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Sample records for affect dna binding

  1. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  2. Diethyl pyrocarbonate reaction with the lactose repressor protein affects both inducer and DNA binding

    SciTech Connect

    Sams, C.F.; Matthews, K.S.

    1988-04-05

    Modification of the lactose repressor protein of Escherichia coli with diethyl pyrocarbonate (DPC) results in decreased inducer binding as well as operator and nonspecific DNA binding. Spectrophotometric measurements indicated a maximum of three histidines per subunit was modified, and quantitation of lysine residues with trinitrobenzenesulfonate revealed the modification of one lysine residue. The loss of DNA binding, both operator and nonspecific, was correlated with histidine modification; removal of the carbethoxy groups from the histidines by hydroxylamine was accompanied by significant recovery of DNA binding function. The presence of inducing sugars during the DPC reaction had no effect on histidine modification or the loss of DNA binding activity. In contrast, inducer binding was not recovered upon reversal of the histidine modification. However, the presence of inducer during reaction protected lysine from reaction and also prevented the decrease in inducer binding; these results indicate that reaction of the lysine residue(s) may correlate to the loss of sugar binding activity. Since no difference in incorporation of radiolabeled carbethoxy was observed following reaction with diethyl pyrocarbonate in the presence or absence of inducer, the reagent appears to function as a catalyst in the modification of the lysine. The formation of an amide bond between the affected lysine and a nearby carboxylic acid moiety provides a possible mechanism for the activity loss. Reaction of the isolated NH2-terminal domain resulted in loss of DNA binding with modification of the single histidine at position 29. Results from the modification of core domain paralleled observations with intact repressor.

  3. The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

    PubMed

    Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

    2013-11-01

    We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.

  4. How hormone receptor-DNA binding affects nucleosomal DNA: the role of symmetry.

    PubMed Central

    Bishop, T C; Kosztin, D; Schulten, K

    1997-01-01

    Molecular dynamics simulations have been employed to determine the optimal conformation of an estrogen receptor DNA binding domain dimer bound to a consensus response element, ds(AGGTCACAGTGACCT), and to a nonconsensus response element, ds(AGAACACAGTGACCT). The structures simulated were derived from a crystallographic structure and solvated by a sphere (45-A radius) of explicit water and counterions. Long-range electrostatic interactions were accounted for during 100-ps simulations by means of a fast multipole expansion algorithm combined with a multiple time-step scheme in the molecular dynamics package NAMD. The simulations demonstrate that the dimer induces a bent and underwound (10.7 bp/turn) conformation in the DNA. The bending reflects the dyad symmetry of the receptor dimer and can be described as an S-shaped curve in the helical axis of DNA when projected onto a plane. A similar bent and underwound conformation is observed for nucleosomal DNA near the nucleosome's dyad axis that reflects the symmetry of the histone octamer. We propose that when a receptor dimer binds to a nucleosome, the most favorable dimer-DNA and histone-DNA interactions are achieved if the respective symmetry axes are aligned. Such positioning of a receptor dimer over the dyad of nucleosome B in the mouse mammary tumor virus promoter is in agreement with experiment. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 9 FIGURE 11 PMID:9129808

  5. Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

    PubMed Central

    Patel, A.; Hashimoto, H.; Zhang, X.; Cheng, X.

    2016-01-01

    Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides. PMID:27372763

  6. "DNA Binding Region" of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint.

    PubMed

    Masuda, Takaaki; Xu, Xiaoling; Dimitriadis, Emilios K; Lahusen, Tyler; Deng, Chu-Xia

    2016-01-01

    The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress.

  7. DNA Binding Region” of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint

    PubMed Central

    Masuda, Takaaki; Xu, Xiaoling; Dimitriadis, Emilios K.; Lahusen, Tyler; Deng, Chu-Xia

    2016-01-01

    The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress. PMID:26884712

  8. Mutations that affect phosphorylation of the adenovirus DNA-binding protein alter its ability to enhance its own synthesis.

    PubMed Central

    Morin, N; Delsert, C; Klessig, D F

    1989-01-01

    The multifunctional adenovirus single-strand DNA-binding protein (DBP) is highly phosphorylated. Its phosphorylation sites are located in the amino-terminal domain of the protein, and its DNA- and RNA-binding activity resides in the carboxy-terminal half of the polypeptide. We have substituted cysteine or alanine for up to 10 of these potential phosphorylation sites by using oligonucleotide-directed mutagenesis. Alteration of one or a few of these sites had little effect on the viability of virus containing the mutated DBP. However, when eight or more sites were altered, viral growth decreased significantly. This suggests that the overall phosphorylation state of the protein was more important than whether any particular site was modified. The reduction in growth correlated with both depressed DNA replication and expression of late genes. This reduction was probably the result of lower DBP accumulation in mutant-infected cells. Interestingly, although the stability of the mutated DBP was not affected, DBP synthesis and the level of its mRNA were depressed 5- to 10-fold for the underphosphorylated protein. These results suggest that DBP enhances its own expression and imply that phosphorylation of the DBP may be important for this function. Similarities to several eucaryotic transcriptional activators, which are composed of negatively charged activating domains and separate binding domains, are discussed. Images PMID:2585602

  9. Phosphorylation Affects DNA-Binding of the Senescence-Regulating bZIP Transcription Factor GBF1

    PubMed Central

    Smykowski, Anja; Fischer, Stefan M.; Zentgraf, Ulrike

    2015-01-01

    Massive changes in the transcriptome of Arabidopsis thaliana during onset and progression of leaf senescence imply a central role for transcription factors. While many transcription factors are themselves up- or down-regulated during senescence, the bZIP transcription factor G-box-binding factor 1 (GBF1/bZIP41) is constitutively expressed in Arabidopsis leaf tissue but at the same time triggers the onset of leaf senescence, suggesting posttranscriptional mechanisms for senescence-specific GBF1 activation. Here we show that GBF1 is phosphorylated by the threonine/serine CASEIN KINASE II (CKII) in vitro and that CKII phosphorylation had a negative effect on GBF1 DNA-binding to G-boxes of two direct target genes, CATALASE2 and RBSCS1a. Phosphorylation mimicry at three serine positions in the basic region of GBF1 also had a negative effect on DNA-binding. Kinase assays revealed that CKII phosphorylates at least one serine in the basic domain but has additional phosphorylation sites outside this domain. Two different ckII α subunit1 and one α subunit2 T-DNA insertion lines showed no visible senescence phenotype, but in all lines the expression of the senescence marker gene SAG12 was remarkably diminished. A model is presented suggesting that senescence-specific GBF1 activation might be achieved by lowering the phosphorylation of GBF1 by CKII. PMID:27135347

  10. TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia

    PubMed Central

    Yamazaki, Jumpei; Jelinek, Jaroslav; Lu, Yue; Cesaroni, Matteo; Madzo, Jozef; Neumann, Frank; He, Rong; Taby, Rodolphe; Vasanthakumar, Aparna; Macrae, Trisha; Ostler, Kelly R.; Kantarjian, Hagop M.; Liang, Shoudan; Estecio, Marcos R.; Godley, Lucy A.; Issa, Jean-Pierre J.

    2015-01-01

    TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently-modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here we report on DNA methylomes in TET2 wild type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites (28,114 sites in CpG islands (CGIs) and 57,020 in non-CpG islands (NCGIs)). TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. By contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and non-promoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1, and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor binding sites. PMID:25972343

  11. Mutations proximal to the minor groove-binding track of human immunodeficiency virus type 1 reverse transcriptase differentially affect utilization of RNA versus DNA as template.

    PubMed

    Fisher, Timothy S; Darden, Tom; Prasad, Vinayaka R

    2003-05-01

    Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), like all retroviral RTs, is a versatile DNA polymerase that can copy both RNA and DNA templates. In spite of extensive investigations into the structure-function of this enzyme, the structural basis for this dual template specificity is poorly understood. Biochemical studies with two mutations in HIV-1 RT that affect residues contacting the template-primer now provide some insight into this specialized property. The mutations are N255D and N265D, both adjoining the minor groove-binding track, in the thumb region. The N265D substitution led to a loss of processive polymerization on DNA but not on RNA, whereas N255D drastically reduced processive synthesis on both templates. This differential template usage was accompanied by a rapid dissociation of the N265D variant on DNA but not RNA templates, whereas the N255D variant rapidly dissociated from both templates. Molecular dynamics modeling suggested that N265D leads to a loss of template strand-specific hydrogen bonding, indicating that this is a key determinant of the differential template affinity. The N255D substitution caused local changes in conformation and a consequent loss of interaction with the primer, leading to a loss of processive synthesis with both templates. We conclude that N265 is part of a subset of template-enzyme contacts that enable RT to utilize DNA templates in addition to RNA templates and that such residues play an important role in facilitating processive DNA synthesis on both RNA and DNA templates.

  12. Mutations affecting the BHLHA9 DNA-binding domain cause MSSD, mesoaxial synostotic syndactyly with phalangeal reduction, Malik-Percin type.

    PubMed

    Malik, Sajid; Percin, Ferda E; Bornholdt, Dorothea; Albrecht, Beate; Percesepe, Antonio; Koch, Manuela C; Landi, Antonio; Fritz, Barbara; Khan, Rizwan; Mumtaz, Sara; Akarsu, Nurten A; Grzeschik, Karl-Heinz

    2014-12-04

    Mesoaxial synostotic syndactyly, Malik-Percin type (MSSD) (syndactyly type IX) is a rare autosomal-recessive nonsyndromic digit anomaly with only two affected families reported so far. We previously showed that the trait is genetically distinct from other syndactyly types, and through autozygosity mapping we had identified a locus on chromosome 17p13.3 for this unique limb malformation. Here, we extend the number of independent pedigrees from various geographic regions segregating MSSD to a total of six. We demonstrate that three neighboring missense mutations affecting the highly conserved DNA-binding region of the basic helix-loop-helix A9 transcription factor (BHLHA9) are associated with this phenotype. Recombinant BHLHA9 generated by transient gene expression is shown to be located in the cytoplasm and the cell nucleus. Transcription factors 3, 4, and 12, members of the E protein (class I) family of helix-loop-helix transcription factors, are highlighted in yeast two-hybrid analysis as potential dimerization partners for BHLHA9. In the presence of BHLHA9, the potential of these three proteins to activate expression of an E-box-regulated target gene is reduced considerably. BHLHA9 harboring one of the three substitutions detected in MSSD-affected individuals eliminates entirely the transcription activation by these class I bHLH proteins. We conclude that by dimerizing with other bHLH protein monomers, BHLHA9 could fine tune the expression of regulatory factors governing determination of central limb mesenchyme cells, a function made impossible by altering critical amino acids in the DNA binding domain. These findings identify BHLHA9 as an essential player in the regulatory network governing limb morphogenesis in humans.

  13. Characterization of the DNA binding properties of polyomavirus capsid protein

    NASA Technical Reports Server (NTRS)

    Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

  14. How Do Structure and Charge Affect Metal-Complex Binding to DNA? An Upper-Division Integrated Laboratory Project Using Cyclic Voltammetry

    ERIC Educational Resources Information Center

    Kulczynska, Agnieszka; Johnson, Reed; Frost, Tony; Margerum, Lawrence D.

    2011-01-01

    An advanced undergraduate laboratory project is described that integrates inorganic, analytical, physical, and biochemical techniques to reveal differences in binding between cationic metal complexes and anionic DNA (herring testes). Students were guided to formulate testable hypotheses based on the title question and a list of different metal…

  15. Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

    PubMed Central

    Ktistaki, E; Ktistakis, N T; Papadogeorgaki, E; Talianidis, I

    1995-01-01

    Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7568236

  16. Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

    PubMed

    Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

    2016-05-06

    Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers.

  17. New DNA-binding radioprotectors

    NASA Astrophysics Data System (ADS)

    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

  18. Difference in DNA-binding abilities of Fur-homolog DNA binding protein from Neisseria gonorrhoeae.

    PubMed

    Bagchi, Angshuman

    2016-10-01

    Gonorrhea is a severe disease infecting both men and women worldwide. The causative agent of the disease is Neisseria gonorrhoeae. The organism mostly affects human beings in iron restricted environments. In such an environment the organism produces a set of proteins which are mostly absent in iron rich environments. The expressions of the genes for the proteins are regulated by the transcription factor (TF) belonging to the Fur family. Interestingly, the same TF acts as the activator and repressor of genes. In this present work, an attempt has been made to analyze the molecular details of the differential DNA-binding activities of the TF from Neisseria gonorrhoeae to come up with a plausible molecular reason behind the difference DNA binding activities of the same TF. Computational modelling technique was used to build the three dimensional structure of the TF. Molecular docking and molecular dynamics simulations were employed to determine the binding interactions between the TF and the promoter DNA. With the help of the computational techniques, the biochemical reason behind the different modes of DNA binding by the TF was analyzed. Results from this analysis may be useful to future drug development endeavours to curtail the spread of Gonorrhea.

  19. The Sunscreen Octyl Methoxycinnamate Binds to DNA

    NASA Astrophysics Data System (ADS)

    Norrell, Johannes; Vohra, Shikhar; Nordlund, T. M.

    2000-03-01

    Sunscreens are designed to prevent skin cancer by absorbing ultraviolet radiation from the sun before it gets to the DNA in skin cells. The purpose of this work is to determine whether or not octyl methoxycinnamate, an active ingredient in many sunscreens, will bind to DNA. If so, the sunscreen could transfer the energy it absorbed from the sun to the DNA and cause damage. To determine this, we prepared samples with varying concentrations of cinnamate added to herring sperm DNA, sonicating the mixture to disperse the hydrophobic sunscreen into solution. Absorption and fluorescence spectra of the mixtures showed (i) much more sunscreen was dispersed into solution when DNA was present, and (ii) the spectra of both DNA and sunscreen differed from those of the separate solutions. We conclude that the octyl methoxycinnamate can indeed bind to DNA in aqueous solution. Energy transfer experiments from DNA to sunscreen and from sunscreen to 2-aminopurine- (a fluorescent DNA base) labeled DNA will be presented.

  20. DNA binding studies of tartrazine food additive.

    PubMed

    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).

  1. Potency of carcinogens derived from covalent DNA binding and stimulation of DNA synthesis in rat liver

    SciTech Connect

    Lutz, W.K.; Buesser, M.T.; Sagelsdorff, P.

    1984-01-01

    In order to investigate the role of the stimulation of cell division for the initiation (and possibly promotion) of liver tumors by chemical carcinogens, the incorporation of radiolabelled thymidine into liver DNA was determined in male rats. Single doses of various levels of aflatoxin B1, benzidine and carbon tetrachloride (all known to be genotoxic via DNA binding) did not affect cell division, whereas several hepatocarcinogens known not to bind to DNA (alpha-HCH, clofibrate, and 2,3,7,8-tetrachlorodibenzo-p-dioxin) gave rise to a dose-dependent stimulation of liver DNA synthesis within 24 h. An equation combining the influences of mitotic stimulation, expressed as dose required to double the control level of DNA synthesis, and DNA binding potency, expressed as the Covalent Binding Index, correlated well with the carcinogenic potency for both classes of hepatocarcinogens.

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

    PubMed

    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.

  3. Free-radical-mediated DNA binding.

    PubMed Central

    O'Brien, P J

    1985-01-01

    Free-radical metabolites can be generated metabolically by a one-electron reductase-catalyzed reaction or a "peroxidase" catalyzed oxidation or by photoactivation of a wide variety of aromatic xenobiotics. Radicals may also be generated during lipid peroxidation. Some radicals can react with DNA or bind covalently or noncovalently as a dismutation product or as a dimer, trimer or polymeric product. Modification to the DNA can result in single-strand breaks, loss of template activity, and crosslinking. The binding can prevent enzymic digestion. In some cases, the radicals react with oxygen, resulting before conversion to DNA reactive oxygen species. Most radicals probably do not interact with DNA. PMID:3007090

  4. Copper(I) and nickel(II) complexes with 1:1 vs. 1:2 coordination of ferrocenyl hydrazone ligands: do the geometry and composition of complexes affect DNA binding/cleavage, protein binding, antioxidant and cytotoxic activities?

    PubMed

    Krishnamoorthy, Paramasivam; Sathyadevi, Palanisamy; Butorac, Rachel R; Cowley, Alan H; Bhuvanesh, Nattamai S P; Dharmaraj, Nallasamy

    2012-04-21

    A new series of geometrically different complexes containing ferrocenyl hydrazone ligands were synthesised by reacting suitable precursor complex [MCl(2)(PPh(3))(2)] with the ligands HL(1) or HL(2) (where M = Cu(II) or Ni(II); HL(1) = [Cp(2)Fe(CH=N-NH-CO-C(6)H(5))] (1) and HL(2) = [Cp(2)Fe(CH=N-NH-CO-C(5)H(4)N)]) (2). The new complexes of the composition [Cu(L(1))(PPh(3))(2)], (3) [Cu(L(2))(PPh(3))(2)] (4), [Ni(L(1))(2)] (5) and [Ni(L(2))(2)] (6) were characterised by various spectral studies. Among them, complexes 3 and 5 characterised by single crystal X-ray diffraction showed a distorted tetrahedral structure for the former with 1:1 metal-ligand stoichiometry, but a distorted square planar geometry with 1:2 metal-ligand stoichiometry in the case of the latter. Systematic biological investigations like DNA binding, DNA cleavage, protein binding, free radical scavenging and cytotoxicity activities were carried out using all the synthesised compounds and the results obtained were explained on the basis of structure-activity relationships. The binding constant (K(b)) values of the synthesised compounds are found to be in the order of magnitude 10(3)-10(5) M(-1) and also they exhibit significant cleavage of supercoiled (SC) pUC19 DNA in the presence of H(2)O(2) as co-oxidant. The conformational changes of bovine serum albumin (BSA) upon binding with the above complexes were also studied. In addition, concentration dependent free radical scavenging potential of all the synthesised compounds (1-6) was also carried out under in vitro conditions. Assays on the cytotoxicity of the above complexes against HeLa and A431 tumor cells and NIH 3T3 normal cells were also carried out.

  5. Direct DNA binding by Brca1

    PubMed Central

    Paull, Tanya T.; Cortez, David; Bowers, Blair; Elledge, Stephen J.; Gellert, Martin

    2001-01-01

    The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein–DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription. PMID:11353843

  6. Importance of DNA stiffness in protein-DNA binding specificity

    NASA Astrophysics Data System (ADS)

    Hogan, M. E.; Austin, R. H.

    1987-09-01

    From the first high-resolution structure of a repressor bound specifically to its DNA recognition sequence1 it has been shown that the phage 434 repressor protein binds as a dimer to the helix. Tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. The centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there2. This observation might be explained should the structure of the intervening DNA segment vary with its sequence, or if DNA at the centre of the operator resists the torsional and bending deformation necessary for complex formation in a sequence dependent fashion. We have considered the second hypothesis by demonstrating that DNA stiffness is sequence dependent. A method is formulated for calculating the stiffness of any particular DNA sequence, and we show that this predicted relationship between sequence and stiffness can explain the repressor binding data in a quantitative manner. We propose that the elastic properties of DNA may be of general importance to an understanding of protein-DNA binding specificity.

  7. Synthesis and DNA-binding properties of novel DNA cyclo-intercalators containing purine-glucuronic acid hybrids.

    PubMed

    Zhang, Renshuai; Chen, Shaopeng; Wang, Xueting; Yu, Rilei; Li, Mingjing; Ren, Sumei; Jiang, Tao

    2016-06-24

    Novel DNA cyclo-intercalators, which incorporated two intercalator subunits linked by two bridges, were synthesized. Binding of the compounds to calf-thymus DNA was studied by fluorescence spectroscopy, and docking simulations were used to predict the binding modes of these cyclic compounds. The spectral data demonstrated that all of these compounds can interact with CT-DNA. The sugar moiety played an important role in the process of binding between the intercalators containing glucuronic acid and DNA. The length and flexibility of the connecting bridges affected the binding affinity of the resultant cyclo-intercalators. Docking simulations showed that compounds 7 and 8 interact with DNA as mono-intercalators.

  8. The R215W mutation in NBS1 impairs {gamma}-H2AX binding and affects DNA repair: molecular bases for the severe phenotype of 657del5/R215W Nijmegen breakage syndrome patients

    SciTech Connect

    Masi, Alessandra di Viganotti, Mara; Polticelli, Fabio; Ascenzi, Paolo; Tanzarella, Caterina; Antoccia, Antonio

    2008-05-09

    Nijmegen breakage syndrome (NBS) is a genetic disorder characterized by chromosomal instability and hypersensitivity to ionising radiation. Compound heterozygous 657del5/R215W NBS patients display a clinical phenotype more severe than the majority of NBS patients homozygous for the 657del5 mutation. The NBS1 protein, mutated in NBS patients, contains a FHA/BRCT domain necessary for the DNA-double strand break (DSB) damage response. Recently, a second BRCT domain has been identified, however, its role is still unknown. Here, we demonstrate that the R215W mutation in NBS1 impairs histone {gamma}-H2AX binding after induction of DNA damage, leading to a delay in DNA-DSB rejoining. Molecular modelling reveals that the 215 residue of NBS1 is located between the two BRCT domains, affecting their relative orientation that appears critical for {gamma}-H2AX binding. Present data represent the first evidence for the role of NBS1 tandem BRCT domains in {gamma}-H2AX recognition, and could explain the severe phenotype observed in 657del5/R215W NBS patients.

  9. DNA Origami Seesaws as Comparative Binding Assay

    PubMed Central

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

    2016-01-01

    Abstract 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. PMID:27038073

  10. Biophysical characterization of DNA binding from single molecule force measurements

    NASA Astrophysics Data System (ADS)

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-09-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.

  11. Biophysical characterization of DNA binding from single molecule force measurements

    PubMed Central

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-01-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function. PMID:20576476

  12. Curcumin binding to DNA and RNA.

    PubMed

    Nafisi, Shohreh; Adelzadeh, Maryam; Norouzi, Zeinab; Sarbolouki, Mohammad Nabi

    2009-04-01

    Curcumin, the yellow pigment from the rhizoma of Curcuma longa, is a widely studied phytochemical with a variety of biological activities. The ongoing research and clinical trials have proved that this natural phenolic compound has great and diverse pharmacological potencies. Beside its effective antioxidant, antiinflammatory, and antimicrobial/antiviral properties, curcumin is also considered as a cancer chemopreventive agent. While the antioxidant activity of curcumin is well documented, its interaction with DNA and RNA is not fully investigated. This study was designed to examine the interactions of curcumin with calf thymus DNA and yeast RNA in aqueous solution at physiological conditions, using constant DNA and RNA concentration (6.25 mM) and various curcumin/polynucleotide (phosphate) ratios of 1/120, 1/80, 1/40, 1/20, and 1/10. Fourier transform infrared (FTIR) and UV-visible spectroscopic methods were used to determine the ligand binding modes, the binding constants, and the stability of curcumin-DNA and curcumin-RNA complexes in aqueous solution. Spectroscopic evidence showed that curcumin binds to the major and minor grooves of DNA duplex and to RNA bases as well as to the back bone phosphate group with overall binding constants of K(curcumin-DNA) = 4.255 x 10(4) M(-1) and K(curcumin-RNA) = 1.262 x 10(4) M(-1). Major DNA and RNA aggregation occurred at high pigment concentration. No conformational changes were observed upon curcumin interaction with these biopolymers; that is, DNA remains in the B, and RNA retains its A-family structure.

  13. HTLV-1 Tax Protein Stimulation of DNA Binding of bZIP Proteins by Enhancing Dimerization

    NASA Astrophysics Data System (ADS)

    Wagner, Susanne; Green, Michael R.

    1993-10-01

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

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

    SciTech Connect

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

    2010-10-22

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

  15. Duplex structural differences and not 2'-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA.

    PubMed

    Olimpo, Jeffrey T; DeStefano, Jeffrey J

    2010-07-01

    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 hydroxyls and RT amino acids do not stabilize binding. RT's K(d) (k(off)/k(on)) for DNA-DNA and RNA-DNA were similar, although k(off) differed almost 40-fold, suggesting a faster k(on) for DNA-DNA. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA-DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA-DNA and DNA-DNA, respectively, allow the former to conform more easily to HIV-RT's binding cleft, leading to more stable binding. Biologically, the ability of RT to form a more stable complex on RNA-DNA may aid in degradation of RNA fragments that remain after DNA synthesis.

  16. Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype.

    PubMed

    Chennathukuzhi, V M; Kurihara, Y; Bray, J D; Yang, J; Hecht, N B

    2001-11-01

    The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by approximately 50% and the poorly hydrolyzed GTP analog, GTPgammaS, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP(GTP) and TB-RBP(GTP) no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP(GTP) will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP(GTP) into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP(GTP) in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.

  17. Survey of variation in human transcription factors reveals prevalent DNA binding changes

    PubMed Central

    Barrera, Luis A.; Rogers, Julia M.; Gisselbrecht, Stephen S.; Rossin, Elizabeth J.; Woodard, Jaie; Mariani, Luca; Kock, Kian Hong; Inukai, Sachi; Siggers, Trevor; Shokri, Leila; Gordân, Raluca; Sahni, Nidhi; Cotsapas, Chris; Hao, Tong; Yi, Song; Kellis, Manolis; Daly, Mark J.; Vidal, Marc; Hill, David E.; Bulyk, Martha L.

    2016-01-01

    Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA-binding activity and used universal protein binding microarrays to assay sequence-specific DNA-binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA-binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA-binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA-binding activities, which may contribute to phenotypic variation. PMID:27013732

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

    PubMed Central

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

    2011-01-01

    Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to >1300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel ‘recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4–Met28–Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNA-binding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity. PMID:22146299

  19. Mechanisms for Binding between Methylene Blue and DNA

    NASA Astrophysics Data System (ADS)

    Vardevanyan, P. O.; Antonyan, A. P.; Parsadanyan, M. A.; Shahinyan, M. A.; Hambardzumyan, L. A.

    2013-09-01

    We have used absorption and fl uorimetric methods to study the interaction between methylene blue (MB) and calfthymus DNA. Based on Scatchard analysis of the experimental data, we plotted the methylene blue-DNA binding curve. This curve consists of two linear sections, which indicates two types of interaction, for which we determined the constants K and the number of binding sites n for binding of this ligand to DNA. Comparison of the data obtained with analogous values found for interaction between ethidium bromide and DNA allowed us to conclude that there are two modes of interaction between methylene blue and DNA: strong binding (semi-intercalation) and weak binding (electrostatic).

  20. DNA binding studies of Vinca alkaloids: experimental and computational evidence.

    PubMed

    Pandya, Prateek; Gupta, Surendra P; Pandav, Kumud; Barthwal, Ritu; Jayaram, B; Kumar, Surat

    2012-03-01

    Fluorescence studies on the indole alkaloids vinblastine sulfate, vincristine sulfate, vincamine and catharanthine have demonstrated the DNA binding ability of these molecules. The binding mode of these molecules in the minor groove of DNA is non-specific. A new parameter of the purine-pyrimidine base sequence specificty was observed in order to define the non-specific DNA binding of ligands. Catharanthine had shown 'same' pattern of 'Pu-Py' specificity while evaluating its DNA binding profile. The proton resonances of a DNA decamer duplex were assigned. The models of the drug:DNA complexes were analyzed for DNA binding features. The effect of temperature on the DNA binding was also evaluated.

  1. Binding characteristics of salbutamol with DNA by spectral methods.

    PubMed

    Bi, Shuyun; Pang, Bo; Zhao, Tingting; Wang, Tianjiao; Wang, Yu; Yan, Lili

    2013-07-01

    Salbutamol interacting with deoxyribonucleic acid (DNA) was examined by fluorescence, UV absorption, viscosity measurements, and DNA melting techniques. The binding constants and binding sites were obtained at different temperatures by fluorescence quenching. The Stern-Volmer plots showed that the quenching of fluorescence of salbutamol by DNA was a static quenching. To probe the binding mode, various analytical methods were performed and the results were as follows: hyperchromic effect was shown in the absorption spectra of salbutamol upon addition of DNA; there was no appreciable increase in melting temperature of DNA when salbutamol was presented in DNA solution; the fluorescence intensity of salbutamol-DNA decrease with the increasing ionic strength; the relative viscosity of DNA did not change in the presence of salbutamol; the binding constant of salbutamol with double strand DNA (dsDNA) was much higher than that of it with single strand DNA (ssDNA). All these results indicated that the binding mode of salbutamol to DNA should be groove binding. The thermodynamic parameters suggested that hydrogen bond or van der Waals force might play an important role in salbutamol binding to DNA. According to the Förster energy transference theory, the binding distance between the acceptor and donor was 3.70 nm.

  2. Binding characteristics of salbutamol with DNA by spectral methods

    NASA Astrophysics Data System (ADS)

    Bi, Shuyun; Pang, Bo; Zhao, Tingting; Wang, Tianjiao; Wang, Yu; Yan, Lili

    2013-07-01

    Salbutamol interacting with deoxyribonucleic acid (DNA) was examined by fluorescence, UV absorption, viscosity measurements, and DNA melting techniques. The binding constants and binding sites were obtained at different temperatures by fluorescence quenching. The Stern-Volmer plots showed that the quenching of fluorescence of salbutamol by DNA was a static quenching. To probe the binding mode, various analytical methods were performed and the results were as follows: hyperchromic effect was shown in the absorption spectra of salbutamol upon addition of DNA; there was no appreciable increase in melting temperature of DNA when salbutamol was presented in DNA solution; the fluorescence intensity of salbutamol-DNA decrease with the increasing ionic strength; the relative viscosity of DNA did not change in the presence of salbutamol; the binding constant of salbutamol with double strand DNA (dsDNA) was much higher than that of it with single strand DNA (ssDNA). All these results indicated that the binding mode of salbutamol to DNA should be groove binding. The thermodynamic parameters suggested that hydrogen bond or van der Waals force might play an important role in salbutamol binding to DNA. According to the Förster energy transference theory, the binding distance between the acceptor and donor was 3.70 nm.

  3. Characterization of single stranded telomeric DNA-binding proteins in cultured soybean (Glycine max) cells.

    PubMed

    Kwon, Chian; Kwon, Kisang; Chung, In Kwon; Kim, Soon Young; Cho, Myeon Haeng; Kang, Bin Goo

    2004-06-30

    We have identified and characterized a protein factor in soybean (Glycine max) nuclear extracts that binds to plant single stranded telomeric DNA repeats. A single DNA-protein complex was detected in gel retardation assays using synthetic telomeres and nuclear extracts. The protein forming this complex was designated soy-bean (Glycine max) single stranded telomeric DNA-binding protein (Gm-STBP). Gm-STBP binds to single stranded telomeric DNA containing more than two repeats. It does not bind to Tetrahymena, human or mutated plant telomere sequences, and its binding activity is not affected by RNase treatment. Gm-STBP activity gradually decreased after suspension cultures entered stationary phase. A slower migrating band was formed with extracts of earlier and later phases of soybean suspension cultures. Our findings suggest that binding of Gm-STBP to plant single stranded telomeric DNA may play a role in the proper functioning of telomeres during development.

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

    PubMed

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

    2012-09-01

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

  5. Noncovalent Binding to DNA: Still a Target in Developing Anticancer Agents.

    PubMed

    Portugal, José; Barceló, Francisca

    2016-01-01

    DNA-binding compounds are of extraordinary importance in medicine, accounting for a substantial portion of antitumor drugs in clinical usage. However, their mechanisms of action remain sometimes incompletely understood. This review critically examines two broad classes of molecules that bind noncovalently to DNA: intercalators and groove binders. Intercalators bind to DNA by inserting their chromophore moiety between two consecutive base pairs, whereas groove binders fit into the grooves of DNA. Noncovalent DNAinteractive drugs can recognize certain supramolecular DNA structures such as the Gquadruplexes found in telomeres and in numerous gene promoters, and they can act as topoisomerase I and II poisons. We discuss how DNA-binding compounds affect transcription and compete with protein factors for binding to consensus binding sites in gene promoters both in vitro and in cultured cancer cells. Moreover, we comment on the design of molecules that can tightly and specifically bind to any desired target DNA, such as various hairpin polyamides which efficacy as chemotherapeutic agents is being evaluated. At present, genome-wide studies, which provide details of events that may influence both cancer progression and therapeutic outcome, are a common way used to analyze the effects of DNA-binding compounds. A conclusive feature that emerges from reviewing the information on DNA-binding compounds is that both natural sources and chemical approaches can be productively used to obtain drugs to manipulate gene expression in cancer cells.

  6. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner.

    PubMed Central

    Miller, S D; Farmer, G; Prives, C

    1995-01-01

    The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that is necessary for the G1 arrest of many cell types. Consistent with its role as a cell cycle checkpoint factor, p53 has been shown to be capable of both transcriptional activation and repression. Here we show a new potential role for p53 as a DNA-binding-dependent regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the late side of the polyomavirus origin were used in in vitro replication assays. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a mutant version of the RGC site was not affected by p53. Several tumor-derived mutant p53 proteins were unable to inhibit replication of the construct with wild-type RGC sites. Additionally, the transactivator GAL4-VP16 was unable to inhibit replication of a construct containing GAL4 binding sites adjacent to the polyomavirus origin. We also show that the inhibition by p53 can occur from sites cloned as far as 600 bp from the origin. Preincubation experiments suggest that p53 inhibits replication at a step mediated by ATP, possibly by inhibiting the binding of polyomavirus T antigen to the core origin. The presence of an endogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition. PMID:8524220

  7. Prediction of zinc finger DNA binding protein.

    PubMed

    Nakata, K

    1995-04-01

    Using the neural network algorithm with back-propagation training procedure, we analysed the zinc finger DNA binding protein sequences. We incorporated the characteristic patterns around the zinc finger motifs TFIIIA type (Cys-X2-5-Cys-X12-13-His-X2-5-His) and the steroid hormone receptor type (Cys-X2-5-Cys-X12-15-Cys-X2-5-Cys-X15-16-Cys-X4-5-Cys-X8-10- Cys-X2-3-Cys) in the neural network algorithm. The patterns used in the neural network were the amino acid pattern, the electric charge and polarity pattern, the side-chain chemical property and subproperty patterns, the hydrophobicity and hydrophilicity patterns and the secondary structure propensity pattern. Two consecutive patterns were also considered. Each pattern was incorporated in the single layer perceptron algorithm and the combinations of patterns were considered in the two-layer perceptron algorithm. As for the TFIIIA type zinc finger DNA binding motifs, the prediction results of the two-layer perceptron algorithm reached up to 96.9% discrimination, and the prediction results of the discriminant analysis using the combination of several characters reached up to 97.0%. As for the steroid hormone receptor type zinc finger, the prediction results of neural network algorithm and the discriminant analyses reached up to 96.0%.

  8. Selective binding of single-stranded DNA-binding proteins onto DNA molecules adsorbed on single-walled carbon nanotubes.

    PubMed

    Nii, Daisuke; Hayashida, Takuya; Yamaguchi, Yuuki; Ikawa, Shukuko; Shibata, Takehiko; Umemura, Kazuo

    2014-09-01

    Single-stranded DNA-binding (SSB) proteins were treated with hybrids of DNA and single-walled carbon nanotubes (SWNTs) to examine the biological function of the DNA molecules adsorbed on the SWNT surface. When single-stranded DNA (ssDNA) was used for the hybridization, significant binding of the SSB molecules to the ssDNA-SWNT hybrids was observed by using atomic force microscopy (AFM) and agarose gel electrophoresis. When double-stranded DNA (dsDNA) was used, the SSB molecules did not bind to the dsDNA-SWNT hybrids in most of the conditions that we evaluated. A specifically modified electrophoresis procedure was used to monitor the locations of the DNA, SSB, and SWNT molecules. Our results clearly showed that ssDNA/dsDNA molecules on the SWNT surfaces retained their single-stranded/double-stranded structures.

  9. E1 initiator DNA binding specificity is unmasked by selective inhibition of non-specific DNA binding

    PubMed Central

    Stenlund, Arne

    2003-01-01

    Initiator proteins are critical components of the DNA replication machinery and mark the site of initiation. This activity probably requires highly selective DNA binding; however, many initiators display modest specificity in vitro. We demonstrate that low specificity of the papillomavirus E1 initiator results from the presence of a non-specific DNA-binding activity, involved in melting, which masks the specificity intrinsic to the E1 DNA-binding domain. The viral factor E2 restores specificity through a physical interaction with E1 that suppresses non-specific binding. We propose that this arrangement, where one DNA-binding activity tethers the initiator to ori while another alters DNA structure, is a characteristic of other viral and cellular initiator proteins. This arrangement would provide an explanation for the low selectivity observed for DNA binding by initiator proteins. PMID:12574131

  10. Duplex structural differences and not 2′-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA

    PubMed Central

    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 hydroxyls and RT amino acids do not stabilize binding. RT’s Kd (koff/kon) for DNA–DNA and RNA–DNA were similar, although koff differed almost 40-fold, suggesting a faster kon for DNA–DNA. Avian myeloblastosis and Moloney murine leukemia virus RTs also bound more stably to RNA–DNA, but the difference was less pronounced than with HIV-RT. We propose that the H- versus B-form structures of RNA–DNA and DNA–DNA, respectively, allow the former to conform more easily to HIV-RT’s binding cleft, leading to more stable binding. Biologically, the ability of RT to form a more stable complex on RNA–DNA may aid in degradation of RNA fragments that remain after DNA synthesis. PMID:20338878

  11. Mutant p53 proteins bind DNA in a DNA structure-selective mode

    PubMed Central

    Göhler, Thomas; Jäger, Stefan; Warnecke, Gabriele; Yasuda, Hideyo; Kim, Ella; Deppert, Wolfgang

    2005-01-01

    Despite the loss of sequence-specific DNA binding, mutant p53 (mutp53) proteins can induce or repress transcription of mutp53-specific target genes. To date, the molecular basis for transcriptional modulation by mutp53 is not understood, but increasing evidence points to the possibility that specific interactions of mutp53 with DNA play an important role. So far, the lack of a common denominator for mutp53 DNA binding, i.e. the existence of common sequence elements, has hampered further characterization of mutp53 DNA binding. Emanating from our previous discovery that DNA structure is an important determinant of wild-type p53 (wtp53) DNA binding, we analyzed the binding of various mutp53 proteins to oligonucleotides mimicking non-B DNA structures. Using various DNA-binding assays we show that mutp53 proteins bind selectively and with high affinity to non-B DNA. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, mutp53 DNA binding to non-B DNA is solely dependent on the stereo-specific configuration of the DNA, and not on DNA sequence. We propose that DNA structure-selective binding of mutp53 proteins is the basis for the well-documented interaction of mutp53 with MAR elements and for transcriptional activities mediates by mutp53. PMID:15722483

  12. The fluorescence properties and binding mechanism of SYTOX green, a bright, low photo-damage DNA intercalating agent.

    PubMed

    Thakur, Shreyasi; Cattoni, Diego I; Nöllmann, Marcelo

    2015-07-01

    DNA intercalators are widely used in cancer therapeutics, to probe protein-DNA interactions and to investigate the statistical-mechanical properties of DNA. Here, we employ single-molecule fluorescence microscopy, magnetic tweezers, and ensemble-binding assays to investigate the fluorescence properties and binding mechanism of SYTOX green, a DNA labeling dye previously used for staining dead cells and becoming of common use for single-molecule methodologies. Specifically, we show that SYTOX green presents several advantages with respect to other dyes: (1) binds DNA rapidly and with high affinity; (2) has a good signal-to-noise ratio even at low concentrations; (3) exhibits a low photobleaching rate; and (4) induces lower light-induced DNA degradation. Finally, we show that SYTOX green is a DNA intercalator that binds DNA cooperatively with a binding site of 3.5 bp, increasing the DNA length upon binding by 43%, while not affecting its mechanical properties.

  13. HTLV-I Tax protein stimulation of DNA binding of bZIP proteins by enhancing dimerization.

    PubMed

    Wagner, S; Green, M R

    1993-10-15

    The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

  14. Flavonoid-DNA binding studies and thermodynamic parameters

    NASA Astrophysics Data System (ADS)

    Janjua, Naveed Kausar; Shaheen, Amber; Yaqub, Azra; Perveen, Fouzia; Sabahat, Sana; Mumtaz, Misbah; Jacob, Claus; Ba, Lalla Aicha; Mohammed, Hamdoon A.

    2011-09-01

    Interactional studies of new flavonoid derivatives (Fl) with chicken blood ds.DNA were investigated spectrophotometrically in DMSO-H 2O (9:1 v/v) at various temperatures. Spectral parameters suggest considerable binding between the flavonoid derivatives studied and ds.DNA. The binding constant values lie in the enhanced-binding range. Thermodynamic parameters obtained from UV studies also point to strong spontaneous binding of Fl with ds.DNA. Viscometric studies complimented the UV results where a small linear increase in relative viscosity of the DNA solution was observed with added optimal flavonoid concentration. An overall mixed mode of interaction (intercalative plus groove binding) is proposed between DNA and flavonoids. Conclusively, investigated flavonoid derivatives are found to be strong DNA binders and seem to be promising drug candidates like their natural analogues.

  15. Conserved Cysteine Residue in the DNA-Binding Domain of the Bovine Papillomavirus Type 1 E2 Protein Confers Redox Regulation of the DNA- Binding Activity in Vitro

    NASA Astrophysics Data System (ADS)

    McBride, Alison A.; Klausner, Richard D.; Howley, Peter M.

    1992-08-01

    The bovine papillomavirus type 1 E2 open reading frame encodes three proteins involved in viral DNA replication and transcriptional regulation. These polypeptides share a carboxyl-terminal domain with a specific DNA-binding activity; through this domain the E2 polypeptides form dimers. In this study, we demonstrate the inhibition of E2 DNA binding in vitro by reagents that oxidize or otherwise chemically modify the free sulfydryl groups of reactive cysteine residues. However, these reagents had no effect on DNA-binding activity when the E2 polypeptide was first bound to DNA, suggesting that the free sulfydryl group(s) may be protected by DNA binding. Sensitivity to sulfydryl modification was mapped to a cysteine residue at position 340 in the E2 DNA-binding domain, an amino acid that is highly conserved among the E2 proteins of different papillomaviruses. Replacement of this residue with other amino acids abrogated the sensitivity to oxidation-reduction changes but did not affect the DNA-binding property of the E2 protein. These results suggest that papillomavirus DNA replication and transcriptional regulation could be modulated through the E2 proteins by changes in the intracellular redox environment. Furthermore, a motif consisting of a reactive cysteine residue carboxyl-terminal to a lysine residue in a basic region of the DNA-binding domain is a feature common to a number of transcriptional regulatory proteins that, like E2, are subject to redox regulation. Thus, posttranslational regulation of the activity of these proteins by the intracellular redox environment may be a general phenomenon.

  16. MCM ring hexamerization is a prerequisite for DNA-binding

    DOE PAGES

    Froelich, Clifford A.; Nourse, Amanda; Enemark, Eric J.

    2015-09-13

    The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings to show that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in themore » hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.« less

  17. MCM ring hexamerization is a prerequisite for DNA-binding

    SciTech Connect

    Froelich, Clifford A.; Nourse, Amanda; Enemark, Eric J.

