in transcriptional regulation. The association of both an N-terminal and a C-terminal basic region with a single Cys2–His2 zinc finger represents a novel DNA-binding motif suggesting that the mechanism of DNA recognition adopted by the SUP protein is different from that described so far in other zinc finger proteins. PMID:12433998

  14. Nucleolin binds specifically to an AP-1 DNA sequence and represses AP1-dependent transactivation of the matrix metalloproteinase-13 gene.

    Science.gov (United States)

    Samuel, Shaija; Twizere, Jean-Claude; Beifuss, Katherine K; Bernstein, Lori R

    2008-01-01

    Transcriptional regulation via activator protein-1 (AP-1) protein binding to AP-1 binding sites within gene promoter regions of AP-1 target genes plays a key role in controlling cellular invasion, proliferation, and oncogenesis, and is important to pathogenesis of arthritis and cardiovascular disease. To identify new proteins that interact with the AP-1 DNA binding site, we performed the DNA affinity chromatography-based Nucleotide Affinity Preincubation Specificity TEst of Recognition (NAPSTER) assay, and discovered a 97 kDa protein that binds in vitro to a minimal AP-1 DNA sequence element. Mass spectrometric fragmentation sequencing determined that p97 is nucleolin. Immunoblotting of DNA affinity-purified material with anti-nucleolin antibodies confirmed this identification. Nucleolin also binds the AP-1 site in gel shift assays. Nucleolin interacts in NAPSTER with the AP-1 site within the promoter sequence of the metalloproteinase-13 gene (MMP-13), and binds in vivo in chromatin immunoprecipitation assays in the vicinity of the AP-1 site in the MMP-13 promoter. Overexpression of nucleolin in human HeLa cervical carcinoma cells significantly represses AP-1 dependent gene transactivation of a minimal AP-1 reporter construct and of an MMP-13 promoter reporter sequence. This is the first report of nucleolin binding and transregulation at the AP-1 site. (c) 2007 Wiley-Liss, Inc.

  15. The interaction of membrane DNA-binding protein with DNA

    Science.gov (United States)

    Gabrielyan, A. G.; Arakhelyan, H. H.; Zakharyan, R. A.

    1994-07-01

    A 31-kDa protein specifically binding to double-stranded DNA (ds-DNA) was isolated from plasmatic membranes of rat liver cells by means of affinity chromatography and high performance liquid chromatography (HPLC). Some of the properties of this protein were determined. Judging by the UV and circular dichroism spectroscopic data, the protein forms a complex with DNA, stabilizing its native structure, mainly in the regions rich in AT pairs. The 31-kDa protein-pAO3 plasmid DNA binding constant was determined by nitrocellulose filter analysis of protein labelled DNA complexes. The results obtained correspond to cooperative binding with DNA molecules of extended interacting ligands, having AT specificity. A possible role of the 31-kDa protein in DNA transmembrane transition processes is discussed.

  16. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E; Krogsdam, A M; Jorgensen, H F

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

  17. Methylation-targeted specificity of the DNA binding proteins R.DpnI and MeCP2 studied by molecular dynamics simulations.

    Science.gov (United States)

    Shanak, Siba; Ulucan, Ozlem; Helms, Volkhard

    2017-05-01

    DNA methylation plays a major role in organismal development and the regulation of gene expression. Methylation of cytosine bases and the cellular roles of methylated cytosine in eukaryotes are well established, as well as methylation of adenine bases in bacterial genomes. Still lacking, however, is a general mechanistic understanding, in structural and thermodynamic terms, of how proteins recognize methylated DNA. Toward this aim, we present the results of molecular dynamics simulations, alchemical free energy perturbation, and MM-PBSA calculations to explain the specificity of the R.DpnI enzyme from Streptococcus pneumonia in binding to adenine-methylated DNA with both its catalytic and winged-helix domains. We found that adenine-methylated DNA binds more favorably to the catalytic subunit of R.DpnI (-4 kcal mol-1) and to the winged-helix domain (-1.6 kcal mol-1) than non-methylated DNA. In particular, N6-adenine methylation is found to enthalpically stabilize binding to R.DpnI. In contrast, C5-cytosine methylation entropically favors complexation by the MBD domain of the human MeCP2 protein with almost no contribution of the binding enthalpy.

  18. Subunit-specific protein footprinting reveals significant structural rearrangements and a role for N-terminal Lys-14 of HIV-1 Integrase during viral DNA binding.

    Science.gov (United States)

    Zhao, Zhuojun; McKee, Christopher J; Kessl, Jacques J; Santos, Webster L; Daigle, Janet E; Engelman, Alan; Verdine, Gregory; Kvaratskhelia, Mamuka

    2008-02-29

    To identify functional contacts between HIV-1 integrase (IN) and its viral DNA substrate, we devised a new experimental strategy combining the following two methodologies. First, disulfide-mediated cross-linking was used to site-specifically link select core and C-terminal domain amino acids to respective positions in viral DNA. Next, surface topologies of free IN and IN-DNA complexes were compared using Lys- and Arg-selective small chemical modifiers and mass spectrometric analysis. This approach enabled us to dissect specific contacts made by different monomers within the multimeric complex. The foot-printing studies for the first time revealed the importance of a specific N-terminal domain residue, Lys-14, in viral DNA binding. In addition, a DNA-induced conformational change involving the connection between the core and C-terminal domains was observed. Site-directed mutagenesis experiments confirmed the importance of the identified contacts for recombinant IN activities and virus infection. These new findings provided major constraints, enabling us to identify the viral DNA binding channel in the active full-length IN multimer. The experimental approach described here has general application to mapping interactions within functional nucleoprotein complexes.

  19. Role of cysteines in the stability and DNA-binding activity of the hypochlorite-specific transcription factor HypT.

    Directory of Open Access Journals (Sweden)

    Adrian Drazic

    Full Text Available Reactive oxygen species are important components of the immune response. Hypochlorite (HOCl is produced by neutrophils to kill invading microorganisms. The bactericidal activity of HOCl is due to proteome-wide unfolding and oxidation of proteins at cysteine and methionine residues. Escherichia coli cells are protected from HOCl-killing by the previously identified dodecameric transcription factor HypT (YjiE. Here, we aimed to unravel whether HOCl activates HypT directly or via a reaction product of HOCl with a cellular component. Bacterial viability assays and analysis of target gene regulation indicate that HypT is highly specific to activation by HOCl and that no reaction products of HOCl such as monochloramine, hydroxyl radicals, or methionine sulfoxide activate HypT in vivo. Surprisingly, purified HypT lost its DNA-binding activity upon incubation with HOCl or reaction products that oxidize HypT to form a disulfide-linked dimer, and regained DNA-binding activity upon reduction. Thus, we postulate that the cysteines in HypT contribute to control the DNA-binding activity of HypT in vitro. HypT contains five cysteine residues; a HypT mutant with all cysteines substituted by serine is aggregation-prone and forms tetramers in addition to the typical dodecamers. Using single and multiple cysteine-to-serine mutants, we identified Cys150 to be required for stability and Cys4 being important for oligomerization of HypT to dodecamers. Further, oxidation of Cys4 is responsible for the loss of DNA-binding of HypT upon oxidation in vitro. It appears that Cys4 oxidation upon conditions that are insufficient to stimulate the DNA-binding activity of HypT prevents unproductive interactions of HypT with DNA. Thus, Cys4 oxidation may be a check point in the activation process of HypT.

  20. An improved SELEX-Seq strategy for characterizing DNA-binding specificity of transcription factor: NF-κB as an example.

    Directory of Open Access Journals (Sweden)

    Guangming Gu

    Full Text Available SELEX-Seq is now the optimal high-throughput technique for characterizing DNA-binding specificities of transcription factors. In this study, we introduced an improved EMSA-based SELEX-Seq strategy with several advantages. The improvements of this strategy included: (1 using a FAM-labeled probe to track protein-DNA complex in polyacrylamide gel for rapidly recovering the protein-bound dsDNA without relying on traditional radioactive labeling or ethidium bromide staining; (2 monitoring the specificity of SELEX selection by detecting a positive and negative sequence doped into the input DNAs used in each round with PCR amplification; (3 using nested PCR to ensure the specificity of PCR amplification of the selected DNAs after each round; (4 using the nucleotides added at the 5' end of the nested PCR primers as the split barcode to code DNAs from various rounds for multiplexing sequencing samples. The split barcode minimized selection times and thus greatly simplified the current SELEX-Seq procedure. The reliability of the strategy was demonstrated by performing a successful SELEX-Seq of a well-known transcription factor, NF-κB. Therefore, this study provided a useful SELEX-Seq strategy for characterizing DNA-binding specificities of transcription factors.

  1. Improved detection of deeply invasive candidiasis with DNA aptamers specific binding to (1→3)-β-D-glucans from Candida albicans.

    Science.gov (United States)

    Tang, X-L; Hua, Y; Guan, Q; Yuan, C-H

    2016-04-01

    Deeply invasive or disseminated candidiasis is a serious and often fatal complication that can occur frequently in immuno-compromised individuals. However, conventional diagnostic methods of Candida albicans display low sensitivity and lack of specificity; the development of rapid and accurate detection methods remains a high priority. Aptamers are single-strand DNA or RNA oligonucleotides that specifically bind to target molecules with high affinity. In this study, we sought to screen high-affinity DNA aptamers that specifically bound to (1→3)-β-D-glucans from cell wall of Candida albicans using a systematic evolution of ligands by exponential enrichment (SELEX) technique, and further evaluate the diagnostic potential for invasive or disseminated candidiasis with selected aptamers. (1→3)-β-D-glucans was purified from Candida albicans, and two single DNA aptamers (designated as AU1 and AD1) were selected. Analysis of dissociation constants and binding domains further revealed that these two selected single DNA aptamers (AU1 and AD1) showed high binding affinity (AD1: Kd = 79.76 nM, AD1: Kd = 103.7 nM) and did not bind to the same domain of (1→3)-β-D-glucans. Next, we further detected (1→3)-β-D-glucans in serum samples from different groups of patients with Candida albicans infection or simple bacterial infection by using a double-aptamer sandwich enzyme-linked oligonucleotide assay (ELONA). The results showed that the sensitivity and specificity of this aptamer-based sandwich ELONA were 92.31 % and 91.94 % respectively. Thus, our study suggests that AU1 and AD1 have potential application for the differentiate diagnosis of deeply invasive candidiasis and provide valuable clues for designing diagnostic agents for the identification of invasive fungal infection.

  2. Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses.

    Science.gov (United States)

    Niyonzima, Nixon; Lambert, Abigail R; Werther, Rachel; De Silva Feelixge, Harshana; Roychoudhury, Pavitra; Greninger, Alexander L; Stone, Daniel; Stoddard, Barry L; Jerome, Keith R

    2017-07-01

    The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while binding its cognate target site with a slightly lower, but still sub-nanomolar affinity. When incorporated into otherwise identical megaTAL protein scaffolds, these two nucleases display significantly different activity and toxicity profiles in cellulo. The structural basis for reprogrammed DNA cleavage specificity was further examined via high-resolution X-ray crystal structures of both enzymes. This analysis illustrated the altered protein-DNA contacts produced by mutagenesis and selection, that resulted both in altered readout of those based and a necessary reduction in DNA binding affinity that were necessary to improve specificity across the target site. The results of this study provide an illustrative example of the potential (and the challenges) associated with the use of surface display and flow cytometry for the retargeting and optimization of enzymes that act on nucleic acid substrates in a sequence-specific manner. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  3. Decorin binding proteins of Borrelia burgdorferi promote arthritis development and joint specific post-treatment DNA persistence in mice.

    Directory of Open Access Journals (Sweden)

    Jemiina Salo

    Full Text Available Decorin binding proteins A and B (DbpA and B of Borrelia burgdorferi are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of B. burgdorferi after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with B. burgdorferi strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the B. burgdorferi strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were B. burgdorferi culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, B. burgdorferi DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on B. burgdorferi surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.

  4. Decorin binding proteins of Borrelia burgdorferi promote arthritis development and joint specific post-treatment DNA persistence in mice.

    Science.gov (United States)

    Salo, Jemiina; Jaatinen, Annukka; Söderström, Mirva; Viljanen, Matti K; Hytönen, Jukka

    2015-01-01

    Decorin binding proteins A and B (DbpA and B) of Borrelia burgdorferi are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of B. burgdorferi after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with B. burgdorferi strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the B. burgdorferi strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were B. burgdorferi culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, B. burgdorferi DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on B. burgdorferi surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.

  5. SEARCHPATTOOL: a new method for mining the most specific frequent patterns for binding sites with application to prokaryotic DNA sequences

    Directory of Open Access Journals (Sweden)

    Nason Martha

    2007-09-01

    Full Text Available Abstract Background Computational methods to predict transcription factor binding sites (TFBS based on exhaustive algorithms are guaranteed to find the best patterns but are often limited to short ones or impose some constraints on the pattern type. Many patterns for binding sites in prokaryotic species are not well characterized but are known to be large, between 16–30 base pairs (bp and contain at least 2 conserved bases. The length of prokaryotic species promoters (about 400 bp and our interest in studying a small set of genes that could be a cluster of co-regulated genes from microarray experiments led to the development of a new exhaustive algorithm targeting these large patterns. Results We present Searchpattool, a new method to search for and select the most specific (conservative frequent patterns. This method does not impose restrictions on the density or the structure of the pattern. The best patterns (motifs are selected using several statistics, including a new application of a z-score based on the number of matching sequences. We compared Searchpattool against other well known algorithms on a Bacillus subtilis group of 14 input sequences and found that in our experiments Searchpattool always performed the best based on performance scores. Conclusion Searchpattool is a new method for pattern discovery relative to transcription factor binding sites for species or genes with short promoters. It outputs the most specific significant patterns and helps the biologist to choose the best candidates.

  6. Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. Methods of increasing binding affinity and sequence specificity of peptide nucleic acids...

  7. SUMO-1 possesses DNA binding activity

    Directory of Open Access Journals (Sweden)

    Wieruszeski Jean-Michel

    2010-05-01

    Full Text Available Abstract Background Conjugation of small ubiquitin-related modifiers (SUMOs is a frequent post-translational modification of proteins. SUMOs can also temporally associate with protein-targets via SUMO binding motifs (SBMs. Protein sumoylation has been identified as an important regulatory mechanism especially in the regulation of transcription and the maintenance of genome stability. The precise molecular mechanisms by which SUMO conjugation and association act are, however, not understood. Findings Using NMR spectroscopy and protein-DNA cross-linking experiments, we demonstrate here that SUMO-1 can specifically interact with dsDNA in a sequence-independent fashion. We also show that SUMO-1 binding to DNA can compete with other protein-DNA interactions at the example of the regulatory domain of Thymine-DNA Glycosylase and, based on these competition studies, estimate the DNA binding constant of SUMO1 in the range 1 mM. Conclusion This finding provides an important insight into how SUMO-1 might exert its activity. SUMO-1 might play a general role in destabilizing DNA bound protein complexes thereby operating in a bottle-opener way of fashion, explaining its pivotal role in regulating the activity of many central transcription and DNA repair complexes.

  8. B-Myb promotes S-phase independently of its sequence-specific DNA binding activity and interacts with polymerase delta-interacting protein 1 (Pdip1)

    Science.gov (United States)

    Werwein, Eugen; Schmedt, Thore; Hoffmann, Heiko; Usadel, Clemens; Obermann, Nora; Singer, Jeffrey D.; Klempnauer, Karl-Heinz

    2012-01-01

    B-Myb is a highly conserved member of the Myb transcription factor family, which plays an essential role in cell cycle progression by regulating the transcription of genes at the G2/M-phase boundary. The role of B-Myb in other parts of the cell cycle is less well-understood. By employing siRNA-mediated silencing of B-Myb expression, we found that B-Myb is required for efficient entry into S-phase. Surprisingly, a B-Myb mutant that lacks sequence-specific DNA-binding activity and is unable to activate transcription of B-Myb target genes is able to rescue the S-phase defect observed after B-Myb knockdown. Moreover, we have identified polymerase delta-interacting protein 1 (Pdip1), a BTB domain protein known to bind to the DNA replication and repair factor PCNA as a novel B-Myb interaction partner. We have shown that Pdip1 is able to interact with B-Myb and PCNA simultaneously. In addition, we found that a fraction of endogenous B-Myb can be co-precipitated via PCNA, suggesting that B-Myb might be involved in processes related to DNA replication or repair. Taken together, our work suggests a novel role for B-Myb in S-phase that appears to be independent of its sequence-specific DNA-binding activity and its ability to stimulate the expression of bona fide B-Myb target genes. PMID:23032261

  9. Single-Nucleotide-Specific Targeting of the Tf1 Retrotransposon Promoted by the DNA-Binding Protein Sap1 of Schizosaccharomyces pombe.

    Science.gov (United States)

    Hickey, Anthony; Esnault, Caroline; Majumdar, Anasuya; Chatterjee, Atreyi Ghatak; Iben, James R; McQueen, Philip G; Yang, Andrew X; Mizuguchi, Takeshi; Grewal, Shiv I S; Levin, Henry L

    2015-11-01

    Transposable elements (TEs) constitute a substantial fraction of the eukaryotic genome and, as a result, have a complex relationship with their host that is both adversarial and dependent. To minimize damage to cellular genes, TEs possess mechanisms that target integration to sequences of low importance. However, the retrotransposon Tf1 of Schizosaccharomyces pombe integrates with a surprising bias for promoter sequences of stress-response genes. The clustering of integration in specific promoters suggests that Tf1 possesses a targeting mechanism that is important for evolutionary adaptation to changes in environment. We report here that Sap1, an essential DNA-binding protein, plays an important role in Tf1 integration. A mutation in Sap1 resulted in a 10-fold drop in Tf1 transposition, and measures of transposon intermediates support the argument that the defect occurred in the process of integration. Published ChIP-Seq data on Sap1 binding combined with high-density maps of Tf1 integration that measure independent insertions at single-nucleotide positions show that 73.4% of all integration occurs at genomic sequences bound by Sap1. This represents high selectivity because Sap1 binds just 6.8% of the genome. A genome-wide analysis of promoter sequences revealed that Sap1 binding and amounts of integration correlate strongly. More important, an alignment of the DNA-binding motif of Sap1 revealed integration clustered on both sides of the motif and showed high levels specifically at positions +19 and -9. These data indicate that Sap1 contributes to the efficiency and position of Tf1 integration. Copyright © 2015 by the Genetics Society of America.

  10. The inhibition of anti-DNA binding to DNA by nucleic acid binding polymers.

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

    Full Text Available Antibodies to DNA (anti-DNA are the serological hallmark of systemic lupus erythematosus (SLE and can mediate disease pathogenesis by the formation of immune complexes. Since blocking immune complex formation can attenuate disease manifestations, the effects of nucleic acid binding polymers (NABPs on anti-DNA binding in vitro were investigated. The compounds tested included polyamidoamine dendrimer, 1,4-diaminobutane core, generation 3.0 (PAMAM-G3, hexadimethrine bromide, and a β-cylodextrin-containing polycation. As shown with plasma from patients with SLE, NABPs can inhibit anti-DNA antibody binding in ELISA assays. The inhibition was specific since the NABPs did not affect binding to tetanus toxoid or the Sm protein, another lupus autoantigen. Furthermore, the polymers could displace antibody from preformed complexes. Together, these results indicate that NABPs can inhibit the formation of immune complexes and may represent a new approach to treatment.

  11. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

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    Steven W Paugh

    2016-02-01

    Full Text Available MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16 for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.

  12. Adenovirus DNA binding protein: helix destabilising properties.

    OpenAIRE

    Monaghan, A; Webster, A; Hay, R T

    1994-01-01

    Adenovirus DNA binding protein is a multifunctional protein essential for viral DNA replication. To investigate the role of the DNA binding protein in this process its interaction with partial DNA duplexes was examined. Duplex regions of DNA, created when a short DNA strand is annealed to its complementary sequence present in the single stranded form of M13 phage DNA, were efficiently unwound by DNA binding protein in a reaction that required neither ATP nor MgCl2. The unwinding activity of D...

  13. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

    Full Text Available Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.

  14. Kinetics of signaling-DNA-aptamer-ATP binding

    Science.gov (United States)

    Nakamura, Issei; Shi, An-Chang; Nutiu, Razvan; Yu, Jasmine M. Y.; Li, Yingfu

    2009-03-01

    DNA aptamers are molecular biosensors consisting of single functionalized DNA molecules, which can bind to specific targets or complementary DNA sequences. The binding kinetics of DNA aptamers is studied by fluorescence quenching at 23°C . A kinetic model for the binding reaction of DNA aptamer, antisense DNA, and ATP target is developed to describe experimental observations. The approach leads to a simple procedure to deduce relevant kinetic reactions and their rate constants. A comparison between theory and experiments indicates that the previously established bimolecular DNA-ATP binding does not provide a complete description of the experimental data. Side reactions such as trimolecular complexation are proposed. Rate constants of the model are determined by comparing the model predictions and experiments. Good agreements between the model and experiments have been obtained. Possible blocking reactions by the misfolded DNA aptamer are also discussed.

  15. New DNA-binding radioprotectors

    Science.gov (United States)

    Martin, Roger

    The normal tissue damage associated with cancer radiotherapy has motivated the development at Peter Mac of a new class of DNA-binding radioprotecting drugs that could be applied top-ically to normal tissues at risk. Methylproamine (MP), the lead compound, reduces radiation induced cell kill at low concentrations. For example, experiments comparing the clonogenic survival of transformed human keratinocytes treated with 30 micromolar MP before and dur-ing various doses of ionising radiation, with the radiation dose response for untreated cells, indicate a dose reduction factor (DRF) of 2. Similar survival curve experiments using various concentrations of MP, with parallel measurements of uptake of MP into cell nuclei, have en-abled the relationship between drug uptake and extent of radioprotection to be established. Radioprotection has also been demonstrated after systemic administration to mice, for three different endpoints, namely lung, jejunum and bone marrow (survival at 30 days post-TBI). The results of pulse radiolysis studies indicated that the drugs act by reduction of transient radiation-induced oxidative species on DNA. This hypothesis was substantiated by the results of experiments in which MP radioprotection of radiation-induced DNA double-strand breaks, assessed as -H2AX foci, in the human keratinocyte cell line. For both endpoints, the extent of radioprotection increased with MP concentration up to a maximal value. These results are consistent with the hypothesis that radioprotection by MP is mediated by attenuation of the extent of initial DNA damage. However, although MP is a potent radioprotector, it becomes cytotoxic at higher concentrations. This limitation has been addressed in an extensive program of lead optimisation and some promising analogues have emerged from which the next lead will be selected. Given the clinical potential of topical radioprotection, the new analogues are being assessed in terms of delivery to mouse oral mucosa. This is

  16. Specific binding of MobA, a plasmid-encoded protein involved in the initiation and termination of conjugal DNA transfer, to single-stranded oriT DNA.

    Science.gov (United States)

    Bhattacharjee, M K; Meyer, R J

    1993-09-25

    MobA protein, encoded by the broad host-range plasmid R1162, is required for conjugal mobilization of this plasmid. The protein is an essential part of the relaxosome, and is also necessary for the termination of strand transfer. In vitro, MobA is a nuclease specific for one of the two DNA strands of the origin of transfer (oriT). The protein can cleave this strand at the same site that is nicked in the relaxosome, and can also ligate the DNA. We show here that purified MobA protein forms a complex that is specific for this single oriT strand. The complex is unusually stable, with a half-life of approximately 95 min, is not disrupted by hybridization with the complementary strand, and reforms rapidly after boiling. Both the inverted repeat within oriT, and the eight bases between this repeat and the site cleaved by MobA, are required for binding by the protein. Mutations reducing base complementarity between the arms of the inverted repeat also decrease binding. This effect is partially suppressed by second-site mutations restoring complementarity. These results parallel the effects of these mutations on termination. Footprinting experiments with P1 nuclease indicate that the DNA between the inverted repeat and the nick site is protected by MobA, but that pairing between the arms of the repeat, which occurs in the absence of protein, is partially disrupted. Our results suggest that termination of strand transfer during conjugation involves tight binding of the MobA protein to the inverted repeat and adjacent oriT DNA. This complex positions the protein for ligation of the ends of the transferred strand, to reform a circular plasmid molecule.

  17. A missense mutation in damage-specific DNA binding protein 2 is a genetic risk factor for limbal squamous cell carcinoma in horses.

    Science.gov (United States)

    Bellone, Rebecca R; Liu, Jiayin; Petersen, Jessica L; Mack, Maura; Singer-Berk, Moriel; Drögemüller, Cord; Malvick, Julia; Wallner, Barbara; Brem, Gottfried; Penedo, M Cecilia; Lassaline, Mary

    2017-07-15

    Squamous cell carcinoma (SCC) is the most common cancer of the equine eye, frequently originating at the limbus, with the potential to invade the cornea, cause visual impairment, and result in loss of the eye. Several breeds of horses have a high occurrence of limbal SCC implicating a genetic basis for limbal SCC predisposition. Pedigree analysis in the Haflinger breed supports a simple recessive mode of inheritance and a genome-wide association study (N = 23) identified a 1.5 Mb locus on ECA12 significantly associated with limbal SCC (Pcorrected = 0.04). Sequencing the most physiologically relevant gene from this locus, damage specific DNA binding protein 2 (DDB2), identified a missense mutation (c.1013 C > T p.Thr338Met) that was strongly associated with limbal SCC (P = 3.41 × 10(-10) ). Genotyping 42 polymorphisms narrowed the ECA12 candidate interval to 483 kb but did not identify another variant that was more strongly associated. DDB2 binds to ultraviolet light damaged DNA and recruits other proteins to perform global genome nucleotide excision repair. Computational modeling predicts this mutation to be deleterious by altering conformation of the β loop involved in photolesion recognition. This DDB2 variant was also detected in two other closely related breeds with reported cases of ocular SCC, the Belgian and the Percheron, suggesting it may also be a SCC risk factor in these breeds. Furthermore, in humans xeroderma pigmentosum complementation group E, a disease characterized by sun sensitivity and increased risk of cutaneous SCC and melanomas, is explained by mutations in DDB2. Cross-species comparison remains to be further evaluated. © 2017 UICC.

  18. RNA recognition by the DNA end-binding Ku heterodimer.

    Science.gov (United States)

    Dalby, Andrew B; Goodrich, Karen J; Pfingsten, Jennifer S; Cech, Thomas R

    2013-06-01

    Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.

  19. DNA binding site analysis of Burkholderia thailandensis response regulators.

    Science.gov (United States)

    Nowak-Lovato, Kristy L; Hickmott, Alexana J; Maity, Tuhin S; Bulyk, Martha L; Dunbar, John; Hong-Geller, Elizabeth

    2012-07-01

    Bacterial response regulators (RR) that function as transcription factors in two component signaling pathways are crucial for ensuring tight regulation and coordinated expression of the genome. Currently, consensus DNA binding sites in the promoter for very few bacterial RRs have been identified. A systematic method to characterize these DNA binding sites for RRs would enable prediction of specific gene expression patterns in response to extracellular stimuli. To identify RR DNA binding sites, we functionally activated RRs using beryllofluoride and applied them to a protein-binding microarray (PBM) to discover DNA binding motifs for RRs expressed in Burkholderia, a Gram-negative bacterial genus. We identified DNA binding motifs for conserved RRs in Burkholderia thailandensis, including KdpE, RisA, and NarL, as well as for a previously uncharacterized RR at locus BTH_II2335 and its ortholog in the human pathogen Burkholderia pseudomallei at locus BPSS2315. We further demonstrate RR binding of predicted genomic targets for the two orthologs using gel shift assays and reveal a pattern of RR regulation of expression of self and other two component systems. Our studies illustrate the use of PBMs to identify DNA binding specificities for bacterial RRs and enable prediction of gene regulatory networks in response to two component signaling. Published by Elsevier B.V.

  20. Hx, a novel fluorescent, minor groove and sequence specific recognition element: design, synthesis, and DNA binding properties of p-anisylbenzimidazole-imidazole/pyrrole-containing polyamides.

    Science.gov (United States)

    Chavda, Sameer; Liu, Yang; Babu, Balaji; Davis, Ryan; Sielaff, Alan; Ruprich, Jennifer; Westrate, Laura; Tronrud, Christopher; Ferguson, Amanda; Franks, Andrew; Tzou, Samuel; Adkins, Chandler; Rice, Toni; Mackay, Hilary; Kluza, Jerome; Tahir, Sharjeel A; Lin, Shicai; Kiakos, Konstantinos; Bruce, Chrystal D; Wilson, W David; Hartley, John A; Lee, Moses

    2011-04-19

    With the aim of incorporating a recognition element that acts as a fluorescent probe upon binding to DNA, three novel pyrrole (P) and imidazole (I)-containing polyamides were synthesized. The compounds contain a p-anisylbenzimidazolecarboxamido (Hx) moiety attached to a PP, IP, or PI unit, giving compounds HxPP (2), HxIP (3), and HxPI (4), respectively. These fluorescent hybrids were tested against their complementary nonfluorescent, non-formamido tetraamide counterparts, namely, PPPP (5), PPIP (6), and PPPI (7) (cognate sequences 5'-AAATTT-3', 5'-ATCGAT-3', and 5'-ACATGT-3', respectively). The binding affinities for both series of polyamides for their cognate and noncognate sequences were ascertained by surface plasmon resonance (SPR) studies, which revealed that the Hx-containing polyamides gave binding constants in the 10(6) M(-1) range while little binding was observed for the noncognates. The binding data were further compared to the corresponding and previously reported formamido-triamides f-PPP (8), f-PIP (9), and f-PPI (10). DNase I footprinting studies provided additional evidence that the Hx moiety behaved similarly to two consecutive pyrroles (PP found in 5-7), which also behaved like a formamido-pyrrole (f-P) unit found in distamycin and many formamido-triamides, including 8-10. The biophysical characterization of polyamides 2-7 on their binding to the abovementioned DNA sequences was determined using thermal melts (ΔT(M)), circular dichroism (CD), and isothermal titration calorimetry (ITC) studies. Density functional calculations (B3LYP) provided a theoretical framework that explains the similarity between PP and Hx on the basis of molecular electrostatic surfaces and dipole moments. Furthermore, emission studies on polyamides 2 and 3 showed that upon excitation at 322 nm binding to their respective cognate sequences resulted in an increase in fluorescence at 370 nm. These low molecular weight polyamides show promise for use as probes for monitoring

  1. Probing the binding mode of psoralen to calf thymus DNA.

    Science.gov (United States)

    Zhou, Xiaoyue; Zhang, Guowen; Wang, Langhong

    2014-06-01

    The binding properties between psoralen (PSO) and calf thymus DNA (ctDNA) were predicted by molecular docking, and then determined with the use of UV-vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, coupled with DNA melting and viscosity measurements. The data matrix obtained from UV-vis spectra was resolved by multivariate curve resolution-alternating least squares (MCR-ALS) approach. The pure spectra and the equilibrium concentration profiles for PSO, ctDNA and PSO-ctDNA complex extracted from the highly overlapping composite response were obtained simultaneously to evaluate the PSO-ctDNA interaction. The intercalation mode of PSO binding to ctDNA was supported by the results from the melting studies, viscosity measurements, iodide quenching and fluorescence polarization experiments, competitive binding investigations and CD analysis. The molecular docking prediction showed that the specific binding most likely occurred between PSO and adenine bases of ctDNA. FT-IR spectra studies further confirmed that PSO preferentially bound to adenine bases, and this binding decreased right-handed helicity of ctDNA and enhanced the degree of base stacking with the preservation of native B-conformation. The calculated thermodynamic parameters indicated that hydrogen bonds and van der Waals forces played a major role in the binding process. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. DNA binding studies of tartrazine food additive.

    Science.gov (United States)

    Kashanian, Soheila; Zeidali, Sahar Heidary

    2011-07-01

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

  3. Measuring Equilibrium Binding Constants for the WT1-DNA Interaction Using a Filter Binding Assay.

    Science.gov (United States)

    Romaniuk, Paul J

    2016-01-01

    Equilibrium binding of WT1 to specific sites in DNA and potentially RNA molecules is central in mediating the regulatory roles of this protein. In order to understand the functional effects of mutations in the nucleic acid-binding domain of WT1 proteins and/or mutations in the DNA- or RNA-binding sites, it is necessary to measure the equilibrium constant for formation of the protein-nucleic acid complex. This chapter describes the use of a filter binding assay to make accurate measurements of the binding of the WT1 zinc finger domain to the consensus WT1-binding site in DNA. The method described is readily adapted to the measurement of the effects of mutations in either the WT1 zinc finger domain or the putative binding sites within a promoter element or cellular RNA.

  4. Structural insight into the specificity of the B3 DNA-binding domains provided by the co-crystal structure of the C-terminal fragment of BfiI restriction enzyme.

    Science.gov (United States)

    Golovenko, Dmitrij; Manakova, Elena; Zakrys, Linas; Zaremba, Mindaugas; Sasnauskas, Giedrius; Gražulis, Saulius; Siksnys, Virginijus

    2014-04-01

    The B3 DNA-binding domains (DBDs) of plant transcription factors (TF) and DBDs of EcoRII and BfiI restriction endonucleases (EcoRII-N and BfiI-C) share a common structural fold, classified as the DNA-binding pseudobarrel. The B3 DBDs in the plant TFs recognize a diverse set of target sequences. The only available co-crystal structure of the B3-like DBD is that of EcoRII-N (recognition sequence 5'-CCTGG-3'). In order to understand the structural and molecular mechanisms of specificity of B3 DBDs, we have solved the crystal structure of BfiI-C (recognition sequence 5'-ACTGGG-3') complexed with 12-bp cognate oligoduplex. Structural comparison of BfiI-C-DNA and EcoRII-N-DNA complexes reveals a conserved DNA-binding mode and a conserved pattern of interactions with the phosphodiester backbone. The determinants of the target specificity are located in the loops that emanate from the conserved structural core. The BfiI-C-DNA structure presented here expands a range of templates for modeling of the DNA-bound complexes of the B3 family of plant TFs.

  5. In vivo protein-DNA interactions on a glucocorticoid response unit of a liver-specific gene: hormone-induced transcription factor binding to constitutively open chromatin.

    Science.gov (United States)

    Zimmermann, P L; Pierreux, C E; Rigaud, G; Rousseau, G G; Lemaigre, F P

    1997-06-01

    Transcription from the liver promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) gene depends on the presence of glucocorticoids that act via a glucocorticoid response unit (GRU) located in the first intron. The promoter and the GRU are in a constitutively open chromatin configuration. To determine how glucocorticoids would affect factor binding to the GRU in absence of chromatin remodeling, we have used a combination of in vitro DNA-binding assays and in vivo genomic footprinting in rat hepatocytes and hepatoma cells. We found that, in the absence of glucocorticoids, the GRU binds nuclear factor-I (NF-I). Glucocorticoid treatment modified factor binding to the NF-I site and induced the binding of hepatocyte nuclear factor-3 (HNF-3). Transfection assays showed that HNF-3 cooperates with the glucocorticoid receptor in stimulating transcription. In contrast with the lack of effect of glucocorticoids on factor binding to constitutively open GRUs of other genes, HNF-3 binding to the open PFK-2 GRU was hormone-dependent. Therefore, the PFK-2 GRU behaves as a novel type of GRU.

  6. Aptamer-Binding Directed DNA Origami Pattern for Logic Gates.

    Science.gov (United States)

    Yang, Jing; Jiang, Shuoxing; Liu, Xiangrong; Pan, Linqiang; Zhang, Cheng

    2016-12-14

    In this study, an aptamer-substrate strategy is introduced to control programmable DNA origami pattern. Combined with DNA aptamer-substrate binding and DNAzyme-cutting, small DNA tiles were specifically controlled to fill into the predesigned DNA origami frame. Here, a set of DNA logic gates (OR, YES, and AND) are performed in response to the stimuli of adenosine triphosphate (ATP) and cocaine. The experimental results are confirmed by AFM imaging and time-dependent fluorescence changes, demonstrating that the geometric patterns are regulated in a controllable and programmable manner. Our approach provides a new platform for engineering programmable origami nanopatterns and constructing complex DNA nanodevices.

  7. The pancreatic and duodenal homeobox protein PDX-1 regulates the ductal specific keratin 19 through the degradation of MEIS1 and DNA binding.

    Directory of Open Access Journals (Sweden)

    Johannes von Burstin

    Full Text Available BACKGROUND: Pancreas organogenesis is the result of well-orchestrated and balanced activities of transcription factors. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. However, the activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. PBX and MEIS proteins belong to the family of three amino acid loop extension (TALE homeodomain proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt19. In this study, we investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt19 regulation. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate that PDX-1 exerts a dual manner of regulation of Krt19 transcriptional activity. Deletion studies highlight that the NH(2-terminus of PDX-1 is functionally relevant for the down-regulation of Krt19, as it is required for DNA binding of PDX-1 to the Krt19 promoter. Moreover, this effect occurs independently of PBX. Second, we provide insight on how PDX-1 regulates the Hox co-factor MEIS1 post-transcriptionally. We find specific binding of MEIS1 and MEIS2 to the Krt19 promoter using IP-EMSA, and siRNA mediated silencing of Meis1, but not Meis2, reduces transcriptional activation of Krt19 in primary pancreatic ductal cells. Over-expression of PDX-1 leads to a decreased level of MEIS1 protein, and this decrease is prevented by inhibition of the proteasome. CONCLUSIONS/SIGNIFICANCE: Taken together, our data provide evidence for a dual mechanism of how PDX-1 negatively regulates Krt19 ductal specific gene expression. These findings imply that transcription factors may efficiently regulate target gene expression through diverse, non

  8. Werner Helicase Wings DNA Binding

    OpenAIRE

    Hoadley, Kelly A.; Keck, James L.

    2010-01-01

    In this issue of Structure, Kitano et al. describe the structure of the DNA-bound winged-helix domain from the Werner helicase. This structure of a RecQ/DNA complex offers insights into the DNA unwinding mechanisms of RecQ family helicases.

  9. Werner helicase wings DNA binding.

    Science.gov (United States)

    Hoadley, Kelly A; Keck, James L

    2010-02-10

    In this issue of Structure, Kitano et al. describe the structure of the DNA-bound winged-helix domain from the Werner helicase. This structure of a RecQ/DNA complex offers insights into the DNA-unwinding mechanisms of RecQ family helicases. Copyright 2010 Elsevier Inc. All rights reserved.

  10. Binding mode and affinity studies of DNA-binding agents using topoisomerase I DNA unwinding assay.

    Science.gov (United States)

    McKnight, Ruel E; Gleason, Aaron B; Keyes, James A; Sahabi, Sadia

    2007-02-15

    A topoisomerase I DNA unwinding assay has been used to determine the relative DNA-binding affinities of a model pair of homologous naphthalene diimides. Binding affinity data were corroborated using calorimetric (ITC) and spectrophotometric (titration and T(m)) studies, with substituent size playing a significant role in binding. The assay was also used to investigate the mode of binding adopted by several known DNA-binding agents, including SYBR Green and PicoGreen. Some of the compounds exhibited unexpected binding modes.

  11. Effects of spectator ligands on the specific recognition of intrastrand platinum-DNA cross-links by high mobility group box and TATA-binding proteins.

    Science.gov (United States)

    Wei, M; Cohen, S M; Silverman, A P; Lippard, S J

    2001-10-19

    The results presented describe the effects of various spectator ligands, attached to a platinum 1,2-intrastand d(GpG) cross-link in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding protein (TBP). In addition to cisplatin-modified DNA, 15-base pair DNA probes modified by [Pt(1R,2R-diaminocyclohexane)](2+), cis-[Pt(NH(3))(cyclohexylamine)](2+), [Pt(ethylenediamine)](2+), cis-[Pt(NH(3))(cyclobutylamine)](2+), and cis-[Pt(NH(3))(2-picoline)](2+) were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discriminate between different platinum-DNA adducts. HMGB1 domain A is the most sensitive to the nature of the spectator ligands on platinum. The effect of the spectator ligands on protein binding also depends highly on the base pairs flanking the platinated d(GpG) site. Double-stranded oligonucleotides containing the AG*G*C sequence, where the asterisks denote the sites of platination, with different spectator ligands are only moderately discriminated by the HMGB proteins and TBP, but the recognition of dsTG*G*A is highly dependent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl(2)] and cis-[Pt(NH(3))(cyclohexylamine)Cl(2)] were also examined. The results suggest that HMGB1 protein levels influence the cellular processing of cis-[Pt(NH(3))- (cyclohexylamine)](2+), but not [Pt((1R,2R)-diaminocyclohexane)](2+), DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTG*G*A probes but not with the binding affinity of HMGB1a and HMGB1 to platinum-damaged dsAG*G*C oligonucleotides. These experiments reinforce the importance of sequence context in platinum-DNA lesion recognition by cellular proteins.

  12. Predicting target DNA sequences of DNA-binding proteins based on unbound structures.

    Directory of Open Access Journals (Sweden)

    Chien-Yu Chen

    Full Text Available DNA-binding proteins such as transcription factors use DNA-binding domains (DBDs to bind to specific sequences in the genome to initiate many important biological functions. Accurate prediction of such target sequences, often represented by position weight matrices (PWMs, is an important step to understand many biological processes. Recent studies have shown that knowledge-based potential functions can be applied on protein-DNA co-crystallized structures to generate PWMs that are considerably consistent with experimental data. However, this success has not been extended to DNA-binding proteins lacking co-crystallized structures. This study aims at investigating the possibility of predicting the DNA sequences bound by DNA-binding proteins from the proteins' unbound structures (structures of the unbound state. Given an unbound query protein and a template complex, the proposed method first employs structure alignment to generate synthetic protein-DNA complexes for the query protein. Once a complex is available, an atomic-level knowledge-based potential function is employed to predict PWMs characterizing the sequences to which the query protein can bind. The evaluation of the proposed method is based on seven DNA-binding proteins, which have structures of both DNA-bound and unbound forms for prediction as well as annotated PWMs for validation. Since this work is the first attempt to predict target sequences of DNA-binding proteins from their unbound structures, three types of structural variations that presumably influence the prediction accuracy were examined and discussed. Based on the analyses conducted in this study, the conformational change of proteins upon binding DNA was shown to be the key factor. This study sheds light on the challenge of predicting the target DNA sequences of a protein lacking co-crystallized structures, which encourages more efforts on the structure alignment-based approaches in addition to docking- and homology

  13. Analysis and classification of DNA-binding sites in single-stranded and double-stranded DNA-binding proteins using protein information.

    Science.gov (United States)

    Wang, Wei; Liu, Juan; Xiong, Yi; Zhu, Lida; Zhou, Xionghui

    2014-08-01

    Single-stranded DNA-binding proteins (SSBs) and double-stranded DNA-binding proteins (DSBs) play different roles in biological processes when they bind to single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA). However, the underlying binding mechanisms of SSBs and DSBs have not yet been fully understood. Here, the authors firstly constructed two groups of ssDNA and dsDNA specific binding sites from two non-redundant sets of SSBs and DSBs. They further analysed the relationship between the two classes of binding sites and a newly proposed set of features (residue charge distribution, secondary structure and spatial shape). To assess and utilise the predictive power of these features, they trained a classification model using support vector machine to make predictions about the ssDNA and the dsDNA binding sites. The author's analysis and prediction results indicated that the two classes of binding sites can be distinguishable by the three types of features, and the final classifier using all the features achieved satisfactory performance. In conclusion, the proposed features will deepen their understanding of the specificity of proteins which bind to ssDNA or dsDNA.

  14. Retinoblastoma-binding Protein 1 Has an Interdigitated Double Tudor Domain with DNA Binding Activity*

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-01-01

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10–100 μm; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1. PMID:24379399

  15. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    Science.gov (United States)

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  16. The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates.

    Science.gov (United States)

    Whitaker, Neal; Chen, Yuqing; Jakubowski, Simon J; Sarkar, Mayukh K; Li, Feng; Christie, Peter J

    2015-07-01

    Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AADPcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. For conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are largely undefined. Here

  17. DNA Origami Seesaws as Comparative Binding Assay.

    Science.gov (United States)

    Nickels, Philipp C; Høiberg, Hans C; Simmel, Stephanie S; Holzmeister, Phil; Tinnefeld, Philip; Liedl, Tim

    2016-06-16

    The application of commonly used force spectroscopy in biological systems is often limited by the need for an invasive tether connecting the molecules of interest to a bead or cantilever tip. Here we present a DNA origami-based prototype in a comparative binding assay. It has the advantage of in situ readout without any physical connection to the macroscopic world. The seesaw-like structure has a lever that is able to move freely relative to its base. Binding partners on each side force the structure into discrete and distinguishable conformations. Model experiments with competing DNA hybridisation reactions yielded a drastic shift towards the conformation with the stronger binding interaction. With reference DNA duplexes of tuneable length on one side, this device can be used to measure ligand interactions in comparative assays. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  18. LNA effects on DNA binding and conformation

    DEFF Research Database (Denmark)

    Pabon-Martinez, Y Vladimir; Xu, You; Villa, Alessandra

    2017-01-01

    The anti-gene strategy is based on sequence-specific recognition of double-strand DNA by triplex forming (TFOs) or DNA strand invading oligonucleotides to modulate gene expression. To be efficient, the oligonucleotides (ONs) should target DNA selectively, with high affinity. Here we combined hybr...