    2015-09-13

    The hexameric Minichromosome Maintenance (MCM) protein complex forms a ring that unwinds DNA at the replication fork in eukaryotes and archaea. Our recent crystal structure of an archaeal MCM N-terminal domain bound to single-stranded DNA (ssDNA) revealed ssDNA associating across tight subunit interfaces but not at the loose interfaces, indicating that DNA-binding is governed not only by the DNA-binding residues of the subunits (MCM ssDNA-binding motif, MSSB) but also by the relative orientation of the subunits. We now extend these findings to show that DNA-binding by the MCM N-terminal domain of the archaeal organism Pyrococcus furiosus occurs specifically in the hexameric oligomeric form. We show that mutants defective for hexamerization are defective in binding ssDNA despite retaining all the residues observed to interact with ssDNA in the crystal structure. One mutation that exhibits severely defective hexamerization and ssDNA-binding is at a conserved phenylalanine that aligns with the mouse Mcm4(Chaos3) mutation associated with chromosomal instability, cancer, and decreased intersubunit association.

  18. DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC–DNA binding

    PubMed Central

    Remus, Dirk; Beall, Eileen L; Botchan, Michael R

    2004-01-01

    Drosophila origin recognition complex (ORC) localizes to defined positions on chromosomes, and in follicle cells the chorion gene amplification loci are well-studied examples. However, the mechanism of specific localization is not known. We have studied the DNA binding of DmORC to investigate the cis-requirements for DmORC:DNA interaction. DmORC displays at best six-fold differences in the relative affinities to DNA from the third chorion locus and to random fragments in vitro, and chemical probing and DNase1 protection experiments did not identify a discrete binding site for DmORC on any of these fragments. The intrinsic DNA-binding specificity of DmORC is therefore insufficient to target DmORC to origins of replication in vivo. However, the topological state of the DNA significantly influences the affinity of DmORC to DNA. We found that the affinity of DmORC for negatively supercoiled DNA is about 30-fold higher than for either relaxed or linear DNA. These data provide biochemical evidence for the notion that origin specification in metazoa likely involves mechanisms other than simple replicator–initiator interactions and that in vivo other proteins must determine ORC's localization. PMID:14765124

  19. Identifying DNA Binding Motifs by Combining Data from Different Sources

    SciTech Connect

    Mao, Linyong; Resat, Haluk; Nagib Callaos; Katsuhisa Horimoto; Jake Chen; Amy Sze Chan

    2004-07-19

    A transcription factor regulates the expression of its target genes by binding to their operator regions. It functions by affecting the interactions between RNA polymerases and the gene's promoter. Many transcription factors bind to their targets by recognizing a specific DNA sequence pattern, which is referred to as a consensus sequence or a motif. Since it would remove the possible biases, combining biological data from different sources can be expected to improve the quality of the information extracted from the biological data. We analyzed the microarray gene expression data and the organism's genome sequence jointly to determine the transcription factor recognition sequences with more accuracy. Utilizing such a data integration approach, we have investigated the regulation of the photosynthesis genes of the purple non-sulphur photosynthetic bacterium Rhodobacter sphaeroides. The photosynthesis genes in this organism are tightly regulated as a function of environmental growth conditions by three major regulatory systems, PrrB/PrrA, AppA/PpsR and FnrL. In this study, we have detected a previously undefined PrrA consensus sequence, improved the previously known DNA-binding motif of PpsR, and confirmed the consensus sequence of the global regulator FnrL.

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

    SciTech Connect

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

    2011-12-09

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

  1. Equilibrium binding of single-stranded DNA to the secondary DNA binding site of the bacterial recombinase RecA.

    PubMed

    Gourves, A S; Defais, M; Johnson, N P

    2001-03-30

    The bacterial recombinase RecA forms a nucleoprotein filament in vitro with single-stranded DNA (ssDNA) at its primary DNA binding site, site I. This filament has a second site, site II, which binds ssDNA and double-stranded DNA. We have investigated the binding of ssDNA to the RecA protein in the presence of adenosine 5'-O-(thiotriphosphate) cofactor using fluorescence anisotropy. The RecA protein carried out DNA strand exchange with a 5'-fluorescein-labeled 32-mer oligonucleotide. The anisotropy signal was shown to measure oligonucleotide binding to RecA, and the relationship between signal and binding density was determined. Binding of ssDNA to site I of RecA was stable at high NaCl concentrations. Binding to site II could be described by a simple two-state equilibrium, K = 4.5 +/- 1.5 x 10(5) m(-1) (37 degrees C, 150 mm NaCl, pH 7.4). The reaction was enthalpy-driven and entropy-opposed. It depended on salt concentration and was sensitive to the type of monovalent anion, suggesting that anion-dependent protein conformations contribute to ssDNA binding at site II.

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

    PubMed

    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).

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

    SciTech Connect

    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% NP-40.

  4. Binding of globular proteins to DNA from surface tension measurement.

    PubMed

    Mitra, A; Chattoraj, D K; Chakraborty, P

    2001-10-01

    Extent of binding (gammap) of globular proteins to calf-thymus DNA have been measured in mole per mole of nucleotide as function of equilibrium protein concentration. We have exploited measurement of the surface tension of the protein solution in the presence and absence of DNA to calculate the binding ration (gammap). Interaction of bovine serum albumin with DNA has been studied at different pH. Interaction of bovine serum albumin with DNA has been studied at different pH, ionic strength and in presence of Ca2+. Interaction of BSA with denatured DNA has also been investigated. Binding isotherms for other globular proteins like beta-lactoglobulin, alpha-lactalbumin and lysozyme have been compared under identical physicochemical condition. It has been noted with considerable interest that globular form of protein is important to some extent in protein-DNA interaction. An attempt has been made to explain the significance of difference in binding ratios of these two biopolymers in aqueous medium for different systems in the light of electrostatic and hydrophobic effects. Values of maximum binding ration (gammap(m)) at saturated level for different systems have been also presented. The Gibb's free energy decrease (-deltaG0) of the binding of proteins to DNA has been compared more precisely for the saturation of binding sites in the DNA with the change of activity of protein in solution from zero to unity in the rational mole fraction scale.

  5. Molecular mechanisms of conformational specificity: A study of Hox in vivo target DNA binding specificities and the structure of a Ure2p mutation that affects fibril formation rates

    NASA Astrophysics Data System (ADS)

    Bauer, William Joseph, Jr.

    The fate of an individual cell, or even an entire organism, is often determined by minute, yet very specific differences in the conformation of a single protein species. Very often, proteins take on alternate folds or even side chain conformations to deal with different situations present within the cell. These differences can be as large as a whole domain or as subtle as the alteration of a single amino acid side chain. Yet, even these seemingly minor side chain conformational differences can determine the development of a cell type during differentiation or even dictate whether a cell will live or die. Two examples of situations where minor conformational differences within a specific protein could lead to major differences in the life cycle of a cell are described herein. The first example describes the variations seen in DNA conformations which can lead to slightly different Hox protein binding conformations responsible for recognizing biologically relevant regulatory sites. These specific differences occur in the minor groove of the bound DNA and are limited to the conformation of only two side chains. The conformation of the bound DNA, however, is not solely determined by the sequence of the DNA, as multiple sequences can result in the same DNA conformation. The second example takes place in the context of a yeast prion protein which contains a mutation that decreases the frequency at which fibrils form. While the specific interactions leading to this physiological change were not directly detected, it can be ascertained from the crystal structure that the structural changes are subtle and most likely involve another binding partner. In both cases, these conformational changes are very slight but have a profound effect on the downstream processes.

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

    PubMed

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-02-15

    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 CONH2 groups (CONH2-SWNT) exhibited very strong interactions between the CONH2-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.

  7. The quinobenzoxazines: relationship between DNA binding and biological activity.

    PubMed

    Kwok, Y; Sun, D; Clement, J J; Hurley, L H

    1999-10-01

    The quinobenzoxazine compounds, derived from antibacterial quinolones, is active in vitro and in vivo against murine and human tumors. In this contribution, we show that the relative DNA binding affinity of the quinobenzoxazine compounds correlates with their cytotoxicity, their ability to inhibit gyrase-DNA complex formation, and the decatenation of kinetoplast DNA by human topoisomerase II. DNA binding studies with the descarboxy-A-62176 analogue indicate that the beta-keto acid moiety of the quinobenzoxazine compounds plays an important role in their interaction with DNA.

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

    PubMed

    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.

  9. Variola type IB DNA topoisomerase: DNA binding and supercoil unwinding using engineered DNA minicircles.

    PubMed

    Anderson, Breeana G; Stivers, James T

    2014-07-08

    Type IB topoisomerases unwind positive and negative DNA supercoils and play a key role in removing supercoils that would otherwise accumulate at replication and transcription forks. An interesting question is whether topoisomerase activity is regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The type IB enzyme from variola virus (vTopo) has proven to be useful in addressing mechanistic questions about topoisomerase function because it forms a reversible 3'-phosphotyrosyl adduct with the DNA backbone at a specific target sequence (5'-CCCTT-3') from which DNA unwinding can proceed. We have synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site that provides highly defined substrates for exploring the effects of supercoil density on DNA binding, strand cleavage and ligation, and unwinding. We observed no topological dependence for binding of vTopo to these supercoiled MC DNAs, indicating that affinity-based targeting to supercoiled DNA regions by vTopo is unlikely. Similarly, the cleavage and religation rates of the MCs were not topologically dependent, but topoisomers with low superhelical densities were found to unwind more slowly than highly supercoiled topoisomers, suggesting that reduced torque at low superhelical densities leads to an increased number of cycles of cleavage and ligation before a successful unwinding event. The K271E charge reversal mutant has an impaired interaction with the rotating DNA segment that leads to an increase in the number of supercoils that were unwound per cleavage event. This result provides evidence that interactions of the enzyme with the rotating DNA segment can restrict the number of supercoils that are unwound. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to be important in

  10. [Features of binding of proflavine to DNA at different DNA-ligand concentration ratios].

    PubMed

    Berezniak, E G; gladkovskaia, N A; Khrebtova, A S; Dukhopel'nikov, E V; Zinchenko, A V

    2009-01-01

    The binding of proflavine to calf thymus DNA has been studied using the methods of differential scanning calorimetry and spectrophotometry. It was shown that proflavine can interact with DNA by at least 3 binding modes. At high DNA-ligand concentration ratios (P/D), proflavine intercalates into both GC- and AT-sites, with a preference to GC-rich sequences. At low P/D ratios proflavine interacts with DNA by the external binding mode. From spectrophotometric concentration dependences, the parameters of complexing of proflavine with DNA were calculated. Thermodynamic parameters of DNA melting were calculated from differential scanning calorimetry data.

  11. Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

    PubMed Central

    Pakula, T M; Caldentey, J; Serrano, M; Gutierrez, C; Hermoso, J M; Salas, M; Bamford, D H

    1990-01-01

    Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication. Images PMID:2251117

  12. Fibronectin inhibits cytokine production induced by CpG DNA in macrophages without direct binding to DNA.

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23610127

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

    SciTech Connect

    Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; Chattopadhyay, Debasish

    2015-06-02

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

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

    DOE PAGES

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

    2015-06-02

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

  17. The yeast telomere-binding protein RAP1 binds to and promotes the formation of DNA quadruplexes in telomeric DNA.

    PubMed Central

    Giraldo, R; Rhodes, D

    1994-01-01

    The protein RAP1 is essential for the maintenance of the telomeres of Saccharomyces cerevisiae and binds in vitro to multiple sites found within the TG1-3 telomeric repeats. We show here that, in addition to its known binding activity for double-stranded DNA, RAP1 binds sequence-specifically to the GT-strands. This indicates that RAP1 is the protein that binds to the telomeric terminal GT-tails. Furthermore, we have found that RAP1 binds to and promotes the formation of G-tetrads, i.e. DNA quadruplexes, in GT-strand oligonucleotides at nanomolar concentrations. The formation of DNA quadruplexes appears to involve the intermolecular association of GT-strands. The minimal DNA-binding domain of RAP1 (DBD) binds only to double-stranded DNA, so that the novel DNA-binding activity we have found involves regions of the protein located outside of the DBD. The finding that a telomeric protein promotes the formation of G-tetrads argues for the use of DNA quadruplexes in telomere association. Images PMID:8194531

  18. Crystal structure of the adenovirus DNA binding protein reveals a hook-on model for cooperative DNA binding.

    PubMed Central

    Tucker, P A; Tsernoglou, D; Tucker, A D; Coenjaerts, F E; Leenders, H; van der Vliet, P C

    1994-01-01

    The adenovirus single-stranded DNA binding protein (Ad DBP) is a multifunctional protein required, amongst other things, for DNA replication and transcription control. It binds to single- and double-stranded DNA, as well as to RNA, in a sequence-independent manner. Like other single-stranded DNA binding proteins, it binds ssDNA, cooperatively. We report the crystal structure, at 2.6 A resolution, of the nucleic acid binding domain. This domain is active in DNA replication. The protein contains two zinc atoms in different, novel coordinations. The zinc atoms appear to be required for the stability of the protein fold rather than being involved in direct contacts with the DNA. The crystal structure shows that the protein contains a 17 amino acid C-terminal extension which hooks onto a second molecule, thereby forming a protein chain. Deletion of this C-terminal arm reduces cooperativity in DNA binding, suggesting a hook-on model for cooperativity. Based on this structural work and mutant studies, we propose that DBP forms a protein core around which the single-stranded DNA winds. Images PMID:8039495

  19. Physical factors affecting chloroquine binding to melanin.

    PubMed

    Schroeder, R L; Pendleton, P; Gerber, J P

    2015-10-01

    Chloroquine is an antimalarial drug but is also prescribed for conditions such as rheumatoid arthritis. Long-term users risk toxic side effects, including retinopathy, thought to be caused by chloroquine accumulation on ocular melanin. Although the binding potential of chloroquine to melanin has been investigated previously, our study is the first to demonstrate clear links between chloroquine adsorption by melanin and system factors including temperature, pH, melanin type, and particle size. In the current work, two Sepia melanins were compared with bovine eye as a representative mammalian melanin. Increasing the surface anionic character due to a pH change from 4.7 to 7.4 increased each melanin's affinity for chloroquine. Although the chloroquine isotherms exhibited an apparently strong interaction with each melanin, isosteric heat analysis indicated a competitive interaction. Buffer solution cations competed effectively at low surface coverage; chloroquine adsorption occurs via buffer cation displacement and is promoted by temperature-influenced secondary structure swelling.

  20. DNA Topoisomerase Iα Affects the Floral Transition1[OPEN

    PubMed Central

    Gong, Ximing; Shen, Lisha; Peng, Ya Zhi; Gan, Yinbo

    2017-01-01

    DNA topoisomerases modulate DNA topology to maintain chromosome superstructure and genome integrity, which is indispensable for DNA replication and RNA transcription. Their function in plant development still remains largely unknown. Here, we report a hitherto unidentified role of Topoisomerase Iα (TOP1α) in controlling flowering time in Arabidopsis (Arabidopsis thaliana). Loss of function of TOP1α results in early flowering under both long and short days. This is attributed mainly to a decrease in the expression of a central flowering repressor, FLOWERING LOCUS C (FLC), and its close homologs, MADS AFFECTING FLOWERING4 (MAF4) and MAF5, during the floral transition. TOP1α physically binds to the genomic regions of FLC, MAF4, and MAF5 and promotes the association of RNA polymerase II complexes to their transcriptional start sites. These correlate with the changes in histone modifications but do not directly affect nucleosome occupancy at these loci. Our results suggest that TOP1α mediates DNA topology to facilitate the recruitment of RNA polymerase II at FLC, MAF4, and MAF5 in conjunction with histone modifications, thus facilitating the expression of these key flowering repressors to prevent precocious flowering in Arabidopsis. PMID:27837087

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

    NASA Astrophysics Data System (ADS)

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

    PubMed

    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.

  3. A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA.

    PubMed

    Yang, William C; Swartz, James R

    2011-08-15

    We present a rapid method for quantifying the apparent DNA binding affinity and capacity of recombinant transcription factors (TFs). We capture His6-tagged TFs using nickel-nitrilotriacetic acid (Ni-NTA) agarose and incubate the immobilized TFs with fluorescently labeled cognate DNA probes. After washing, the strength of the fluorescence signal indicates the extent of DNA binding. The assay was validated using two pluripotency-regulating TFs: SOX2 and NANOG. Using competitive binding analysis with nonlabeled competitor DNA, we show that SOX2 and NANOG specifically bind to their consensus sequences. We also determined the apparent affinity of SOX2 and NANOG for their consensus sequences to be 54.2±9 and 44.0±6nM, respectively, in approximate agreement with literature values. Our assay does not require radioactivity, but radioactively labeling the TFs enables the measurement of absolute amounts of immobilized SOX2 and NANOG and, hence, a DNA-to-protein binding ratio. SOX2 possesses a 0.95 DNA-to-protein binding ratio, whereas NANOG possesses a 0.44 ratio, suggesting that most of the SOX2 and approximately half of the NANOG are competent for DNA binding. Alternatively, the NANOG dimer may be capable of binding only one DNA target. This flexible DNA binding assay enables the analysis of crude or purified samples with or without radioactivity.

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

    PubMed

    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.

  5. CCAAT box binding protein NF-Y facilitates in vivo recruitment of upstream DNA binding transcription factors.

    PubMed Central

    Wright, K L; Vilen, B J; Itoh-Lindstrom, Y; Moore, T L; Li, G; Criscitiello, M; Cogswell, P; Clarke, J B; Ting, J P

    1994-01-01

    NF-Y binds a CCAAT motif found in many eukaryotic polymerase II-dependent promoters. In the HLA-DRA promoter it has been demonstrated that stereo-specific alignment between this motif and the upstream elements X1 and X2 is required for activation. To study the underlying mechanism for this requirement, a panel of transfected cell lines that maintained integrated, wild-type and mutant promoters were analyzed by in vivo genomic footprinting. Cell lines harboring a mutated CCAAT element exhibited a loss of interactions at the CCAAT site, as expected, and no transcriptional activity. Most importantly, mutation of the CCAAT sequence nearly abolished in vivo binding at the X1 and X2 sites, while mutations of X1 and X2 had little effect on CCAAT box binding. However, X1 and X2 binding was interdependent. In vitro, X1 binding activities are known to be stabilized by NF-Y binding. Interaction between NF-Y and X box binding proteins was demonstrated by reciprocal co-immunoprecipitation in the absence of DNA and co-affinity purification in the presence of DNA. Collectively, these studies indicate that occupancy of the CCAAT element represents an early event affecting other protein-DNA interactions and suggest that NF-Y stabilizes and interacts with X box factors to mediate this function. These findings may represent a common theme among promoters containing a CCAAT element. Images PMID:8076600

  6. Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ)

    PubMed Central

    Kim, Doyoun; Hur, Jeonghwan; Park, Kwangsoo; Bae, Sangsu; Shin, Donghyuk; Ha, Sung Chul; Hwang, Hye-Yeon; Hohng, Sungchul; Lee, Joon-Hwa; Lee, Sangho; Kim, Yang-Gyun; Kim, Kyeong Kyu

    2014-01-01

    Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Zα) of PKZ from Carassius auratus (caZαPKZ). The 1.7-Å resolution crystal structure of caZαPKZ:Z-DNA revealed that caZαPKZ shares the overall fold with other Zα, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZαPKZ and its mutants revealed that caZαPKZ mediates the fastest B-to-Z transition of DNA among Zα, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZαPKZ. Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the β-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZαPKZ further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the β-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate. PMID:24682817

  7. Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures.

    PubMed

    Aktas, Gülsen Betül; Skouridou, Vasso; Masip, Lluis

    2017-03-22

    A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the

  8. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, S.; Richardson, C.

    1997-03-25

    A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.

  9. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  10. Mechanochemical regulations of RPA's binding to ssDNA

    NASA Astrophysics Data System (ADS)

    Chen, Jin; Le, Shimin; Basu, Anindita; Chazin, Walter J.; Yan, Jie

    2015-03-01

    Replication protein A (RPA) is a ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein that serves to protect ssDNA from degradation and annealing, and as a template for recruitment of many downstream factors in virtually all DNA transactions in cell. During many of these transactions, DNA is tethered and is likely subject to force. Previous studies of RPA's binding behavior on ssDNA were conducted in the absence of force; therefore the RPA-ssDNA conformations regulated by force remain unclear. Here, using a combination of atomic force microscopy imaging and mechanical manipulation of single ssDNA tethers, we show that force mediates a switch of the RPA bound ssDNA from amorphous aggregation to a much more regular extended conformation. Further, we found an interesting non-monotonic dependence of the binding affinity on monovalent salt concentration in the presence of force. In addition, we discovered that zinc in micromolar concentrations drives ssDNA to a unique, highly stiff and more compact state. These results provide new mechanochemical insights into the influences and the mechanisms of action of RPA on large single ssDNA.

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

    SciTech Connect

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

    2011-09-20

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

  12. Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy

    ERIC Educational Resources Information Center

    Healy, Eamonn F.

    2007-01-01

    The effective use of fluorescence spectroscopy for determining the binding of the intercalcating agent crhidium bromide to DNA is being described. The analysis used simple measurement techniques and hence can be easily adopted by the students for a better understanding.

  13. Nitropyrene: DNA binding and adduct formation in respiratory tissues.

    PubMed Central

    Jackson, M A; King, L C; Ball, L M; Ghayourmanesh, S; Jeffrey, A M; Lewtas, J

    1985-01-01

    Binding of 1-nitro (14C)pyrene (NP) or its metabolites to cellular DNA and protein in cultures of rabbit alveolar macrophages, lung tissue, and tracheal tissue was examined. DNA binding in tracheal tissue (136 +/- 18.3 pmole NP/mg DNA) was four to five times the levels measured in either lung tissue (38 +/- 9.4 pmole NP/mg DNA) or macrophages (26 +/- 7.5 pmole NP/mg DNA). Adduct analysis of DNA isolated from lung tissue incubated with 1-nitro[H3]pyrene in vitro resulted in the identification of 2 to 5% of the NP adducts as C8-deoxyguanosine 1-aminopyrene. NP was also bound to cellular protein in tracheal tissue and lung tissue, and at a lower level in macrophages. Cocultivation of the macrophages with lung and tracheal tissue decreased the DNA binding in tracheal tissue by 45%. Following intratracheal instillation of diesel particles (5 mg) vapor-coated with 14C-NP (380 ppm, 0.085 muCi/mg) particles into rats, 5-8% of the radioactivity remained in the lungs after 20 hr. Most of the diesel particles were also deposited in the lung. Examination of DNA and protein binding in this tissue showed 5 to 12% of the pulmonary 14C bound to protein and no detectable levels of 14C bound to DNA. PMID:3841313

  14. Structural basis for DNA binding by replication initiator Mcm10

    SciTech Connect

    Warren, Eric M.; Vaithiyalingam, Sivaraja; Haworth, Justin; Greer, Briana; Bielinsky, Anja-Katrin; Chazin, Walter J.; Eichman, Brandt F.

    2009-06-30

    Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae result in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.

  15. Structures of apo IRF-3 and IRF-7 DNA binding domains: effect of loop L1 on DNA binding

    SciTech Connect

    De Ioannes, Pablo; Escalante, Carlos R.; Aggarwal, Aneel K.

    2013-11-20

    Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-{beta} (IFN-{beta}) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-{beta} promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures of IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.

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

    PubMed Central

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

    2016-01-01

    Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling. PMID:27604871

  17. Thermodynamics of cationic lipid binding to DNA and DNA condensation: roles of electrostatics and hydrophobicity.

    PubMed

    Matulis, Daumantas; Rouzina, Ioulia; Bloomfield, Victor A

    2002-06-26

    Alkylammonium binding to DNA was studied by isothermal titration calorimetry. Experimental data, obtained as functions of alkyl chain length, salt concentration, DNA concentration, and temperature, provided a detailed thermodynamic description of lipid-DNA binding reactions leading to DNA condensation. Lipid binding, counterion displacement, and DNA condensation were highly cooperative processes, driven by a large increase in entropy and opposed by a relatively small endothermic enthalpy at room temperature. Large negative heat capacity change indicated a contribution from hydrophobic interactions between aliphatic tails. An approximation of lipid-DNA binding as dominated by two factors-ionic and hydrophobic interactions-yielded a model that was consistent with experimental data. Chemical group contributions to the energetics of binding were determined and could be used to predict energetics of other lipid binding to DNA. Electrostatic and hydrophobic contributions to Gibbs free energy, enthalpy, entropy, and heat capacity could be distinguished by applying additivity principles. Binding of lipids with two, three, and four aliphatic tails was investigated and compared to single-tailed lipid binding. Structurally, the model suggests that lipid cationic headgroups and aliphatic tails distribute evenly and lay down on DNA surface without the formation of micelles.

  18. Erythroblast transformation by FLI-1 depends upon its specific DNA binding and transcriptional activation properties.

    PubMed

    Ano, Sabine; Pereira, Rui; Pironin, Martine; Lesault, Isabelle; Milley, Caroline; Lebigot, Ingrid; Quang, Christine Tran; Ghysdael, Jacques

    2004-01-23

    FLI-1 is a transcriptional regulator of the ETS family of proteins. Insertional activation at the FLI-1 locus is an early event in F-murine leukemia virus-induced erythroleukemia. Consistent with its essential role in erythroid transformation, enforced expression of FLI-1 in primary erythroblasts strongly impairs the response of these cells to erythropoietin (Epo), a cytokine essential to erythropoiesis. We show here that point mutations in the ETS domain that abolished FLI-1 binding to specific DNA elements (ETS-binding sites) suppressed the ability of FLI-1 to transform erythroblasts. The exchange of the entire ETS domain (DNA binding domain) of FLI-1 for that of PU.1 changed the DNA binding specificity of FLI-1 for that of PU.1 and impaired FLI-1 transforming properties. In contrast, ETS domain swapping mutants that maintained the DNA binding specificity of FLI-1 did not affect the ability of FLI-1 to transform erythroblasts. Deletion and swapping mutants that failed to inhibit the DNA binding activity of FLI-1 but impaired its transcriptional activation properties were also transformation-defective. Taken together, these results show that both the ability of FLI-1 to inhibit Epo-induced differentiation of erythroblasts and to confer enhanced cell survival in the absence of Epo critically depend upon FLI-1 ETS-binding site-dependent transcriptional activation properties.

  19. ssDNA binding reveals the atomic structure of graphene.

    PubMed

    Husale, By Sudhir; Sahoo, Sangeeta; Radenovic, Aleksandra; Traversi, Floriano; Annibale, Paolo; Kis, Andras

    2010-12-07

    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 SiO(2) substrate, confirming that the binding energy is mainly due to the π-π stacking interaction. Furthermore, deposited ssDNA will map the graphene underlying structure. We also quantify the π-π stacking interaction by correlating the amount of deposited DNA with the graphene layer thickness. Our findings agree with reported electrostatic force microscopy (EFM) measurements. Finally, we inspected the suitability of using a graphene as a substrate for DNA origami-based nanostructures.

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

    PubMed

    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.

  1. DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

    PubMed Central

    Salas, Margarita; Holguera, Isabel; Redrejo-Rodríguez, Modesto; de Vega, Miguel

    2016-01-01

    Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5′ ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3′–5′ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and

  2. Defining a minimal estrogen receptor DNA binding domain.

    PubMed Central

    Mader, S; Chambon, P; White, J H

    1993-01-01

    The estrogen receptor (ER) is a transcriptional regulator which binds to cognate palindromic DNA sequences known as estrogen response elements (EREs). A 66 amino acid core region which contains two zinc fingers and is highly conserved among the nuclear receptors is essential for site specific DNA recognition. However, it remains unclear how many flanking amino acids in addition to the zinc finger core are required for DNA binding. Here, we have characterized the minimal DNA binding region of the human ER by analysing the DNA binding properties of a series of deletion mutants expressed in bacteria. We find that the 66 amino acid zinc finger core of the DBD fails to bind DNA, and that the C-terminal end of the minimal ER DBD required for binding to perfectly palindromic EREs corresponds to the limit of 100% amino acid homology between the chicken and human receptors, which represents the boundary between regions C and D in the ER. Moreover, amino acids of region D up to 30 residues C-terminal to the zinc fingers greatly stabilize DNA binding by the DBD to perfectly palindromic EREs and are absolutely required for formation of gel retardation complexes by the DBD on certain physiological imperfectly palindromic EREs. These results indicate that in addition to the zinc finger core, amino acids C-terminal to the core in regions C and D play a key role in DNA binding by the ER, particularly to imperfectly palindromic response elements. The ER DBD expressed in E. coli binds as a dimer to ERE palindromes in a highly cooperative manner and forms only low levels of monomeric protein-DNA complexes on either palindromic or half-palindromic response elements. Conversion of ER amino acids 222 to 226, which lie within region C, to the corresponding residues of the human RAR alpha abolishes formation of dimeric protein-DNA complexes. Conversely, replacement of the same region of RAR alpha with ER residues 222 to 226 creates a derivative that, unlike the RAR alpha DBD, binds

  3. DNA Conforming Dynamics and Protein Binding

    DTIC Science & Technology

    2006-12-01

    spectroscopy". We repeat this Introduction here for completeness. The Watson - Crick double- helix is the thermodynamically stable configuration of a DNA ...molecule under physiological conditions (normal salt and room/body temperature). This stability is effected (a) by Watson - Crick H-bonding, that is...contribution to DNA helix stability comes from base-stacking between neighboring base pairs: through hydrophobic interactions between the planar aromatic

  4. Zinc-binding Domain of the Bacteriophage T7 DNA Primase Modulates Binding to the DNA Template*

    PubMed Central

    Lee, Seung-Joo; Zhu, Bin; Akabayov, Barak; Richardson, Charles C.

    2012-01-01

    The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis. PMID:23024359

  5. DNA-binding properties of ARID family proteins

    PubMed Central

    Patsialou, Antonia; Wilsker, Deborah; Moran, Elizabeth

    2005-01-01

    The ARID (A–T Rich Interaction Domain) is a helix–turn–helix motif-based DNA-binding domain, conserved in all eukaryotes and diagnostic of a family that includes 15 distinct human proteins with important roles in development, tissue-specific gene expression and proliferation control. The 15 human ARID family proteins can be divided into seven subfamilies based on the degree of sequence identity between individual members. Most ARID family members have not been characterized with respect to their DNA-binding behavior, but it is already apparent that not all ARIDs conform to the pattern of binding AT-rich sequences. To understand better the divergent characteristics of the ARID proteins, we undertook a survey of DNA-binding properties across the entire ARID family. The results indicate that the majority of ARID subfamilies (i.e. five out of seven) bind DNA without obvious sequence preference. DNA-binding affinity also varies somewhat between subfamilies. Site-specific mutagenesis does not support suggestions made from structure analysis that specific amino acids in Loop 2 or Helix 5 are the main determinants of sequence specificity. Most probably, this is determined by multiple interacting differences across the entire ARID structure. PMID:15640446

  6. Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences

    PubMed Central

    Wright, Elisé P.; Day, Henry A.; Ibrahim, Ali M.; Kumar, Jeethendra; Boswell, Leo J. E.; Huguin, Camille; Stevenson, Clare E. M.; Pors, Klaus; Waller, Zoë A. E.

    2016-01-01

    There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences. PMID:28004744

  7. Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences

    NASA Astrophysics Data System (ADS)

    Wright, Elisé P.; Day, Henry A.; Ibrahim, Ali M.; Kumar, Jeethendra; Boswell, Leo J. E.; Huguin, Camille; Stevenson, Clare E. M.; Pors, Klaus; Waller, Zoë A. E.

    2016-12-01

    There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences.

  8. Protein-DNA binding in high-resolution

    PubMed Central

    Mahony, Shaun; Pugh, B. Franklin

    2015-01-01

    Recent advances in experimental and computational methodologies are enabling ultra-high resolution genome-wide profiles of protein-DNA binding events. For example, the ChIP-exo protocol precisely characterizes protein-DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5′ → 3′ exonuclease digestion. Similarly, deeply sequenced chromatin accessibility assays (e.g. DNase-seq and ATACseq) enable the detection of protected footprints at protein-DNA binding sites. With these techniques and others, we have the potential to characterize the individual nucleotides that interact with transcription factors, nucleosomes, RNA polymerases, and other regulatory proteins in a particular cellular context. In this review, we explain the experimental assays and computational analysis methods that enable high-resolution profiling of protein-DNA binding events. We discuss the challenges and opportunities associated with such approaches. PMID:26038153

  9. Effect of clustered peptide binding on DNA condensation.

    PubMed

    Haley, Jennifer; Kabiru, Paul; Geng, Yan

    2010-01-01

    DNA condensation in-vitro has been studied as a model system to reveal common principles underlying gene packaging in biology, and as the critical first step towards the development of non-viral gene delivery vectors. In this study, we use a bio-inspired approach, where small DNA-binding peptides are controllably clustered by an amphiphilic block copolymer scaffold, to reveal the effect of clustered peptide binding on the energetics, size, shape and physical properties of DNA condensation in-vitro. This provides insights into the general architectural effect of gene-binding proteins on DNA condensation process. Moreover, the versatility afforded by regulating the clustering density and composition of peptides may provide a novel design platform for gene delivery applications in the future.

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

    PubMed

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

    2011-05-01

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

  11. Structure-based Analysis to Hu-DNA Binding

    SciTech Connect

    Swinger,K.; Rice, P.

    2007-01-01

    HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from 10-14.5 kcal/mol, representing K{sub d} values in the nanomolar to low picomolar range, and a maximum stabilization of at least 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.

  12. Structure-based analysis of HU-DNA binding.

    PubMed

    Swinger, Kerren K; Rice, Phoebe A

    2007-01-26

    HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently published Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from approximately 10-14.5 kcal/mol, representing K(d) values in the nanomolar to low picomolar range, and a maximum stabilization of at least approximately 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.

  13. Effects of DNA-binding drugs on T4 DNA ligase.

    PubMed Central

    Montecucco, A; Pedrali-Noy, G; Spadari, S; Lestingi, M; Ciarrocchi, G

    1990-01-01

    A number of DNA intercalating and externally binding drugs have been found to inhibit nick sealing, cohesive and blunt end ligation, AMP-dependent DNA topoisomerization and EDTA-induced DNA nicking mediated by bacteriophage T4 DNA ligase. The inhibition seems to arise from drug-substrate interaction so that formation of active DNA-Mg2(+)-AMP-enzyme complex is impaired while assembled and active complexes are not disturbed by drug binding to the substrate. Images Fig. 2. Fig. 4. Fig. 5. PMID:2156493

  14. SA1 and TRF1 synergistically bind to telomeric DNA and promote DNA-DNA pairing

    NASA Astrophysics Data System (ADS)

    Wang, Hong; Lin, Jiangguo; Countryman, Preston; Pan, Hai; Parminder Kaur Team; Robert Riehn Team; Patricia Opresko Team; Jane Tao Team; Susan Smith Team

    Impaired telomere cohesion leads to increased aneuploidy and early onset of tumorigenesis. Cohesion is thought to occur through the entrapment of two DNA strands within tripartite cohesin ring(s), along with a fourth subunit (SA1/SA2). Surprisingly, cohesion rings are not essential for telomere cohesion, which instead requires SA1 and shelterin proteins including TRF1. However, neither this unique cohesion mechanism at telomeres or DNA-binding properties of SA1 is understood. Here, using single-molecule fluorescence imaging of quantum dot-labeled proteins on DNA we discover that while SA1 diffuses across multiple telomeric and non-telomeric regions, the diffusion mediated through its N-terminal domain is slower at telomeric regions. However, addition of TRF1 traps SA1 within telomeric regions, which form longer DNA-DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy. Together, these experimental results and coarse-grained molecular dynamics simulations suggest that TRF1 and SA1 synergistically interact with DNA to support telomere cohesion without cohesin rings.

  15. Novel DNA-binding properties of the RNA-binding protein TIAR.

    PubMed

    Suswam, Esther A; Li, Yan Yan; Mahtani, Harry; King, Peter H

    2005-01-01

    TIA-1 related protein binds avidly to uridine-rich elements in mRNA and pre-mRNAs of a wide range of genes, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). The protein has diverse regulatory roles, which in part depend on the locus of binding within the transcript, including translational control, splicing and apoptosis. Here, we observed selective and potent inhibition of TIAR-RNP complex formation with IL-8 and VEGF 3'-untranslated regions (3'-UTRs) using thymidine-rich deoxyoligonucleotide (ODN) sequences derived from the VEFG 3'-UTR. We show by ultraviolet crosslinking and electrophoretic mobility shift assays that TIAR can bind directly to single-stranded, thymidine-rich ODNs but not to double-stranded ODNs containing the same sequence. TIAR had a nearly 6-fold greater affinity for DNA than RNA (K(d)app = 1.6x10(-9) M versus 9.4 x 10(-9) M). Truncation of TIAR indicated that the high affinity DNA-binding site overlaps with the RNA-binding site involving RNA recognition motif 2 (RRM2). However, RRM1 alone could also bind to DNA. Finally, we show that TIAR can be displaced from single-stranded DNA by active transcription through the binding site. These results provide a potential mechanism by which TIAR can shuttle between RNA and DNA ligands.

  16. High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein.

    PubMed

    Morten, Michael J; Gamsjaeger, Roland; Cubeddu, Liza; Kariawasam, Ruvini; Peregrina, Jose; Penedo, J Carlos; White, Malcolm F

    2017-03-01

    Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.

  17. Dynamics of nucleosome invasion by DNA binding proteins.

    PubMed

    Tims, Hannah S; Gurunathan, Kaushik; Levitus, Marcia; Widom, Jonathan

    2011-08-12

    Nucleosomes sterically occlude their wrapped DNA from interacting with many large protein complexes. How proteins gain access to nucleosomal DNA target sites in vivo is not known. Outer stretches of nucleosomal DNA spontaneously unwrap and rewrap with high frequency, providing rapid and efficient access to regulatory DNA target sites located there; however, rates for access to the nucleosome interior have not been measured. Here we show that for a selected high-affinity nucleosome positioning sequence, the spontaneous DNA unwrapping rate decreases dramatically with distance inside the nucleosome. The rewrapping rate also decreases, but only slightly. Our results explain the previously known strong position dependence on the equilibrium accessibility of nucleosomal DNA, which is characteristic of both selected and natural sequences. Our results point to slow nucleosome conformational fluctuations as a potential source of cell-cell variability in gene activation dynamics, and they reveal the dominant kinetic path by which multiple DNA binding proteins cooperatively invade a nucleosome.

  18. Postranslational modifications significantly alter the binding-folding pathways of proteins associating with DNA

    NASA Astrophysics Data System (ADS)

    Papoian, Garegin

    2012-02-01

    Many important regulators of gene activity are natively disordered, but fully or partially order when they bind to their targets on DNA. Interestingly, the ensembles of disordered states for such free proteins are not structurally featureless, but can qualitatively differ from protein to protein. In particular, in random coil like states the chains are swollen, making relatively few contacts, while in molten globule like states a significant collapse occurs, with ensuing high density of intra-protein interactions. Furthermore, since many DNA binding proteins are positively charged polyelectrolytes, the electrostatic self-repulsion also influences the degree of collapse of the chain and its conformational preferences in the free state and upon binding to DNA. In our work, we have found that the nature of the natively disordered ensemble significantly affects the way the protein folds upon binding to DNA. In particular, we showed that posttranslational modifications of amino acid residues, such as lysine acetylation, can alter the degree of collapse and conformational preferences for a free protein, and also profoundly impact the binding affinity and pathways for the protein DNA association. These trends will be discussed in the context of DNA interacting with various histone tails and the p53 protein.

  19. Predicting DNA-Binding Specificities of Eukaryotic Transcription Factors

    PubMed Central

    Schröder, Adrian; Eichner, Johannes; Supper, Jochen; Eichner, Jonas; Wanke, Dierk; Henneges, Carsten; Zell, Andreas

    2010-01-01

    Today, annotated amino acid sequences of more and more transcription factors (TFs) are readily available. Quantitative information about their DNA-binding specificities, however, are hard to obtain. Position frequency matrices (PFMs), the most widely used models to represent binding specificities, are experimentally characterized only for a small fraction of all TFs. Even for some of the most intensively studied eukaryotic organisms (i.e., human, rat and mouse), roughly one-sixth of all proteins with annotated DNA-binding domain have been characterized experimentally. Here, we present a new method based on support vector regression for predicting quantitative DNA-binding specificities of TFs in different eukaryotic species. This approach estimates a quantitative measure for the PFM similarity of two proteins, based on various features derived from their protein sequences. The method is trained and tested on a dataset containing 1 239 TFs with known DNA-binding specificity, and used to predict specific DNA target motifs for 645 TFs with high accuracy. PMID:21152420

  20. pH-dependent specific binding and combing of DNA.

    PubMed Central

    Allemand, J F; Bensimon, D; Jullien, L; Bensimon, A; Croquette, V

    1997-01-01

    Recent developments in the rapid sequencing, mapping, and analysis of DNA rely on the specific binding of DNA to specially treated surfaces. We show here that specific binding of DNA via its unmodified extremities can be achieved on a great variety of surfaces by a judicious choice of the pH. On hydrophobic surfaces the best binding efficiency is reached at a pH of approximately 5.5. At that pH a approximately 40-kbp DNA is 10 times more likely to bind by an extremity than by a midsegment. A model is proposed to account for the differential adsorption of the molecule extremities and midsection as a function of pH. The pH-dependent specific binding can be used to align anchored DNA molecules by a receding meniscus, a process called molecular combing. The resulting properties of the combed molecules will be discussed. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 6 FIGURE 7 PMID:9336201

  1. Recombination hotspots and single-stranded DNA binding proteins couple DNA translocation to DNA unwinding by the AddAB helicase-nuclease.

    PubMed

    Yeeles, Joseph T P; van Aelst, Kara; Dillingham, Mark S; Moreno-Herrero, Fernando

    2011-06-24

    AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase assays and our understanding of helicase mechanisms in general.

  2. Flow cytometric fluorescence lifetime analysis of DNA binding fluorochromes

    SciTech Connect

    Crissman, Harry A.; Cui, H. H.; 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

  3. Prediction of DNA-binding proteins from relational features

    PubMed Central

    2012-01-01

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

  4. Asymmetric DNA binding by a homodimeric bHLH protein.

    PubMed

    Winston, R L; Ehley, J A; Baird, E E; Dervan, P B; Gottesfeld, J M

    2000-08-08

    Protein-DNA interactions that lie outside of the core recognition sequence for the Drosophila bHLH transcription factor Deadpan (Dpn) were investigated using minor groove binding pyrrole-imidazole polyamides. Electrophoretic mobility shift assays and DNase I footprinting demonstrate that hairpin polyamides bound immediately upstream, but not immediately downstream of the Dpn homodimer selectively inhibit protein-DNA complex formation. Mutation of the Dpn consensus binding site from the asymmetric sequence 5'-CACGCG-3' to the palindromic sequence 5'-CACGTG-3' abolishes asymmetric inhibition. A Dpn mutant containing the unnatural amino acid norleucine in place of lysine at position 80 in the bHLH loop region is not inhibited by the polyamide, suggesting that the epsilon amino group at this position is responsible for DNA contacts outside the major groove. We conclude that the nonpalindromic Dpn recognition site imparts binding asymmetry by providing unique contacts to the basic region of each monomer in the bHLH homodimer.