  19. Methylated DNA-binding protein is present in various mammalian cell types

    Energy Technology Data Exchange (ETDEWEB)

    Supakar, P.C.; Weist, D.; Zhang, D.; Inamdar, N.; Zhang, Xianyang; Khan, R.; Ehrlich, M. (Tulane Medical School, New Orleans, LA (USA)); Ehrlich, K.C. (Department of Agriculture, New Orleans, LA (USA))

    1988-08-25

    A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m{sup 5}C) residues at specific positions. The authors found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.

  20. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    Directory of Open Access Journals (Sweden)

    Anna Schrader

    Full Text Available The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

  1. Hepatoma derived growth factor binds DNA through the N-terminal PWWP domain

    Directory of Open Access Journals (Sweden)

    Everett Allen D

    2007-10-01

    Full Text Available Abstract Background Hepatoma Derived Growth Factor (HDGF is a nuclear protein with nuclear targeting required for mitogenic activity. Recently we demonstrated that HDGF is a transcriptional repressor, but whether HDGF binds DNA, the specificity of DNA binding and what protein domain is required are still unknown. In this study, we aimed to identify if HDGF is a DNA binding protein, map the functional DNA binding domain and DNA binding element for HDGF. Results Using chromatin immunoprecipitation (ChIP of human DNA, we isolated 10 DNA sequences sharing a conserved ~200 bp element. Homology analysis identified the binding sequences as a motif within the promoter of the SMYD1 gene, a HDGF target gene. Electrophoretic Mobility Shift Assays (EMSA confirmed the binding of HDGF to this conserved sequence. As a result, an 80 bp conserved sequence located in the SMYD1 promoter bound GST-HDGF tightly. The binding core sequence for HDGF was narrowed down to 37 bp using a deletion mapping strategy from both the 5' and 3' ends. Moreover, ChIP and DNase I footprinting analysis revealed that HDGF binds this 80 bp DNA fragment specifically. Functionally overexpression of HDGF represses a reporter gene which is controlled by an SV-40 promoter containing the 80 bp DNA element. Using serial truncations of GST-HDGF, we mapped the DNA binding domain of HDGF to the N-terminal PWWP domain. Conclusion HDGF is a DNA binding protein, binds DNA specifically, and prefers a minimum of 37 bp long DNA fragment. The N-terminal PWWP domain of HDGF is required for DNA binding. HDGF exerts its transcription repressive effect through binding to a conserved DNA element in the promoter of target genes.

  2. Binding Interaction of HMGB4 with Cisplatin-Modified DNA

    Science.gov (United States)

    Park, Semi; Lippard, Stephen J.

    2012-01-01

    Proteins in the HMG family are important transcription factors. They recognize cisplatin-damaged DNA lesions with a structure-specific preference and account for more than 70% of all proteins that interact with the cisplatin 1,2-intrastrand d(GpG) cross-link. HMGB4, a new member of the mammalian HMGB protein family expressed preferentially in the testis, was generated recombinantly and its interactions with cisplatin-modified DNA were investigated in vitro. The binding affinities of the two individual DNA-binding domains of HMGB4 to DNA carrying a cisplatin 1,2-intrastrand d(GpG) cross-link are weaker than those of the DNA-binding domains of HMGB1. Full-length HMGB4, however, has a 28-fold stronger binding affinity (Kd = 4.35 nM) for the platinated adduct compared to that of HMGB1 (Kd = 120 nM), presumably because the former lacks a C-terminal acidic tail. The residue Phe37 plays a critical role in stabilizing the binding complex of HMGB4 with the cisplatin-modified DNA, as it does for HMGB1. Hydroxyl radical footprinting analysis of the HMGB4/platinated DNA complex reveals a very different footprinting pattern from that of HMGB1, however, revealing very little binding asymmetry with respect to the platinated lesion. An in vitro repair assay revealed that HMGB4, at 1 µM concentration, interferes with repair of cisplatin 1,2-intrastrand cross-link damage by >90% compared to control, whereas HMGB1 at the same concentration inhibits repair by 45% This repair inhibition capability is highly dependent on both the binding affinity and size of the proteins. The putative role of HMGB4 in the mechanism of action of cisplatin, and especially its potential relevance to the hypersensitivity of testicular germ cell tumors to cisplatin, are discussed. PMID:22901013

  3. IFI16 Preferentially Binds to DNA with Quadruplex Structure and Enhances DNA Quadruplex Formation.

    Science.gov (United States)

    Hároníková, Lucia; Coufal, Jan; Kejnovská, Iva; Jagelská, Eva B; Fojta, Miroslav; Dvořáková, Petra; Muller, Petr; Vojtesek, Borivoj; Brázda, Václav

    2016-01-01

    Interferon-inducible protein 16 (IFI16) is a member of the HIN-200 protein family, containing two HIN domains and one PYRIN domain. IFI16 acts as a sensor of viral and bacterial DNA and is important for innate immune responses. IFI16 binds DNA and binding has been described to be DNA length-dependent, but a preference for supercoiled DNA has also been demonstrated. Here we report a specific preference of IFI16 for binding to quadruplex DNA compared to other DNA structures. IFI16 binds to quadruplex DNA with significantly higher affinity than to the same sequence in double stranded DNA. By circular dichroism (CD) spectroscopy we also demonstrated the ability of IFI16 to stabilize quadruplex structures with quadruplex-forming oligonucleotides derived from human telomere (HTEL) sequences and the MYC promotor. A novel H/D exchange mass spectrometry approach was developed to assess protein interactions with quadruplex DNA. Quadruplex DNA changed the IFI16 deuteration profile in parts of the PYRIN domain (aa 0-80) and in structurally identical parts of both HIN domains (aa 271-302 and aa 586-617) compared to single stranded or double stranded DNAs, supporting the preferential affinity of IFI16 for structured DNA. Our results reveal the importance of quadruplex DNA structure in IFI16 binding and improve our understanding of how IFI16 senses DNA. IFI16 selectivity for quadruplex structure provides a mechanistic framework for IFI16 in immunity and cellular processes including DNA damage responses and cell proliferation.

  4. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit...

  5. Cytotoxic, DNA binding, DNA cleavage and antibacterial studies of ...

    Indian Academy of Sciences (India)

    Six new Ru(II) and Ru(III) complexes have been synthesized and characterized by elemental analysis, LC-MS, electronic spectra, IR spectra and magnetic moment measurements. DNA-binding properties of Ru complexes have been studied by means of absorption spectrophotometry and viscosity measurements as well as ...

  6. Cytotoxic, DNA binding, DNA cleavage and antibacterial studies of ...

    Indian Academy of Sciences (India)

    Abstract. Six new Ru(II) and Ru(III) complexes have been synthesized and characterized by elemental ana- lysis, LC-MS, electronic spectra, IR spectra and magnetic moment measurements. DNA-binding properties of. Ru complexes have been studied by means of absorption spectrophotometry and viscosity measurements ...

  7. A robust assay to measure DNA topology-dependent protein binding affinity.

    Science.gov (United States)

    Litwin, Tamara R; Solà, Maria; Holt, Ian J; Neuman, Keir C

    2015-04-20

    DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by US Government employees and is in the public domain in the US.

  8. Structure and Cellular Dynamics of Deinococcus radiodurans Single-stranded DNA (ssDNA)-binding Protein (SSB)-DNA Complexes*

    Science.gov (United States)

    George, Nicholas P.; Ngo, Khanh V.; Chitteni-Pattu, Sindhu; Norais, Cédric A.; Battista, John R.; Cox, Michael M.; Keck, James L.

    2012-01-01

    The single-stranded DNA (ssDNA)-binding protein from the radiation-resistant bacterium Deinococcus radiodurans (DrSSB) functions as a homodimer in which each monomer contains two oligonucleotide-binding (OB) domains. This arrangement is exceedingly rare among bacterial SSBs, which typically form homotetramers of single-OB domain subunits. To better understand how this unusual structure influences the DNA binding and biological functions of DrSSB in D. radiodurans radiation resistance, we have examined the structure of DrSSB in complex with ssDNA and the DNA damage-dependent cellular dynamics of DrSSB. The x-ray crystal structure of the DrSSB-ssDNA complex shows that ssDNA binds to surfaces of DrSSB that are analogous to those mapped in homotetrameric SSBs, although there are distinct contacts in DrSSB that mediate species-specific ssDNA binding. Observations by electron microscopy reveal two salt-dependent ssDNA-binding modes for DrSSB that strongly resemble those of the homotetrameric Escherichia coli SSB, further supporting a shared overall DNA binding mechanism between the two classes of bacterial SSBs. In vivo, DrSSB levels are heavily induced following exposure to ionizing radiation. This accumulation is accompanied by dramatic time-dependent DrSSB cellular dynamics in which a single nucleoid-centric focus of DrSSB is observed within 1 h of irradiation but is dispersed by 3 h after irradiation. These kinetics parallel those of D. radiodurans postirradiation genome reconstitution, suggesting that DrSSB dynamics could play important organizational roles in DNA repair. PMID:22570477

  9. Effects of Cu(II) and cisplatin on the stability of Specific protein 1 (Sp1)-DNA binding: Insights into the regulation of copper homeostasis and platinum drug transport.

    Science.gov (United States)

    Yan, Dong; Aiba, Isamu; Chen, Helen H W; Kuo, Macus Tien

    2016-08-01

    The human high-affinity copper transporter 1 (hCtr1) transports both Cu(I) and cisplatin (cDDP). Because Cu deficiency is lethal yet Cu overload is poisonous, hCtr1 expression is transcriptionally upregulated in response to Cu deficiency but is downregulated under Cu replete conditions in controlling Cu homeostasis. The up- and down-regulation of hCtr1 is regulated by Specific protein 1 (Sp1), which itself is also correspondingly regulated under these Cu conditions. hCtr1 expression is also upregulated by cDDP via upregulation of Sp1. The underlying mechanisms of these regulations are unknown. Using gel-electrophoretic mobility shift assays, we demonstrated here that Sp1-DNA binding affinity is reduced under Cu replete conditions but increased under reduced Cu conditions. Similarly, Sp1-DNA binding affinity is increased by cDDP treatment. This in vitro system demonstrated, for the first time, that regulation of Sp1/hCtr1 expression by these agents is modulated by the stability of Sp1-DNA binding, the first step in the Sp1-mediated transcriptional regulation process. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

    Science.gov (United States)

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

    2015-01-01

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

  11. RNA-binding specificity of Y-box protein 1.

    Science.gov (United States)

    Dong, Jinjiang; Akcakanat, Argun; Stivers, David N; Zhang, Jiexin; Kim, Doyil; Meric-Bernstam, Funda

    2009-01-01

    Y-box protein 1 (YB-1) is a multifunctional DNA/RNA-binding protein that regulates transcription and translation. The specificity of YB-1's RNA binding and its consequences are unknown. Because expression and subcellular localization of YB-1 have been reported to be important in breast cancer, we determined the specificity and functional impact of YB-1 mRNA-binding in MCF7 breast cancer cells. We used YB-1 antibodies to immunoprecipitate YB-1 and microarray profiling to compare YB-1-bound and total poly(A) RNA. We demonstrated that YB-1 mRNA-binding was preferential. Transcript sequences significantly associated with this binding had high GC content. Selected YB-1 mRNA-binding targets were confirmed by QRT-PCR. However, downregulation of YB-1 levels by siRNA did not affect their RNA or protein expression. Thus, YB-1 has RNA-binding specificity; however, YB-1 binding does not necessarily regulate the stability or translation of its mRNA targets. Further study is needed to determine the functional consequences of selective YB-1 mRNA binding.

  12. Steps in the process of DNA binding and entry in transformation. [Pneumococcus

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S

    1978-01-01

    The DNA uptake phase of genetic transformation in S. pheumoniae is reviewed with emphasis on molecular interactions at each step. An initial, reversible binding appears to be dependent on the molecular concentration of donor DNA. Subsequent irreversible binding, limited to the numbr of molecules corresponding to a fixed number of receptor sites, requires potassium ions and energy. Competition of different DNAs for uptake occurs at the initial step, but depends on the size of the DNA as well as its molecular concentration. Single-strand breakage accompanies irreversible binding of DNA. The frequency of breaks does not appear to depend on DNA concentration. Entry of DNA follows irreversible binding. The entry step, in which donor DNA is converted to single strands, requires action of a membrane nuclease. In the membrane this nuclease is part ofa specific multiprotein structure, which may function as a unit in DNA binding and entry.

  13. T3: Targeted Proteomics of DNA-Binding Proteins

    Science.gov (United States)

    Nagore, Linda I.; Jarrett, Harry W.

    2015-01-01

    A technique that allows the inclusion of a specific DNA to enrich and direct proteomic identification of transcription factors (TF) while providing a route for high throughput screening on a single platform would be valuable in investigations of gene expression and regulation. Polyvinylpyrrolidone binds DNA avidly while binding negligible amounts of protein. This observation is used in a proof-of-concept method to enrich for TF by combining nuclear extract with a specific DNA sequence and immobilizing the DNA-protein complex on a PVP-coated MALDI plate. Any unbound proteins are washed away and further processed for analysis in a MALDI-TOF/TOF mass spectrometer. Enrichment on a PVP-coated plate gives the unique advantage of purification, enzymatic digestion and analysis on a single platform. The method is termed T3 as it combines Targeted purification on a Target plate with Targeted proteomics. Validation was achieved in model experiments with a chimeric fusion protein, green fluorescent protein-CAAT enhancer binding protein (GFP-C/EBP) with an oligonucleotide containing the CAAT sequence. Both domains were identified with an expectation value of less than 10−15 and over 15% sequence coverage. The same oligonucleotide mixed with HEK293 cell nuclear extract allowed the unambiguous identification of native human C/EBP alpha with 24.3% sequence coverage. PMID:25644705

  14. In Vitro Selection of DNA Aptamers that Binds Geniposide

    Directory of Open Access Journals (Sweden)

    Aozhe Zhang

    2017-02-01

    Full Text Available Geniposide is a key iridoid glycoside from Gardenia jasminoides fructus widely used in traditional Chinese herbal medicine. However, detection of this small molecule represents a significant challenge mostly due to the lack of specific molecular recognition elements. In this study, we have performed in vitro selection experiments to isolate DNA aptamers that can specifically bind geniposide. Using a stringent selection procedure, we have isolated DNA aptamers that can distinguish geniposide from genipin and glucose, two structural analogs of geniposide. Two top aptamers exhibit low micromolar binding affinity towards geniposide, but show significantly reduced affinity to genipin and glucose. These aptamers have the potential to be further developed into analytical tools for the detection of geniposide.

  15. Artificial zinc finger DNA binding domains: versatile tools for genome engineering and modulation of gene expression.

    Science.gov (United States)

    Hossain, Mir A; Barrow, Joeva J; Shen, Yong; Haq, Md Imdadul; Bungert, Jörg

    2015-11-01

    Genome editing and alteration of gene expression by synthetic DNA binding activities gained a lot of momentum over the last decade. This is due to the development of new DNA binding molecules with enhanced binding specificity. The most commonly used DNA binding modules are zinc fingers (ZFs), TALE-domains, and the RNA component of the CRISPR/Cas9 system. These binding modules are fused or linked to either nucleases that cut the DNA and induce DNA repair processes, or to protein domains that activate or repress transcription of genes close to the targeted site in the genome. This review focuses on the structure, design, and applications of ZF DNA binding domains (ZFDBDs). ZFDBDs are relatively small and have been shown to penetrate the cell membrane without additional tags suggesting that they could be delivered to cells without a DNA or RNA intermediate. Advanced algorithms that are based on extensive knowledge of the mode of ZF/DNA interactions are used to design the amino acid composition of ZFDBDs so that they bind to unique sites in the genome. Off-target binding has been a concern for all synthetic DNA binding molecules. Thus, increasing the specificity and affinity of ZFDBDs will have a significant impact on their use in analytical or therapeutic settings. © 2015 Wiley Periodicals, Inc.

  16. Identification of procollagen promoter DNA-binding proteins: effects of dexamethasone

    Energy Technology Data Exchange (ETDEWEB)

    Sweeney, C.; Cutroneo, K.R.

    1987-05-01

    Glucocorticoids selectively decrease procollagen synthesis by decreasing procollagen mRNA transcription. Dexamethasone coordinately decreased total cellular type I and type III procollagen mRNAs in mouse embryonic skin fibroblasts. Since sequence specific DNA-binding proteins are known to modulate eukaryotic gene expression the authors identified in mouse fibroblasts nuclear proteins which bind to types I and III procollagen promoter DNAs. Nuclear proteins were electrophoresed, blotted onto nitrocellulose and probed with /sup 32/P-end-labeled type I and type III procollagen promoter DNAs in the presence of equimolar amounts of /sup 32/P-end-labeled vector DNA. Differences in total DNA binding were noted by the densitometric scans of the nuclear proteins. Dexamethasone treatment enhanced total DNA binding. Increasing the NaCl concentration decreased the number of promoter DNA-binding proteins without altering the relative specificity for the promoter DNAs. Promoter DNA binding to nuclear proteins was also inhibited by increasing concentrations of E. coli DNA. The number of DNA-binding proteins was greater for type III procollagen promoter DNA. The effect of dexamethasone treatment on promoter DNA binding to nuclear proteins was determined.

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

    Science.gov (United States)

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

    2012-01-01

    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[LSSSSSS]3+ and (P)-Yb[LRRRRRR]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. PMID:22675072

  18. A Cationic Smart Copolymer for DNA Binding

    Directory of Open Access Journals (Sweden)

    Tânia Ribeiro

    2017-11-01

    Full Text Available A new block copolymer with a temperature-responsive block and a cationic block was prepared by reversible addition-fragmentation chain transfer (RAFT polymerization, with good control of its size and composition. The first block is composed by di(ethylene glycol methyl ether methacrylate (DEGMA and oligo(ethylene glycol methyl ether methacrylate (OEGMA, with the ratio DEGMA/OEGMA being used to choose the volume phase transition temperature of the polymer in water, tunable from ca. 25 to above 90 °C. The second block, of trimethyl-2-methacroyloxyethylammonium chloride (TMEC, is positively charged at physiological pH values and is used for DNA binding. The coacervate complexes between the block copolymer and a model single strand DNA are characterized by fluorescence correlation spectroscopy and fluorescence spectroscopy. The new materials offer good prospects for biomedical application, for example in controlled gene delivery.

  19. Binding dynamics of single-stranded DNA binding proteins to fluctuating bubbles in breathing DNA

    Energy Technology Data Exchange (ETDEWEB)

    Ambjoernsson, Tobias; Metzler, Ralf [NORDITA-Nordic Institute for Theoretical Physics, Blegdamsvej 17, DK-2100 Copenhagen Oe (Denmark)

    2005-05-25

    We investigate the dynamics of a single local denaturation zone in a DNA molecule, a so-called DNA bubble, in the presence of single-stranded DNA binding proteins (SSBs). In particular, we develop a dynamical description of the process in terms of a two-dimensional master equation for the time evolution of the probability distribution of having a bubble of size m with n bound SSBs, for the case when m and n are the slowest variables in the system. We derive explicit expressions for the equilibrium statistical weights for a given m and n, which depend on the statistical weight u associated with breaking a base-pair interaction, the loop closure exponent c, the cooperativity parameter {sigma}{sub 0}, the SSB size {lambda}, and binding strength {kappa}. These statistical weights determine, through the detailed balance condition, the transfer coefficient in the master equation. For the case of slow and fast binding dynamics the problem can be reduced to one-dimensional master equations. In the latter case, we perform explicitly the adiabatic elimination of the fast variable n. Furthermore, we find that for the case that the loop closure is neglected and the binding dynamics is vanishing (but with arbitrary {sigma}{sub 0}) the eigenvalues and the eigenvectors of the master equation can be obtained analytically, using an orthogonal polynomial approach. We solve the general case numerically (i.e., including SSB binding and the loop closure) as a function of statistical weight u, binding protein size {lambda}, and binding strength {kappa}, and compare to the fast and slow binding limits. In particular, we find that the presence of SSBs in general increases the relaxation time, compared to the case when no binding proteins are present. By tuning the parameters, we can drive the system from regular bubble fluctuation in the absence of SSBs to full denaturation, reflecting experimental and in vivo situations.

  20. Specificity of Baculorivus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation

    NARCIS (Netherlands)

    Wang, M.; Tuladhar, E.; Shen, S.; Wang, H.; Oers, van M.M.; Vlak, J.M.; Westenberg, M.

    2010-01-01

    The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9

  1. Binding specificity of Escherichia coli trigger factor

    Science.gov (United States)

    Patzelt, Holger; Rüdiger, Stefan; Brehmer, Dirk; Kramer, Günter; Vorderwülbecke, Sonja; Schaffitzel, Elke; Waitz, Andreas; Hesterkamp, Thomas; Dong, Liying; Schneider-Mergener, Jens; Bukau, Bernd; Deuerling, Elke

    2001-01-01

    The ribosome-associated chaperone trigger factor (TF) assists the folding of newly synthesized cytosolic proteins in Escherichia coli. Here, we determined the substrate specificity of TF by examining its binding to 2842 membrane-coupled 13meric peptides. The binding motif of TF was identified as a stretch of eight amino acids, enriched in basic and aromatic residues and with a positive net charge. Fluorescence spectroscopy verified that TF exhibited a comparable substrate specificity for peptides in solution. The affinity to peptides in solution was low, indicating that TF requires ribosome association to create high local concentrations of nascent polypeptide substrates for productive interaction in vivo. Binding to membrane-coupled peptides occurred through the central peptidyl-prolyl-cis/trans isomerase (PPIase) domain of TF, however, independently of prolyl residues. Crosslinking experiments showed that a TF fragment containing the PPIase domain linked to the ribosome via the N-terminal domain is sufficient for interaction with nascent polypeptide substrates. Homology modeling of the PPIase domain revealed a conserved FKBP(FK506-binding protein)-like binding pocket composed of exposed aromatic residues embedded in a groove with negative surface charge. The features of this groove complement well the determined substrate specificity of TF. Moreover, a mutation (E178V) in this putative substrate binding groove known to enhance PPIase activity also enhanced TF's association with a prolyl-free model peptide in solution and with nascent polypeptides. This result suggests that both prolyl-independent binding of peptide substrates and peptidyl-prolyl isomerization involve the same binding site. PMID:11724963

  2. A monoclonal antibody to triplex DNA binds to eucaryotic chromosomes.

    Science.gov (United States)

    Lee, J S; Burkholder, G D; Latimer, L J; Haug, B L; Braun, R P

    1987-01-01

    A monoclonal antibody (Jel 318) was produced by immunizing mice with poly[d(TmC)].poly[d(GA)].poly[d(mCT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly[d(TG)].poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g. poly[d(TC)].poly[d(GmA)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes was assessed by immunofluorescent microscopy of mouse myeloma cells which had been fixed in methanol/acetic acid. An antibody specific for duplex DNA (Jel 239) served as a control. The fluorescence due to Jel 318 was much weaker than that of Jel 239 but binding to metaphase chromosomes and interphase nuclei was observed. The staining by Jel 318 was unaffected by addition of E. coli DNA but it was obliterated in the presence of triplex. Since an acid pH favours triplex formation, nuclei were also prepared from mouse melanoma cells by fixation in cold acetone. Again Jel 318 showed weak but consistent staining of the nuclei. Therefore it seems likely that triplexes are an inherent feature of the structure of eucaryotic DNA. Images PMID:2434928

  3. Spectral characterization and DNA binding properties of lanthanide(III)

    African Journals Online (AJOL)

    ... with CT-DNA was investigated using absorption spectrophotometry. Based on spectral changes, groove binding of complexes to DNA is suggested. KEY WORDS: Lanthanide(III) complexes, 2-Acetylpyridine isonicotinoylhydrazone, Spectral characterization, DNA binding. Bull. Chem. Soc. Ethiop. 2016, 30(2), 221-230.

  4. Nonspecific DNA Binding and Bending by HUαβ: Interfaces of the Three Binding Modes Characterized by Salt Dependent Thermodynamics

    Science.gov (United States)

    Koh, Junseock; Shkel, Irina; Saecker, Ruth M.; Record, M. Thomas

    2011-01-01

    Previous ITC and FRET studies demonstrated that Escherichia coli HUαβ binds nonspecifically to duplex DNA in three different binding modes: a tighter-binding 34 bp mode which interacts with DNA in large (>34 bp) gaps between bound proteins, reversibly bending it 140° and thereby increasing its flexibility, and two weaker, modestly cooperative small-site-size modes (10 bp, 6 bp) useful for filling gaps between bound proteins shorter than 34 bp. Here we use ITC to determine the thermodynamics of these binding modes as a function of salt concentration, and deduce that DNA in the 34 bp mode is bent around but not wrapped on the body of HU, in contrast to specific binding of IHF. Analyses of binding isotherms (8, 15, 34 bp DNA) and initial binding heats (34, 38, 160 bp DNA) reveal that all three modes have similar log-log salt concentration derivatives of the binding constants (Ski) even though their binding site sizes differ greatly; most probable values of Ski on 34 bp or larger DNA are − 7.5 ± 0.5. From the similarity of Ski values, we conclude that binding interfaces of all three modes involve the same region of the arms and saddle of HU. All modes are entropy-driven, as expected for nonspecific binding driven by the polyelectrolyte effect. The bent-DNA 34 bp mode is most endothermic, presumably because of the cost of HU-induced DNA bending, while the 6 bp mode is modestly exothermic at all salt concentrations examined. Structural models consistent with the observed Ski values are proposed. PMID:21513716

  5. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  6. The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

    Directory of Open Access Journals (Sweden)

    Vida Vafaizadeh

    2013-08-01

    Full Text Available The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment.

  7. Comparative footprinting of DNA-binding proteins.

    Science.gov (United States)

    Contreras-Moreira, Bruno; Collado-Vides, Julio

    2006-07-15

    Comparative modelling is a computational method used to tackle a variety of problems in molecular biology and biotechnology. Traditionally it has been applied to model the structure of proteins on their own or bound to small ligands, although more recently it has also been used to model protein-protein interfaces. This work is the first to systematically analyze whether comparative models of protein-DNA complexes could be built and be useful for predicting DNA binding sites. First, we describe the structural and evolutionary conservation of protein-DNA interfaces, and the limits they impose on modelling accuracy. Second, we find that side-chains from contacting residues can be reasonably modeled and therefore used to identify contacting nucleotides. Third, the DNASITE protocol is implemented and different parameters are benchmarked on a set of 85 regulators from Escherichia coli. Results show that comparative footprinting can make useful predictions based solely on structural data, depending primarily on the interface identity with respect to the template used. DNASITE code available on request from the authors.

  8. DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein.

    Science.gov (United States)

    Morin, José A; Cerrón, Fernando; Jarillo, Javier; Beltran-Heredia, Elena; Ciesielski, Grzegorz L; Arias-Gonzalez, J Ricardo; Kaguni, Laurie S; Cao, Francisco J; Ibarra, Borja

    2017-07-07

    Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB-DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during 'in situ' DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Structures of DNA-binding mutant zinc finger domains: implications for DNA binding.

    Science.gov (United States)

    Hoffman, R C; Horvath, S J; Klevit, R E

    1993-06-01

    Studies of Cys2-His2 zinc finger domains have revealed that the structures of individual finger domains in solution determined by NMR spectroscopy are strikingly similar to the structure of fingers bound to DNA determined by X-ray diffraction. Therefore, detailed structural analyses of single finger domains that contain amino acid substitutions known to affect DNA binding in the whole protein can yield information concerning the structural ramifications of such mutations. We have used this approach to study two mutants in the N-terminal finger domain of ADR1, a yeast transcription factor that contains two Cys2-His2 zinc finger sequences spanning residues 102-159. Two point mutants at position 118 in the N-terminal zinc finger (ADR1b: 102-130) that adversely affect the DNA-binding activity of ADR1 have previously been identified: H118A and H118Y. The structures of wild-type ADR1b and the two mutant zinc finger domains were determined using two-dimensional nuclear magnetic resonance spectroscopy and distance geometry and were refined using a complete relaxation matrix method approach (REPENT) to improve agreement between the models and the nuclear Overhauser effect spectroscopy data from which they were generated. The molecular architecture of the refined wild-type ADR1b domain is presented in detail. Comparisons of wild-type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation. The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which has been postulated to make a direct DNA contact in the wild-type ADR1 protein. The results suggest that the identity of the side chain at the middle DNA contact position in Cys2-His2 zinc fingers may be changed with impunity regarding the domain structure and can affect the affinity of the protein-DNA interaction.

  10. Bacillus halodurans RecA-DNA binding and RecAmediated ...

    African Journals Online (AJOL)

    This is critical for normal cellular function under conditions that lead to altered. DNA metabolism and DNA damage. The RecA proteins of E. coli and Bacillus halodurans both can bind to DNA and catalyze the specific proteolytic cleavage of LexA and lambda repressor which induces. SOS response. At neutral pH ...

  11. MOCCS: Clarifying DNA-binding motif ambiguity using ChIP-Seq data.

    Science.gov (United States)

    Ozaki, Haruka; Iwasaki, Wataru

    2016-08-01

    As a key mechanism of gene regulation, transcription factors (TFs) bind to DNA by recognizing specific short sequence patterns that are called DNA-binding motifs. A single TF can accept ambiguity within its DNA-binding motifs, which comprise both canonical (typical) and non-canonical motifs. Clarification of such DNA-binding motif ambiguity is crucial for revealing gene regulatory networks and evaluating mutations in cis-regulatory elements. Although chromatin immunoprecipitation sequencing (ChIP-seq) now provides abundant data on the genomic sequences to which a given TF binds, existing motif discovery methods are unable to directly answer whether a given TF can bind to a specific DNA-binding motif. Here, we report a method for clarifying the DNA-binding motif ambiguity, MOCCS. Given ChIP-Seq data of any TF, MOCCS comprehensively analyzes and describes every k-mer to which that TF binds. Analysis of simulated datasets revealed that MOCCS is applicable to various ChIP-Seq datasets, requiring only a few minutes per dataset. Application to the ENCODE ChIP-Seq datasets proved that MOCCS directly evaluates whether a given TF binds to each DNA-binding motif, even if known position weight matrix models do not provide sufficient information on DNA-binding motif ambiguity. Furthermore, users are not required to provide numerous parameters or background genomic sequence models that are typically unavailable. MOCCS is implemented in Perl and R and is freely available via https://github.com/yuifu/moccs. By complementing existing motif-discovery software, MOCCS will contribute to the basic understanding of how the genome controls diverse cellular processes via DNA-protein interactions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Tuning genetic clocks employing DNA binding sites.

    Directory of Open Access Journals (Sweden)

    Shridhar Jayanthi

    Full Text Available Periodic oscillations play a key role in cell physiology from the cell cycle to circadian clocks. The interplay of positive and negative feedback loops among genes and proteins is ubiquitous in these networks. Often, delays in a negative feedback loop and/or degradation rates are a crucial mechanism to obtain sustained oscillations. How does nature control delays and kinetic rates in feedback networks? Known mechanisms include proper selection of the number of steps composing a feedback loop and alteration of protease activity, respectively. Here, we show that a remarkably simple means to control both delays and effective kinetic rates is the employment of DNA binding sites. We illustrate this design principle on a widely studied activator-repressor clock motif, which is ubiquitous in natural systems. By suitably employing DNA target sites for the activator and/or the repressor, one can switch the clock "on" and "off" and precisely tune its period to a desired value. Our study reveals a design principle to engineer dynamic behavior in biomolecular networks, which may be largely exploited by natural systems and employed for the rational design of synthetic circuits.

  13. Carcinogenic adducts induce distinct DNA polymerase binding orientations

    Science.gov (United States)

    Vrtis, Kyle B.; Markiewicz, Radoslaw P.; Romano, Louis J.; Rueda, David

    2013-01-01

    DNA polymerases must accurately replicate DNA to maintain genome integrity. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), covalently bind DNA bases and promote mutagenesis near the adduct site. The mechanism by which carcinogenic adducts inhibit DNA synthesis and cause mutagenesis remains unclear. Here, we measure interactions between a DNA polymerase and carcinogenic DNA adducts in real-time by single-molecule fluorescence. We find the degree to which an adduct affects polymerase binding to the DNA depends on the adduct location with respect to the primer terminus, the adduct structure and the nucleotides present in the solution. Not only do the adducts influence the polymerase dwell time on the DNA but also its binding position and orientation. Finally, we have directly observed an adduct- and mismatch-induced intermediate state, which may be an obligatory step in the DNA polymerase proofreading mechanism. PMID:23814187

  14. A comprehensive comparative review of sequence-based predictors of DNA- and RNA-binding residues.

    Science.gov (United States)

    Yan, Jing; Friedrich, Stefanie; Kurgan, Lukasz

    2016-01-01

    Motivated by the pressing need to characterize protein-DNA and protein-RNA interactions on large scale, we review a comprehensive set of 30 computational methods for high-throughput prediction of RNA- or DNA-binding residues from protein sequences. We summarize these predictors from several significant perspectives including their design, outputs and availability. We perform empirical assessment of methods that offer web servers using a new benchmark data set characterized by a more complete annotation that includes binding residues transferred from the same or similar proteins. We show that predictors of DNA-binding (RNA-binding) residues offer relatively strong predictive performance but they are unable to properly separate DNA- from RNA-binding residues. We design and empirically assess several types of consensuses and demonstrate that machine learning (ML)-based approaches provide improved predictive performance when compared with the individual predictors of DNA-binding residues or RNA-binding residues. We also formulate and execute first-of-its-kind study that targets combined prediction of DNA- and RNA-binding residues. We design and test three types of consensuses for this prediction and conclude that this novel approach that relies on ML design provides better predictive quality than individual predictors when tested on prediction of DNA- and RNA-binding residues individually. It also substantially improves discrimination between these two types of nucleic acids. Our results suggest that development of a new generation of predictors would benefit from using training data sets that combine both RNA- and DNA-binding proteins, designing new inputs that specifically target either DNA- or RNA-binding residues and pursuing combined prediction of DNA- and RNA-binding residues. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  15. Multiple DNA binding proteins contribute to timing of chromosome replication in E. coli

    DEFF Research Database (Denmark)

    Riber, Leise; Frimodt-Møller, Jakob; Charbon, Godefroid

    2016-01-01

    Chromosome replication in Escherichia coli is initiated from a single origin, oriC. Initiation involves a number of DNA binding proteins, but only DnaA is essential and specific for the initiation process. DnaA is an AAA+ protein that binds both ATP and ADP with similar high affinities. Dna...... replication is initiated, or the time window in which all origins present in a single cell are initiated, i.e. initiation synchrony, or both. Overall, these DNA binding proteins modulate the initiation frequency from oriC by: (i) binding directly to oriC to affect DnaA binding, (ii) altering the DNA topology...... in or around oriC, (iii) altering the nucleotide bound status of DnaA by interacting with non-coding chromosomal sequences, distant from oriC, that are important for DnaA activity. Thus, although DnaA is the key protein for initiation of replication, other DNA-binding proteins act not only on ori...

  16. An Overview of the Prediction of Protein DNA-Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

    Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  17. Heterogeneous dynamics in DNA site discrimination by the structurally homologous DNA-binding domains of ETS-family transcription factors

    Science.gov (United States)

    He, Gaofei; Tolic, Ana; Bashkin, James K.; Poon, Gregory M. K.

    2015-01-01

    The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs’ binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs. PMID:25824951

  18. DNA minor groove binding of small molecules: Experimental and ...

    Indian Academy of Sciences (India)

    Eight indole derivatives were studied for their DNA binding ability using fluorescence quenching and molecular docking methods. These indole compounds have structural moieties similar as in few indole alkaloids. Experimental and theoretical studies have suggested that indole derivatives bind in the minor groove of DNA.

  19. Synthesis, DNA binding and cytotoxic evaluation of aminoquinoline ...

    Indian Academy of Sciences (India)

    DNA binding studies of selected isomeric compounds showed interaction withDNA via intercalation mode with higher binding affinity of 4-substituted ... Universiti Sains Malaysia, Minden 11800, Penang, Malaysia; New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy College, Alwar, ...

  20. A role for the weak DnaA binding sites in bacterial replication origins

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Løbner-Olesen, Anders

    2011-01-01

    DnaA initiates the chromosomal DNA replication in nearly all bacteria, and replication origins are characterized by binding sites for the DnaA protein (DnaA-boxes) along with an ‘AT-rich’ region. However, great variation in number, spatial organization and specificity of DnaA-boxes is observed...... between species. In the study by Taylor et al. (2011), new and unexpectedly weak DnaA-boxes were identified within the Caulobacter crescentus origin of replication (Cori). The position of weak and stronger DnaA-boxes follows a pattern seen in Escherichia coli oriC. This raises the possibility...

  1. DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

    Directory of Open Access Journals (Sweden)

    Marx Kenneth A

    2006-03-01

    Full Text Available Abstract Background The centromeres in yeast (S. cerevisiae are organized by short DNA sequences (125 bp on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants. Results In the CDE I and CDEIII core binding regions an increase in the magnitude of change in deformability of chromosome 6 single base mutants with respect to the wild type correlates to an increase in the measured chromosome loss rate. These correlations were found to be significant relative to 105 Monte Carlo randomizations of the dinucleotide PD-scale applied to the same calculation. A net loss of deformability also tends to increase the loss rate. Binding site position specific, 4 data-point correlations were also

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

    Science.gov (United States)

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

    2013-03-05

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

  3. Screening of Threading Bis-Intercalators Binding to Duplex DNA by Electrospray Ionization Tandem Mass Spectrometry

    Science.gov (United States)

    Mazzitelli, Carolyn L.; Chu, Yongjun; Reczek, Joseph J.; Iverson, Brent L.

    2007-01-01

    The DNA binding of novel threading bis-intercalators V1, trans-D1, and cis-C1, which contain two naphthalene diimide (NDI) intercalation units connected by a scaffold, was evaluated using electrospray ionization mass spectrometry (ESI-MS) and DNAse footprinting techniques. ESI-MS experiments confirmed that V1, the ligand containing the –Gly3-Lys-peptide scaffold, binds to a DNA duplex containing the 5'-GGTACC-3' specific binding site identified in previous NMR-based studies. The ligand formed complexes with a ligand/DNA binding stoichiometry of 1:1, even when there was excess ligand in solution. Trans-D1 and cis-C1 are new ligands containing a rigid spiro-tricyclic scaffold in the trans- and cis- orientations, respectively. Preliminary DNAse footprinting experiments identified possible specific binding sites of 5'-CAGTGA-5' for trans-D1 and 5'-GGTACC-3' for cis-C1. ESI-MS experiments revealed that both ligands bound to DNA duplexes containing the respective specific binding sequences, with cis-C1 exhibiting the most extensive binding based on a higher fraction of bound DNA value. Cis-C1 formed complexes with a dominant 1:1 binding stoichiometry, whereas trans-D1 was able to form 2:1 complexes at ligand/DNA molar ratios ≥ 1 which is suggestive of non-specific binding. Collisional activated dissociation (CAD) experiments indicate that DNA complexes containing V1, trans-D1, and cis-C1 have a unique fragmentation pathway, which was also observed for complexes containing the commercially available bisintercalator echinomycin, as a result of similar binding interactions, marked by intercalation in addition to hydrogen bonding by the scaffold with the DNA major or minor groove. PMID:17098442

  4. DNase I footprinting of small molecule binding sites on DNA.

    Science.gov (United States)

    Bailly, Christian; Kluza, Jérôme; Martin, Christopher; Ellis, Thomas; Waring, Michael J

    2005-01-01

    Nuclease footprinting techniques were initially developed to investigate protein-deoxyribonucleic acid (DNA) interactions but these tools of molecular biology have also become instrumental for probing sequence-selective binding of small molecules to DNA. Here, the method is described and technical details are given for performing deoxyribonuclease (DNase) I footprinting with DNA-binding drugs. An example is presented where DNase I is used (as well as DNase II and micrococcal nuclease) to probe the patterns of sequence-selective recognition of DNA by the anticancer antibiotic actinomycin D. DNase I is a convenient endonuclease for detecting and locating the position of actinomycin-binding sites within GC-rich sequences.

  5. Binding of 8-methoxypsoralen to DNA in vitro: Monitoring by spectroscopic and chemometrics approaches

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Xiaoyue; Zhang, Guowen, E-mail: gwzhang@ncu.edu.cn; Wang, Langhong

    2014-10-15

    8-Methoxypsoralen (8-MOP) is a naturally occurring furanocoumarin with a variety of biological and pharmacological activities. The binding mechanism of 8-MOP to calf thymus DNA (ctDNA) at physiological pH was investigated by multi-spectroscopic techniques including UV–vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy along with DNA melting studies and viscosity measurements. The multivariate curve resolution-alternating least squares (MCR-ALS) chemometrics approach was introduced to resolve the expanded UV–vis spectral data matrix, and both the pure spectra and the equilibrium concentration profiles for the components (8-MOP, ctDNA and 8-MOP-ctDNA complex) in the system were successfully obtained to monitor the 8-MOP-ctDNA interaction. The results suggested that 8-MOP could bind to ctDNA via intercalation binding as evidenced by significant increases in melting and relative viscosity of ctDNA and competitive study using acridine orange (AO) as a fluorescence probe. The positive values of enthalpy and entropy change suggested that hydrogen bonds and van der Waals forces played a predominant role in the binding process. Further, FT-IR and CD spectra analysis indicated that 8-MOP preferentially bound to A–T base pairs with no major perturbation in ctDNA double helix conformation. Moreover, molecular docking was employed to exhibit the specific binding mode of 8-MOP to ctDNA intuitively. - Highlights: • The interaction processes of 8-MOP with ctDNA was monitored by MCR-ALS approach. • The binding mode of 8-MOP to ctDNA was an intercalation. • 8-MOP most likely bound to adenine and thymine base pairs of ctDNA. • Molecular docking illustrated the specific binding.

  6. [Design of artificial DNA binding proteins toward control and elucidation of cellular functions].

    Science.gov (United States)

    Imanishi, Miki

    2012-01-01

    An artificial transcription factor that can regulate the expression of specific genes at a desired time is very useful for research in chemical biology, cell biology, and future gene therapy. A C2H2 zinc finger motif, one of zinc-containing proteins, is known as the most ubiquitous DNA binding motif. The motif is attractive for designing artificial transcription factors with desired DNA binding specificities because of its characteristic DNA binding properties: (1) recognition of 3 bp per motif, (2) tandemly connected modular structure, and (3) binding to non-palindrome sequences as a monomer. Taking advantage of these properties, artificial DNA binding proteins with new DNA binding characteristics have been designed. By changing the linker region between two 3-zinc finger domains, artificial 6-zinc finger proteins were developed and shown to skip DNA sequences. Zinc-responsive transcription factors were created by altering one of the zinc ligands. An artificial zinc finger transcription factor targeting a core clock gene induced phase shifts of the cellular "circadian rhythm". Herein, I will summarize creation and function of the above-mentioned artificial zinc finger-type DNA binding proteins and transcription factors.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1996-05-01

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

  8. Adsorption of DNA binding proteins to functionalized carbon nanotube surfaces with and without DNA wrapping.

    Science.gov (United States)

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-09-01

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH 2 groups (CONH 2 -SWNT) exhibited very strong interactions between the CONH 2 -SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  9. DNA and protein footprinting analysis of the modulation of DNA binding by the N-terminal domain of the Saccharomyces cerevisiae TATA binding protein.

    Science.gov (United States)

    Gupta, Sayan; Cheng, Huiyong; Mollah, A K M M; Jamison, Elizabeth; Morris, Stephanie; Chance, Mark R; Khrapunov, Sergei; Brenowitz, Michael

    2007-09-04

    Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by "protein footprinting" with hydroxyl radical (*OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.

  10. DNA and Protein Footprinting Analysis of the Modulation of DNA Binding by the N-Terminal Domain of the Saccharomyces cervisiae TATA Binding Protein

    Energy Technology Data Exchange (ETDEWEB)

    Gupta,S.; Cheng, H.; Mollah, A.; Jamison, E.; Morris, S.; Chance, M.; Khrapunov, S.; Brenowitz, M.