  5. Solution structure and binding specificity of the p63 DNA binding domain

    PubMed Central

    Enthart, Andreas; Klein, Christian; Dehner, Alexander; Coles, Murray; Gemmecker, Gerd; Kessler, Horst; Hagn, Franz

    2016-01-01

    p63 is a close homologue of p53 and, together with p73, is grouped into the p53 family of transcription factors. p63 is known to be involved in the induction of controlled apoptosis important for differentiation processes, germ line integrity and development. Despite its high homology to p53, especially within the DNA binding domain (DBD), p63-DBD does not show cooperative DNA binding properties and is significantly more stable against thermal and chemical denaturation. Here, we determined the solution structure of p63-DBD and show that it is markedly less dynamic than p53-DBD. In addition, we also investigate the effect of a double salt bridge present in p53-DBD, but not in p63-DBD on the cooperative binding behavior and specificity to various DNA sites. Restoration of the salt bridges in p63-DBD by mutagenesis leads to enhanced binding affinity to p53-specific, but not p63-specific response elements. Furthermore, we show that p63-DBD is capable of binding to anti-apoptotic BclxL via its DNA binding interface, a feature that has only been shown for p53 so far. These data suggest that all p53 family members - despite alterations in the specificity and binding affinity - are capable of activating pro-apoptotic pathways in a tissue specific manner. PMID:27225672

  6. Solution structure and binding specificity of the p63 DNA binding domain.

    PubMed

    Enthart, Andreas; Klein, Christian; Dehner, Alexander; Coles, Murray; Gemmecker, Gerd; Kessler, Horst; Hagn, Franz

    2016-05-26

    p63 is a close homologue of p53 and, together with p73, is grouped into the p53 family of transcription factors. p63 is known to be involved in the induction of controlled apoptosis important for differentiation processes, germ line integrity and development. Despite its high homology to p53, especially within the DNA binding domain (DBD), p63-DBD does not show cooperative DNA binding properties and is significantly more stable against thermal and chemical denaturation. Here, we determined the solution structure of p63-DBD and show that it is markedly less dynamic than p53-DBD. In addition, we also investigate the effect of a double salt bridge present in p53-DBD, but not in p63-DBD on the cooperative binding behavior and specificity to various DNA sites. Restoration of the salt bridges in p63-DBD by mutagenesis leads to enhanced binding affinity to p53-specific, but not p63-specific response elements. Furthermore, we show that p63-DBD is capable of binding to anti-apoptotic BclxL via its DNA binding interface, a feature that has only been shown for p53 so far. These data suggest that all p53 family members - despite alterations in the specificity and binding affinity - are capable of activating pro-apoptotic pathways in a tissue specific manner.

  7. Chromatin landscape dictates HSF binding to target DNA elements.

    PubMed

    Guertin, Michael J; Lis, John T

    2010-09-09

    Sequence-specific transcription factors (TFs) are critical for specifying patterns and levels of gene expression, but target DNA elements are not sufficient to specify TF binding in vivo. In eukaryotes, the binding of a TF is in competition with a constellation of other proteins, including histones, which package DNA into nucleosomes. We used the ChIP-seq assay to examine the genome-wide distribution of Drosophila Heat Shock Factor (HSF), a TF whose binding activity is mediated by heat shock-induced trimerization. HSF binds to 464 sites after heat shock, the vast majority of which contain HSF Sequence-binding Elements (HSEs). HSF-bound sequence motifs represent only a small fraction of the total HSEs present in the genome. ModENCODE ChIP-chip datasets, generated during non-heat shock conditions, were used to show that inducibly bound HSE motifs are associated with histone acetylation, H3K4 trimethylation, RNA Polymerase II, and coactivators, compared to HSE motifs that remain HSF-free. Furthermore, directly changing the chromatin landscape, from an inactive to an active state, permits inducible HSF binding. There is a strong correlation of bound HSEs to active chromatin marks present prior to induced HSF binding, indicating that an HSE's residence in "active" chromatin is a primary determinant of whether HSF can bind following heat shock.

  8. Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

    PubMed Central

    Singh, K K; Samson, L

    1995-01-01

    Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7761422

  9. Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

    PubMed Central

    Bulyk, Martha L.; Huang, Xiaohua; Choo, Yen; Church, George M.

    2001-01-01

    A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA–protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites. PMID:11404456

  10. Rhodopsin targeted transcriptional silencing by DNA-binding.

    PubMed

    Botta, Salvatore; Marrocco, Elena; de Prisco, Nicola; Curion, Fabiola; Renda, Mario; Sofia, Martina; Lupo, Mariangela; Carissimo, Annamaria; Bacci, Maria Laura; Gesualdo, Carlo; Rossi, Settimio; Simonelli, Francesca; Surace, Enrico Maria

    2016-03-14

    Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations.

  11. Rhodopsin targeted transcriptional silencing by DNA-binding

    PubMed Central

    Botta, Salvatore; Marrocco, Elena; de Prisco, Nicola; Curion, Fabiola; Renda, Mario; Sofia, Martina; Lupo, Mariangela; Carissimo, Annamaria; Bacci, Maria Laura; Gesualdo, Carlo; Rossi, Settimio; Simonelli, Francesca; Surace, Enrico Maria

    2016-01-01

    Transcription factors (TFs) operate by the combined activity of their DNA-binding domains (DBDs) and effector domains (EDs) enabling the coordination of gene expression on a genomic scale. Here we show that in vivo delivery of an engineered DNA-binding protein uncoupled from the repressor domain can produce efficient and gene-specific transcriptional silencing. To interfere with RHODOPSIN (RHO) gain-of-function mutations we engineered the ZF6-DNA-binding protein (ZF6-DB) that targets 20 base pairs (bp) of a RHOcis-regulatory element (CRE) and demonstrate Rho specific transcriptional silencing upon adeno-associated viral (AAV) vector-mediated expression in photoreceptors. The data show that the 20 bp-long genomic DNA sequence is necessary for RHO expression and that photoreceptor delivery of the corresponding cognate synthetic trans-acting factor ZF6-DB without the intrinsic transcriptional repression properties of the canonical ED blocks Rho expression with negligible genome-wide transcript perturbations. The data support DNA-binding-mediated silencing as a novel mode to treat gain-of-function mutations. DOI: http://dx.doi.org/10.7554/eLife.12242.001 PMID:26974343

  12. Topological friction strongly affects viral DNA ejection

    PubMed Central

    Marenduzzo, Davide; Micheletti, Cristian; Orlandini, Enzo; Sumners, De Witt

    2013-01-01

    Bacteriophages initiate infection by releasing their double-stranded DNA into the cytosol of their bacterial host. However, what controls and sets the timescales of DNA ejection? Here we provide evidence from stochastic simulations which shows that the topology and organization of DNA packed inside the capsid plays a key role in determining these properties. Even with similar osmotic pressure pushing out the DNA, we find that spatially ordered DNA spools have a much lower effective friction than disordered entangled states. Such spools are only found when the tendency of nearby DNA strands to align locally is accounted for. This topological or conformational friction also depends on DNA knot type in the packing geometry and slows down or arrests the ejection of twist knots and very complex knots. We also find that the family of (2, 2k+1) torus knots unravel gradually by simplifying their topology in a stepwise fashion. Finally, an analysis of DNA trajectories inside the capsid shows that the knots formed throughout the ejection process mirror those found in gel electrophoresis experiments for viral DNA molecules extracted from the capsids. PMID:24272939

  13. Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

    PubMed Central

    Stuiver, M H; van der Vliet, P C

    1990-01-01

    The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI. Images PMID:2293667

  14. Intercalative DNA binding of the marine anticancer drug variolin B

    PubMed Central

    Canals, Albert; Arribas-Bosacoma, Raquel; Albericio, Fernando; Álvarez, Mercedes; Aymamí, Joan; Coll, Miquel

    2017-01-01

    Variolin B is a rare marine alkaloid that showed promising anti-cancer activity soon after its isolation. It acts as a cyclin-dependent kinase inhibitor, although the precise mechanism through which it exerts the cytotoxic effects is still unknown. The crystal structure of a variolin B bound to a DNA forming a pseudo-Holliday junction shows that this compound can also contribute, through intercalative binding, to either the formation or stabilization of multi-stranded DNA forms. PMID:28051169

  15. Binding stability of a cross-linked drug: Calculation of an anticancer drug cisplatin-DNA complex

    NASA Astrophysics Data System (ADS)

    Chen, Y. Z.; Zhang, Yong-Li; Prohofsky, E. W.

    1997-05-01

    One of the binding modes of anticancer and antibiotic drugs bound to DNA is the formation of a cross link, i.e., binding is made through the formation of covalent bonds between a binding drug and DNA. In this work we present a computational method to calculate the binding stability of a drug cross linked to DNA. Our method is based on the modified self-consistent harmonic approach in which the disruption probabil- ity of the cross-linked bonds as well as hydrogen bonds is calculated from a statistical analysis of micro- scopic thermal fluctuational motions. A Morse potential with appropriate parameters is used to model the cross-linked covalent bonds. Our method is applied to an anticancer drug cisplatin-DNA oligomer d(CTCTAGTGCTCAC).d(GTGAGCACTAGAG) complex. We calculated the equilibrium binding constant of a cisplatin bound to this DNA oligomer. Our method can also be used to analyze the effect of drug binding on DNA base-pair thermal stability. We find that, despite the disruption of certain interbase H bonds, the thermal fluctuational opening probability Pop of base pairs in the cisplatin binding region is enhanced by the formation of non-Watson-Crick H bonds as well as cross-linked covalent bonds. Although the entire DNA helix is bent by cisplatin binding, the stability of the base pairs outside the binding region is only slightly affected by this deformation.

  16. DNA scissors device used to measure MutS binding to DNA mis-pairs.

    PubMed

    Gu, Hongzhou; Yang, Wei; Seeman, Nadrian C

    2010-03-31

    MutS is a DNA repair protein that recognizes unpaired and bulged bases. When it binds to DNA, it bends the double helix. We have developed a novel DNA-based nanomechanical device that measures the amount of work that a DNA-bending protein can do when it binds to the double helix. The device we report here is a scissors-like device consisting of two double-crossover (DX) molecules connected to each other by a flexible Holliday junction. The two DX components are connected by a double helix that contains the binding site for MutS; when the binding site duplex is bent, the scissors contracts. The two DX molecules are also joined by sticky ends on an edge adjacent to the binding site; the sticky ends can be disrupted if the protein binds with sufficient free energy. Those sticky ends are flanked by a pair of dyes; when the sticky ends are disrupted, the dyes separate, and the fluorescence resonance energy transfer signal can monitor the disruption. The strength of the sticky ends is readily varied, so that the ability of the protein to disrupt them can be quantitated. We use this device to measure work in conjunction with a second device that measures the bending angle resulting from protein binding, so as to calibrate the system. Our data are in good agreement with previous measurements of MutS binding, indicating that this device is able to measure the strength of binding correctly.

  17. Dynamics of the Competition Between Nucleosome Unwrapping and DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Bundschuh, Ralf

    2015-03-01

    In eukaryotic organisms DNA is tightly wrapped into nucleosomes. This bears the question how this DNA can be accessed in order to be copied, transcribed, or repaired. A process that allows access to the DNA is transient unwrapping of the DNA from the histone proteins. We have developed a quantitative model of this unwrapping process which we calibrate by comparison to nucleosome unzipping experiments by the Wang group. We then apply this model to quantitatively explain the dynamics of transcription factor binding within nucleosomal DNA. In this context, it has been well known that nucleosomes reduce the affinity for transcription factors to binding sites covered by the nucleosome. It has been assumed that this is due to a reduction in on-rate since a transcription factor can only bind when a rare thermal fluctuation of the nucleosome makes the DNA accessible. However, recent experimental data surprisingly shows that the off-rate of transcription factors is also strongly affected in the presence of a nucleosome. The application of our nucleosome unwrapping free energy landscape demonstrates that this increase in off-rate by several orders of magnitude is a consequence of a competition between partial binding events of dimeric transcription factors and the nucleosome. This material is based upon work supported by the National Science Foundation under Grant Nos. 1105458 and 1410172.

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Cell specificity in DNA binding and repair of chemical carcinogens.

    PubMed Central

    Swenberg, J A; Rickert, D E; Baranyi, B L; Goodman, J I

    1983-01-01

    Many animal models for organ specific neoplasia have been developed and used to study the pathogenesis of cancer. Morphologic studies have usually concentrated on the response of target cells, whereas biochemical investigations have usually employed whole organ homogenates. Since hepatocytes comprise nearly 90% of the liver's mass and 70-80% of its DNA, alterations in DNA replication, covalent binding and DNA repair of nonparenchymal cells are usually obscured when whole organ homogenates are used. By utilizing cell separation methods, we have been able to demonstrate differences between hepatocyte and nonparenchymal cell replication. DNA damage and repair following exposure to a variety of hepatocarcinogen. Differences in removal of simple O6-alkylguanine and DNA replication correlate with cell specific carcinogenesis of simply alkylating agents. For several other procarcinogens, including 2-acetylaminofluorene and dinitroluene, cell specificity appears to reside primarily in the differential metabolic competence of hepatocytes and nonparenchymal cells. This results in greater covalent binding of the carcinogen to hepatocyte DNA, although the DNA adducts are removed at a similar rate in both cell types. Images FIGURE 1. PMID:6832089

  20. Preparation of (32)P-end-labeled DNA fragments for performing DNA-binding experiments.

    PubMed

    Carey, Michael F; Peterson, Craig L; Smale, Stephen T

    2013-05-01

    The generation of a uniquely (32)P-end-labeled DNA fragment is essential for DNA-binding experiments such as DNase I footprinting and ethylation interference. We describe here a protocol for end-labeling a restriction fragment. For a plasmid DNA bearing a region containing the binding site of interest, cleaving with a single restriction endonuclease generates a 5' overhang containing a phosphate. This is generally necessary for both common forms of fragment end-labeling: phosphorylation with polynucleotide kinase and "filling in the end" with DNA polymerases (e.g., Klenow fragment). For the phosphorylation reaction, as described here, the phosphate is removed with calf intestinal phosphatase or bacterial alkaline phosphatase, and the resulting free 5'-OH is phosphorylated with polynucleotide kinase and [γ-(32)P]ATP. This generates a plasmid labeled at each end with γ-(32)P. The molar amount of plasmid DNA must be below the amount of ATP added to the reaction and the ATP must be of sufficiently high specific activity to generate a fragment labeled to the extent necessary for many DNA-binding experiments. To generate a uniquely end-labeled DNA fragment, the labeled plasmid is heat-treated to inactivate any remaining kinase and recleaved with a second endonuclease, releasing a short DNA fragment and a longer vector fragment. The DNA fragment is purified from the labeled vector on a 5%-8% native polyacrylamide gel. The preparation and labeling of DNA restriction fragments typically takes 1-2 d.

  1. Structural modeling for DNA binding to antioxidants resveratrol, genistein and curcumin.

    PubMed

    N'soukpoé-Kossi, C N; Bourassa, P; Mandeville, J S; Bekale, L; Tajmir-Riahi, H A

    2015-10-01

    Several models are presented here for the bindings of the antioxidant polyphenols resveratrol, genistein and curcumin with DNA in aqueous solution at physiological conditions. Multiple spectroscopic methods and molecular modeling were used to locate the binding sites of these polyphenols with DNA duplex. Structural models showed that intercalation is more stable for resveratrol and genistein than groove bindings, while curcumin interaction is via DNA grooves. Docking showed more stable complexes formed with resveratrol and genistein than curcumin with the free binding energies of -4.62 for resveratrol-DNA (intercalation), -4.28 for resveratrol-DNA (groove binding), -4.54 for genistein-DNA (intercalation), -4.38 for genistein-DNA (groove binding) and -3.84 kcal/mol for curcumin-DNA (groove binding). The free binding energies show polyphenol-DNA complexation is spontaneous at room temperature. At high polyphenol concentration a major DNA aggregation occurred, while biopolymer remained in B-family structure.

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

    PubMed

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

    2014-05-02

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

  3. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner

    SciTech Connect

    Miller, S.D.; Farmer, G.; Prives, C.

    1995-12-01

    This report discusses new findings that the tumor supressor gene product p53 may play a role as a DNA-binding-dependent regulator of DNA replication. The results were obtained using polyomavirus in replication assays. Details regarding effects on cell growth arrest and transcriptional activation are discussed. 61 refs., 7 figs.

  4. Insight into the cooperative DNA binding of the O⁶-alkylguanine DNA alkyltransferase.

    PubMed

    Tessmer, Ingrid; Fried, Michael G

    2014-08-01

    The O(6)-alkylguanine DNA alkyltransferase (AGT) is a highly conserved protein responsible for direct repair of alkylated guanine and to a lesser degree thymine bases. While specific DNA lesion-bound complexes in crystal structures consist of monomeric AGT, several solution studies have suggested that cooperative DNA binding plays a role in the physiological activities of AGT. Cooperative AGT-DNA complexes have been described by theoretical models, which can be tested by atomic force microscopy (AFM). Direct access to structural features of AGT-DNA complexes at the single molecule level by AFM imaging revealed non-specifically bound, cooperative complexes with limited cluster length. Implications of cooperative binding in AGT-DNA interactions are discussed.

  5. Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

    SciTech Connect

    Choi, Yoo Seong; Pack, Seung Pil; Yoo, Young Je . E-mail: yjyoo@snu.ac.kr

    2005-04-22

    A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.

  6. Cooperative binding of Ets-1 and core binding factor to DNA.

    PubMed Central

    Wotton, D; Ghysdael, J; Wang, S; Speck, N A; Owen, M J

    1994-01-01

    Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bound individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to beta E2 and beta E3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increased the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appeared to be little constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA-binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant. Ets and core sites are found in several T-cell-specific enhancers, suggesting that this interaction is of general importance in T-cell-specific transcription. Images PMID:8264651

  7. Differential impact of ionic and coordinate covalent chromium (Cr)-DNA binding on DNA replication.

    PubMed

    Fornsaglio, Jamie L; O'Brien, Travis J; Patierno, Steven R

    2005-11-01

    The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA. These Cr(III)-DNA interactions consist of Cr-DNA monoadducts, Cr-DNA ternary adducts, and Cr-DNA interstrand cross-links (Cr-ICLs), the latter of which are DNA polymerase arresting lesions (PALs). We sought to determine the impact of Cr-DNA interactions on the formation of replication blocking lesions in S. cerevisiae using a PCR-based method. We found that target sequence (TS) amplification using DNA isolated from Cr(VI)-treated yeast actually increased as a function of Cr(VI) concentration. Moreover, the enhanced TS amplification was reproduced in vitro using Cr(III)-treated DNA. In contrast, PCR amplification of TS from DNA isolated from yeast exposed to equitoxic doses of the inorganic DNA cross-linking agent cisplatin (CDDP), was decreased in a concentration-dependent manner. This paradox suggested that a specific Cr-DNA interaction, such as an ionic Cr-DNA complex, was responsible for the enhanced TS amplification, thereby masking the replication-blocking effect of certain ternary Cr-DNA adducts (i.e. interstrand cross-links). To test this possibility, we removed ionically associated Cr from the DNA using salt extraction prior to PCR analysis. This procedure obviated the increased amplification and revealed a dose-dependent decrease in TS amplification and an increase in Cr-PALs. These data from DNA analyzed ex vivo after treatment of intact cells indicate that ionic interactions of Cr with DNA result in increased DNA amplification whereas coordinate-covalent Cr-DNA complexes lead to formation of Cr-PALs. Thus, these results suggest that treatment of living cells with Cr(VI) leads to two modes of Cr-binding, which may have conflicting effects on DNA replication.

  8. Universal protein binding microarrays for the comprehensive characterization of the DNA binding specificities of transcription factors

    PubMed Central

    Berger, Michael F.; Bulyk, Martha L.

    2010-01-01

    Protein binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF binding specificities at high resolution using such ‘all 10-mer’ universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray, and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day. PMID:19265799

  9. Nanoparticles inhibit DNA replication by binding to DNA: modeling and experimental validation.

    PubMed

    Li, Kungang; Zhao, Xiaonan; K Hammer, Brian; Du, Songyan; Chen, Yongsheng

    2013-11-26

    Predictive models are beneficial tools for researchers to use in prioritizing nanoparticles (NPs) for toxicological tests, but experimental evaluation can be time-consuming and expensive, and thus, priority should be given to tests that identify the NPs most likely to be harmful. For characterization of NPs, the physical binding of NPs to DNA molecules is important to measure, as interference with DNA function may be one cause of toxicity. Here, we determined the interaction energy between 12 types of NPs and DNA based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) model and then predicted the affinity of the NPs for DNA. Using the single-molecule imaging technique known as atomic force microscopy (AFM), we experimentally determined the binding affinity of those NPs for DNA. Theoretical predictions and experimental observations of the binding affinity agreed well. Furthermore, the effect of NPs on DNA replication in vitro was investigated with the polymerase chain reaction (PCR) technique. The results showed that NPs with a high affinity for DNA strongly inhibited DNA replication, whereas NPs with low affinity had no or minimal effects on DNA replication. The methodology here is expected to benefit the genotoxicological testing of NPs as well as the design of safe NPs.

  10. Z-DNA binding protein from chicken blood nuclei

    NASA Technical Reports Server (NTRS)

    Herbert, A. G.; Spitzner, J. R.; Lowenhaupt, K.; Rich, A.

    1993-01-01

    A protein (Z alpha) that appears to be highly specific for the left-handed Z-DNA conformer has been identified in chicken blood nuclear extracts. Z alpha activity is measured in a band-shift assay by using a radioactive probe consisting of a (dC-dG)35 oligomer that has 50% of the deoxycytosines replaced with 5-bromodeoxycytosine. In the presence of 10 mM Mg2+, the probe converts to the Z-DNA conformation and is bound by Z alpha. The binding of Z alpha to the radioactive probe is specifically blocked by competition with linear poly(dC-dG) stabilized in the Z-DNA form by chemical bromination but not by B-form poly(dC-dG) or boiled salmon-sperm DNA. In addition, the binding activity of Z alpha is competitively blocked by supercoiled plasmids containing a Z-DNA insert but not by either the linearized plasmid or by an equivalent amount of the parental supercoiled plasmid without the Z-DNA-forming insert. Z alpha can be crosslinked to the 32P-labeled brominated probe with UV light, allowing us to estimate that the minimal molecular mass of Z alpha is 39 kDa.

  11. Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure.

    PubMed Central

    Thayer, M M; Ahern, H; Xing, D; Cunningham, R P; Tainer, J A

    1995-01-01

    The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins. Images PMID:7664751

  12. Predicting DNA-binding proteins and binding residues by complex structure prediction and application to human proteome.

    PubMed

    Zhao, Huiying; Wang, Jihua; Zhou, Yaoqi; Yang, Yuedong

    2014-01-01

    As more and more protein sequences are uncovered from increasingly inexpensive sequencing techniques, an urgent task is to find their functions. This work presents a highly reliable computational technique for predicting DNA-binding function at the level of protein-DNA complex structures, rather than low-resolution two-state prediction of DNA-binding as most existing techniques do. The method first predicts protein-DNA complex structure by utilizing the template-based structure prediction technique HHblits, followed by binding affinity prediction based on a knowledge-based energy function (Distance-scaled finite ideal-gas reference state for protein-DNA interactions). A leave-one-out cross validation of the method based on 179 DNA-binding and 3797 non-binding protein domains achieves a Matthews correlation coefficient (MCC) of 0.77 with high precision (94%) and high sensitivity (65%). We further found 51% sensitivity for 82 newly determined structures of DNA-binding proteins and 56% sensitivity for the human proteome. In addition, the method provides a reasonably accurate prediction of DNA-binding residues in proteins based on predicted DNA-binding complex structures. Its application to human proteome leads to more than 300 novel DNA-binding proteins; some of these predicted structures were validated by known structures of homologous proteins in APO forms. The method [SPOT-Seq (DNA)] is available as an on-line server at http://sparks-lab.org.

  13. Independent versus Cooperative Binding in Polyethylenimine–DNA and Poly(L-lysine)–DNA Polyplexes

    PubMed Central

    Ketola, Tiia-Maaria; Hanzlíková, Martina; Leppänen, Linda; Raviña, Manuela; Bishop, Corey J.; Green, Jordan J.; Urtti, Arto; Lemmetyinen, Helge; Yliperttula, Marjo; Vuorimaa-Laukkanen, Elina

    2013-01-01

    The mechanism of polyethylenimine–DNA and poly(L-lysine)–DNA complex formation at pH 5.2 and 7.4 was studied by a time-resolved spectroscopic method. The formation of a polyplex core was observed to be complete at approximately N/P = 2, at which point nearly all DNA phosphate groups were bound by polymer amine groups. The data were analyzed further both by an independent binding model and by a cooperative model for multivalent ligand binding to multisubunit substrate. At pH 5.2, the polyplex formation was cooperative at all N/P ratios, whereas for pH 7.4 at N/P < 0.6 the polyplex formation followed independent binding changing to cooperative binding at higher N/Ps. PMID:23941196

  14. Mg NMR in DNA solutions: Dominance of site binding effects.

    PubMed

    Rose, D M; Bleam, M L; Record, M T; Bryant, R G

    1980-11-01

    (25)Mg NMR spectroscopy is applied to a study of magnesium ion interactions with DNA, which is considered as a model for a linear polyelectrolyte. It is demonstrated that the magnesium ion spectrum is complicated by a non-Lorent-zian line shape and is dominated by the effects of chemical exchange with macromolecule binding sites. A distinction is made between specific-site interactions in which the magnesium ion loses a water molecule from the first coordination sphere on binding and those interactions, referred to as territorial binding, in which the ion maintains its first coordination sphere complement of solvent. The first type of site-binding interactions are shown to dominate the magnesium ion NMR spectrum, based on a consideration of the magnitudes of the observed (25)Mg relaxation rates compared with (23)Na relaxation rates, the clear contributions of chemical exchange-limited relaxation, and an ion displacement experiment employing sodium.

  15. Weak operator binding enhances simulated Lac repressor-mediated DNA looping.

    PubMed

    Colasanti, Andrew V; Grosner, Michael A; Perez, Pamela J; Clauvelin, Nicolas; Lu, Xiang-Jun; Olson, Wilma K

    2013-12-01

    The 50th anniversary of Biopolymers coincides closely with the like celebration of the discovery of the Escherichia coli (lac) lactose operon, a classic genetic system long used to illustrate the influence of biomolecular structure on function. The looping of DNA induced by the binding of the Lac repressor protein to sequentially distant operator sites on DNA continues to serve as a paradigm for understanding long-range genomic communication. Advances in analyses of DNA structures and in incorporation of proteins in computer simulations of DNA looping allow us to address long-standing questions about the role of protein-mediated DNA loop formation in transcriptional control. Here we report insights gained from studies of the sequence-dependent contributions of the natural lac operators to Lac repressor-mediated DNA looping. Novel superposition of the ensembles of protein-bound operator structures derived from NMR measurements reveals variations in DNA folding missed in conventional structural alignments. The changes in folding affect the predicted ease with which the repressor induces loop formation and the ways that DNA closes between the protein headpieces. The peeling of the auxiliary operators away from the repressor enhances the formation of loops with the 92-bp wildtype spacing and hints of a structural reason behind their weak binding.

  16. Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding.

    PubMed Central

    Khoroshilova, N; Beinert, H; Kiley, P J

    1995-01-01

    In the facultative anaerobe Escherichia coli, the transcription factor FNR (fumarate nitrate reduction) regulates gene expression in response to oxygen deprivation. To investigate how the activity of FNR is regulated by oxygen availability, two mutant proteins, DA154 and LH28-DA154, which have enhanced in vivo activity in the presence of oxygen, were purified and compared. Unlike other previously examined FNR preparations, the absorption spectrum of LH28-DA154 had two maxima at 324 nm and 419 nm, typical of iron-sulfur (Fe-S)-containing proteins. Consistent with these data, metal analysis showed that only the LH28-DA154 protein contained a significant amount of iron and acid-labile sulfide, and, by low temperature EPR spectroscopy, a signal typical of a [3Fe-4S]+ cluster was detected. The LH28-DA154 protein that contained the Fe-S cluster also contained a higher proportion of dimers and had a 3- to 4-fold higher apparent affinity for the target DNA than the DA154 protein. In agreement with this, we found that when the LH28-DA154 protein was treated with an iron chelator (alpha,alpha'-dipyridyl), it lost its characteristic absorption and the apparent affinity for DNA was reduced 6-fold. However, increased DNA binding and the characteristic absorption spectrum could be restored by in vitro reconstitution of the Fe-S center. DNA binding of the LH28-DA154 protein was also affected by the redox state of the Fe-S center, since protein exposed to oxygen bound 1/10th as much DNA as the protein reduced anaerobically with dithionite. The observation that DNA binding is enhanced when the Fe-S center is reduced indicates that the redox state of the Fe-S center affects the DNA-binding activity of this protein and suggests a possible mechanism for regulation of the wild-type protein. Images Fig. 4 PMID:7708673

  17. T antigen origin-binding domain of simian virus 40: determinants of specific DNA binding.

    PubMed

    Bradshaw, Elizabeth M; Sanford, David G; Luo, Xuelian; Sudmeier, James L; Gurard-Levin, Zachary A; Bullock, Peter A; Bachovchin, William W

    2004-06-08

    To better understand origin recognition and initiation of DNA replication, we have examined by NMR complexes formed between the origin-binding domain of SV40 T antigen (T-ag-obd), the initiator protein of the SV40 virus, and cognate and noncognate DNA oligomers. The results reveal two structural effects associated with "origin-specific" binding that are absent in nonspecific DNA binding. The first is the formation of a hydrogen bond (H-bond) involving His 203, a residue that genetic studies have previously identified as crucial to both specific and nonspecific DNA binding in full-length T antigen. In free T-ag-obd, the side chain of His 203 has a pK(a) value of approximately 5, titrating to the N(epsilon)(1)H tautomer at neutral pH (Sudmeier, J. L., et al. (1996) J. Magn. Reson., Ser. B 113, 236-247). In complexes with origin DNA, His 203 N(delta)(1) becomes protonated and remains nontitrating as the imidazolium cation at all pH values from 4 to 8. The H-bonded N(delta1)H resonates at 15.9 ppm, an unusually large N-H proton chemical shift, of a magnitude previously observed only in the catalytic triad of serine proteases at low pH. The formation of this H-bond requires the middle G/C base pair of the recognition pentanucleotide, GAGGC. The second structural effect is a selective distortion of the A/T base pair characterized by a large (0.6 ppm) upfield chemical-shift change of its Watson-Crick proton, while nearby H-bonded protons remain relatively unaffected. The results indicate that T antigen, like many other DNA-binding proteins, may employ "catalytic" or "transition-state-like" interactions in binding its cognate DNA (Jen-Jacobson, L. (1997) Biopolymers 44, 153-180), which may be the solution to the well-known paradox between the relatively modest DNA-binding specificity exhibited by initiator proteins and the high specificity of initiation.

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

    SciTech Connect

    Ryzhikov, Mikhail; Koroleva, Olga; Postnov, Dmitri; Tran, Andrew; Korolev, Sergey

    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.

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

    PubMed

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

    1989-12-26

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

  20. Eukaryotic damaged DNA-binding proteins: DNA repair proteins or transcription factors?

    SciTech Connect

    Protic, M.

    1994-12-31

    Recognition and removal of structural defects in the genome, caused by diverse physical and chemical agents, are among the most important cell functions. Proteins that recognize and bind to modified DNA, and thereby initiate damage-induced recovery processes, have been identified in prokaryotic and eukaryotic cells. Damaged DNA-binding (DDB) proteins from prokaryotes are either DNA repair enzymes or noncatalytic subunits of larger DNA repair complexes that participate in excision repair, or in recombinational repair and SOS-mutagenesis. Although the methods employed may not have allowed detection of all eukaryotic DDB proteins and identification of their functions, it appears that during evolution cells have developed a wide array of DDB proteins that can discriminate among the diversity of DNA conformations found in the eukaryotic nucleus, as well as a gene-sharing feature found in DDB proteins that also act as transcription factors.

  1. Dendritic star polymers for efficient DNA binding and stimulus-dependent DNA release.

    PubMed

    Yin, Meizhen; Ding, Ke; Gropeanu, Radu A; Shen, Jie; Berger, Rüdiger; Weil, Tanja; Müllen, Klaus

    2008-11-01

    Water-soluble core-shell star polymers consisting of a dendritic polyphenylene core and an outer shell containing a defined number of amino groups have been synthesized via atom transfer radical polymerization (ATRP). All macromolecules efficiently interacted with a diverse set of DNA fragments, and stable complexes were formed and visualized by atomic force microscopy. The observed tight binding of DNA, which was found in the sub-nanomolar range, was mainly attributed to strong electrostatic interactions. Complex stoichiometries between the polyelectrolytes were controlled via the number of amino groups of the star polymers, and well-defined nanoscopic architectures were formed. DNA was released from the complexes after treatment with high concentrations of sodium chloride in aqueous solution. Such star polymers, which allow the binding and release of DNA, represent attractive candidates for the development of novel anion-exchange resins for DNA purification or as nonviral vector systems for gene delivery.

  2. Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

    PubMed

    Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

    2013-10-01

    Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

  3. Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

    PubMed Central

    Jose, Davis; Weitzel, Steven E.; Baase, Walter A.; Michael, Miya M.; von Hippel, Peter H.

    2015-01-01

    We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex. PMID:26275774

  4. Differential DNA binding of Ku antigen determines its involvement in DNA replication.

    PubMed

    Schild-Poulter, Caroline; Matheos, Diamanto; Novac, Olivia; Cui, Bo; Giffin, Ward; Ruiz, Marcia T; Price, Gerald B; Zannis-Hadjopoulos, Maria; Haché, Robert J G

    2003-02-01

    Ku antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.

  5. Specific enrichment of prokaryotic DNA using a recombinant DNA-binding protein.

    PubMed

    Sandetskaya, Natalia; Naumann, Andreas; Hennig, Katharina; Kuhlmeier, Dirk

    2014-06-01

    Targeted enrichment of DNA is often necessary for its detection and characterization in complex samples. We describe the development and application of the novel molecular tool for the specific enrichment of prokaryotic DNA. A fused protein comprising the DNA-binding subunit of the bacterial topoisomerase II, gyrase, was expressed, purified, and immobilized on magnetic particles. We demonstrated the specific affinity of the immobilized protein towards bacterial DNA and investigated its efficiency in the samples with high background of eukaryotic DNA. The reported approach allowed for the selective isolation and further detection of as few as 5 pg Staphylococcus aureus DNA from the sample with 4 × 10(6)-fold surplus of human DNA. This method is a promising approach for the preparation of such type of samples, for example in molecular diagnostics of sepsis.

  6. Molecular spectroscopy evidence of berberine binding to DNA: comparative binding and thermodynamic profile of intercalation.

    PubMed

    Li, Xiao-Ling; Hu, Yan-Jun; Wang, Hong; Yu, Bing-Qiong; Yue, Hua-Li

    2012-03-12

    Berberine (BH) is an important traditional medicinal herb endowed with diverse pharmacological and biological activities. In this work, the binding characteristics and molecular mechanism of the interaction between the BH and herring sperm DNA were explored by UV-vis absorbance and fluorescence spectroscopy. In the mechanism discussion, fluorescence quenching, absorption spectra, competition experiment, and iodide quenching experiment studies hinted at an intercalative mode of binding for BH to DNA. Fluorescence studies revealed the binding constant (K) of BH-DNA was ∼10(4) L·mol(-1). The effects of temperature, chemical denaturants, thermal denaturation, and pH were studied to show the factors of the interaction and provided further support for the intercalative binding mode. The results of thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures indicated that the hydrogen bonds and van der Waals interactions played major roles in the reaction, and the effect of ionic strength indicated that electrostatic attraction between the BH and DNA was also a component of the interaction.

  7. Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions

    PubMed Central

    Nakayama, Yuki; Yamaguchi, Hiromi; Einaga, Naoki; Esumi, Mariko

    2016-01-01

    The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for

  8. Facile dimer synthesis for DNA-binding polyamide ligands.

    PubMed

    Wetzler, Modi; Wemmer, David E

    2010-08-06

    Pyrrole-imidazole polyamide ligands are highly sequence specific synthetic DNA-binding ligands that bind with high affinity. To counter the synthetic difficulties associated with coupling the electron-rich heterocyclic acids to the electron-deficient nucleophilic imidazole amine, a novel approach is described for synthesis of Fmoc-protected dimers for solid-phase peptide synthesis (SPPS). This method produces the dimers in high yields, is broadly applicable to other heterocyclic-containing polyamides, and results in improved ligand yields and synthesis times.

  9. DBD2BS: connecting a DNA-binding protein with its binding sites

    PubMed Central

    Chien, Ting-Ying; Lin, Chih-Kang; Lin, Chih-Wei; Weng, Yi-Zhong; Chen, Chien-Yu; Chang, Darby Tien-Hao

    2012-01-01

    By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes can be summarized as a PWM. This technique provides an effective alternative when the chromatin immunoprecipitation data are unavailable for PWM inference. To facilitate the procedure of predicting PWMs based on protein–DNA complexes or even structures of the unbound state, the web server, DBD2BS, is presented in this study. The DBD2BS uses an atom-level knowledge-based potential function to predict PWMs characterizing the sequences to which the query DBD structure can bind. For unbound queries, a list of 1066 DBD–DNA complexes (including 1813 protein chains) is compiled for use as templates for synthesizing bound structures. The DBD2BS provides users with an easy-to-use interface for visualizing the PWMs predicted based on different templates and the spatial relationships of the query protein, the DBDs and the DNAs. The DBD2BS is the first attempt to predict PWMs of DBDs from unbound structures rather than from bound ones. This approach increases the number of existing protein structures that can be exploited when analyzing protein–DNA interactions. In a recent study, the authors showed that the kernel adopted by the DBD2BS can generate PWMs consistent with those obtained from the experimental data. The use of DBD2BS to predict PWMs can be incorporated with sequence-based methods to discover binding sites in genome-wide studies. Available at: http://dbd2bs.csie.ntu.edu.tw/, http://dbd2bs.csbb.ntu.edu.tw/, and http://dbd2bs.ee.ncku.edu.tw. PMID:22693214

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

    NASA Astrophysics Data System (ADS)

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

    2009-06-01

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

  11. DNA-binding and fluorescence properties of the DNA bisintercalating purple oxazole dimer POPO-1

    NASA Astrophysics Data System (ADS)

    Winter, Stefan; Loeber, Gunter

    1997-01-01

    Dimers of the fluorescent DNA intercalators oxazole yellow and thiazole orange are used for high-sensitivity DNA detection due to their excellent fluorescence properties. Fluorescence lifetime techniques and absorption spectroscopy were used to investigate the DNA binding properties of POPO- 1 [4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis-4-(3- methyl-2,3-dihydrobenzo-1,3-oxazolyl)-2-methylidene] with the double-stranded homopurine-homopyrimidine polynucleotides poly(dA(DOT)dT), poly(dG(DOT)dC) and calf thymus DNA. The coexistence of different binding modes of POPO-1 with polynucleotides such as bisintercalation and monointercalation was found in connection with minor groove binding as well as electrostatic attachment. At high excess of polynucleotides, bisintercalation is the only existing form of binding whereas an increasing amount of POPO-1 leads to the coexistence of bis- and monointercalated dye molecules. The amount of bound dye increases with decreasing ionic strength of the buffer and is dependent on the polynucleotide itself. The best binding conditions were found with calf thymus DNA, followed by poly(dA(DOT)dT) and poly(dG(DOT)dC).

  12. DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication.

    PubMed Central

    Yamaguchi, M; DePamphilis, M L

    1986-01-01

    Efficient initiation of DNA replication in the absence of nonspecific DNA repair synthesis was obtained by using a modification of the system developed by J.J. Li and T.J. Kelly [(1984) Proc. Natl. Acad. Sci. USA 81, 6973-6977]. Circular double-stranded DNA plasmids replicated in extracts of CV-1 cells only when the plasmids contained the cis-acting origin sequence for simian virus 40 DNA replication (ori) and the extract contained simian virus 40 large tumor antigen. Competition between plasmids containing ori and plasmids carrying deletions in and about ori served to identify a sequence that binds the rate-limiting factor(s) required to initiate DNA replication. The minimum binding site (nucleotides 72-5243) encompassed one-half of the simian virus 40 ori sequence that is required for initiation of replication (ori-core) plus the contiguous sequence on the late gene side of ori-core containing G + C-rich repeats that facilitates initiation (ori-auxiliary). This initiation factor binding site was specific for the simian virus 40 ori region, even though it excluded the high-affinity large tumor antigen DNA binding sites. Images PMID:3006062

  13. Specific glucocorticoid receptor binding to DNA reconstituted in a nucleosome.

    PubMed Central

    Perlmann, T; Wrange, O

    1988-01-01

    We have reconstituted a nucleosome with core histones from rat liver using a restriction fragment containing a sequence from the mouse mammary tumour virus (MTV) long terminal repeat (LTR). This sequence harbours glucocorticoid responsive elements (GREs) which mediate glucocorticoid hormone induction of transcription from the MTV promoter via glucocorticoid receptor (GR) binding. Exonuclease III and DNase I footprinting demonstrated that the reconstituted nucleosome was specifically located between positions -219 and -76. A nucleosome was previously shown to be located at a similar or identical position in the MTV promoter in situ and to be structurally altered upon glucocorticoid hormone induction. We demonstrated, by DNase I footprinting, that GR is able to bind sequence specifically to the DNA in the in vitro assembled nucleosome. No evidence for unfolding of the nucleosome was obtained, but the DNase I footprinting pattern demonstrated GR induced local alterations in the DNA. Images PMID:2846275

  14. Nitric oxide inhibits c-Jun DNA binding by specifically targeted S-glutathionylation.

    PubMed

    Klatt, P; Molina, E P; Lamas, S

    1999-05-28

    This study addresses potential molecular mechanisms underlying the inhibition of the transcription factor c-Jun by nitric oxide. We show that in the presence of the physiological sulfhydryl glutathione nitric oxide modifies the two cysteine residues contained in the DNA binding module of c-Jun in a selective and distinct way. Although nitric oxide induced the formation of an intermolecular disulfide bridge between cysteine residues in the leucine zipper site of c-Jun monomers, this same radical directed the covalent incorporation of stoichiometric amounts of glutathione to a single conserved cysteine residue in the DNA-binding site of the protein. We found that covalent dimerization of c-Jun apparently did not affect its DNA binding activity, whereas the formation of a mixed disulfide with glutathione correlated well with the inhibition of transcription factor binding to DNA. Furthermore, we provide experimental evidence that nitric oxide-induced S-glutathionylation and inhibition of c-Jun involves the formation of S-nitrosoglutathione. In conclusion, our results support the reversible formation of a mixed disulfide between glutathione and c-Jun as a potential mechanism by which nitrosative stress may be transduced into a functional response at the level of transcription.

  15. Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process

    PubMed Central

    Reshetnikov, Roman V.; Sponer, Jiri; Rassokhina, Olga I.; Kopylov, Alexei M.; Tsvetkov, Philipp O.; Makarov, Alexander A.; Golovin, Andrey V.

    2011-01-01

    A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange. PMID:21893589

  16. Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process.

    PubMed

    Reshetnikov, Roman V; Sponer, Jiri; Rassokhina, Olga I; Kopylov, Alexei M; Tsvetkov, Philipp O; Makarov, Alexander A; Golovin, Andrey V

    2011-12-01

    A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 µs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange.

  17. Binding energies of nucleobase complexes: Relevance to homology recognition of DNA

    NASA Astrophysics Data System (ADS)

    León, Sergio Cruz; Prentiss, Mara; Fyta, Maria

    2016-06-01

    The binding energies of complexes of DNA nucleobase pairs are evaluated using quantum mechanical calculations at the level of dispersion corrected density functional theory. We begin with Watson-Crick base pairs of singlets, duplets, and triplets and calculate their binding energies. At a second step, mismatches are incorporated into the Watson-Crick complexes in order to evaluate the variation in the binding energy with respect to the canonical Watson-Crick pairs. A linear variation of this binding energy with the degree of mismatching is observed. The binding energies for the duplets and triplets containing mismatches are further compared to the energies of the respective singlets in order to assess the degree of collectivity in these complexes. This study also suggests that mismatches do not considerably affect the energetics of canonical base pairs. Our work is highly relevant to the recognition process in DNA promoted through the RecA protein and suggests a clear distinction between recognition in singlets, and recognition in duplets or triplets. Our work assesses the importance of collectivity in the homology recognition of DNA.

  18. Differential DNA binding properties of three human homeodomain proteins.

    PubMed Central

    Corsetti, M T; Briata, P; Sanseverino, L; Daga, A; Airoldi, I; Simeone, A; Palmisano, G; Angelini, C; Boncinelli, E; Corte, G

    1992-01-01

    The products of three human homeobox containing (HOX) genes, 2C, 3C and 4B, were produced in insect cells using the Baculovirus expression system and purified to near homogeneity. In this system we observed that the DNA binding forms of the three proteins are not glycosylated. HOX 3C and 4B are phosphorylated in insect cells, while HOX 2C is not. The three HOX proteins bind to a DNA sequence known to be a target site for Antennapedia protein with a very similar affinity (Kd = 1-2 x 10(-9) M). We then measured their binding properties to four human sequences present in the HOX 3D, 4C, 1C and 4B promoters. Two of these sequences have been reported to be binding sites for HOX proteins. HOX 2C, 3C and 4B behaved quite differently, showing low affinity for promoters of genes located upstream from their own gene in the HOX clusters and a higher affinity for regulatory sequences of their own gene and downstream HOX genes. Images PMID:1357628

  19. Computational redesign of endonuclease DNA binding and cleavage specificity

    NASA Astrophysics Data System (ADS)

    Ashworth, Justin; Havranek, James J.; Duarte, Carlos M.; Sussman, Django; Monnat, Raymond J.; Stoddard, Barry L.; Baker, David

    2006-06-01

    The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ~10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

  20. The shape of the DNA minor groove directs binding by the DNA-bending protein Fis

    SciTech Connect

    Stella, Stefano; Cascio, Duilio; Johnson, Reid C.

    2010-06-21

    The bacterial nucleoid-associated protein Fis regulates diverse reactions by bending DNA and through DNA-dependent interactions with other control proteins and enzymes. In addition to dynamic nonspecific binding to DNA, Fis forms stable complexes with DNA segments that share little sequence conservation. Here we report the first crystal structures of Fis bound to high- and low-affinity 27-base-pair DNA sites. These 11 structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the minor groove and sequence-dependent induced fits over adjacent major groove interfaces. The DNA shows an overall curvature of {approx}65{sup o}, and the unprecedented close spacing between helix-turn-helix motifs present in the apodimer is accommodated by severe compression of the central minor groove. In silico DNA structure models show that only the roll, twist, and slide parameters are sufficient to reproduce the changes in minor groove widths and recreate the curved Fis-bound DNA structure. Models based on naked DNA structures suggest that Fis initially selects DNA targets with intrinsically narrow minor grooves using the separation between helix-turn-helix motifs in the Fis dimer as a ruler. Then Fis further compresses the minor groove and bends the DNA to generate the bound structure.

  1. Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents.

    PubMed

    Pham, Xuan Hoi; Tuteja, Narendra

    2002-06-07

    Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.

  2. Predicting the sequence specificities of DNA- and RNA-binding proteins by deep learning.

    PubMed

    Alipanahi, Babak; Delong, Andrew; Weirauch, Matthew T; Frey, Brendan J

    2015-08-01

    Knowing the sequence specificities of DNA- and RNA-binding proteins is essential for developing models of the regulatory processes in biological systems and for identifying causal disease variants. Here we show that sequence specificities can be ascertained from experimental data with 'deep learning' techniques, which offer a scalable, flexible and unified computational approach for pattern discovery. Using a diverse array of experimental data and evaluation metrics, we find that deep learning outperforms other state-of-the-art methods, even when training on in vitro data and testing on in vivo data. We call this approach DeepBind and have built a stand-alone software tool that is fully automatic and handles millions of sequences per experiment. Specificities determined by DeepBind are readily visualized as a weighted ensemble of position weight matrices or as a 'mutation map' that indicates how variations affect binding within a specific sequence.

  3. Binding of the Vestigial co-factor switches the DNA-target selectivity of the Scalloped selector protein.

    PubMed

    Halder, G; Carroll, S B

    2001-09-01

    The formation and identity of organs and appendages are regulated by specific selector genes that encode transcription factors that regulate potentially large sets of target genes. The DNA-binding domains of selector proteins often exhibit relatively low DNA-binding specificity in vitro. It is not understood how the target selectivity of most selector proteins is determined in vivo. The Scalloped selector protein controls wing development in Drosophila by regulating the expression of numerous target genes and forming a complex with the Vestigial protein. We show that binding of Vestigial to Scalloped switches the DNA-binding selectivity of Scalloped. Two conserved domains of the Vestigial protein that are not required for Scalloped binding in solution are required for the formation of the heterotetrameric Vestigial-Scalloped complex on DNA. We suggest that Vestigial affects the conformation of Scalloped to create a wing cell-specific DNA-binding selectivity. The modification of selector protein DNA-binding specificity by co-factors appears to be a general mechanism for regulating their target selectivity in vivo.

  4. Fluorescence-determined preferential binding of quinacrine to DNA.

    PubMed Central

    Baldini, G; Doglia, S; Dolci, S; Sassi, G

    1981-01-01

    Quinacrine complexes with native DNA (Calf thymus, Micrococcus lysodeikticus, Escherichia coli, Bacillus subtilis, and Colstridium perfringens) and synthetic polynucleotides (poly(dA) . poly(dT), poly[d(A-T)] . poly[d(A-T)], poly(dG) . poly(dC) and poly[d(G-C)] . poly[d(G-C)]) has been investigated in solution at 0.1 M NaCl, 0.05 M Tris HCl, 0.001 M EDTA, pH 7.5, at 20 degrees C. Fluorescence excitation spectra of complexes with dye concentration D = 5-30 microM and DNA phosphate concentration P = 400 microM have been examined from 300 to 500 nm, while collecting the emission above 520 nm. The amounts of free and bound quinacrine in the dye-DNA complexes have been determined by means of equilibrium dialysis experiments. Different affinities have been found for the various DNAs and their values have been examined with a model that assumes that the binding constants associated with alternating purine and pyrimidine sequences are larger than those relative to nonalternating ones. Among the alternating nearest neighbor base sequences, the Pyr(3'-5')Pur sequences, i.e., C-G, T-G, C-A and T-A seem to bind quinacrine stronger than the remaining sequences. In particular the three sites, where a G . C base pair is involved, are found to display higher affinities. Good agreement is found with recent calculations on the energetics of intercalation sites in DNA. The analysis of the equilibrium shows also that the strength of the excitation spectrum of bound dye depends strongly upon the ratio of bound quinacrine to DNA. This effect can be attributed to dye-dye energy transfer along DNA. PMID:7326321

  5. DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding.

    PubMed

    Szambowska, Anna; Tessmer, Ingrid; Kursula, Petri; Usskilat, Christian; Prus, Piotr; Pospiech, Helmut; Grosse, Frank

    2014-02-01

    The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.

  6. Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions

    PubMed Central

    Brown, Maxwell W.; Kim, Yoori; Williams, Gregory M.; Huck, John D.; Surtees, Jennifer A.; Finkelstein, Ilya J.

    2016-01-01

    DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2–Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2–Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2–Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2–Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2–Msh3 and Msh2–Msh6 navigate on a crowded genome and suggest how Msh2–Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin. PMID:26837705

  7. OGG1-DNA interactions facilitate NF-κB binding to DNA targets

    PubMed Central

    Pan, Lang; Hao, Wenjing; Zheng, Xu; Zeng, Xianlu; Ahmed Abbasi, Adeel; Boldogh, Istvan; Ba, Xueqing

    2017-01-01

    DNA repair protein counteracting oxidative promoter lesions may modulate gene expression. Oxidative DNA bases modified by reactive oxygen species (ROS), primarily as 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG), which is repaired by 8-oxoguanine DNA glycosylase1 (OGG1) during base excision repair (BER) pathway. Because cellular response to oxidative challenge is accompanied by DNA damage repair, we tested whether the repair by OGG1 is compatible with transcription factor binding and gene expression. We performed electrophoretic mobility shift assay (EMSA) using wild-type sequence deriving from Cxcl2 gene promoter and the same sequence bearing a single synthetic 8-oxoG at defined 5′ or 3′ guanine in runs of guanines to mimic oxidative effects. We showed that DNA occupancy of NF-κB present in nuclear extracts from tumour necrosis factor alpha (TNFα) exposed cells is OGG1 and 8-oxoG position dependent, importantly, OGG1 counteracting 8-oxoG outside consensus motif had a profound influence on purified NF-κB binding to DNA. Furthermore, OGG1 is essential for NF-κB dependent gene expression, prior to 8-oxoG excised from DNA. These observations imply that pre-excision step(s) during OGG1 initiated BER evoked by ROS facilitates NF-κB DNA occupancy and gene expression. PMID:28266569

  8. The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

    PubMed Central

    Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

    2016-01-01

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize 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. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

  9. DNABP: Identification of DNA-Binding Proteins Based on Feature Selection Using a Random Forest and Predicting Binding Residues

    PubMed Central

    Guo, Jing; Sun, Xiao

    2016-01-01

    DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information. In this paper, a novel predictor, DNABP (DNA-binding proteins), was designed to predict DNA-binding proteins using the random forest (RF) classifier with a hybrid feature. The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability. Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance (mRMR) method combined with incremental feature selection (IFS) was carried out during the model construction. The results showed that the DNABP model could achieve 86.90% accuracy, 83.76% sensitivity, 90.03% specificity and a Matthews correlation coefficient of 0.727. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information. The DNABP web server system is freely available at http://www.cbi.seu.edu.cn/DNABP/. PMID:27907159

  10. The HRDC domain of E. coli RecQ helicase controls single-stranded DNA translocation and double-stranded DNA unwinding rates without affecting mechanoenzymatic coupling.

    PubMed

    Harami, Gábor M; Nagy, Nikolett T; Martina, Máté; Neuman, Keir C; Kovács, Mihály

    2015-06-11

    DNA-restructuring activities of RecQ-family helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom's syndrome (BLM) helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme.

  11. Molecular beacons for DNA binding proteins: an emerging technology for detection of DNA binding proteins and their ligands.

    PubMed

    Dummitt, Benjamin; Chang, Yie-Hwa

    2006-06-01

    Quantitation of the level or activity of specific proteins is one of the most commonly performed experiments in biomedical research. Protein detection has historically been difficult to adapt to high throughput platforms because of heavy reliance upon antibodies for protein detection. Molecular beacons for DNA binding proteins is a recently developed technology that attempts to overcome such limitations. Protein detection is accomplished using inexpensive, easy-to-synthesize oligonucleotides, accompanied by a fluorescence readout. Importantly, detection of the protein and reporting of the signal occur simultaneously, allowing for one-step protocols and increased potential for use in high throughput analysis. While the initial iteration of the technology allowed only for the detection of sequence-specific DNA binding proteins, more recent adaptations allow for the possibility of development of beacons for any protein, independent of native DNA binding activity. Here, we discuss the development of the technology, the mechanism of the reaction, and recent improvements and modifications made to improve the assay in terms of sensitivity, potential for multiplexing, and broad applicability.

  12. Inhibition of NF-kappaB DNA binding by nitric oxide.

    PubMed Central

    Matthews, J R; Botting, C H; Panico, M; Morris, H R; Hay, R T

    1996-01-01

    It has been suggested that the NF-kappaB transcription factor family may mediate expression of the gene encoding the cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric oxide (NO) could in turn affect activity of NF-kappaB, the ability of NO-donor compounds to influence NF-kappaB DNA binding activity in vitro was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibited the DNA binding activity of recombinant NF-kappaB p50 and p65 homodimers and of p50-p65 heterodimers. Inhibition of NF-kappaB p50 DNA binding by NO-donor compounds involved modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant was significantly more resistant to SNP-mediated inactivation. Non-reducing SDS-polyacrylamide gel electrophoresis demonstrated that SNP could inhibit p50 DNA binding by mechanisms other than the formation of intersubunit disulphide bonds involving p50 residue C62. Electrospray ionization mass spectrometry of a synthetic NF-kappaB p5O peptide containing the C62 residue suggested that NO gas can modify C62 by S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins, suggesting that cellular NO provides another control mechanism for modulating the expression of NF-kappaB-responsive genes. PMID:8710491

  13. A Novel DNA Binding Mechanism for maf Basic Region-Leucine Zipper Factors Inferred from a MafA-DNA Complex Structure and Binding Specificities

    SciTech Connect

    Lu, Xun; Guanga, Gerald P; Wan, Cheng; Rose, Robert B

    2012-11-13

    MafA is a proto-oncoprotein and is critical for insulin gene expression in pancreatic β-cells. Maf proteins belong to the AP1 superfamily of basic region-leucine zipper (bZIP) transcription factors. Residues in the basic helix and an ancillary N-terminal domain, the Extended Homology Region (EHR), endow maf proteins with unique DNA binding properties: binding a 13 bp consensus site consisting of a core AP1 site (TGACTCA) flanked by TGC sequences and binding DNA stably as monomers. To further characterize maf DNA binding, we determined the structure of a MafA–DNA complex. MafA forms base-specific hydrogen bonds with the flanking G–5C–4 and central C0/G0 bases, but not with the core-TGA bases. However, in vitro binding studies utilizing a pulse–chase electrophoretic mobility shift assay protocol revealed that mutating either the core-TGA or flanking-TGC bases dramatically increases the binding off rate. Comparing the known maf structures, we propose that DNA binding specificity results from positioning the basic helix through unique phosphate contacts. The EHR does not contact DNA directly but stabilizes DNA binding by contacting the basic helix. Collectively, these results suggest a novel multistep DNA binding process involving a conformational change from contacting the core-TGA to contacting the flanking-TGC bases.

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

    PubMed

    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.

  15. Phase Behavior of DNA in the Presence of DNA-Binding Proteins

    PubMed Central

    Le Treut, Guillaume; Képès, François; Orland, Henri

    2016-01-01

    To characterize the thermodynamical equilibrium of DNA chains interacting with a solution of nonspecific binding proteins, we implemented a Flory-Huggins free energy model. We explored the dependence on DNA and protein concentrations of the DNA collapse. For physiologically relevant values of the DNA-protein affinity, this collapse gives rise to a biphasic regime with a dense and a dilute phase; the corresponding phase diagram was computed. Using an approach based on Hamiltonian paths, we show that the dense phase has either a molten globule or a crystalline structure, depending on the DNA bending rigidity, which is influenced by the ionic strength. These results are valid at the thermodynamical equilibrium and therefore should be consistent with many biological processes, whose characteristic timescales range typically from 1 ms to 10 s. Our model may thus be applied to biological phenomena that involve DNA-binding proteins, such as DNA condensation with crystalline order, which occurs in some bacteria to protect their chromosome from detrimental factors; or transcription initiation, which occurs in clusters called transcription factories that are reminiscent of the dense phase characterized in this study. PMID:26745409

  16. N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein

    SciTech Connect

    Ruyechan, W.T. )

    1988-03-01

    The major herpes simplex virus DNA-binding protein, designated ICP8, binds tightly to single-stranded DNA and is required for replication of viral DNA. The sensitivity of the DNA-binding activity of ICP8 to the action of the sulfhydryl reagent N-ethylmaleimide has been examined by using nitrocellulose filter-binding and agarose gel electrophoresis assays. Incubation of ICP8 with N-ethylmaleimide results in a rapid loss of DNA-binding activity. Preincubation of ICP8 with single-stranded DNA markedly inhibits this loss of binding activity. These results imply that a free sulfhydryl group is involved in the interaction of ICP8 with single-stranded DNA and that this sulfhydryl group becomes less accessible to the environment upon binding. Agarose gel electrophoretic analysis of the binding interaction in the presence and absence of N-ethylmaleimide indicates that the cooperative binding exhibited by ICP8 is lost upon treatment with this reagent but that some residual noncooperative binding may remain. This last result was confirmed by equilibrium dialysis experiments with the {sup 32}P-labeled oligonucleotide dT{sub 10} and native and N-ethylmaleimide-treated ICP8.

  17. Role of Single-Stranded DNA Binding Activity of T Antigen in Simian Virus 40 DNA Replication

    PubMed Central

    Wu, Chunxiao; Roy, Rupa; Simmons, Daniel T.

    2001-01-01

    We have previously mapped the single-stranded DNA binding domain of large T antigen to amino acid residues 259 to 627. By using internal deletion mutants, we show that this domain most likely begins after residue 301 and that the region between residues 501 and 550 is not required. To study the function of this binding activity, a series of single-point substitutions were introduced in this domain, and the mutants were tested for their ability to support simian virus 40 (SV40) replication and to bind to single-stranded DNA. Two replication-defective mutants (429DA and 460EA) were grossly impaired in single-stranded DNA binding. These two mutants were further tested for other biochemical activities needed for viral DNA replication. They bound to origin DNA and formed double hexamers in the presence of ATP. Their ability to unwind origin DNA and a helicase substrate was severely reduced, although they still had ATPase activity. These results suggest that the single-stranded DNA binding activity is involved in DNA unwinding. The two mutants were also very defective in structural distortion of origin DNA, making it likely that single-stranded DNA binding is also required for this process. These data show that single-stranded DNA binding is needed for at least two steps during SV40 DNA replication. PMID:11222709

  18. The biotin repressor: thermodynamic coupling of corepressor binding, protein assembly, and sequence-specific DNA binding.

    PubMed

    Streaker, Emily D; Gupta, Aditi; Beckett, Dorothy

    2002-12-03

    The Escherichia coli biotin repressor, an allosteric transcriptional regulator, is activated for binding to the biotin operator by the small molecule biotinyl-5'-AMP. Results of combined thermodynamic, kinetic, and structural studies of the protein have revealed that corepressor binding results in disorder to order transitions in the protein monomer that facilitate tighter dimerization. The enhanced stability of the dimer leads to stabilization of the resulting biotin repressor-biotin operator complex. It is not clear, however, that the allosteric response in the system is transmitted solely through the protein-protein interface. In this work, the allosteric mechanism has been quantitatively probed by measuring the biotin operator binding and dimerization properties of three biotin repressor species: the apo or unliganded form, the biotin-bound form, and the holo or bio-5'-AMP-bound form. Comparisons of the pairwise differences in the bioO binding and dimerization energetics for the apo and holo species reveal that the enhanced DNA binding energetics resulting from adenylate binding track closely with the enhanced assembly energetics. However, when the results for repressor pairs that include the biotin-bound species are compared, no such equivalence is observed.

  19. Calculation of cooperativity and equilibrium constants of ligands binding to G-quadruplex DNA in solution.

    PubMed

    Kudrev, A G

    2013-11-15

    Equilibrium model of a ligand binding with DNA oligomer has been considered as a process of small molecule adsorption onto a lattice of multiple binding sites. An experimental example has been used to verify the assertion that during saturation of the macromolecule by a ligand should expect effect of cooperativity due to changes in DNA conformation or the mutual influence between bound ligands. Such phenomenon cannot be entirely described by the classical stepwise complex formation model. To evaluate a ligand binding affinity and cooperativity of ligand-oligomer complex formation the statistical approach has been proposed. This new computational approach used to re-examine previously studded ligand binding towards DNA quadruplexes targets with multiple binding sites. The intrinsic equilibrium constants K1-3 of the mesotetrakis-(N-methyl-4-pyridyl)-porphyrin (TMPyP4) binding with the [d(T4G4)]4 and with the [AG3(T2AG3)3] quadruplexes and the correction for the mutual influence between bound ligands (cooperativity parameters ω) was determined from the Job plots based upon the nonlinear least-squares fitting procedure. The re-examination of experimental curves reveals that the equilibrium is affected by the positive cooperative (ω>1) binding of the TMPyP4 ligand with tetramolecular [d(T4G4)]4. However for an intramolecular antiparallel-parallel hybrid structure [AG3(T2AG3)3] the weak anti-cooperativity of TMPyP4 accommodation (ω<1) onto two from three nonidentical sites was detected.

  20. Coupling between ATP Binding and DNA Cleavage by DNA Topoisomerase II

    PubMed Central

    Mueller-Planitz, Felix; Herschlag, Daniel

    2008-01-01

    DNA topoisomerase II is a molecular machine that couples ATP hydrolysis to the transport of one DNA segment through a transient break in another segment. To learn about the energetic connectivity that underlies this coupling, we investigated how the ATPase domains exert control over DNA cleavage. We dissected the DNA cleavage reaction by measuring rate and equilibrium constants for the individual reaction steps utilizing defined DNA duplexes in the presence and absence of the nonhydrolyzable ATP analog 5′-adenylyl-β,γ-imidodiphosphate (AMPPNP). Our results revealed the existence of two enzyme conformations whose relative abundance is sensitive to the presence of nucleotides. The predominant species in the absence of nucleotides binds DNA at a diffusion limited rate but cannot efficiently cleave DNA. In the presence of AMPPNP, most of the enzyme is converted to a state in which DNA binding and release is extremely slow but which allows DNA cleavage. A minimal kinetic and thermodynamic framework is established that accounts for the cooperativity of cleavage of the two DNA strands in the presence and absence of bound AMPPNP and includes conformational steps revealed in the kinetic studies. The model unifies available kinetic, thermodynamic, and structural data to provide a description for the reaction in terms of the order and rate of individual reaction steps and the physical nature of the species on the reaction path. Furthermore, this reaction framework provides a foundation for a future in-depth analysis of energy transduction by topoisomerase II, for guiding and interpreting future structural studies, and for analyzing the mechanism of drugs that convert topoisomerase into a cellular poison. PMID:18403371

  1. Interaction of zanamivir with DNA and RNA: Models for drug DNA and drug RNA bindings

    NASA Astrophysics Data System (ADS)

    Nafisi, Shohreh; Kahangi, Fatemeh Ghoreyshi; Azizi, Ebrahim; Zebarjad, Nader; Tajmir-Riahi, Heidar-Ali

    2007-03-01

    Zanamivir (ZAN) is the first of a new generation of influenza virus-specific drugs known as neuraminidase inhibitors, which acts by interfering with life cycles of influenza viruses A and B. It prevents the virus spreading infection to other cells by blocking the neuraminidase enzyme present on the surface of the virus. The aim of this study was to examine the stability and structural features of calf thymus DNA and yeast RNA complexes with zanamivir in aqueous solution, using constant DNA or RNA concentration (12.5 mM) and various zanamivir/polynucleotide ( P) ratios of 1/20, 1/10, 1/4, and 1/2. FTIR and UV-visible spectroscopy are used to determine the drug external binding modes, the binding constant and the stability of zanamivir-DNA and RNA complexes in aqueous solution. Structural analysis showed major interaction of zanamivir with G-C (major groove) and A-T (minor groove) base pairs and minor perturbations of the backbone PO 2 group with overall binding constants of Kzanamivir-DNA = 1.30 × 10 4 M -1 and Kzanamivir-RNA = 1.38 × 10 4 M -1. The drug interaction induces a partial B to A-DNA transition, while RNA remains in A-conformation.

  2. BindUP: a web server for non-homology-based prediction of DNA and RNA binding proteins.

    PubMed

    Paz, Inbal; Kligun, Efrat; Bengad, Barak; Mandel-Gutfreund, Yael

    2016-07-08

    Gene expression is a multi-step process involving many layers of regulation. The main regulators of the pathway are DNA and RNA binding proteins. While over the years, a large number of DNA and RNA binding proteins have been identified and extensively studied, it is still expected that many other proteins, some with yet another known function, are awaiting to be discovered. Here we present a new web server, BindUP, freely accessible through the website http://bindup.technion.ac.il/, for predicting DNA and RNA binding proteins using a non-homology-based approach. Our method is based on the electrostatic features of the protein surface and other general properties of the protein. BindUP predicts nucleic acid binding function given the proteins three-dimensional structure or a structural model. Additionally, BindUP provides information on the largest electrostatic surface patches, visualized on the server. The server was tested on several datasets of DNA and RNA binding proteins, including proteins which do not possess DNA or RNA binding domains and have no similarity to known nucleic acid binding proteins, achieving very high accuracy. BindUP is applicable in either single or batch modes and can be applied for testing hundreds of proteins simultaneously in a highly efficient manner.

  3. Base specific binding of deoxyguanylate and deoxycytidylate antibodies to double stranded DNA.

    PubMed Central

    Jacob, A; Jacob, T M

    1982-01-01

    Antibodies raised in rabbits against deoxyguanylate and deoxycytidylate bind to 3H-lambda double stranded DNA and the binding is base specific. The concentrations of antibody populations that bind to double stranded DNA are much less than those binding to denatured DNA. Due to their low concentrations, these antibodies were not detected in earlier studies. These antibodies are expected to be useful to probe the conformational flexibilities of double stranded DNAs. PMID:6217448

  4. DNA sensing by a Eu-binding peptide containing a proflavine unit.

    PubMed

    Ancel, Laetitia; Gateau, Christelle; Lebrun, Colette; Delangle, Pascale

    2013-01-18

    Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

  5. A designed DNA binding motif that recognizes extended sites and spans two adjacent major grooves†

    PubMed Central

    Rodríguez, Jéssica; Mosquera, Jesús; García-Fandiño, Rebeca; Vázquez, M. Eugenio; Mascareñas, José L.

    2016-01-01

    We report the rational design of a DNA-binding peptide construct composed of the DNA-contacting regions of two transcription factors (GCN4 and GAGA) linked through an AT-hook DNA anchor. The resulting chimera, which represents a new, non-natural DNA binding motif, binds with high affinity and selectivity to a long composite sequence of 13 base pairs (TCAT-AATT-GAGAG). PMID:27252825

  6. DNA Mutagenic Activity and Capacity for HIV-1 Restriction of the Cytidine Deaminase APOBEC3G Depends on Whether DNA or RNA Binds to Tyrosine 315.

    PubMed

    Polevoda, Bogdan; Joseph, Rebecca; Friedman, Alan E; Bennett, Ryan P; Greiner, Rebecca; De Zoysa, Thareendra; Stewart, Ryan A; Smith, Harold C

    2017-04-05

    APOBEC3G (A3G) belongs to the AID/APOBEC protein family of cytidine deaminases (CDA) that bind to nucleic acids. A3G mutates the HIV genome by deamination of dC to dU, leading to accumulation of virus-inactivating mutations. Binding to cellular RNAs inhibits A3G binding to substrate single-stranded (ss) DNA and CDA activity. RNA and ssDNA bind to the same three A3G tryptic peptides (amino acids 181-194, 314-320, and 345-374) that form parts of a continuously exposed protein surface extending from the catalytic domain in the C-terminus of A3G to its N-terminus. We show here that the A3G tyrosines 181 and 315 directly cross-link ssDNA. Binding experiments showed that a Y315A mutation alone significantly reduced A3G binding to both ssDNA and RNA, whereas Y181A and Y182A mutations only moderately affected A3G nucleic acid binding. Consistent with these findings, the Y315A mutant exhibited little to no deaminase activity in an E. coli DNA mutator reporter, while Y181A and Y182A mutants retained ~50% of wild-type A3G activity. The Y315A mutant also showed a markedly reduced ability to assemble into viral particles and had reduced antiviral activity. In uninfected cells, the impaired RNA-binding capacity of Y315A was evident by a shift of A3G from high-molecular-mass ribonucleoprotein complexes to low-molecular-mass complexes. We conclude that Y315 is essential for coordinating ssDNA interaction with or entry to the deaminase domain and hypothesize that RNA bound to Y315 may be sufficient to competitively inhibit ssDNA deaminase-dependent antiviral activity.

  7. Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif.

    PubMed Central

    Sharrocks, A D; Gille, H; Shaw, P E

    1993-01-01

    The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure. Images PMID:8417320

  8. Dynamical DNA accessibility induced by chromatin remodeling and protein binding

    NASA Astrophysics Data System (ADS)

    Montel, F.; Faivre-Moskalenko, C.; Castelnovo, M.

    2014-11-01

    Chromatin remodeling factors are enzymes being able to alter locally chromatin structure at the nucleosomal level and they actively participate in the regulation of gene expression. Using simple rules for individual nucleosome motion induced by a remodeling factor, we designed simulations of the remodeling of oligomeric chromatin, in order to address quantitatively collective effects in DNA accessibility upon nucleosome mobilization. Our results suggest that accessibility profiles are inhomogeneous thanks to borders effects like protein binding. Remarkably, we show that the accessibility lifetime of DNA sequence is roughly doubled in the vicinity of borders as compared to its value in bulk regions far from the borders. These results are quantitatively interpreted as resulting from the confined diffusion of a large nucleosome depleted region.

  9. Methyl-binding DNA capture Sequencing for Patient Tissues

    PubMed Central

    Jadhav, Rohit R.; Wang, Yao V.; Hsu, Ya-Ting; Liu, Joseph; Garcia, Dawn; Lai, Zhao; Huang, Tim H. M.; Jin, Victor X.

    2016-01-01

    Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System). PMID:27842364

  10. Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.

    PubMed

    Martinez, A; Kolter, R

    1997-08-01

    Reactive oxygen species can damage most cellular components, but DNA appears to be the most sensitive target of these agents. Here we present the first evidence of DNA protection against the toxic and mutagenic effects of oxidative damage in metabolically active cells: direct protection of DNA by Dps, an inducible nonspecific DNA-binding protein from Escherichia coli. We demonstrate that in a recA-deficient strain, expression of Dps from an inducible promoter prior to hydrogen peroxide challenge increases survival and reduces the number of chromosomal single-strand breaks. dps mutants exhibit increased levels of the G x C-->T x A mutations characteristic of oxidative damage after treatment with hydrogen peroxide. In addition, expression of Dps from the inducible plasmid reduces the frequency of spontaneous G x C-->T x A and A x T-->T x A mutations and can partially suppress the mutator phenotype of mutM (fpg) and mutY alleles. In a purified in vitro system, Dps reduces the number of DNA single-strand breaks and Fpg-sensitive sites introduced by hydrogen peroxide treatment, indicating that the protection observed in vivo is a direct effect of DNA binding by Dps. The widespread conservation of Dps homologs among prokaryotes suggests that this may be a general strategy for coping with oxidative stress.

  11. Recombinant human MDM2 oncoprotein shows sequence composition selectivity for binding to both RNA and DNA.

    PubMed

    Challen, Christine; Anderson, John J; Chrzanowska-Lightowlers, Zofia M A; Lightowlers, Robert N; Lunec, John

    2012-03-01

    MDM2 is a 90 kDa nucleo-phosphoprotein that binds p53 and other proteins contributing to its oncogenic properties. Its structure includes an amino proximal p53 binding site, a central acidic domain and a carboxy region which incorporates Zinc and Ring Finger domains suggestive of nucleic acid binding or transcription factor function. It has previously been reported that a bacculovirus expressed MDM2 protein binds RNA in a sequence-specific manner through the Ring Finger domain, however, its ability to bind DNA has yet to be examined. We report here that a bacterially expressed human MDM2 protein binds both DNA as well as the previously defined RNA consensus sequence. DNA binding appears selective and involves the carboxy-terminal domain of the molecule. RNA binding is inhibited by an MDM2 specific antibody, which recognises an epitope within the carboxy region of the protein. Selection cloning and sequence analysis of MDM2 DNA binding sequences, unlike RNA binding sequences, revealed no obvious DNA binding consensus sequence, but preferential binding to oligopurine:pyrimidine-rich stretches. Our results suggest that the observed preferential DNA binding may occur through the Zinc Finger or in a charge-charge interaction through the Ring Finger, thereby implying potentially different mechanisms for DNA and RNA MDM2 binding.

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

    PubMed

    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

  13. Characterization of a mitochondrial protein binding to single-stranded DNA.

    PubMed Central

    Mignotte, B; Barat, M; Mounolou, J C

    1985-01-01

    A DNA-binding protein from Xenopus laevis oocyte mitochondria which has been found associated with the D-loop also shows a strong preference for single-stranded DNA. The binding to polynucleotides is dependent on the base composition, but no sequence specificity was found. This protein, called mtSSB, binds tightly and cooperatively to single-stranded DNA. By its amino-acid composition and its binding properties it appears to be similar to the single-stranded DNA-binding proteins found in prokaryotes. PMID:4039816

  14. Structural identification of DnaK binding sites within bovine and sheep bactenecin Bac7.

    PubMed

    Zahn, Michael; Kieslich, Bjorn; Berthold, Nicole; Knappe, Daniel; Hoffmann, Ralf; Strater, Norbert

    2014-04-01

    Bacterial resistance against common antibiotics is an increasing health problem. New pharmaceuticals for the treatment of infections caused by resistant pathogens are needed. Small proline-rich antimicrobial peptides (PrAMPs) from insects are known to bind intracellularly to the conventional substrate binding cleft of the E. coli Hsp70 chaperone DnaK. Furthermore, bactenecins from mammals, members of the cathelicidin family, also contain potential DnaK binding sites. Crystal structures of bovine and sheep Bac7 in complex with the DnaK substrate binding domain show that the peptides bind in the forward binding mode with a leucine positioned in the central hydrophobic pocket. In most structures, proline and arginine residues preceding leucine occupy the hydrophobic DnaK binding sites -1 and -2. Within bovine Bac7, four potential DnaK binding sites were identified.

  15. Molecular simulations of polycation-DNA binding exploring the effect of peptide chemistry and sequence in nuclear localization sequence based polycations.

    PubMed

    Elder, Robert M; Jayaraman, Arthi

    2013-10-10

    Gene therapy relies on the delivery of DNA into cells, and polycations are one class of vectors enabling efficient DNA delivery. Nuclear localization sequences (NLS), cationic oligopeptides that target molecules for nuclear entry, can be incorporated into polycations to improve their gene delivery efficiency. We use simulations to study the effect of peptide chemistry and sequence on the DNA-binding behavior of NLS-grafted polycations by systematically mutating the residues in the grafts, which are based on the SV40 NLS (peptide sequence PKKKRKV). Replacing arginine (R) with lysine (K) reduces binding strength by eliminating arginine-DNA interactions, but placing R in a less hindered location (e.g., farther from the grafting point to the polycation backbone) has surprisingly little effect on polycation-DNA binding strength. Changing the positions of the hydrophobic proline (P) and valine (V) residues relative to the polycation backbone changes hydrophobic aggregation within the polycation and, consequently, changes the conformational entropy loss that occurs upon polycation-DNA binding. Since conformational entropy loss affects the free energy of binding, the positions of P and V in the grafts affect DNA binding affinity. The insight from this work guides synthesis of polycations with tailored DNA binding affinity and, in turn, efficient DNA delivery.

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

    SciTech Connect

    Supakar, P.C.; Weist, D.; Zhang, D.; Inamdar, N.; Zhang, Xianyang; Khan, R.; Ehrlich, M. ); Ehrlich, K.C. )

    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.

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

    PubMed

    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.

  18. Investigations of the CLOCK and BMAL1 Proteins Binding to DNA: A Molecular Dynamics Simulation Study

    PubMed Central

    Xue, Tuo; Song, Chunnian; Wang, Qing; Wang, Yan; Chen, Guangju

    2016-01-01

    The circadian locomotor output cycles kaput (CLOCK), and brain and muscle ARNT-like 1 (BMAL1) proteins are important transcriptional factors of the endogenous circadian clock. The CLOCK and BMAL1 proteins can regulate the transcription-translation activities of the clock-related genes through the DNA binding. The hetero-/homo-dimerization and DNA combination of the CLOCK and BMAL1 proteins play a key role in the positive and negative transcriptional feedback processes. In the present work, we constructed a series of binary and ternary models for the bHLH/bHLH-PAS domains of the CLOCK and BMAL1 proteins, and the DNA molecule, and carried out molecular dynamics simulations, free energy calculations and conformational analysis to explore the interaction properties of the CLOCK and BMAL1 proteins with DNA. The results show that the bHLH domains of CLOCK and BMAL1 can favorably form the heterodimer of the bHLH domains of CLOCK and BMAL1 and the homodimer of the bHLH domains of BMAL1. And both dimers could respectively bind to DNA at its H1-H1 interface. The DNA bindings of the H1 helices in the hetero- and homo-bHLH dimers present the rectangular and diagonal binding modes, respectively. Due to the function of the α-helical forceps in these dimers, the tight gripping of the H1 helices to the major groove of DNA would cause the decrease of interactions at the H1-H2 interfaces in the CLOCK and BMAL1 proteins. The additional PAS domains in the CLOCK and BMAL1 proteins affect insignificantly the interactions of the CLOCK and BMAL1 proteins with the DNA molecule due to the flexible and long loop linkers located at the middle of the PAS and bHLH domains. The present work theoretically explains the interaction mechanisms of the bHLH domains of the CLOCK and BMAL1 proteins with DNA. PMID:27153104

  19. Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: gp32 monomer binding

    PubMed Central

    Jose, Davis; Weitzel, Steven E.; Baase, Walter A.; von Hippel, Peter H.

    2015-01-01

    Combining biophysical measurements on T4 bacteriophage replication complexes with detailed structural information can illuminate the molecular mechanisms of these ‘macromolecular machines’. Here we use the low energy circular dichroism (CD) and fluorescent properties of site-specifically introduced base analogues to map and quantify the equilibrium binding interactions of short (8 nts) ssDNA oligomers with gp32 monomers at single nucleotide resolution. We show that single gp32 molecules interact most directly and specifically near the 3′-end of these ssDNA oligomers, thus defining the polarity of gp32 binding with respect to the ssDNA lattice, and that only 2–3 nts are directly involved in this tight binding interaction. The loss of exciton coupling in the CD spectra of dimer 2-AP (2-aminopurine) probes at various positions in the ssDNA constructs, together with increases in fluorescence intensity, suggest that gp32 binding directly extends the sugar-phosphate backbone of this ssDNA oligomer, particularly at the 3′-end and facilitates base unstacking along the entire 8-mer lattice. These results provide a model (and ‘DNA map’) for the isolated gp32 binding to ssDNA targets, which serves as the nucleation step for the cooperative binding that occurs at transiently exposed ssDNA sequences within the functioning T4 DNA replication complex. PMID:26275775

  20. The DNA-remodelling activity of DnaD is the sum of oligomerization and DNA-binding activities on separate domains

    PubMed Central

    Carneiro, Maria J. V. M.; Zhang, Wenke; Ioannou, Charikleia; Scott, David J.; Allen, Stephanie; Roberts, Clive J.; Soultanas, Panos

    2011-01-01

    Summary The Bacillus subtilis DnaD protein is an essential protein that has been implicated in the primosomal step of DNA replication, and recently in global DNA remodelling. Here we show that DnaD consists of two domains with distinct activities; an N-terminal domain (Nd) with oligomerization activity, and a C-terminal domain (Cd) with DNA-binding activity and a second DNA-induced oligomerization activity. Although Cd can bind to DNA and form large nucleoprotein complexes, it does not exhibit global DNA-remodelling activity. The presence of separate Nd does not restore this activity. Our data suggest that the global DNA-remodelling activity of DnaD is the sum of three separate oligomerization and DNA-binding activities residing on two distinct but linked domains. PMID:16677303

  1. Further characterization of functional domains of PerA, role of amino and carboxy terminal domains in DNA binding.

    PubMed

    Ibarra, J Antonio; García-Zacarias, Claudia M; Lara-Ochoa, Cristina; Carabarin-Lima, Alejandro; Tecpanecatl-Xihuitl, J Sergio; Perez-Rueda, Ernesto; Martínez-Laguna, Ygnacio; Puente, José L

    2013-01-01

    PerA is a key regulator of virulence genes in enteropathogenic E. coli. PerA is a member of the AraC/XylS family of transcriptional regulators that directly regulates the expression of the bfp and per operons in response to different environmental cues. Here, we characterized mutants in both the amino (NTD) and carboxy (CTD) terminal domains of PerA that affect its ability to activate the expression of the bfp and per promoters. Mutants at residues predicted to be important for DNA binding within the CTD had a significant defect in their ability to bind to the regulatory regions of the bfp and per operons and, consequently, in transcriptional activation. Notably, mutants in specific NTD residues were also impaired to bind to DNA suggesting that this domain is involved in structuring the protein for correct DNA recognition. Mutations in residues E116 and D168, located in the vicinity of the putative linker region, significantly affected the activation of the perA promoter, without affecting PerA binding to the per or bfp regulatory sequences. Overall these results provide additional evidence of the importance of the N-terminal domain in PerA activity and suggest that the activation of these promoters involves differential interactions with the transcriptional machinery. This study further contributes to the characterization of the functional domains of PerA by identifying critical residues involved in DNA binding, differential promoter activation and, potentially, in the possible response to environmental cues.

  2. R248Q mutation--Beyond p53-DNA binding.

    PubMed

    Ng, Jeremy W K; Lama, Dilraj; Lukman, Suryani; Lane, David P; Verma, Chandra S; Sim, Adelene Y L

    2015-12-01

    R248 in the DNA binding domain (DBD) of p53 interacts directly with the minor groove of DNA. Earlier nuclear magnetic resonance (NMR) studies indicated that the R248Q mutation resulted in conformation changes in parts of DBD far from the mutation site. However, how information propagates from the mutation site to the rest of the DBD is still not well understood. We performed a series of all-atom molecular dynamics (MD) simulations to dissect sterics and charge effects of R248 on p53-DBD conformation: (i) wild-type p53 DBD; (ii) p53 DBD with an electrically neutral arginine side-chain; (iii) p53 DBD with R248A; (iv) p53 DBD with R248W; and (v) p53 DBD with R248Q. Our results agree well with experimental observations of global conformational changes induced by the R248Q mutation. Our simulations suggest that both charge- and sterics are important in the dynamics of the loop (L3) where the mutation resides. We show that helix 2 (H2) dynamics is altered as a result of a change in the hydrogen bonding partner of D281. In turn, neighboring L1 dynamics is altered: in mutants, L1 predominantly adopts the recessed conformation and is unable to interact with the major groove of DNA. We focused our attention the R248Q mutant that is commonly found in a wide range of cancer and observed changes at the zinc-binding pocket that might account for the dominant negative effects of R248Q. Furthermore, in our simulations, the S6/S7 turn was more frequently solvent exposed in R248Q, suggesting that there is a greater tendency of R248Q to partially unfold and possibly lead to an increased aggregation propensity. Finally, based on the observations made in our simulations, we propose strategies for the rescue of R248Q mutants.

  3. In vitro DNA binding of the archaeal protein Sso7d induces negative supercoiling at temperatures typical for thermophilic growth.