    2007-01-01

    Recombinant full-length Saccharomyces cerevisiae TATA binding protein (TBP) and its isolated C-terminal conserved core domain (TBPc) were prepared with measured high specific DNA-binding activities. Direct, quantitative comparison of TATA box binding by TBP and TBPc reveals greater affinity by TBPc for either of two high-affinity sequences at several different experimental conditions. TBPc associates more rapidly than TBP to TATA box bearing DNA and dissociates more slowly. The structural origins of the thermodynamic and kinetic effects of the N-terminal domain on DNA binding by TBP were explored in comparative studies of TBPc and TBP by 'protein footprinting' with hydroxyl radical ({center_dot}OH) side chain oxidation. Some residues within TBPc and the C-terminal domain of TBP are comparably protected by DNA, consistent with solvent accessibility changes calculated from core domain crystal structures. In contrast, the reactivity of some residues located on the top surface and the DNA-binding saddle of the C-terminal domain differs between TBP and TBPc in both the presence and absence of bound DNA; these results are not predicted from the crystal structures. A strikingly different pattern of side chain oxidation is observed for TBP when a nonionic detergent is present. Taken together, these results are consistent with the N-terminal domain actively modulating TATA box binding by TBP and nonionic detergent modulating the interdomain interaction.

  11. Peptide Nucleic Acids Having Enhanced Binding Affinity, Sequence Specificity and Solubility

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from a group consisting of naturally......-occurring nucleobases and non-naturally-occurring nucleobases attached to a polyamide backbone, and contain C1-C8 alkylamine side chains. Methods of enhancing the solubility, binding affinity and sequence specificity of PNAs are provided....

  12. Specific triplex binding capacity of mixed base sequence duplex nucleic acids used for single-nucleotide polymorphism detection.

    Science.gov (United States)

    Daksis, Jasmine I; Erikson, Glen H

    2005-01-01

    Specific base recognition and binding between native double-stranded DNA (dsDNA) and complementary single-stranded DNA (ssDNA) of mixed base sequence is presented. Third-strand binding, facilitated and stabilized by a DNA intercalator, YOYO-1, occurs within 5 min at room temperature. This triplex binding capability has been used to develop a homogeneous assay that accurately detects 1-, 2-, or 3-bp mutations or deletions in the dsDNA target. Every type of 1-bp mismatch can be identified. The assay can reliably distinguish homozygous from heterozygous polymerase chain reaction (PCR)-amplified genomic dsDNA, thus providing a highly sensitive clinical diagnostic assay.

  13. Plasmodium vivax Duffy binding protein peptides specifically bind to reticulocytes.

    Science.gov (United States)

    Ocampo, Marisol; Vera, Ricardo; Eduardo Rodriguez, Luis; Curtidor, Hernando; Urquiza, Mauricio; Suarez, Jorge; Garcia, Javier; Puentes, Alvaro; Lopez, Ramsés; Trujillo, Mary; Torres, Elizabeth; Patarroyo, Manuel Elkin

    2002-01-01

    Plasmodium vivax Duffy Binding Protein (Pv-DBP) is essential during merozoite invasion of reticulocytes. Reticulocyte binding region identification is important for understanding Pv-DBP reticulocyte recognition. Fifty 20 mer non-overlapping peptides, spanning Pv-DBP sequences, were tested in erythrocyte and reticulocyte binding assays. Ten HARBPs, mainly located in region II (Kd 50-130 nM), were High Activity Reticulocyte Binding Peptides (HARBPs); one bound to erythrocytes. Reticulocyte trypsin-, chymotrypsin- or neuraminidase- treatment affects HARBP binding differently, suggesting that these peptides have different reticulocyte-binding-sites. Some peptides bound to a Coomasie non-stainable 40 Kda band. Some HARBPs were able to block recombinant PvRII binding (Pv-DBP region II) to Duffy positive reticulocytes.

  14. Extended HSR/CARD domain mediates AIRE binding to DNA

    Energy Technology Data Exchange (ETDEWEB)

    Maslovskaja, Julia, E-mail: julia.maslovskaja@ut.ee; Saare, Mario; Liiv, Ingrid; Rebane, Ana; Peterson, Pärt

    2015-12-25

    Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved in AIRE binding to DNA. - Highlights: • Promoter and mRNA processing elements are not important for AIRE to activate gene expression from reporter plasmids. • AIRE protein fragment aa 1–138 mediates direct binding to DNA. • Integrity of the HSR/CARD domain is needed for AIRE binding to DNA.

  15. Cloning and characterization of a cDNA encoding a male-specific serum protein of the Mediterranean fruit fly, Ceratitis capitata, with sequence similarity to odourant-binding proteins.

    Science.gov (United States)

    Thymianou, S; Mavroidis, M; Kokolakis, G; Komitopoulou, K; Zacharopoulou, A; Mintzas, A C

    1998-11-01

    Male-specific serum proteins (MSSPs) are low molecular weight proteins which accumulate in high amounts in the haemolymph of adult males of the medfly Ceratitis capitata. By screening an expression library with anti-MSSP antibodies, we have isolated and determined the nucleotide sequence of a cDNA clone coding for one of the male-specific polypeptides (MSSP-alpha). The MSSP-alpha mRNA encodes a polypeptide of 144 amino acids with a secretory signal sequence of sixteen amino acids. Southern analysis indicated that there are multiple copies of MSSP genes in the medfly genome. Northern analysis showed that the MSSP mRNAs are synthesized only in adult males. The accumulation pattern of these mRNAs during development suggests that the expression of the MSSP genes is developmentally regulated at both transcriptional and translational levels. The predicted peptide sequence of MSSP-alpha shows significant similarity to a group of pheromone- and general odourant-binding proteins of insects.

  16. In vivo footprinting and DNA affinity chromatography for analysis of p53 DNA binding ability.

    Science.gov (United States)

    Molina, Maria Patricia; Cain, Christine; Bargonetti, Jill

    2003-01-01

    p53 is a sequence-specific DNA binding protein. The p53 consensus is two copies of 5'- RRRC(A/T)(T/A)GYYY-3'. The interaction of p53 with specific DNA binding sites (DBS) has been analyzed extensively using electrophoretic mobility shift analysis (EMSA). These studies do not address the interaction of p53 with nuclear chromatin or the stability of p53-DBS interactions. In vivo footprinting examines the dynamic interactions of p53 protein in nuclear chromatin. p53 DBS affinity chromatography compares the stability of p53 from different cellular extracts with different DBS. Isogenic strains expressing high p53 levels, and deleted for p53, are required for controlled experiments using both methods. Different systems can be used to generate sufficient p53 protein (including DNA damage), and this results in the analysis of different forms of p53. A comparison of different cellular sources of high levels of p53 (in the presence and absence of DNA damage) vs different p53 DBS is required to appreciate the complexity of the regulation. Methods for comparing p53 from three different cellular sources with different DBS are presented here. The p53 research community needs to expand this analysis to complete the picture of how p53 differentially regulates transcription of target genes in nuclear chromatin.

  17. CRITERIA FOR AN UPDATED CLASSIFICATION OF HUMAN TRANSCRIPTION FACTOR DNA-BINDING DOMAINS

    NARCIS (Netherlands)

    Wingender, Edgar

    By binding to cis-regulatory elements in a sequence-specific manner, transcription factors regulate the activity of nearby genes. Here, we discuss the criteria for a comprehensive classification of human TFs based on their DNA-binding domains. In particular, classification of basic leucine zipper

  18. Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair.

    Directory of Open Access Journals (Sweden)

    Corentin Claeys Bouuaert

    2017-05-01

    Full Text Available Mlh1-Mlh3 (MutLγ is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ and, surprisingly, single-stranded DNA (ssDNA, which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced

  19. Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair

    Science.gov (United States)

    2017-01-01

    Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of

  20. Nanopore-based DNA-probe sequence-evolution method unveiling characteristics of protein-DNA binding phenomena in a nanoscale confined space.

    Science.gov (United States)

    Liu, Nannan; Yang, Zekun; Lou, Xiaoding; Wei, Benmei; Zhang, Juntao; Gao, Pengcheng; Hou, Ruizuo; Xia, Fan

    2015-04-07

    Almost all of the important functions of DNA are realized by proteins which interact with specific DNA, which actually happens in a limited space. However, most of the studies about the protein-DNA binding are in an unconfined space. Here, we propose a new method, nanopore-based DNA-probe sequence-evolution (NDPSE), which includes up to 6 different DNA-probe systems successively designed in a nanoscale confined space which unveil the more realistic characteristics of protein-DNA binding phenomena. There are several features; for example, first, the edge-hindrance and core-hindrance contribute differently for the binding events, and second, there is an equilibrium between protein-DNA binding and DNA-DNA hybridization.

  1. DNA-Based Nanostructures: Changes of Mechanical Properties of DNA upon Ligand Binding

    Science.gov (United States)

    Nechipurenko, Yury; Grokhovsky, Sergey; Gursky, Georgy; Nechipurenko, Dmitry; Polozov, Robert

    The formation of DNA-based nanostructures involves the binding of different kinds of ligands to DNA as well as the interaction of DNA molecules with each other. Complex formation between ligand and DNA can alter physicochemical properties of the DNA molecule. In the present work, the accessibility of DNA-ligand complexes to cleavage by DNase I are considered, and the exact algorithms for analysis of diagrams of DNase I footprinting for ligand-DNA complexes are obtained. Changes of mechanical properties of the DNA upon ligand binding are also demonstrated by the cleavage patterns generated upon ultrasound irradiation of cis-platin-DNA complexes. Propagation of the mechanical perturbations along DNA in the presence of bound ligands is considered in terms of a string model with a heterogeneity corresponding to the position of a bound ligand on DNA. This model can reproduce qualitatively the cleavage patterns obtained upon ultrasound irradiation of cis-platin-DNA complexes.

  2. Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans.

    Science.gov (United States)

    Warfel, Jaycob D; LiCata, Vince J

    2015-07-01

    Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Maternal low-protein diet affects epigenetic regulation of hepatic mitochondrial DNA transcription in a sex-specific manner in newborn piglets associated with GR binding to its promoter.

    Directory of Open Access Journals (Sweden)

    Yimin Jia

    Full Text Available Mitochondrial oxidative phosphorylation (OXPHOS plays an important role in energy homeostasis by controlling electron transfer and ATP generation. Maternal malnutrition during pregnancy affects mitochondrial (mt DNA-encoded OXPHOS activity in offspring, yet it is unknown whether epigenetic mechanism is involved in the transcriptional regulation of mtDNA-encoded OXPHOS genes. In this study, 14 primiparous purebred Meishan sows were fed either standard- (SP, 12% crude protein or low-protein (LP; 6% crude protein diets throughout gestation, and the hepatic expression and transcriptional regulation of mtDNA-encoded OXPHOS genes were analyzed in newborn piglets. Maternal low protein diet decreased hepatic mtDNA copy number in males, but not in females. LP male piglets had significantly higher hepatic AMP concentration and low energy charge, which was accompanied by enhanced mRNA expression of NADH dehydrogenase subunits 6, cytochrome c oxidase subunit 1, 2, 3 and cytochrome b, as well as increased cytochrome c oxidase enzyme activity. In contrast, LP female piglets showed significantly lower hepatic AMP concentrations and higher energy charge with no alterations in OXPHOS gene expression. Moreover, LP males demonstrated higher glucocorticoid receptor (GR binding to the mtDNA promoter compared with SP males, which was accompanied by lower cytosine methylation and hydroxymethylation on mtDNA promoter. Interestingly, opposite changes were seen in females, which showed diminished GR binding and enriched cytosine methylation and hydroxymethylation on mtDNA promoter. These results suggest that maternal low protein diet during pregnancy causes sex-dependent epigenetic alterations in mtDNA-encoded OXPHOS gene expression, possibly GR is involved in mtDNA transcription regulation.

  4. Novel approach for selecting the best predictor for identifying the binding sites in DNA binding proteins.

    Science.gov (United States)

    Nagarajan, R; Ahmad, Shandar; Gromiha, M Michael

    2013-09-01

    Protein-DNA complexes play vital roles in many cellular processes by the interactions of amino acids with DNA. Several computational methods have been developed for predicting the interacting residues in DNA-binding proteins using sequence and/or structural information. These methods showed different levels of accuracies, which may depend on the choice of data sets used in training, the feature sets selected for developing a predictive model, the ability of the models to capture information useful for prediction or a combination of these factors. In many cases, different methods are likely to produce similar results, whereas in others, the predictors may return contradictory predictions. In this situation, a priori estimates of prediction performance applicable to the system being investigated would be helpful for biologists to choose the best method for designing their experiments. In this work, we have constructed unbiased, stringent and diverse data sets for DNA-binding proteins based on various biologically relevant considerations: (i) seven structural classes, (ii) 86 folds, (iii) 106 superfamilies, (iv) 194 families, (v) 15 binding motifs, (vi) single/double-stranded DNA, (vii) DNA conformation (A, B, Z, etc.), (viii) three functions and (ix) disordered regions. These data sets were culled as non-redundant with sequence identities of 25 and 40% and used to evaluate the performance of 11 different methods in which online services or standalone programs are available. We observed that the best performing methods for each of the data sets showed significant biases toward the data sets selected for their benchmark. Our analysis revealed important data set features, which could be used to estimate these context-specific biases and hence suggest the best method to be used for a given problem. We have developed a web server, which considers these features on demand and displays the best method that the investigator should use. The web server is freely available at

  5. The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange.

    Science.gov (United States)

    Qian, Yufeng; Johnson, Kenneth A

    2017-08-04

    The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB-ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB) 30 and (SSB) 60 , defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB) 60 and (SSB) 30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 10 9 m -1 s -1 ) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Binding properties of pendimethalin herbicide to DNA: multispectroscopic and molecular docking approaches.

    Science.gov (United States)

    Ahmad, Irshad; Ahmad, Ajaz; Ahmad, Masood

    2016-03-07

    Pendimethalin (PND) is a systemic herbicide widely used on rice, cotton, peas, wheat, potatoes, fruits, nuts and other residential and non-residential crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-targeted animals including humans. Most likely, the genotoxicity of agrochemicals/drugs is modulated through cellular distribution of bound DNA. Therefore, the in vitro interaction of PND with calf thymus DNA (ctDNA) has been investigated using various sensitive biophysical techniques to ascertain its binding mechanism. UV-visible absorption and fluorescence spectra suggested the formation of a complex between PND and ctDNA. The binding constant of the PND-ctDNA complex was found to be around 10(4) M(-1) using steady state fluorescence titration. The calculated positive values of enthalpy and entropy changes suggested that the binding reaction was predominantly driven by hydrophobic interactions. Competitive displacement studies using acridine orange (AO), ethidium bromide (EB) and Hoechst dye suggested intercalation of PND molecules into the double helix of ctDNA by replacing the bound AO and EB probes. An increase in the viscosity and melting temperature of ctDNA and a decrease in iodine-quenching also support the intercalative binding of PND with ctDNA. Molecular docking analysis further confirmed the specific binding mode of PND between adjacent 'G-C' base pairs of ctDNA.

  7. The structural basis for dynamic DNA binding and bridging interactions which condense the bacterial centromere.

    Science.gov (United States)

    Fisher, Gemma Lm; Pastrana, César L; Higman, Victoria A; Koh, Alan; Taylor, James A; Butterer, Annika; Craggs, Timothy; Sobott, Frank; Murray, Heath; Crump, Matthew P; Moreno-Herrero, Fernando; Dillingham, Mark S

    2017-12-15

    The ParB protein forms DNA bridging interactions around parS to condense DNA and earmark the bacterial chromosome for segregation. The molecular mechanism underlying the formation of these ParB networks is unclear. We show here that while the central DNA binding domain is essential for anchoring at parS , this interaction is not required for DNA condensation. Structural analysis of the C-terminal domain reveals a dimer with a lysine-rich surface that binds DNA non-specifically and is essential for DNA condensation in vitro. Mutation of either the dimerisation or the DNA binding interface eliminates ParB-GFP foci formation in vivo. Moreover, the free C-terminal domain can rapidly decondense ParB networks independently of its ability to bind DNA. Our work reveals a dual role for the C-terminal domain of ParB as both a DNA binding and bridging interface, and highlights the dynamic nature of ParB networks in Bacillus subtilis .

  8. Identification of a Bipartite Jasmonate-Responsive Promoter Element in the Catharanthus roseus ORCA3 Transcription Factor Gene That Interacts Specifically with AT-Hook DNA-Binding Proteins1[W

    Science.gov (United States)

    Vom Endt, Débora; Soares e Silva, Marina; Kijne, Jan W.; Pasquali, Giancarlo; Memelink, Johan

    2007-01-01

    Jasmonates are plant signaling molecules that play key roles in defense against certain pathogens and insects, among others, by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the APETALA2-domain transcription factor ORCA3 is involved in the jasmonate-responsive activation of terpenoid indole alkaloid biosynthetic genes. ORCA3 gene expression is itself induced by jasmonate. By loss- and gain-of-function experiments, we located a 74-bp region within the ORCA3 promoter, which contains an autonomous jasmonate-responsive element (JRE). The ORCA3 JRE is composed of two important sequences: a quantitative sequence responsible for a high level of expression and a qualitative sequence that appears to act as an on/off switch in response to methyl jasmonate. We isolated 12 different DNA-binding proteins having one of four different types of DNA-binding domains, using the ORCA3 JRE as bait in a yeast (Saccharomyces cerevisiae) one-hybrid transcription factor screening. The binding of one class of proteins bearing a single AT-hook DNA-binding motif was affected by mutations in the quantitative sequence within the JRE. Two of the AT-hook proteins tested had a weak activating effect on JRE-mediated reporter gene expression, suggesting that AT-hook family members may be involved in determining the level of expression of ORCA3 in response to jasmonate. PMID:17496112

  9. Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation.

    Science.gov (United States)

    Clark, Andrew G; Naufer, M Nabuan; Westerlund, Fredrik; Lincoln, Per; Rouzina, Ioulia; Paramanathan, Thayaparan; Williams, Mark C

    2018-01-05

    Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Crystal structure of the mineralocorticoid receptor DNA binding domain in complex with DNA.

    Science.gov (United States)

    Hudson, William H; Youn, Christine; Ortlund, Eric A

    2014-01-01

    The steroid hormone receptors regulate important physiological functions such as reproduction, metabolism, immunity, and electrolyte balance. Mutations within steroid receptors result in endocrine disorders and can often drive cancer formation and progression. Despite the conserved three-dimensional structure shared among members of the steroid receptor family and their overlapping DNA binding preference, activation of individual steroid receptors drive unique effects on gene expression. Here, we present the first structure of the human mineralocorticoid receptor DNA binding domain, in complex with a canonical DNA response element. The overall structure is similar to the glucocorticoid receptor DNA binding domain, but small changes in the mode of DNA binding and lever arm conformation may begin to explain the differential effects on gene regulation by the mineralocorticoid and glucocorticoid receptors. In addition, we explore the structural effects of mineralocorticoid receptor DNA binding domain mutations found in type I pseudohypoaldosteronism and multiple types of cancer.

  12. Convolutional neural network architectures for predicting DNA-protein binding.

    Science.gov (United States)

    Zeng, Haoyang; Edwards, Matthew D; Liu, Ge; Gifford, David K

    2016-06-15

    Convolutional neural networks (CNN) have outperformed conventional methods in modeling the sequence specificity of DNA-protein binding. Yet inappropriate CNN architectures can yield poorer performance than simpler models. Thus an in-depth understanding of how to match CNN architecture to a given task is needed to fully harness the power of CNNs for computational biology applications. We present a systematic exploration of CNN architectures for predicting DNA sequence binding using a large compendium of transcription factor datasets. We identify the best-performing architectures by varying CNN width, depth and pooling designs. We find that adding convolutional kernels to a network is important for motif-based tasks. We show the benefits of CNNs in learning rich higher-order sequence features, such as secondary motifs and local sequence context, by comparing network performance on multiple modeling tasks ranging in difficulty. We also demonstrate how careful construction of sequence benchmark datasets, using approaches that control potentially confounding effects like positional or motif strength bias, is critical in making fair comparisons between competing methods. We explore how to establish the sufficiency of training data for these learning tasks, and we have created a flexible cloud-based framework that permits the rapid exploration of alternative neural network architectures for problems in computational biology. All the models analyzed are available at http://cnn.csail.mit.edu gifford@mit.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

  13. Binding of nucleoid-associated protein fis to DNA is regulated by DNA breathing dynamics.

    Directory of Open Access Journals (Sweden)

    Kristy Nowak-Lovato

    Full Text Available Physicochemical properties of DNA, such as shape, affect protein-DNA recognition. However, the properties of DNA that are most relevant for predicting the binding sites of particular transcription factors (TFs or classes of TFs have yet to be fully understood. Here, using a model that accurately captures the melting behavior and breathing dynamics (spontaneous local openings of the double helix of double-stranded DNA, we simulated the dynamics of known binding sites of the TF and nucleoid-associated protein Fis in Escherichia coli. Our study involves simulations of breathing dynamics, analysis of large published in vitro and genomic datasets, and targeted experimental tests of our predictions. Our simulation results and available in vitro binding data indicate a strong correlation between DNA breathing dynamics and Fis binding. Indeed, we can define an average DNA breathing profile that is characteristic of Fis binding sites. This profile is significantly enriched among the identified in vivo E. coli Fis binding sites. To test our understanding of how Fis binding is influenced by DNA breathing dynamics, we designed base-pair substitutions, mismatch, and methylation modifications of DNA regions that are known to interact (or not interact with Fis. The goal in each case was to make the local DNA breathing dynamics either closer to or farther from the breathing profile characteristic of a strong Fis binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by gel-shift assay, changed in accordance with our expectations. We conclude that Fis binding is associated with DNA breathing dynamics, which in turn may be regulated by various nucleotide modifications.

  14. New non detrimental DNA binding mutants of the Escherichia coli initiator protein DnaA

    DEFF Research Database (Denmark)

    Asklund, Marlene; Atlung, Tove

    2004-01-01

    gave rise to 30 single amino acid substitutions and, including double substitutions, more than 100 mutants functional in initiation of chromosome replication were characterized. The analysis indicated that all regions of the DNA-binding domain are involved in DNA binding, but the most important amino......The initiator protein DnaA has several unique DNA-binding features. It binds with high affinity as a monomer to the nonamer DnaA box. In the ATP form, DnaA binds cooperatively to the low-affinity ATP-DnaA boxes, and to single-stranded DNA in the 13mer region of the origin. We have carried out...... an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection...

  15. DNA binding and cleavage activity of a structurally characterized Ni ...

    Indian Academy of Sciences (India)

    The binding constant (Kb) and the linear Stern-Volmer quenching constant (Ksv) of the complex have been determined as 9.23 × 10 4 M−1 and 2.0 × 10 4 M−1, respectively. Spectroscopic and hydrodynamic investigations revealed groove or electrostatic nature of binding of 1 with DNA. 1 is also found to induce oxidative ...

  16. Ligand-binding pocket bridges DNA-binding and dimerization domains of the urate-responsive MarR homologue MftR from Burkholderia thailandensis.

    Science.gov (United States)

    Gupta, Ashish; Grove, Anne

    2014-07-15

    Members of the multiple antibiotic resistance regulator (MarR) family often regulate gene activity by responding to a specific ligand. In the absence of ligand, most MarR proteins function as repressors, while ligand binding causes attenuated DNA binding and therefore increased gene expression. Previously, we have shown that urate is a ligand for MftR (major facilitator transport regulator), which is encoded by the soil bacterium Burkholderia thailandensis. We show here that both mftR and the divergently oriented gene mftP encoding a major facilitator transport protein are upregulated in the presence of urate. MftR binds two cognate sites in the mftR-mftP intergenic region with equivalent affinity and sensitivity to urate. Mutagenesis of four conserved residues previously reported to be involved in urate binding to Deinococcus radiodurans HucR and Rhizobium radiobacter PecS significantly reduced protein stability and DNA binding affinity but not ligand binding. These data suggest that residues equivalent to those implicated in ligand binding to HucR and PecS serve structural roles and that MftR relies on distinct residues for ligand binding. MftR exhibits a two-step melting transition suggesting independent unfolding of the dimerization and DNA-binding regions; urate binding or mutations in the predicted ligand-binding sites result in one-step unfolding transitions. We suggest that MftR binds the ligand in a cleft between the DNA-binding lobes and the dimer interface but that the mechanism of ligand-mediated attenuation of DNA binding differs from that proposed for other urate-responsive MarR homologues. Since DNA binding by MftR is attenuated at 37 °C, our data also suggest that MftR responds to both ligand and a thermal upshift by attenuated DNA binding and upregulation of the genes under its control.

  17. β -Cyclodextrin polymer binding to DNA: Modulating the physicochemical parameters

    Science.gov (United States)

    Rocha, J. C. B.; Silva, E. F.; Oliveira, M. F.; Sousa, F. B.; Teixeira, A. V. N. C.; Rocha, M. S.

    2017-05-01

    Cyclodextrins and cyclodextrins-modified molecules have interesting and appealing properties due to their capacity to host components that are normally insoluble or poorly soluble in water. In this work, we investigate the interaction of a β -cyclodextrin polymer (poly-β -CD) with λ -DNA. The polymers are obtained by the reaction of β -CD with epichlorohydrin in alkaline conditions. We have used optical tweezers to characterize the changes of the mechanical properties of DNA molecules by increasing the concentration of poly-β -CD in the sample. The physical chemistry of the interaction is then deduced from these measurements by using a recently developed quenched-disorder statistical model. It is shown that the contour length of the DNA does not change in the whole range of poly-β -CD concentration (binding modes corresponding to the clustering of ˜2.6 and ˜14 polymer molecules along the DNA double helix, depending on the polymer concentration. Comparing these results with the ones obtained for monomeric β -CD, it was observed that the concentration of CD that alters the DNA persistence length is considerably smaller when in the polymeric form. Also, the binding constant of the polymer-DNA interaction is three orders of magnitude higher than the one found for native (monomeric) β -CD. These results show that the polymerization of the β -CD strongly increases its binding affinity to the DNA molecule. This property can be wisely used to modulate the binding of cyclodextrins to the DNA double helix.

  18. Quantitative modeling of gene expression using DNA shape features of binding sites.

    Science.gov (United States)

    Peng, Pei-Chen; Sinha, Saurabh

    2016-07-27

    Prediction of gene expression levels driven by regulatory sequences is pivotal in genomic biology. A major focus in transcriptional regulation is sequence-to-expression modeling, which interprets the enhancer sequence based on transcription factor concentrations and DNA binding specificities and predicts precise gene expression levels in varying cellular contexts. Such models largely rely on the position weight matrix (PWM) model for DNA binding, and the effect of alternative models based on DNA shape remains unexplored. Here, we propose a statistical thermodynamics model of gene expression using DNA shape features of binding sites. We used rigorous methods to evaluate the fits of expression readouts of 37 enhancers regulating spatial gene expression patterns in Drosophila embryo, and show that DNA shape-based models perform arguably better than PWM-based models. We also observed DNA shape captures information complimentary to the PWM, in a way that is useful for expression modeling. Furthermore, we tested if combining shape and PWM-based features provides better predictions than using either binding model alone. Our work demonstrates that the increasingly popular DNA-binding models based on local DNA shape can be useful in sequence-to-expression modeling. It also provides a framework for future studies to predict gene expression better than with PWM models alone. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

    Science.gov (United States)

    Upadhyay, Santosh Kumar

    2017-03-01

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

  20. Determining DNA Sequence Specificity of Natural and Artificial Transcription Factors by Cognate Site Identifier Analysis

    Science.gov (United States)

    Ozers, Mary S.; Warren, Christopher L.; Ansari, Aseem Z.

    Artificial transcription factors (ATFs) are designed to mimic natural transcription factors in the control of gene expression and are comprised of domains for DNA binding and gene regulation. ATF domains are modular, interchangeable, and can be composed of protein-based or nonpeptidic moieties, yielding DNA-interacting regulatory molecules that can either activate or inhibit transcription. Sequence-specific targeting is a key determinant in ATF activity, and DNA-binding domains such as natural zinc fingers and synthetic polyamides have emerged as useful DNA targeting molecules. Defining the comprehensive DNA binding specificity of these targeting molecules for accurate manipulations of the genome can be achieved using cognate site identifier DNA microarrays to explore the entire sequence space of binding sites. Design of ATFs that regulate gene expression with temporal control will generate important molecular tools to probe cell- and tissue-specific gene regulation and to function as potential therapeutic agents.

  1. Measuring p53 Binding to Single DNA Molecules in a Nanofluidic Device

    Science.gov (United States)

    Whelsky, Amber; Gonzalez, Nicholas, Jr.; Gal, Susannah; Levy, Stephen

    2012-02-01

    Protein-DNA binding is central to several important cellular processes, for instance, the transfer of genetic information into proteins. The p53 protein plays a central role in regulating several major cell cycle pathways, in part by binding to well-characterized DNA sequences in the promoters of specific genes. Recent studies show that the most common mutation to the protein occurs in the region responsible for its binding to DNA. We have fabricated slit-like nanofluidic devices that allow us to trap and stretch single molecules of DNA containing a known recognition sequence of p53. We use fluorescent microscopy to observe the diffusion of a single p53 protein as it searches for its DNA recognition site. We measure the reaction rates of binding to selected DNA sequences as well as the one-dimensional, non-sequence specific diffusion of p53 along a stretched DNA molecule as a function of salt concentration. The mechanism of facilitated diffusion attempts to explain how proteins seem able to find their DNA target sequences much more quickly than would be expected from three-dimensional diffusion alone. We compare the observed search mechanism used by normal and mutated p53 from cancer cells to predictions based on this theory.

  2. ssDNA Binding Reveals the Atomic Structure of Graphene

    OpenAIRE

    Husale S; Sahoo S; Radenovic A; Traversi F; Annibale P; Kis A

    2010-01-01

    We used AFM to investigate the interaction of polyelectrolytes such as ssDNA and dsDNA molecules with graphene as a substrate. Graphene is an appropriate substrate due to its planarity, relatively large surfaces that are detectable via an optical microscope, and straightforward identification of the number of layers. We observe that in the absence of the screening ions deposited ssDNA will bind only to the graphene and not to the SiO2 substrate, confirming that the binding energy is mainly du...

  3. The Positively Charged Surface of Herpes Simplex Virus UL42 Mediates DNA Binding*S

    Science.gov (United States)

    Komazin-Meredith, Gloria; Santos, Webster L.; Filman, David J.; Hogle, James M.; Verdine, Gregory L.; Coen, Donald M.

    2010-01-01

    Herpes simplex virus DNA polymerase is a heterodimer composed of UL30, a catalytic subunit, and UL42, a processivity subunit. Mutations that decrease DNA binding by UL42 decrease long chain DNA synthesis by the polymerase. The crystal structure of UL42 bound to the C terminus of UL30 revealed an extensive positively charged surface (“back face”). We tested two hypotheses, 1) the C terminus of UL30 affects DNA binding and 2) the positively charged back face mediates DNA binding. Addressing the first hypothesis, we found that the presence of a peptide corresponding to the UL30 C terminus did not result in altered binding of UL42 to DNA. Addressing the second hypothesis, previous work showed that substitution of four conserved arginine residues on the basic face with alanines resulted in decreased DNA affinity. We tested the affinities for DNA and the stimulation of long chain DNA synthesis of mutants in which the four conserved arginine residues were substituted individually or together with lysines and also a mutant in which a conserved glutamine residue was substituted with an arginine to increase positive charge on the back face. We also engineered cysteines onto this surface to permit disulfide cross-linking studies. Last, we assayed the effects of ionic strength on DNA binding by UL42 to estimate the number of ions released upon binding. Our results taken together strongly suggest that the basic back face of UL42 contacts DNA and that positive charge on this surface is important for this interaction. PMID:18178550

  4. Structural basis of DNA recognition by PCG2 reveals a novel DNA binding mode for winged helix-turn-helix domains

    Science.gov (United States)

    Liu, Junfeng; Huang, Jinguang; Zhao, Yanxiang; Liu, Huaian; Wang, Dawei; Yang, Jun; Zhao, Wensheng; Taylor, Ian A.; Peng, You-Liang

    2015-01-01

    The MBP1 family proteins are the DNA binding subunits of MBF cell-cycle transcription factor complexes and contain an N terminal winged helix-turn-helix (wHTH) DNA binding domain (DBD). Although the DNA binding mechanism of MBP1 from Saccharomyces cerevisiae has been extensively studied, the structural framework and the DNA binding mode of other MBP1 family proteins remains to be disclosed. Here, we determined the crystal structure of the DBD of PCG2, the Magnaporthe oryzae orthologue of MBP1, bound to MCB–DNA. The structure revealed that the wing, the 20-loop, helix A and helix B in PCG2–DBD are important elements for DNA binding. Unlike previously characterized wHTH proteins, PCG2–DBD utilizes the wing and helix-B to bind the minor groove and the major groove of the MCB–DNA whilst the 20-loop and helix A interact non-specifically with DNA. Notably, two glutamines Q89 and Q82 within the wing were found to recognize the MCB core CGCG sequence through making hydrogen bond interactions. Further in vitro assays confirmed essential roles of Q89 and Q82 in the DNA binding. These data together indicate that the MBP1 homologue PCG2 employs an unusual mode of binding to target DNA and demonstrate the versatility of wHTH domains. PMID:25550425

  5. Molecular mechanism of DNA association with single-stranded DNA binding protein.

    Science.gov (United States)

    Maffeo, Christopher; Aksimentiev, Aleksei

    2017-12-01

    During DNA replication, the single-stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA) with high affinity to protect it from degradation and prevent secondary structure formation. Although SSB binds ssDNA tightly, it can be repositioned along ssDNA to follow the advancement of the replication fork. Using all-atom molecular dynamics simulations, we characterized the molecular mechanism of ssDNA association with SSB. Placed in solution, ssDNA-SSB assemblies were observed to change their structure spontaneously; such structural changes were suppressed in the crystallographic environment. Repeat simulations of the SSB-ssDNA complex under mechanical tension revealed a multitude of possible pathways for ssDNA to come off SSB punctuated by prolonged arrests at reproducible sites at the SSB surface. Ensemble simulations of spontaneous association of short ssDNA fragments with SSB detailed a three-dimensional map of local affinity to DNA; the equilibrium amount of ssDNA bound to SSB was found to depend on the electrolyte concentration but not on the presence of the acidic tips of the SSB tails. Spontaneous formation of ssDNA bulges and their diffusive motion along SSB surface was directly observed in multiple 10-µs-long simulations. Such reptation-like motion was confined by DNA binding to high-affinity spots, suggesting a two-step mechanism for SSB diffusion. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. DNA sequencing using polymerase substrate-binding kinetics.

    Science.gov (United States)

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-23

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications.

  7. Lack of Ligand-Selective Binding of the Aryl Hydrocarbon Receptor to Putative DNA Binding Sites Regulating Expression of Bax and Paraoxonase 1 Genes

    OpenAIRE

    DeGroot, Danica E.; Hayashi, Ai; Michael S Denison

    2013-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals through its ability to bind specific DNA recognition sites (dioxin responsive elements (DREs)), and activate transcription of adjacent genes. While the DRE has a highly conserved consensus sequence, it has been suggested that the nucleotide specificity of AhR DNA binding may be ligand-dependent. The upstream regulatory regions ...

  8. Regulation of DNA Metabolism by DNA-Binding Proteins Probed by Single Molecule Spectroscopy

    Science.gov (United States)

    2006-12-05

    Recent advances in single - molecule force spectroscopy of DNA make it possible to study the thermodynamics and kinetics of DNA binding proteins under...to transient single-stranded DNA regions due to thermal fluctuations. The model is used to analyze recent single - molecule spectroscopy data of this system.

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

    Science.gov (United States)

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

    2012-01-01

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

  10. Competition for DNA binding sites using Promega DNA IQ™ paramagnetic beads.

    Science.gov (United States)

    Frégeau, Chantal J; De Moors, Anick

    2012-09-01

    The Promega DNA IQ™ system is easily amenable to automation and has been an integral part of standard operating procedures for many forensic laboratories including those of the Royal Canadian Mounted Police (RCMP) since 2004. Due to some failure to extract DNA from samples that should have produced DNA using our validated automated DNA IQ™-based protocol, the competition for binding sites on the DNA IQ™ magnetic beads was more closely examined. Heme from heavily blooded samples interfered slightly with DNA binding. Increasing the concentration of Proteinase K during lysis of these samples did not enhance DNA recovery. However, diluting the sample lysate following lysis prior to DNA extraction overcame the reduction in DNA yield and preserved portions of the lysates for subsequent manual or automated extraction. Dye/chemicals from black denim lysates competed for binding sites on the DNA IQ™ beads and significantly reduced DNA recovery. Increasing the size or number of black denim cuttings during lysis had a direct adverse effect on DNA yield from various blood volumes. The dilution approach was successful on these samples and permitted the extraction of high DNA yields. Alternatively, shortening the incubation time for cell lysis to 30 min instead of the usual overnight at 56 °C prevented competition from black denim dye/chemicals and increased DNA yields. Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.

  11. Building an automated classification of DNA-binding protein domains.

    Science.gov (United States)

    Ponomarenko, Julia V; Bourne, Philip E; Shindyalov, Ilya N

    2002-01-01

    Intensive growth in 3D structure data on DNA-protein complexes as reflected in the Protein Data Bank (PDB) demands new approaches to the annotation and characterization of these data and will lead to a new understanding of critical biological processes involving these data. These data and those from other protein structure classifications will become increasingly important for the modeling of complete proteomes. We propose a fully automated classification of DNA-binding protein domains based on existing 3D-structures from the PDB. The classification, by domain, relies on the Protein Domain Parser (PDP) and the Combinatorial Extension (CE) algorithm for structural alignment. The approach involves the analysis of 3D-interaction patterns in DNA-protein interfaces, assignment of structural domains interacting with DNA, clustering of domains based on structural similarity and DNA-interacting patterns. Comparison with existing resources on describing structural and functional classifications of DNA-binding proteins was used to validate and improve the approach proposed here. In the course of our study we defined a set of criteria and heuristics allowing us to automatically build a biologically meaningful classification and define classes of functionally related protein domains. It was shown that taking into consideration interactions between protein domains and DNA considerably improves the classification accuracy. Our approach provides a high-throughput and up-to-date annotation of DNA-binding protein families which can be found at http://spdc.sdsc.edu.

  12. Synthesis, DNA-binding and photocleavage studies of Ru(II ...

    Indian Academy of Sciences (India)

    Administrator

    molecules to DNA are very important in the deve- lopment of new therapeutic reagents and DNA molecular probes. 15–18. Polypyridyl ruthenium(II) complexes can bind to DNA in a non-covalent inter- actions fashion such as electrostatic binding, groove binding,. 19 intercalative binding and partial intercala- tive binding. 20.

  13. BRCA1 DNA-binding activity is stimulated by BARD1.

    Science.gov (United States)

    Simons, Amanda M; Horwitz, Andrew A; Starita, Lea M; Griffin, Karen; Williams, R Scott; Glover, J N Mark; Parvin, Jeffrey D

    2006-02-15

    The breast- and ovarian-specific tumor suppressor BRCA1 has been implicated in numerous cellular processes, including transcription, ubiquitination, and DNA repair. Its tumor suppression activity is tightly linked to that of BARD1, a protein that heterodimerizes with BRCA1. It has been previously shown that BRCA1 binds to DNA, an interesting functional observation in light of the genetic data linking BRCA1 to DNA repair pathways. In this work, we reexamine the DNA-binding properties of BRCA1, comparing them with the DNA-binding properties of the BRCA1/BARD1 heterodimer. Because nuclear BRCA1 exists as a heterodimer with BARD1, it is likely that in vitro studies of the heterodimer will provide a more accurate model of physiologic conditions. Our results indicate that whereas BARD1 cannot directly bind DNA, it does enhance DNA binding by BRCA1. This is a surprising observation as both DNA-binding domains are distal to the BARD1-interacting RING domain of BRCA1. Further analysis of the dimerization reveals that the BRCA1/BARD1 interaction is not limited to the amino-terminal RING domains of each protein. The carboxyl terminus of BRCA1 contributes significantly to the stability of the heterodimer. We also show that the presence of BARD1 has a secondary effect, as autoubiquitination of BRCA1/BARD1 heterodimers additionally enhances the affinity of BRCA1 for DNA. Together, these data suggest that BRCA1 and BARD1 heterodimerization is stabilized via domains not previously thought to interact and that BARD1 acts in both ubiquitination-dependent and ubiquitination-independent ways to influence the role of BRCA1 in DNA repair.

  14. Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites.

    Science.gov (United States)

    Anisenko, Andrey N; Knyazhanskaya, Ekaterina S; Zatsepin, Timofey S; Gottikh, Marina B

    2017-01-01

    Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described. However, Ku70 subunit is known to have independent DNA binding capability, which is less characterized. RNA binding properties of Ku70 have not been yet specially studied. We have prepared recombinant full-length Ku70 and a set of its truncated mutants in E. coli, and studied their interactions with nucleic acids of various structures: linear single- and double-stranded DNA and RNA, as well as closed circular DNA and hairpin RNA. Ku70 has demonstrated a high affinity binding to double stranded DNA and hairpin RNA with a certain structure only. Interestingly, in contrast to the Ku heterodimer, Ku70 is found to interact with closed circular DNA. We also show for the first time that Ku70 employs two different sites for DNA and RNA binding. The double-stranded DNA is recognized by the C-terminal part of Ku70 including SAP domain as it has been earlier demonstrated, whereas hairpin RNA binding is provided by amino acids 251-438. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. MARs Wars: heterogeneity and clustering of DNA-binding domains in the nuclear matrix

    Directory of Open Access Journals (Sweden)

    Ioudinkova E. S.

    2009-12-01

    Full Text Available Aim. CO326 is a chicken nuclear scaffold/matrix attachment region (MAR associated with the nuclear matrix in several types of chicken cells. It contains a binding site for a sequence-specific DNA-binding protein, F326. We have studied its interaction with the nuclear matrix. Methods. We have used an in vitro MAR assay with isolated matrices from chicken HD3 cells. Results. We have found that an oligonucleotide binding site for the F326 inhibits binding of the CO326 to the nuclear matrix. At the same time, the binding of heterologous MARs is enhanced. Conclusions. Taken together, these data suggest that there exist several classes of MARs and MAR-binding domains and that the MAR-binding proteins may be clustered in the nuclear matrix.

  16. Binding of Harmine Derivatives to DNA: A Spectroscopic Investigation

    Directory of Open Access Journals (Sweden)

    Bruno Pagano

    2017-10-01

    Full Text Available Harmine belongs to a group of β-carboline alkaloids endowed with antitumor properties. Harmine and its derivatives are thought to bind to DNA and interfere with topoisomerase activities. We investigated the base-dependent binding of harmine, and three of its synthetic anticancer-active derivatives to the genomic DNA from calf thymus and two synthetic 20-mer double helices, the poly(dG-dC·poly(dG-dC and the poly(dA-dT·poly(dA-dT, by means of UV-Vis and circular dichroism (CD spectroscopies. The data show that the DNA binding and stabilising properties of the investigated derivatives are base pair-dependent. These results could be used as a guide to design and develop further bioactive analogues.

  17. Drosophila DNA-Binding Proteins in Polycomb Repression

    Directory of Open Access Journals (Sweden)

    Maksim Erokhin

    2018-01-01

    Full Text Available The formation of individual gene expression patterns in different cell types is required during differentiation and development of multicellular organisms. Polycomb group (PcG proteins are key epigenetic regulators responsible for gene repression, and dysregulation of their activities leads to developmental abnormalities and diseases. PcG proteins were first identified in Drosophila, which still remains the most convenient system for studying PcG-dependent repression. In the Drosophila genome, these proteins bind to DNA regions called Polycomb response elements (PREs. A major role in the recruitment of PcG proteins to PREs is played by DNA-binding factors, several of which have been characterized in detail. However, current knowledge is insufficient for comprehensively describing the mechanism of this process. In this review, we summarize and discuss the available data on the role of DNA-binding proteins in PcG recruitment to chromatin.

  18. Enhanced peptide nucleic acid binding to supercoiled DNA: possible implications for DNA "breathing" dynamics

    DEFF Research Database (Denmark)

    Bentin, T; Nielsen, Peter E.

    1996-01-01

    to that of relaxed DNA. The pseudo-first-order rate constant [k psi (sigma)] obeys an exponential function, k psi (sigma) = k psi (lin)e-sigma delta, where delta is a constant of 105 and k psi lin is the rate of PNA binding to linear DNA (sigma = 0). The activation energy [Ea(sigma)] was determined as approximately...... efficient with supercoiled than with linear DNA. In the presence of 140 mM KCI, the PNA binding rate was reduced but, notably, highly dependent on template topology. Negative supercoiling (mean superhelix density, sigma approximately -0.051) increased the rate of binding by 2 orders of magnitude compared...