    PubMed Central

    López-García, P; Knapp, S; Ladenstein, R; Forterre, P

    1998-01-01

    The topological state of DNA in hyperthermophilic archaea appears to correspond to a linking excess in comparison with DNA in mesophilic organisms. Since DNA binding proteins often contribute to the control of DNA topology by affecting DNA geometry in the presence of DNA topoisomerases, we tested whether the histone-like protein Sso7d from the hyperthermophilic archaeon Sulfolobus solfataricus alters DNA conformation. In ligase-mediated supercoiling assays carried out at 37, 60, 70, 80 and 90 degrees C we found that DNA binding of increasing amounts of Sso7d led to a progressive decrease in plasmid linking number (Lk), producing negative supercoiling. Identical unwinding effects were observed when recombinant non-methylated Sso7d was used. For a given Sso7d concentration the DNA unwinding induced was augmented with increasing temperature. However, after correction for the overwinding effect of high temperature on DNA, plasmids ligated at 60-90 degrees C exhibited similar sigma values at the highest Sso7d concentrations assayed. These results suggest that Sso7d may play a compensatory role in vivo by counteracting the overwinding effect of high temperature on DNA. Additionally, Sso7d unwinding could be involved in the topological changes observed during thermal stress (heat and cold shock), playing an analogous role in crenarchaeal cells to that proposed for HU in bacteria. PMID:9580681

  4. The quorum sensing transcriptional regulator TraR has separate binding sites for DNA and the anti-activator

    SciTech Connect

    Zheng, Zhida; Fuqua, Clay; Chen, Lingling

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer Quorum sensing transcription factor TraR is inhibited by forming TraR-TraM complex. Black-Right-Pointing-Pointer K213 is a key DNA binding residue, but not involved in interaction with TraM. Black-Right-Pointing-Pointer Mutations of TraM-interacting TraR residues did not affect DNA-binding of TraR. Black-Right-Pointing-Pointer Mutations of TraR residues reduced the TraR-TraM interaction more than those of TraM. Black-Right-Pointing-Pointer TraM inhibition on DNA-binding of TraR is driven by thermodynamics. -- Abstract: Quorum sensing represents a mechanism by which bacteria control their genetic behaviors via diffusible signals that reflect their population density. TraR, a quorum sensing transcriptional activator in the Rhizobiaceae family, is regulated negatively by the anti-activator TraM via formation of a TraR-TraM heterocomplex. Prior structural analysis suggests that TraM and DNA bind to TraR in distinct sites. Here we combined isothermal titration calorimetry (ITC) and electrophoretic mobility shift assays (EMSA) to investigate roles of TraR residues from Rhizobium sp. NGR234 in binding of both TraM and DNA. We found that K213A mutation of TraR{sub NGR} abolished DNA binding, however, did not alter TraM binding. Mutations of TraM-interfacing TraR{sub NGR} residues decreased the TraR-TraM interaction, but did not affect the DNA-binding activity of TraR{sub NGR}. Thus, our biochemical studies support the independent binding sites on TraR for TraM and DNA. We also found that point mutations in TraR{sub NGR} appeared to decrease the TraR-TraM interaction more effectively than those in TraM{sub NGR}, consistent with structural observations that individual TraR{sub NGR} residues contact with more TraM{sub NGR} residues than each TraM{sub NGR} residues with TraR{sub NGR} residues. Finally, we showed that TraM inhibition on DNA-binding of TraR was driven thermodynamically. We discussed subtle mechanistic differences in Tra

  5. A systematic survey of the Cys2His2 zinc finger DNA-binding landscape

    PubMed Central

    Persikov, Anton V.; Wetzel, Joshua L.; Rowland, Elizabeth F.; Oakes, Benjamin L.; Xu, Denise J.; Singh, Mona; Noyes, Marcus B.

    2015-01-01

    Cys2His2 zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain's well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain–DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain–DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities. PMID:25593323

  6. Interaction of 6 Mercaptopurine with Calf Thymus DNA – Deciphering the Binding Mode and Photoinduced DNA Damage

    PubMed Central

    Rehman, Sayeed Ur; Yaseen, Zahid; Husain, Mohammed Amir; Sarwar, Tarique; Ishqi, Hassan Mubarak; Tabish, Mohammad

    2014-01-01

    DNA is one of the major intracellular targets for a wide range of anticancer and antibiotic drugs. Elucidating the binding between small molecules and DNA provides great help in understanding drug-DNA interactions and in designing of new and promising drugs for clinical use. The ability of small molecules to bind and interfere with DNA replication and transcription provides further insight into how the drugs control the expression of genes. Interaction of an antimetabolite anticancer drug 6mercaptopurine (6MP) with calf thymus DNA was studied using various approaches like UV-visible spectroscopy, fluorescence spectroscopy, CD, viscosity and molecular docking. UV-visible spectroscopy confirmed 6MP-DNA interaction. Steady state fluorescence experiments revealed a moderate binding constant of 7.48×103 M−1 which was consistent with an external binding mode. Competitive displacement assays further confirmed a non-intercalative binding mode of 6MP which was further confirmed by CD and viscosity experiments. Molecular docking further revealed the minimum energy conformation (−119.67 kJ/mole) of the complex formed between DNA and 6MP. Hence, the biophysical techniques and in-silico molecular docking approaches confirmed the groove binding/electrostatic mode of interaction between 6MP and DNA. Further, photo induced generation of ROS by 6MP was studied spectrophotometrically and DNA damage was assessed by plasmid nicking and comet assay. There was a significant increase in ROS generation and consequent DNA damage in the presence of light. PMID:24718609

  7. DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.

    PubMed Central

    Myrset, A H; Bostad, A; Jamin, N; Lirsac, P N; Toma, F; Gabrielsen, O S

    1993-01-01

    The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a 'recognition' helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2. Images PMID:8223472

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

    PubMed

    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.

  9. TP53 codon 72 polymorphism affects accumulation of mtDNA damage in human cells

    PubMed Central

    Altilia, Serena; Santoro, Aurelia; Malagoli, Davide; Lanzarini, Catia; Álvarez, Josué Adolfo Ballesteros; Galazzo, Gianluca; Porter, Donald Carl; Crocco, Paolina; Rose, Giuseppina; Passarino, Giuseppe; Roninson, Igor Boris; Franceschi, Claudio; Salvioli, Stefano

    2012-01-01

    Human TP53 gene is characterised by a polymorphism at codon 72 leading to an Arginine-to-Proline (R/P) substitution. The two resulting p53 isoforms have a different subcellular localisation after stress (more nuclear or more mitochondrial for the P or R isoform, respectively). p53P72 variant is more efficient than p53R72 in inducing the expression of genes involved in nuclear DNA repair. Since p53 is involved also in mitochondrial DNA (mtDNA) maintenance, we wondered whether these p53 isoforms are associated with different accumulation of mtDNA damage. We observed that cells bearing p53R72 accumulate lower amount of mtDNA damage upon rotenone stress with respect to cells bearing p53P72, and that p53R72 co-localises with polymerase gamma more than p53P72. We also analysed the in vivo accumulation of heteroplasmy in a 300 bp fragment of mtDNA D-loop of 425 aged subjects. We observed that subjects with heteroplasmy higher than 5% are significantly less than expected in the p53R72/R72 group. On the whole, these data suggest that the polymorphism of TP53 at codon 72 affects the accumulation of mtDNA mutations, likely through the different ability of the two p53 isoforms to bind to polymerase gamma, and may contribute to in vivo accumulation of mtDNA mutations. PMID:22289634

  10. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    NASA Astrophysics Data System (ADS)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  11. Histones and DNA Compete for Binding Polyphosphoinositides in Bilayers

    PubMed Central

    Lete, Marta G.; Sot, Jesús; Ahyayauch, Hasna; Fernández-Rivero, Noelia; Prado, Adelina; Goñi, Félix M.; Alonso, Alicia

    2014-01-01

    Recent discoveries on the presence and location of phosphoinositides in the eukaryotic cell nucleoplasm and nuclear membrane prompted us to study the putative interaction of chromatin components with these lipids in model membranes (liposomes). Turbidimetric studies revealed that a variety of histones and histone combinations (H1, H2AH2B, H3H4, octamers) caused a dose-dependent aggregation of phosphatidylcholine vesicles (large unilamellar vesicle or small unilamellar vesicle) containing negatively charged phospholipids. 5 mol % phosphatidylinositol-4-phosphate (PIP) was enough to cause extensive aggregation under our conditions, whereas with phosphatidylinositol (PI) at least 20 mol % was necessary to obtain a similar effect. Histone binding to giant unilamellar vesicle and vesicle aggregation was visualized by confocal microscopy. Histone did not cause vesicle aggregation in the presence of DNA, and the latter was able to disassemble the histone-vesicle aggregates. At DNA/H1 weight ratios 0.1–0.5 DNA- and PIP-bound H1 appear to coexist. Isothermal calorimetry studies revealed that the PIP-H1 association constant was one order of magnitude higher than that of PI-H1, and the corresponding lipid/histone stoichiometries were ∼0.5 and ∼1, respectively. The results suggest that, in the nucleoplasm, a complex interplay of histones, DNA, and phosphoinositides may be taking place, particularly at the nucleoplasmic reticula that reach deep within the nucleoplasm, or during somatic and nonsomatic nuclear envelope assembly. The data described here provide a minimal model for analyzing and understanding the mechanism of these interactions. PMID:24606933

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

    PubMed

    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.

  13. Determinants of the DNA binding specificity of class I and class II TCP transcription factors.

    PubMed

    Viola, Ivana L; Reinheimer, Renata; Ripoll, Rodrigo; Manassero, Nora G Uberti; Gonzalez, Daniel H

    2012-01-02

    TCP proteins constitute a family of plant transcription factors with more than 20 members in angiosperms. They can be divided in two classes based on sequence homology and the presence of an insertion within the basic region of the TCP DNA binding and dimerization domain. Here, we describe binding site selection studies with the class I protein TCP16, showing that its DNA binding preferences are similar to those of class II proteins. Through sequence comparison and the analysis of mutants and chimeras of TCP16, TCP20 (class I), and TCP4 (class II), we established that the identity of residue 11 of the class I TCP domain or the equivalent residue 15 of the class II domain, whether it is Gly or Asp, determines a preference for a class I or a class II sequence, respectively. Footprinting analysis indicated that specific DNA contacts related to these preferences are established with one of the strands of DNA. The dimerization motif also influences the selectivity of the proteins toward class I and class II sequences and determines a requirement of an extended basic region in proteins with Asp-15. We postulate that differences in orientation of base-contacting residues brought about by the presence of either Gly or Asp are responsible for the binding site preferences of TCP proteins. Expression of repressor forms of TCP16 with Asp-11 or Gly-11 differently affects leaf development. TCP16-like proteins with Asp-11 in the TCP domain arose in rosids and may be related to developmental characteristics of this lineage of eudicots.

  14. Design of Sequence-Specific DNA Binding Molecules for DNA Methyltransferase Inhibition

    PubMed Central

    2015-01-01

    The CpG dyad, an important genomic feature in DNA methylation and transcriptional regulation, is an attractive target for small molecules. To assess the utility of minor groove binding oligomers for CpG recognition, we screened a small library of hairpin pyrrole-imidazole polyamides targeting the sequence 5′-CGCG-3′ and assessed their sequence specificity using an unbiased next-generation sequencing assay. Our findings indicate that hairpin polyamide of sequence PyImβIm-γ-PyImβIm (1), previously identified as a high affinity 5′-CGCG-3′ binder, favors 5′-GCGC-3′ in an unanticipated reverse binding orientation. Replacement of one β alanine with Py to afford PyImPyIm-γ-PyImβIm (3) restores the preference for 5′-CGCG-3′ binding in a forward orientation. The minor groove binding hairpin 3 inhibits DNA methyltransferase activity in the major groove at its target site more effectively than 1, providing a molecular basis for design of sequence-specific antagonists of CpG methylation. PMID:24502234

  15. An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase.

    PubMed

    Windgassen, Tricia A; Keck, James L

    2016-11-16

    Helicases couple ATP hydrolysis to nucleic acid binding and unwinding via molecular mechanisms that remain poorly defined for most enzyme subfamilies within the superfamily 2 (SF2) helicase group. A crystal structure of the PriA SF2 DNA helicase, which governs restart of prematurely terminated replication processes in bacteria, revealed the presence of an aromatic-rich loop (ARL) on the presumptive DNA-binding surface of the enzyme. The position and sequence of the ARL was similar to loops known to couple ATP hydrolysis with DNA binding in a subset of other SF2 enzymes, however, the roles of the ARL in PriA had not been investigated. Here, we show that changes within the ARL sequence uncouple PriA ATPase activity from DNA binding. In vitro protein-DNA crosslinking experiments define a residue- and nucleotide-specific interaction map for PriA, showing that the ARL binds replication fork junctions whereas other sites bind the leading or lagging strands. We propose that DNA binding to the ARL allosterically triggers ATP hydrolysis in PriA. Additional SF2 helicases with similarly positioned loops may also couple DNA binding to ATP hydrolysis using related mechanisms.

  16. An aromatic-rich loop couples DNA binding and ATP hydrolysis in the PriA DNA helicase

    PubMed Central

    Windgassen, Tricia A.; Keck, James L.

    2016-01-01

    Helicases couple ATP hydrolysis to nucleic acid binding and unwinding via molecular mechanisms that remain poorly defined for most enzyme subfamilies within the superfamily 2 (SF2) helicase group. A crystal structure of the PriA SF2 DNA helicase, which governs restart of prematurely terminated replication processes in bacteria, revealed the presence of an aromatic-rich loop (ARL) on the presumptive DNA-binding surface of the enzyme. The position and sequence of the ARL was similar to loops known to couple ATP hydrolysis with DNA binding in a subset of other SF2 enzymes, however, the roles of the ARL in PriA had not been investigated. Here, we show that changes within the ARL sequence uncouple PriA ATPase activity from DNA binding. In vitro protein-DNA crosslinking experiments define a residue- and nucleotide-specific interaction map for PriA, showing that the ARL binds replication fork junctions whereas other sites bind the leading or lagging strands. We propose that DNA binding to the ARL allosterically triggers ATP hydrolysis in PriA. Additional SF2 helicases with similarly positioned loops may also couple DNA binding to ATP hydrolysis using related mechanisms. PMID:27484483

  17. Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR

    PubMed Central

    Schumacher, Maria A.; Miller, Marshall C.; Grkovic, Steve; Brown, Melissa H.; Skurray, Ronald A.; Brennan, Richard G.

    2002-01-01

    The Staphylococcus aureus multidrug-binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by multiple structurally dissimilar drugs. QacR is a member of the TetR/CamR family of transcriptional regulators, which share highly homologous N-terminal DNA-binding domains connected to seemingly non-homologous ligand-binding domains. Unlike other TetR members, which bind ∼15 bp operators, QacR recognizes an unusually long 28 bp operator, IR1, which it appears to bind cooperatively. To elucidate the DNA-binding mechanism of QacR, we determined the 2.90 Å resolution crystal structure of a QacR–IR1 complex. Strikingly, our data reveal that the DNA recognition mode of QacR is distinct from TetR and involves the binding of a pair of QacR dimers. In this unique binding mode, recognition at each IR1 half-site is mediated by a complement of DNA contacts made by two helix–turn–helix motifs. The inferred cooperativity does not arise from cross-dimer protein–protein contacts, but from the global undertwisting and major groove widening elicited by the binding of two QacR dimers. PMID:11867549

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

    PubMed

    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.

  19. New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA

    PubMed Central

    Zucchelli, Chiara; Ferrari, Elena; Blasi, Francesco; Musco, Giovanna; Bruckmann, Chiara

    2017-01-01

    PREP1 and PBX1 are homeodomain (HD) transcription factors that play crucial roles in embryonic development. Here, we present the first biophysical characterization of a PREP1 HD, and the NMR spectroscopic study of its DNA binding pocket. The data show that residues flanking the HD participate in DNA binding. The kinetic parameters for DNA binding of individual PREP1 and PBX1 HDs, and of their combination, show that isolated PREP1 and PBX1 HDs bind to DNA in a cooperative manner. A novel PREP1 motif, flanking the HD at the C-terminus, is required for cooperativity. PMID:28094776

  20. Redox regulation of c-Jun DNA binding by reversible S-glutathiolation.

    PubMed

    Klatt, P; Molina, E P; De Lacoba, M G; Padilla, C A; Martinez-Galesteo, E; Barcena, J A; Lamas, S

    1999-09-01

    Redox control of the transcription factor c-Jun maps to a single cysteine in its DNA binding domain. However, the nature of the oxidized state of this cysteine and, thus, the potential molecular mechanisms accounting for the redox regulation of c-Jun DNA binding remain unclear. To address this issue, we have analyzed the purified recombinant c-Jun DNA binding domain for redox-dependent thiol modifications and concomitant changes in DNA binding activity. We show that changes in the ratio of reduced to oxidized glutathione provide the potential to oxidize c-Jun sulfhydryls by mechanisms that include both protein disulfide formation and S-glutathiolation. We provide evidence that S-glutathiolation, which is specifically targeted to the cysteine residue located in the DNA binding site of the protein, may account for the reversible redox regulation of c-Jun DNA binding. Furthermore, based on a molecular model of the S-glutathiolated protein, we discuss the structural elements facilitating S-glutathiolation and how this modification interferes with DNA binding. Given the structural similarities between the positively charged cysteine-containing DNA binding motif of c-Jun and the DNA binding site of related oxidant-sensitive transcriptional activators, the unprecedented phenomenon of redox-triggered S-thiolation of a transcription factor described in this report suggests a novel role for protein thiolation in the redox control of transcription.

  1. NonO enhances the association of many DNA-binding proteins to their targets.

    PubMed

    Yang, Y S; Yang, M C; Tucker, P W; Capra, J D

    1997-06-15

    NonO is an unusual nucleic acid binding protein not only in that it binds both DNA and RNA but that it does so via functionally separable domains. Here we document that NonO enhances the binding of some (E47, OTF-1 and OTF-2) but not all (PEA3) conventional sequence-specific transcription factors to their recognition sites in artificial substrates as well as in an immunoglobulin VHpromoter. We also show that NonO induces the binding of the Ku complex to DNA ends. Ku has no known DNA sequence specificity. These enhancement of binding effects are NonO concentration dependent. Using the E box activity of E47 as a model, kinetic studies demonstrate that the association rate of the protein-DNA complex increases in the presence of NonO while the dissociation rate remains the same, thereby increasing the sum total of the interaction. Oligo competition experiments indicate that NonO does not contact the target DNA in order to enhance the binding activity of DNA binding proteins. Rather, methylation interference analysis reveals that the induced E47 binding-activity has the same DNA-binding sequence specificity as the normal binding. This result suggests that one of the effects of NonO is to induce a true protein-DNA interaction. In this way, it might be possible for NonO to play a crucial role in gene regulation.

  2. A Key Evolutionary Mutation Enhances DNA Binding of the FOXP2 Forkhead Domain.

    PubMed

    Morris, Gavin; Fanucchi, Sylvia

    2016-04-05

    Forkhead box (FOX) transcription factors share a conserved forkhead DNA binding domain (FHD) and are key role players in the development of many eukaryotic species. Their involvement in various congenital disorders and cancers makes them clinically relevant targets for novel therapeutic strategies. Among them, the FOXP subfamily of multidomain transcriptional repressors is unique in its ability to form DNA binding homo and heterodimers. The truncated FOXP2 FHD, in the absence of the leucine zipper, exists in equilibrium between monomeric and domain-swapped dimeric states in vitro. As a consequence, determining the DNA binding properties of the FOXP2 FHD becomes inherently difficult. In this work, two FOXP2 FHD hinge loop mutants have been generated to successfully prevent both the formation (A539P) and the dissociation (F541C) of the homodimers. This allows for the separation of the two species for downstream DNA binding studies. Comparison of DNA binding of the different species using electrophoretic mobility shift assay, fluorescence anisotropy and isothermal titration calorimetry indicates that the wild-type FOXP2 FHD binds DNA as a monomer. However, comparison of the DNA-binding energetics of the monomer and wild-type FHD, reveals that there is a difference in the mechanism of binding between the two species. We conclude that the naturally occurring reverse mutation (P539A) seen in the FOXP subfamily increases DNA binding affinity and may increase the potential for nonspecific binding compared to other FOX family members.

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

    SciTech Connect

    Strickland, Devin; Moffat, Keith; Sosnick, Tobin R.

    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.

  4. Recognition of DNA sequencing through binding of nucleobases to graphene

    NASA Astrophysics Data System (ADS)

    Zaffino, Valentina

    Graphene is one of the most promising materials in nanotechnology. Its large surface to volume ratio, high conductivity and electron mobility at room temperature are outstanding properties for use in DNA sensors. For this study, we used Density Functional Theory (DFT), ?with and without the inclusion of van der Waals (vdW) interactions, ?to investigate the adsorption of nucleobases (cytosine, guanine, adenine, thymine, and uracil) on pristine graphene and graphene with defects (Divacancy and Stone-Wales). We investigated the performance of two types of vdW-DF functional (optB86b-vdW and rPW86-vdW), as well as the PBE functional, and their description of the adsorption geometry and electronic structure of the nucleobase-graphene systems.The inclusion of defects results in an increase in binding energy, closer adsorption of the molecule to graphene and greater buckling in both the graphene structure and nucleobase.

  5. DNA sequencing using polymerase substrate-binding kinetics

    PubMed Central

    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

    2015-01-01

    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. PMID:25612848

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

    SciTech Connect

    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.

  7. Determination of the drug-DNA binding modes using fluorescence-based assays.

    PubMed

    Williams, Alicia K; Dasilva, Sofia Cheliout; Bhatta, Ankit; Rawal, Baibhav; Liu, Melinda; Korobkova, Ekaterina A

    2012-03-15

    Therapeutic drugs and environmental pollutants may exhibit high reactivity toward DNA bases and backbone. Understanding the mechanisms of drug-DNA binding is crucial for predicting their potential genotoxicity. We developed a fluorescence analytical method for the determination of the preferential binding mode for drug-DNA interactions. Two nucleic acid dyes were employed in the method: TO-PRO-3 iodide (TP3) and 4',6-diamidino-2-phenylindole (DAPI). TP3 binds DNA by intercalation, whereas DAPI exhibits minor groove binding. Both dyes exhibit significant fluorescence magnification on binding to DNA. We evaluated the DNA binding constant, K(b), for each dye. We also performed fluorescence quenching experiments with 11 molecules of various structures and measured a C(50) value for each compound. We determined preferential binding modes for the aforementioned molecules and found that they bound to DNA consistently, as indicated by other studies. The values of the likelihood of DNA intercalation were correlated with the partition coefficients of the molecules. In addition, we performed nuclear magnetic resonance (NMR) studies of the interactions with calf thymus DNA for the three molecules. The results were consistent with the fluorescence method described above. Thus, we conclude that the fluorescence method we developed provides a reliable determination of the likelihoods of the two different DNA binding modes.

  8. Differentiated, Promoter-specific Response of [4Fe-4S] NsrR DNA Binding to Reaction with Nitric Oxide*

    PubMed Central

    Crack, Jason C.; Svistunenko, Dimitri A.; Munnoch, John; Thomson, Andrew J.; Hutchings, Matthew I.; Le Brun, Nick E.

    2016-01-01

    NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ∼2 NO per cluster, although for the hmpA1 and nsrR promoters, ∼4 and ∼8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8–10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ∼2 NO per cluster, along with two further intermediates at ∼4 and ∼6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes. PMID:26887943

  9. Structure, mechanics, and binding mode heterogeneity of LEDGF/p75-DNA nucleoprotein complexes revealed by scanning force microscopy

    NASA Astrophysics Data System (ADS)

    Vanderlinden, Willem; Lipfert, Jan; Demeulemeester, Jonas; Debyser, Zeger; de Feyter, Steven

    2014-04-01

    LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease.LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease. Electronic supplementary information (ESI) available: SFM topographs of phage lambda DNA in situ, in the absence and presence of LEDGF/p75; model-independent tests for DNA chain equilibration in 2D; SFM topographs of

  10. Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9

    SciTech Connect

    Zhang, Ruoxi; Fang, Liurong; Wang, Dang; Cai, Kaimei; Zhang, Huan; Xie, Lilan; Li, Yi; Chen, Huanchun; Xiao, Shaobo

    2015-11-15

    To subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN signaling pathway. Porcine bocavirus (PBoV), a newly identified porcine parvovirus, has received attention because it shows clinically high co-infection prevalence with other pathogens in post-weaning multisystemic wasting syndrome (PWMS) and diarrheic piglets. In this study, we screened the structural and non-structural proteins encoded by PBoV and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element (ISRE) activity and subsequent IFN-stimulated gene (ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3 (STAT1/STAT2/IRF9). Detailed analysis demonstrated that PBoV NP1 blocked the ISGF3 DNA-binding activity by combining with the DNA-binding domain (DBD) of IRF9. In summary, these results indicate that PBoV NP1 interferes with type I IFN signaling pathway by blocking DNA binding of ISGF3 to attenuate innate immune responses. - Highlights: • Porcine bocavirus (PBoV) NP1 interferes with the IFN α/β signaling pathway. • PBoV NP1 does not prevent STAT1/STAT2 phosphorylation and nuclear translocation. • PBoV NP1 inhibits the DNA-binding activity of ISGF3. • PBoV NP1 interacts with the DNA-binding domain of IRF9.

  11. Binding of DNA-binding alkaloids berberine and palmatine to tRNA and comparison to ethidium: Spectroscopic and molecular modeling studies

    NASA Astrophysics Data System (ADS)

    Islam, Md. Maidul; Pandya, Prateek; Chowdhury, Sebanti Roy; Kumar, Surat; Kumar, Gopinatha Suresh

    2008-11-01

    The interaction of two natural protoberberine plant alkaloids berberine and palmatine with tRNA phe was studied using various biophysical techniques and molecular modeling and the data were compared with the binding of the classical DNA intercalator, ethidium. Circular dichroic studies revealed that the tRNA conformation was moderately perturbed on binding of the alkaloids. The cooperative binding of both the alkaloids and ethidium to tRNA was revealed from absorbance and fluorescence studies. Fluorescence quenching studies advanced a conclusion that while berberine and palmatine are partially intercalated, ethidium is fully intercalated on the tRNA molecule. The binding of the alkaloids as well as ethidium stabilized the tRNA melting, and the binding constant evaluated from the averaged optical melting temperature data was in agreement with fluorescence spectral-binding data. Differential scanning calorimetry revealed that the tRNA melting showed three close transitions that were affected on binding of these small molecules. Molecular docking calculations performed showed the preferred regions of binding of these small molecules on the tRNA. Taken together, the results suggest that the binding of the alkaloids berberine and palmatine on the tRNA structure appears to be mostly by partial intercalation while ethidium intercalates fully on the tRNA. These results further advance our knowledge on the molecular aspects on the interaction of these alkaloids to tRNA.

  12. The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition.

    PubMed

    Wienk, Hans; Slootweg, Jack C; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2013-07-01

    To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition.

  13. Unveiling the effects of berenil, a DNA-binding drug, on Trypanosoma cruzi: implications for kDNA ultrastructure and replication.

    PubMed

    Zuma, Aline Araujo; Cavalcanti, Danielle Pereira; Zogovich, Marcelo; Machado, Ana Carolina Loyola; Mendes, Isabela Cecília; Thiry, Marc; Galina, Antonio; de Souza, Wanderley; Machado, Carlos Renato; Motta, Maria Cristina Machado

    2015-02-01

    Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a single mitochondrion with an enlarged portion termed kinetoplast. This unique structure harbors the mitochondrial DNA (kDNA), composed of interlocked molecules: minicircles and maxicircles. kDNA is a hallmark of kinetoplastids and for this reason constitutes a valuable target in chemotherapeutic and cell biology studies. In the present work, we analyzed the effects of berenil, a minor-groove-binding agent that acts preferentially at the kDNA, thereby affecting cell proliferation, ultrastructure, and mitochondrial activity of T. cruzi epimastigote form. Our results showed that berenil promoted a reduction on parasite growth when high concentrations were used; however, cell viability was not affected. This compound caused significant changes in kDNA arrangement, including the appearance of membrane profiles in the network and electron-lucent areas in the kinetoplast matrix, but nuclear ultrastructure was not modified. The use of the TdT technique, which specifically labels DNA, conjugated to atomic force microscopy analysis indicates that berenil prevents the minicircle decatenation of the network, thus impairing DNA replication and culminating in the appearance of dyskinetoplastic cells. Alterations in the kinetoplast network may be associated with kDNA lesions, as suggested by the quantitative PCR (qPCR) technique. Furthermore, parasites treated with berenil presented higher levels of reactive oxygen species and a slight decrease in the mitochondrial membrane potential and oxygen consumption. Taken together, our results reveal that this DNA-binding drug mainly affects kDNA topology and replication, reinforcing the idea that the kinetoplast represents a potential target for chemotherapy against trypanosomatids.

  14. Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

    PubMed Central

    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

  15. NMR chemical shift perturbation mapping of DNA binding by a zinc-finger domain from the yeast transcription factor ADR1.

    PubMed Central

    Schmiedeskamp, M.; Rajagopal, P.; Klevit, R. E.

    1997-01-01

    Mutagenesis studies have revealed that the minimal DNA-binding domain of the yeast transcription factor ADR1 consists of two Cys2-His2 zinc fingers plus an additional 20 residues proximal and N-terminal to the fingers. We have assigned NMR 1H, 15N, and 13C chemical shifts for the entire minimal DNA-binding domain of ADR1 both free and bound to specific DNA. 1H chemical shift values suggest little structural difference between the zinc fingers in this construct and in single-finger constructs, and 13C alpha chemical shift index analysis indicates little change in finger structure upon DNA binding. 1H chemical shift perturbations upon DNA binding are observed, however, and these are mapped to define the protein-DNA interface. The two zinc fingers appear to bind DNA with different orientations, as the entire helix of finger 1 is perturbed, while only the extreme N-terminus of the finger 2 helix is affected. Furthermore, residues N-terminal to the first finger undergo large chemical shift changes upon DNA binding suggesting a role at the protein-DNA interface. A striking correspondence is observed between the protein-DNA interface mapped by chemical shift changes and that previously mapped by mutagenesis. PMID:9300483

  16. GAGA factor binding to DNA via a single trinucleotide sequence element.

    PubMed Central

    Wilkins, R C; Lis, J T

    1998-01-01

    GAGA transcription factor (GAF) is an essential protein in Drosophila , important for the transcriptional regulation of numerous genes. GAF binds to GA repeats in the promoters of these genes via a DNA-binding domain containing a single zinc finger. While GAF binding sites are typically composed of 3.5 GA repeats, the Drosophila hsp70 gene contains much smaller elements, some of which are as little as three bases (GAG) in length. Interestingly, the binding of GAF to more distant trinucleotide elements is relatively strong and not appreciably affected by the removal of larger GA arrays in the promoter. Moreover, a simple synthetic GAG sequence is sufficient to bind GAF in vitro . Here we directly compare the affinity of GAF for different sequence elements by immunoprecipitation and gel mobility shift analysis. Furthermore, our measures of the concentration of GAF in vivo indicate that it is a highly abundant nuclear protein, prevalent enough to occupy a sizable fraction of correspondingly abundant trinucleotide sites. PMID:9592153

  17. Sequence-specific binding of simian virus 40 A protein to nonorigin and cellular DNA.

    PubMed Central

    Wright, P J; DeLucia, A L; Tegtmeyer, P

    1984-01-01

    The simian virus 40 A protein (T antigen) recognized and bound to the consensus sequence 5'-GAGGC-3' in DNA from many sources. Sequence-specific binding to single pentanucleotides in randomly chosen DNA predominated over binding to nonspecific sequences. The asymmetric orientation of protein bound to nonorigin recognition sequences also resembled that of protein bound to the origin region of simian virus 40 DNA. Sequence variations in the DNA adjacent to single pentanucleotides influenced binding affinities even though methylation interference and protection studies did not reveal specific interactions outside of pentanucleotides. Thus, potential locations of A protein bound to any DNA can be predicted although the determinants of binding affinity are not yet understood. Sequence-specific binding of A protein to cellular DNA would provide a mechanism for specific alterations of host gene expression that facilitate viral function. Images PMID:6570189

  18. Theoretical studies on binding modes of copper-based nucleases with DNA.

    PubMed

    Liu, Chunmei; Zhu, Yanyan; Tang, Mingsheng

    2016-03-01

    In the present work, molecular simulations were performed for the purpose of predicting the binding modes of four types of copper nucleases (a total 33 compounds) with DNA. Our docking results accurately predicted the groove binding and electrostatic interaction for some copper nucleases with B-DNA. The intercalation modes were also reproduced by "gap DNA". The obtained results demonstrated that the ligand size, length, functional groups and chelate ring size bound to the copper center could influence the binding affinities of copper nucleases. The binding affinities obtained from the docking calculations herein also replicated results found using MM-PBSA approach. The predicted DNA binding modes of copper nucleases with DNA will ultimately help us to better understand the interaction of copper compounds with DNA.

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

    PubMed

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

    2015-04-30

    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.

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

    PubMed

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

    2016-11-01

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

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

    PubMed

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

    2016-12-17

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

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

    PubMed

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

    2016-12-01

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

  3. Specific DNA replication mutations affect telomere length in Saccharomyces cerevisiae.

    PubMed Central

    Adams, A K; Holm, C

    1996-01-01

    To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length. PMID:8756617

  4. Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

    NASA Astrophysics Data System (ADS)

    Qian, Long; Kussell, Edo

    2016-10-01

    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.

  5. Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators.

    PubMed

    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.

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

    SciTech Connect

    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.

  7. Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis.

    PubMed

    Kasho, Kazutoshi; Tanaka, Hiroyuki; Sakai, Ryuji; Katayama, Tsutomu

    2017-01-27

    Timely initiation of replication in Escherichia coli requires functional regulation of the replication initiator, ATP-DnaA. The cellular level of ATP-DnaA increases just before initiation, after which its level decreases through hydrolysis of DnaA-bound ATP, yielding initiation-inactive ADP-DnaA. Previously, we reported a novel DnaA-ATP hydrolysis system involving the chromosomal locus datA and named it datA-dependent DnaA-ATP hydrolysis (DDAH). The datA locus contains a binding site for a nucleoid-associating factor integration host factor (IHF) and a cluster of three known DnaA-binding sites, which are important for DDAH. However, the mechanisms underlying the formation and regulation of the datA-IHF·DnaA complex remain unclear. We now demonstrate that a novel DnaA box within datA is essential for ATP-DnaA complex formation and DnaA-ATP hydrolysis. Specific DnaA residues, which are important for interaction with bound ATP and for head-to-tail inter-DnaA interaction, were also required for ATP-DnaA-specific oligomer formation on datA Furthermore, we show that negative DNA supercoiling of datA stabilizes ATP-DnaA oligomers, and stimulates datA-IHF interaction and DnaA-ATP hydrolysis. Relaxation of DNA supercoiling by the addition of novobiocin, a DNA gyrase inhibitor, inhibits datA function in cells. On the basis of these results, we propose a mechanistic model of datA-IHF·DnaA complex formation and DNA supercoiling-dependent regulation for DDAH.

  8. Intermembrane contact affects calcium binding to phospholipid vesicles.

    PubMed Central

    Ekerdt, R; Papahadjopoulos, D

    1982-01-01

    Binding of Ca2+ to liposomes composed of phosphatidylserine (PtdSer) was analyzed by potentiometric titrations. Ca2+ binding to large unilamellar PtdSer vesicles was saturable at a stoichiometry of 1:2 (Ca2+/PtdSer). At approximately 6 X 10(-4) M [Ca2+]free, the binding curve exhibited a discontinuity that can be attributed to the formation of a Ca2+/PtdSer complex with a higher affinity. When both Ca2+ and Mg2+ are present, depending on the relative concentrations, Mg2+ can either complete or can enhance Ca2+ binding. Concomitant to the enhanced binding, the vesicle suspension was found to aggregate, suggesting that close contact of membranes is a prerequisite for the abrupt change in affinity. This concept was tested by binding studies with liposomes of mixed composition. It was found that the incorporation of 50 mol% phosphatidylethanolamine (PtdEtn) into PtdSer liposomes produced a similar binding pattern to that of pure PtdSer with a saturable stoichiometry of 1:2 (Ca2+/PtdSer). However, incorporation of 50 mol% phosphatidylcholine (PtdCho) completely abolished the discontinuous shift in affinity and apparent saturation was reached at a stoichiometry of 1:4 (Ca2+/PtdSer). In addition, Ca2+ binding to PtdSer liposomes with 10 mol% galactosylcerebroside was not altered when compared to pure PtdSer, whereas 10 mol% of the glycolipid GL-4 abolished the increased binding. The results are closely correlated with recent findings on the role of the membrane composition in Ca2+-induced fusion of liposomes and argue in favor of a specific Ca2+/PtdSer complex (with 1:2 stoichiometry) forming only at points of close contact between membranes and serving as the trigger for membrane fusion. PMID:6954538

  9. G =  MAT: linking transcription factor expression and DNA binding data.

    PubMed

    Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

    2011-01-31

    Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/.

  10. G = MAT: Linking Transcription Factor Expression and DNA Binding Data

    PubMed Central

    Tretyakov, Konstantin; Laur, Sven; Vilo, Jaak

    2011-01-01

    Transcription factors are proteins that bind to motifs on the DNA and thus affect gene expression regulation. The qualitative description of the corresponding processes is therefore important for a better understanding of essential biological mechanisms. However, wet lab experiments targeted at the discovery of the regulatory interplay between transcription factors and binding sites are expensive. We propose a new, purely computational method for finding putative associations between transcription factors and motifs. This method is based on a linear model that combines sequence information with expression data. We present various methods for model parameter estimation and show, via experiments on simulated data, that these methods are reliable. Finally, we examine the performance of this model on biological data and conclude that it can indeed be used to discover meaningful associations. The developed software is available as a web tool and Scilab source code at http://biit.cs.ut.ee/gmat/. PMID:21297945

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

    NASA Astrophysics Data System (ADS)

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

    2002-09-01

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

  12. De-novo protein function prediction using DNA binding and RNA binding proteins as a test case

    PubMed Central

    Peled, Sapir; Leiderman, Olga; Charar, Rotem; Efroni, Gilat; Shav-Tal, Yaron; Ofran, Yanay

    2016-01-01

    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. PMID:27869118

  13. De-novo protein function prediction using DNA binding and RNA binding proteins as a test case.

    PubMed

    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.

  14. Evolution of DNA-Binding Sites of a Floral Master Regulatory Transcription Factor

    PubMed Central

    Muiño, Jose M.; de Bruijn, Suzanne; Pajoro, Alice; Geuten, Koen; Vingron, Martin; Angenent, Gerco C.; Kaufmann, Kerstin

    2016-01-01

    Flower development is controlled by the action of key regulatory transcription factors of the MADS-domain family. The function of these factors appears to be highly conserved among species based on mutant phenotypes. However, the conservation of their downstream processes is much less well understood, mostly because the evolutionary turnover and variation of their DNA-binding sites (BSs) among plant species have not yet been experimentally determined. Here, we performed comparative ChIP (chromatin immunoprecipitation)-seq experiments of the MADS-domain transcription factor SEPALLATA3 (SEP3) in two closely related Arabidopsis species: Arabidopsis thaliana and A. lyrata which have very similar floral organ morphology. We found that BS conservation is associated with DNA sequence conservation, the presence of the CArG-box BS motif and on the relative position of the BS to its potential target gene. Differences in genome size and structure can explain that SEP3 BSs in A. lyrata can be located more distantly to their potential target genes than their counterparts in A. thaliana. In A. lyrata, we identified transposition as a mechanism to generate novel SEP3 binding locations in the genome. Comparative gene expression analysis shows that the loss/gain of BSs is associated with a change in gene expression. In summary, this study investigates the evolutionary dynamics of DNA BSs of a floral key-regulatory transcription factor and explores factors affecting this phenomenon. PMID:26429922

  15. DNA Binding of Centromere Protein C (CENPC) Is Stabilized by Single-Stranded RNA

    PubMed Central

    Du, Yaqing; Topp, Christopher N.; Dawe, R. Kelly

    2010-01-01

    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. PMID:20140237

  16. Binding interaction between sorafenib and calf thymus DNA: Spectroscopic methodology, viscosity measurement and molecular docking

    NASA Astrophysics Data System (ADS)

    Shi, Jie-Hua; Chen, Jun; Wang, Jing; Zhu, Ying-Yao

    2015-02-01

    The binding interaction of sorafenib with calf thymus DNA (ct-DNA) was studied using UV-vis absorption spectroscopy, fluorescence emission spectroscopy, circular dichroism (CD), viscosity measurement and molecular docking methods. The experimental results revealed that there was obvious binding interaction between sorafenib and ct-DNA. The binding constant (Kb) of sorafenib with ct-DNA was 5.6 × 103 M-1 at 298 K. The enthalpy and entropy changes (ΔH0 and ΔS0) in the binding process of sorafenib with ct-DNA were -27.66 KJ mol-1 and -21.02 J mol-1 K-1, respectively, indicating that the main binding interaction forces were van der Waals force and hydrogen bonding. The docking results suggested that sorafenib preferred to bind on the minor groove of A-T rich DNA and the binding site of sorafenib was 4 base pairs long. The conformation change of sorafenib in the sorafenib-DNA complex was obviously observed and the change was close relation with the structure of DNA, implying that the flexibility of sorafenib molecule played an important role in the formation of the stable sorafenib-ct-DNA complex.

  17. Identification and properties of the crenarchaeal single-stranded DNA binding protein from Sulfolobus solfataricus

    PubMed Central

    Wadsworth, Ross I. M.; White, Malcolm F.

    2001-01-01

    Single-stranded DNA binding proteins (SSBs) play central roles in cellular and viral processes involving the generation of single-stranded DNA. These include DNA replication, homologous recombination and DNA repair pathways. SSBs bind DNA using four ‘OB-fold’ (oligonucleotide/oligosaccharide binding fold) domains that can be organised in a variety of overall quaternary structures. Thus eubacterial SSBs are homotetrameric whilst the eucaryal RPA protein is a heterotrimer and euryarchaeal proteins vary significantly in their subunit compositions. We demonstrate that the crenarchaeal SSB protein is an abundant protein with a unique structural organisation, existing as a monomer in solution and multimerising on DNA binding. The protein binds single-stranded DNA distributively with a binding site size of ~5 nt per monomer. Sulfolobus SSB lacks the zinc finger motif found in the eucaryal and euryarchaeal proteins, possessing instead a flexible C-terminal tail, sensitive to trypsin digestion, that is not required for DNA binding. In comparison with Escherichia coli SSB, the tail may play a role in protein–protein interactions during DNA replication and repair. PMID:11160923

  18. Methylation-independent DNA Binding Modulates Specificity of Repressor of Silencing 1 (ROS1) and Facilitates Demethylation in Long Substrates*

    PubMed Central

    Ponferrada-Marín, María Isabel; Martínez-Macías, María Isabel; Morales-Ruiz, Teresa; Roldán-Arjona, Teresa; Ariza, Rafael R.

    2010-01-01

    DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5-meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC, we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short N-terminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA and is required for efficient activity on 5-meC·G, but not for T·G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5-meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly paired base such as 5-meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases. PMID:20489198

  19. SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks

    PubMed Central

    Lopez-Mosqueda, Jaime; Maddi, Karthik; Prgomet, Stefan; Kalayil, Sissy; Marinovic-Terzic, Ivana; Terzic, Janos; Dikic, Ivan

    2016-01-01

    Ruijs-Aalfs syndrome is a segmental progeroid syndrome resulting from mutations in the SPRTN gene. Cells derived from patients with SPRTN mutations elicit genomic instability and people afflicted with this syndrome developed hepatocellular carcinoma. Here we describe the molecular mechanism by which SPRTN contributes to genome stability and normal cellular homeostasis. We show that SPRTN is a DNA-dependent mammalian protease required for resolving cytotoxic DNA-protein crosslinks (DPCs)— a function that had only been attributed to the metalloprotease Wss1 in budding yeast. We provide genetic evidence that SPRTN and Wss1 function distinctly in vivo to resolve DPCs. Upon DNA and ubiquitin binding, SPRTN can elicit proteolytic activity; cleaving DPC substrates and itself. SPRTN null cells or cells derived from patients with Ruijs-Aalfs syndrome are impaired in the resolution of covalent DPCs in vivo. Collectively, SPRTN is a mammalian protease required for resolving DNA-protein crosslinks in vivo whose function is compromised in Ruijs-Aalfs syndrome patients. DOI: http://dx.doi.org/10.7554/eLife.21491.001 PMID:27852435

  20. Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme.

    PubMed Central

    Bouhamdan, M; Benichou, S; Rey, F; Navarro, J M; Agostini, I; Spire, B; Camonis, J; Slupphaug, G; Vigne, R; Benarous, R; Sire, J

    1996-01-01

    The role of the accessory gene product Vpr during human immunodeficiency virus type 1 infection remains unclear. We have used the yeast two-hybrid system to identify cellular proteins that interact with Vpr and could be involved in its function. A cDNA clone which encodes the human uracil DNA glycosylase (UNG), a DNA repair enzyme involved in removal of uracil in DNA, has been isolated. Interaction between Vpr and UNG has been demonstrated by in vitro protein-protein binding assays using translated, radiolabeled Vpr and UNG recombinant proteins expressed as a glutathione S-transferase fusion protein. Conversely, purified UNG has been demonstrated to interact with Vpr recombinant protein expressed as a glutathione S-transferase fusion protein. Coimmunoprecipitation experiments confirmed that Vpr and UNG are associated within cells expressing Vpr. By using a panel of C- and N-terminally deleted Vpr mutants, we have determined that the core protein of Vpr, spanning amino acids 15 to 77, is involved in the interaction with UNG. We also demonstrate by in vitro experiments that the enzymatic activity of UNG is retained upon interaction with Vpr. PMID:8551605

  1. STRUCTURE OF THE DNA REPAIR HELICASE HEL308 REVEALS DNA BINDING AND AUTOINHIBITORY DOMAINS

    PubMed Central

    Richards, Jodi; Johnson, Ken; Liu, Huanting; Oke, Stephen McMahon. Muse; Carter, Lester; Naismith, James H; White, Malcolm F

    2012-01-01

    Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest, and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high-resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as a molecular brake, clamping the ssDNA extruded through the central pore of the helicase structure to limit the enzyme’s helicase activity. This provides an elegant mechanism to tune the enzyme’s processivity to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, suggesting that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates. PMID:18056710

  2. Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA–DNA pairing

    PubMed Central

    Lin, Jiangguo; Countryman, Preston; Chen, Haijiang; Pan, Hai; Fan, Yanlin; Jiang, Yunyun; Kaur, Parminder; Miao, Wang; Gurgel, Gisele; You, Changjiang; Piehler, Jacob; Kad, Neil M.; Riehn, Robert; Opresko, Patricia L.; Smith, Susan; Tao, Yizhi Jane; Wang, Hong

    2016-01-01

    Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA–DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA–DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding. PMID:27298259

  3. Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

    PubMed Central

    Adams, C C; Workman, J L

    1995-01-01

    To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo. PMID:7862134

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

    PubMed

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

    2016-12-15

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

  5. High-resolution mapping of architectural DNA binding protein facilitation of a DNA repression loop in Escherichia coli.

    PubMed

    Becker, Nicole A; Maher, L James

    2015-06-09

    Double-stranded DNA is a locally inflexible polymer that resists bending and twisting over hundreds of base pairs. Despite this, tight DNA bending is biologically important for DNA packaging in eukaryotic chromatin and tight DNA looping is important for gene repression in prokaryotes. We and others have previously shown that sequence nonspecific DNA kinking proteins, such as Escherichia coli heat unstable and Saccharomyces cerevisiae non-histone chromosomal protein 6A (Nhp6A), facilitate lac repressor (LacI) repression loops in E. coli. It has been unknown if this facilitation involves direct protein binding to the tightly bent DNA loop or an indirect effect promoting global negative supercoiling of DNA. Here we adapt two high-resolution in vivo protein-mapping techniques to demonstrate direct binding of the heterologous Nhp6A protein at a LacI repression loop in living E. coli cells.

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

    PubMed

    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.

  7. Molecular dynamics study of DNA binding by INT-DBD under a polarized force field.

    PubMed

    Yao, Xue X; Ji, Chang G; Xie, Dai Q; Zhang, John Z H

    2013-05-15

    The DNA binding domain of transposon Tn916 integrase (INT-DBD) binds to DNA target site by positioning the face of a three-stranded antiparallel β-sheet within the major groove. As the negatively charged DNA directly interacts with the positively charged residues (such as Arg and Lys) of INT-DBD, the electrostatic interaction is expected to play an important role in the dynamical stability of the protein-DNA binding complex. In the current work, the combined use of quantum-based polarized protein-specific charge (PPC) for protein and polarized nucleic acid-specific charge (PNC) for DNA were employed in molecular dynamics simulation to study the interaction dynamics between INT-DBD and DNA. Our study shows that the protein-DNA structure is stabilized by polarization and the calculated protein-DNA binding free energy is in good agreement with the experimental data. Furthermore, our study revealed a positive correlation between the measured binding energy difference in alanine mutation and the occupancy of the corresponding residue's hydrogen bond. This correlation relation directly relates the contribution of a specific residue to protein-DNA binding energy to the strength of the hydrogen bond formed between the specific residue and DNA.

  8. A new protein domain for binding to DNA through the minor groove.

    PubMed Central

    Freire, R; Salas, M; Hermoso, J M

    1994-01-01

    Protein p6 of the Bacillus subtilis phage phi 29 binds with low sequence specificity to DNA through the minor groove, forming a multimeric nucleoprotein complex that activates the initiation of phi 29 DNA replication. Deletion analysis suggested that the N-terminal part of protein p6, predicted to form an amphipathic alpha-helix, is involved in DNA binding. We have constructed site-directed mutants at the polar side of the putative alpha-helix. DNA binding and activation of initiation of phi 29 DNA replication were impaired in most of the mutant proteins obtained. A 19 amino acid peptide comprising the N-terminus of protein p6 interacted with a DNA fragment containing high-affinity signals for protein p6 binding with approximately 50-fold higher affinity than the peptide corresponding to an inactive mutant. Both wild-type peptide and protein p6 recognized the same sequences in this DNA fragment. This result, together with distamycin competition experiments, suggested that the wild-type peptide also binds to DNA through the minor groove. In addition, CD spectra of the wild-type peptide showed an increase in the alpha-helical content when bound to DNA. All these results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. Images PMID:7925279

  9. DNA-binding dependent and independent functions of WT1 protein during human hematopoiesis

    SciTech Connect

    Svensson, Emelie; Eriksson, Helena; Gekas, Christos; Olofsson, Tor; Richter, Johan; Gullberg, Urban . E-mail: urban.gullberg@hematologi.lu.se

    2005-08-01

    The Wilms tumor gene 1 (WT1) encodes a zinc-finger-containing transcription factor highly expressed in immature hematopoietic progenitor cells. Overexpression and presence of somatic mutations in acute leukemia indicate a role for WT1 in the pathogenesis of leukemia. CD34{sup +} progenitor cells were transduced with one splice variant of human WT1 without the KTS insert in the zinc-finger domain, WT1(+/-), and with a deleted mutant of WT1 lacking the entire zinc-finger region, WT1(delZ), thus incapable of binding DNA. We show that inhibition of erythroid colony formation and differentiation is absolutely dependent on the DNA-binding zinc-finger domain of WT1. Unexpectedly, however, WT1(delZ) was equally effective as wild type protein in the reduction of myeloid clonogenic growth as well as in stimulation of myeloid differentiation, as judged by the expression of cell surface CD11b. Expression of neither WT1(+/-) nor WT1(delZ) upregulated mRNA for the cdk inhibitor p21{sup Waf1/Cip1} or p27{sup Kip1}. Our results demonstrate that WT1 affects proliferation and differentiation in erythroid and myeloid cells by different molecular mechanisms, and suggest that mutations affecting the zinc-finger domain of WT1 could interfere with normal differentiation in the pathogenesis of leukemia.

  10. Guiding the design of synthetic DNA-binding molecules with massively parallel sequencing.

    PubMed

    Meier, Jordan L; Yu, Abigail S; Korf, Ian; Segal, David J; Dervan, Peter B

    2012-10-24

    Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing β/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5'-WCGCGW-3' from 5'-WGCGCW-3'. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.

  11. Differential sensitivity to methylated DNA by ETS-family transcription factors is intrinsically encoded in their DNA-binding domains.

    PubMed

    Stephens, Dominique C; Poon, Gregory M K

    2016-10-14

    Transactivation by the ETS family of transcription factors, whose members share structurally conserved DNA-binding domains, is variably sensitive to methylation of their target genes. The mechanism by which DNA methylation controls ETS proteins remains poorly understood. Uncertainly also pervades the effects of hemi-methylated DNA, which occurs following DNA replication and in response to hypomethylating agents, on site recognition by ETS proteins. To address these questions, we measured the affinities of two sequence-divergent ETS homologs, PU.1 and Ets-1, to DNA sites harboring a hemi- and fully methylated CpG dinucleotide. While the two proteins bound unmethylated DNA with indistinguishable affinity, their affinities to methylated DNA are markedly heterogeneous and exhibit major energetic coupling between the two CpG methylcytosines. Analysis of simulated DNA and existing co-crystal structures revealed that hemi-methylation induced non-local backbone and groove geometries that were not conserved in the fully methylated state. Indirect readout of these perturbations was differentially achieved by the two ETS homologs, with the distinctive interfacial hydration in PU.1/DNA binding moderating the inhibitory effects of DNA methylation on binding. This data established a biophysical basis for the pioneering properties associated with PU.1, which robustly bound fully methylated DNA, but not Ets-1, which was substantially inhibited.

  12. Novel signal amplification approach for HRP-based colorimetric genosensors using DNA binding protein tags.

    PubMed

    Aktas, Gülsen Betül; Skouridou, Vasso; Masip, Lluis

    2015-12-15

    The need for sensitive detection of DNA is growing as more specific DNA sequences are being correlated to gene markers for disease diagnosis, food safety and other security related applications. Detection in hybridization-based assays is usually achieved with target-specific ssDNA probes conjugated directly to enzyme labels like HRP that provide signal amplification or with nanoparticles functionalized with DNA and multiple HRP molecules. In order to overcome some of the drawbacks presented by these approaches, we developed a unique DNA sensing platform based on an HRP-DNA binding protein tag conjugate and a hybrid ssDNA-dsDNA detection probe. Specifically, in this work we describe the preparation and characterization of an HRP conjugate with scCro DNA binding protein tag and its application for the detection of a model ssDNA target sequence. By using the HRP-scCro conjugate together with a hybrid detection probe containing three scCro-specific dsDNA binding sites, we demonstrate an improvement by over 3-fold in both sensitivity and limit of detection of high-risk human papillomavirus (HPV16), compared to the standard ssDNA-HRP conjugate. These results show that the HRP-DNA binding protein tag conjugate can be used as an alternative and universal tool for signal enhancement in enzyme-linked assays suitable for integration in point-of-care systems.

  13. Quantum dot binding to DNA: single-molecule imaging with atomic force microscopy.

    PubMed

    Li, Kungang; Zhang, Wen; Chen, Yongsheng

    2013-01-01

    The interaction between nanoparticles (NPs) and DNA is of significance for both application and implication research of NPs. In this study, a single-molecule imaging technique based on atomic force microscopy (AFM) was employed to probe the NP-DNA interactions with quantum dots (QDs) as model NPs. Reproducible high-quality images of single DNA molecules in air and in liquids were acquired on mica by optimizing sample preparation conditions. Furthermore, the binding of QDs to DNA was explored using AFM. The DNA concentration was found to be a key factor influencing AFM imaging quality. In air and liquids, the optimal DNA concentration for imaging DNA molecules was approximately 2.5 and 0.25 μg/mL, and that for imaging DNA binding with QDs was 0.5 and 0.25 μg/mL, respectively. In the presence of QDs, the DNA conformation was altered with the formation of DNA condensates. Finally, the fine conformation of QD-DNA binding sites was examined to analyze the binding mechanisms. This work will benefit investigations of NP-DNA interactions and the understanding of the structure of NP-DNA bioconjugates. See accompanying article by Wang DOI: 10.1002/biot.201200309.

  14. Mechanistic aspects of thioflavin-T self-aggregation and DNA binding: evidence for dimer attack on DNA grooves.

    PubMed

    Biancardi, A; Biver, T; Burgalassi, A; Mattonai, M; Secco, F; Venturini, M

    2014-10-07

    Thioflavin-T (TFT) is a fluorescent marker widely employed in biomedical research but the mechanism of its binding to polynucleotides has been poorly understood. This paper presents a study of the mechanisms of TFT self-aggregation and binding to DNA. Relaxation kinetics of TFT solutions show that the cyanine undergoes dimerization followed by dimer isomerisation. The interaction of TFT with DNA has been investigated using static methods, such as spectrophotometric and spectrofluorometric titrations under different conditions (salt content, temperature), fluorescence quenching, viscometric experiments and the T-jump relaxation method. The combined use of these techniques enabled us to show that the TFT monomer undergoes intercalation between the DNA base pairs and external binding according to a branched mechanism. Moreover, it has also been observed that, under dye excess conditions, the TFT dimer binds to the DNA grooves. The molecular structures of intercalated TFT and the groove-bound TFT dimer are obtained by performing QM/MM MD simulations.

  15. Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

    SciTech Connect

    Kikuchi, M.; Ishii, S. )

    1989-12-01

    By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

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

    PubMed

    Udayakumar, Durga; Dynan, William S

    2015-08-07

    Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA double-strand breaks. We have previously shown that a complex of SFPQ (PSF) and NONO (p54(nrb)) cooperates with Ku protein at an early step of NHEJ, forming a committed preligation complex and stimulating end-joining activity by 10-fold or more. SFPQ and NONO show no resemblance to other repair factors, and their mechanism of action is uncertain. Here, we use an optimized microwell-based assay to characterize the in vitro DNA binding behavior of the native SFPQ·NONO complex purified from human (HeLa) cells. SFPQ·NONO and Ku protein bind independently to DNA, with little evidence of cooperativity and only slight mutual interference at high concentration. Whereas Ku protein requires free DNA ends for binding, SFPQ·NONO does not. Both Ku and SFPQ·NONO have pairing activity, as measured by the ability of DNA-bound protein to capture a second DNA fragment in a microwell-based assay. Additionally, SFPQ·NONO stimulates DNA-dependent protein kinase autophosphorylation, consistent with the ability to promote formation of a synaptic complex formation without occluding the DNA termini proper. These findings suggest that SFPQ·NONO promotes end joining by binding to internal DNA sequences and cooperating with other repair proteins to stabilize a synaptic pre-ligation complex.

  17. Luminescence and binding properties of two isoquinoline alkaloids chelerythrine and sanguinarine with ctDNA

    NASA Astrophysics Data System (ADS)

    Li, Junfen; Li, Baohong; Wu, Yanbo; Shuang, Shaomin; Dong, Chuan; Choi, Martin M. F.

    2012-09-01

    The binding mode and mechanism of the interactions between two planar cationic alkaloids chelerythrine (Che) and sanguinarine (San) with calf thymus DNA (ctDNA) were systematically investigated at pH 5.40 using UV-vis absorption spectroscopy, fluorescence spectroscopy and cyclic voltammetry. Che and San show strong fluorescence at 570 and 589 nm, respectively. Che displays fluorescence enhancement with ctDNA whereas the fluorescence of San is quenched on interaction with ctDNA. In addition, UV-vis spectra of both alkaloids show apparent hypochromicity and are bathochromic shifted, indicating that they could intercalate into ctDNA bases. The fluorescence polarization of Che and San increases in the presence of ctDNA, again implying the intercalation of two alkaloids with ctDNA. This conclusion was also supported by the results obtained from anion quenching and cyclic voltammetry. The binding constants of both alkaloids with ctDNA were calculated in the order of 105 L/mol. San binds with ctDNA 3-fold stronger than Che. The stoichiometric bindings are five nucleotides per Che or San. Electrostatic binding also exists between the alkaloids and DNA helix. Finally, theoretical calculations show that only certain parts of Che and San molecules intercalate into the DNA helix.

  18. Determination of the cationic amphiphilic drug-DNA binding mode and DNA-assisted fluorescence resonance energy transfer amplification

    NASA Astrophysics Data System (ADS)

    Yaseen, Zahid; Banday, Abdul Rouf; Hussain, Mohammed Aamir; Tabish, Mohammad; Kabir-ud-Din

    2014-03-01

    Understanding the mechanism of drug-DNA binding is crucial for predicting the potential genotoxicity of drugs. Agarose gel electrophoresis, absorption, steady state fluorescence, and circular dichroism have been used in exploring the interaction of cationic amphiphilic drugs (CADs) such as amitriptyline hydrochloride (AMT), imipramine hydrochloride (IMP), and promethazine hydrochloride (PMT) with calf thymus or pUC19 DNA. Agarose gel electrophoresis assay, along with absorption and steady state fluorescence studies, reveal interaction between the CADs and DNA. A comparative study of the drugs with respect to the effect of urea, iodide induced quenching, and ethidium bromide (EB) exclusion assay reflects binding of CADs to the DNA primarily in an intercalative fashion. Circular dichroism data also support the intercalative mode of binding. Besides quenching, there is fluorescence exchange energy transfer (FRET) in between CADs and EB using DNA as a template.

  19. TAR DNA-binding protein 43 in neurodegenerative disease

    PubMed Central

    Chen-Plotkin, Alice S.; Lee, Virginia M.-Y.; Trojanowski, John Q.

    2010-01-01

    In 2006, TAR DNA-binding protein 43 (TDP-43), a highly conserved nuclear protein, was identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and in the most common variant of frontotemporal lobar degeneration (FTLD), FTLD-U, which is characterized by cytoplasmic inclusions that stain positive for ubiquitin but negative for tau and α-synuclein. Since then, rapid advances have been made in our understanding of the physiological function of TDP-43 and the role of this protein in neurodegeneration. These advances link ALS and FTLD-U (now designated FTLD-TDP) to a shared mechanism of disease. In this Review, we summarize the current evidence regarding the normal function of TDP-43 and the TDP-43 pathology observed in FTLD-TDP, ALS, and other neurodegenerative diseases wherein TDP-43 pathology co-occurs with other disease-specific lesions (for example, with amyloid plaques and neurofibrillary tangles in Alzheimer disease). Moreover, we discuss the accumulating data that support our view that FTLD-TDP and ALS represent two ends of a spectrum of primary TDP-43 proteinopathies. Finally, we comment on the importance of recent advances in TDP-43-related research to neurological practice, including the new opportunities to develop better diagnostics and disease-modifying therapies for ALS, FTLD-TDP, and related disorders exhibiting TDP-43 pathology. PMID:20234357

  20. Drosophila Mad binds to DNA and directly mediates activation of vestigial by Decapentaplegic.

    PubMed

    Kim, J; Johnson, K; Chen, H J; Carroll, S; Laughon, A

    1997-07-17

    The TGF-beta (transforming growth factor-beta)-related signalling proteins, including Decapentaplegic (Dpp) in Drosophila-and bone morphogenic proteins and activin in vertebrates, affect the growth and patterning of a great variety of structures. However, the mechanisms by which these ligands regulate gene expression are not understood. Activation of complexes of type I with type II receptors results in the phosphorylation and nuclear localization of members of the SMAD protein family, which are thought to act as co-activators of transcription, perhaps in conjunction with sequence-specific cofactors. Here we show that the amino-terminal domain of the Drosophila Mothers against dpp protein (Mad), a mediator of Dpp signalling, possesses a sequence-specific DNA-binding activity that becomes apparent when carboxy-terminal residues are removed. Mad binds to and is required for the activation of an enhancer within the vestigial wing-patterning gene in cells across the entire developing wing blade. Mad also binds to Dpp-response elements in other genes. These results suggest that Dpp signalling regulates gene expression by activating Mad binding to target gene enhancers.

  1. A polyplex qPCR-based binding assay for protein-DNA interactions.

    PubMed

    Moreau, Morgane J J; Schaeffer, Patrick M

    2012-09-21

    The measurement of protein-DNA interactions is difficult and often involves radioisotope-labelled DNA to obtain the desired assay sensitivity. More recently, high-throughput proteomic approaches were developed but they generally lack sensitivity. For these methods, the level of technical difficulties involved is high due to the need for specialised facilities or equipment and training. The new qPCR-based DNA-binding assay involves immunoprecipitation of a GFP-tagged DNA-binding protein in complex with various DNA targets (Ter sites) followed by qPCR quantification, affording a very sensitive and quantitative method that can be performed in polyplex. Using a single binding reaction, the binding specificity of the DNA replication terminator protein Tus for ten termination sites TerA-J could be obtained for the first time in just a few hours. This new qPCR DNA-binding assay can easily be adapted to determine the binding specificity of virtually any soluble and functional epitope-tagged DNA-binding protein.

  2. A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

    NASA Technical Reports Server (NTRS)

    Reddy, A. S.; Reddy, V. S.; Golovkin, M.

    2000-01-01

    Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

  3. Yeast general transcription factor GFI: sequence requirements for binding to DNA and evolutionary conservation.

    PubMed Central

    Dorsman, J C; van Heeswijk, W C; Grivell, L A

    1990-01-01

    GFI is an abundant DNA binding protein in the yeast S. cerevisiae. The protein binds to specific sequences in both ARS elements and the upstream regions of a large number of genes and is likely to play an important role in yeast cell growth. To get insight into the relative strength of the various GFI-DNA binding sites within the yeast genome, we have determined dissociation rates for several GFI-DNA complexes and found them to vary over a 70-fold range. Strong binding sites for GFI are present in the upstream activating sequences of the gene encoding the 40 kDa subunit II of the QH2:cytochrome c reductase, the gene encoding ribosomal protein S33 and in the intron of the actin gene. The binding site in the ARS1-TRP1 region is of intermediate strength. All strong binding sites conform to the sequence 5' RTCRYYYNNNACG-3'. Modification interference experiments and studies with mutant binding sites indicate that critical bases for GFI recognition are within the two elements of the consensus DNA recognition sequence. Proteins with the DNA binding specificities of GFI and GFII can also be detected in the yeast K. lactis, suggesting evolutionary conservation of at least the respective DNA-binding domains in both yeasts. Images PMID:2187179

  4. Clickable tyrosine binding bifunctional linkers for preparation of DNA-protein conjugates.

    PubMed

    Bauer, Dennis M; Ahmed, Ishtiaq; Vigovskaya, Antonina; Fruk, Ljiljana

    2013-06-19

    We have prepared bifunctional linkers containing clickable functional groups that enable preparation of protein-DNA conjugates through binding onto tyrosine residues. Mild conjugation strategy was demonstrated using two proteins, streptavidin(STV) and myoglobin (Mb) and it resulted in conjugates with preserved functionality of both the proteins and DNA strands. Furthermore, we show that protein-DNA conjugates can be successfully immobilized onto solid surface containing complementary DNA strands and the enzymatic activity of Mb-DNA conjugates is even higher than that of corresponding conjugates prepared through Lys binding.

  5. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

    PubMed Central

    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

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

    PubMed

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

    2011-07-01

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

  7. Synthesis, DNA Binding, and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives.

    PubMed

    de Almeida, Sinara Mônica Vitalino; Lafayette, Elizabeth Almeida; da Silva, Lúcia Patrícia Bezerra Gomes; Amorim, Cézar Augusto da Cruz; de Oliveira, Tiago Bento; Ruiz, Ana Lucia Tasca Gois; de Carvalho, João Ernesto; de Moura, Ricardo Olímpio; Beltrão, Eduardo Isidoro Carneiro; de Lima, Maria do Carmo Alves; de Carvalho Júnior, Luiz Bezerra

    2015-06-09

    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 × 10(4) to 1.0 × 10(6) M(-1) and quenching constants from -0.2 × 10(4) to 2.18 × 10(4) 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.

  8. Deciphering the intercalative binding modes of benzoyl peroxide with calf thymus DNA.

    PubMed

    Xia, Kaixin; Zhang, Guowen; Gong, Deming

    2017-01-24

    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.

  9. A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

    NASA Technical Reports Server (NTRS)

    Yang, Tianbao; Poovaiah, B. W.

    2002-01-01

    We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

  10. Molecular modeling and spectroscopic studies of semustine binding with DNA and its comparison with lomustine-DNA adduct formation.

    PubMed

    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.

  11. Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1.

    PubMed

    Miroshnikova, A D; Kuznetsova, A A; Kuznetsov, N A; Fedorova, O S

    2016-01-01

    Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.

  12. RecA Binding to a Single Double-Stranded DNA Molecule: A Possible Role of DNA Conformational Fluctuations

    NASA Astrophysics Data System (ADS)

    Leger, J. F.; Robert, J.; Bourdieu, L.; Chatenay, D.; Marko, J. F.

    1998-10-01

    Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.

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

    PubMed

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

    2012-10-01

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

  14. Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay.

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  16. Protein Phosphatase 2A (PP2A) Regulates Low Density Lipoprotein Uptake through Regulating Sterol Response Element-binding Protein-2 (SREBP-2) DNA Binding*

    PubMed Central

    Rice, Lyndi M.; Donigan, Melissa; Yang, Muhua; Liu, Weidong; Pandya, Devanshi; Joseph, Biny K.; Sodi, Valerie; Gearhart, Tricia L.; Yip, Jenny; Bouchard, Michael; Nickels, Joseph T.

    2014-01-01

    LDL-cholesterol (LDL-C) uptake by Ldlr is regulated at the transcriptional level by the cleavage-dependent activation of membrane-associated sterol response element-binding protein (SREBP-2). Activated SREBP-2 translocates to the nucleus, where it binds to an LDLR promoter sterol response element (SRE), increasing LDLR gene expression and LDL-C uptake. SREBP-2 cleavage and translocation steps are well established. Several SREBP-2 phosphorylation sites have been mapped and functionally characterized. The phosphatases dephosphorylating these sites remain elusive. The phosphatase(s) regulating SREBP-2 represents a novel pharmacological target for treating hypercholesterolemia. Here we show that protein phosphatase 2A (PP2A) promotes SREBP-2 LDLR promoter binding in response to cholesterol depletion. No binding to an LDLR SRE was observed in the presence of the HMG-CoA reductase inhibitor, lovastatin, when PP2A activity was inhibited by okadaic acid or depleted by siRNA methods. SREBP-2 cleavage and nuclear translocation were not affected by loss of PP2A. PP2A activity was required for SREBP-2 DNA binding. In response to cholesterol depletion, PP2A directly interacted with SREBP-2 and altered its phosphorylation state, causing an increase in SREBP-2 binding to an LDLR SRE site. Increased binding resulted in induced LDLR gene expression and increased LDL uptake. We conclude that PP2A activity regulates cholesterol homeostasis and LDL-C uptake. PMID:24770487

  17. Investigation of DNA binding, DNA photocleavage, topoisomerase I inhibition and antioxidant activities of water soluble titanium(IV) phthalocyanine compounds.

    PubMed

    Özel, Arzu; Barut, Burak; Demirbaş, Ümit; Biyiklioglu, Zekeriya

    2016-04-01

    The binding mode of water soluble peripherally tetra-substituted titanium(IV) phthalocyanine (Pc) compounds Pc1, Pc2 and Pc3 with calf thymus (CT) DNA was investigated by using UV-Vis spectroscopy and thermal denaturation studies in this work. The results of DNA binding constants (Kb) and the changes in the thermal denaturation profile of DNA with the addition of Pc compounds indicated that Pc1, Pc2 and Pc3 are able to bind to CT-DNA with different binding affinities. DNA photocleavage studies of Pc compounds were performed in the absence and presence of oxidizing agents such as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-mercaptoethanol (ME) using the agarose gel electrophoresis method at irradiation 650 nm. According to the results of electrophoresis studies, Pc1, Pc2 and Pc3 cleaved of supercoiled pBR322 DNA via photocleavage pathway. The Pc1, Pc2 and Pc3 compounds were examined for topoisomerase I inhibition by measuring the relaxation of supercoiled pBR322 DNA. The all of Pc compounds inhibited topoisomerase I at 20 μM concentration. A series of antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, superoxide radical scavenging (SOD) assay and metal chelating effect assay were performed for Pc1, Pc2 and Pc3 compounds. The results of antioxidant assays indicated that Pc1, Pc2 and Pc3 compounds have remarkable superoxide radical scavenging activities, moderate 2,2-diphenyl-1-picrylhydrazyl activities and metal chelating effect activities. All the experimental studies showed that Pc1, Pc2 and Pc3 compounds bind to CT-DNA via minor groove binding, cleave of supercoiled pBR322 DNA via photocleavage pathway, inhibit topoisomerase I and have remarkable superoxide radical scavenging activities. Thanks to these properties the Pc1, Pc2 and Pc3 compounds are suitable agents for photo dynamic therapy.

  18. Yeast TATA Binding Protein Interaction with DNA: Fluorescence Determination of Oligomeric State, Equilibrium Binding, On-Rate, and Dissociation Kinetics†

    PubMed Central

    Perez-Howard, Gina M.; Weil, P. Anthony

    2010-01-01

    A combination of steady-state, stopped-flow, and time-resolved fluorescence of intrinsic tryptophan and extrinsically labeled fluorescent DNA is utilized to examine the interaction of yeast TATA binding protein (TBP) with DNA. TBP is composed of two structural domains, the carboxy domain (residues 61–240), which is responsible for DNA binding and initiation of basal level transcription, and an amino terminal domain (residues 1–60), whose function is currently unknown. The steady-state fluorescence emission spectrum of the single tryptophan in the amino terminal domain of TBP undergoes a huge (30–40 nm) red-shift upon interaction with stoichiometric amounts of TATA box containing DNA. From time-resolved tryptophan fluorescence anisotropy studies, we demonstrate that, in the absence of DNA, the protein exists as a multimer in solution and it contains (at least) two primary conformations, one with the amino terminus associated tightly with the protein(s) in a hydrophobic environment and one with the amino terminus decoupled away from the rest of the protein and solvent-exposed. Upon binding DNA, the protein dissociates into a monomeric complex, upon which only the solvent-exposed amino terminus conformation remains. Kinetic and equilibrium binding studies were performed on TATA box containing DNA which was extrinsically labeled with a fluorescent probe Rhodamine-X at the 5′-end. This “fluorescent” DNA allowed for the collection of quantitative spectroscopic binding, kinetic on-rate, and kinetic off-rate data at physiological concentrations. Global analysis of equilibrium binding studies performed from 500 pM to 50 nM DNA reveals a single dissociation constant (Kd) of approximately 5 nM. Global analysis of stopped-flow anisotropy on-rate experiments, with millisecond timing resolution and TBP concentrations ranging from 20 to 600 nM (20 nM DNA), can be perfectly described by a single second-order rate constant of 1.66 × 105 M−1 s−1. These measurements

  19. Unraveling the motion of single-stranded DNA binding proteins on DNA using force and fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Ha, Taekjip

    2012-02-01

    Single-stranded DNA binding (SSB) proteins bind to and control the accessibility of single stranded (ss) DNA generated as a transient intermediate during a variety of cellular processes. For subsequent DNA processing, however, such a tightly wrapped, high-affinity protein--DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. Here we show, using single-molecule two- and three-colour fluorescence resonance energy transfer, that SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes. In addition, using an optomechanical tool combining single-molecule fluorescence and force methods, we probed how proteins with such a large binding site size (˜ 65 nucleotides) can migrate rapidly on DNA and how protein-protein interactions and tension may modulate the motion. We observed force-induced progressive unravelling of ssDNA from the SSB surface between 1 and 6 pN, followed by SSB dissociation at ˜10 pN, and obtained experimental evidence of a reptation mechanism for protein movement along DNA wherein a protein slides via DNA bulge formation and propagation. SSB diffusion persists even when bound with RecO, and at forces under which the fully wrapped state is perturbed, suggesting that even in crowded cellular conditions SSB can act as a sliding platform to recruit and carry its interacting proteins for use in DNA replication, recombination and repair.

  20. Protein-protein and protein-DNA interaction regions within the DNA end-binding protein Ku70-Ku86.

    PubMed Central

    Wu, X; Lieber, M R

    1996-01-01

    DNA ends are generated during double-strand-break repair and recombination. A p70-p86 heterodimer, Ku, accounts for the DNA end binding activity in eukaryotic cell extracts. When one or both subunits of Ku are missing, mammalian cells are deficient in double-strand-break repair and in specialized recombination, such as V(D)J recombination. Little is known of which regions of Ku70 and Ku86 bind to each other to form the heterodimeric complex or of which regions are important for DNA end binding. We have done genetic and biochemical studies to examine the domains within the two subunits important for protein assembly and for DNA end binding. We found that the C-terminal 20-kDa region of Ku70 and the C-terminal 32-kDa region of Ku86 are important for subunit-subunit interaction. For DNA binding, full-length individual subunits are inactive, indicating that heterodimer assembly precedes DNA binding. DNA end binding activity by the heterodimer requires the C-terminal 40-kDa region of Ku70 and the C-terminal 45-kDa region of Ku86. Leucine zipper-like motifs in both subunits that have been suggested as the Ku70-Ku86 interaction domains do not appear to be the sites of such interaction because these are dispensable for both assembly and DNA end binding. On the basis of these studies, we have organized Ku70 into nine sequence regions conserved between Saccharomyces cerevisiae, Drosophila melanogaster, mice, and humans; only the C-terminal three regions are essential for assembly (amino acids [aa] 439 to 609), and the C-terminal four regions appear to be essential for DNA end binding (aa 254 to 609). Within the minimal active fragment of Ku86 necessary for subunit interaction (aa 449 to 732) and DNA binding (aa 334 to 732), a proline-rich region is the only defined motif. PMID:8756676

  1. Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations.

    PubMed

    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.

  2. Association of the Adenovirus DNA-Binding Protein with RNA Both in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Cleghon, Vaughn G.; Klessig, Daniel F.

    1986-12-01

    The multifunctional DNA-binding protein (DBP) encoded by human adenovirus binds RNA. The association of purified DBP with RNA in vitro was demonstrated by using either a gel filtration or a filter binding assay. This association is sensitive to ionic strength and exhibits no apparent sequence specificity. DBP also interacts with RNA in vivo; it can be crosslinked to polyadenylylated RNA by UV-irradiation of intact cells during the late phase of adenovirus infections. The 46-kDa carboxyl-terminal domain of DBP binds RNA in vitro and was found to be associated with polyadenylylated RNA in vivo. This is the same domain that interacts with DNA. However, the differences in sensitivity of DBP to trypsin when bound to RNA versus DNA suggest that RNA and DNA either bind at different sites within this domain or induce different conformational changes within the protein.

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

    NASA Astrophysics Data System (ADS)

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

    2009-12-01

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

  4. Factors affecting binding of galacto ligands to Actinomyces viscosus lectin.

    PubMed Central

    Heeb, M J; Marini, A M; Gabriel, O

    1985-01-01

    The specificity requirements for the binding of Actinomyces viscosus T14V were examined by testing simple sugars, oligopeptides, and glycoproteins as inhibitors of the aggregation of glycoprotein-coated latex beads and washed A. viscosus cells. Lactose was the most inhibitory simple sugar; D-fucose and D-galactose were equally inhibitory, methyl-alpha-D-fucoside was slightly less inhibitory, and L-fucose and raffinose were not inhibitory. The concentration of galactose residues required for 50% inhibition of aggregation was 15 times higher in the form of lactose than in the form of asialoglycoprotein, suggesting an enhancement of lectin binding when galactose residues are clustered. However, when the inhibitory power of bi-, tri-, and tetraantennary asialooligopeptides of alpha 1-acid glycoprotein was compared with that of equivalent concentrations of galactose in the form of lactose, the biantennary form was slightly less effective than lactose, the triantennary form was approximately as effective as lactose, and the tetraantennary form was slightly more effective than lactose. Steric interference may prevent this type of clustering from enhancing lectin binding. The O-linked asialooligopeptides of asialofetuin were 10 times more inhibitory than an equivalent concentration of galactose in the form of N-linked asialooligopeptides. Thus, galactose beta-1----3 linked to N-acetylgalactosamine exhibits greater specificity for the A. viscosus lectin than does galactose beta-1----4 linked to N-acetylglucosamine. These results, taken together with previously reported data, are consistent with a lectin of low affinity, binding enhanced by multivalency, and specificity for beta-linked galactose. PMID:2578122

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

    SciTech Connect

    Fagan, Patricia A.

    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.

  6. Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis.

    PubMed

    Kassouf, Mira T; Chagraoui, Hedia; Vyas, Paresh; Porcher, Catherine

    2008-08-15

    Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding-independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

  7. Context influences on TALE-DNA binding revealed by quantitative profiling.

    PubMed

    Rogers, Julia M; Barrera, Luis A; Reyon, Deepak; Sander, Jeffry D; Kellis, Manolis; Joung, J Keith; Bulyk, Martha L

    2015-06-11

    Transcription activator-like effector (TALE) proteins recognize DNA using a seemingly simple DNA-binding code, which makes them attractive for use in genome engineering technologies that require precise targeting. Although this code is used successfully to design TALEs to target specific sequences, off-target binding has been observed and is difficult to predict. Here we explore TALE-DNA interactions comprehensively by quantitatively assaying the DNA-binding specificities of 21 representative TALEs to ∼5,000-20,000 unique DNA sequences per protein using custom-designed protein-binding microarrays (PBMs). We find that protein context features exert significant influences on binding. Thus, the canonical recognition code does not fully capture the complexity of TALE-DNA binding. We used the PBM data to develop a computational model, Specificity Inference For TAL-Effector Design (SIFTED), to predict the DNA-binding specificity of any TALE. We provide SIFTED as a publicly available web tool that predicts potential genomic off-target sites for improved TALE design.

  8. Dimerization-induced corepressor binding and relaxed DNA-binding specificity are critical for PML/RARA-induced immortalization.

    PubMed

    Zhou, Jun; Pérès, Laurent; Honoré, Nicole; Nasr, Rihab; Zhu, Jun; de Thé, Hugues

    2006-06-13

    The pathogenesis of acute promyelocytic leukemia involves the transcriptional repression of master genes of myeloid differentiation by the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARA) oncogene. PML-enforced RARA homodimerization allows the tighter binding of corepressors, silencing RARA target genes. In addition, homodimerization dramatically extends the spectrum of DNA-binding sites of the fusion protein compared with those of normal RARA. Yet, any contribution of these two properties of PML/RARA to differentiation arrest and immortalization of primary mouse hematopoietic progenitors was unknown. We demonstrate that dimerization-induced silencing mediator of retinoid and thyroid receptors (SMRT)-enhanced binding and relaxed DNA-binding site specificity are both required for efficient immortalization. Thus, enforced RARA dimerization is critical not only for triggering transcriptional repression but also for extending the repertoire of target genes. Our studies exemplify how dimerization-induced gain of functions converts an unessential transcription factor into a dominant oncogenic protein.

  9. Twist- and Tension-Mediated Elastic Coupling between DNA-Binding Proteins

    NASA Astrophysics Data System (ADS)

    Koslover, Elena F.; Spakowitz, Andrew J.

    2009-05-01

    We study the effective interaction between DNA-binding proteins that arises from elastic stresses in the DNA when tension is applied. Using the wormlike chain model, we calculate the free energy cost of introducing multiple nearby bends in the DNA. We find that the bend deformation energy promotes aggregation to straighten the linker DNA, while twist resistance of the linker leads to damped oscillations in the coupling free energy between two proteins. We calculate the mean first encounter time for proteins sliding along DNA, indicating, in some cases, an optimal applied tension for protein assembly. Our results highlight the need to consider DNA twist even when no torsion is applied and the DNA ends are free to rotate. The variable-range oscillatory coupling between DNA-binding proteins may provide a versatile mechanism for tension-mediated gene regulation.

  10. Conformation of nanoconfined DNA as a function of ATP, AMP, CTP, Mg2+, and dye binding

    NASA Astrophysics Data System (ADS)

    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+.

  11. PARP activation regulates the RNA-binding protein NONO in the DNA damage response to DNA double-strand breaks.

    PubMed

    Krietsch, Jana; Caron, Marie-Christine; Gagné, Jean-Philippe; Ethier, Chantal; Vignard, Julien; Vincent, Michel; Rouleau, Michèle; Hendzel, Michael J; Poirier, Guy G; Masson, Jean-Yves

    2012-11-01

    After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR) in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until date poorly understood.

  12. Structure of a Thyroid Hormone Receptor DNA-Binding Domain Homodimer Bound to an Inverted Palindrome DNA Response Element

    SciTech Connect

    Chen, Yi; Young, Matthew A.

    2010-10-22

    Thyroid hormone receptor (TR), as a member of the nuclear hormone receptor family, can recognize and bind different classes of DNA response element targets as either a monomer, a homooligomer, or a heterooligomer. We report here the first crystal structure of a homodimer TR DNA-binding domain (DBD) in complex with an inverted repeat class of thyroid response element (TRE). The structure shows a nearly symmetric structure of the TR DBD assembled on the F2 TRE where the base recognition contacts in the homodimer DNA complex are conserved relative to the previously published structure of a TR-9-cis-retinoic acid receptor heterodimer DNA complex. The new structure also reveals that the T-box region of the DBD can function as a structural hinge that enables a large degree of flexibility in the position of the C-terminal extension helix that connects the DBD to the ligand-binding domain. Although the isolated TR DBDs exist as monomers in solution, we have measured highly cooperative binding of the two TR DBD subunits onto the inverted repeat DNA sequence. This suggests that elements of the DBD can influence the specific TR oligomerization at target genes, and it is not just interactions between the ligand-binding domains that are responsible for TR oligomerization at target genes. Mutational analysis shows that intersubunit contacts at the DBD C terminus account for some, but not all, of the cooperative homodimer TR binding to the inverted repeat class TRE.

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

    PubMed

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

    2014-01-01

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

  14. DNA-induced dimerization of the single-stranded DNA binding telomeric protein Pot1 from Schizosaccharomyces pombe

    PubMed Central

    Nandakumar, Jayakrishnan; Cech, Thomas R.

    2012-01-01

    Eukaryotic chromosome ends are protected from illicit DNA joining by protein–DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA. PMID:21911358

  15. A DNA-binding protein containing two widely separated zinc finger motifs that recognize the same DNA sequence.

    PubMed

    Fan, C M; Maniatis, T

    1990-01-01

    We have isolated a full-length cDNA clone encoding a protein (PRDII-BF1) that binds specifically to a positive regulatory domain (PRDII) of the human IFN-beta gene promoter, and to a similar sequence present in a number of other promoters and enhancers. The sequence of this protein reveals two novel structural features. First, it is the largest sequence-specific DNA-binding protein reported to date (298 kD). Second, it contains two widely separated sets of C2-H2-type zinc fingers. Remarkably, each set of zinc fingers binds to the same DNA sequence motif with similar affinities and methylation interference patterns. Thus, this protein may act by binding simultaneously to reiterated copies of the same recognition sequence. Although the function of PRDII-BF1 is not known, the level of its mRNA is inducible by serum and virus, albeit with different kinetics.