  19. Thermodynamic characterization of specific interactions between the human Lon protease and G-quartet DNA.

    Science.gov (United States)

    Chen, Si-Han; Suzuki, Carolyn K; Wu, Shih-Hsiung

    2008-03-01

    Lon is an ATP-powered protease that binds DNA. However, the function of DNA binding by Lon remains elusive. Studies suggest that human Lon (hLon) binds preferentially to a G-rich single-stranded DNA (ssDNA) sequence overlapping the light strand promoter of mitochondrial DNA. This sequence is contained within a 24-base oligonucleotide referred to as LSPas. Here, we use biochemical and biophysical approaches to elucidate the structural properties of ssDNAs bound by hLon, as well as the thermodynamics of DNA binding by hLon. Electrophoretic mobility shift assay and circular dichroism show that ssDNAs with a propensity for forming parallel G-quartets are specifically bound by hLon. Isothermal titration calorimetry demonstrates that hLon binding to LSPas is primarily driven by enthalpy change associated with a significant reduction in heat capacity. Differential scanning calorimetry pinpoints an excess heat capacity upon hLon binding to LSPas. By contrast, hLon binding to an 8-base G-rich core sequence is entropically driven with a relatively negligible change in heat capacity. A considerable enhancement of thermal stability accompanies hLon binding to LSPas as compared to the G-rich core. Taken together, these data support the notion that hLon binds G-quartets through rigid-body binding and that binding to LSPas is coupled with structural adaptation.

  20. Thermodynamic characterization of specific interactions between the human Lon protease and G-quartet DNA

    Science.gov (United States)

    Chen, Si-Han; Suzuki, Carolyn K.; Wu, Shih-Hsiung

    2008-01-01

    Lon is an ATP-powered protease that binds DNA. However, the function of DNA binding by Lon remains elusive. Studies suggest that human Lon (hLon) binds preferentially to a G-rich single-stranded DNA (ssDNA) sequence overlapping the light strand promoter of mitochondrial DNA. This sequence is contained within a 24-base oligonucleotide referred to as LSPas. Here, we use biochemical and biophysical approaches to elucidate the structural properties of ssDNAs bound by hLon, as well as the thermodynamics of DNA binding by hLon. Electrophoretic mobility shift assay and circular dichroism show that ssDNAs with a propensity for forming parallel G-quartets are specifically bound by hLon. Isothermal titration calorimetry demonstrates that hLon binding to LSPas is primarily driven by enthalpy change associated with a significant reduction in heat capacity. Differential scanning calorimetry pinpoints an excess heat capacity upon hLon binding to LSPas. By contrast, hLon binding to an 8-base G-rich core sequence is entropically driven with a relatively negligible change in heat capacity. A considerable enhancement of thermal stability accompanies hLon binding to LSPas as compared to the G-rich core. Taken together, these data support the notion that hLon binds G-quartets through rigid-body binding and that binding to LSPas is coupled with structural adaptation. PMID:18174225

  1. Targeting Transcription Factor Binding to DNA by Competing with DNA Binders as an Approach for Controlling Gene Expression.

    Science.gov (United States)

    Bouhlel, Mohamed Amine; Lambert, Melanie; David-Cordonnier, Marie-Helene

    2015-01-01

    Transcription factors are recognized as the master regulators of gene expression. Interestingly, about 10% of the transcription factors described in mammals are up to date directly implicated in a very large number of human diseases. With the exception of ligand-inducible nuclear receptors, transcription factors have longtime been considered as "undruggable" targets for therapeutics. However, the significant breakthroughs in their protein biochemistry and interactions with DNA at the structural level, together with increasing needs for new targeted-approaches particularly in cancers, has changed this postulate and opened the way for targeting transcription factors. Along with a better knowledge of their specific DNA binding sequences by genome wide and high throughput sequencing assay, these informations make possible the potent targeting of the transcription factors by three approaches dependently of their mechanism of action. In this review, we discuss the different physicochemical interactions between the transcription factors and the DNA helix, and the protein/protein interactions within a transcription factor complex and their impacts on the DNA structure. In order to impair transcription factor activities, small molecules compounds can either act by direct interaction on the transcription factor, or by blocking the protein/protein interactions in a transcription complex, or by competing with the transcription factor itself and specifically targeting its cognate binding sequence. For this latter mode of transcription targeting, we pay special attention to the DNA intercalating, alkylating or groove binders for transcription factor/DNA binding modulation.

  2. Structure and DNA binding of alkylation response protein AidB

    Energy Technology Data Exchange (ETDEWEB)

    Bowles, Timothy; Metz, Audrey H.; O' Quin, Jami; Wawrzak, Zdzislaw; Eichman, Brandt F. (Vanderbilt); (NWU)

    2009-01-12

    Exposure of Escherichia coli to alkylating agents activates expression of AidB in addition to DNA repair proteins Ada, AlkA, and AlkB. AidB was recently shown to possess a flavin adenine dinucleotide (FAD) cofactor and to bind to dsDNA, implicating it as a flavin-dependent DNA repair enzyme. However, the molecular mechanism by which AidB acts to reduce the mutagenic effects of specific DNA alkylators is unknown. We present a 1.7-{angstrom} crystal structure of AidB, which bears superficial resemblance to the acyl-CoA dehydrogenase superfamily of flavoproteins. The structure reveals a unique quaternary organization and a distinctive FAD active site that provides a rationale for AidB's limited dehydrogenase activity. A highly electropositive C-terminal domain not present in structural homologs was identified by mutational analysis as the DNA binding site. Structural analysis of the DNA and FAD binding sites provides evidence against AidB-catalyzed DNA repair and supports a model in which AidB acts to prevent alkylation damage by protecting DNA and destroying alkylating agents that have yet to reach their DNA target.

  3. A New Design Strategy and Diagnostic to Tailor the DNA-Binding Mechanism of Small Organic Molecules and Drugs.

    Science.gov (United States)

    Doan, Phi; Pitter, Demar R G; Kocher, Andrea; Wilson, James N; Goodson, Theodore

    2016-11-18

    The classical model for DNA groove binding states that groove binding molecules should adopt a crescent shape that closely matches the helical groove of DNA. Here, we present a new design strategy that does not obey this classical model. The DNA-binding mechanism of small organic molecules was investigated by synthesizing and examining a series of novel compounds that bind with DNA. This study has led to the emergence of structure-property relationships for DNA-binding molecules and/or drugs, which reveals that the structure can be designed to either intercalate or groove bind with calf thymus dsDNA by modifying the electron acceptor properties of the central heterocyclic core. This suggests that the electron accepting abilities of the central core play a key role in the DNA-binding mechanism. These small molecules were characterized by steady-state and ultrafast nonlinear spectroscopies. Bioimaging experiments were performed in live cells to evaluate cellular uptake and localization of the novel small molecules. This report paves a new route for the design and development of small organic molecules, such as therapeutics, targeted at DNA as their performance and specificity is dependent on the DNA-binding mechanism.

  4. Multiple DNA-binding modes for the ETS family transcription factor PU.1.

    Science.gov (United States)

    Esaki, Shingo; Evich, Marina G; Erlitzki, Noa; Germann, Markus W; Poon, Gregory M K

    2017-09-29

    The eponymous DNA-binding domain of ETS ( E 26 t ransformation- s pecific) transcription factors binds a single sequence-specific site as a monomer over a single helical turn. Following our previous observation by titration calorimetry that the ETS member PU.1 dimerizes sequentially at a single sequence-specific DNA-binding site to form a 2:1 complex, we have carried out an extensive spectroscopic and biochemical characterization of site-specific PU.1 ETS complexes. Whereas 10 bp of DNA was sufficient to support PU.1 binding as a monomer, additional flanking bases were required to invoke sequential dimerization of the bound protein. NMR spectroscopy revealed a marked loss of signal intensity in the 2:1 complex, and mutational analysis implicated the distal surface away from the bound DNA as the dimerization interface. Hydroxyl radical DNA footprinting indicated that the site-specifically bound PU.1 dimers occupied an extended DNA interface downstream from the 5'-GGAA-3' core consensus relative to its 1:1 counterpart, thus explaining the apparent site size requirement for sequential dimerization. The site-specifically bound PU.1 dimer resisted competition from nonspecific DNA and showed affinities similar to other functionally significant PU.1 interactions. As sequential dimerization did not occur with the ETS domain of Ets-1, a close structural homolog of PU.1, 2:1 complex formation may represent an alternative autoinhibitory mechanism in the ETS family at the protein-DNA level. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Flow cytometric fluorescence lifetime analysis of DNA binding fluorochromes

    Energy Technology Data Exchange (ETDEWEB)

    Crissman, Harry A.; Cui, H. H. (H. Helen); Steinkamp, J. A.

    2002-01-01

    Most flow cytometry (FCM) applications monitor fluorescence intensity to quantitate the various cellular parameters; however, the fluorescence emission also contains information relative to the fluorescence lifetime. Recent developments in FCM (Pinsky et al., 1993; Steinkamp & Crissman, 1993; Steinkamp et al., 1993), provide for the measurement of fluorescence lifetime which is also commonly referred to as fluorescence decay, or the time interval in which a fluorochrome remains in the excited state. Many unbound fluorochromes have characteristic lifetime values that are determined by their molecular structure; however, when the probe becomes bound, the lifetime value is influenced by a number of factors that affect the probe interaction with a target molecule. Monitoring the changes in the lifetime of the probe yields information relating to the molecular conformation, the functional state or activity of the molecular target. In addition, the lifetime values can be used as signatures to resolve the emissions of multiple fluorochrome labels with overlapping emission spectra that cannot be resolved by conventional FCM methodology. Such strategies can increase the number of fluorochrome combinations used in a flow cytometer with a single excitation source. Our studies demonstrate various applications of lifetime measurements for the analysis of the binding of different fluorochromes to DNA in single cells. Data presented in this session will show the utility of lifetime measurements for monitoring changes in chromatin structure associated with cell cycle progression, cellular differentiation, or DNA damage, such as induced during apoptosis. Several studies show that dyes with specificity for nucleic acids display different lifetime values when bound to DNA or to dsRNA. The Phase Sensitive Flow Cytometer is a multiparameter instrument, capable of performing lifetime measurements in conjunction with all the conventional FCM measurements. Future modifications of this

  6. Biochemical characterization of DNA-binding proteins from Pyrobaculum aerophilum and Aeropyrum pernix.

    Science.gov (United States)

    Hardy, Christine D; Martin, Patrick K

    2008-03-01

    Several representatives of the Crenarchaeal branch of the Archaea contain highly abundant, small, positively charged proteins exemplified by the Sso7d protein from Sulfolobus solfataricus. These proteins bind to DNA in a non-sequence-specific manner. Using publicly available genomic sequence information, we identified a second class of small Crenarchaeal DNA-binding proteins represented by the Pyrobaculum aerophilum open reading frame 3192-encoded (Pae3192) protein and its paralogs. We investigated the biochemical properties of the Pae3192 protein and an orthologous protein (Ape1322b) from Aeropyrum pernix in side-by-side experiments with the Sso7d protein. We demonstrate that the recombinant Ape1322b, Pae3192 and Sso7d proteins bind to DNA and that the DNA-protein complexes formed are slightly different for each protein. We show that like Sso7d, Pae3192 constrains negative supercoils in DNA. In addition, we show that all three proteins raise the melting temperature of duplex DNA upon binding. Finally, we present the equilibrium affinity constants and kinetic association constants of each protein for single-stranded and double-stranded DNA.

  7. Chromosome number9 specific repetitive DNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    Joste, N.E.; Cram, L.S.; Hildebrand, C.E.; Jones, M.; Longmire, J.; Robinson, T.; Moyzis, R.K.

    1986-05-01

    Human repetitive DNA libraries have been constructed and various recombinant DNA clones isolated that are likely candidates for chromosome specific sequences. The first clone tested (pHuR 98; plasmid human repeat 98) was biotinylated and hybridized to human chromosomes in situ. The hybridized recombinant probe was detected with fluoresceinated avidin, and chromosomes were counter-stained with either propidium iodide or distamycin-DAPI. Specific hybridization to chromosome band 9q1 was obtained. The localization was confirmed by hybridizing radiolabeled pHuR 98 DNA to human chromosomes sorted by flow cytometry. Various methods, including orthogonal field pulsed gel electrophoresis analysis indicate that 75 kilobase blocks of this sequence are interspersed with other repetitive DNA sequences in this chromosome band. This study is the first to report a human repetitive DNA sequence uniquely localized to a specific chromosome. This clone provides an easily detected and highly specific chromosomal marker for molecular cytogenetic analyses in numerous basic research and clinical studies.

  8. A plasmonic multi-logic gate platform based on sequence-specific binding of estrogen receptors and gold nanorods.

    Science.gov (United States)

    Pallares, Roger M; Bosman, Michel; Thanh, Nguyen T K; Su, Xiaodi

    2016-12-08

    A hybrid system made of gold nanorods (AuNRs) and double-stranded DNA (dsDNA) is used to build a versatile multi-logic gate platform, capable of performing six different logic operations. The sequence-specific binding of transcription factors to the DNA drives the optical response of the design.

  9. Autoinhibition of ETV6 DNA Binding Is Established by the Stability of Its Inhibitory Helix.

    Science.gov (United States)

    De, Soumya; Okon, Mark; Graves, Barbara J; McIntosh, Lawrence P

    2016-04-24

    The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge-dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    Science.gov (United States)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  11. DNA familial binding profiles made easy: comparison of various motif alignment and clustering strategies.

    Science.gov (United States)

    Mahony, Shaun; Auron, Philip E; Benos, Panayiotis V

    2007-03-30

    Transcription factor (TF) proteins recognize a small number of DNA sequences with high specificity and control the expression of neighbouring genes. The evolution of TF binding preference has been the subject of a number of recent studies, in which generalized binding profiles have been introduced and used to improve the prediction of new target sites. Generalized profiles are generated by aligning and merging the individual profiles of related TFs. However, the distance metrics and alignment algorithms used to compare the binding profiles have not yet been fully explored or optimized. As a result, binding profiles depend on TF structural information and sometimes may ignore important distinctions between subfamilies. Prediction of the identity or the structural class of a protein that binds to a given DNA pattern will enhance the analysis of microarray and ChIP-chip data where frequently multiple putative targets of usually unknown TFs are predicted. Various comparison metrics and alignment algorithms are evaluated (a total of 105 combinations). We find that local alignments are generally better than global alignments at detecting eukaryotic DNA motif similarities, especially when combined with the sum of squared distances or Pearson's correlation coefficient comparison metrics. In addition, multiple-alignment strategies for binding profiles and tree-building methods are tested for their efficiency in constructing generalized binding models. A new method for automatic determination of the optimal number of clusters is developed and applied in the construction of a new set of familial binding profiles which improves upon TF classification accuracy. A software tool, STAMP, is developed to host all tested methods and make them publicly available. This work provides a high quality reference set of familial binding profiles and the first comprehensive platform for analysis of DNA profiles. Detecting similarities between DNA motifs is a key step in the comparative study

  12. DNA familial binding profiles made easy: comparison of various motif alignment and clustering strategies.

    Directory of Open Access Journals (Sweden)

    Shaun Mahony

    2007-03-01

    Full Text Available Transcription factor (TF proteins recognize a small number of DNA sequences with high specificity and control the expression of neighbouring genes. The evolution of TF binding preference has been the subject of a number of recent studies, in which generalized binding profiles have been introduced and used to improve the prediction of new target sites. Generalized profiles are generated by aligning and merging the individual profiles of related TFs. However, the distance metrics and alignment algorithms used to compare the binding profiles have not yet been fully explored or optimized. As a result, binding profiles depend on TF structural information and sometimes may ignore important distinctions between subfamilies. Prediction of the identity or the structural class of a protein that binds to a given DNA pattern will enhance the analysis of microarray and ChIP-chip data where frequently multiple putative targets of usually unknown TFs are predicted. Various comparison metrics and alignment algorithms are evaluated (a total of 105 combinations. We find that local alignments are generally better than global alignments at detecting eukaryotic DNA motif similarities, especially when combined with the sum of squared distances or Pearson's correlation coefficient comparison metrics. In addition, multiple-alignment strategies for binding profiles and tree-building methods are tested for their efficiency in constructing generalized binding models. A new method for automatic determination of the optimal number of clusters is developed and applied in the construction of a new set of familial binding profiles which improves upon TF classification accuracy. A software tool, STAMP, is developed to host all tested methods and make them publicly available. This work provides a high quality reference set of familial binding profiles and the first comprehensive platform for analysis of DNA profiles. Detecting similarities between DNA motifs is a key step in the

  13. Chromosome-specific DNA Repeat Probes

    Energy Technology Data Exchange (ETDEWEB)

    Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

    2006-03-16

    In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

  14. Evidence for an Important Role of WRKY DNA Binding Proteins in the Regulation of NPR1 Gene Expression

    National Research Council Canada - National Science Library

    Yu, Diqiu; Chen, Chunhong; Chen, Zhixiang

    2001-01-01

    .... In the present study, we report the identification of W-box sequences in the promoter region of the NPR1 gene that are recognized specifically by SA-induced WRKY DNA binding proteins from Arabidopsis...

  15. nDNA-Prot: identification of DNA-binding proteins based on unbalanced classification.

    Science.gov (United States)

    Song, Li; Li, Dapeng; Zeng, Xiangxiang; Wu, Yunfeng; Guo, Li; Zou, Quan

    2014-09-08

    DNA-binding proteins are vital for the study of cellular processes. In recent genome engineering studies, the identification of proteins with certain functions has become increasingly important and needs to be performed rapidly and efficiently. In previous years, several approaches have been developed to improve the identification of DNA-binding proteins. However, the currently available resources are insufficient to accurately identify these proteins. Because of this, the previous research has been limited by the relatively unbalanced accuracy rate and the low identification success of the current methods. In this paper, we explored the practicality of modelling DNA binding identification and simultaneously employed an ensemble classifier, and a new predictor (nDNA-Prot) was designed. The presented framework is comprised of two stages: a 188-dimension feature extraction method to obtain the protein structure and an ensemble classifier designated as imDC. Experiments using different datasets showed that our method is more successful than the traditional methods in identifying DNA-binding proteins. The identification was conducted using a feature that selected the minimum Redundancy and Maximum Relevance (mRMR). An accuracy rate of 95.80% and an Area Under the Curve (AUC) value of 0.986 were obtained in a cross validation. A test dataset was tested in our method and resulted in an 86% accuracy, versus a 76% using iDNA-Prot and a 68% accuracy using DNA-Prot. Our method can help to accurately identify DNA-binding proteins, and the web server is accessible at http://datamining.xmu.edu.cn/~songli/nDNA. In addition, we also predicted possible DNA-binding protein sequences in all of the sequences from the UniProtKB/Swiss-Prot database.

  16. A single-step competitive binding assay for mapping of single DNA molecules.

    Science.gov (United States)

    Nyberg, Lena K; Persson, Fredrik; Berg, Johan; Bergström, Johanna; Fransson, Emelie; Olsson, Linnea; Persson, Moa; Stålnacke, Antti; Wigenius, Jens; Tegenfeldt, Jonas O; Westerlund, Fredrik

    2012-01-06

    Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence. Copyright © 2011 Elsevier Inc. All rights reserved.

  17. Extensive mapping of PPAR binding to genomic DNA

    DEFF Research Database (Denmark)

    Nielsen, Ronni; Pedersen, Thomas Åskov; Trindade, Luisa

    The peroxisome proliferator-activated receptor (PPAR) transcription factors a, d and g are members of the nuclear hormone receptor super family. The PPARs bind regulatory DNA elements (PPREs) as heterodimers with the retinoid X receptor (RXR) and thereby induce transcription in response to ligand...

  18. Synthesis, spectral properties and DNA binding and nuclease ...

    Indian Academy of Sciences (India)

    Ce(BPBH)2(NO3)3] leads to a supramolecular arrangement in its network. The binding properties of these complexes with calf-thymus. DNA have been investigated by viscosity measurements. The complexes show more nuclease activity in the.

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

    Energy Technology Data Exchange (ETDEWEB)

    Pröpper, Kevin [University of Göttingen, (Germany); Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Meindl, Kathrin; Sammito, Massimo [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Dittrich, Birger; Sheldrick, George M. [University of Göttingen, (Germany); Pohl, Ehmke, E-mail: ehmke.pohl@durham.ac.uk [Durham University, (United Kingdom); Usón, Isabel, E-mail: ehmke.pohl@durham.ac.uk [Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), (Spain); Institucio Catalana de Recerca i Estudis Avancats (ICREA), (Spain); University of Göttingen, (Germany)

    2014-06-01

    The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite the fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.

  20. Characterization of antibodies specific for UV-damaged DNA by ELISA

    Energy Technology Data Exchange (ETDEWEB)

    Eggset, G.; Volden, G.; Krokan, H.

    1987-04-01

    The specificity of affinity purified antibodies raised against UV-irradiated DNA was examined using an enzyme-linked immunosorbent assay. DNA irradiated with UV doses higher than needed for saturation with pyrimidine dimers bound increasing amounts of antibody. Photosensitized DNA, containing high amounts of pyrimidine dimers, showed very poor binding of antibody. When UV-irradiated DNA was given a second dose of 340-nm UV light, the binding of antibodies was inhibited. Taken together, this indicates a major specificity for (6-4)-photoproducts, which are photochemically reversed by UV light in the 340-nm region. The antibodies also showed little but detectable binding to pyrimidine glycols produced in DNA by oxidation with OsO/sub 4/. Previously, we have used these antibodies for the detection of UV-induced DNA damage and its repair in human skin in vivo. These findings indicate that (6-4)-photoproducts, considered highly mutagenic, are repaired in human skin.

  1. Cloning of two sea urchin DNA-binding proteins involved in mitochondrial DNA replication and transcription.

    Science.gov (United States)

    Loguercio Polosa, Paola; Megli, Fiammetta; Di Ponzio, Barbara; Gadaleta, Maria Nicola; Cantatore, Palmiro; Roberti, Marina

    2002-03-06

    The cloning of the cDNA for two mitochondrial proteins involved in sea urchin mtDNA replication and transcription is reported here. The cDNA for the mitochondrial D-loop binding protein (mtDBP) from the sea urchin Strongylocentrotus purpuratus has been cloned by a polymerase chain reaction-based approach. The protein displays a very high similarity with the Paracentrotus lividus homologue as it contains also the two leucine zipper-like domains which are thought to be involved in intramolecular interactions needed to expose the two DNA binding domains in the correct position for contacting DNA. The cDNA for the mitochondrial single-stranded DNA-binding protein (mtSSB) from P. lividus has been also cloned by a similar approach. The precursor protein is 146 amino acids long with a presequence of 16 residues. The deduced amino acid sequence shows the highest homology with the Xenopus laevis protein and the lowest with the Drosophila mtSSB. The computer modeling of the tertiary structure of P. lividus mtSSB shows a structure very similar to that experimentally determined for human mtSSB, with the conservation of the main residues involved in protein tetramerization and in DNA binding.

  2. Extended HSR/CARD domain mediates AIRE binding to DNA.

    Science.gov (United States)

    Maslovskaja, Julia; Saare, Mario; Liiv, Ingrid; Rebane, Ana; Peterson, Pärt

    2015-12-25

    Autoimmune regulator (AIRE) activates the transcription of many genes in an unusual promiscuous and stochastic manner. The mechanism by which AIRE binds to the chromatin and DNA is not fully understood, and the regulatory elements that AIRE target genes possess are not delineated. In the current study, we demonstrate that AIRE activates the expression of transiently transfected luciferase reporters that lack defined promoter regions, as well as intron and poly(A) signal sequences. Our protein-DNA interaction experiments with mutated AIRE reveal that the intact homogeneously staining region/caspase recruitment domain (HSR/CARD) and amino acids R113 and K114 are key elements involved in AIRE binding to DNA. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Quantifying the Impact of Non-coding Variants on Transcription Factor-DNA Binding.

    Science.gov (United States)

    Zhao, Jingkang; Li, Dongshunyi; Seo, Jungkyun; Allen, Andrew S; Gordân, Raluca

    2017-05-01

    Many recent studies have emphasized the importance of genetic variants and mutations in cancer and other complex human diseases. The overwhelming majority of these variants occur in non-coding portions of the genome, where they can have a functional impact by disrupting regulatory interactions between transcription factors (TFs) and DNA. Here, we present a method for assessing the impact of non-coding mutations on TF-DNA interactions, based on regression models of DNA-binding specificity trained on high-throughput in vitro data. We use ordinary least squares (OLS) to estimate the parameters of the binding model for each TF, and we show that our predictions of TF-binding changes due to DNA mutations correlate well with measured changes in gene expression. In addition, by leveraging distributional results associated with OLS estimation, for each predicted change in TF binding we also compute a normalized score (z-score) and a significance value (p-value) reflecting our confidence that the mutation affects TF binding. We use this approach to analyze a large set of pathogenic non-coding variants, and we show that these variants lead to significant differences in TF binding between alleles, compared to a control set of common variants. Thus, our results indicate that there is a strong regulatory component to the pathogenic non-coding variants identified thus far.

  4. Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

    DEFF Research Database (Denmark)

    Kenchappa, Chandra; Heiðarsson, Pétur Orri; Kragelund, Birthe

    2013-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds...... specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal...... in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys(7) and Cys(28) enhancing high thermal stability of Cbp2(Hb) through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2...

  5. Spore Photoproduct (SP) Lyase from Bacillus subtilis Specifically Binds to and Cleaves SP (5-Thyminyl-5,6-Dihydrothymine) but Not Cyclobutane Pyrimidine Dimers in UV-Irradiated DNA

    Science.gov (United States)

    Slieman, Tony A.; Rebeil, Roberto; Nicholson, Wayne L.

    2000-01-01

    The predominant photolesion in the DNA of UV-irradiated dormant bacterial spores is the thymine dimer 5-thyminyl-5,6-dihydrothymine, commonly referred to as spore photoproduct (SP). A major determinant of SP repair during spore germination is its direct reversal by the enzyme SP lyase, encoded by the splB gene in Bacillus subtilis. SplB protein containing an N-terminal tag of six histidine residues [(6His)SplB] was purified from dormant B. subtilis spores and shown to efficiently cleave SP but not cyclobutane cis,syn thymine-thymine dimers in vitro. In contrast, SplB protein containing an N-terminal 10-histidine tag [(10His)SplB] purified from an Escherichia coli overexpression system was incompetent to cleave SP unless the 10-His tag was first removed by proteolysis at an engineered factor Xa site. To assay the parameters of binding of SplB protein to UV-damaged DNA, a 35-bp double-stranded oligonucleotide was constructed which carried a single pair of adjacent thymines on one strand. Irradiation of the oligonucleotide in aqueous solution or at 10% relative humidity resulted in formation of cyclobutane pyrimidine dimers (Py◊Py) or SP, respectively. (10His)SplB was assayed for oligonucleotide binding using a DNase I protection assay. In the presence of (10His)SplB, the SP-containing oligonucleotide was selectively protected from DNase I digestion (half-life, >60 min), while the Py◊Py-containing oligonucleotide and the unirradiated oligonucleotide were rapidly digested by DNase I (half-lives, 6 and 9 min, respectively). DNase I footprinting of (10His)SplB bound to the artificial substrate was carried out utilizing the 32P end-labeled 35-bp oligonucleotide containing SP. DNase I footprinting showed that SplB protected at least a 9-bp region surrounding SP from digestion with DNase I with the exception of two DNase I-hypersensitive sites within the protected region. (10His)SplB also caused significant enhancement of DNase I digestion of the SP

  6. Diverse p53/DNA binding modes expand the repertoire of p53 response elements.

    Science.gov (United States)

    Vyas, Pratik; Beno, Itai; Xi, Zhiqun; Stein, Yan; Golovenko, Dmitrij; Kessler, Naama; Rotter, Varda; Shakked, Zippora; Haran, Tali E

    2017-10-03

    The tumor suppressor protein p53 acts as a transcription factor, binding sequence-specifically to defined DNA sites, thereby activating the expression of genes leading to diverse cellular outcomes. Canonical p53 response elements (REs) are made of two decameric half-sites separated by a variable number of base pairs (spacers). Fifty percent of all validated p53 REs contain spacers between 1 and 18 bp; however, their functional significance is unclear at present. Here, we show that p53 forms two different tetrameric complexes with consensus or natural REs, both with long spacers: a fully specific complex where two p53 dimers bind to two specific half-sites, and a hemispecific complex where one dimer binds to a specific half-site and the second binds to an adjacent spacer sequence. The two types of complexes have comparable binding affinity and specificity, as judged from binding competition against bulk genomic DNA. Structural analysis of the p53 REs in solution shows that these sites are not bent in both their free and p53-bound states when the two half-sites are either abutting or separated by spacers. Cell-based assay supports the physiological relevance of our findings. We propose that p53 REs with long spacers comprise separate specific half-sites that can lead to several different tetrameric complexes. This finding expands the universe of p53 binding sites and demonstrates that even isolated p53 half-sites can form tetrameric complexes. Moreover, it explains the manner in which p53 binds to clusters of more than one canonical binding site, common in many natural REs.

  7. NeuroD Factors Discriminate Mineralocorticoid From Glucocorticoid Receptor DNA Binding in the Male Rat Brain.

    Science.gov (United States)

    van Weert, Lisa T C M; Buurstede, Jacobus C; Mahfouz, Ahmed; Braakhuis, Pamela S M; Polman, J Annelies E; Sips, Hetty C M; Roozendaal, Benno; Balog, Judit; de Kloet, E Ronald; Datson, Nicole A; Meijer, Onno C

    2017-05-01

    In the limbic brain, mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) both function as receptors for the naturally occurring glucocorticoids (corticosterone/cortisol) but mediate distinct effects on cellular physiology via transcriptional mechanisms. The transcriptional basis for specificity of these MR- vs GR-mediated effects is unknown. To address this conundrum, we have identified the extent of MR/GR DNA-binding selectivity in the rat hippocampus using chromatin immunoprecipitation followed by sequencing. We found 918 and 1450 nonoverlapping binding sites for MR and GR, respectively. Furthermore, 475 loci were co-occupied by MR and GR. De novo motif analysis resulted in a similar binding motif for both receptors at 100% of the target loci, which matched the known glucocorticoid response element (GRE). In addition, the Atoh/NeuroD consensus sequence was found in co-occurrence with all MR-specific binding sites but was absent for GR-specific or MR-GR overlapping sites. Basic helix-loop-helix family members Neurod1, Neurod2, and Neurod6 showed hippocampal expression and were hypothesized to bind the Atoh motif. Neurod2 was detected at rat hippocampal MR binding sites but not at GR-exclusive sites. All three NeuroD transcription factors acted as DNA-binding-dependent coactivators for both MR and GR in reporter assays in heterologous HEK293 cells, likely via indirect interactions with the receptors. In conclusion, a NeuroD family member binding to an additional motif near the GRE seems to drive specificity for MR over GR binding at hippocampal binding sites. Copyright © 2017 Endocrine Society.

  8. Selective DNA binding of (N-alkylamine)-substituted naphthalene imides and diimides to G+C-rich DNA.

    Science.gov (United States)

    Liu, Z R; Hecker, K H; Rill, R L

    1996-12-01

    Alkylamine-substituted naphthalene imides and diimides bind DNA by intercalation and have applications as anticancer agents. The unique structures of these imides in which two adjacent carbonyl groups lie coplanar to an extended aromatic ring system allow the possibility of sequence-selective interactions between the intercalated chromophore and guanine amino groups situated in the DNA minor groove. The binding affinities of N-[3-(dimethylamino)propyl amine]-1,8-naphthalenedicarboxylic imide (N-DMPrNI) and N,N'-bis [3,3'-(dimethylamino)propylamine]-naphthalene-1,4,5,8-tetracarboxylic diimide (N-BDMPrNDI) for natural DNAs of differing base composition were determined spectroscopically and by equilibrium dialysis. In agreement with the above proposition, binding studies indicated that both the naphthalene imide and diimide strongly prefer to intercalate into steps containing at least one G:C base pair. The dependencies of association constants on DNA base composition are consistent with a requirement for one G:C pair in the binding site of the monomide, and two G:C pairs in binding sites of the diimide. These selectivities are comparable to or exceed that of actinomycin D, a classic G:C-selective drug. Protection footprinting with DNase I confirmed that the naphthalene monoiimide (N-DMPrNI) prefers to bind adjacent to G:C base pairs, with a most consistent preference for "mixed" steps containing both a G:C and an A:T pair, excepting GA:TC. Several 5'-CG-3' steps were also good binding sites as indicated by nuclease protection, but few GC:GC or GG:CC steps were protected. The naphthalene diimide inhibited DNase I digestion, but did not yield a footprint. The base recognition ability and versatile chemistry make naphthalene imides and diimides attractive building blocks for design of highly sequence-specific, DNA-directed drug candidates including conjugated oligonucleotides or oligopeptides.

  9. Solution structure and DNA binding of the zinc-finger domain from DNA ligase IIIalpha.

    Science.gov (United States)

    Kulczyk, Arkadiusz W; Yang, Ji-Chun; Neuhaus, David

    2004-08-13

    DNA ligase IIIalpha carries out the final ligation step in the base excision repair (BER) and single strand break repair (SSBR) mechanisms of DNA repair. The enzyme recognises single-strand nicks and other damage features in double-stranded DNA, both through the catalytic domain and an N-terminal domain containing a single zinc finger. The latter is homologous to other zinc fingers that recognise damaged DNA, two in the N terminus of poly(adenosine-ribose)polymerase and three in the N terminus of the Arabidopsis thaliana nick-sensing DNA 3'-phosphoesterase. Here, we present the solution structure of the zinc-finger domain of human DNA ligase IIIalpha, the first structure of a finger from this group. It is related to that of the erythroid transcription factor GATA-1, but has an additional N-terminal beta-strand and C-terminal alpha-helix. Chemical shift mapping using a DNA ligand containing a single-stranded break showed that the DNA-binding surface of the DNA-ligase IIIalpha zinc finger is substantially different from that of GATA-1, consistent with the fact that the two proteins recognise very different features in the DNA. Likely implications for DNA binding are discussed.

  10. Position specific variation in the rate of evolution intranscription factor binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  11. Position specific variation in the rate of evolution in transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Kellis Manolis

    2003-08-01

    Full Text Available Abstract Background The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Results Here we analyse the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikatae to study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artefacts of computational motif finding algorithms. Conclusion As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will

  12. Predicting DNA-binding sites of proteins from amino acid sequence

    Directory of Open Access Journals (Sweden)

    Wu Feihong

    2006-05-01

    Full Text Available Abstract Background Understanding the molecular details of protein-DNA interactions is critical for deciphering the mechanisms of gene regulation. We present a machine learning approach for the identification of amino acid residues involved in protein-DNA interactions. Results We start with a Naïve Bayes classifier trained to predict whether a given amino acid residue is a DNA-binding residue based on its identity and the identities of its sequence neighbors. The input to the classifier consists of the identities of the target residue and 4 sequence neighbors on each side of the target residue. The classifier is trained and evaluated (using leave-one-out cross-validation on a non-redundant set of 171 proteins. Our results indicate the feasibility of identifying interface residues based on local sequence information. The classifier achieves 71% overall accuracy with a correlation coefficient of 0.24, 35% specificity and 53% sensitivity in identifying interface residues as evaluated by leave-one-out cross-validation. We show that the performance of the classifier is improved by using sequence entropy of the target residue (the entropy of the corresponding column in multiple alignment obtained by aligning the target sequence with its sequence homologs as additional input. The classifier achieves 78% overall accuracy with a correlation coefficient of 0.28, 44% specificity and 41% sensitivity in identifying interface residues. Examination of the predictions in the context of 3-dimensional structures of proteins demonstrates the effectiveness of this method in identifying DNA-binding sites from sequence information. In 33% (56 out of 171 of the proteins, the classifier identifies the interaction sites by correctly recognizing at least half of the interface residues. In 87% (149 out of 171 of the proteins, the classifier correctly identifies at least 20% of the interface residues. This suggests the possibility of using such classifiers to identify

  13. ATP-independent cooperative binding of yeast Isw1a to bare and nucleosomal DNA.

    Directory of Open Access Journals (Sweden)

    Anne De Cian

    Full Text Available Among chromatin remodeling factors, the ISWI family displays a nucleosome-enhanced ATPase activity coupled to DNA translocation. While these enzymes are known to bind to DNA, their activity has not been fully characterized. Here we use TEM imaging and single molecule manipulation to investigate the interaction between DNA and yeast Isw1a. We show that Isw1a displays a highly cooperative ATP-independent binding to and bridging between DNA segments. Under appropriate tension, rare single nucleation events can sometimes be observed and loop DNA with a regular step. These nucleation events are often followed by binding of successive complexes bridging between nearby DNA segments in a zipper-like fashion, as confirmed by TEM observations. On nucleosomal substrates, we show that the specific ATP-dependent remodeling activity occurs in the context of cooperative Isw1a complexes bridging extranucleosomal DNA. Our results are interpreted in the context of the recently published partial structure of Isw1a and support its acting as a "protein ruler" (with possibly more than one tick.

  14. Single-stranded DNA binding by F TraI relaxase and helicase domains is coordinately regulated.

    Science.gov (United States)

    Dostál, Lubomír; Schildbach, Joel F

    2010-07-01

    Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal approximately 300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.

  15. Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated▿

    Science.gov (United States)

    Dostál, Lubomír; Schildbach, Joel F.

    2010-01-01

    Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal ∼300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer. PMID:20435720

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

    Science.gov (United States)

    Kachhap, Sangita; Singh, Balvinder

    2015-01-01

    In most of homeodomain-DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1-DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein-DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein-DNA complexes. The order of stability of protein-DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein-DNA complexes. Among specific protein-DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.

  17. Cation specific binding with protein surface charges.

    Science.gov (United States)

    Hess, Berk; van der Vegt, Nico F A

    2009-08-11

    Biological organization depends on a sensitive balance of noncovalent interactions, in particular also those involving interactions between ions. Ion-pairing is qualitatively described by the law of "matching water affinities." This law predicts that cations and anions (with equal valence) form stable contact ion pairs if their sizes match. We show that this simple physical model fails to describe the interaction of cations with (molecular) anions of weak carboxylic acids, which are present on the surfaces of many intra- and extracellular proteins. We performed molecular simulations with quantitatively accurate models and observed that the order K(+) < Na(+) < Li(+) of increasing binding affinity with carboxylate ions is caused by a stronger preference for forming weak solvent-shared ion pairs. The relative insignificance of contact pair interactions with protein surfaces indicates that thermodynamic stability and interactions between proteins in alkali salt solutions is governed by interactions mediated through hydration water molecules.

  18. Zinc binding to lambda phage DNA studied by voltammetric techniques

    Directory of Open Access Journals (Sweden)

    Souza Jurandir R. de

    2000-01-01

    Full Text Available Binding of zinc to lambda phage DNA was investigated by differential pulse voltammetry and cyclic voltammetry. These methods rely on the direct monitoring of reduction and oxidation current of zinc in the absence and presence of this virion DNA. Titration graphs of Zn2+ with DNA were obtained in the concentration ranges from 3.57 x 10-12 to 3.92 x 10-11 mol L-1 and 6.97 x 10-12 to 5.56 x 10-11 mol L-1. These data were used to calculate the dissociation constant of the complex and the stoichiometry. The mechanism of this reaction was studied by cyclic voltammetry through curves I (oxidation current of Zn2+ versus v½ (square root of scan rate. These curves showed that the oxidation-reduction process of Zn2+ was not controlled by diffusion in the presence of lambda phage DNA.

  19. Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Ryzhikov, Mikhail; Koroleva, Olga; Postnov, Dmitri; Tran, Andrew; Korolev, Sergey (St. Louis-MED)

    2011-08-25

    RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.

  20. BuD, a helix–loop–helix DNA-binding domain for genome modification

    Energy Technology Data Exchange (ETDEWEB)

    Stella, Stefano [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark); Molina, Rafael; López-Méndez, Blanca [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Campos-Olivas, Ramon [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Duchateau, Phillippe [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Montoya, Guillermo, E-mail: guillermo.montoya@cpr.ku.dk [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark)

    2014-07-01

    Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

  1. Nuclear receptors from the ctenophore Mnemiopsis leidyi lack a zinc-finger DNA-binding domain: lineage-specific loss or ancestral condition in the emergence of the nuclear receptor superfamily?

    Directory of Open Access Journals (Sweden)

    Reitzel Adam M

    2011-02-01

    Full Text Available Abstract Background Nuclear receptors (NRs are an ancient superfamily of metazoan transcription factors that play critical roles in regulation of reproduction, development, and energetic homeostasis. Although the evolutionary relationships among NRs are well-described in two prominent clades of animals (deuterostomes and protostomes, comparatively little information has been reported on the diversity of NRs in early diverging metazoans. Here, we identified NRs from the phylum Ctenophora and used a phylogenomic approach to explore the emergence of the NR superfamily in the animal kingdom. In addition, to gain insight into conserved or novel functions, we examined NR expression during ctenophore development. Results We report the first described NRs from the phylum Ctenophora: two from Mnemiopsis leidyi and one from Pleurobrachia pileus. All ctenophore NRs contained a ligand-binding domain and grouped with NRs from the subfamily NR2A (HNF4. Surprisingly, all the ctenophore NRs lacked the highly conserved DNA-binding domain (DBD. NRs from Mnemiopsis were expressed in different regions of developing ctenophores. One was broadly expressed in the endoderm during gastrulation. The second was initially expressed in the ectoderm during gastrulation, in regions corresponding to the future tentacles; subsequent expression was restricted to the apical organ. Phylogenetic analyses of NRs from ctenophores, sponges, cnidarians, and a placozoan support the hypothesis that expansion of the superfamily occurred in a step-wise fashion, with initial radiations in NR family 2, followed by representatives of NR families 3, 6, and 1/4 originating prior to the appearance of the bilaterian ancestor. Conclusions Our study provides the first description of NRs from ctenophores, including the full complement from Mnemiopsis. Ctenophores have the least diverse NR complement of any animal phylum with representatives that cluster with only one subfamily (NR2A. Ctenophores and

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

    Directory of Open Access Journals (Sweden)

    Mudasir Mudasir

    2010-06-01

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

  3. Drug-binding properties of rat alpha-foetoprotein. Specificities of the phenylbutazone-binding and warfarin-binding sites.

    Science.gov (United States)

    Hervé, F; Rajkowski, K M; Martin, M T; Dessen, P; Cittanova, N

    1986-01-01

    Rat alpha-foetoprotein (alpha-FP) strongly binds the drugs warfarin and phenylbutazone, as does albumin; however, the binding sites for the two drugs seemed to be different. This possibility and the specificity of this/these drug-binding site(s) of rat alpha-FP were investigated by competitive protein-binding experiments with a variety of drugs, representing different pharmacological groups, and biomolecules that are strongly bound by the foetal protein and that are suspected to play a specific role during foetal development. The binding mechanisms were further investigated by using comparisons between computer-derived theoretical displacement curves and experimental points in order to distinguish different possible binding models. The results indicate: that warfarin and phenylbutazone are bound at two distinct sites on rat alpha-FP and that a negative modulatory effect is exerted between the two sites; that the degree of specificity of these two drug-binding sites is different, since the warfarin-binding site appears to be specific for the binding of coumarinic and anthranilic drugs whereas that for phenylbutazone is able to bind substances of very varied chemical structure and is more hydrophobic; that the phenylbutazone-binding site is the site that binds oestrogens that thyroid hormones and, probably, fatty acids and bilirubin are bound at (an)other site(s) but exert negative modulatory effects on phenylbutazone binding. The nature of the different binding areas of rat alpha-FP is compared with that of those already proposed for albumin. The potential risks of toxicity of such interactions between drugs and/or biomolecules on foetal development are also discussed. PMID:2434073

  4. Specific binding of neoglycoproteins to Toxoplasma gondii tachyzoites.

    Science.gov (United States)

    Robert, R; de la Jarrige, P L; Mahaza, C; Cottin, J; Marot-Leblond, A; Senet, J M

    1991-01-01

    Several studies have shown that protozoa bind to glycoproteins or neoglycoproteins. Here we report that Toxoplasma gondii binds strongly to bovine serum albumin-glucosamide. The binding was rapid, time dependent, partially reversible, saturable, and specific. Scatchard analysis showed about 40,000 molecules of bovine serum albumin-glucosamide per toxoplasma cell. The apparent dissociation constant was found to be 4.46 x 10(-8) M. Images PMID:1937826

  5. Selection of DNA aptamers specific for live Pseudomonas aeruginosa.

    Directory of Open Access Journals (Sweden)

    Jennifer Soundy

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen that causes significant morbidity and mortality in immunocompromised patients, particular cystic fibrosis sufferers, burns victims, diabetics and neonates. It thrives in moist places where it forms biofilms that are exceedingly difficult to eradicate on hospital surfaces, in water supplies and implanted biomaterials. Using a live cell SELEX approach we selected DNA aptamers to P. aeruginosa grown as biofilms in microfluidic cells. From a pool of aptamer candidates showing tight binding a stem-loop structure was identified as being important for binding. Enhanced binding and increased specificity was achieved by truncating structures and generating chimeric aptamers from the pool of top candidates. The top candidates have low nanomolar binding constants and high discrimination for P. aeruginosa over other Gram-negative bacteria. The aptamers bind both planktonic grown and biofilm grown cells. They do not have intrinsic bacteriostatic or bactericidal activity, but are ideal candidates for modification for use as aptamer-drug conjugates and in biosensors.