  16. A simple, high-resolution method for establishing DNA binding affinity and sequence selectivity.

    PubMed

    Boger, D L; Fink, B E; Brunette, S R; Tse, W C; Hedrick, M P

    2001-06-27

    Full details of the development of a simple, nondestructive, and high-throughput method for establishing DNA binding affinity and sequence selectivity are described. The method is based on the loss of fluorescence derived from the displacement of ethidium bromide or thiazole orange from the DNA of interest or, in selected instances, the change in intrinsic fluorescence of a DNA binding agent itself and is applicable for assessing relative or absolute DNA binding affinities. Enlisting a library of hairpin deoxyoligonucleotides containing all five base pair (512 hairpins) or four base pair (136 hairpins) sequences displayed in a 96-well format, a compound's rank order binding to all possible sequences is generated, resulting in a high-resolution definition of its sequence selectivity using this fluorescent intercalator displacement (FID) assay. As such, the technique complements the use of footprinting or affinity cleavage for the establishment of DNA binding selectivity and provides the information at a higher resolution. The merged bar graphs generated by this rank order binding provide a qualitative way to compare, or profile, DNA binding affinity and selectivity. The 96-well format assay (512 hairpins) can be conducted at a minimal cost (presently ca. $100 for hairpin deoxyoligonucleotides/assay with ethiduim bromide or less with thiazole orange), with a rapid readout using a fluorescent plate reader (15 min), and is adaptable to automation (Tecan Genesis Workstation 100 robotic system). Its use in generating a profile of DNA binding selectivity for several agents including distamycin A, netropsin, DAPI, Hoechst 33258, and berenil is described. Techniques for establishing binding constants from quantitative titrations are compared, and recommendations are made for use of a Scatchard or curve fitting analysis of the titration binding curves as a reliable means to quantitate the binding affinity.

  17. CtBP Levels Control Intergenic Transcripts, PHO/YY1 DNA Binding, and PcG Recruitment to DNA

    PubMed Central

    Basu, Arindam; Atchison, Michael L.

    2013-01-01

    Carboxy-terminal binding protein (CtBP) is a well-known corepressor of several DNA binding transcription factors in Drosophila as well as in mammals. CtBP is implicated in Polycomb Group (PcG) complex-mediated transcriptional repression because it can bind to some PcG proteins, and mutation of the ctbp gene in flies results in lost PcG protein recruitment to Polycomb Response Elements (PREs) and lost PcG repression. However, the mechanism of reduced PcG DNA binding in CtBP mutant backgrounds is unknown. We show here that in a Drosophila CtBP mutant background, intergenic transcripts are induced across several PRE sequences and this corresponds to reduced DNA binding by PcG proteins Pleiohomeotic (PHO) and Polycomb (Pc), and reduced trimethylation of histone H3 on lysine 27, a hallmark of PcG repression. Restoration of CtBP levels by expression of a CtBP transgene results in repression of intergenic transcripts, restored PcG binding, and elevated trimethylation of H3 on lysine 27. Our results support a model in which CtBP regulates expression of intergenic transcripts that controls DNA binding by PcG proteins and subsequent histone modifications and transcriptional activity. PMID:20082324

  18. Quest for the binding mode of tetrabromobisphenol A with Calf thymus DNA

    NASA Astrophysics Data System (ADS)

    Wang, Yan-Qing; Zhang, Hong-Mei; Cao, Jian

    2014-10-01

    The binding interaction of tetrabromobisphenol A with Calf thymus DNA was studied by multi-spectroscopic and molecular modeling methods. The UV-vis study revealed that an obvious interaction between tetrabromobisphenol A and Calf thymus DNA happened. The π-π∗ transitions and the electron cloud of tetrabromobisphenol A might be changed by entering the groove of Calf thymus DNA. From the fluorescence spectral and thermodynamics studies, it was concluded that the hydrogen bonds and hydrophobic force played a major role in the binding of tetrabromobisphenol A to Calf thymus DNA. The molecular modeling study showed that the possible sites of tetrabromobisphenol A in the groove of DNA. Circular dichroism study also depicted that tetrabromobisphenol A bond to DNA. These above results would further advance our knowledge on the molecular mechanism of the binding interactions of brominated flame-retardants with nucleic acid.

  19. Deciphering the groove binding modes of tau-fluvalinate and flumethrin with calf thymus DNA

    NASA Astrophysics Data System (ADS)

    Tao, Mo; Zhang, Guowen; Pan, Junhui; Xiong, Chunhong

    2016-02-01

    Tau-fluvalinate (TFL) and flumethrin (FL), widely used in agriculture and a class of synthetic pyrethroid pesticides with a similar structure, may cause a potential security risk. Herein, the modes of binding in vitro of TFL and FL with calf thymus DNA (ctDNA) were characterized by fluorescence, UV-vis absorption, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy with the aid of viscosity measurements, melting analyses and molecular docking studies. The fluorescence titration indicated that both TFL and FL bound to ctDNA forming complexes through hydrogen bonding and van der Waals forces. The binding constants of TFL and FL with ctDNA were in the range of 104 L mol- 1, and FL exhibited a higher binding propensity than TFL. The iodide quenching effect, single/double-stranded DNA effects, and ctDNA melting and viscosity measurements demonstrated that the binding of both TFL and FL to ctDNA was groove mode. The FT-IR analyses suggested the A-T region of the minor groove of ctDNA as the preferential binding for TFL and FL, which was confirmed by the displacement assays with Hoechst 33258 probe, and the molecular docking visualized the specific binding. The changes in CD spectra indicated that both FL and TFL induced the perturbation on the base stacking and helicity of B-DNA, but the disturbance caused by FL was more obvious. Gel electrophoresis analyses indicated that both TFL and FL did not cause significant DNA cleavage. This study provides novel insights into the binding properties of TFL/FL with ctDNA and its toxic mechanisms.

  20. Experimental factors affecting the robustness of DNA methylation analysis

    PubMed Central

    Pharo, Heidi D.; Honne, Hilde; Vedeld, Hege M.; Dahl, Christina; Andresen, Kim; Liestøl, Knut; Jeanmougin, Marine; Guldberg, Per; Lind, Guro E.

    2016-01-01

    Diverging methylation frequencies are often reported for the same locus in the same disease, underscoring the need for limiting technical variability in DNA methylation analyses. We have investigated seven likely sources of variability at different steps of bisulfite PCR-based DNA methylation analyses using a fully automated quantitative methylation-specific PCR setup of six gene promoters across 20 colon cancer cell lines. Based on >15,000 individual PCRs, all tested parameters affected the normalized percent of methylated reference (PMR) differences, with a fourfold varying magnitude. Additionally, large variations were observed across the six genes analyzed. The highest variation was seen using single-copy genes as reference for normalization, followed by different amounts of template in the PCR, different amounts of DNA in the bisulfite reaction, and storage of bisulfite converted samples. Finally, when a highly standardized pipeline was repeated, the difference in PMR value for the same assay in the same cell line was on average limited to five (on a 0–100 scale). In conclusion, a standardized pipeline is essential for consistent methylation results, where parameters are kept constant for all samples. Nevertheless, a certain level of variation in methylation values must be expected, underscoring the need for careful interpretation of data. PMID:27671843

  1. Intranuclear DNA density affects chromosome condensation in metazoans.

    PubMed

    Hara, Yuki; Iwabuchi, Mari; Ohsumi, Keita; Kimura, Akatsuki

    2013-08-01

    Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or "intranuclear DNA density." Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans.

  2. Transactive Response DNA-Binding Protein 43 Burden in Familial Alzheimer Disease and Down Syndrome

    PubMed Central

    Lippa, Carol F.; Rosso, Andrea L.; Stutzbach, Lauren D.; Neumann, Manuela; Lee, Virginia M.-Y.; Trojanowski, John Q.

    2010-01-01

    Objective To assess the transactive response DNA-binding protein 43 (TDP-43) burden in familial forms of Alzheimer disease (FAD) and Down syndrome (DS) to determine whether TDP-43 inclusions are also present. Design Using standard immunohistochemical techniques, we examined brain tissue samples from 42 subjects with FAD and 14 with DS. Results We found pathological TDP-43 aggregates in 14.0% of participants (6 of 42 and 2 of 14 participants with FAD and DS, respectively). In both FAD and DS, TDP-43 immunoreactivity did not colocalize with neurofibrillary tangles. Occasionally participants with FAD or DS had TDP-43–positive neuropil threads or dots. Overall, the amygdala was most commonly affected, followed by the hippocampus, with no TDP-43 pathology in neocortical regions. A similar distribution of TDP-43 inclusions is seen in sporadic Alzheimer disease, but it differs from that seen in amyotrophic lateral sclerosis and frontotemporal dementia. Conclusions Transactive response DNA-binding protein 43 pathology occurs in FAD and DS, similar to that observed in sporadic Alzheimer disease. Thus, pathological TDP-43 may contribute the cognitive impairments in familial and sporadic forms of Alzheimer disease. PMID:20008652

  3. Structure of the RAG1 Nonamer Binding Domain with DNA Reveals a Dimer that Mediates DNA Synapsis

    SciTech Connect

    Yin, F.; Bailey, S; Innis, C; Ciubotaru, M; Kamtekar, S; Steitz, T; Schatz, D

    2009-01-01

    The products of recombination-activating genes RAG1 and RAG2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2-DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1 and RAG2.

  4. Structure of the RAG1 nonamer-binding domain with DNA reveals a dimer that mediates DNA synapsis

    PubMed Central

    Fang Yin, Fang; Bailey, Scott; Innis, C. Axel; Ciubotaru, Mihai; Kamtekar, Satwik; Steitz, Thomas A.; Schatz, David G.

    2009-01-01

    The products of recombination activating genes (RAG) 1 and 2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA-binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer-binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2-DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1/2. PMID:19396172

  5. The ecdysone receptor (ScEcR-A) binds DNA puffs at the start of DNA amplification in Sciara coprophila

    PubMed Central

    Liew, Gerald M.; Foulk, Michael S.

    2014-01-01

    The steroid hormone ecdysone induces DNA amplification and subsequent DNA puff formation in late fourth larval instar salivary gland polytene chromosomes of the fungus fly, Sciara coprophila. Previous in vitro studies on DNA puff II/9A in Sciara demonstrated that the ecdysone receptor (ScEcR-A) efficiently binds an ecdysone response element adjacent to the origin recognition complex binding site within the II/9A amplification origin, implying a role for ScEcR-A in amplification. Here, we extrapolate themolecular details from locus II/9A to the rest of the genome using immunofluorescence with a ScEcR-A-specific antibody. ScEcR-A binds all DNA puff sites just as amplification begins and persists throughout the processes of amplification, transcription, and puffing. Ecdysone injections into pre-amplification stage larvae prematurely induce both DNA amplification and ScEcR-A binding to DNA puff sites. These data are consistent with a direct role for ScEcR-A in DNA amplification. PMID:23737076

  6. Reagentless, Electrochemical Approach for the Specific Detection of Double- and Single-Stranded DNA Binding Proteins

    PubMed Central

    Ricci, Francesco; Bonham, Andrew J.; Mason, Aaron C.; Reich, Norbert O.; Plaxco, Kevin W.

    2009-01-01

    Here we demonstrate a reagentless, electrochemical platform for the specific detection of proteins that bind to single- or double-stranded DNA. The sensor is composed of a double- or single-stranded, redox-tagged DNA probe which is covalently attached to an interrogating electrode. Upon protein binding the current arising from the redox tag is suppressed, indicating the presence of the target. Using this approach we have fabricated sensors against the double-stranded DNA binding proteins TATA-box binding protein and M.HhaI methyltransferase, and against the single-strand binding proteins Escherichia coli SSBP and replication protein A. All four targets are detected at nanomolar concentrations, in minutes, and in a convenient, general, readily reusable, electrochemical format. The approach is specific; we observed no significant cross-reactivity between the sensors. Likewise the approach is selective; it supports, for example, the detection of single strand binding protein directly in crude nuclear extracts. The generality of our approach (including its ability to detect both double- and single-strand binding proteins) and a strong, non-monotonic dependence of signal gain on probe density support a collisional signaling mechanism in which binding alters the collision efficiency, and thus electron transfer efficiency, of the attached redox tag. Given the ubiquity with which protein binding will alter the collisional dynamics of an oligonucleotide, we believe this approach may prove of general utility in the detection of DNA and RNA binding proteins. PMID:19199570

  7. Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Chang, D.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

  8. Metabolism and binding to DNA of a novel hepatocarcinogen: methapyrilene

    SciTech Connect

    Lampe, M.A.

    1987-01-01

    The purpose of this study was to characterize the metabolism and macromolecular binding of (/sup 14/C)-MPH. When rats were treated for up to sixteen weeks with 1000 ppm MPH, they excreted at least six MPH related compounds in their urine including 2-hydroxymethyl-thiophene, N-2-pyridyl-N'-dimethylethylenediamine, (5-hydroxypyridyl)methapyrilene, (5-hydroxypyridyl)N-2-pyridyl-N'-dimethylethylenediamine and MPH. In vitro metabolism is also affected by chronic treatment of rats with MPH, with the most remarkable change observed being an increase in the mass balance to approximately 100% after 8 or 16 weeks pretreatment. Metabolism of MPH by rat liver microsomes is dependent on the activity of cytochrome P-450 enzymes, as metabolism in vitro was inhibited by the addition 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) and 2-diethylaminoethyl-2,2-diphenylvalerate (SKF-525A). Treatment of rats with phenobarbital or 3-methylcholanthrene induced metabolism of MPH while pretreatment of rats with isosafole or pregnenolone-16..cap alpha..-carbonitrile had a more selective effect on MPH metabolism, and only increased the formation of methapyrilene-amide.

  9. Differential DNA binding by the androgen and glucocorticoid receptors involves the second Zn-finger and a C-terminal extension of the DNA-binding domains.

    PubMed Central

    Schoenmakers, E; Alen, P; Verrijdt, G; Peeters, B; Verhoeven, G; Rombauts, W; Claessens, F

    1999-01-01

    The androgen and glucocorticoid hormones evoke specific in vivo responses by activating different sets of responsive genes. Although the consensus sequences of the glucocorticoid and androgen response elements are very similar, this in vivo specificity can in some cases be explained by differences in DNA recognition between both receptors. This has clearly been demonstrated for the androgen response element PB-ARE-2 described in the promoter of the rat probasin gene. Swapping of different fragments between the androgen- and glucocorticoid-receptor DNA-binding domains demonstrates that (i) the first Zn-finger module is not involved in this sequence selectivity and (ii) that residues in the second Zn-finger as well as a C-terminal extension of the DNA-binding domain from the androgen receptor are required. For specific and high-affinity binding to response elements, the DNA-binding domains of the androgen and glucocorticoid receptors need a different C-terminal extension. The glucocorticoid receptor requires 12 C-terminal amino acids for high affinity DNA binding, while the androgen receptor only involves four residues. However, for specific recognition of the PB-ARE-2, the androgen receptor also requires 12 C-terminal residues. Our data demonstrate that the mechanism by which the androgen receptor binds selectively to the PB-ARE-2 is different from that used by the glucocorticoid receptor to bind a consensus response element. We would like to suggest that the androgen receptor recognizes response elements as a direct repeat rather than the classical inverted repeat. PMID:10417312

  10. The DNA Binding Domain of a Papillomavirus E2 Protein Programs a Chimeric Nuclease To Cleave Integrated Human Papillomavirus DNA in HeLa Cervical Carcinoma Cells▿

    PubMed Central

    Horner, Stacy M.; DiMaio, Daniel

    2007-01-01

    Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA binding domain of the bovine papillomavirus type 1 (BPV1) E2 protein to the catalytic domain of the FokI restriction endonuclease, generating a BPV1 E2-FokI chimeric nuclease (BEF). BEF introduced DNA double-strand breaks on both sides of an E2 binding site in vitro, whereas DNA binding or catalytic mutants of BEF did not. After expression of BEF in HeLa cervical carcinoma cells, we detected cleavage at E2 binding sites in the integrated HPV18 DNA in these cells and also at an E2 binding site in cellular DNA. BEF-expressing cells underwent senescence, which required the DNA binding activity of BEF, but not its nuclease activity. These results demonstrate that DNA binding domains of viral proteins can target effector molecules to cognate binding sites in virally infected cells. PMID:17392356

  11. Nbs1-dependent binding of Mre11 to adenovirus E4 mutant viral DNA is important for inhibiting DNA replication

    SciTech Connect

    Mathew, Shomita S.; Bridge, Eileen

    2008-04-25

    Adenovirus (Ad) infections stimulate the activation of cellular DNA damage response and repair pathways. Ad early regulatory proteins prevent activation of DNA damage responses by targeting the MRN complex, composed of the Mre11, Rad50 and Nbs1 proteins, for relocalization and degradation. In the absence of these viral proteins, Mre11 colocalizes with viral DNA replication foci. Mre11 foci formation at DNA damage induced by ionizing radiation depends on the Nbs1 component of the MRN complex and is stabilized by the mediator of DNA damage checkpoint protein 1 (Mdc1). We find that Nbs1 is required for Mre11 localization at DNA replication foci in Ad E4 mutant infections. Mre11 is important for Mdc1 foci formation in infected cells, consistent with its role as a sensor of DNA damage. Chromatin immunoprecipitation assays indicate that both Mre11 and Mdc1 are physically bound to viral DNA, which could account for their localization in viral DNA containing foci. Efficient binding of Mre11 to E4 mutant DNA depends on the presence of Nbs1, and is correlated with a significant E4 mutant DNA replication defect. Our results are consistent with a model in which physical interaction of Mre11 with viral DNA is mediated by Nbs1, and interferes with viral DNA replication.

  12. Novel DNA motif binding activity observed in vivo with an estrogen receptor α mutant mouse.

    PubMed

    Hewitt, Sylvia C; Li, Leping; Grimm, Sara A; Winuthayanon, Wipawee; Hamilton, Katherine J; Pockette, Brianna; Rubel, Cory A; Pedersen, Lars C; Fargo, David; Lanz, Rainer B; DeMayo, Francesco J; Schütz, Günther; Korach, Kenneth S

    2014-06-01

    Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the "EAAE" ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo.

  13. Novel DNA Motif Binding Activity Observed In Vivo With an Estrogen Receptor α Mutant Mouse

    PubMed Central

    Li, Leping; Grimm, Sara A.; Winuthayanon, Wipawee; Hamilton, Katherine J.; Pockette, Brianna; Rubel, Cory A.; Pedersen, Lars C.; Fargo, David; Lanz, Rainer B.; DeMayo, Francesco J.; Schütz, Günther; Korach, Kenneth S.

    2014-01-01

    Estrogen receptor α (ERα) interacts with DNA directly or indirectly via other transcription factors, referred to as “tethering.” Evidence for tethering is based on in vitro studies and a widely used “KIKO” mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERα binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERα has HRE DNA-binding activity, we evaluated the “EAAE” ERα, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERα null–like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERα tethering, because KIKO ERα effectively stimulates transcription using HRE motifs. The EAAE-ERα DNA-binding domain mutant mouse demonstrates that ERα DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERα tethering may not contribute to estrogen responsiveness in vivo. PMID:24713037

  14. The single-stranded DNA-binding protein of Escherichia coli.

    PubMed Central

    Meyer, R R; Laine, P S

    1990-01-01

    The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake. PMID:2087220

  15. DAPI binding to the DNA minor groove: a continuum solvent analysis.

    PubMed

    De Castro, L F Pineda; Zacharias, M

    2002-01-01

    A continuum solvent model based on the generalized Born (GB) or finite-difference Poisson-Boltzmann (FDPB) approaches has been employed to compare the binding of 4'-6-diamidine-2-phenyl indole (DAPI) to the minor groove of various DNA sequences. Qualitative agreement between the results of GB and FDPB approaches as well as between calculated and experimentally observed trends regarding the sequence specificity of DAPI binding to B-DNA was obtained. Calculated binding energies were decomposed into various contributions to solvation and DNA-ligand interaction. DNA conformational adaptation was found to make a favorable contribution to the calculated total interaction energy but did not change the DAPI binding affinity ranking of different DNA sequences. The calculations indicate that closed complex formation is mainly driven by nonpolar contributions and was found to be disfavored electrostatically due to a desolvation penalty that outbalances the attractive Coulomb interaction. The calculated penalty was larger for DAPI binding to GC-rich sequences compared with AT-rich target sequences and generally larger for the FDPB vs the GB continuum model. A radial interaction profile for DAPI at different distances from the DNA minor groove revealed an electrostatic energy minimum a few Angstroms farther away from the closed binding geometry. The calculated electrostatic interaction up to this distance is attractive and it may stabilize a nonspecific binding arrangement.

  16. The SV40 large T-antigen origin binding domain directly participates in DNA unwinding.

    PubMed

    Foster, Erin C; Simmons, Daniel T

    2010-03-16

    The origin binding domain (OBD) of SV40 large T-ag serves a critical role during initiation of DNA replication to position T-ag on the origin. After origin recognition, T-ag forms a double hexamer over the origin. Within each hexamer, the OBD adopts a lock washer structure where the origin recognizing A1 and B2 loops face toward the helicase domain and likely become unavailable for binding DNA. In this study, we investigated the role of the central channel of the OBD hexamer in DNA replication by analyzing the effects of mutations of residues lining the channel. All mutants showed binding defects with origin DNA and ssDNA especially at low protein concentrations, but only half were defective at supporting DNA replication in vitro. All mutants were normal in unwinding linear origin DNA fragments. However, replication defective mutants failed to unwind a small origin containing circular DNA whereas replication competent mutants did so normally. The presence of RPA and/or pol/prim restored circular DNA unwinding activity of compromised mutants probably by interacting with the separated DNA strands on the T-ag surface. We interpret these results to indicate a role for the OBD central channel in binding and threading ssDNA during unwinding of circular SV40 DNA. Mixing experiments suggested that only one monomer in an OBD hexamer was necessary for DNA unwinding. We present a model of DNA threading through the T-ag complex illustrating how single-stranded DNA could pass close to a trough generated by key residues in one monomer of the OBD hexamer.

  17. Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor

    PubMed Central

    Kim, Minsik; Kim, Hee Jung; Son, Sang Hyeon; Yoon, Hye Jin; Lim, Youngbin; Lee, Jong Woo; Seok, Yeong-Jae; Jin, Kyeong Sik; Yu, Yeon Gyu; Kim, Seong Keun; Ryu, Sangryeol; Lee, Hyung Ho

    2016-01-01

    DNA-binding repressors are involved in transcriptional repression in many organisms. Disabling a repressor is a crucial step in activating expression of desired genes. Thus, several mechanisms have been identified for the removal of a stably bound repressor (Rep) from the operator. Here, we describe an uncharacterized mechanism of noncanonical DNA binding and induction by a Rep from the temperate Salmonella phage SPC32H; this mechanism was revealed using the crystal structures of homotetrameric Rep (92–198) and a hetero-octameric complex between the Rep and its antirepressor (Ant). The canonical method of inactivating a repressor is through the competitive binding of the antirepressor to the operator-binding site of the repressor; however, these studies revealed several noncanonical features. First, Ant does not compete for the DNA-binding region of Rep. Instead, the tetrameric Ant binds to the C-terminal domains of two asymmetric Rep dimers. Simultaneously, Ant facilitates the binding of the Rep N-terminal domains to Ant, resulting in the release of two Rep dimers from the bound DNA. Second, the dimer pairs of the N-terminal DNA-binding domains originate from different dimers of a Rep tetramer (trans model). This situation is different from that of other canonical Reps, in which two N-terminal DNA-binding domains from the same dimeric unit form a dimer upon DNA binding (cis model). On the basis of these observations, we propose a noncanonical model for the reversible inactivation of a Rep by an Ant. PMID:27099293

  18. FANCI protein binds to DNA and interacts with FANCD2 to recognize branched structures.

    PubMed

    Yuan, Fenghua; El Hokayem, Jimmy; Zhou, Wen; Zhang, Yanbin

    2009-09-04

    In this study, we report that the purified wild-type FANCI (Fanconi anemia complementation group I) protein directly binds to a variety of DNA substrates. The DNA binding domain roughly encompasses residues 200-1000, as suggested by the truncation study. When co-expressed in insect cells, a small fraction of FANCI forms a stable complex with FANCD2 (Fanconi anemia complementation group D2). Intriguingly, the purified FANCI-FANCD2 complex preferentially binds to the branched DNA structures when compared with either FANCI or FANCD2 alone. Co-immunoprecipitation with purified proteins indicates that FANCI interacts with FANCD2 through its C-terminal amino acid 1001-1328 fragment. Although the C terminus of FANCI is dispensable for direct DNA binding, it seems to be involved in the regulation of DNA binding activity. This notion is further enhanced by two C-terminal point mutations, R1285Q and D1301A, which showed differentiated DNA binding activity. We also demonstrate that FANCI forms discrete nuclear foci in HeLa cells in the absence or presence of exogenous DNA damage. The FANCI foci are colocalized perfectly with FANCD2 and partially with proliferating cell nuclear antigen irrespective of mitomycin C treatment. An increased number of FANCI foci form and become resistant to Triton X extraction in response to mitomycin C treatment. Our data suggest that the FANCI-FANCD2 complex may participate in repair of damaged replication forks through its preferential recognition of branched structures.

  19. SiteOut: An Online Tool to Design Binding Site-Free DNA Sequences.

    PubMed

    Estrada, Javier; Ruiz-Herrero, Teresa; Scholes, Clarissa; Wunderlich, Zeba; DePace, Angela H

    2016-01-01

    DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/.

  20. The secretion ATPase ComGA is required for the binding and transport of transforming DNA

    PubMed Central

    Briley, Kenneth; Dorsey-Oresto, Angella; Prepiak, Peter; Dias, Miguel J.; Mann, Jessica M.; Dubnau, David

    2011-01-01

    Summary Transformation requires specialized proteins to facilitate the binding and uptake of DNA. The genes of the B. subtilis comG operon (comGA–G) are required for transformation and to assemble a structure, the pseudopilus, in the cell envelope. No role for the pseudopilus has been established and the functions of the individual comG genes are unknown. We show that among the comG genes, only comGA is absolutely required for DNA binding to the cell surface. ComEA, an integral membrane DNA binding protein plays a minor role in the initial binding step, while an unidentified protein which communicates with ComGA must be directly responsible for binding to the cell. We show that the use of resistance to DNAse to measure “DNA uptake” reflects the movement of transforming DNA to a protected state in which it is not irreversibly associated with the protoplast, and presumably resides outside the cell membrane, in the periplasm or associated with the cell wall. We suggest that ComGA is needed for the acquisition of DNAse-resistance as well as for the binding of DNA to the cell surface. Finally, we show that the pseudopilus is required for DNA uptake and we offer a revised model for the transformation process. PMID:21707789

  1. SiteOut: An Online Tool to Design Binding Site-Free DNA Sequences

    PubMed Central

    Scholes, Clarissa; Wunderlich, Zeba; DePace, Angela H.

    2016-01-01

    DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/. PMID:26987123

  2. Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins

    PubMed Central

    Xu, Zhixiong; Meng, Xianzhang; Cai, Ying; Liang, Hong; Nagarajan, Lalitha; Brandt, Stephen J.

    2007-01-01

    The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and β-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins. PMID:17437998

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

    PubMed

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

    2012-11-01

    DNA interaction studies of two mononuclear [1:1(1); 1:2(2)] copper(II) complexes of curcumin have been studied. The interaction of these complexes with CT-DNA has been explored by physical methods to propose modes of DNA binding of the complexes. Absorption spectral titrations of complex 1 with CT-DNA shows a red-shift of 3 nm with the DNA binding affinity of K(b), 5.21×10(4)M(-1) that are higher than that obtained for 2 (red-shift, 2 nm; K(b), 1.73×10(4)M(-1)) reveal that the binding occurs in grooves as a result of the interaction is via exterior phosphates. The CD spectra of these Cu(II) complexes show a red shift of 3-10nm in the positive band with increase in intensities. This spectral change of induced CD due to the hydrophobic interaction of copper complexes with DNA is the characteristic of B to A conformational change. The EB displacement assay also reveals the same trend as observed in UV-Vis spectral titration. The addition of complexes 1 and 2 to the DNA bound ethidium bromide (EB) solutions causes an obvious reduction in emission intensities indicating that these complexes competitively bind to DNA with EB. The positive shift of both the E(pc) and E(0)' accompanied by reduction of peak currents in differential pulse voltammogram (DPV), upon adding different concentrations of DNA to the metal complexes, are obviously in favor of strong binding to DNA. The super coiled plasmid pUC18 DNA cleavage ability of Cu(II) complexes in the presence of reducing agent reveals the single strand DNA cleavage (ssDNA) is observed. The hydroxyl radical (HO()) and the singlet oxygen are believed to be the reactive species responsible for the cleavage.

  4. From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces

    PubMed Central

    Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

    2011-01-01

    Protein nucleic acid interactions play a critical role in all steps of the gene expression pathway. Nucleic acid (NA) binding proteins interact with their partners, DNA or RNA, via distinct regions on their surface that are characterized by an ensemble of chemical, physical and geometrical properties. In this study, we introduce a novel methodology based on differential geometry, commonly used in face recognition, to characterize and predict NA binding surfaces on proteins. Applying the method on experimentally solved three-dimensional structures of proteins we successfully classify double-stranded DNA (dsDNA) from single-stranded RNA (ssRNA) binding proteins, with 83% accuracy. We show that the method is insensitive to conformational changes that occur upon binding and can be applicable for de novo protein-function prediction. Remarkably, when concentrating on the zinc finger motif, we distinguish successfully between RNA and DNA binding interfaces possessing the same binding motif even within the same protein, as demonstrated for the RNA polymerase transcription-factor, TFIIIA. In conclusion, we present a novel methodology to characterize protein surfaces, which can accurately tell apart dsDNA from an ssRNA binding interfaces. The strength of our method in recognizing fine-tuned differences on NA binding interfaces make it applicable for many other molecular recognition problems, with potential implications for drug design. PMID:21693557

  5. Threading polyintercalators with extremely slow dissociation rates and extended DNA binding sites

    PubMed Central

    Smith, Amy Rhoden; Iverson, Brent L.

    2013-01-01

    The development of small molecules that bind DNA sequence specifically has the potential to modulate gene expression in a general way. One mode of DNA binding is intercalation, or the insertion of molecules between DNA base pairs. We have developed a modular polyintercalation system in which intercalating naphthalene diimide (NDI) units are connected by flexible linkers that alternate between the minor and major grooves of DNA when bound. We recently reported a threading tetraintercalator with a dissociation half-life of 16 days, the longest reported to date, from its preferred 14 bp binding site. Herein, three new tetraintercalator derivatives were synthesized with one, two, and three additional methylene units in the central major groove-binding linker. These molecules displayed dissociation half-lives of 57, 27, and 18 days, respectively, from the 14 bp site. The optimal major groove-binding linker was used in the design of an NDI hexaintercalator that was analyzed by gel-shift assays, DNase I footprinting, and UV-visible spectroscopy. The hexaintercalator bound its entire 22 bp binding site, the longest reported specific binding site for a synthetic, non-nucleic acid based DNA binding molecule, but with a significantly faster dissociation rate compared to the tetraintercalators. PMID:23919778

  6. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

    PubMed

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

    2016-01-01

    Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  7. Binding mechanism of PicoGreen to DNA characterized by magnetic tweezers and fluorescence spectroscopy.

    PubMed

    Wang, Ying; Schellenberg, Helene; Walhorn, Volker; Toensing, Katja; Anselmetti, Dario

    2017-03-01

    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.

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

    NASA Astrophysics Data System (ADS)

    Mahalakshmi, Rajkumar; Raman, Natarajan

    2013-08-01

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

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

    PubMed

    Mahalakshmi, Rajkumar; Raman, Natarajan

    2013-08-01

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

  10. The amplification effect of functionalized gold nanoparticles on the binding of anticancer drug dacarbazine to DNA and DNA bases

    NASA Astrophysics Data System (ADS)

    Shen, Qin; Wang, Xuemei; Fu, Degang

    2008-11-01

    The promising application of functionalized gold nanoparticles to amplify the performance of biosensors and relevant biomolecular recognition processes has been explored in this paper. Our observations illustrate the apparent enhancement effect of the gold nanoparticles on the electrochemical response of the anticancer drug dacarbazine (DTIC) binding to DNA and DNA bases, indicating that these functionalized gold nanoparticles could readily facilitate the specific interactions between DTIC and DNA/DNA bases. This raises the potential valuable applications of these biocompatible nanoparticles in the promising biosensors and biomedical engineering.

  11. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    PubMed Central

    Liu, Jiajian; Stormo, Gary D

    2005-01-01

    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. PMID:16014175

  12. The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η

    PubMed Central

    Roy, Upasana; Mukherjee, Shivam; Sharma, Anjali; Frank, Ekaterina G.; Schärer, Orlando D.

    2016-01-01

    Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases. PMID:27257072

  13. Interaction of coumarin with calf thymus DNA: deciphering the mode of binding by in vitro studies.

    PubMed

    Sarwar, Tarique; Rehman, Sayeed Ur; Husain, Mohammed Amir; Ishqi, Hassan Mubarak; Tabish, Mohammad

    2015-02-01

    DNA is the major target for a wide range of therapeutic substances. Thus, there has been considerable interest in the binding studies of small molecules with DNA. Interaction between small molecules and DNA provides a structural guideline in rational drug designing and in the synthesis of new and improved drugs with enhanced selective activity and greater clinical efficacy. Plant derived polyphenolic compounds have a large number of biological and pharmacological properties. Coumarin is a polyphenolic compound which has been extensively studied for its diverse pharmacological properties. However, its mode of interaction with DNA has not been elucidated. In the present study, we have attempted to ascertain the mode of binding of coumarin with calf thymus DNA (Ct-DNA) through various biophysical techniques. Analysis of UV-visible absorbance spectra and fluorescence spectra indicates the formation of complex between coumarin and Ct-DNA. Several other experiments such as effect of ionic strength, iodide induced quenching, competitive binding assay with ethidium bromide, acridine orange and Hoechst 33258 reflected that coumarin possibly binds to the minor groove of the Ct-DNA. These observations were further supported by CD spectral analysis, viscosity measurements, DNA melting studies and in silico molecular docking.

  14. Simulation of the type of coralin alkaloid-DNA binding

    NASA Astrophysics Data System (ADS)

    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.

  15. RecA-ssDNA filaments supercoil in the presence of single-stranded DNA-binding protein

    SciTech Connect

    Shi Weixian; Larson, Ronald G. . E-mail: rlarson@umich.edu

    2007-06-08

    Using atomic force microscopy (AFM), we find that RecA-single-stranded DNA (RecA-ssDNA) filaments, in the presence of single-stranded DNA-binding (SSB) protein, organize into left-handed bundles, which differ from the previously reported disordered aggregates formed when SSB is excluded from the reaction. In addition, we see both left- and right-handedness on bundles of two filaments. These two-filament supercoils, individual filaments, and other smaller bundles further organize into more complicated bundles, showing overall left-handedness which cannot be explained by earlier arguments that presumed supercoiling is absent in RecA-ssDNA filaments. This novel finding and our previous results regarding supercoiling of RecA-double-stranded DNA (RecA-dsDNA) filaments are, however, consistent with each other and can possibly be explained by the intrinsic tendency of RecA-DNA filaments, in their fully coated form, to order themselves into helical bundles, independent of the DNA inside the filaments (ssDNA or dsDNA). RecA-RecA interactions may dominate the bundling process, while the original conformation of DNA inside filaments and other factors (mechanical properties of filaments, concentration of filaments, and Mg{sup 2+} concentration) could contribute to the variation in the appearance and pitch of supercoils. The tendency of RecA-DNA filaments to form ordered supercoils and their presence during strand exchange suggest a possible biological importance of supercoiled filaments.

  16. Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

    PubMed Central

    Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.

    2015-01-01

    Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site. PMID:25793507

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

    PubMed Central

    Blackwell, L J; Borowiec, J A

    1994-01-01

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

  18. Overcoming transcription activator-like effector (TALE) DNA binding domain sensitivity to cytosine methylation.

    PubMed

    Valton, Julien; Dupuy, Aurélie; Daboussi, Fayza; Thomas, Séverine; Maréchal, Alan; Macmaster, Rachel; Melliand, Kevin; Juillerat, Alexandre; Duchateau, Philippe

    2012-11-09

    Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it.

  19. Transcriptional Regulation in Mammalian Cells by Sequence-Specific DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Mitchell, Pamela J.; Tjian, Robert

    1989-07-01

    The cloning of genes encoding mammalian DNA binding transcription factors for RNA polymerase II has provided the opportunity to analyze the structure and function of these proteins. This review summarizes recent studies that define structural domains for DNA binding and transcriptional activation functions in sequence-specific transcription factors. The mechanisms by which these factors may activate transcriptional initiation and by which they may be regulated to achieve differential gene expression are also discussed.

  20. Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

    PubMed Central

    Putterman, D G; Casadevall, A; Boyle, P D; Yang, H L; Frangione, B; Day, L A

    1984-01-01

    The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, f1) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNA-binding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses. Images PMID:6422463

  1. A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide

    NASA Technical Reports Server (NTRS)

    Herbert, A. G.; Rich, A.

    1993-01-01

    An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.

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

    PubMed

    Psomas, George

    2008-09-01

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

  3. Mannose-binding lectin may affect pregnancy outcome.

    PubMed

    Çalkavur, Şebnem; Erdemir, Gülin; Onay, Hüseyin; Altun Köroğlu, Özge; Yalaz, Mehmet; Zekioğlu, Osman; Aksu, Güzide; Özkınay, Ferda; Akercan, Fuat; Kültürsay, Nilgün

    2015-01-01

    Mannose-binding lectin (MBL) is a component of the innate immune system and acts as a complement activator through the lectin pathway. Genetic variations of MBL and low MBL levels cause several infection problems, which may also be related to pregnancy problems. We aimed to investigate the role of MBL gene codon 54 polymorphism and serum MBL levels in pregnancy problems and premature delivery. In this prospective study, MBL gene codon 54 polymorphism and serum MBL levels were studied in 45 mothers who delivered earlier than 35 gestational weeks. The frequency of MBL gene codon 54 variant allele B was much higher (homozygous 4.4% and heterozygous 33.3%) in the study group mothers than the previously reported frequency in the healthy Turkish population (homozygous 2-6%, heterozygous 12-20%). MBL variant allele B frequency was closely related to low MBL levels (<0.1 μg/ml), vaginitis and increased IL-6 levels. The median MBL levels were lower than the critical level of 0.1 μg/ ml in study mothers who had recurrent miscarriage, infertility, preeclampsia, gestational diabetes mellitus, preterm premature rupture of membranes with duration of longer than 72 hours, tocolysis, histological chorioamnionitis, urinary tract infection and vaginitis. MBL gene codon 54 variant allele B is related to low serum MBL levels, increased IL-6 levels, genitourinary infections and may cause pregnancy-related problems such as infertility, recurrent miscarriage and preterm delivery.

  4. Minor groove binding of the food colorant carmoisine to DNA: spectroscopic and calorimetric characterization studies.

    PubMed

    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.

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

    PubMed

    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.

  6. Effect of DNA binding on geminate CO recombination kinetics in CooA

    NASA Astrophysics Data System (ADS)

    Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan; Poulos, Thomas; Champion, Paul

    2012-02-01

    CooA proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding to RrCooA. The effects of DNA binding and the truncation of the DNA binding domain on the CO geminate recombination kinetics were investigated. The CO rebinding kinetics in these CooA complexes takes place on ultrafast timescales but remains 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 timescale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in RrCooA 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. Analysis of our data 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.

  7. Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

    PubMed

    Bauer, Amy L; Hlavacek, William S; Unkefer, Pat J; Mu, Fangping

    2010-11-18

    An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF). Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

  8. The activation of a specific DNA binding protein by neutron irradiation

    SciTech Connect

    Teale, B.; Singh, S.; Cohen, D.

    1995-08-30

    The purpose of this investigation was to determine whether the quality of ionizing radiation is critical for activation of a radiation-specific DNA binding protein. We have previously shown that after exposing Epstein Barr virus-transformed lymphoblastoid cells to ionizing radiation, a specific DNA binding factor appears in the nucleus apparently as a result of translocation from the cytoplasm. This protein binds to a number of different genomic sequences and a consensus motif has been identified. Because the protein was not activated by UV light, it was of interest whether high linear energy transfer (LET) radiation was capable of activation. We describe here the activation of a specific DNA binding protein by high LET neutron radiation. The protein binds a region adjacent to and overlapping with the distal repeat within a 179 base-pair fragment of the well-characterized Simian Virus (SV40) bidirectional promoter/enhancer element. The appearance of the DNA binding activity was dose dependent and reached a maximum level by 90 min postirradiation. A reduction in DNA binding activity was evident at later times after irradiation. The specific nature of this response and the rapidity of activation may indicate a pivotal role for this protein in repair or in some other aspect of the cellular response to radiation damage. 22 refs., 4 figs.

  9. The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA.

    PubMed Central

    Kota, R S; Runge, K W

    1998-01-01

    TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA. PMID:9490802

  10. Determining the binding mode and binding affinity constant of tyrosine kinase inhibitor PD153035 to DNA using optical tweezers

    SciTech Connect

    Cheng, Chih-Ming; Lee, Yuarn-Jang; Wang, Wei-Ting; Hsu, Chien-Ting; Tsai, Jing-Shin; Wu, Chien-Ming; Ou, Keng-Liang; and others

    2011-01-07

    Research highlights: {yields} PD153035 is a DNA intercalator and intercalation occurs only under very low salt concentration. {yields} The minimum distance between adjacent bound PD153035 {approx} 11 bp. {yields} Binding affinity constant for PD153035 is 1.18({+-}0.09) x 10{sup 4} (1/M). {yields} The change of binding free energy of PD153035-DNA interaction is -5.49 kcal mol{sup -1} at 23 {+-} 0.5 {sup o}C. -- Abstract: Accurately predicting binding affinity constant (K{sub A}) is highly required to determine the binding energetics of the driving forces in drug-DNA interactions. Recently, PD153035, brominated anilinoquinazoline, has been reported to be not only a highly selective inhibitor of epidermal growth factor receptor but also a DNA intercalator. Here, we use a dual-trap optical tweezers to determining K{sub A} for PD153035, where K{sub A} is determined from the changes in B-form contour length (L) of PD153035-DNA complex. Here, L is fitted using a modified wormlike chain model. We found that a noticeable increment in L in 1 mM sodium cacodylate was exhibited. Furthermore, our results showed that K{sub A} = 1.18({+-}0.09) x 10{sup 4} (1/M) at 23 {+-} 0.5 {sup o}C and the minimum distance between adjacent bound PD153035 {approx} 11 bp. We anticipate that by using this approach we can determine the complete thermodynamic profiles due to the presence of DNA intercalators.