  6. The Tomato Nucleotide-binding Leucine-rich Repeat (NLR) Immune Receptor I-2 Couples DNA-Binding to Nucleotide-Binding Domain Nucleotide Exchange

    NARCIS (Netherlands)

    Fenyk, S.; Dixon, C.H.; Gittens, W.H.; Townsend, P.D.; Sharples, G.J.; Pålsson, L.O.; Takken, F.L.W.; Cann, M.J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognise and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception.

  7. Processing Binding Relations in Specific Language Impairment

    Science.gov (United States)

    Schwartz, Richard G.; Hestvik, Arild; Seiger-Gardner, Liat; Almodovar, Diana

    2016-01-01

    Purpose: This sentence processing experiment examined the abilities of children with specific language impairment (SLI) and children with typical language development (TD) to establish relations between pronouns or reflexives and their antecedents in real time. Method: Twenty-two children with SLI and 24 age-matched children with TD (7;3-10;11…

  8. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    Directory of Open Access Journals (Sweden)

    Toshitsugu Fujita

    2015-09-01

    Full Text Available Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE proteins and the clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR-associated proteins (Cas (CRISPR/Cas system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing.

  9. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

    Science.gov (United States)

    Pai, Dave A.; Kaplan, Craig D.; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C.; Engelke, David R.

    2014-01-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template. PMID:24614752

  10. Thermodynamic effects of multiple protein conformations on stability and DNA binding.

    Science.gov (United States)

    Inaba, Satomi; Fukada, Harumi; Ikegami, Takahisa; Oda, Masayuki

    2013-09-15

    The side-chain conformations of amino acids in the hydrophobic core are important for protein folding and function. A previous NMR study has shown that a mutant protein of transcriptional activator c-Myb, I155L/I181L R3, has multiple conformations and increased fluctuation in comparison with the wild type. To elucidate the quantitative correlation of structural fluctuation with stability and function, we analyzed the thermodynamic effects of I155L and I181L mutations, using R2R3 that encompasses the minimum specific DNA-binding region. Circular dichroism and differential scanning calorimetry measurements showed that the mutation of I155L had little effect on stability, while the I181L mutation significantly destabilized the protein. It is noteworthy that the decreased stability resulting from the I181L mutation was mainly due to decreased enthalpy change, which is partially compensated by decreased entropy change. Isothermal titration calorimetry measurements showed that the specific DNA-binding affinity was decreased owing to the I181L mutation, which was due to decreased binding entropy change. Entropy in the folded state, which corresponds to the DNA-free state, increases due to the I181L mutation because of the increased conformational fluctuation observed in I155L/I181L mutant of R2R3 by CLEANEX-PM NMR analysis, which in turn results in decreased folding entropy and DNA-binding entropy changes. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

    Science.gov (United States)

    Pai, Dave A; Kaplan, Craig D; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C; Engelke, David R

    2014-05-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

  12. CRISPR Outsourcing: Commissioning IHF for Site-Specific Integration of Foreign DNA at the CRISPR Array.

    Science.gov (United States)

    Wei, Yunzhou; Terns, Michael P

    2016-06-16

    In this issue of Molecular Cell, Nuñez et al. (2016) report that site-specific integration of foreign DNA into CRISPR loci by the Cas1-Cas2 integrase complex is promoted by a host factor, IHF (integration host factor), that binds and bends CRISPR leader DNA. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Unexpected regiospecific formation and DNA binding of new 3 ...

    Indian Academy of Sciences (India)

    DOI 10.1007/s12039-015-1023-7. Unexpected regiospecific formation and DNA binding of new. 3-(acridin-9-yl)methyl-2-iminothiazolidin-4-ones. JÁN IMRICHa∗, DANICA SABOLOVÁb, MÁRIA VILKOVÁa and JÚLIA KUDLÁ ˇCOVÁb. aDepartment of Organic Chemistry bDepartment of Biochemistry, Institute of Chemistry,.

  14. DNA-binding determinants promoting NHEJ by human Polµ

    Science.gov (United States)

    Martin, Maria Jose; Juarez, Raquel; Blanco, Luis

    2012-01-01

    Non-homologous end-joining (NHEJ), the preferred pathway to repair double-strand breaks (DSBs) in higher eukaryotes, relies on a collection of molecular tools to process the broken ends, including specific DNA polymerases. Among them, Polµ is unique as it can catalyze DNA synthesis upon connection of two non-complementary ends. Here, we demonstrate that this capacity is intrinsic to Polµ, not conferred by other NHEJ factors. To understand the molecular determinants of its specific function in NHEJ, the interaction of human Polµ with DNA has been directly visualized by electromobility shift assay and footprinting assays. Stable interaction with a DNA gap requires the presence of a recessive 5′-P, thus orienting the catalytic domain for primer and nucleotide binding. Accordingly, recognition of the 5′-P is crucial to align the two DNA substrates of the NHEJ reaction. Site-directed mutagenesis demonstrates the relevance of three specific residues (Lys249, Arg253 and Arg416) in stabilizing the primer strand during end synapsis, allowing a range of microhomology-induced distortions beneficial for NHEJ. Moreover, our results suggest that the Polµ BRCT domain, thought to be exclusively involved in interaction with NHEJ core factors, has a direct role in binding the DNA region neighbor to the 5′-P, thus boosting Polµ-mediated NHEJ reactions. PMID:23034807

  15. The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Sass, Ehud; Suski, Catherine

    2014-01-01

    Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus...... as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications....

  16. Relaxed specificity of prokaryotic DNA methyltransferases results in DNA site-specific modification of RNA/DNA heteroduplexes.

    Science.gov (United States)

    Wons, Ewa; Mruk, Iwona; Kaczorowski, Tadeusz

    2015-11-01

    RNA/DNA hybrid duplexes regularly occur in nature, for example in transcriptional R loops. Their susceptibility to modification by DNA-specific or RNA-specific enzymes is, thus, a biologically relevant question, which, in addition, has possible biotechnological implications. In this study, we investigated the activity of four isospecific DNA methyltransferases (M.EcoVIII, M.LlaCI, M.HindIII, M.BstZ1II) toward an RNA/DNA duplex carrying one 5'-AAGCUU-3'/3'-TTCGAA-5' target sequence. The analyzed enzymes belong to the β-group of adenine N6-methyltransferases and recognize the palindromic DNA sequence 5'-AAGCTT-3'/3'-TTCGAA-5'. Under standard conditions, none of these isospecific enzymes could detectibly methylate the RNA/DNA duplex. However, the addition of agents that generally relax specificity, such as dimethyl sulfoxide (DMSO) and glycerol, resulted in substantial methylation of the RNA/DNA duplex by M.EcoVIII and M.LlaCI. Only the DNA strand of the RNA/DNA duplex was methylated. The same was not observed for M.HindIII or M.BstZ1II. This is, to our knowledge, the first report that demonstrates such activity by prokaryotic DNA methyltransferases. Possible applications of these findings in a laboratory practice are also discussed.

  17. TRF2 binds branched DNA to safeguard telomere integrity.

    Science.gov (United States)

    Schmutz, Isabelle; Timashev, Leonid; Xie, Wei; Patel, Dinshaw J; de Lange, Titia

    2017-09-01

    Although t-loops protect telomeres, they are at risk of cleavage by Holliday junction (HJ) resolvases if branch migration converts the three-way t-loop junction into four-way HJs. T-loop cleavage is repressed by the TRF2 basic domain, which binds three- and four-way junctions and protects HJs in vitro. By replacing the basic domain with bacterial-protein domains binding three- and four-way junctions, we demonstrated the in vivo relevance of branched-DNA binding. Branched-DNA binding also repressed PARP1, presumably by masking the PARP1 site in the t-loop junction. Although PARP1 recruits HJ resolvases and promotes t-loop cleavage, PARP1 activation alone did not result in t-loop cleavage, thus suggesting that the basic domain also prevents formation of HJs. Concordantly, removal of HJs by BLM helicase mitigated t-loop cleavage in response to loss of the basic domain. We propose that TRF2 masks and stabilizes the t-loop three-way junction, thereby protecting telomeres from detrimental deletions and PARP1 activation.

  18. Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

    Energy Technology Data Exchange (ETDEWEB)

    Serck-Hanssen, G.; Soevik, O.

    1987-12-28

    Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of /sup 125/I-insulin was carried out at 15/sup 0/C for 3 hrs in the absence or presence of excess unlabeled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. The authors conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells. 24 references, 2 figures, 1 table.

  19. Binding specificity of the G1/S transcriptional regulators in budding yeast.

    Directory of Open Access Journals (Sweden)

    Michael R Harris

    Full Text Available BACKGROUND: G1/S transcriptional regulation in the budding yeast Saccharomyces cerevisiae depends on three main transcriptional components, Swi4, Swi6 and Mbp1. These proteins constitute two transcription factor complexes that regulate over 300 G1/S transcripts, namely SBF (Swi4-Swi6 and MBF (Mbp1-Swi6. SBF and MBF are involved in regulating largely non-overlapping sets of G1/S genes via clearly distinct mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Here we establish and confirm protein-protein and protein-DNA interactions using specific polyclonal antisera to whole Swi6 and to the C-terminal domains of related proteins Swi4 and Mbp1. Our data confirm the protein-protein binding specificity of Swi4 and Mbp1 to Swi6 but not to each other, and support the binding specificity of the transcriptional inhibitor Whi5 to SBF and of the corepressor Nrm1 to MBF. We also show the DNA binding preference of Swi4 to the CLN2 promoter and Mbp1 to the RNR1 promoter, while Swi6 binds both promoters. Finally, we establish the binding dynamics of Swi4 and Whi5 to the CLN2 promoter during the cell cycle. CONCLUSIONS/SIGNIFICANCE: These data confirm the binding specificity of the G1/S transcriptional regulators. Whereas previous observations were made using tagged Swi4, Swi6 and Mbp1, here we use specific polyclonal antisera to reestablish the protein-protein and protein-DNA interactions of these G1/S transcriptional components. Our data also reveal the dynamic changes in promoter binding of Swi4 during the cell cycle, which suggests a possible positive feedback loop involving Swi4.

  20. Human TFIID binds to core promoter DNA in a reorganized structural state

    Science.gov (United States)

    Cianfrocco, Michael A.; Kassavetis, George A.; Grob, Patricia; Fang, Jie; Juven-Gershon, Tamar; Kadonaga, James T.; Nogales, Eva

    2012-01-01

    A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single particle electron microscopy to visualize human TFIID with promoter DNA. This analysis revealed that TFIID co-exists in two predominant and distinct structural states that differ by a 100Å translocation of TFIID’s lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a novel rearranged state of TFIID capable of promoter recognition and binding. DNA-labeling and footprinting, together with cryo-EM studies, mapped the locations of the TATA, Inr, MTE, and DPE promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors. PMID:23332750

  1. Characterization of the single stranded DNA binding protein SsbB encoded in the Gonoccocal Genetic Island.

    Directory of Open Access Journals (Sweden)

    Samta Jain

    Full Text Available Most strains of Neisseria gonorrhoeae carry a Gonococcal Genetic Island which encodes a type IV secretion system involved in the secretion of ssDNA. We characterize the GGI-encoded ssDNA binding protein, SsbB. Close homologs of SsbB are located within a conserved genetic cluster found in genetic islands of different proteobacteria. This cluster encodes DNA-processing enzymes such as the ParA and ParB partitioning proteins, the TopB topoisomerase, and four conserved hypothetical proteins. The SsbB homologs found in these clusters form a family separated from other ssDNA binding proteins.In contrast to most other SSBs, SsbB did not complement the Escherichia coli ssb deletion mutant. Purified SsbB forms a stable tetramer. Electrophoretic mobility shift assays and fluorescence titration assays, as well as atomic force microscopy demonstrate that SsbB binds ssDNA specifically with high affinity. SsbB binds single-stranded DNA with minimal binding frames for one or two SsbB tetramers of 15 and 70 nucleotides. The binding mode was independent of increasing Mg(2+ or NaCl concentrations. No role of SsbB in ssDNA secretion or DNA uptake could be identified, but SsbB strongly stimulated Topoisomerase I activity.We propose that these novel SsbBs play an unknown role in the maintenance of genetic islands.

  2. The conformational state of the nucleosome entry–exit site modulates TATA box-specific TBP binding

    Science.gov (United States)

    Hieb, Aaron R.; Gansen, Alexander; Böhm, Vera; Langowski, Jörg

    2014-01-01

    The TATA binding protein (TBP) is a critical transcription factor used for nucleating assembly of the RNA polymerase II machinery. TBP binds TATA box elements with high affinity and kinetic stability and in vivo is correlated with high levels of transcription activation. However, since most promoters use less stable TATA-less or TATA-like elements, while also competing with nucleosome occupancy, further mechanistic insight into TBP's DNA binding properties and ability to access chromatin is needed. Using bulk and single-molecule FRET, we find that TBP binds a minimal consensus TATA box as a two-state equilibrium process, showing no evidence for intermediate states. However, upon addition of flanking DNA sequence, we observe non-specific cooperative binding to multiple DNA sites that compete for TATA-box specificity. Thus, we conclude that TBP binding is defined by a branched pathway, wherein TBP initially binds with little sequence specificity and is thermodynamically positioned by its kinetic stability to the TATA box. Furthermore, we observed the real-time access of TBP binding to TATA box DNA located within the DNA entry–exit site of the nucleosome. From these data, we determined salt-dependent changes in the nucleosome conformation regulate TBP's access to the TATA box, where access is highly constrained under physiological conditions, but is alleviated by histone acetylation and TFIIA. PMID:24829456

  3. The conformational state of the nucleosome entry-exit site modulates TATA box-specific TBP binding.

    Science.gov (United States)

    Hieb, Aaron R; Gansen, Alexander; Böhm, Vera; Langowski, Jörg

    2014-07-01

    The TATA binding protein (TBP) is a critical transcription factor used for nucleating assembly of the RNA polymerase II machinery. TBP binds TATA box elements with high affinity and kinetic stability and in vivo is correlated with high levels of transcription activation. However, since most promoters use less stable TATA-less or TATA-like elements, while also competing with nucleosome occupancy, further mechanistic insight into TBP's DNA binding properties and ability to access chromatin is needed. Using bulk and single-molecule FRET, we find that TBP binds a minimal consensus TATA box as a two-state equilibrium process, showing no evidence for intermediate states. However, upon addition of flanking DNA sequence, we observe non-specific cooperative binding to multiple DNA sites that compete for TATA-box specificity. Thus, we conclude that TBP binding is defined by a branched pathway, wherein TBP initially binds with little sequence specificity and is thermodynamically positioned by its kinetic stability to the TATA box. Furthermore, we observed the real-time access of TBP binding to TATA box DNA located within the DNA entry-exit site of the nucleosome. From these data, we determined salt-dependent changes in the nucleosome conformation regulate TBP's access to the TATA box, where access is highly constrained under physiological conditions, but is alleviated by histone acetylation and TFIIA. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

    DEFF Research Database (Denmark)

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan

    2012-01-01

    Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered...... by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation of the aptamers and thereby to maximize anticoagulant activity in functional assays. By judicious engineering...... of the DNA nanostructures, we have created a novel, functional DNA nanostructure, which is a multi-aptamer inhibitor with activity eightfold higher than free aptamer. Reversal of the thrombin inhibition was also achieved by the use of single-stranded DNA antidotes, thus enabling significant control over...

  5. Coupled binding-bending-folding: The complex conformational dynamics of protein-DNA binding studied by atomistic molecular dynamics simulations.

    Science.gov (United States)

    van der Vaart, Arjan

    2015-05-01

    Protein-DNA binding often involves dramatic conformational changes such as protein folding and DNA bending. While thermodynamic aspects of this behavior are understood, and its biological function is often known, the mechanism by which the conformational changes occur is generally unclear. By providing detailed structural and energetic data, molecular dynamics simulations have been helpful in elucidating and rationalizing protein-DNA binding. This review will summarize recent atomistic molecular dynamics simulations of the conformational dynamics of DNA and protein-DNA binding. A brief overview of recent developments in DNA force fields is given as well. Simulations have been crucial in rationalizing the intrinsic flexibility of DNA, and have been instrumental in identifying the sequence of binding events, the triggers for the conformational motion, and the mechanism of binding for a number of important DNA-binding proteins. Molecular dynamics simulations are an important tool for understanding the complex binding behavior of DNA-binding proteins. With recent advances in force fields and rapid increases in simulation time scales, simulations will become even more important for future studies. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Copyright © 2014. Published by Elsevier B.V.

  6. Bombyx mori nucleopolyhedrovirus encodes a DNA-binding protein capable of destabilizing duplex DNA.

    Science.gov (United States)

    Mikhailov, V S; Mikhailova, A L; Iwanaga, M; Gomi, S; Maeda, S

    1998-04-01

    A DNA-binding protein (designated DBP) with an apparent molecular mass of 38 kDa was purified to homogeneity from BmN cells (derived from Bombyx mori) infected with the B. mori nucleopolyhedrovirus (BmNPV). Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. The determined amino acid sequences indicated that DBP was encoded by an open reading frame (ORF16) located at nucleotides (nt) 16189 to 17139 in the BmNPV genome (GenBank accession no. L33180). This ORF (designated dbp) is a homolog of Autographa californica multicapsid NPV ORF25, whose product has not been identified. BmNPV DBP is predicted to contain 317 amino acids (calculated molecular mass of 36.7 kDa) and to have an isoelectric point of 7.8. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA. When bound to oligonucleotides, DBP protected them from hydrolysis by phage T4 DNA polymerase-associated 3'-->5' exonuclease. The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. DBP, but not BmNPV LEF-3, was capable of unwinding partial DNA duplexes in an in vitro system. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.

  7. The interplay of primer-template DNA phosphorylation status and single-stranded DNA binding proteins in directing clamp loaders to the appropriate polarity of DNA.

    Science.gov (United States)

    Hayner, Jaclyn N; Douma, Lauren G; Bloom, Linda B

    2014-01-01

    Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3'OH (3'DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3'DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5'phosphate (5'P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5'DNA). The 5'P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3'DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5'DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3'DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. A molecular code dictates sequence-specific DNA recognition by homeodomains.

    Science.gov (United States)

    Damante, G; Pellizzari, L; Esposito, G; Fogolari, F; Viglino, P; Fabbro, D; Tell, G; Formisano, S; Di Lauro, R

    1996-09-16

    Most homeodomains bind to DNA sequences containing the motif 5'-TAAT-3'. The homeodomain of thyroid transcription factor 1 (TTF-1HD) binds to sequences containing a 5'-CAAG-3' core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF-1HD by both structural and functional approaches. As already suggested by the three-dimensional structure of TTF-1HD, the DNA binding specificities of the TTF-1, Antennapedia and Engrailed homeodomains, either wild-type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3' end of the core motif 5'-NAAN-3'. The nucleotide at the 5' position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF-1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule.

  9. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair

    NARCIS (Netherlands)

    W.L. de Laat (Wouter); E. Appeldoorn (Esther); K. Sugasawa (Kaoru); E.P.W.C. Weterings (Eric); J.H.J. Hoeijmakers (Jan); N.G.J. Jaspers (Nicolaas)

    1998-01-01

    textabstractThe human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a

  10. DPI-ELISA: a fast and versatile method to specify the binding of plant transcription factors to DNA in vitro

    Directory of Open Access Journals (Sweden)

    Chaban Christina

    2010-11-01

    Full Text Available Abstract Background About 10% of all genes in eukaryote genomes are predicted to encode transcription factors. The specific binding of transcription factors to short DNA-motifs influences the expression of neighbouring genes. However, little is known about the DNA-protein interaction itself. To date there are only a few suitable methods to characterise DNA-protein-interactions, among which the EMSA is the method most frequently used in laboratories. Besides EMSA, several protocols describe the effective use of an ELISA-based transcription factor binding assay e.g. for the analysis of human NFκB binding to specific DNA sequences. Results We provide a unified protocol for this type of ELISA analysis, termed DNA-Protein-Interaction (DPI-ELISA. Qualitative analyses with His-epitope tagged plant transcription factors expressed in E. coli revealed that EMSA and DPI-ELISA result in comparable and reproducible data. The binding of AtbZIP63 to the C-box and AtWRKY11 to the W2-box could be reproduced and validated by both methods. We next examined the physical binding of the C-terminal DNA-binding domains of AtWRKY33, AtWRKY50 and AtWRKY75 to the W2-box. Although the DNA-binding domain is highly conserved among the WRKY proteins tested, the use of the DPI-ELISA discloses differences in W2-box binding properties between these proteins. In addition to these well-studied transcription factor families, we applied our protocol to AtBPC2, a member of the so far uncharacterised plant specific Basic Pentacysteine transcription factor family. We could demonstrate binding to GA/TC-dinucleotide repeat motifs by our DPI-ELISA protocol. Different buffers and reaction conditions were examined. Conclusions We successfully applied our DPI-ELISA protocol to investigate the DNA-binding specificities of three different classes of transcription factors from Arabidopsis thaliana. However, the analysis of the binding affinity of any DNA-binding protein to any given DNA

  11. Binding by the hepatitis C virus NS3 helicase partially melts duplex DNA.

    Science.gov (United States)

    Raney, Veronica M; Reynolds, Kimberly A; Harrison, Melody K; Harrison, David K; Cameron, Craig E; Raney, Kevin D

    2012-09-25

    Binding of NS3 helicase to DNA was investigated by footprinting with KMnO(4), which reacts preferentially with thymidine residues in single-stranded DNA (ssDNA) compared to those in double-stranded DNA (dsDNA). A distinct pattern of reactivity was observed on ssDNA, which repeated every 8 nucleotides (nt) and is consistent with the known binding site size of NS3. Binding to a DNA substrate containing a partial duplex was also investigated. The DNA contained a 15 nt overhang made entirely of thymidine residues adjacent to a 22 bp duplex that contained thymidine at every other position. Surprisingly, the KMnO(4) reactivity pattern extended from the ssDNA into the dsDNA region of the substrate. Lengthening the partial duplex to 30 bp revealed a similar pattern extending from the ssDNA into the dsDNA, indicating that NS3 binds within the duplex region. Increasing the length of the ssDNA portion of the partial duplex by 4 nt resulted in a shift in the footprinting pattern for the ssDNA by 4 nt, which is consistent with binding to the 3'-end of the ssDNA. However, the footprinting pattern in the dsDNA region was shifted by only 1-2 bp, indicating that binding to the ssDNA-dsDNA region was preferred. Footprinting performed as a function of time indicated that NS3 binds to the ssDNA rapidly, followed by slower binding to the duplex. Hence, multiple molecules of NS3 can bind along a ssDNA-dsDNA partial duplex by interacting with the ssDNA as well as the duplex DNA.

  12. Miz-1 activates gene expression via a novel consensus DNA binding motif.

    Directory of Open Access Journals (Sweden)

    Bonnie L Barrilleaux

    Full Text Available The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput in vitro technique, Bind-n-Seq, to identify two Miz-1 consensus DNA binding motif sequences--ATCGGTAATC and ATCGAT (Mizm1 and Mizm2--bound by full-length Miz-1 and its zinc finger domain, respectively. We validated these sequences directly as high affinity Miz-1 binding motifs. Competition assays using mutant probes indicated that the binding affinity of Miz-1 for Mizm1 and Mizm2 is highly sequence-specific. Miz-1 strongly activates gene expression through the motifs in a Myc-independent manner. MEME-ChIP analysis of Miz-1 ChIP-seq data in two different cell types reveals a long motif with a central core sequence highly similar to the Mizm1 motif identified by Bind-n-Seq, validating the in vivo relevance of the findings. Miz-1 ChIP-seq peaks containing the long motif are predominantly located outside of proximal promoter regions, in contrast to peaks without the motif, which are highly concentrated within 1.5 kb of the nearest transcription start site. Overall, our results indicate that Miz-1 may be directed in vivo to the novel motif sequences we have identified, where it can recruit its specific binding partners to control gene expression and ultimately regulate cell fate.

  13. Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami.

    Science.gov (United States)

    Jungmann, Ralf; Steinhauer, Christian; Scheible, Max; Kuzyk, Anton; Tinnefeld, Philip; Simmel, Friedrich C

    2010-11-10

    DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with <30 nm resolution. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.

  14. Plasmodium falciparum normocyte binding protein (PfNBP-1) peptides bind specifically to human erythrocytes.

    Science.gov (United States)

    Valbuena, John Jairo; Vera, Ricardo; García, Javier; Puentes, Alvaro; Curtidor, Hernando; Ocampo, Marisol; Urquiza, Mauricio; Rivera, Zuly; Guzmán, Fanny; Torres, Elizabeth; Patarroyo, Manuel Elkin

    2003-07-01

    Plasmodium falciparum normocyte binding protein-1 (PfNBP-1), a Plasmodium vivax RBP-1 orthologue is expressed in the apical merozoite area. PfNBP-1 binds directly to human erythrocyte membrane in a sialic acid-dependent but trypsin-resistant way. Erythrocyte binding assays were done with synthetic peptides covering the sequence reported as PfNBP-1. Two specific erythrocyte high activity binding peptides were found: 101VFINDLDTYQYEYFYEWNQ(120), peptide 26332, and 181NTKETYLKELNKKKMLQNKK(200), peptide 26336. These two peptides' binding was saturable and presenting nanomolar affinity constants. The critical binding residues (those residues underlined and highlighted in bold) were determined by competition assays with glycine-scan analogue peptides. These peptides were able to block merozoite in vitro invasion of erythrocytes.

  15. CSI-Tree: a regression tree approach for modeling binding properties of DNA-binding molecules based on cognate site identification (CSI) data.

    Science.gov (United States)

    Keleş, Sündüz; Warren, Christopher L; Carlson, Clayton D; Ansari, Aseem Z

    2008-06-01

    The identification and characterization of binding sites of DNA-binding molecules, including transcription factors (TFs), is a critical problem at the interface of chemistry, biology and molecular medicine. The Cognate Site Identification (CSI) array is a high-throughput microarray platform for measuring comprehensive recognition profiles of DNA-binding molecules. This technique produces datasets that are useful not only for identifying binding sites of previously uncharacterized TFs but also for elucidating dependencies, both local and nonlocal, between the nucleotides at different positions of the recognition sites. We have developed a regression tree technique, CSI-Tree, for exploring the spectrum of binding sites of DNA-binding molecules. Our approach constructs regression trees utilizing the CSI data of unaligned sequences. The resulting model partitions the binding spectrum into homogeneous regions of position specific nucleotide effects. Each homogeneous partition is then summarized by a position weight matrix (PWM). Hence, the final outcome is a binding intensity rank-ordered collection of PWMs each of which spans a different region in the binding spectrum. Nodes of the regression tree depict the critical position/nucleotide combinations. We analyze the CSI data of the eukaryotic TF Nkx-2.5 and two engineered small molecule DNA ligands and obtain unique insights into their binding properties. The CSI tree for Nkx-2.5 reveals an interaction between two positions of the binding profile and elucidates how different nucleotide combinations at these two positions lead to different binding affinities. The CSI trees for the engineered DNA ligands exhibit a common preference for the dinucleotide AA in the first two positions, which is consistent with preference for a narrow and relatively flat minor groove. We carry out a reanalysis of these data with a mixture of PWMs approach. This approach is an advancement over the simple PWM model and accommodates position

  16. Role of teh Rad52 Amino-terminal DNA Binding Activity in DNA Strand Capture in Homologous Recombination

    DEFF Research Database (Denmark)

    Shi, Idina; Hallwyl, Swee Chuang Lim; Seong, Changhyun

    2009-01-01

    -terminal DNA binding domain, is capable of Rad51 delivery to DNA but is deficient in DNA annealing. Results from chromatin immunoprecipitation experiments find that rad52-R70A associates with DNA double-strand breaks and promotes recruitment of Rad51 as efficiently as wild-type Rad52. Analysis of gene...... conversion intermediates reveals that rad52-R70A cells can mediate DNA strand invasion but are unable to complete the recombination event. These results provide evidence that DNA binding by the evolutionarily conserved amino terminus of Rad52 is needed for the capture of the second DNA end during homologous...

  17. Sequence-selective binding of C8-conjugated pyrrolobenzodiazepines (PBDs) to DNA.

    Science.gov (United States)

    Basher, Mohammad A; Rahman, Khondaker Miraz; Jackson, Paul J M; Thurston, David E; Fox, Keith R

    2017-11-01

    DNA footprinting and melting experiments have been used to examine the sequence-specific binding of C8-conjugates of pyrrolobenzodiazepines (PBDs) and benzofused rings including benzothiophene and benzofuran, which are attached using pyrrole- or imidazole-containing linkers. The conjugates modulate the covalent attachment points of the PBDs, so that they bind best to guanines flanked by A/T-rich sequences on either the 5'- or 3'-side. The linker affects the binding, and pyrrole produces larger changes than imidazole. Melting studies with 14-mer oligonucleotide duplexes confirm covalent attachment of the conjugates, which show a different selectivity to anthramycin and reveal that more than one ligand molecule can bind to each duplex. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Asmita; Yakovleva, Lyudmila; Shuman, Stewart; Mondragón, Alfonso (NWU); (SKI)

    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.

  19. Neutralizing the function of a β-globin-associated cis-regulatory DNA element using an artificial zinc finger DNA-binding domain.

    Science.gov (United States)

    Barrow, Joeva J; Masannat, Jude; Bungert, Jörg

    2012-10-30

    Gene expression is primarily regulated by cis-regulatory DNA elements and trans-interacting proteins. Transcription factors bind in a DNA sequence-specific manner and recruit activities that modulate the association and activity of transcription complexes at specific genes. Often, transcription factors belong to families of related proteins that interact with similar DNA sequences. Furthermore, genes are regulated by multiple, sometimes redundant, cis-regulatory elements. Thus, the analysis of the role of a specific DNA regulatory sequence and the interacting proteins in the context of intact cells is challenging. In this study, we designed and functionally characterized an artificial DNA-binding domain that neutralizes the function of a cis-regulatory DNA element associated with adult β-globin gene expression. The zinc finger DNA-binding domain (ZF-DBD), comprising six ZFs, interacted specifically with a CACCC site located 90 bp upstream of the transcription start site (-90 β-ZF-DBD), which is normally occupied by KLF1, a major regulator of adult β-globin gene expression. Stable expression of the -90 β-ZF-DBD in mouse erythroleukemia cells reduced the binding of KLF1 with the β-globin gene, but not with locus control region element HS2, and led to reduced transcription. Transient transgenic embryos expressing the -90 β-ZF-DBD developed normally but revealed reduced expression of the adult β-globin gene. These results demonstrate that artificial DNA-binding proteins lacking effector domains are useful tools for studying and modulating the function of cis-regulatory DNA elements.

  20. A single amino acid substitution in the DNA-binding domain of Aeropyrum pernix DNA ligase impairs its interaction with proliferating cell nuclear antigen.

    Science.gov (United States)

    Kiyonari, Shinichi; Kamigochi, Toru; Ishino, Yoshizumi

    2007-09-01

    Proliferating cell nuclear antigen (PCNA) is known as a DNA sliding clamp that acts as a platform for the assembly of enzymes involved in DNA replication and repair. Previously, it was reported that a crenarchaeal PCNA formed a heterotrimeric structure, and that each PCNA subunit has distinct binding specificity to PCNA-binding proteins. Here we describe the PCNA-binding properties of a DNA ligase from the hyperthermophilic crenarchaeon Aeropyrum pernix K1. Based on our findings on the Pyrococcus furiosus DNA ligase-PCNA interaction, we predicted that the aromatic residue, Phe132, in the DNA-binding domain of A. pernix DNA ligase (ApeLig) would play a critical role in binding to A. pernix PCNA (ApePCNA). Surface plasmon resonance analyses revealed that the ApeLig F132A mutant does not interact with an immobilized subunit of ApePCNA. Furthermore, we could not detect any stimulation of the ligation activity of the ApeLig F132A protein by ApePCNA in vitro. These results indicated that the phenylalanine, which is located in our predicted PCNA-binding region in ApeLig, has a critical role for the physical and functional interaction with ApePCNA.

  1. Cyclic perylene diimide: Selective ligand for tetraplex DNA binding over double stranded DNA.

    Science.gov (United States)

    Vasimalla, Suresh; Sato, Shinobu; Takenaka, Fuminori; Kurose, Yui; Takenaka, Shigeori

    2017-12-15

    Synthesized cyclic perylene diimide, cPDI, showed the binding constant of 6.3 × 10 6  M -1 with binding number of n = 2 with TA-core as a tetraplex DNA in 50 mM Tris-HCl buffer (pH = 7.4) containing 100 mM KCl using Schatchard analysis and showed a higher preference for tetraplex DNA than for double stranded DNA with over 10 3 times. CD spectra showed that TA-core induced its antiparallel conformation upon addition of cPDI in the absence or presence of K + or Na + ions. The cPDI inhibits the telomerase activity with IC 50 of 0.3 µM using TRAP assay which is potential anti-cancer drug with low side effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Specific binding and mineralization of calcified surfaces by small peptides.

    Science.gov (United States)

    Yarbrough, Daniel K; Hagerman, Elizabeth; Eckert, Randal; He, Jian; Choi, Hyewon; Cao, Nga; Le, Karen; Hedger, Jennifer; Qi, Fengxia; Anderson, Maxwell; Rutherford, Bruce; Wu, Ben; Tetradis, Sotiris; Shi, Wenyuan

    2010-01-01

    Several small (dentin phosphoprotein, one of the major noncollagenous proteins thought to be involved in the mineralization of the dentin extracellular matrix during tooth development. These peptides, consisting of multiple repeats of the tripeptide aspartate-serine-serine (DSS), bind with high affinity to calcium phosphate compounds and, when immobilized, can recruit calcium phosphate to peptide-derivatized polystyrene beads or to demineralized human dentin surfaces. The affinity of binding to hydroxyapatite surfaces increases with the number of (DSS)(n) repeats, and though similar repeated sequences-(NTT)(n), (DTT)(n), (ETT)(n), (NSS)(n), (ESS)(n), (DAA)(n), (ASS)(n), and (NAA)(n)-also showed HA binding activity, it was generally not at the same level as the natural sequence. Binding of the (DSS)(n) peptides to sectioned human teeth was shown to be tissue-specific, with high levels of binding to the mantle dentin, lower levels of binding to the circumpulpal dentin, and little or no binding to healthy enamel. Phosphorylation of the serines of these peptides was found to affect the avidity, but not the affinity, of binding. The potential utility of these peptides in the detection of carious lesions, the delivery of therapeutic compounds to mineralized tissues, and the modulation of remineralization is discussed.

  3. Identification of DNA-binding protein target sequences by physical effective energy functions: free energy analysis of lambda repressor-DNA complexes.

    Directory of Open Access Journals (Sweden)

    Caselle Michele

    2007-09-01

    Full Text Available Abstract Background Specific binding of proteins to DNA is one of the most common ways gene expression is controlled. Although general rules for the DNA-protein recognition can be derived, the ambiguous and complex nature of this mechanism precludes a simple recognition code, therefore the prediction of DNA target sequences is not straightforward. DNA-protein interactions can be studied using computational methods which can complement the current experimental methods and offer some advantages. In the present work we use physical effective potentials to evaluate the DNA-protein binding affinities for the λ repressor-DNA complex for which structural and thermodynamic experimental data are available. Results The binding free energy of two molecules can be expressed as the sum of an intermolecular energy (evaluated using a molecular mechanics forcefield, a solvation free energy term and an entropic term. Different solvation models are used including distance dependent dielectric constants, solvent accessible surface tension models and the Generalized Born model. The effect of conformational sampling by Molecular Dynamics simulations on the computed binding energy is assessed; results show that this effect is in general negative and the reproducibility of the experimental values decreases with the increase of simulation time considered. The free energy of binding for non-specific complexes, estimated using the best energetic model, agrees with earlier theoretical suggestions. As a results of these analyses, we propose a protocol for the prediction of DNA-binding target sequences. The possibility of searching regulatory elements within the bacteriophage λ genome using this protocol is explored. Our analysis shows good prediction capabilities, even in absence of any thermodynamic data and information on the naturally recognized sequence. Conclusion This study supports the conclusion that physics-based methods can offer a completely complementary

  4. Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains.

    Directory of Open Access Journals (Sweden)

    Nicolas Siaud

    2011-12-01

    Full Text Available The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR. Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations.

  5. Synthesis, Characterization, and DNA Binding Studies of Nanoplumbagin

    Directory of Open Access Journals (Sweden)

    Sheik Dawood Shahida Parveen

    2014-01-01

    Full Text Available The traditional anticancer medicine plumbagin (PLN was prepared as nanostructured material (nanoplumbagin, NPn1 from its commercial counterparts, simultaneously coencapsulating with cetyltrimethylammonium bromide or cyclodextrin as stabilizers using ultrasonication technique. Surface morphology of NPn analysed from atomic force microscopy (AFM indicates that NPn has tunable size between 75 nm and 100 nm with narrow particle size distribution. Its binding efficiency with herring sperm DNA was studied using spectral and electrochemical techniques and its efficiency was found to be more compared to the commercial microcrystalline plumbagin (PLN. DNA cleavage was also studied by gel electrophoresis. The observed results indicate that NPn1 has better solubility in aqueous medium and hence showed better bioavailability compared to its commercial counterparts.

  6. Cultivated single stranded DNA phages that infect marine Bacteroidetes prove difficult to detect with DNA binding stains

    DEFF Research Database (Denmark)

    Holmfeldt, Karin; Odic, Dusko; Sullivan, Matthew B.

    2012-01-01

    This is the first description of cultivated icosahedral single stranded DNA (ssDNA) phages isolated on heterotrophic marine bacterioplankton and with Bacteroidetes hosts. None of the 8 phages stained well with DNA binding stains, suggesting that in situ abundances of ssDNA phages are drastically...

  7. Deoxyribonuclease I footprinting reveals different DNA binding modes of bifunctional platinum complexes.

    Science.gov (United States)

    Chválová, Katerina; Kaspárková, Jana; Farrell, Nicholas; Brabec, Viktor

    2006-08-01

    Deoxyribonuclease I (DNase I) footprinting methodology was used to analyze oligodeoxyribonucleotide duplexes containing unique and single, site-specific adducts of trinuclear bifunctional platinum compound, [{trans-PtCl(NH3)2}2 mu-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (BBR3464) and the results were compared with DNase I footprints of some adducts of conventional mononuclear cis-diamminedichloroplatinum(II) (cisplatin). These examinations took into account the fact that the local conformation of the DNA at the sites of the contacts of DNase I with DNA phosphates, such as the minor groove width and depth, sequence-dependent flexibility and bendability of the double helix, are important determinants of sequence-dependent binding to and cutting of DNA by DNase I. It was shown that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by DNase I probing. The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I.

  8. Molecular mechanism of DNA recognition by the alpha subunit of the Oxytricha telomere binding protein.

    Science.gov (United States)

    Laporte, L; Benevides, J M; Thomas, G J

    1999-01-12

    Interactions between telomeric DNA and the alpha subunit of the heterodimeric telomere binding protein of Oxytricha nova have been probed by Raman spectroscopy, CD spectroscopy, and nondenaturing gel electrophoresis. Telomeric sequences investigated include the Oxytricha 3' overhang, d(T4G4)2, and the related sequence dT6(T4G4)2, which incorporates a 5'-thymidylate leader. Corresponding nontelomeric isomers, d(TG)8 and dT6(TG)8, have also been investigated. Both d(T4G4)2 and dT6(T4G4)2 form stable hairpins that contain Hoogsteen G.G base pairs [Laporte, L., and Thomas, G. J., Jr. (1998) J. Mol. Biol. 281, 261-270]. The alpha subunit binds specifically and stoichiometrically to the dT6(T4G4)2 hairpin and alters its secondary structure by inducing conformational changes in the 5'-thymidylate leader without extensive disruption of G.G base pairing. Conversely, binding of the alpha subunit to d(T4G4)2 eliminates G.G pairing and unfolds the hairpin. DNA unfolding is accompanied by conformational changes affecting both the backbone and dG residues, as evidenced by Raman and CD spectra. Interestingly, the alpha subunit also forms complexes with the nontelomeric isomers, d(TG)8 and dT6(TG)8, evidenced by altered electrophoretic mobility in nondenaturing gels; however, Raman and CD spectra of complexes of the alpha subunit with nontelomeric DNA suggest no significant changes in backbone or deoxynucleoside conformations. Similarly, the alpha subunit binds to but does not appreciably alter the secondary structure of duplex DNA. The present results show that while the alpha subunit has the capacity to bind to Watson-Crick and different non-Watson-Crick motifs, DNA refolding is specific to the Oxytricha telomeric hairpin and the retention of G.G pairing is specific to the telomeric sequence incorporating the 5' leading sequence. A model is proposed for alpha subunit binding to telomeric DNA, and the physiological role of the alpha subunit in telomere organization is discussed.

  9. Probing the binding of insecticide permethrin to calf thymus DNA by spectroscopic techniques merging with chemometrics method.

    Science.gov (United States)

    Zhang, Yue; Zhang, Guowen; Li, Yu; Hu, Yuting

    2013-03-20

    The binding of permethrin (PE) with calf thymus DNA (ctDNA) in physiological buffer (pH 7.4) was investigated by ultraviolet-visible (UV-vis) absorption, fluorescence, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy merging with multivariate curve resolution-alternating least-squares (MCR-ALS) chemometrics approach. The MCR-ALS was applied to resolve the combined spectroscopic data matrix, which was obtained by UV-vis and fluorescence methods. The concentration profiles of PE, ctDNA, and PE-ctDNA complex and their pure spectra were then successfully obtained. The PE molecular was found to be able to intercalate into the base pairs of ctDNA as evidenced by decreases in resonance light-scattering signal and iodide-quenching effect and increase in ctDNA viscosity. The results of FT-IR spectra indicated that PE was prone to bind to G-C base pairs of ctDNA, and the molecular docking studies were used to validate and clarify the specific binding. The observed changes in CD signals revealed that the DNA turned into a more highly wound form of B-conformation. The calculated thermodynamic parameters, enthalpy change (ΔH°) and entropy change (ΔS°), suggested that hydrogen bonds and van der Waals forces played a predominant role in the binding of PE to ctDNA.

  10. Using FRET to Measure the Angle at Which a Protein Bends DNA: TBP Binding a TATA Box as a Model System

    Science.gov (United States)

    Kugel, Jennifer F.

    2008-01-01

    An undergraduate biochemistry laboratory experiment that will teach the technique of fluorescence resonance energy transfer (FRET) while analyzing protein-induced DNA bending is described. The experiment uses the protein TATA binding protein (TBP), which is a general transcription factor that recognizes and binds specific DNA sequences known as…

  11. The Facilitation of Protein-DNA Search and Recognition by Multiple Modes of Binding

    Science.gov (United States)

    Leith, Jason Suh

    The studies discussed in this thesis unify experimental and theoretical techniques, both established and novel, in investigating the problem of how a protein that binds specific sites on DNA translocates to, recognizes, and stably binds to its target site or sites. The thesis is organized into two parts. Part I outlines the history of the problem and the theory and experiments that have addressed the problem and presents an apparent incompatibility between efficient search and stable, specific binding. To address this problem, we elaborate a model of protein-DNA interaction in which the protein may bind DNA in either a search (S) mode or a recognition (R) mode. The former is characterized by zero or weak sequence-dependence in the binding energy, while the latter is highly sequence-dependent. The protein undergoes a random walk along the DNA in the S mode, and if it encounters its target site, must undergo a conformational transition into the R mode. The model resolves the apparent paradox, and accounts for the observed speed, specificity, and stability in protein-DNA interactions. The model shows internal agreement as regards theoretical and simulated results, as well as external agreement with experimental measurements. Part II reports on research that has tested the applicability of the two-mode model to the tumor suppressor transcription factor p53. It describes in greater depth the experimental techinques and findings up to the present work, and introduces the techinques and biological system used in our research. We employ single-molecule optical microscopy in two projects to study the diffusional kinetics of p53 on DNA. The first project measures the diffusion coefficient of p53 and determines that the protein satisfies a number of requirements for the validity of the two-mode model and for efficient target localization. The second project examines the sequence-dependence in p53's sliding kinetics, and explicitly models the energy landscape it experiences on

  12. A DNA sequence recognition loop on APOBEC3A controls substrate specificity.

    Directory of Open Access Journals (Sweden)

    Eric C Logue

    Full Text Available APOBEC3A (A3A, one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G, deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G. Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.

  13. Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA.

    Science.gov (United States)

    Hashimoto, Hideharu; Wang, Dongxue; Horton, John R; Zhang, Xing; Corces, Victor G; Cheng, Xiaodong

    2017-06-01

    The multidomain CCCTC-binding factor (CTCF), containing a tandem array of 11 zinc fingers (ZFs), modulates the three-dimensional organization of chromatin. We crystallized the human CTCF DNA-binding domain in complex with a known CTCF-binding site. While ZF2 does not make sequence-specific contacts, each finger of ZF3-7 contacts three bases of the 15-bp consensus sequence. Each conserved nucleotide makes base-specific hydrogen bonds with a particular residue. Most of the variable base pairs within the core sequence also engage in interactions with the protein. These interactions compensate for deviations from the consensus sequence, allowing CTCF to adapt to sequence variations. CTCF is sensitive to cytosine methylation at position 2, but insensitive at position 12 of the 15-bp core sequence. These differences can be rationalized structurally. Although included in crystallizations, ZF10 and ZF11 are not visible, while ZF8 and ZF9 span the backbone of the DNA duplex, conferring no sequence specificity but adding to overall binding stability. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.

    Science.gov (United States)

    Duplat, D; Puisségur, M; Bédouet, L; Rousseau, M; Boulzaguet, H; Milet, C; Sellos, D; Van Wormhoudt, A; Lopez, E

    2006-05-01

    Nacre or mother-of-pearl in the shell of Pinctada margaritifera is composed of 95-99% calcium carbonate and 1-5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre-forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)-PCR. Seventy-two specific cDNA sequences have been obtained so far. These include a protein containing two EF-hand Ca2+-binding domains which was completely sequenced after amplification by RACE-PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi-quantitative RT-PCR on NFC and mantle cells.

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

    Indian Academy of Sciences (India)

    Interactions of zinc and cobalt with peptides cysteinylglycine and histidylglycine have been studied. The binding modes were identified and geometry assigned. Stabilities of these complexes and their ability to bind DNA have been investigated. It is demonstrated that only zinc complexes bind DNA as compared to cobalt ...

  16. Variation of the intercalating proline in artificial peptides mimicking the DNA binding and bending IHF protein.

    Science.gov (United States)

    Scholz, S; Liebler, E K; Eickmann, B; Fritz, H-J; Diederichsen, U

    2012-07-01

    The integration host factor (IHF) is a protein which sequence specifically induces a bend of double-stranded DNA by more than 160°. Based on IHF as lead structure, a peptide mimic was introduced resembling the positively charged body of the protein by a lysine dendrimer and the minor groove recognition loop by a cyclopeptide. The proline located close to the tip of the recognition loop intercalates between the base pair plane. It was modified in order to evaluate the influence of the side chain residue with respect to size (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), aromaticity (phenylalanine), conformation of the five-membered ring [(4R)-fluoroproline, (4S)-fluoroproline, 3,4-dehydroproline], and the peptide backbone conformation (α-methylproline) on binding dsDNA and bending the double strand. Binding and bending studies were carried out by fluorescence resonance energy transfer experiments and gel electrophoresis using DNA sequences prepared by PCR with the IHF binding site in central or terminal position. Whereas aromatic residues and α-methylproline were not tolerated as proline substitute, incorporation of (4S)-fluoroproline and 3,4-dehydroproline provided enhanced binding.

  17. Molecular binding and enhanced photoluminescence of bromocresol purple in marine derived DNA

    Science.gov (United States)

    Diggs, Darnell E.; Hagen, Josh; Yu, Zhou; Heckman, Emily; Hopkins, F. Kenneth; Grote, James G.; Steckl, Andrew

    2005-08-01

    Deoxyribonucleic acid (DNA) extracted and purified from salmon roe and milt sacs, a waste product of the fishing industry was studied for molecular binding and photoluminescence effects using bromocresol purple (BCP). Since BCP is both water and alcohol soluble it was investigated for binding efficiency in DNA/water solutions and modified DNA-CTMA/butanol solutions. Circular dichroism studies show that there is a maximum binding concentration of BCP in the DNA/water solution at ~5% by weight of BCP:DNA. In contrast, DNA-CTMA/butanol solutions showed increased binding concentrations up through 10wt% BCP:DNA-CTMA. This apparent binding affinity of DNA-CTMA for BCP also resulted in a significantly higher (6x) photoluminescence in thin film form when compared to BCP:PMMA films of the same doping concentration.

  18. Non-intercalative, deoxyribose binding of boric acid to calf thymus DNA.

    Science.gov (United States)

    Ozdemir, Ayse; Gursaclı, Refiye Tekiner; Tekinay, Turgay

    2014-05-01

    The present study characterizes the effects of the boric acid binding on calf thymus DNA (ct-DNA) by spectroscopic and calorimetric methods. UV-Vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize binding properties. Changes in the secondary structure of ct-DNA were determined by CD spectroscopy. Sizes and morphologies of boric acid-DNA complexes were determined by transmission electron microscopy (TEM). The kinetics of boric acid binding to calf thymus DNA (ct-DNA) was investigated by isothermal titration calorimetry (ITC). ITC results revealed that boric acid exhibits a moderate affinity to ct-DNA with a binding constant (K a) of 9.54 × 10(4) M(-1). FT-IR results revealed that boric acid binds to the deoxyribose sugar of DNA without disrupting the B-conformation at tested concentrations.

  19. Structure of p73 DNA-binding domain tetramer modulates p73 transactivation

    Science.gov (United States)

    Ethayathulla, Abdul S.; Tse, Pui-Wah; Monti, Paola; Nguyen, Sonha; Inga, Alberto; Fronza, Gilberto; Viadiu, Hector

    2012-01-01

    The transcription factor p73 triggers developmental pathways and overlaps stress-induced p53 transcriptional pathways. How p53-family response elements determine and regulate transcriptional specificity remains an unsolved problem. In this work, we have determined the first crystal structures of p73 DNA-binding domain tetramer bound to response elements with spacers of different length. The structure and function of the adaptable tetramer are determined by the distance between two half-sites. The structures with zero and one base-pair spacers show compact p73 DNA-binding domain tetramers with large tetramerization interfaces; a two base-pair spacer results in DNA unwinding and a smaller tetramerization interface, whereas a four base-pair spacer hinders tetramerization. Functionally, p73 is more sensitive to spacer length than p53, with one base-pair spacer reducing 90% of transactivation activity and longer spacers reducing transactivation to basal levels. Our results establish the quaternary structure of the p73 DNA-binding domain required as a scaffold to promote transactivation. PMID:22474346

  20. Characterization of a mitochondrially targeted single-stranded DNA-binding protein in Arabidopsis thaliana.

    Science.gov (United States)

    Edmondson, Andrew C; Song, Daqing; Alvarez, Luis A; Wall, Melisa K; Almond, David; McClellan, David A; Maxwell, Anthony; Nielsen, Brent L

    2005-04-01

    A gene encoding a predicted mitochondrially targeted single-stranded DNA binding protein (mtSSB) was identified in the Arabidopsis thaliana genome sequence. This gene (At4g11060) codes for a protein of 201 amino acids, including a 28-residue putative mitochondrial targeting transit peptide. Protein sequence alignment shows high similarity between the mtSSB protein and single-stranded DNA binding proteins (SSB) from bacteria, including residues conserved for SSB function. Phylogenetic analysis indicates a close relationship between this protein and other mitochondrially targeted SSB proteins. The predicted targeting sequence was fused with the GFP coding region, and the organellar localization of the expressed fusion protein was determined. Specific targeting to mitochondria was observed in in-vitro import experiments and by transient expression of a GFP fusion construct in Arabidopsis leaves after microprojectile bombardment. The mature mtSSB coding region was overexpressed in Escherichia coli and the protein was purified for biochemical characterization. The purified protein binds single-stranded, but not double-stranded, DNA. MtSSB stimulates the homologous strand-exchange activity of E. coli RecA. These results indicate that mtSSB is a functional homologue of the E. coli SSB, and that it may play a role in mitochondrial DNA recombination.

  1. C-terminal diversity within the p53 family accounts for differences in DNA binding and transcriptional activity

    Science.gov (United States)

    Sauer, Markus; Bretz, Anne Catherine; Beinoraviciute-Kellner, Rasa; Beitzinger, Michaela; Burek, Christof; Rosenwald, Andreas; Harms, Gregory S.; Stiewe, Thorsten

    2008-01-01

    The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA-binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity: while p53 and the p73 isoform p73γ have basic CTDs and form weak sequence-specific protein–DNA complexes, the major p73 isoforms have neutral CTDs and bind DNA strongly. A basic CTD has been previously shown to enable sliding along the DNA backbone and to facilitate the search for binding sites in the complex genome. Our experiments, however, reveal that a basic CTD also reduces protein–DNA complex stability, intranuclear mobility, promoter occupancy in vivo, target gene activation and induction of cell cycle arrest or apoptosis. A basic CTD therefore provides both positive and negative regulatory functions presumably to enable rapid switching of protein activity in response to stress. The different DNA-binding characteristics of the p53 family members could therefore reflect their predominant role in the cellular stress response (p53) or developmental processes (p73). PMID:18267967

  2. Polyethyleneimine anchored copper(II) complexes: synthesis, characterization, in vitro DNA binding studies and cytotoxicity studies.

    Science.gov (United States)

    Lakshmipraba, Jagadeesan; Arunachalam, Sankaralingam; Riyasdeen, Anvarbatcha; Dhivya, Rajakumar; Akbarsha, Mohammad Abdulkader

    2015-01-01

    The water soluble polyethyleneimine-copper(II) complexes, [Cu(phen)(L-tyr)BPEI]ClO4 (where phen=1,10-phenanthroline, L-tyr=L-tyrosine and BPEI=branched polyethyleneimine) with various degree of copper(II) complex units in the polymer chain were synthesized and characterized by elemental analysis and electronic, FT-IR, EPR spectroscopic techniques. The binding of these complexes with CT-DNA was studied using UV-visible absorption titration, thermal denaturation, emission, circular dichroism spectroscopy and cyclic voltammetric methods. The changes observed in the physicochemcial properties indicated that the binding between the polymer-copper complexes and DNA was mostly through electrostatic mode of binding. Among these complexes, the polymer-copper(II) complex with the highest degrees of copper(II) complex units (higher degrees of coordination) showed higher binding constant than those with lower copper(II) complex units (lower degrees of coordination) complexes. The complex with the highest number of metal centre bound strongly due to the cooperative binding effect. Therefore, anticancer study was carried out using this complex. The cytotoxic activity for this complex on MCF-7 breast cancer cell line was determined adopting MTT assay, acridine orange/ethidium bromide (AO/EB) staining and comet assay techniques, which revealed that the cells were committed to specific mode of cell death either apoptosis or necrosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Structural and Dynamics Studies of Pax5 Reveal Asymmetry in Stability and DNA Binding by the Paired Domain.

    Science.gov (United States)

    Perez-Borrajero, Cecilia; Okon, Mark; McIntosh, Lawrence P

    2016-06-05

    The eukaryotic transcription factor Pax5 or B-cell specific activator protein (BSAP) is central to B-cell development and has been implicated in a large number of cellular malignancies resulting from loss- or gain-of-function mutations. In this study, we characterized the DNA-binding Paired domain (PD) of Pax5 in its free and DNA-bound forms using NMR spectroscopy. In isolation, the PD folds as two independent helical bundle subdomains separated by a conformationally disordered linker. The two subdomains differ in stability, with the C-terminal subdomain (CTD) being ~10-fold more protected from amide hydrogen exchange (HX) than the N-terminal subdomain (NTD). Upon binding DNA, the linker and an induced N-terminal β-hairpin become ordered with significantly dampened motions and increased HX protection. Both subdomains of the PD contribute to specific DNA binding, resulting in an equilibrium dissociation constant more than three orders of magnitude lower than exhibited by the separate subdomains for their respective half-sites (nM versus μM). The isolated CTD binds non-specific DNA sequences with only ~10-fold weaker affinity than cognate sequences. In contrast, the NTD associates very poorly with non-specific DNA. We propose that the more stable CTD has evolved to provide relatively low affinity non-specific contacts with DNA. In contrast, the more dynamic NTD discriminates between cognate and non-specific sites. The distinct roles of the PD subdomains may enable efficient searching of genomic DNA by Pax5 while retaining specificity for functional regulatory sites. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins.

    Science.gov (United States)

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

    A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (-89° to -60°, -59° to -30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Generation of species-specific DNA probes for Leishmania aethiopica

    NARCIS (Netherlands)

    Laskay, T.; Kiessling, R.; Rinke deWit, T. F.; Wirth, D. F.

    1991-01-01

    We report here the cloning of kinetoplast DNA (kDNA) sequences from Leishmania aethiopica in order to develop a specific and sensitive method for the identification of the parasite. Analysis of the cloned kDNA sequences showed different taxonomic specificities demonstrating sequence diversity within

  6. Describing the Peptide Binding Specificity of HLA-C

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Harndahl, Mikkel Nors; Nielsen, Morten

    . We find preference for hydrophobic residues at the peptide C-terminus for all HLA-C molecules. Most molecules were found to have an additional strong anchor at P2 or P3, with auxiliary anchor observed at P1, P2, P3, and P7. The binding affinity is measured for peptides fitting the specificity matrix...

  7. DNA Bending is Induced in an Enhancer by the DNA-Binding Domain of the Bovine Papillomavirus E2 Protein

    Science.gov (United States)

    Moskaluk, Christopher; Bastia, Deepak

    1988-03-01

    The E2 gene of bovine papillomavirus type 1 has been shown to encode a DNA-binding protein and to trans-activate the viral enhancer. We have localized the DNA-binding domain of the E2 protein to the carboxyl-terminal 126 amino acids of the E2 open reading frame. The DNA-binding domain has been expressed in Escherichia coli and partially purified. Gel retardation and DNase I ``footprinting'' on the bovine papillomavirus type 1 enhancer identify the sequence motif ACCN6GGT (in which N = any nucleotide) as the E2 binding site. Using electrophoretic methods we have shown that the DNA-binding domain changes conformation of the enhancer by inducing significant DNA bending.

  8. The human papillomavirus DNA helicase E1 binds, stimulates, and confers processivity to cellular DNA polymerase epsilon

    Science.gov (United States)

    Chojnacki, Michaelle

    2018-01-01

    Abstract The papillomavirus (PV) helicase protein E1 recruits components of the cellular DNA replication machinery to the PV replication fork, such as Replication Protein A (RPA), DNA polymerase α-primase (pol α) and topoisomerase I (topo I). Here we show that E1 binds to DNA polymerase ϵ (pol ϵ) and dramatically stimulates the DNA synthesis activity of pol ϵ. This stimulation of pol ϵ by E1 is highly specific and occurs even in the absence of the known pol ϵ cofactors Replication Factor C (RFC), Proliferating Cell Nuclear Antigen (PCNA) and RPA. This stimulation is due to an increase in the processivity of pol ϵ and occurs independently of pol ϵ’s replication cofactors. This increase in processivity is dependent on the ability of the E1 helicase to hydrolyze ATP, suggesting it is dependent on E1’s helicase action. In addition, RPA, thought to be vital for processive DNA synthesis by both pol ϵ and pol δ, was found to be dispensable for processive synthesis by pol ϵ in the presence of E1. Overall, E1 appears to be conferring processivity to pol ϵ by directly tethering pol ϵ to the DNA parental strand and towing ϵ behind the E1 helicase as the replication fork progresses; and thereby apparently obviating the need for RPA for leading strand synthesis. Thus far only pol α and pol δ have been implicated in the DNA replication of mammalian viruses; this is the first reported example of a virus recruiting pol ϵ. Furthermore, this demonstrates a unique capacity of a viral helicase having evolved to stimulate a cellular replicative DNA polymerase. PMID:29155954

  9. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  10. The Multiple Carbohydrate Binding Specificities of Helicobacter pylori

    Science.gov (United States)

    Teneberg, Susann

    Persistent colonization of the human stomach by Helicobacter pylori is a risk factor for the development of peptic ulcer disease and gastric cancer. Adhesion of microbes to the target tissue is an important determinant for successful initiation, establishment and maintenance of infection, and a variety of different candidate carbohydrate receptors for H. pylori have been identified. Here the different the binding specifities, and their potential role in adhesion to human gastric epithelium are described. Finally, recent findings on the roles of sialic acid binding SabA adhesin in interactions with human neutrophils and erythrocytes are discussed.

  11. Quantification of specific bindings of biomolecules by magnetorelaxometry

    Directory of Open Access Journals (Sweden)

    Steinhoff Uwe

    2008-03-01

    Full Text Available Abstract The binding reaction of the biomolecules streptavidin and anti-biotin antibody, both labelled by magnetic nanoparticles (MNP, to biotin coated on agarose beads, was quantified by magnetorelaxometry (MRX. Highly sensitive SQUID-based MRX revealed the immobilization of the MNP caused by the biotin-streptavidin coupling. We found that about 85% of streptavidin-functionalised MNP bound specifically to biotin-agarose beads. On the other hand only 20% of antibiotin-antibody functionalised MNP were specifically bound. Variation of the suspension medium revealed in comparison to phosphate buffer with 0.1% bovine serum albumin a slight change of the binding behaviour in human serum, probably due to the presence of functioning (non heated serum proteins. Furthermore, in human serum an additional non-specific binding occurs, being independent from the serum protein functionality. The presented homogeneous bead based assay is applicable in simple, uncoated vials and it enables the assessment of the binding kinetics in a volume without liquid flow. The estimated association rate constant for the MNP-labelled streptavidin is by about two orders of magnitude smaller than the value reported for free streptavidin. This is probably due to the relatively large size of the magnetic markers which reduces the diffusion of streptavidin. Furthermore, long time non-exponential kinetics were observed and interpreted as agglutination of the agarose beads.

  12. Structural basis for sequence-specific recognition of DNA by TAL effectors

    KAUST Repository

    Deng, Dong

    2012-01-05

    TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.

  13. Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding.

    Science.gov (United States)

    Tubeleviciute, Agne; Skirgaila, Remigijus

    2010-08-01

    The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.

  14. Evaluation of a Solid Phase DNA Binding Matrix for Downstream PCR Analysis

    National Research Council Canada - National Science Library

    Bader, Douglas E; Fisher, Glen R; Stratilo, Chad W

    2005-01-01

    A commercially available solid-phase DNA binding matrix (FTA cards) was evaluated for its ability to capture and release DNA for downstream gene amplification and detection assays using polymerase chain reaction (PCR...

  15. Temperature Dependence and Thermodynamics of Klenow Polymerase Binding to Primed-Template DNA

    National Research Council Canada - National Science Library

    Datta, Kausiki; Wowor, Andy J; Richard, Allison J; LiCata, Vince J

    2006-01-01

    DNA binding of Klenow polymerase has been characterized with respect to temperature to delineate the thermodynamic driving forces involved in the interaction of this polymerase with primed-template DNA...

  16. Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function.

    Science.gov (United States)

    Ciolkowski, Ingo; Wanke, Dierk; Birkenbihl, Rainer P; Somssich, Imre E

    2008-09-01

    WRKY transcription factors have been shown to play a major role in regulating, both positively and negatively, the plant defense transcriptome. Nearly all studied WRKY factors appear to have a stereotypic binding preference to one DNA element termed the W-box. How specificity for certain promoters is accomplished therefore remains completely unknown. In this study, we tested five distinct Arabidopsis WRKY transcription factor subfamily members for their DNA binding selectivity towards variants of the W-box embedded in neighboring DNA sequences. These studies revealed for the first time differences in their binding site preferences, which are partly dependent on additional adjacent DNA sequences outside of the TTGACY-core motif. A consensus WRKY binding site derived from these studies was used for in silico analysis to identify potential target genes within the Arabidopsis genome. Furthermore, we show that even subtle amino acid substitutions within the DNA binding region of AtWRKY11 strongly impinge on its binding activity. Additionally, all five factors were found localized exclusively to the plant cell nucleus and to be capable of trans-activating expression of a reporter gene construct in vivo.

  17. Light-activated DNA binding in a designed allosteric protein

    Energy Technology Data Exchange (ETDEWEB)

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R. (UC)

    2008-09-03

    An understanding of how allostery, the conformational coupling of distant functional sites, arises in highly evolvable systems is of considerable interest in areas ranging from cell biology to protein design and signaling networks. We reasoned that the rigidity and defined geometry of an {alpha}-helical domain linker would make it effective as a conduit for allosteric signals. To test this idea, we rationally designed 12 fusions between the naturally photoactive LOV2 domain from Avena sativa phototropin 1 and the Escherichia coli trp repressor. When illuminated, one of the fusions selectively binds operator DNA and protects it from nuclease digestion. The ready success of our rational design strategy suggests that the helical 'allosteric lever arm' is a general scheme for coupling the function of two proteins.

  18. In silico footprinting of ligands binding to the minor groove of DNA.

    Science.gov (United States)

    Anthony, Nahoum G; Huchet, Guillaume; Johnston, Blair F; Parkinson, John A; Suckling, Colin J; Waigh, Roger D; Mackay, Simon P

    2005-01-01

    The sequence selectivity of small molecules binding to the minor groove of DNA can be predicted by "in silico footprinting". Any potential ligand can be docked in the minor groove and then moved along it using simple simulation techniques. By applying a simple scoring function to the trajectory after energy minimization, the preferred binding site can be identified. We show application to all known noncovalent binding modes, namely 1:1 ligand:DNA binding (including hairpin ligands) and 2:1 side-by-side binding, with various DNA base pair sequences and show excellent agreement with experimental results from X-ray crystallography, NMR, and gel-based footprinting.

  19. Structure of the DNA-binding and RNA-polymerase-binding region of transcription antitermination factor λQ.

    Science.gov (United States)

    Vorobiev, Sergey M; Gensler, Yocheved; Vahedian-Movahed, Hanif; Seetharaman, Jayaraman; Su, Min; Huang, Janet Y; Xiao, Rong; Kornhaber, Gregory; Montelione, Gaetano T; Tong, Liang; Ebright, Richard H; Nickels, Bryce E

    2014-03-04

    The bacteriophage λ Q protein is a transcription antitermination factor that controls expression of the phage late genes as a stable component of the transcription elongation complex. To join the elongation complex, λQ binds a specific DNA sequence element and interacts with RNA polymerase that is paused during early elongation. λQ binds to the paused early-elongation complex through interactions between λQ and two regions of RNA polymerase: region 4 of the σ(70) subunit and the flap region of the β subunit. We present the 2.1 Å resolution crystal structure of a portion of λQ containing determinants for interaction with DNA, interaction with region 4 of σ(70), and interaction with the β flap. The structure provides a framework for interpreting prior genetic and biochemical analysis and sets the stage for future structural studies to elucidate the mechanism by which λQ alters the functional properties of the transcription elongation complex. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Relaxase DNA Binding and Cleavage Are Two Distinguishable Steps in Conjugative DNA Processing That Involve Different Sequence Elements of the nic Site*

    Science.gov (United States)

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-01-01

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR2). Mutation analysis in the nic region indicated that recognition of the IR2 proximal arm and the nucleotides located between IR2 and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR2 cruciform and recognition of the distal IR2 arm and loop were not necessary for these reactions to take place. On the other hand, IR2 was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR2-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place. PMID:20061574

  1. Relaxase DNA binding and cleavage are two distinguishable steps in conjugative DNA processing that involve different sequence elements of the nic site.

    Science.gov (United States)

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-03-19

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR(2)). Mutation analysis in the nic region indicated that recognition of the IR(2) proximal arm and the nucleotides located between IR(2) and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR(2) cruciform and recognition of the distal IR(2) arm and loop were not necessary for these reactions to take place. On the other hand, IR(2) was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR(2)-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.

  2. The energetic contribution of induced electrostatic asymmetry to DNA bending by a site-specific protein.

    Science.gov (United States)

    Hancock, Stephen P; Hiller, David A; Perona, John J; Jen-Jacobson, Linda

    2011-02-18

    DNA bending can be promoted by reducing the net negative electrostatic potential around phosphates on one face of the DNA, such that electrostatic repulsion among phosphates on the opposite face drives bending toward the less negative surface. To provide the first assessment of energetic contribution to DNA bending when electrostatic asymmetry is induced by a site-specific DNA binding protein, we manipulated the electrostatics in the EcoRV endonuclease-DNA complex by mutation of cationic side chains that contact DNA phosphates and/or by replacement of a selected phosphate in each strand with uncharged methylphosphonate. Reducing the net negative charge at two symmetrically located phosphates on the concave DNA face contributes -2.3 kcal mol(-1) to -0.9 kcal mol(-1) (depending on position) to complex formation. In contrast, reducing negative charge on the opposing convex face produces a penalty of +1.3 kcal mol(-1). Förster resonance energy transfer experiments show that the extent of axial DNA bending (about 50°) is little affected in modified complexes, implying that modification affects the energetic cost but not the extent of DNA bending. Kinetic studies show that the favorable effects of induced electrostatic asymmetry on equilibrium binding derive primarily from a reduced rate of complex dissociation, suggesting stabilization of the specific complex between protein and markedly bent DNA. A smaller increase in the association rate may suggest that the DNA in the initial encounter complex is mildly bent. The data imply that protein-induced electrostatic asymmetry makes a significant contribution to DNA bending but is not itself sufficient to drive full bending in the specific EcoRV-DNA complex. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding.

    Science.gov (United States)

    Quax, P H; Grimbergen, J M; Lansink, M; Bakker, A H; Blatter, M C; Belin, D; van Hinsbergh, V W; Verheijen, J H

    1998-05-01

    Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no

  4. Comprehensive Identification and Annotation of Cell Type-Specific and Ubiquitous CTCF-Binding Sites in the Human Genome

    Science.gov (United States)

    Shu, Wenjie; Bo, Xiaochen; Wang, Shengqi

    2012-01-01

    Chromatin insulators are DNA elements that regulate the level of gene expression either by preventing gene silencing through the maintenance of heterochromatin boundaries or by preventing gene activation by blocking interactions between enhancers and promoters. CCCTC-binding factor (CTCF), a ubiquitously expressed 11-zinc-finger DNA-binding protein, is the only protein implicated in the establishment of insulators in vertebrates. While CTCF has been implicated in diverse regulatory functions, CTCF has only been studied in a limited number of cell types across human genome. Thus, it is not clear whether the identified cell type-specific differences in CTCF-binding sites are functionally significant. Here, we identify and characterize cell type-specific and ubiquitous CTCF-binding sites in the human genome across 38 cell types designated by the Encyclopedia of DNA Elements (ENCODE) consortium. These cell type-specific and ubiquitous CTCF-binding sites show uniquely versatile transcriptional functions and characteristic chromatin features. In addition, we confirm the insulator barrier function of CTCF-binding and explore the novel function of CTCF in DNA replication. These results represent a critical step toward the comprehensive and systematic understanding of CTCF-dependent insulators and their versatile roles in the human genome. PMID:22829947

  5. In silico engineering and optimization of Transcription Activator-Like Effectors and their derivatives for improved DNA binding predictions.

    KAUST Repository

    Piatek, Marek J.

    2015-12-01

    Transcription Activator-Like Effectors (TALEs) can be used as adaptable DNAbinding modules to create site-specific chimeric nucleases or synthetic transcriptional regulators. The central repeat domain mediates specific DNA binding via hypervariable repeat di-residues (RVDs). This DNA-Binding Domain can be engineered to bind preferentially to any user-selected DNA sequence if engineered appropriately. Therefore, TALEs and their derivatives have become indispensable molecular tools in site-specific manipulation of genes and genomes. This thesis revolves around two problems: in silico design and improved binding site prediction of TALEs. In the first part, a study is shown where TALEs are successfully designed in silico and validated in laboratory to yield the anticipated effects on selected genes. Software is developed to accompany the process of designing and prediction of binding sites. I expanded the functionality of the software to be used as a more generic set of tools for the design, target and offtarget searching. Part two contributes a method and associated toolkit developed to allow users to design in silico optimized synthetic TALEs with user-defined specificities for various experimental purposes. This method is based on a mutual relationship of three consecutive tandem repeats in the DNA-binding domain. This approach revealed positional and compositional bias behind the binding of TALEs to DNA. In conclusion, I developed methods, approaches, and software to enhance the functionality of synthetic TALEs, which should improve understanding of TALEs biology and will further advance genome-engineering applications in various organisms and cell types.

  6. Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Aimee; Higgins, Darren E.; Panne, Daniel; (Harvard-Med); (EMBL)

    2009-07-22

    The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

  7. 9-Hydroxyellipticine alters the conformation and DNA binding characteristics of mutated p53 protein.

    Science.gov (United States)

    Sugikawa, E; Tsunoda, S; Nakanishi, N; Ohashi, M

    2001-01-01

    The tumor suppressor protein p53 is a phosphoprotein which shows growth and transformation suppression functions. Mutational loss of p53 function is the most frequently detected genetic event in human cancers. We examined whether 9-hydroxyellipticine (9HE), a cytotoxic agent, affected the tertiary structure of mutant p53 and DNA binding characteristics. Although several types of p53 mutants were resistant to degradation by calpain, the p53 mutants treated with 9HE were markedly sensitive to calpain as well as wild-type p53. Furthermore, mutant p53 proteins isolated from 9HE-treated cells regained the ability to bind a wild-type-specific p53 DNA consensus sequence. Wild-type p53 proteins prepared from both untreated and 9HE-treated cells bound the p53 consensus sequence and were degradaded by calpain equally well. These results suggest that 9HE affects the tertiary structure of mutated p53, which results in the restoration of DNA binding characteristics.

  8. A Structural Analysis of DNA Binding by Myelin Transcription Factor 1 Double Zinc Fingers*

    Science.gov (United States)

    Gamsjaeger, Roland; O'Connell, Mitchell R.; Cubeddu, Liza; Shepherd, Nicholas E.; Lowry, Jason A.; Kwan, Ann H.; Vandevenne, Marylene; Swanton, Michael K.; Matthews, Jacqueline M.; Mackay, Joel P.

    2013-01-01

    Myelin transcription factor 1 (MyT1/NZF2), a member of the neural zinc-finger (NZF) protein family, is a transcription factor that plays a central role in the developing central nervous system. It has also recently been shown that, in combination with two other transcription factors, the highly similar paralog MyT1L is able to direct the differentiation of murine and human stem cells into functional neurons. MyT1 contains seven zinc fingers (ZFs) that are highly conserved throughout the protein and throughout the NZF family. We recently presented a model for the interaction of the fifth ZF of MyT1 with a DNA sequence derived from the promoter of the retinoic acid receptor (RARE) gene. Here, we have used NMR spectroscopy, in combination with surface plasmon resonance and data-driven molecular docking, to delineate the mechanism of DNA binding for double ZF polypeptides derived from MyT1. Our data indicate that a two-ZF unit interacts with the major groove of the entire RARE motif and that both fingers bind in an identical manner and with overall two-fold rotational symmetry, consistent with the palindromic nature of the target DNA. Several key residues located in one of the irregular loops of the ZFs are utilized to achieve specific binding. Analysis of the human and mouse genomes based on our structural data reveals three putative MyT1 target genes involved in neuronal development. PMID:24097990

  9. Single molecule characterization of DNA binding and strand displacement reactions on lithographic DNA origami microarrays.

    Science.gov (United States)

    Scheible, Max B; Pardatscher, Günther; Kuzyk, Anton; Simmel, Friedrich C

    2014-03-12

    The combination of molecular self-assembly based on the DNA origami technique with lithographic patterning enables the creation of hierarchically ordered nanosystems, in which single molecules are positioned at precise locations on multiple length scales. Based on a hybrid assembly protocol utilizing DNA self-assembly and electron-beam lithography on transparent glass substrates, we here demonstrate a DNA origami microarray, which is compatible with the requirements of single molecule fluorescence and super-resolution microscopy. The spatial arrangement allows for a simple and reliable identification of single molecule events and facilitates automated read-out and data analysis. As a specific application, we utilize the microarray to characterize the performance of DNA strand displacement reactions localized on the DNA origami structures. We find considerable variability within the array, which results both from structural variations and stochastic reaction dynamics prevalent at the single molecule level.

  10. The function of DNA binding protein nucleophosmin in AAV replication.

    Science.gov (United States)

    Satkunanathan, Stifani; Thorpe, Robin; Zhao, Yuan

    2017-10-01

    Adeno-associated viruses (AAV) contain minimal viral proteins necessary for their replication. During virus assembly, AAV acquire, inherently and submissively, various cellular proteins. Our previous studies identified the association of AAV vectors with the DNA binding protein nucleophosmin (NPM1). Nucleophosmin has been reported to enhance AAV infection by mobilizing AAV capsids into and out of the nucleolus, indicating the importance of NPM1 in the AAV life cycle; however the role of NPM1 in AAV production remains unknown. In this study, we systematically investigated NPM1 function on AAV production using NPM1 knockdown cells and revealing for the first time the presence of G-quadruplex DNA sequences (GQRS) in the AAV genome, the synergistic NPM1-GQRS function in AAV production and the significant enhancement of NPM1 gene knockdown on AAV vector production. Understanding the role of cellular proteins in the AAV life cycle will greatly facilitate high titre production of AAV vectors for clinical use. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  11. RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins

    Science.gov (United States)

    Lambert, Nicole; Robertson, Alex; Jangi, Mohini; McGeary, Sean; Sharp, Phillip A.; Burge, Christopher B.

    2014-01-01

    Summary Specific protein-RNA interactions guide post-transcriptional gene regulation. Here we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1 and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity. PMID:24837674

  12. Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

    Science.gov (United States)

    Das, Theerthankar; Kutty, Samuel K.; Tavallaie, Roya; Ibugo, Amaye I.; Panchompoo, Janjira; Sehar, Shama; Aldous, Leigh; Yeung, Amanda W. S.; Thomas, Shane R.; Kumar, Naresh; Gooding, J. Justin; Manefield, Mike

    2015-01-01

    Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation. PMID:25669133

  13. Immobilized sialyloligo-macroligand and its protein binding specificity.

    Science.gov (United States)

    Narla, Satya Nandana; Sun, Xue-Long

    2012-05-14

    We report a chemoenzymatic synthesis of chain-end functionalized sialyllactose-containing glycopolymers with different linkages and their oriented immobilization for glycoarray and SPR-based glyco-biosensor applications. Specifically, O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were synthesized by enzymatic α2,3- and α2,6-sialylation of a lactose-containing glycopolymer that was synthesized by cyanoxyl-mediated free radical polymerization. (1)H NMR showed almost quantitative α2,3- and α2,6-sialylation. The O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were printed onto amine-functionalized glass slides via isourea bond formation for glycoarray formation. Specific protein binding activity of the arrays was confirmed with α2,3- and α2,6-sialyl specific binding lectins together with inhibition assays. Further, immobilizing O-cyanate chain-end functionalized sialyllactose-containing glycopolymers onto amine-modified SPR chip via isourea bond formation afforded SPR-based glyco-biosensor, which showed specific binding activity for lectins and influenza viral hemagglutinins (HA). These sialyloligo-macroligand derived glycoarray and SPR-based glyco-biosensor are closely to mimic 3D nature presentation of sialyloligosaccharides and will provide important high-throughput tools for virus diagnosis and potential antiviral drug candidates screening applications.

  14. DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein.

    Science.gov (United States)

    Duan, Ming-Rui; Nan, Jie; Liang, Yu-He; Mao, Peng; Lu, Lu; Li, Lanfen; Wei, Chunhong; Lai, Luhua; Li, Yi; Su, Xiao-Dong

    2007-01-01

    WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 A resolution has revealed that this domain is composed of a globular structure with five beta strands, forming an antiparallel beta-sheet. A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at beta2 and beta3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.

  15. Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

    Directory of Open Access Journals (Sweden)

    Maher A Noureddine

    2009-05-01

    Full Text Available The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs. Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

  16. Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

    Directory of Open Access Journals (Sweden)

    Long Qian

    2016-10-01

    Full Text Available The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

  17. Mutagenesis of the HMGB (high-mobility group B) protein Cmb1 (cytosine-mismatch binding 1) of Schizosaccharomyces pombe: effects on recognition of DNA mismatches and damage.

    OpenAIRE

    Kunz, Christophe; Zurbriggen, Karin; Fleck, Oliver

    2003-01-01

    Cmb1 (cytosine-mismatch binding 1) is a high-mobility group (HMG) protein of Schizosaccharomyces pombe, which consists of 223 amino acids and has a single HMG domain at the C-terminal end. We have created several mutant and deletion forms of the Cmb1 protein and studied the effects on general DNA binding and specific binding to DNA mismatches and damaged DNA. Cmb1Delta41 (i.e. Cmb1 from which the 41 N-terminal amino acids have been deleted) bound specifically to cytosine-containing mismatches...

  18. DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

    Energy Technology Data Exchange (ETDEWEB)

    Lafaye, Céline; Barbier, Ewa; Miscioscia, Audrey; Saint-Pierre, Christine [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Kraut, Alexandra; Couté, Yohann [Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S_1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France); Plo, Isabelle [INSERM, U1009, Institut Gustave Roussy, Université Paris 11, 114 rue Edouard Vaillant, Villejuif F-94805 (France); Gasparutto, Didier; Ravanat, Jean-Luc [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Breton, Jean, E-mail: jean.breton@cea.fr [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France)

    2014-03-28

    Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.

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

    Science.gov (United States)

    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.

  20. Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

    DEFF Research Database (Denmark)

    Izvolsky, K I; Demidov, V V; Nielsen, P E

    2000-01-01

    I restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate...... DNA duplexes in a virtually sequence-unrestricted manner....

  1. An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.

    Science.gov (United States)

    Perez-Arnaiz, Patricia; Kaplan, Daniel L

    2016-11-20

    Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. The nucleoid protein Dps binds genomic DNA of Escherichia coli in a non-random manner

    Science.gov (United States)

    Kondrashov, F. A.; Toshchakov, S. V.; Dominova, I.; Shvyreva, U. S.; Vrublevskaya, V. V.; Morenkov, O. S.; Panyukov, V. V.

    2017-01-01

    Dps is a multifunctional homododecameric protein that oxidizes Fe2+ ions accumulating them in the form of Fe2O3 within its protein cavity, interacts with DNA tightly condensing bacterial nucleoid upon starvation and performs some other functions. During the last two decades from discovery of this protein, its ferroxidase activity became rather well studied, but the mechanism of Dps interaction with DNA still remains enigmatic. The crucial role of lysine residues in the unstructured N-terminal tails led to the conventional point of view that Dps binds DNA without sequence or structural specificity. However, deletion of dps changed the profile of proteins in starved cells, SELEX screen revealed genomic regions preferentially bound in vitro and certain affinity of Dps for artificial branched molecules was detected by atomic force microscopy. Here we report a non-random distribution of Dps binding sites across the bacterial chromosome in exponentially growing cells and show their enrichment with inverted repeats prone to form secondary structures. We found that the Dps-bound regions overlap with sites occupied by other nucleoid proteins, and contain overrepresented motifs typical for their consensus sequences. Of the two types of genomic domains with extensive protein occupancy, which can be highly expressed or transcriptionally silent only those that are enriched with RNA polymerase molecules were preferentially occupied by Dps. In the dps-null mutant we, therefore, observed a differentially altered expression of several targeted genes and found suppressed transcription from the dps promoter. In most cases this can be explained by the relieved interference with Dps for nucleoid proteins exploiting sequence-specific modes of DNA binding. Thus, protecting bacterial cells from different stresses during exponential growth, Dps can modulate transcriptional integrity of the bacterial chromosome hampering RNA biosynthesis from some genes via competition with RNA polymerase

  3. Characterization of DNA binding sites of the ComE response regulator from Streptococcus mutans.

    Science.gov (United States)

    Hung, David C I; Downey, Jennifer S; Ayala, Eduardo A; Kreth, Jens; Mair, Richard; Senadheera, Dilani B; Qi, Fengxia; Cvitkovitch, Dennis G; Shi, Wenyuan; Goodman, Steven D

    2011-07-01

    In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.

  4. De-novo protein function prediction using DNA binding and RNA binding proteins as a test case.

    Science.gov (United States)

    Peled, Sapir; Leiderman, Olga; Charar, Rotem; Efroni, Gilat; Shav-Tal, Yaron; Ofran, Yanay

    2016-11-21

    Of the currently identified protein sequences, 99.6% have never been observed in the laboratory as proteins and their molecular function has not been established experimentally. Predicting the function of such proteins relies mostly on annotated homologs. However, this has resulted in some erroneous annotations, and many proteins have no annotated homologs. Here we propose a de-novo function prediction approach based on identifying biophysical features that underlie function. Using our approach, we discover DNA and RNA binding proteins that cannot be identified based on homology and validate these predictions experimentally. For example, FGF14, which belongs to a family of secreted growth factors was predicted to bind DNA. We verify this experimentally and also show that FGF14 is localized to the nucleus. Mutating the predicted binding site on FGF14 abrogated DNA binding. These results demonstrate the feasibility of automated de-novo function prediction based on identifying function-related biophysical features.

  5. Cell-specific DNA methylation patterns of retina-specific genes.

    Directory of Open Access Journals (Sweden)

    Shannath L Merbs

    Full Text Available Many studies have demonstrated that epigenetic mechanisms are important in the regulation of gene expression during embryogenesis, gametogenesis, and other forms of tissue-specific gene regulation. We sought to explore the possible role of epigenetics, specifically DNA methylation, in the establishment and maintenance of cell type-restricted gene expression in the retina. To assess the relationship between DNA methylation status and expression level of retinal genes, bisulfite sequence analysis of the 1000 bp region around the transcription start sites (TSS of representative rod and cone photoreceptor-specific genes and gene expression analysis were performed in the WERI and Y79 human retinoblastoma cell lines. Next, the homologous genes in mouse were bisulfite sequenced in the retina and in non-expressing tissues. Finally, bisulfite sequencing was performed on isolated photoreceptor and non-photoreceptor retinal cells isolated by laser capture microdissection. Differential methylation of rhodopsin (RHO, retinal binding protein 3 (RBP3, IRBP cone opsin, short-wave-sensitive (OPN1SW, cone opsin, middle-wave-sensitive (OPN1MW, and cone opsin, long-wave-sensitive (OPN1LW was found in the retinoblastoma cell lines that inversely correlated with gene expression levels. Similarly, we found tissue-specific hypomethylation of the promoter region of Rho and Rbp3 in mouse retina as compared to non-expressing tissues, and also observed hypomethylation of retinal-expressed microRNAs. The Rho and Rbp3 promoter regions were unmethylated in expressing photoreceptor cells and methylated in non-expressing, non-photoreceptor cells from the inner nuclear layer. A third regional hypomethylation pattern of photoreceptor-specific genes was seen in a subpopulation of non-expressing photoreceptors (Rho in cones from the Nrl -/- mouse and Opn1sw in rods. These results demonstrate that a number of photoreceptor-specific genes have cell-specific differential DNA

  6. Defining the plasticity of transcription factor binding sites by Deconstructing DNA consensus sequences: the PhoP-binding sites among gamma/enterobacteria.

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    Oscar Harari

    2010-07-01

    Full Text Available Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg(2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs using a machine learning method inspired by the "Divide & Conquer" strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target

  7. DNA binding of centromere protein C (CENPC is stabilized by single-stranded RNA.

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    Yaqing Du

    2010-02-01

    Full Text Available Centromeres are the attachment points between the genome and the cytoskeleton: centromeres bind to kinetochores, which in turn bind to spindles and move chromosomes. Paradoxically, the DNA sequence of centromeres has little or no role in perpetuating kinetochores. As such they are striking examples of genetic information being transmitted in a manner that is independent of DNA sequence (epigenetically. It has been found that RNA transcribed from centromeres remains bound within the kinetochore region, and this local population of RNA is thought to be part of the epigenetic marking system. Here we carried out a genetic and biochemical study of maize CENPC, a key inner kinetochore protein. We show that DNA binding is conferred by a localized region 122 amino acids long, and that the DNA-binding reaction is exquisitely sensitive to single-stranded RNA. Long, single-stranded nucleic acids strongly promote the binding of CENPC to DNA, and the types of RNAs that stabilize DNA binding match in size and character the RNAs present on kinetochores in vivo. Removal or replacement of the binding module with HIV integrase binding domain causes a partial delocalization of CENPC in vivo. The data suggest that centromeric RNA helps to recruit CENPC to the inner kinetochore by altering its DNA binding characteristics.

  8. Roles of the human Rad51 L1 and L2 loops in DNA binding.

    Science.gov (United States)

    Matsuo, Yusuke; Sakane, Isao; Takizawa, Yoshimasa; Takahashi, Masayuki; Kurumizaka, Hitoshi

    2006-07-01

    The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.

  9. Structural basis for sequence-specific DNA recognition by an Arabidopsis WRKY transcription factor.

    Science.gov (United States)

    Yamasaki, Kazuhiko; Kigawa, Takanori; Watanabe, Satoru; Inoue, Makoto; Yamasaki, Tomoko; Seki, Motoaki; Shinozaki, Kazuo; Yokoyama, Shigeyuki

    2012-03-02

    The WRKY family transcription factors regulate plant-specific reactions that are mostly related to biotic and abiotic stresses. They share the WRKY domain, which recognizes a DNA element (TTGAC(C/T)) termed the W-box, in target genes. Here, we determined the solution structure of the C-terminal WRKY domain of Arabidopsis WRKY4 in complex with the W-box DNA by NMR. A four-stranded β-sheet enters the major groove of DNA in an atypical mode termed the β-wedge, where the sheet is nearly perpendicular to the DNA helical axis. Residues in the conserved WRKYGQK motif contact DNA bases mainly through extensive apolar contacts with thymine methyl groups. The importance of these contacts was verified by substituting the relevant T bases with U and by surface plasmon resonance analyses of DNA binding.

  10. Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription.

    Science.gov (United States)

    Kamenova, Ivanka; Warfield, Linda; Hahn, Steven

    2014-08-01

    Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  11. Sequence-specific electron injection into DNA from an intermolecular electron donor.

    Science.gov (United States)

    Morinaga, Hironobu; Takenaka, Tomohiro; Hashiya, Fumitaka; Kizaki, Seiichiro; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

    2013-04-01

    Electron transfer in DNA has been intensively studied to elucidate its biological roles and for applications in bottom-up DNA nanotechnology. Recently, mechanisms of electron transfer to DNA have been investigated; however, most of the systems designed are intramolecular. Here, we synthesized pyrene-conjugated pyrrole-imidazole polyamides (PPIs) to achieve sequence-specific electron injection into DNA in an intermolecular fashion. Electron injection from PPIs into DNA was detected using 5-bromouracil as an electron acceptor. Twelve different 5-bromouracil-containing oligomers were synthesized to examine the electron-injection ability of PPI. Product analysis demonstrated that the electron transfer from PPIs was localized in a range of 8 bp from the binding site of the PPIs. These results demonstrate that PPIs can be a useful tool for sequence-specific electron injection.

  12. Single-molecule kinetic analysis of HP1-chromatin binding reveals a dynamic network of histone modification and DNA interactions.

    Science.gov (United States)

    Bryan, Louise C; Weilandt, Daniel R; Bachmann, Andreas L; Kilic, Sinan; Lechner, Carolin C; Odermatt, Pascal D; Fantner, Georg E; Georgeon, Sandrine; Hantschel, Oliver; Hatzimanikatis, Vassily; Fierz, Beat

    2017-10-13

    Chromatin recruitment of effector proteins involved in gene regulation depends on multivalent interaction with histone post-translational modifications (PTMs) and structural features of the chromatin fiber. Due to the complex interactions involved, it is currently not understood how effectors dynamically sample the chromatin landscape. Here, we dissect the dynamic chromatin interactions of a family of multivalent effectors, heterochromatin protein 1 (HP1) proteins, using single-molecule fluorescence imaging and computational modeling. We show that the three human HP1 isoforms are recruited and retained on chromatin by a dynamic exchange between histone PTM and DNA bound states. These interactions depend on local chromatin structure, the HP1 isoforms as well as on PTMs on HP1 itself. Of the HP1 isoforms, HP1α exhibits the longest residence times and fastest binding rates due to DNA interactions in addition to PTM binding. HP1α phosphorylation further increases chromatin retention through strengthening of multivalency while reducing DNA binding. As DNA binding in combination with specific PTM recognition is found in many chromatin effectors, we propose a general dynamic capture mechanism for effector recruitment. Multiple weak protein and DNA interactions result in a multivalent interaction network that targets effectors to a specific chromatin modification state, where their activity is required. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators.

    Science.gov (United States)

    Perera, Inoka C; Grove, Anne

    2010-10-01

    Bacteria and archaea encode members of the large multiple antibiotic resistance regulator (MarR) family of transcriptional regulators. Generally, MarR homologs regulate activity of genes involved in antibiotic resistance, stress responses, virulence or catabolism of aromatic compounds. They constitute a diverse group of transcriptional regulators that includes both repressors and activators, and the conventional mode of regulation entails a genetic locus in which the MarR homolog and a gene under its regulation are encoded divergently; binding of the MarR homolog to the intergenic region typically represses transcription of both genes, while binding of a specific ligand to the transcription factor results in attenuated DNA binding and hence activated gene expression. For many homologs, the natural ligand is unknown. Crystal structures reveal a common architecture with a characteristic winged helix domain for DNA binding, and recent structural information of homologs solved both in the absence and presence of their respective ligands, as well as biochemical data, is finally converging to illuminate the mechanisms by which ligand-binding causes attenuated DNA binding. As MarR homologs regulate pathways that are critical to bacterial physiology, including virulence, a molecular understanding of mechanisms by which ligands affect a regulation of gene activity is essential. Specifying the position of ligand-binding pockets further has the potential to aid in identifying the ligands for MarR homologs for which the ligand remains unknown.

  14. Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA.

    Science.gov (United States)

    Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan; Poulos, Thomas L; Champion, Paul M

    2012-06-22

    Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in the CooA proteins relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases, and the distribution of the conformations available in the heme domain becomes restricted. The kinetic studies also offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role.

  15. Effect of DNA Binding on Geminate CO Recombination Kinetics in CO-sensing Transcription Factor CooA*

    Science.gov (United States)

    Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan; Poulos, Thomas L.; Champion, Paul M.

    2012-01-01

    Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in the CooA proteins relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases, and the distribution of the conformations available in the heme domain becomes restricted. The kinetic studies also offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role. PMID:22544803

  16. Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs.

    Science.gov (United States)

    Ma, Yunmei; Lieber, Michael R

    2002-03-29

    The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK(cs), to that end. The Ku.DNA-PK(cs) complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP(6)) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP(6) bound to DNA-PK(cs) (not to Ku). Here we clearly show that, in fact, IP(6) associates not with DNA-PK(cs), but rather with Ku. Furthermore, the binding of DNA ends and IP(6) to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.

  17. Human single-stranded DNA binding proteins are essential for maintaining genomic stability

    Science.gov (United States)

    2013-01-01

    The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance. PMID:23548139

  18. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    Science.gov (United States)

    Ray, Sujay

    observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Finally, we studied another protein-GQ system where a protein complex works synergistically with a GQ to suppress DNA damage signals by preventing RPA to bind to telomeric DNA. Human telomeres that terminate with a single-stranded 3' G-overhang can be recognized as a DNA damage site by RPA. The protection of telomere-1 (POT1) and POT1-interacting protein (TPP1) heterodimer, binds specifically to telomeric DNA and protects it against RPA binding. Using model telomeric DNA, we studied the competition between POT1/TPP1 and RPA to access telomeric GQs in vitro. Under physiological salt and pH conditions, POT1/TPP1 stably load to a minimal DNA sequence adjacent to a folded GQ and unfolds the anti-parallel GQ as the parallel conformation remains folded. We showed that GQ formation of telomeres enhances the ability of POT1/TPP1 to block RPA's access to telomeres by two orders of magnitude and contributes to suppress DNA damage signals.

  19. High-Affinity LNA-DNA Mixmer Probes for Detection of Chromosome-Specific Polymorphisms of 5S rDNA Repeats in Arabidopsis thaliana.

    Science.gov (United States)

    Simon, Lauriane; Probst, Aline V

    2018-01-01

    Fluorescence in situ hybridization is a standard technique to visualize specific DNA sequences by hybridization with fluorescent probes and, most commonly, relies on DNA probes generated by nick translation. In this chapter, we describe the use of directly labeled LNA-DNA mixmer probes for the rapid detection of repetitive sequences on Arabidopsis thaliana nuclei spreads. We further demonstrate that due to the high thermal stability of the heteroduplexes and the resulting elevated binding affinity of LNA-DNA mixmer probes for their target DNA, these probes can be used to discriminate between repetitive sequences differing by only a few single nucleotide polymorphisms.

  20. Fibronectin inhibits cytokine production induced by CpG DNA in macrophages without direct binding to DNA.

    Science.gov (United States)

    Yoshida, Hiroyuki; Nishikawa, Makiya; Yasuda, Sachiyo; Toyota, Hiroyasu; Kiyota, Tsuyoshi; Takahashi, Yuki; Takakura, Yoshinobu

    2012-10-01

    Fibronectin (FN) is known to have four DNA-binding domains although their physiological significance is unknown. Primary murine peritoneal macrophages have been shown to exhibit markedly lower responsiveness to CpG motif-replete plasmid DNA (pDNA), Toll-like receptor-9 (TLR9) ligand, compared with murine macrophage-like cell lines. The present study was conducted to examine whether FN having DNA-binding domains is involved in this phenomenon. The expression of FN was significantly higher in primary macrophages than in a macrophage-like cell line, RAW264.7, suggesting that abundant FN might suppress the responsiveness in the primary macrophages. However, electrophoretic analysis revealed that FN did not bind to pDNA in the presence of a physiological concentration of divalent cations. Surprisingly, marked tumor necrosis factor - (TNF-)α production from murine macrophages upon CpG DNA stimulation was significantly reduced by exogenously added FN in a concentration-dependent manner but not by BSA, laminin or collagen. FN did not affect apparent pDNA uptake by the cells. Moreover, FN reduced TNF-α production induced by polyI:C (TLR3 ligand), and imiquimod (TLR7 ligand), but not by LPS (TLR4 ligand), or a non-CpG pDNA/cationic liposome complex. The confocal microscopic study showed that pDNA was co-localized with FN in the same intracellular compartment in RAW264.7, suggesting that FN inhibits cytokine signal transduction in the endosomal/lysosomal compartment. Taken together, the results of the present study has revealed, for the first time, a novel effect of FN whereby the glycoprotein modulates cytokine signal transduction via CpG-DNA/TLR9 interaction in macrophages without direct binding to DNA through its putative DNA-binding domains. Copyright © 2012 Elsevier Ltd. All rights reserved.

  1. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  2. The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence.

    Science.gov (United States)

    Li, Shijun; Tan, Min; Juillard, Franceline; Ponnusamy, Rajesh; Correia, Bruno; Simas, J Pedro; Carrondo, Maria A; McVey, Colin E; Kaye, Kenneth M

    2015-11-20

    Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Synthetic Nucleotides as Probes of DNA Polymerase Specificity

    Directory of Open Access Journals (Sweden)

    Jason M. Walsh

    2012-01-01

    Full Text Available The genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example, Escherichia coli DNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides.

  4. The structural basis of the kinetic mechanism of a gap-filling X-family DNA polymerase that binds Mg(2+)-dNTP before binding to DNA.

    Science.gov (United States)

    Nakane, Shuhei; Ishikawa, Hirohito; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

    2012-03-30

    DNA with single-nucleotide (1-nt) gaps can arise during various DNA processing events. These lesions are repaired by X-family DNA polymerases (PolXs) with high gap-filling activity. Some PolXs can bind productively to dNTPs in the absence of DNA and fill these 1-nt gaps. Although PolXs have a crucial role in efficient gap filling, currently, little is known of the kinetic and structural details of their productive dNTP binding. Here, we show that Thermus thermophilus HB8 PolX (ttPolX) had strong binding affinity for Mg(2+)-dNTPs in the absence of DNA and that it follows a Theorell-Chance (hit-and-run) mechanism with nucleotide binding first. Comparison of the intermediate crystal structures of ttPolX in a binary complex with dGTP and in a ternary complex with 1-nt gapped DNA and Mg(2+)-ddGTP revealed that the conformation of the incoming nucleotide depended on whether or not DNA was present. Furthermore, the Lys263 residue located between two guanosine conformations was essential to the strong binding affinity of the enzyme. The ability to bind to either syn-dNTP or anti-dNTP and the involvement of a Theorell-Chance mechanism are key aspects of the strong nucleotide-binding and efficient gap-filling activities of ttPolX. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Interactions between the R2R3-MYB transcription factor, AtMYB61, and target DNA binding sites.

    Directory of Open Access Journals (Sweden)

    Michael B Prouse

    Full Text Available Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing. The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators.

  6. Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

    2014-01-01

    The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies...... have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding...... yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells....

  7. Single-Molecule Studies of ssDNA-Binding Proteins Exchange.

    Science.gov (United States)

    Yang, Olivia; Ha, Taekjip

    2018-01-01

    Single-stranded DNA-binding protein (SSB) is important not only for the protection of single-stranded DNA (ssDNA) but also for the recruitment of other proteins for DNA replication, recombination, and repair. The interaction of SSB with ssDNA is highly dynamic as it exists as an intermediate during cellular processes that unwind dsDNA. It has been proposed that SSB redistributes itself among multiple ssDNA segments, but transient intermediates are difficult to observe in bulk experiments. We can use single-molecule FRET microscopy to observe intermediates of the transfer of a single Escherichia coli SSB from one ssDNA strand to another or exchange of one SSB for another on a single ssDNA in real time. This single-molecule approach can be further applicable to understand relative binding affinities and competitive dynamics for other SSBs and variants across various systems. © 2018 Elsevier Inc. All rights reserved.

  8. Statistical-mechanical lattice models for protein-DNA binding in chromatin

    CERN Document Server

    Teif, Vladimir B

    2010-01-01

    Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibriums measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quant...

  9. RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

    Science.gov (United States)

    Liu, Shaofeng; Xu, Zhiyun; Leng, He; Zheng, Pu; Yang, Jiayi; Chen, Kaifu; Feng, Jianxun; Li, Qing

    2017-01-27

    DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication. Copyright © 2017, American Association for the Advancement of Science.

  10. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

    Science.gov (United States)

    de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; Gomes da Silva, Lúcia Patrícia Bezerra; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Gois Ruiz, Ana Lucia Tasca; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

    2015-01-01

    In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties. PMID:26068233

  11. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

    Directory of Open Access Journals (Sweden)

    Sinara Mônica Vitalino de Almeida

    2015-06-01

    Full Text Available In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide derivatives (3a–h were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z-2-(acridin-9-ylmethylene-N- (4-chlorophenyl hydrazinecarbothioamide (3f, while the most active compound in antiproliferative assay was (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide (3a. There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

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

    Indian Academy of Sciences (India)

    activity, anti-cancer and anti-microbial activities.9 12. The design of small complexes that bind and react with DNA is one of the interesting activities of bioinor- ganic chemist. Hence it is important to investigate more efficient drugs that target DNA. In recent years,13 15 there is some interest towards synthesis, DNA interac-.

  13. cDNA cloning and characterization of a mannose-binding lectin from ...

    Indian Academy of Sciences (India)

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a ...

  14. Deciphering the intercalative binding modes of benzoyl peroxide with calf thymus DNA.

    Science.gov (United States)

    Xia, Kaixin; Zhang, Guowen; Gong, Deming

    2017-09-01

    The binding of benzoyl peroxide (BPO), a flour brightener, with calf thymus DNA (ctDNA) was predicted by molecular simulation, and this were confirmed using multi-spectroscopic techniques and a chemometrics algorithm. The molecular docking result showed that BPO could insert into the base pairs of ctDNA, and the adenine bases were the preferential binding sites which were validated by the analysis of Fourier transform infrared spectra. The mode of binding of BPO with ctDNA was an intercalation as supported by the results from ctDNA melting and viscosity measurements, iodide quenching effects and competitive binding investigations. The circular dichroism and DNA cleavage assays indicated that BPO induced a conformational change from B-like DNA structure towards to A-like form, but did not lead to significant damage in the DNA. The complexation was driven mainly by hydrogen bonds and hydrophobic interactions. Moreover, the ultraviolet-visible (UV-vis) spectroscopic data matrix was resolved by a multivariate curve resolution-alternating least-squares algorithm. The equilibrium concentration profiles for the components (BPO, ctDNA and BPO-ctDNA complex) were extracted from the highly overlapping composite response to quantitatively monitor the BPO-ctDNA interaction. This study has provided insights into the mechanism of the interaction of BPO with ctDNA and potential hazards of the food additive. Copyright © 2017 John Wiley & Sons, Ltd.

  15. Duplex structural differences and not 2′-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA

    OpenAIRE

    Olimpo, Jeffrey T.; DeStefano, Jeffrey J.

    2010-01-01

    Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA–DNA versus DNA–DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3′ recessed DNA base) are required for stable binding to RNA–DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA–DNA. Replacing 2′- hydroxyls on pivotal RNA bases with 2′-O-methyls did not affect stability, indicating that interactions between hy...

  16. Regulation of Bacterial DNA Packaging in Early Stationary Phase by Competitive DNA Binding of Dps and IHF.

    Science.gov (United States)

    Lee, Sin Yi; Lim, Ci Ji; Dröge, Peter; Yan, Jie

    2015-12-14

    The bacterial nucleoid, a bacterial genome packed by nucleoid binding proteins, forms the physical basis for cellular processes such as gene transcription and DNA replication. Bacteria need to dynamically modulate their nucleoid structures at different growth phases and in response to environmental changes. At the nutrients deficient stationary phase, DNA-binding proteins from starved cells (Dps) and Integration host factors (IHF) are the two most abundant nucleoid associated proteins in E. coli. Yet, it remains unclear how the nucleoid architecture is controlled by the interplay between these two proteins, as well as the nucleoid's response to environmental changes. This question is addressed here using single DNA manipulation approach. Our results reveal that the two proteins are differentially selected for DNA binding, which can be tuned by changing environmental factors over physiological ranges including KCl (50-300 mM), MgCl2 (0-10 mM), pH (6.5-8.5) and temperature (23-37 °C). Increasing pH and MgCl2 concentrations switch from Dps-binding to IHF-binding. Stable Dps-DNA and IHF-DNA complexes are insensitive to temperature changes for the range tested. The environment dependent selection between IHF and Dps results in different physical organizations of DNA. Overall, our findings provide important insights into E. coli nucleoid architecture.

  17. Structural insights into single-stranded DNA binding and cleavage by F factor TraI.

    Science.gov (United States)

    Datta, Saumen; Larkin, Chris; Schildbach, Joel F

    2003-11-01

    Conjugative plasmid transfer between bacteria disseminates antibiotic resistance and diversifies prokaryotic genomes. Relaxases, proteins essential for conjugation, cleave one plasmid strand sequence specifically prior to transfer. Cleavage occurs through a Mg(2+)-dependent transesterification involving a tyrosyl hydroxyl and a DNA phosphate. The structure of the F plasmid TraI relaxase domain, described here, is a five-strand beta sheet flanked by alpha helices. The protein resembles replication initiator protein AAV-5 Rep but is circularly permuted, yielding a different topology. The beta sheet forms a binding cleft lined with neutral, nonaromatic residues, unlike most single-stranded DNA binding proteins which use aromatic and charged residues. The cleft contains depressions, suggesting base recognition occurs in a knob-into-hole fashion. Unlike most nucleases, three histidines but no acidic residues coordinate a Mg(2+) located near the catalytic tyrosine. The full positive charge on the Mg(2+) and the architecture of the active site suggest multiple roles for Mg(2+) in DNA cleavage.

  18. iDNA-Prot|dis: identifying DNA-binding proteins by incorporating amino acid distance-pairs and reduced alphabet profile into the general pseudo amino acid composition.

    Science.gov (United States)

    Liu, Bin; Xu, Jinghao; Lan, Xun; Xu, Ruifeng; Zhou, Jiyun; Wang, Xiaolong; Chou, Kuo-Chen

    2014-01-01

    Playing crucial roles in various cellular processes, such as recognition of specific nucleotide sequences, regulation of transcription, and regulation of gene expression, DNA-binding proteins are essential ingredients for both eukaryotic and prokaryotic proteomes. With the avalanche of protein sequences generated in the postgenomic age, it is a critical challenge to develop automated methods for accurate and rapidly identifying DNA-binding proteins based on their sequence information alone. Here, a novel predictor, called "iDNA-Prot|dis", was established by incorporating the amino acid distance-pair coupling information and the amino acid reduced alphabet profile into the general pseudo amino acid composition (PseAAC) vector. The former can capture the characteristics of DNA-binding proteins so as to enhance its prediction quality, while the latter can reduce the dimension of PseAAC vector so as to speed up its prediction process. It was observed by the rigorous jackknife and independent dataset tests that the new predictor outperformed the existing predictors for the same purpose. As a user-friendly web-server, iDNA-Prot|dis is accessible to the public at http://bioinformatics.hitsz.edu.cn/iDNA-Prot_dis/. Moreover, for the convenience of the vast majority of experimental scientists, a step-by-step protocol guide is provided on how to use the web-server to get their desired results without the need to follow the complicated mathematic equations that are presented in this paper just for the integrity of its developing process. It is anticipated that the iDNA-Prot|dis predictor may become a useful high throughput tool for large-scale analysis of DNA-binding proteins, or at the very least, play a complementary role to the existing predictors in this regard.

  19. Specificity and function of Archaeal DNA replication initiator proteins

    DEFF Research Database (Denmark)

    Samson, Rachel Y.; Xu, Yanqun; Gadelha, Catarina

    2013-01-01

    Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins...... to investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels...... the protein's structure rather than that of the DNA template....

  20. Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay.

    Science.gov (United States)

    Hendrickson, W; Schleif, R F

    1984-09-25

    DNA binding properties of the proteins required for induction of the Escherichia coli L-arabinose operon were measured using a polyacrylamide gel electrophoresis assay. The mechanisms of induction and repression were studied by observing the multiple interactions of RNA polymerase, cyclic AMP receptor protein and araC protein with short DNA fragments containing either the araC or araBAD promoter regions. These studies show that binding of araC protein to the operator site, araO1, directly blocks RNA polymerase binding at the araC promoter, pC. We find that cyclic AMP receptor protein and araC protein do not bind co-operatively at their respective sites to linear DNA fragments containing the pBAD promoter. Nevertheless, both these positive effectors must be present on the DNA to stimulate binding of RNA polymerase. Additionally, binding of the proteins to the DNA is not sufficient; araC protein must also be in the inducing state, for RNA polymerase to bind. Equilibrium binding constraints and kinetics were determined for araC protein binding to the araI and the araO1 sites. In the presence of inducer, L-arabinose, araC protein binds with equal affinity to DNA fragments containing either of these sites. In the presence of anti-inducer, D-fucose, the affinity for both sites is reduced 40-fold. The apparent equilibrium binding constants for both states of the protein vary in parallel with the buffer salt concentration. This result suggests that the inducing and repressing forms of araC protein displace a similar number of cations upon binding DNA.

  1. Molecular modeling and spectroscopic studies of semustine binding with DNA and its comparison with lomustine-DNA adduct formation.

    Science.gov (United States)

    Agarwal, Shweta; Chadha, Deepti; Mehrotra, Ranjana

    2015-01-01

    Chloroethyl nitrosoureas constitute an important family of cancer chemotherapeutic agents, used in the treatment of various types of cancer. They exert antitumor activity by inducing DNA interstrand cross-links. Semustine, a chloroethyl nitrosourea, is a 4-methyl derivative of lomustine. There exist some interesting reports dealing with DNA-binding properties of chloroethyl nitrosoureas; however, underlying mechanism of cytotoxicity caused by semustine has not been precisely and completely delineated. The present work focuses on understanding semustine-DNA interaction to comprehend its anti-proliferative action at molecular level using various spectroscopic techniques. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy is used to determine the binding site of semustine on DNA. Conformational transition in DNA after semustine complexation is investigated using circular dichroism (CD) spectroscopy. Stability of semustine-DNA complexes is determined using absorption spectroscopy. Results of the present study demonstrate that semustine performs major-groove-directed DNA alkylation at guanine residues in an incubation-time-drug-concentration-dependent manner. CD spectral outcomes suggest partial transition of DNA from native B-conformation to C-form. Calculated binding constants (Ka) for semustine and lomustine interactions with DNA are 1.53 × 10(3) M(-1) and 8.12 × 10(3) M(-1), respectively. Moreover, molecular modeling simulation is performed to predict preferential binding orientation of semustine with DNA that corroborates well with spectral outcomes. Results based on comparative study of DNA-binding properties of semustine and lomustine, presented here, may establish a correlation between molecular structure and cytotoxicity of chloroethyl nitrosoureas that may be instrumental in the designing and synthesis of new nitrosourea therapeutics possessing better efficacy and fewer side effects.

  2. In vitro selection of DNA aptamers binding pesticide fluoroacetamide.

    Science.gov (United States)

    Cao, Fangqi; Lu, Xinwei; Hu, Xiaolong; Zhang, Yurong; Zeng, Libo; Chen, Liankang; Sun, Meiqi

    2016-05-01

    Fluoroacetamide (Mw = 77.06) is a lethal rodenticide to humans and animals which is still frequently abused in food storage somewhere in China. The production of antibodies for fluoroacetamide is difficult due to its high toxicity to animals, which limits the application of immunoassay method in poison detection. In this work, aptamers targeting N-fluoroacetyl glycine as an analog of fluoroacetamide were selected by a specific systematic evolution of ligands by exponential enrichment (SELEX) strategy. The binding ability of the selected aptamers to fluoroacetamide was identified using surface plasmon resonance (SPR)-based assay. The estimated KD values in the low micromolar range showed a good affinity of these aptamers to the target. Our work verified that the SELEX strategy has the potential for developing aptamers targeted to small molecular toxicants and aptamers can be employed as new recognition elements instead of antibodies for poison detection.

  3. Structure and flexibility adaptation in nonspecific and specific protein-DNA complexes

    NARCIS (Netherlands)

    Kalodimos, Ch.; Biris, N.; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238; Levandoski, M.M.; Guennuegues, M.; Boelens, R.|info:eu-repo/dai/nl/070151407; Kaptein, R.|info:eu-repo/dai/nl/074334603

    2004-01-01

    Interaction of regulatory DNA binding proteins with their target sites is usually preceded by binding to nonspecific DNA. This speeds up the search for the target site by several orders of magnitude. We report the solution structure and dynamics of the complex of a dimeric lac repressor DNA binding

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

    Science.gov (United States)

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

    2009-12-01

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

  5. Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations.

    Science.gov (United States)

    Mi, Ran; Tu, Bao; Bai, Xiao-Ting; Chen, Jun; Ouyang, Yu; Hu, Yan-Jun

    2015-12-01

    Palmatine, an isoquinoline alkaloid, is an important medicinal herbal extract with diverse pharmacological and biological properties. In this work, spectroscopic and molecular modeling approaches were employed to reveal the interaction between palmatine and DNA isolated from herring sperm. The absorption spectra and iodide quenching results indicated that groove binding was the main binding mode of palmatine to DNA. Fluorescence studies indicated that the binding constant (K) of palmatine and DNA was ~ 10(4)L·mol(-1). The associated thermodynamic parameters, ΔG, ΔH, and ΔS, indicated that hydrogen bonds and van der Waals forces played major roles in the interaction. The effects of chemical denaturant, thermal denaturation and pH on the interaction were investigated and provided further support for the groove binding mode. In addition to experimental approaches, molecular modeling was conducted to verify binding pattern of palmatine-DNA. Copyright © 2015 John Wiley & Sons, Ltd.

  6. Unreplicated DNA in mitosis precludes condensin binding and chromosome condensation in S. cerevisiae

    Science.gov (United States)

    Dulev, Stanimir; Aragon, Luis; Strunnikov, Alexander

    2009-01-01

    Condensin is the core activity responsible for chromosome condensation in mitosis. In the yeast S. cerevisiae, condensin binding is enriched at the regions where DNA replication terminates. Therefore, we investigated whether DNA replication completion determines the condensin-binding proficiency of chromatin. In order to fulfill putative mitotic requirements for condensin activity we analyzed chromosome condensation and condensin binding to unreplicated chromosomes in mitosis. For this purpose we used pGAL:CDC6 cdc15-ts cells that are known to enter mitosis without DNA replication if CDC6 transcription is repressed prior to S-phase. Both the condensation of nucleolar chromatin and proper condensin targeting to rDNA sites failed when unreplicated chromosomes were driven in mitosis. We propose that the DNA replication results in structural and/or biochemical changes to replicated chromatin, which are required for two-phase condensin binding and proper chromosome condensation. PMID:18508626

  7. DNA binding affinity of a macrocyclic copper(II) complex: Spectroscopic and molecular docking studies.

    Science.gov (United States)

    Shahabadi, Nahid; Hakimi, Mohammad; Morovati, Teimoor; Fatahi, Navid

    2017-08-03

    The interaction of a novel macrocyclic copper(II) complex, ([CuL(ClO 4 ) 2 ] that L is 1,3,6,10,12,15-hexaazatricyclo[13.3.1.1 6,10 ]eicosane) with calf thymus DNA (ct-DNA) was investigated by various physicochemical techniques and molecular docking at simulated physiological conditions (pH = 7.4). The absorption spectra of the Cu(II) complex with ct-DNA showed a marked hyperchroism with 10 nm blue shift. The intrinsic binding constant (K b ) was determined as 1.25 × 10 4 M -1 , which is more in keeping with the groove binding with DNA. Furthermore, competitive fluorimetric studies with Hoechst33258 have shown that Cu(II) complex exhibits the ability to displace the ct-DNA-bound Hoechst33258 indicating that it binds to ct-DNA in strong competition with Hoechst33258 for the groove binding. Also, no change in the relative viscosity of ct-DNA and fluorescence intensity of ct-DNA-MB complex in the present of Cu(II) complex is another evidence to groove binding. 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 binding reaction. The experimental results were in agreement with the results obtained via molecular docking study.

  8. The identification of FANCD2 DNA binding domains reveals nuclear localization sequences.

    Science.gov (United States)

    Niraj, Joshi; Caron, Marie-Christine; Drapeau, Karine; Bérubé, Stéphanie; Guitton-Sert, Laure; Coulombe, Yan; Couturier, Anthony M; Masson, Jean-Yves

    2017-08-21

    Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Spectral analysis of naturally occurring methylxanthines (theophylline, theobromine and caffeine) binding with DNA.

    Science.gov (United States)

    Johnson, Irudayam Maria; Prakash, Halan; Prathiba, Jeyaguru; Raghunathan, Raghavachary; Malathi, Raghunathan

    2012-01-01

    Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR) spectroscopic methods, and especially monitored their binding affinity in the presence of Mg(2+) and during helix-coil transitions of DNA by temperature (T(m)) or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophylline = 3.5×10(3) M(-1), DNA-theobromine = 1.1×10(3) M(-1), and DNA-Caffeine = 3.8×10(3) M(-1). On the other hand T(m)/pH melting profiles showed 24-35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C) and phosphate group through hydrogen bond (H-bond) interaction. In the presence of Mg(2+), methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg(2+). The spectral analyses indicated the order of binding affinity as "caffeine≥theophylline>theobromine" to the native double helical DNA, and "theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions.

  10. Spectral analysis of naturally occurring methylxanthines (theophylline, theobromine and caffeine binding with DNA.

    Directory of Open Access Journals (Sweden)

    Irudayam Maria Johnson

    Full Text Available Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR spectroscopic methods, and especially monitored their binding affinity in the presence of Mg(2+ and during helix-coil transitions of DNA by temperature (T(m or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophylline = 3.5×10(3 M(-1, DNA-theobromine = 1.1×10(3 M(-1, and DNA-Caffeine = 3.8×10(3 M(-1. On the other hand T(m/pH melting profiles showed 24-35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C and phosphate group through hydrogen bond (H-bond interaction. In the presence of Mg(2+, methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg(2+. The spectral analyses indicated the order of binding affinity as "caffeine≥theophylline>theobromine" to the native double helical DNA, and "theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions.

  11. Spectral Analysis of Naturally Occurring Methylxanthines (Theophylline, Theobromine and Caffeine) Binding with DNA

    Science.gov (United States)

    Johnson, Irudayam Maria; Prakash, Halan; Prathiba, Jeyaguru; Raghunathan, Raghavachary; Malathi, Raghunathan

    2012-01-01

    Nucleic acids exist in a dynamic equilibrium with a number of molecules that constantly interact with them and regulate the cellular activities. The inherent nature of the structure and conformational integrity of these macromolecules can lead to altered biological activity through proper targeting of nucleic acids binding ligands or drug molecules. We studied the interaction of naturally occurring methylxanthines such as theophylline, theobromine and caffeine with DNA, using UV absorption and Fourier transform infrared (FTIR) spectroscopic methods, and especially monitored their binding affinity in the presence of Mg2+ and during helix-coil transitions of DNA by temperature (Tm) or pH melting profiles. The study indicates that all these molecules effectively bind to DNA in a dose dependent manner. The overall binding constants of DNA-theophylline = 3.5×103 M−1, DNA-theobromine = 1.1×103 M−1, and DNA-Caffeine = 3.8×103 M−1. On the other hand Tm/pH melting profiles showed 24–35% of enhanced binding activity of methylxanthines during helix-coil transitions of DNA rather than to its native double helical structure. The FTIR analysis divulged that theophylline, theobromine and caffeine interact with all the base pairs of DNA (A-T; G-C) and phosphate group through hydrogen bond (H-bond) interaction. In the presence of Mg2+, methylxanthines altered the structure of DNA from B to A-family. However, the B-family structure of DNA remained unaltered in DNA-methylxanthines complexes or in the absence of Mg2+. The spectral analyses indicated the order of binding affinity as “caffeine≥theophylline>theobromine” to the native double helical DNA, and “theophylline≥theobromine>caffeine to the denatured form of DNA and in the presence of divalent metal ions. PMID:23236361

  12. Analysis of sequence variation underlying tissue-specific transcription factor binding and gene expression.

    Science.gov (United States)

    Lower, Karen M; De Gobbi, Marco; Hughes, Jim R; Derry, Christopher J; Ayyub, Helena; Sloane-Stanley, Jacqueline A; Vernimmen, Douglas; Garrick, David; Gibbons, Richard J; Higgs, Douglas R

    2013-08-01

    Although mutations causing monogenic disorders most frequently lie within the affected gene, sequence variation in complex disorders is more commonly found in noncoding regions. Furthermore, recent genome- wide studies have shown that common DNA sequence variants in noncoding regions are associated with "normal" variation in gene expression resulting in cell-specific and/or allele-specific differences. The mechanism by which such sequence variation causes changes in gene expression is largely unknown. We have addressed this by studying natural variation in the binding of key transcription factors (TFs) in the well-defined, purified cell system of erythropoiesis. We have shown that common polymorphisms frequently directly perturb the binding sites of key TFs, and detailed analysis shows how this causes considerable (~10-fold) changes in expression from a single allele in a tissue-specific manner. We also show how a SNP, located at some distance from the recognized TF binding site, may affect the recruitment of a large multiprotein complex and alter the associated chromatin modification of the variant regulatory element. This study illustrates the principles by which common sequence variation may cause changes in tissue-specific gene expression, and suggests that such variation may underlie an individual's propensity to develop complex human genetic diseases. © 2013 WILEY PERIODICALS, INC.

  13. Conformation of nanoconfined DNA as a function of ATP, AMP, CTP, Mg2+, and dye binding

    Science.gov (United States)

    Roushan, Maedeh; Riehn, Robert

    2014-03-01

    DNA molecules stretch in nanochannels with a channel cross-section of 100x100 nm2, thereby allowing analysis by observation of a fluorescent dye. The length and configuration of DNA can be directly observed, and the effect of different DNA-binding proteins on DNA configuration can be studied. Recently, we reported on the ability of T4 ligase to transiently manipulate DNA as a function of ATP and magnesium exposure. In this process we have extensively probed the interactions of dyes and enzyme co-factors with DNA under nanoconfinement. We find negligible effects if DNA is visualized using groove-binding dyes such as DAPI. However, if an intercalating dye (YOYO-1) is used, we find a significant shortening of the DNA in the presence of ATP that we attribute to an interaction of dye and ATP (as well as AMP and CTP). We did not record a noticeable effect due to Mg2+.

  14. Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.

    Science.gov (United States)

    Hishiki, Asami; Hara, Kodai; Ikegaya, Yuzu; Yokoyama, Hideshi; Shimizu, Toshiyuki; Sato, Mamoru; Hashimoto, Hiroshi

    2015-05-22

    HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3'-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3'-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. A Comparative Structure/Function Analysis of Two Type IV Pilin DNA Receptors Defines a Novel Mode of DNA Binding.

    Science.gov (United States)

    Berry, Jamie-Lee; Xu, Yingqi; Ward, Philip N; Lea, Susan M; Matthews, Stephen J; Pelicic, Vladimir

    2016-06-07

    DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  16. Deep convolutional neural networks for pan-specific peptide-MHC class I binding prediction.

    Science.gov (United States)

    Han, Youngmahn; Kim, Dongsup

    2017-12-28

    Computational scanning of peptide candidates that bind to a specific major histocompatibility complex (MHC) can speed up the peptide-based vaccine development process and therefore various methods are being actively developed. Recently, machine-learning-based methods have generated successful results by training large amounts of experimental data. However, many machine learning-based methods are generally less sensitive in recognizing locally-clustered interactions, which can synergistically stabilize peptide binding. Deep convolutional neural network (DCNN) is a deep learning method inspired by visual recognition process of animal brain and it is known to be able to capture meaningful local patterns from 2D images. Once the peptide-MHC interactions can be encoded into image-like array(ILA) data, DCNN can be employed to build a predictive model for peptide-MHC binding prediction. In this study, we demonstrated that DCNN is able to not only reliably predict peptide-MHC binding, but also sensitively detect locally-clustered interactions. Nonapeptide-HLA-A and -B binding data were encoded into ILA data. A DCNN, as a pan-specific prediction model, was trained on the ILA data. The DCNN showed higher performance than other prediction tools for the latest benchmark datasets, which consist of 43 datasets for 15 HLA-A alleles and 25 datasets for 10 HLA-B alleles. In particular, the DCNN outperformed other tools for alleles belonging to the HLA-A3 supertype. The F1 scores of the DCNN were 0.86, 0.94, and 0.67 for HLA-A*31:01, HLA-A*03:01, and HLA-A*68:01 alleles, respectively, which were significantly higher than those of other tools. We found that the DCNN was able to recognize locally-clustered interactions that could synergistically stabilize peptide binding. We developed ConvMHC, a web server to provide user-friendly web interfaces for peptide-MHC class I binding predictions using the DCNN. ConvMHC web server can be accessible via http://jumong.kaist.ac.kr:8080/convmhc

  17. Strong physical constraints on sequence-specific target location by proteins on DNA molecules

    DEFF Research Database (Denmark)

    Flyvbjerg, H.; Keatch, S.A.; Dryden, D.T.F

    2006-01-01

    of the loading bay and allows an estimation of the size of the footprint on the DNA of the sequence-specific protein by assaying protein binding or function in the presence of increasing concentrations of non-specific ligand. Assaying for function gives an 'activity footprint'; the minimum length of DNA required...... for function rather than the more commonly measured physical footprint. Assaying the complex type I restriction enzyme, EcoKI, gives an activity footprint of similar to 66 bp for ATP hydrolysis and 300 bp for the DNA cleavage function which is intimately linked with translocation of DNA by EcoKI. Furthermore......Sequence-specific binding to DNA in the presence of competing non-sequence-specific ligands is a problem faced by proteins in all organisms. It is akin to the problem of parking a truck at a loading bay by the side of a road in the presence of cars parked at random along the road. Cars even...

  18. Evolutionary rates and patterns for human transcription factor binding sites derived from repetitive DNA

    Directory of Open Access Journals (Sweden)

    McDonald John F

    2008-05-01

    Full Text Available Abstract Background The majority of human non-protein-coding DNA is made up of repetitive sequences, mainly transposable elements (TEs. It is becoming increasingly apparent that many of these repetitive DNA sequence elements encode gene regulatory functions. This fact has important evolutionary implications, since repetitive DNA is the most dynamic part of the genome. We set out to assess the evolutionary rate and pattern of experimentally characterized human transcription factor binding sites (TFBS that are derived from repetitive versus non-repetitive DNA to test whether repeat-derived TFBS are in fact rapidly evolving. We also evaluated the position-specific patterns of variation among TFBS to look for signs of functional constraint on TFBS derived from repetitive and non-repetitive DNA. Results We found numerous experimentally characterized TFBS in the human genome, 7–10% of all mapped sites, which are derived from repetitive DNA sequences including simple sequence repeats (SSRs and TEs. TE-derived TFBS sequences are far less conserved between species than TFBS derived from SSRs and non-repetitive DNA. Despite their rapid evolution, several lines of evidence indicate that TE-derived TFBS are functionally constrained. First of all, ancient TE families, such as MIR and L2, are enriched for TFBS relative to younger families like Alu and L1. Secondly, functionally important positions in TE-derived TFBS, specifically those residues thought to physically interact with their cognate protein binding factors (TF, are more evolutionarily conserved than adjacent TFBS positions. Finally, TE-derived TFBS show position-specific patterns of sequence variation that are highly distinct from random patterns and similar to the variation seen for non-repeat derived sequences of the same TFBS. Conclusion The abundance of experimentally characterized human TFBS that are derived from repetitive DNA speaks to the substantial regulatory effects that this class of

  19. Overproduction, purification and structural characterization of the functional N-terminal DNA-binding domain of the fru repressor from Escherichia coli K-12.

    Science.gov (United States)

    Scarabel, M; Penin, F; Bonod-Bidaud, C; Nègre, D; Cozzone, A J; Cortay, J C

    1995-02-03

    A DNA fragment encoding the DNA-binding domain (amino acids 1-60) of the Escherichia coli fru transcriptional regulator was cloned into the pGEX-KT vector and expressed in frame with the fused gene encoding glutathione S-transferase. The fusion protein was purified to homogeneity by affinity chromatography on immobilized glutathione, and then cleaved with thrombin. After separation by a cation-exchange chromatography step, the DNA-binding domain exhibited proper folding, as shown by proton NMR analysis. Furthermore, it showed specific interaction with the operator region of the ace operon, as checked by gel retardation and DNA methylation-protection experiments.

  20. Improving specificity of DNA hybridization-based methods

    OpenAIRE

    Chalaya, Tatyana; Gogvadze, Elena; Buzdin, Anton; Kovalskaya, Elena; Sverdlov, Eugene D.

    2004-01-01

    Methods based on DNA reassociation in solution with the subsequent PCR amplification of certain hybrid molecules, such as coincidence cloning and subtractive hybridization, all suffer from a common imperfection: cross-hybridization between various types of paralogous repetitive DNA fragments. Although the situation can be slightly improved by the addition of repeat-specific competitor DNA into the hybridization mixture, the cross-hybridization outcome is a significant number of background chi...

  1. DNA minicircles clarify the specific role of DNA structure on retroviral integration

    OpenAIRE

    Pasi, Marco; Mornico, Damien; Volant, Stevenn; Juchet, Anna; Batisse, Julien; Bouchier, Christiane; Parissi, Vincent; Ruff, Marc; Lavery, Richard; Lavigne, Marc

    2016-01-01

    International audience; Chromatin regulates the selectivity of retroviral integration into the genome of infected cells. At the nucleosome level, both histones and DNA structure are involved in this regulation. We propose a strategy that allows to specifically study a single factor: the DNA distortion induced by the nucleosome. This strategy relies on mimicking this distortion using DNA minicircles (MCs) having a fixed rotational orientation of DNA curvature, coupled with atomic-resolution mo...

  2. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A. [Brookhaven National Lab., Upton, NY (United States). Biology Dept.; Lees-Miller, S.P.; Lintott, L.G. [Univ. of Calgary, Alberta (Canada). Dept. of Biological Sciences; Sakaguchi, Kazuyasu; Appella, E. [National Institutes of Health, Bethesda, MD (United States). Lab. of Cell Biology

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  3. Linchpin DNA-binding residues serve as go/no-go controls in the replication factor C-catalyzed clamp-loading mechanism.

    Science.gov (United States)

    Liu, Juan; Zhou, Yayan; Hingorani, Manju M

    2017-09-22

    DNA polymerases depend on circular sliding clamps for processive replication. Clamps must be loaded onto primer-template DNA (ptDNA) by clamp loaders that open and close clamps around ptDNA in an ATP-fueled reaction. All clamp loaders share a core structure in which five subunits form a spiral chamber that binds the clamp at its base in a twisted open form and encloses ptDNA within, while binding and hydrolyzing ATP to topologically link the clamp and ptDNA. To understand how clamp loaders perform this complex task, here we focused on conserved arginines that might play a central coordinating role in the mechanism because they can alternately contact ptDNA or Walker B glutamate in the ATPase site and lie close to the clamp loader-clamp-binding interface. We mutated Arg-84, Arg-88, and Arg-101 in the ATPase-active B, C, and D subunits of Saccharomyces cerevisiae replication factor C (RFC) clamp loader, respectively, and assessed the impact on multiple transient events in the reaction: proliferating cell nuclear antigen (PCNA) clamp binding/opening/closure/release, ptDNA binding/release, and ATP hydrolysis/product release. The results show that these arginines relay critical information between the PCNA-binding, DNA-binding, and ATPase sites at all steps of the reaction, particularly at a checkpoint before RFC commits to ATP hydrolysis. Moreover, their actions are subunit-specific with RFC-C Arg-88 serving as an accelerator that enables rapid ATP hydrolysis upon contact with ptDNA and RFC-D Arg-101 serving as a brake that confers specificity for ptDNA as the correct substrate for loading PCNA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. A new mode of DNA binding distinguishes Capicua from other HMG-box factors and explains its mutation patterns in cancer.

    Science.gov (United States)

    Forés, Marta; Simón-Carrasco, Lucía; Ajuria, Leiore; Samper, Núria; González-Crespo, Sergio; Drosten, Matthias; Barbacid, Mariano; Jiménez, Gerardo

    2017-03-01

    HMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This binding, however, is relatively weak and both Sox and TCF factors employ distinct mechanisms for enhancing their affinity and specificity for DNA. Here we report that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. We find that, contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. We demonstrate that this binding mechanism operates throughout Drosophila development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors, while being preserved in oncogenic CIC-DUX4 fusion chimeras associated to Ewing-like sarcomas.

  5. Prospects of nanoparticle-DNA binding and its implications in medical biotechnology.

    Science.gov (United States)

    An, Hongjie; Jin, Bo

    2012-01-01

    Bio-nanotechnology is a new interdisciplinary R&D area that integrates engineering and physical science with biology through the development of multifunctional devices and systems, focusing biology inspired processes or their applications, in particular in medical biotechnology. DNA based nanotechnology, in many ways, has been one of the most intensively studied fields in recent years that involves the use and the creation of bio-inspired materials and their technologies for highly selective biosensing, nanoarchitecture engineering and nanoelectronics. Increasing researches have been offered to a fundamental understanding how the interactions between the nanoparticles and DNA molecules could alter DNA molecular structure and its biochemical activities. This minor review describes the mechanisms of the nanoparticle-DNA binding and molecular interactions. We present recent discoveries and research progresses how the nanoparticle-DNA binding could vary DNA molecular structure, DNA detection, and gene therapy. We report a few case studies associated with the application of the nanoparticle-DNA binding devices in medical detection and biotechnology. The potential impacts of the nanoparticles via DNA binding on toxicity of the microorganisms are briefly discussed. The nanoparticle-DNA interactions and their impact on molecular and microbial functionalities have only drown attention in recent a few years. The information presented in this review can provide useful references for further studies on biomedical science and technology. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Identification of DNA-binding proteins using support vector machines and evolutionary profiles

    Directory of Open Access Journals (Sweden)

    Gromiha Michael M

    2007-11-01

    Full Text Available Abstract Background Identification of DNA-binding proteins is one of the major challenges in the field of genome annotation, as these proteins play a crucial role in gene-regulation. In this paper, we developed various SVM modules for predicting DNA-binding domains and proteins. All models were trained and tested on multiple datasets of non-redundant proteins. Results SVM models have been developed on DNAaset, which consists of 1153 DNA-binding and equal number of non DNA-binding proteins, and achieved the maximum accuracy of 72.42% and 71.59% using amino acid and dipeptide compositions, respectively. The performance of SVM model improved from 72.42% to 74.22%, when evolutionary information in form of PSSM profiles was used as input instead of amino acid composition. In addition, SVM models have been developed on DNAset, which consists of 146 DNA-binding and 250 non-binding chains/domains, and achieved the maximum accuracy of 79.80% and 86.62% using amino acid composition and PSSM profiles. The SVM models developed in this study perform better than existing methods on a blind dataset. Conclusion A highly accurate method has been developed for predicting DNA-binding proteins using SVM and PSSM profiles. This is the first study in which evolutionary information in form of PSSM profiles has been used successfully for predicting DNA-binding proteins. A web-server DNAbinder has been developed for identifying DNA-binding proteins and domains from query amino acid sequences http://www.imtech.res.in/raghava/dnabinder/.

  7. Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX.

    Science.gov (United States)

    Dwivedi, Hari P; Smiley, R Derike; Jaykus, Lee-Ann

    2010-08-01

    The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d) value) of 292.8 +/- 53.1 nM with 47.27 +/- 5.58% cells fluorescent (bound) in a 1.48-microM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25-36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1-5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.

  8. Stepwise bending of DNA by a single TATA box binding protein

    DEFF Research Database (Denmark)

    Tolic-Nørrelykke, Simon F; Rasmussen, Mette B; Pavone, Francesco S

    2006-01-01

    cerevisiae TBPs interacting with single DNA molecules containing a TATA-box. Using video microscopy, we observed the Brownian motion of the beads tethered by short surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the DNA changes and the amplitude of Brownian motion of the tehtered......-defined free and bound classes of Brownian motion, we observed another two classes of motion. These extra classes were identified with intermediate states of a three-step, linear binding pathway. Biological implications of the intermediate states are discussed....

  9. DNA binding during expanded bed adsorption and factors affecting adsorbent aggregation

    DEFF Research Database (Denmark)

    Arpanaei, Ayyoob; Mathiasen, N.; Hobley, Timothy John

    2008-01-01

    DNA-induced aggregation and contraction of expanded bed adsorption chromatography beds have been examined using strong anion exchanger Q HyperZ and calf thymus DNA in buffers containing added NaCl. Two batches of adsorbent with different ionic capacities were used allowing the effects of different...... tolerance of anion exchangers when binding DNA. However, more importantly. with the adsorbents examined here. attempts to reduce bed aggregation by feedstock conditioning with added salt may increase DNA binding leading to a reduction in expanded bed adsorption performance compromising protein capture...

  10. Mapping arm-DNA-binding domain interactions in AraC.

    Science.gov (United States)

    Wu, M; Schleif, R

    2001-04-06

    AraC protein, the regulator of the l-arabinose operon in Escherichia coli has been postulated to function by a light switch mechanism. According to this mechanism, it should be possible to find mutations in the DNA-binding domain of AraC that result in weaker arm-DNA-binding domain interactions and which make the protein constitutive, that is, it no longer requires arabinose to activate transcription. We isolated such mutations by randomizing three contiguous leucine residues in the DNA-binding domain, and then by systematically scanning surface residues of the DNA-binding domain with alanine and glutamic acid. As a result, a total of 20 constitutive mutations were found at ten different positions. They form a contiguous trail on the DNA-distal face of the DNA-binding domain, and likely define the region where the N-terminal arm that extends from the N-terminal dimerization domain contacts the C-terminal DNA-binding domain. Copyright 2001 Academic Press.

  11. Imaging chromatin nanostructure with binding-activated localization microscopy based on DNA structure fluctuations.

    Science.gov (United States)

    Szczurek, Aleksander; Klewes, Ludger; Xing, Jun; Gourram, Amine; Birk, Udo; Knecht, Hans; Dobrucki, Jurek W; Mai, Sabine; Cremer, Christoph

    2017-05-05

    Advanced light microscopy is an important tool for nanostructure analysis of chromatin. In this report we present a general concept for Single Molecule localization Microscopy (SMLM) super-resolved imaging of DNA-binding dyes based on modifying the properties of DNA and the dye. By careful adjustment of the chemical environment leading to local, reversible DNA melting and hybridization control over the fluorescence signal of the DNA-binding dye molecules can be introduced. We postulate a transient binding as the basis for our variation of binding-activated localization microscopy (BALM). We demonstrate that several intercalating and minor-groove binding DNA dyes can be used to register (optically isolate) only a few DNA-binding dye signals at a time. To highlight this DNA structure fluctuation-assisted BALM (fBALM), we applied it to measure, for the first time, nanoscale differences in nuclear architecture in model ischemia with an anticipated structural resolution of approximately 50 nm. Our data suggest that this approach may open an avenue for the enhanced microscopic analysis of chromatin nano-architecture and hence the microscopic analysis of nuclear structure aberrations occurring in various pathological conditions. It may also become possible to analyse nuclear nanostructure differences in different cell types, stages of development or environmental stress conditions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Structural basis of Ets1 cooperative binding to widely separated sites on promoter DNA.

    Directory of Open Access Journals (Sweden)

    Nigar D Babayeva

    Full Text Available Ets1 is a member of the Ets family of transcription factors. Ets1 is expressed in autoinhibited form and its DNA binding depends on partner proteins bound to adjacent sequences or the relative positioning of a second Ets-binding site (EBS. The autoinhibition of Ets1 is mediated by structural coupling of regions flanking the DNA-binding domain. The NMR structure of Ets1 revealed that the inhibitory regions comprised of helices HI1 and HI2 and H4 are packed together on the Ets domain to form an inhibitory module. The crystal structure of Ets1 unexpectedly revealed a homodimer in which homodimerisation occurs via swapping of HI1 helices. Modeling of DNA binding indicates that the Ets1 dimer can bind to two antiparallel pieces of DNA. To verify this, we crystallized and solved the structure of the complex comprised of Ets1 dimer and two pieces of DNA. DNA binding by Ets1 dimer resulted in formation of additional intermolecular protein•DNA interactions, implying that the complex formation is cooperative.

  13. Structural basis of Ets1 cooperative binding to widely separated sites on promoter DNA.

    Science.gov (United States)

    Babayeva, Nigar D; Baranovskaya, Oxana I; Tahirov, Tahir H

    2012-01-01

    Ets1 is a member of the Ets family of transcription factors. Ets1 is expressed in autoinhibited form and its DNA binding depends on partner proteins bound to adjacent sequences or the relative positioning of a second Ets-binding site (EBS). The autoinhibition of Ets1 is mediated by structural coupling of regions flanking the DNA-binding domain. The NMR structure of Ets1 revealed that the inhibitory regions comprised of helices HI1 and HI2 and H4 are packed together on the Ets domain to form an inhibitory module. The crystal structure of Ets1 unexpectedly revealed a homodimer in which homodimerisation occurs via swapping of HI1 helices. Modeling of DNA binding indicates that the Ets1 dimer can bind to two antiparallel pieces of DNA. To verify this, we crystallized and solved the structure of the complex comprised of Ets1 dimer and two pieces of DNA. DNA binding by Ets1 dimer resulted in formation of additional intermolecular protein•DNA interactions, implying that the complex formation is cooperative.

  14. DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

    Science.gov (United States)

    Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

    2017-09-01

    Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB).

    Science.gov (United States)

    van Loon, Barbara; Samson, Leona D

    2013-03-01

    Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair. Copyright © 2012. Published by Elsevier B.V.

  16. Accurate Estimation of the Standard Binding Free Energy of Netropsin with DNA

    Directory of Open Access Journals (Sweden)

    Hong Zhang

    2018-01-01

    Full Text Available DNA is the target of chemical compounds (drugs, pollutants, photosensitizers, etc., which bind through non-covalent interactions. Depending on their structure and their chemical properties, DNA binders can associate to the minor or to the major groove of double-stranded DNA. They can also intercalate between two adjacent base pairs, or even replace one or two base pairs within the DNA double helix. The subsequent biological effects are strongly dependent on the architecture of the binding motif. Discriminating between the different binding patterns is of paramount importance to predict and rationalize the effect of a given compound on DNA. The structural characterization of DNA complexes remains, however, cumbersome at the experimental level. In this contribution, we employed all-atom molecular dynamics simulations to determine the standard binding free energy of DNA with netropsin, a well-characterized antiviral and antimicrobial drug, which associates to the minor groove of double-stranded DNA. To overcome the sampling limitations of classical molecular dynamics simulations, which cannot capture the large change in configurational entropy that accompanies binding, we resort to a series of potentials of mean force calculations involving a set of geometrical restraints acting on collective variables.

  17. Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence spectroscopy.

    Science.gov (United States)

    Wang, Ying; Schellenberg, Helene; Walhorn, Volker; Toensing, Katja; Anselmetti, Dario

    2017-09-01

    Fluorescent dyes are broadly used in many biotechnological applications to detect and visualize DNA molecules. However, their binding to DNA alters the structural and nanomechanical properties of DNA and, thus, interferes with associated biological processes. In this work we employed magnetic tweezers and fluorescence spectroscopy to investigate the binding of PicoGreen to DNA at room temperature in a concentration-dependent manner. PicoGreen is an ultrasensitive quinolinium nucleic acid stain exhibiting hardly any background signal from unbound dye molecules. By means of stretching and overwinding single, torsionally constrained, nick-free double-stranded DNA molecules, we acquired force-extension and supercoiling curves which allow quantifying DNA contour length, persistence length and other thermodynamical binding parameters, respectively. The results of our magnetic tweezers single-molecule binding study were well supported through analyzing the fluorescent spectra of stained DNA. On the basis of our work, we could identify a concentration-dependent bimodal binding behavior, where, apparently, PicoGreen associates to DNA as an intercalator and minor-groove binder simultaneously.

  18. Solution-state NMR investigation of DNA binding interactions in Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg): a dynamic description of the DNA/protein interface.

    Science.gov (United States)

    Buchko, Garry W; McAteer, Kathleen; Wallace, Susan S; Kennedy, Michael A

    2005-03-02

    Formamidopyrimidine-DNA glycosylase (Fpg) is a base excision repair (BER) protein that removes oxidative DNA lesions. Recent crystal structures of Fpg bound to DNA revealed residues involved in damage recognition and enzyme catalysis, but failed to shed light on the dynamic nature of the processes. To examine the structural and dynamic changes that occur in solution when Fpg binds DNA, NMR spectroscopy was used to study Escherichia coli Fpg free in solution and bound to a double-stranded DNA oligomer containing 1,3-propanediol (13-PD), a non-hydrolyzable abasic-site analogue. Only 209 out of a possible 251 (83%) free-precession 15N/1H HSQC cross peaks were observed and 180 of these were assignable, indicating that approximately 30% of the residues undergo intermediate motion on the NMR timescale, broadening the resonances beyond detection or making them intractable in backbone assignment experiments. The majority of these affected residues were in the polypeptide linker region and the interface between the N- and C-terminal domains. DNA titration experiments revealed line broadening and chemical shift perturbations for backbone amides nearby and distant from the DNA binding surface, but failed to quench the intermediate timescale motion observed for free Fpg, including those residues directly involved in DNA binding, notwithstanding a nanomolar dissociation constant for 13-PD binding. Indeed, after binding to 13-PD, at least approximately 40% of the Fpg residues undergo intermediate timescale motion even though all other residues exhibit tight DNA binding characteristic of slow exchange. CPMG-HSQC experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that were quenched upon binding 13-PD. In free Fpg, heteronuclear 1H-15N NOE experiments detected picosecond timescale backbone motion in the alphaF-beta9 loop, the region primarily responsible for chemically discriminating 8-oxoguanine (8-oxoG) over normal guanine, that was

  19. Designing DNA interstrand lock for locus-specific methylation detection in a nanopore

    Science.gov (United States)

    Kang, Insoon; Wang, Yong; Reagan, Corbin; Fu, Yumei; Wang, Michael X.; Gu, Li-Qun

    2013-10-01

    DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg2+) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg2+ binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5'-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer.

  20. DNA binding by Corynebacterium glutamicum TetR-type transcription regulator AmtR

    Directory of Open Access Journals (Sweden)

    Sticht Heinrich

    2009-07-01

    Full Text Available Abstract Background The TetR family member AmtR is the central regulator of nitrogen starvation response in Corynebacterium glutamicum. While the AmtR regulon was physiologically characterized in great detail up to now, mechanistic questions of AmtR binding were not addressed. This study presents a characterization of functionally important amino acids in the DNA binding domain of AmtR and of crucial nucleotides in the AmtR recognition motif. Results Site-directed mutagenesis, the characterization of corresponding mutant proteins by gel retardation assays and surface plasmon resonance and molecular modelling revealed several amino acids, which are directly involved in DNA binding, while others have more structural function. Furthermore, we could show that the spacing of the binding motif half sites is crucial for repression of transcription by AmtR. Conclusion Although the DNA binding domain of TetR-type repressors is highly conserved and a core binding motif was identified for AmtR and TetR(D, the AmtR binding domain shows individual properties compared to other TetR proteins. Besides by distinct amino acids of AmtR, DNA binding is influenced by nucleotides not only of the conserved binding motif but also by spacing nucleotides in C. glutamicum.

  1. Recognition of methylated DNA through methyl-CpG binding domain proteins

    DEFF Research Database (Denmark)

    Zou, Xueqing; Ma, Wen; Solov'yov, Ilia

    2012-01-01

    the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines...... and by strengthening the interaction between mDNA and MBD proteins. Free-energy perturbation calculations also show that methylation yields favorable contribution to the binding free energy for MBD-mDNA complex.......DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although...

  2. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  3. Comparison of the binding potential of various diisocyanates on DNA in vitro.

    Science.gov (United States)

    Peel, M; Marczynski, B; Baur, X

    1997-12-26

    Inhalation of diisocyanate vapors is associated with immediate-type hypersensitivity reactions and direct toxic responses. The genotoxic effects of diisocyanates have not been clarified. The aim of this study was to examine the changes in DNA following in vitro exposure to three most commonly used diisocyanates (toluene diisocyanate, TDI; methylenediphenyl-4,4'-diisocyanate, MDI; and hexamethylene diisocyanate, HDI) and to compare their binding potential using melting behavior of DNA and electrophoresis studies in DNA. Following incubation of DNA with MDI (pure and mix) and HDI we found no differences in the melting behavior compared to the control calf thymus DNA. However, DNA treated with TDI showed differences in the shape of the native DNA curves due to changes in hyperchromicity and exhibited 14% more DNA reconstitution after renaturation. The small changes in the melting behavior of native DNA do not suggest the formation of DNA intrastrand cross-links but rather conformational changes of single- and double-stranded DNA. These conformational changes were further explored by agarose electrophoresis of native and denatured calf thymus DNA. Control and all diisocyanate-exposed DNA showed no differences in the size of native DNA fragments. Conversely, electrophoresis of TDI mix-incubated DNA, following denaturation, showed a distinct reduction in the double-stranded DNA fragment size compared to the control, MDI-denatured (pure and mix), and HDI-denatured DNA. These findings may help to better understand the mechanisms of the genotoxic effect of TDI.

  4. Visual Detection of Single-Base Mismatches in DNA Using Hairpin Oligonucleotide with Double-Target DNA Binding Sequences and Gold Nanoparticles

    Science.gov (United States)

    He, Yuqing; Zhang, Xibao; Zhang, Sanquan; Kris, Mak Ka Long; Man, Fong Chi; Liu, Guodong

    2014-01-01

    We describe a hairpin oligonucleotide (HO) with double-target DNA binding sequences in the loop and 11-base in the stem for visual detection of single-base mismatches (SBM) in DNA with highly specificity. The thiol-modified HO was immobilized on gold nanoparticle (Au-NP) surface through a self-assembling process. The strategy of detecting SBM depends on the unique molecular recognition properties of HO to the perfect-matched DNA and SBM to generate different quantities of duplex DNA on the Au-NP surface, which are captured on the test zone of lateral flow test strip via the DNA hybridization reaction between the duplex DNA and preimmobilized DNA probe. Accumulation of Au-NPs produces the characteristic red bands, enabling visual detection of SBM. It was found that the ability of HO to differentiate perfect-matched DNA and SBM was increased dramatically by incorporating double-target DNA binding sequences in the loop of HO. The signal ratio between perfect-matched DNA and SBM was up to 28, which is much higher than that of conventional HO or molecular beacon. The approach was applied to detect the mutation sites, Arg142Cys and Gly529Ile, of transglutaminase 1 gene in autosomal recessive congenital ichthyosis. The results presented here show that the new HO is a potential molecular recognition probe for the future development of nucleic acid-based biosensors and bioassays. The approach can be used for point-of-care diagnosis of genetic diseases and detecting infectious agents or warning against bio-warfare agents. PMID:22386491

  5. Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3'-Tail Sequences Are Sufficient for Tight and Complete Binding.

    Science.gov (United States)

    Smaldino, Philip J; Routh, Eric D; Kim, Jung H; Giri, Banabihari; Creacy, Steven D; Hantgan, Roy R; Akman, Steven A; Vaughn, James P

    2015-01-01

    Ends of human chromosomes consist of the six nucleotide repeat d[pTTAGGG]n known as telomeric DNA, which protects chromosomes. We have previously shown that the DHX36 gene product, G4 Resolvase 1 (G4R1), binds parallel G-quadruplex (G4) DNA with an unusually tight apparent Kd. Recent work associates G4R1 with the telomerase holoenzyme, which may allow it to access telomeric G4-DNA. Here we show that G4R1 can tightly bind telomeric G4-DNA, and in the context of the telomeric sequence, we determine length, sequence, and structural requirements sufficient for tight G4R1 telomeric binding. Specifically, G4R1 binds telomeric DNA in the K+-induced "3+1" G4-topology with an apparent Kd = 10 ± 1.9 pM, a value similar as previously found for binding to unimolecular parallel G4-DNA. G4R1 binds to the Na+-induced "2+2" basket G4-structure formed by the same DNA sequence with an apparent Kd = 71 ± 2.2 pM. While the minimal G4-structure is not sufficient for G4R1 binding, a 5' G4-structure with a 3' unstructured tail containing a guanine flanked by adenine(s) is sufficient for maximal binding. Mutations directed to disrupt G4-structure similarly disrupt G4R1 binding; secondary mutations that restore G4-structure also restore G4R1 binding. We present a model showing that a replication fork disrupting a T-loop could create a 5' quadruplex with an opened 3'tail structure that is recognized by G4R1.

  6. Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3’-Tail Sequences Are Sufficient for Tight and Complete Binding

    Science.gov (United States)

    Smaldino, Philip J.; Routh, Eric D.; Kim, Jung H.; Giri, Banabihari; Creacy, Steven D.; Hantgan, Roy R.; Akman, Steven A.; Vaughn, James P.

    2015-01-01

    Ends of human chromosomes consist of the six nucleotide repeat d[pTTAGGG]n known as telomeric DNA, which protects chromosomes. We have previously shown that the DHX36 gene product, G4 Resolvase 1 (G4R1), binds parallel G-quadruplex (G4) DNA with an unusually tight apparent Kd. Recent work associates G4R1 with the telomerase holoenzyme, which may allow it to access telomeric G4-DNA. Here we show that G4R1 can tightly bind telomeric G4-DNA, and in the context of the telomeric sequence, we determine length, sequence, and structural requirements sufficient for tight G4R1 telomeric binding. Specifically, G4R1 binds telomeric DNA in the K+-induced “3+1” G4-topology with an apparent Kd = 10 ±1.9 pM, a value similar as previously found for binding to unimolecular parallel G4-DNA. G4R1 binds to the Na+-induced “2+2” basket G4-structure formed by the same DNA sequence with an apparent Kd = 71 ± 2.2 pM. While the minimal G4-structure is not sufficient for G4R1 binding, a 5’ G4-structure with a 3’ unstructured tail containing a guanine flanked by adenine(s) is sufficient for maximal binding. Mutations directed to disrupt G4-structure similarly disrupt G4R1 binding; secondary mutations that restore G4-structure also restore G4R1 binding. We present a model showing that a replication fork disrupting a T-loop could create a 5’ quadruplex with an opened 3’tail structure that is recognized by G4R1. PMID:26172836

  7. Sequence specificity of DNA alkylation by the antitumor natural product leinamycin.

    Science.gov (United States)

    Zang, Hong; Gates, Kent S

    2003-12-01

    Reaction with thiol converts the antitumor natural product leinamycin to an episulfonium ion that alkylates the N(7)-position of guanine residues in double-stranded DNA. The sequence specificity for DNA alkylation by this structurally novel compound has not previously been examined. It is reported here that leinamycin shows significant (>10-fold) preferences for alkylation at the 5'-G in 5'-GG and 5'-GT sequences. The sequence preferences for activated leinamycin are significantly different from that observed for the structurally simple episulfonium ion generated from 2-chloroethyl ethyl sulfide. DNA alkylation by activated leinamycin is inhibited by addition of salt (100 mM NaClO(4)), although the degree of inhibition is somewhat less than that seen for 2-chloroethyl ethyl sulfide. This result suggests that electrostatic interactions between the activated leinamycin and the N(7)-position of guanine residues facilitate efficient DNA alkylation. However, the observed sequence preferences for DNA alkylation by activated leinamycin do not correlate strongly with calculated sequence-dependent variations in the molecular electrostatic potential at the N(7)-atom of guanine residues in duplex DNA. Thus, electrostatic interactions between activated leinamycin and DNA do not appear to be the primary determinant for sequence specificity. Rather, the results suggest that sequence-specific noncovalent interactions of leinamycin with the DNA double helix on the 3'-side of the alkylated guanine residue play a major role in determining the preferred alkylation sites. Consistent with the notion that noncovalent binding plays an important role in DNA alkylation by leinamycin, experiments with 2'-deoxyoligonucleotide substrates confirm that the natural product does not alkylate single-stranded DNA under conditions where duplex DNA is efficiently alkylated.

  8. Food storage material silver nanoparticles interfere with DNA replication fidelity and bind with DNA

    Science.gov (United States)

    Yang, Wenjuan; Shen, Cenchao; Ji, Qiaoli; An, Hongjie; Wang, Jinju; Liu, Qingdai; Zhang, Zhizhou

    2009-02-01

    Nanosilver is increasingly used in the food industry and biomedical applications. A lot of studies have been done to investigate the potential toxicity of nanosilver. But information on whether or how nanosilver particles bring changes in genetic materials remains scant. In this study, the replication fidelity of the rpsL gene was quantified when nanosilver particles were present in polymerase chain reactions (PCRs) or cell cultures of E. coli transformed with the wild-type rpsL gene. Three types of nanosilver (silver nanopowder, SN; silver-copper nanopowder, SCN; and colloidal silver, CS) were tested. The results showed that the replication fidelity of the rpsL gene was differentially compromised by all three kinds of nanosilver particle compared with that without nanosilver. This assay could be expanded and applied to any other materials to preliminarily assess their potential long-term toxicity as a food additive or biomedical reagent. Moreover, we found that nanosilver materials bind with genomic DNA under atomic force microscopy, and this might be an explanation for the compromised DNA replication fidelity.

  9. Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans-Expression and characterization.

    Directory of Open Access Journals (Sweden)

    Marcin Olszewski

    Full Text Available DNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the Taq DNA Stoffel domain and a single-stranded DNA binding-like protein of Nanoarchaeum equitans in order to significantly improve the properties of DNA polymerase. The DNA coding sequence of Taq Stoffel DNA polymerase and the nonspecific DNA-binding protein of Nanoarchaeum equitans (NeqSSB-like protein were fused. A novel recombinant gene was obtained which was cloned into the pET-30 Ek/LIC vector and introduced into E. coli for expression. The recombinant enzyme was purified and its enzymatic properties including DNA polymerase activity, PCR amplification rate, thermostability, processivity and resistance to inhibitors, were tested. The yield of the target protein reached approximately 18 mg/l after 24 h of the IPTG induction. The specific activity of the polymerase was 2200 U/mg. The recombinant NeqSSB-TaqS exhibited a much higher extension rate (1000 bp template in 20 s, processivity (19 nt, thermostability (half-life 35 min at 95°C and higher tolerance to PCR inhibitors (0.3-1.25% of whole blood, 0.84-13.5 μg of lactoferrin and 4.7-150 ng of heparin than Taq Stoffel DNA polymerase. Furthermore, our studies show that NeqSSB-TaqS DNA polymerase has a high level of flexibility in relation to Mg2+ ions (from 1 to 5 mM and KCl or (NH42SO4 salts (more than 60 mM and 40 mM, respectively. Using NeqSSB-TaqS DNA polymerase instead of the Taq DNA polymerase could be a better choice in many PCR applications.

  10. Binding and interaction of di- and tri-substituted organometallic triptycene palladium complexes with DNA.

    Science.gov (United States)

    Kumari, Rina; Bhowmick, Sourav; Das, Neeladri; Das, Prolay

    2014-10-01

    Two triptycene-based ligands with pendant bromophenyl units have been prepared. These triptycene derivatives have been used as synthons for the synthesis of di and tri nuclear palladium complexes. The organic molecules and their corresponding organometallic complexes have been fully characterized using nuclear magnetic resonance (NMR), infrared (IR) spectroscopy and mass spectrometry. The mode of binding and effect of the complexes on pUC19 plasmid, calf thymus DNA and oligomer duplex DNA have been investigated by a host of analytical methods. The complexes brought about unwinding of supercoiled plasmid and the unwinding angle was found to be related to the binding affinity of the complexes with DNA, where both these parameters were guided by the structure of the complexes. Concentration-dependent inhibition of endonuclease activity of SspI and BamHI by the complexes indicates preference for G/C sequence for binding to DNA. However, neither the complexes did not introduce any cleavage at abasic site in oligomer duplex DNA, nor they created linear form of the plasmid upon co-incubation with the DNA samples. The interactions of the complexes with DNA were found to be strongly guided by the structure of the complexes, where intercalation as well as groove binding was observed, without inflicting any damage to the DNA. The mode of interaction of the complexes with DNA was further confirmed by isothermal calorimetry.

  11. RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency

    Science.gov (United States)

    Qi, Xiaodong; Xie, Mingyi; Brown, Andrew F; Bley, Christopher J; Podlevsky, Joshua D; Chen, Julian J-L

    2012-01-01

    Telomerase synthesizes telomeric DNA repeats onto chromosome termini from an intrinsic RNA template. The processive synthesis of DNA repeats relies on a unique, yet poorly understood, mechanism whereby the telomerase RNA template translocates and realigns with the DNA primer after synthesizing each repeat. Here, we provide evidence that binding of the realigned RNA/DNA hybrid by the active site is an essential step for template translocation. Employing a template-free human telomerase system, we demonstrate that the telomerase active site directly binds to RNA/DNA hybrid substrates for DNA polymerization. In telomerase processivity mutants, the template-translocation efficiency correlates with the affinity for the RNA/DNA hybrid substrate. Furthermore, the active site is unoccupied during template translocation as a 5 bp extrinsic RNA/DNA hybrid effectively reduces the processivity of the template-containing telomerase. This suggests that strand separation and template realignment occur outside the active site, preceding the binding of realigned hybrid to the active site. Our results provide new insights into the ancient RNA/DNA hybrid binding ability of telomerase and its role in template translocation. PMID:21989387

  12. Systematic functional comparative analysis of four single-stranded DNA-binding proteins and their affection on viral RNA metabolism.

    Directory of Open Access Journals (Sweden)

    Haiyan Shi

    Full Text Available The accumulation of single-stranded DNA-binding (SSB proteins is essential for organisms and has various applications. However, no study has simultaneously and systematically compared the characteristics of SSB proteins. In addition, SSB proteins may bind RNA and play an unknown biological role in RNA metabolism. Here, we expressed a novel species of SSB protein derived from Thermococcus kodakarensis KOD1 (KOD, as well as SSB proteins from Thermus thermophilus (TTH, Escherichia coli, and Sulfolobus Solfataricus P2 (SSOB, abbreviated kod, tth, bl21, and ssob, respectively. These SSB proteins could bind ssDNA and viral RNA. bl21 resisted heat treatment for more than 9 h, Ssob and kod could withstand 95°C for 10 h and retain its ssDNA- and RNA-binding ability. Four SSB proteins promoted the specificity of the DNA polymerase in PCR-based 5- and 9-kb genome fragment amplification. kod also increased the amplification of a 13-kb PCR product, and SSB protein-bound RNA resisted Benzonase digestion. The SSB proteins could also enter the host cell bound to RNA, which resulted in modulation of viral RNA metabolism, particularly ssob and bl21.

  13. Reactivation of mutant p53: Constraints on mechanism highlighted by principal component analysis of the DNA binding domain.

    Science.gov (United States)

    Ouaray, Zahra; ElSawy, Karim M; Lane, David P; Essex, Jonathan W; Verma, Chandra

    2016-10-01

    Most p53 mutations associated with cancer are located in its DNA binding domain (DBD). Many structures (X-ray and NMR) of this domain are available in the protein data bank (PDB) and a vast conformational heterogeneity characterizes the various free and complexed states. The major difference between the apo and the holo-complexed states appears to lie in the L1 loop. In particular, the conformations of this loop appear to depend intimately on the sequence of DNA to which it binds. This conclusion builds upon recent observations that implicate the tetramerization and the C-terminal domains (respectively TD and Cter) in DNA binding specificity. Detailed PCA analysis of the most recent collection of DBD structures from the PDB have been carried out. In contrast to recommendations that small molecules/drugs stabilize the flexible L1 loop to rescue mutant p53, our study highlights a need to retain the flexibility of the p53 DNA binding surface (DBS). It is the adaptability of this region that enables p53 to engage in the diverse interactions responsible for its functionality. Proteins 2016; 84:1443-1461. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Synthesis, micellization behavior, antimicrobial and intercalative DNA binding of some novel surfactant copper(II) complexes containing modified phenanthroline ligands.

    Science.gov (United States)

    Nagaraj, Karuppiah; Ambika, Subramanian; Rajasri, Shanmugasundaram; Sakthinathan, Subramanian; Arunachalam, Sankaralingam

    2014-10-01

    The novel surfactant copper(II) complexes, [Cu(ip)2DA](ClO4)21, [Cu(dpqc)2DA](ClO4)22, [Cu(dppn)2DA](ClO4)23, where ip=imidazo[4,5-f][1,10]phenanthroline, dpqc=dipyrido[3,2-a:2',4'-c](6,7,8,9-tetrahydro)phenazine, dppn=benzo[1]dipyrido[3,2-a':2',3'-c]phenazine and DA-dodecylamine, were synthesized and characterized by physico-chemical and spectroscopic methods. In these complexes 1-3, the geometry of copper metal ions was described as square pyramidal. The critical micelle concentration (CMC) value of these surfactant copper(II) complexes in aqueous solution was found out from conductance measurements. Specific conductivity data at different temperatures served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization (ΔGm°, ΔHm° and ΔSm°). The binding interaction of these complexes with DNA (calf thymus DNA) in Tris buffer was studied by physico-chemical techniques. In the presence of the DNA UV-vis spectrum of complexes showed red shift of the absorption band along with significant hypochromicity indicating intercalation of our complexes with nucleic acids. Competitive binding study with ethidium bromide (EB) shows that the complexes exhibit the ability to displace the nucleic acid-bound EB indicating that the complexes bind to nucleic acids in strong competition with EB for the intercalative binding site. Observed changes in the circular dichoric spectra of DNA in the presence of surfactant complexes support the strong binding of complexes with DNA. CV results also confirm this mode of binding. Some significant thermodynamic parameters of the binding of the titled complexes to DNA have also been determined. The results reveal that the extent of DNA binding of 3 was greater than that of 1 and 2. The antibacterial and antifungal screening tests of these complexes have shown good results compared to its precursor chloride complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Identification of a Second DNA Binding Site in the Human Rad52 Protein*S⃞

    OpenAIRE

    Kagawa, Wataru; Kagawa, Ako; Saito, Kengo; Ikawa, Shukuko; Shibata, Takehiko; Kurumizaka, Hitoshi; Yokoyama, Shigeyuki

    2008-01-01

    Rad52 plays essential roles in homology-dependent double-strand break repair. Various studies have established the functions of Rad52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of Rad52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within Rad52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding ...

  16. Simulation of the type of coralin alkaloid-DNA binding

    Science.gov (United States)

    Kulikov, K. G.; Koshlan, T. V.

    2015-05-01

    Interaction between a synthesized coralin protoberberine alkaloid and the DNA double helix of the calf's thymus in a salt solution is studied by optical absorption spectroscopy and spectropolarimetry. The dependence of the spectral characteristics of the alkaloid on a ratio between the DNA base pair concentration and the alkaloid molecule concentration is considered. The parameters of bonds between the coralin alkaloid and the DNA double helix are determined using modified McGhee-von Hippel equations.

  17. ACETYLATION INCREASES EWS-FLI1 DNA BINDING AND TRANSCRIPTIONAL ACTIVITY

    Directory of Open Access Journals (Sweden)

    Silke eSchlottmann

    2012-09-01

    Full Text Available Ewing Sarcoma (ES is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant posttranslational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors, and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with histone deacetylase inhibitors (HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in COS7 cells. However, our data that evaluates the acetylation of ful-length EWS-FLI1 remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

  18. Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides.

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    Muszyńska, Klaudia; Ostrowska, Dominika; Bartnicki, Filip; Kowalska, Ewa; Bodaszewska-Lubaś, Małgorzata; Hermanowicz, Paweł; Faulstich, Heinz; Strzałka, Wojciech

    2017-01-01

    Mushroom foraging is very popular in some regions of the world. Sometimes toxic and edible mushrooms are mistaken by mushroom collectors, leading to serious human poisoning. The group of mushrooms highly dangerous for human health includes Amanita phalloides. This mushroom produces a toxic octapeptide called α-amanitin which is an inhibitor of nuclear RNA polymerase II. The inhibition of this polymerase results in the abortion of mRNA synthesis. The ingestion of A. phalloides causes liver failure due to the fact that most of the toxin is uptaken by hepatocytes. The hospitalization of poisoned patients involves the removal of the toxin from the digestive tract, its dilution in the circulatory system and the administration of therapeutic adjuvants. Since there is no effective antidote against amanitin poisoning, in this study we developed a DNA aptamer exhibiting specific binding to α-amanitin. This aptamer was selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. Next, its ability of toxin removal from aqueous solution was confirmed by pull-down assay. The aptamer region sufficient for α-amanitin binding was determined. Finally, the dissociation constant of the α-amanitin/DNA aptamer complex was calculated.

  19. DNA binding domains in diverse nuclear receptors function as nuclear export signals.

    Science.gov (United States)

    Black, B E; Holaska, J M; Rastinejad, F; Paschal, B M

    2001-11-13

    The nuclear receptor superfamily of transcription factors directs gene expression through DNA sequence-specific interactions with target genes. Nuclear import of these receptors involves recognition of a nuclear localization signal (NLS) by importins, which mediate translocation into the nucleus. Nuclear receptors lack a leucine-rich nuclear export signal (NES), and export is insensitive to leptomycin B, indicating that nuclear export is not mediated by Crm1. We set out to define the NES in the glucocorticoid receptor (GR) and to characterize the export pathway. We found that the 69 amino acid DNA binding domain (DBD) of GR, which is unrelated to any known NES, is necessary and sufficient for export. Mutational analysis revealed that a 15 amino acid sequence between the two zinc binding loops in the GR-DBD confers nuclear export to a GFP reporter protein, and alanine-scanning mutagenesis was used to identify the residues within this sequence that are critical for export. The DBD is highly related (41%-88% identity) in steroid, nonsteroid, and orphan nuclear receptors, and we found that the DBDs from ten different nuclear receptors all function as export signals. DBD-dependent nuclear export is saturable, and prolonged nuclear localization of the GR increases its transcriptional activity. Multiple members of the nuclear receptor superfamily use a common pathway to exit the nucleus. We propose that NLS-mediated import and DBD-mediated export define a shuttling cycle that integrates the compartmentalization and activity of nuclear receptors.

  20. Quantitative analysis of pheromone-binding protein specificity

    OpenAIRE

    Katti, S.; Lokhande, N.; González, D.; Cassill, A.; Renthal, R.

    2012-01-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-p...

  1. EL_PSSM-RT: DNA-binding residue prediction by integrating ensemble learning with PSSM Relation Transformation.

    Science.gov (United States)

    Zhou, Jiyun; Lu, Qin; Xu, Ruifeng; He, Yulan; Wang, Hongpeng

    2017-08-29

    Prediction of DNA-binding residue is important for understanding the protein-DNA recognition mechanism. Many computational methods have been proposed for the prediction, but most of them do not consider the relationships of evolutionary information between residues. In this paper, we first propose a novel residue encoding method, referred to as the Position Specific Score Matrix (PSSM) Relation Transformation (PSSM-RT), to encode residues by utilizing the relationships of evolutionary information between residues. PDNA-62 and PDNA-224 are used to evaluate PSSM-RT and two existing PSSM encoding methods by five-fold cross-validation. Performance evaluations indicate that PSSM-RT is more effective than previous methods. This validates the point that the relationship of evolutionary information between residues is indeed useful in DNA-binding residue prediction. An ensemble learning classifier (EL_PSSM-RT) is also proposed by combining ensemble learning model and PSSM-RT to better handle the imbalance between binding and non-binding residues in datasets. EL_PSSM-RT is evaluated by five-fold cross-validation using PDNA-62 and PDNA-224 as well as two independent datasets TS-72 and TS-61. Performance comparisons with existing predictors on the four datasets demonstrate that EL_PSSM-RT is the best-performing method among all the predicting methods with improvement between 0.02-0.07 for MCC, 4.18-21.47% for ST and 0.013-0.131 for AUC. Furthermore, we analyze the importance of the pair-relationships extracted by PSSM-RT and the results validates the usefulness of PSSM-RT for encoding DNA-binding residues. We propose a novel prediction method for the prediction of DNA-binding residue with the inclusion of relationship of evolutionary information and ensemble learning. Performance evaluation shows that the relationship of evolutionary information between residues is indeed useful in DNA-binding residue prediction and ensemble learning can be used to address the data imbalance

  2. Minor groove binding of the food colorant carmoisine to DNA: spectroscopic and calorimetric characterization studies.

    Science.gov (United States)

    Basu, Anirban; Kumar, Gopinatha Suresh

    2014-01-08

    The interaction of the food additive carmoisine with herring testes DNA was studied by multifaceted biophysical techniques. Carmoisine exhibited hypochromic effects in absorbance, whereas in fluorescence the intensity enhanced upon complexation with DNA. Energy transfer from the DNA base pairs to carmoisine molecules occurred upon complexation. A groove binding model of interaction was envisaged for carmoisine-DNA complexation from 4',6-diamidino-2-phenylindole (DAPI) and Hoechst displacement studies. The binding of carmoisine stabilized the DNA structure against thermal denaturation. The binding induced moderate conformational perturbations in the B-form structure of DNA. The binding affinity (10(4) M(-1)) values, calculated from absorbance and fluorescence data, and calorimetry titrations were in close agreement with each other. The binding was characterized to be exothermic and favored by small negative enthalpic and large positive entropic contributions. Salt-dependent calorimetric studies revealed that the binding reaction was dominated by nonpolyelectrolytic forces. The negative heat capacity value suggested the role of hydrophobic effect in the interaction.

  3. Crystal structures, metal activation, and DNA-binding properties of two-domain IdeR from Mycobacterium tuberculosis.

    Science.gov (United States)

    Wisedchaisri, Goragot; Chou, C James; Wu, Meiting; Roach, Claudia; Rice, Adrian E; Holmes, Randall K; Beeson, Craig; Hol, Wim G J

    2007-01-16

    The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake in Mycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domain of this essential regulator, the metal-binding and DNA-binding properties of two-domain IdeR (2D-IdeR) whose SH3-like domain has been truncated were characterized. The equilibrium dissociation constants for Co2+ and Ni2+ activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinities of 2D-IdeR in the presence of excess metal ions were estimated using fluorescence spectroscopy. 2D-IdeR binds to fxbA operator DNA with similar affinity as full-length IdeR in the presence of excess metal ion. However, the Ni2+ concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co2+ required for activation remains the same. We have determined the crystal structures of Ni2+-activated 2D-IdeR at 1.96 A resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 A and 2.6 A, the highest resolutions for DNA complexes for any structures of iron-dependent regulator family members so far. The 2D-IdeR-DNA complex structures confirm the specificity of Ser37 and Pro39 for thymine bases and suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structures reveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminal His6. This C-terminal tail promotes crystal contacts by forming a beta-sheet with the corresponding tail of neighboring subunits in two unrelated structures of 2D-IdeR, one with and one without DNA. The contact-promoting properties of this C-terminal TEV cleavage sequence may be beneficial for crystallizing other proteins.

  4. Zinc-regulated DNA binding of the yeast Zap1 zinc-responsive activator.

    Directory of Open Access Journals (Sweden)

    Avery G Frey

    Full Text Available The Zap1 transcription factor of Saccharomyces cerevisiae plays a central role in zinc homeostasis by controlling the expression of genes involved in zinc metabolism. Zap1 is active in zinc-limited cells and repressed in replete cells. At the transcriptional level, Zap1 controls its own expression via positive autoregulation. In addition, Zap1's two activation domains are regulated independently of each other by zinc binding directly to those regions and repressing activation function. In this report, we show that Zap1 DNA binding is also inhibited by zinc. DMS footprinting showed that Zap1 target gene promoter occupancy is regulated with or without transcriptional autoregulation. These results were confirmed using chromatin immunoprecipitation. Zinc regulation of DNA binding activity mapped to the DNA