  11. Spermine Attenuates the Action of the DNA Intercalator, Actinomycin D, on DNA Binding and the Inhibition of Transcription and DNA Replication

    PubMed Central

    Chen, Jeremy J. W.; Wu, Wen-Lin; Yuann, Jeu-Ming P.; Su, Wang-Lin; Chuang, Show-Mei; Hou, Ming-Hon

    2012-01-01

    The anticancer activity of DNA intercalators is related to their ability to intercalate into the DNA duplex with high affinity, thereby interfering with DNA replication and transcription. Polyamines (spermine in particular) are almost exclusively bound to nucleic acids and are involved in many cellular processes that require nucleic acids. Until now, the effects of polyamines on DNA intercalator activities have remained unclear because intercalation is the most important mechanism employed by DNA-binding drugs. Herein, using actinomycin D (ACTD) as a model, we have attempted to elucidate the effects of spermine on the action of ACTD, including its DNA-binding ability, RNA and DNA polymerase interference, and its role in the transcription and replication inhibition of ACTD within cells. We found that spermine interfered with the binding and stabilization of ACTD to DNA. The presence of increasing concentrations of spermine enhanced the transcriptional and replication activities of RNA and DNA polymerases, respectively, in vitro treated with ActD. Moreover, a decrease in intracellular polyamine concentrations stimulated by methylglyoxal-bis(guanylhydrazone) (MGBG) enhanced the ACTD-induced inhibition of c-myc transcription and DNA replication in several cancer cell lines. The results indicated that spermine attenuates ACTD binding to DNA and its inhibition of transcription and DNA replication both in vitro and within cells. Finally, a synergistic antiproliferative effect of MGBG and ACTD was observed in a cell viability assay. Our findings will be of significant relevance to future developments in combination with cancer therapy by enhancing the anticancer activity of DNA interactors through polyamine depletion. PMID:23144800

  12. A comprehensive approach to ascertain the binding mode of curcumin with DNA.

    PubMed

    Haris, P; Mary, Varughese; Aparna, P; Dileep, K V; Sudarsanakumar, C

    2017-03-15

    Curcumin is a natural phytochemical from the rhizoma of Curcuma longa, the popular Indian spice that exhibits a wide range of pharmacological properties like antioxidant, anticancer, anti-inflammatory, antitumor, and antiviral activities. In the published literatures we can see different studies and arguments on the interaction of curcumin with DNA. The intercalative binding, groove binding and no binding of curcumin with DNA were reported. In this context, we conducted a detailed study to understand the mechanism of recognition of dimethylsulfoxide-solubilized curcumin by DNA. The interaction of curcumin with calf thymus DNA (ctDNA) was confirmed by agarose gel electrophoresis. The nature of binding and energetics of interaction were studied by Isothermal Titration Calorimetry (ITC), Differential Scanning Calorimetry (DSC), UV-visible, fluorescence and melting temperature (Tm) analysis. The experimental data were compared with molecular modeling studies. Our investigation confirmed that dimethylsulfoxide-solubilized curcumin binds in the minor groove of the ctDNA without causing significant structural alteration to the DNA.

  13. A robust method for determining DNA binding constants using capillary zone electrophoresis.

    PubMed

    Li, C; Martin, L M

    1998-10-01

    Capillary zone electrophoresis (CZE or CE) with on-line UV detection was utilized to measure the binding constants between purified calf thymus DNA and a library of designed tetrapeptides which had been constructed using unnatural amino acids with thiazole ring side chains. Mixtures containing a constant amount of a tetrapeptide, the neutral marker (mesityl oxide), and varying concentrations of DNA were prepared and equilibrated at 8 degreesC for 12 h. CE was then utilized to separate unbound tetrapeptides from the DNA-peptide complex. The UV absorbance of the peak representing unbound tetrapeptide decreased incrementally as a result of increasing the concentration of DNA in the equilibrium mixture. The absorbance of the peak corresponding to the unbound tetrapeptide was obtained directly from the electropherogram and used in the calculation of the DNA-peptide binding constants. The binding constant for each tetrapeptide to calf thymus DNA was obtained from the negative slope of a Scatchard plot and a comparison of the binding constants for different peptides showed that the tetrapeptides in the library have DNA-binding affinities ranging from 10(2) to 10(6) M-1.

  14. A comprehensive approach to ascertain the binding mode of curcumin with DNA

    NASA Astrophysics Data System (ADS)

    Haris, P.; Mary, Varughese; Aparna, P.; Dileep, K. V.; Sudarsanakumar, C.

    2017-03-01

    Curcumin is a natural phytochemical from the rhizoma of Curcuma longa, the popular Indian spice that exhibits a wide range of pharmacological properties like antioxidant, anticancer, anti-inflammatory, antitumor, and antiviral activities. In the published literatures we can see different studies and arguments on the interaction of curcumin with DNA. The intercalative binding, groove binding and no binding of curcumin with DNA were reported. In this context, we conducted a detailed study to understand the mechanism of recognition of dimethylsulfoxide-solubilized curcumin by DNA. The interaction of curcumin with calf thymus DNA (ctDNA) was confirmed by agarose gel electrophoresis. The nature of binding and energetics of interaction were studied by Isothermal Titration Calorimetry (ITC), Differential Scanning Calorimetry (DSC), UV-visible, fluorescence and melting temperature (Tm) analysis. The experimental data were compared with molecular modeling studies. Our investigation confirmed that dimethylsulfoxide-solubilized curcumin binds in the minor groove of the ctDNA without causing significant structural alteration to the DNA.

  15. DNA-binding study of anticancer drug cytarabine by spectroscopic and molecular docking techniques.

    PubMed

    Shahabadi, Nahid; Falsafi, Monireh; Maghsudi, Maryam

    2017-01-02

    The interaction of anticancer drug cytarabine with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multispectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove-binding mode, while the binding constant of UV-vis and the number of binding sites were 4.0 ± 0.2 × 10(4) L mol(-1) and 1.39, respectively. The fluorimetric studies showed that the reaction between the drugs with CT-DNA is exothermic. Circular dichroism spectroscopy was employed to measure the conformational change of DNA in the presence of cytarabine. Furthermore, the drug induces detectable changes in its viscosity for DNA interaction. The molecular modeling results illustrated that cytarabine strongly binds to groove of DNA by relative binding energy of docked structure -20.61 KJ mol(-1). This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the interaction of small molecular pollutants and drugs with biomacromolecules for clarifying the molecular mechanism of toxicity or side effect in vivo.

  16. Multiple specific binding sites for purified glucocorticoid receptors on mammary tumor virus DNA.

    PubMed

    Payvar, F; Firestone, G L; Ross, S R; Chandler, V L; Wrange, O; Carlstedt-Duke, J; Gustafsson, J A; Yamamoto, K R

    1982-01-01

    Glucocorticoid hormones selectively stimulate the rate of transcription of integrated mammary tumor virus (MTV) sequences in infected rat hepatoma cells. Using two independent assays, we find that purified rat liver glucocorticoid receptor protein binds specifically to at least four widely separated regions on pure MTV proviral DNA. One of these specific binding domains, which itself contains at least two distinct receptor binding sites, resides within a fragment of viral DNA that maps 110-449 bp upstream of the promoter for MTV RNA synthesis. Three other binding domains lie downstream of the promoter and within the MTV primary transcription unit. Restriction fragments bearing separate binding domains have been introduced into cultured cells; transformants have been recovered in which the introduced fragments are expressed under glucocorticoid control. Thus, it appears that this assay will be useful for assessing the biological significance of the receptor binding sites detected in vitro.

  17. Electrostatic contributions to heat capacity changes of DNA-ligand binding.

    PubMed Central

    Gallagher, K; Sharp, K

    1998-01-01

    Significant heat capacity changes (DeltaCp) often accompany protein unfolding, protein binding, and specific DNA-ligand binding reactions. Such changes are widely used to analyze contributions arising from hydrophobic and polar hydration. Current models relate the magnitude of DeltaCp to the solvent accessible surface area (ASA) of the molecule. However, for many binding systems-particularly those involving non-peptide ligands-these models predict a DeltaCp that is significantly different from the experimentally measured value. Electrostatic interactions provide a potential source of heat capacity changes and do not scale with ASA. Using finite-difference Poisson-Boltzmann methods (FDPB), we have determined the contribution of electrostatics to the DeltaCp associated with binding for DNA binding reactions involving the ligands DAPI, netropsin, lexitropsin, and the lambda repressor binding domain. PMID:9675178

  18. DNA binding, DNA cleavage and cytotoxicity studies of a new water soluble copper(II) complex: The effect of ligand shape on the mode of binding

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

    The interaction of native calf thymus DNA (CT-DNA) with [Cu(ph 2phen)(phen-dione)Cl]Cl was studied at physiological pH by spectrophotometric, spectrofluorometric, circular dichroism, and viscometric techniques. Considerable hypochromicity and red shift are observed in the UV absorption band of the Cu complex. Binding constants ( Kb) of DNA with the complex were calculated at different temperatures. Thermodynamic parameters, enthalpy and entropy changes were calculated according to Van't Hoff equation, which indicated that reaction is predominantly enthalpically driven. All these results indicate that Cu(II) complex interacts with CT-DNA via intercalative mode. Also, this new complex induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) and human T lymphocyte carcinoma-Jurkat cell lines.

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

    DTIC Science & Technology

    2000-08-01

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

  20. Solution-state NMR Investigation of DNA Binding Interactions in Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg): A Dynamic Description of the DNA/Protein Interface

    SciTech Connect

    Buchko, Garry W.; McAteer, Kathleen; Wallace, Susan S.; Kennedy, Michael A.

    2005-03-02

    Formamidopyrimidine-DNA glycosylase (Fpg) is a base excision repair 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 and bound to a double-stranded DNA oligomer (13-PD) containing propanediol, a non-hydrolyzable abasic-site analogue. Only 209 out of a possible 252 (83%) free-precession HSQC cross peaks were observed and 180 of these were assignable, indicating that ~30% of the residues undergo intermediate timescale motion that makes them intractable in backbone assignment experiments. 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 time-scale motion observed for free Fpg. CPMG-HSQC experiments revealed millisecond to microsecond motion for the backbone amides of D91 and H92 that was quenched upon binding 13-PD. Collectively, these observations reveal that, in solution, Fpg contains highly flexible regions. The dynamic nature of Fpg, especially at the DNA binding surface, may be key to its processive search mechanism.

  1. Theory of site-specific DNA-protein interactions in the presence of conformational fluctuations of DNA binding domains.

    PubMed

    Murugan, R

    2010-07-21

    We develop a theory that explains how the thermally driven conformational fluctuations in the DNA binding domains (DBDs) of the DNA binding proteins (DBPs) are effectively coupled to the one-dimensional searching dynamics of DBPs for their cognate sites on DNA. We show that the rate gammaopt, associated with the flipping of conformational states of DBDs beyond which the maximum search efficiency of DBPs is achieved, varies with the one-dimensional sliding length L as gammaopt proportional, L(-2) and with the number of roadblock protein molecules present on the same DNA m as gammaopt proportional, m2. The required free energy barrier ERTO associated with this flipping transition seems to be varying with L as ERTO proportional, variant ln L2. When the barrier height associated with the conformational flipping of DBDs is comparable with that of the thermal free energy, then the possible value of L under in vivo conditions seems to be L

  2. Discovery of selective inhibitors of tyrosyl-DNA phosphodiesterase 2 by targeting the enzyme DNA-binding cleft.

    PubMed

    Kossmann, Bradley R; Abdelmalak, Monica; Lopez, Sophia; Tender, Gabrielle; Yan, Chunli; Pommier, Yves; Marchand, Christophe; Ivanov, Ivaylo

    2016-07-15

    Tyrosyl-DNA phosphodiesterase 2 (TDP2) processes protein/DNA adducts resulting from abortive DNA topoisomerase II (Top2) activity. TDP2 inhibition could provide synergism with the Top2 poison class of chemotherapeutics. By virtual screening of the NCI diversity small molecule database, we identified selective TDP2 inhibitors and experimentally verified their selective inhibitory activity. Three inhibitors exhibited low-micromolar IC50 values. Molecular dynamics simulations revealed a common binding mode for these inhibitors, involving association to the TDP2 DNA-binding cleft. MM-PBSA per-residue energy decomposition identified important interactions of the compounds with specific TDP2 residues. These interactions could provide new avenues for synthetic optimization of these scaffolds.

  3. Inhibition of DNA helicase, ATPase and DNA-binding activities of E. coli RecQ helicase by chemotherapeutic agents.

    PubMed

    Zhang, Bo; Zhang, Ai-hua; Chen, Lei; Xi, Xu Guang

    2008-06-01

    RecQ helicases play an essential role in maintaining genetic integrity in all organisms from Escherichia coli to humans. Defects to these enzymes are responsible for three distinct human diseases: Werner syndrome, Bloom syndrome and Rothmund-Thomson syndrome. All three diseases are characterized by a predisposition to cancer due to increased genomic instability. Previous studies on the effects of non-covalent DNA modifications on the catalytic activity of purified Werner and Bloom DNA helicases have shown that both enzymes have similar sensitivity profiles to these DNA-binding agents and are most strongly inhibited by the minor groove binder distamycin A. In this study, we show that the sensitivity profiles of E. coli RecQ to a number of DNA-binding ligands are different to those observed for WRN and Bloom helicases. These observations may give insights into the differences in molecular mechanisms underlying efficient motor function of RecQ helicases.

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

    NASA Astrophysics Data System (ADS)

    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.

  5. The Tip of the Tail Needle Affects the Rate of DNA Delivery by Bacteriophage P22

    PubMed Central

    Leavitt, Justin C.; Gogokhia, Lasha; Gilcrease, Eddie B.; Bhardwaj, Anshul; Cingolani, Gino; Casjens, Sherwood R.

    2013-01-01

    The P22-like bacteriophages have short tails. Their virions bind to their polysaccharide receptors through six trimeric tailspike proteins that surround the tail tip. These short tails also have a trimeric needle protein that extends beyond the tailspikes from the center of the tail tip, in a position that suggests that it should make first contact with the host’s outer membrane during the infection process. The base of the needle serves as a plug that keeps the DNA in the virion, but role of the needle during adsorption and DNA injection is not well understood. Among the P22-like phages are needle types with two completely different C-terminal distal tip domains. In the phage Sf6-type needle, unlike the other P22-type needle, the distal tip folds into a “knob” with a TNF-like fold, similar to the fiber knobs of bacteriophage PRD1 and Adenovirus. The phage HS1 knob is very similar to that of Sf6, and we report here its crystal structure which, like the Sf6 knob, contains three bound L-glutamate molecules. A chimeric P22 phage with a tail needle that contains the HS1 terminal knob efficiently infects the P22 host, Salmonella enterica, suggesting the knob does not confer host specificity. Likewise, mutations that should abrogate the binding of L-glutamate to the needle do not appear to affect virion function, but several different other genetic changes to the tip of the needle slow down potassium release from the host during infection. These findings suggest that the needle plays a role in phage P22 DNA delivery by controlling the kinetics of DNA ejection into the host. PMID:23951045

  6. A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells

    SciTech Connect

    Hirschfeld, S.; Levine, A.S.; Ozato, K.; Protic, M. )

    1990-05-01

    Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts.

  7. The genomic DNA immobilization on microcrystalline cellulose and its application to separate DNA-binding proteins from kumquat (Fortunella margarita Swingle).

    PubMed

    Yang, Hua; Li, Huang; Guo, Chun; Li, Meng-Yun; Rao, Li-Qun; Liu, Ting

    2014-01-01

    A method of immobilizing genomic DNA on microcrystalline cellulose was described to isolate DNA-binding proteins. At first, DNA fragments were prepared by sonication and immobilized on cellulose phase. After the immobilization, DNA duplex formation was done. Using this microcrystalline cellulose affinity chromatography technique, DNA-binding proteins from kumquat (Fortunella margarita Swingle) leaf samples were isolated and then analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS). LC-MS/MS analysis showed that twenty-eight kinds of protein mainly including histones, protein-synthetic proteins and other DNA-binding proteins were identified. The identification list consists with the results in previous research on DNA-binding proteins isolation. It further suggests that the technique developed in this study can be applied to the effective isolation of DNA-binding proteins.

  8. Mutation of serum response factor phosphorylation sites and the mechanism by which its DNA-binding activity is increased by casein kinase II.

    PubMed Central

    Manak, J R; Prywes, R

    1991-01-01

    Casein kinase II (CKII) phosphorylates the mammalian transcription factor serum response factor (SRF) on a serine residue(s) located within a region of the protein spanning amino acids 70 to 92, thereby enhancing its DNA-binding activity in vitro. We report here that serine 83 appears to be the residue phosphorylated by CKII but that three other serines in this region can also be involved in phosphorylation and the enhancement of DNA-binding activity. A mutant that contained glutamate residues in place of these serines had only low-level binding activity; however, when the serines were replaced with glutamates and further mutations were made that increased the negative charge of the region, the resulting mutant showed a constitutively high level of binding equal to that achieved by phosphorylation of wild-type SRF. We have investigated the mechanism by which phosphorylation of SRF increases its DNA-binding activity. We have ruled out the possibilities that phosphorylation affects SRF dimerization or relieves inhibition due to masking of the DNA-binding domain by an amino-terminal region of the protein. Rather, using partial proteolysis to probe SRF's structure, we find that the conformation of SRF's DNA-binding domain is altered by phosphorylation. Images PMID:2046671

  9. Calf thymus DNA-binding ability study of anthocyanins from purple sweet potatoes ( Ipomoea batatas L.).

    PubMed

    Wang, Dan; Wang, Xirui; Zhang, Chao; Ma, Yue; Zhao, Xiaoyan

    2011-07-13

    A total of 10 anthocyanin compounds were identified from five purple sweet potato ( Ipomoea batatas L.) varieties, Qunzi, Zishu038, Ji18, Jingshu6, and Ziluolan, by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to assess their calf thymus DNA-binding ability in vitro. The interaction between anthocyanins and calf thymus DNA in Tris-HCl buffer solution (pH 6.9) was evaluated by fluorescence spectroscopy. Using ethidium bromide (EB) as a fluorescence probe, fluorescence quenching of the emission peak was seen in the DNA-EB system when anthocyanins were added, indicating that the anthocyanins bound with DNA. The acylated groups influenced the ability of the interaction with DNA. Anthocyanins from purple sweet potato with more acylated groups in sorphorose have a stronger binding ability with DNA.

  10. Structural Basis for Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA

    SciTech Connect

    Mitchell, M.; Gillis, A; Futahashi, M; Fujiwara, H; Skordalakes, E

    2010-01-01

    Telomerase is a specialized DNA polymerase that extends the 3{prime} ends of eukaryotic linear chromosomes, a process required for genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA. The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA template and motifs 2 and B{prime} position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT-RNA template and TERT-telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases, suggesting common mechanistic aspects of DNA replication between the two families of enzymes.

  11. Applying DNA affinity chromatography to specifically screen for sucrose-related DNA-binding transcriptional regulators of Xanthomonas campestris.

    PubMed

    Leßmeier, Lennart; Alkhateeb, Rabeaa S; Schulte, Fabian; Steffens, Tim; Loka, Tobias Pascal; Pühler, Alfred; Niehaus, Karsten; Vorhölter, Frank-Jörg

    2016-08-20

    At a molecular level, the regulation of many important cellular processes is still obscure in xanthomonads, a bacterial group of outstanding relevance as world-wide plant pathogens and important for biotechnology as producers of the polysaccharide xanthan. Transcriptome analysis indicated a sucrose-dependent regulation of 18 genes in Xanthomonas campestris pv. campestris (Xcc) B100. The expression of 12 of these genes was clearly increased in the presence of sucrose. Only part of these genes was obviously involved in sucrose utilization. To identify regulatory proteins involved in transcriptional regulation, a DNA fragment-specific pull-down approach was established for Xcc. Putative promoter regions were identified and used to isolate DNA-binding proteins, which were separated by SDS PAGE and identified by MALDI-TOF mass spectrometry. This led to the identification of four transcriptional regulators, among them the global transcriptional regulator Clp and a previously identified regulator of sucrose utilization, SuxR, plus a third DNA-binding transcriptional regulator encoded by xcc-b100_2861 and recently shown to interact with a cyclic di-GMP-binding protein. The fourth regulatory protein was encoded by xcc-b100_2791. These results indicate DNA fragment-specific pull-down experiments as promising approaches to screen for specific DNA-binding regulatory proteins in Xcc.

  12. Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

    PubMed Central

    Foster, David A.; Hantzopoulos, Petros; Zubay, Geoffrey

    1982-01-01

    Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion. Images PMID:6809958

  13. The N-terminal domain of DnaT, a primosomal DNA replication protein, is crucial for PriB binding and self-trimerization.

    PubMed

    Huang, Yen-Hua; Huang, Cheng-Yang

    2013-12-13

    DnaT and PriB are replication restart primosomal proteins required for re-initiating chromosomal DNA replication in bacteria. Although the interaction of DnaT with PriB has been proposed, which region of DnaT is involved in PriB binding and self-trimerization remains unknown. In this study, we identified the N-terminal domain in DnaT (aa 1-83) that is important in PriB binding and self-trimerization but not in single-stranded DNA (ssDNA) binding. DnaT and the deletion mutant DnaT42-179 protein can bind to PriB according to native polyacrylamide gel electrophoresis, Western blot analysis, and pull-down assay, whereas DnaT84-179 cannot bind to PriB. In contrast to DnaT, DnaT26-179, and DnaT42-179 proteins, which form distinct complexes with ssDNA of different lengths, DnaT84-179 forms only a single complex with ssDNA. Analysis of DnaT84-179 protein by gel filtration chromatography showed a stable monomer in solution rather than a trimer, such as DnaT, DnaT26-179, and DnaT42-179 proteins. These results constitute a pioneering study of the domain definition of DnaT. Further research can directly focus on determining how DnaT binds to the PriA-PriB-DNA tricomplex in replication restart by the hand-off mechanism.

  14. The structural basis for recognition of base J containing DNA by a novel DNA binding domain in JBP1.

    PubMed

    Heidebrecht, Tatjana; Christodoulou, Evangelos; Chalmers, Michael J; Jan, Sabrina; Ter Riet, Bas; Grover, Rajesh K; Joosten, Robbie P; Littler, Dene; van Luenen, Henri; Griffin, Patrick R; Wentworth, Paul; Borst, Piet; Perrakis, Anastassis

    2011-07-01

    The J-binding protein 1 (JBP1) is essential for biosynthesis and maintenance of DNA base-J (β-d-glucosyl-hydroxymethyluracil). Base-J and JBP1 are confined to some pathogenic protozoa and are absent from higher eukaryotes, prokaryotes and viruses. We show that JBP1 recognizes J-containing DNA (J-DNA) through a 160-residue domain, DB-JBP1, with 10 000-fold preference over normal DNA. The crystal structure of DB-JBP1 revealed a helix-turn-helix variant fold, a 'helical bouquet' with a 'ribbon' helix encompassing the amino acids responsible for DNA binding. Mutation of a single residue (Asp525) in the ribbon helix abrogates specificity toward J-DNA. The same mutation renders JBP1 unable to rescue the targeted deletion of endogenous JBP1 genes in Leishmania and changes its distribution in the nucleus. Based on mutational analysis and hydrogen/deuterium-exchange mass-spectrometry data, a model of JBP1 bound to J-DNA was constructed and validated by small-angle X-ray scattering data. Our results open new possibilities for targeted prevention of J-DNA recognition as a therapeutic intervention for parasitic diseases.

  15. DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

    PubMed Central

    John, M; Leppik, R; Busch, S J; Granger-Schnarr, M; Schnarr, M

    1996-01-01

    We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures. PMID:8948639

  16. GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

    PubMed

    He, Ximiao; Syed, Khund Sayeed; Tillo, Desiree; Mann, Ishminder; Weirauch, Matthew T; Vinson, Charles

    2015-07-16

    To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABPα and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS ((C)/GCGGAA GT: ) and CRE ( GT: GACGTCAC) motifs overlap precisely, producing the ETS↔CRE motif ((C)/GCGGAA GT: GACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABPα and CREB1 upon binding the ETS↔CRE motif. The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes. CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding. We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding.

  17. Switch recombination breakpoints are strictly correlated with DNA recognition motifs for immunoglobulin S gamma 3 DNA-binding proteins

    PubMed Central

    1992-01-01

    The deletion looping out model of switch (S) recombination predicts that the intervening DNA between switch regions will be excised as a circle. Circular excision products of immunoglobulin switch recombination have been recently isolated from lipopolysaccharide (LPS)- stimulated spleen cells. The recombination breakpoints in these large circles were found to fall within switch regions. Since switch recombination is clearly focused on switch regions, we hypothesized that some DNA-binding protein factor might be involved in specifically recognizing and facilitating the alignment of switch regions before recombination. Two DNA-binding proteins that specifically interact with two discrete regions of the S gamma 3 tandem repeat have been identified in crude and partially purified nuclear extracts derived from LPS- and dextran sulfate (DxS)-activated splenic B cells. The first factor has been found indistinguishable from NF-kappa B by mobility shift assays, methylation interference, competition binding studies, and supershift analysis using an antiserum specific for the p50 component. The second appears to be composed of two closely traveling mobilities that do not separate upon partial purification. This second complex is unique and specific for S gamma 3 by methylation interference assays and competition-binding analysis. The sites at which recombination occurs in the S gamma 3 switch region have been analyzed and found to strictly correlate with the binding sites of the S gamma 3 switch binding proteins. PMID:1500850

  18. The DNA binding properties of the Escherichia coli RecQ helicase.

    PubMed

    Dou, Shuo-Xing; Wang, Peng-Ye; Xu, Hou Qiang; Xi, Xu Guang

    2004-02-20

    The RecQ helicase family is highly conserved from bacteria to men and plays a conserved role in the preservation of genome integrity. Its deficiency in human cells leads to a marked genomic instability that is associated with premature aging and cancer. To determine the thermodynamic parameters for the interaction of Escherichia coli RecQ helicase with DNA, equilibrium binding studies have been performed using the thermodynamic rigorous fluorescence titration technique. Steady-state fluorescence anisotropy measurements of fluorescein-labeled oligonucleotides revealed that RecQ helicase bound to DNA with an apparent binding stoichiometry of 1 protein monomer/10 nucleotides. This stoichiometry was not altered in the presence of AMPPNP (adenosine 5'-(beta,gamma-imido) triphosphate) or ADP. Analyses of RecQ helicase interactions with oligonucleotides of different lengths over a wide range of pH, NaCl, and nucleic acid concentrations indicate that the RecQ helicase has a single strong DNA binding site with an association constant at 25 degrees C of K=6.7 +/- 0.95 x 10(6) M(-1) and a cooperativity parameter of omega=25.5 +/- 1.2. Both single-stranded DNA and double-stranded DNA bind competitively to the same site. The intrinsic affinities are salt-dependent, and the formation of DNA-helicase complex is accompanied by a net release of 3-4 ions. Allosteric effects of nucleotide cofactors on RecQ binding to DNA were observed only for single-stranded DNA in the presence of 1.5 mM AMPPNP, whereas both AMPPNP and ADP had no detectable effect on double-stranded DNA binding over a large range of nucleotide cofactor concentrations.

  19. The binding mode of human nucleoside diphosphate kinase B to single-strand DNA.

    PubMed

    Agou, F; Raveh, S; Véron, M

    2000-06-01

    In this paper, we studied the interaction of the human isoform B of nucleoside diphosphate kinase (NDP kinase B) with the nuclease hypersensitive element (NHE) present in the promoter element of the c-myc oncogene. The DNA-binding properties of NDP kinase B and other NDP kinases are compared and the nucleotide requirement for binding are discussed. Using quantitative methods, we identified the DNA-binding sites on the protein and we proposed a structural model for a complex of one hexameric NDP kinase B with an oligonucleotide.

  20. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases.

    PubMed

    Manthei, Kelly A; Hill, Morgan C; Burke, Jordan E; Butcher, Samuel E; Keck, James L

    2015-04-07

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.

  1. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

    PubMed Central

    Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; Butcher, Samuel E.; Keck, James L.

    2015-01-01

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3′ single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures. PMID:25831501

  2. Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

    SciTech Connect

    Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; Butcher, Samuel E.; Keck, James L.

    2015-03-23

    RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. In this paper, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ~90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. Finally, this bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.

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

    PubMed

    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.

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

    PubMed

    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.

  5. Binding Potency of Heparin Immobilized on Activated Charcoal for DNA Antibodies.

    PubMed

    Snezhkova, E A; Tridon, A; Evrard, B; Nikolaev, V G; Uvarov, V Yu; Tsimbalyuk, R S; Ivanuk, A A; Komov, V V; Sakhno, L A

    2016-02-01

    In vitro experiments showed that heparin adsorbed on activated charcoal can bind antibodies raised against native and single-stranded DNA in a diluted sera pool with a high level of these DNA. Thus, heparin used as anticoagulant during hemosorption procedure can demonstrate supplementary therapeutic activity resulting from its interaction with various agents involved in acute and chronic inflammatory reactions such as DNA- and RNA-binding substances, proinflammatory cytokines, complement components, growth factors, etc. Research and development of heparin-containing carbonic adsorbents for the therapy of numerous inflammatory and autoimmune diseases seems to be a promising avenue in hematology.

  6. The p53 Protein is an Unusually Shaped Tetramer that Binds Directly to DNA

    NASA Astrophysics Data System (ADS)

    Friedman, Paula N.; Chen, Xinbin; Bargonetti, Jill; Prives, Carol

    1993-04-01

    We have analyzed the size and structure of native immunopurified human p53 protein. By using a combination of chemical crosslinking, gel filtration chromatography, and zonal velocity gradient centrifugation, we have determined that the predominant form of p53 in such preparations is a tetramer. The behavior of purified p53 in gels and sucrose gradients implies that the protein has an extended shape. Wild-type p53 has been shown to bind specifically to sites in cellular and viral DNA. We show in this study by Southwestern ligand blotting and by analysis of DNA-bound crosslinked p53 that p53 monomers, dimers, and tetramers can bind directly to DNA.

  7. Isolation and characterization of the DNA-binding protein (DBP) of the Autographa californica multiple nucleopolyhedrovirus

    SciTech Connect

    Mikhailov, Victor S. Vanarsdall, Adam L.; Rohrmann, George F.

    2008-01-20

    DNA-binding protein (DBP) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was expressed as an N-terminal His{sub 6}-tag fusion using a recombinant baculovirus and purified to near homogeneity. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein, LEF-3, for binding sites. DBP binding protected ssDNA against hydrolysis by a baculovirus alkaline nuclease AN/LEF-3 complex. Partial proteolysis by trypsin revealed a domain structure of DBP that is required for interaction with DNA and that can be disrupted by thermal treatment. Binding to ssDNA, but not to dsDNA, changed the pattern of proteolytic fragments of DBP indicating adjustments in protein structure upon interaction with ssDNA. DBP was capable of unwinding short DNA duplexes and also promoted the renaturation of long complementary strands of ssDNA into duplexes. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide. A high affinity of DBP for ssDNA and its unwinding and renaturation activities confirmed identification of DBP as a member of the SSB/recombinase family. These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates.

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

    PubMed

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

    2012-08-01

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

  9. A Small Molecule Inhibitor of Pot1 Binding to Telomeric DNA

    PubMed Central

    Altschuler, Sarah E.; Croy, Johnny E.; Wuttke, Deborah S.

    2012-01-01

    Chromosome ends are complex structures, consisting of repetitive DNA sequence terminating in an ssDNA overhang with many associated proteins. Because alteration of these ends is a hallmark of cancer, telomeres and telomere maintenance have been prime drug targets. The universally conserved ssDNA overhang is sequence-specifically bound and regulated by Pot1 (protection of telomeres), and perturbation of Pot1 function has deleterious effects for proliferating cells. The specificity of the Pot1/ssDNA interaction and the key involvement of this protein in telomere maintenance have suggested directed inhibition of Pot1/ssDNA binding as an efficient means of disrupting telomere function. To explore this idea, we developed a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) screen for inhibitors of Pot1/ssDNA interaction. We conducted this screen with the DNA-binding subdomain of S. pombe Pot1 (Pot1pN), which confers the vast majority of Pot1 sequence-specificity and is highly similar to the first domain of human Pot1 (hPOT1). Screening a library of ~20,000 compounds yielded a single inhibitor, which we found interacted tightly with submicromolar affinity. Furthermore, this compound, subsequently identified as the bis-azo dye Congo red, was able to competitively inhibit hPOT1 binding to telomeric DNA. ITC and NMR chemical shift analysis suggest that CR interacts specifically with the ssDNA-binding cleft of Pot1, and that alteration of this surface disrupts CR binding. The identification of a specific inhibitor of ssDNA interaction establishes a new pathway for targeted telomere disruption. PMID:22978652

  10. Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1

    PubMed Central

    Miroshnikova, A. D.; Kuznetsova, A. A.; Kuznetsov, N. A.; Fedorova, O. S.

    2016-01-01

    Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5’-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1’ hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of “crystalline” water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5’-phosphate-2’-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step. PMID:27099790

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

    NASA Astrophysics Data System (ADS)

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

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

  12. Structure and DNA binding of alkylation response protein AidB

    SciTech Connect

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

    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.

  13. Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

    PubMed Central

    Ng, R; Carbon, J

    1987-01-01

    Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo. Images PMID:2830498

  14. Cloning and characterization of seven human forkhead proteins: binding site specificity and DNA bending.

    PubMed Central

    Pierrou, S; Hellqvist, M; Samuelsson, L; Enerbäck, S; Carlsson, P

    1994-01-01

    The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins. We have used PCR and low stringency hybridization to isolate clones from human cDNA and genomic libraries that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from specific for a single tissue to nearly ubiquitous. The DNA binding specificities of four of the FREAC proteins were determined by selection of binding sites from random sequence oligonucleotides. The binding sites for all four FREAC proteins share a core sequence, RTAAAYA, but differ in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as responsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins to their cognate sites results in bending of the DNA at an angle of 80-90 degrees. Images PMID:7957066

  15. Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2.

    PubMed

    Berntsson, Ronnie P-A; Odegrip, Richard; Sehlén, Wilhelmina; Skaar, Karin; Svensson, Linda M; Massad, Tariq; Högbom, Martin; Haggård-Ljungquist, Elisabeth; Stenmark, Pål

    2014-02-01

    The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 Å resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

  16. Mutually Exclusive Binding of Telomerase RNA and DNA by Ku Alters Telomerase Recruitment Model

    PubMed Central

    Pfingsten, Jennifer S.; Goodrich, Karen J.; Taabazuing, Cornelius; Ouenzar, Faissal; Chartrand, Pascal; Cech, Thomas R.

    2012-01-01

    SUMMARY In Saccharomyces cerevisiae, the Ku heterodimer contributes to telomere maintenance as a component of telomeric chromatin and as an accessory subunit of telomerase. How Ku binding to double-stranded DNA (dsDNA) and to telomerase RNA (TLC1) promotes its telomeric functions is incompletely understood. We demonstrate that deletions designed to constrict the DNA-binding ring of Ku80 disrupt non-homologous end-joining (NHEJ), telomeric gene silencing and telomere length maintenance, suggesting that these functions require Ku's DNA end-binding activity. Contrary to the current model, a mutant Ku with low affinity for dsDNA also loses affinity for TLC1 both in vitro and in vivo. Competition experiments reveal that wild-type Ku binds dsDNA and TLC1 mutually exclusively. Cells expressing the mutant Ku are deficient in nuclear accumulation of TLC1, as expected from the RNA-binding defect. These findings force reconsideration of the mechanisms by which Ku assists in recruiting telomerase to natural telomeres and broken chromosome ends. PMID:22365814

  17. Quantifying the DNA binding characteristics of ruthenium based threading intercalator Λ Λ -P with optical tweezers

    NASA Astrophysics Data System (ADS)

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

    Utilizing optical tweezers, biophysics researchers have been able to study drug-DNA interactions on the single molecule level. Binuclear ruthenium complexes are a particular type of drug molecule that have been found to have potential cancer-fighting qualities, due to their high binding affinity and low dissociation rates. These complexes are threading intercalators, meaning that they must thread their bulky side chains through DNA base pairs to allow the central planar moiety to intercalate between the bases. In this study, we explored the binding properties of the binuclear ruthenium complex, ΛΛ -P (ΛΛ -[µ-bidppz(phen)4Ru2]4+) . A single DNA molecule is held at a constant force and the ΛΛ -P solution introduced to the system in varying concentrations until equilibrium is reached. DNA extension data at various concentrations of ΛΛ -P recorded as a function of time provide the DNA binding kinetics and equilibrium binding affinity. Preliminary data analysis suggests that ΛΛ -P exhibits fast binding kinetics compared to the very similar ΔΔ -P. These complexes have the same chemical structure and only differ in their chirality, which suggests that the left handed (ΛΛ) threading moieties require less DNA structural distortion for threading compared with the right handed (ΔΔ) threading moieties.

  18. Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

    PubMed

    Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

    2014-11-01

    DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

  19. Energy coupling between DNA binding and subunit association is responsible for the specificity of DNA-Arc interaction.

    PubMed Central

    Silva, J. L.; Silveira, C. F.

    1993-01-01

    The effects of several DNA molecules on the free energy of subunit association of Arc repressor were measured. The association studies under equilibrium conditions were performed by the dissociating perturbation of hydrostatic pressure. The magnitude of stabilization of the subunit interaction was determined by the specificity of the protein-DNA interaction. Operator DNA stabilized the free energy of association by about 2.2 kcal/mol of monomeric unit, whereas poly(dG-dC) stabilized the subunit interaction by only 0.26 kcal. Measurements of the stabilizing free energy at different DNA concentrations revealed a stoichiometry of two dimers per 21 bp for the operator DNA sequence and for the nonspecific DNA poly(dA-dT). However, the maximum stabilization was much larger for operator sequence (delta p = 1,750 bar) as compared for poly(dA-dT) (delta p = 750 bar). The importance of the free-energy linkage for the recognition process was corroborated by its absence in a mutant Arc protein (PL8) that binds to operator and nonspecific DNA sequences with equal, low affinity. We conclude that the coupling accounts for the high specificity of the Arc-operator DNA interaction. We hypothesize a mutual coupling between the protein subunits and the two DNA strands, in which the much higher persistency of the associated form when Arc is bound to operator would stabilize the interactions between the two DNA strands. PMID:8318899

  20. An RNA aptamer that interferes with the DNA binding of the HSF transcription activator.

    PubMed

    Zhao, Xiaoching; Shi, Hua; Sevilimedu, Aarti; Liachko, Nicole; Nelson, Hillary C M; Lis, John T

    2006-01-01

    Heat shock factor (HSF) is a conserved and highly potent transcription activator. It is involved in a wide variety of important biological processes including the stress response and specific steps in normal development. Reagents that interfere with HSF function would be useful for both basic studies and practical applications. We selected an RNA aptamer that binds to HSF with high specificity. Deletion analysis defined the minimal binding motif of this aptamer to be two stems and one stem-loop joined by a three-way junction. This RNA aptamer interferes with normal interaction of HSF with its DNA element, which is a key regulatory step for HSF function. The DNA-binding domain plus a flanking linker region on the HSF (DL) is essential for the RNA binding. Additionally, this aptamer inhibits HSF-induced transcription in vitro in the complex milieu of a whole cell extract. In contrast to the previously characterized NF-kappaB aptamer, the HSF aptamer does not simply mimic DNA binding, but rather binds to HSF in a manner distinct from DNA binding to HSF.

  1. Lack of in vivo DNA binding of mercaptobenzothiazole to selected tissues of the rat

    SciTech Connect

    Brewster, D.W.; Mirly, K.J.; Wilson, A.G.; Barnett, J.W. Jr. )

    1989-11-30

    In this study, the in vivo binding of {sup 14}C-labelled 2-mercaptobenzothiazole (MBT) to DNA was investigated. Male and female Fischer 344 rats were gavaged with 375 mg MBT/kg body weight and killed 8 hours later. DNA was extracted from the liver, adrenal glands, pituitary gland, pancreas, and bone marrow and the amount of radioactivity associated with the DNA was determined. Results from this study indicate that MBT does not significantly bind to DNA from any of the tissues examined. CBI values for liver for the 3 methods of purification were -1-3 which are on the low end of the covalent binding index. The CBI values for the other tissues were always less than 1. Other chemicals with similar CBI values include estrone and diethylstilbesterol. Strong hepatocarcinogens such as dimethylnitrosamine and aflatoxin have CBI values ranging from 6000 to greater than 20000.

  2. Quantitative Assessment of the Interplay Between DNA Elasticity and Cooperative Binding of Ligands

    NASA Astrophysics Data System (ADS)

    Siman, L.; Carrasco, I. S. S.; da Silva, J. K. L.; de Oliveira, M. C.; Rocha, M. S.; Mesquita, O. N.

    2012-12-01

    Binding of ligands to DNA can be studied by measuring the change of the persistence length of the complex formed, in single-molecule assays. We propose a methodology for persistence length data analysis based on a quenched disorder statistical model and describing the binding isotherm by a Hill-type equation. We obtain an expression for the effective persistence length as a function of the total ligand concentration, which we apply to our data of the DNA-cationic β-cyclodextrin and to the DNA-HU protein data available in the literature, determining the values of the local persistence lengths, the dissociation constant, and the degree of cooperativity for each set of data. In both cases the persistence length behaves nonmonotonically as a function of ligand concentration and based on the results obtained we discuss some physical aspects of the interplay between DNA elasticity and cooperative binding of ligands.

  3. Calf thymus DNA binding studies of the new neodymium-naproxen complex.

    PubMed

    Huo, Ruina; Xu, Guiqing; Jiang, Xiaoying; Ge, Yao; Xue, Zaikun; Cui, Fengling

    2012-05-01

    Fluorescence spectroscopy in combination with UV absorption spectroscopy was carried out to investigate the interaction between the neodymium-naproxen complex (Nd-NAP) and calf thymus DNA (ctDNA). The experimental results showed that Nd-NAP intercalated with the ctDNA base pairs. Analysis of fluorescence quenching data of Nd-NAP by ctDNA at different temperatures using a Stern-Volmer equation revealed that dynamic and static quenching occurred simultaneously. The binding constants and the number of binding sites at 293 and 310 K were obtained as 2.904 × 10(4) L mol(-1), 1.172 and 2.432 × 10(4) L mol(-1), 1.143, respectively. The thermodynamic parameters ΔG, ΔH, and ΔS calculated at different temperatures indicated that hydrogen bonding and van der Waals force were the main binding forces.

  4. Predictive binding geometry of ligands to DNA minor groove: isohelicity and hydrogen-bonding pattern.

    PubMed

    Stockert, Juan C

    2014-01-01

    The interaction of drugs and dyes with nucleic acids, particularly when binding to DNA minor groove occurs, has increasing importance in biomedical sciences. This is due to the resulting biological activity and to the possibility of recognizing AT and GC base pairs. In such cases, DNA binding can be predicted if appropriate helical and hydrogen-bonding parameters are deduced from DNA models, and a simplified geometrical rule in the form of a stencil is then applied on computer-drawn molecules of interest. Relevant structure parameter values for minor groove binders are the length (4.6 < L < 5.4 Å) and angle (152 < σ < 156.5°) between three consecutive units, measured at the level of hydrogen donor or acceptor groups. Application of the stencil shows that predictive methods can aid in the design of new compounds, by checking the possible binding of isohelical sequence-specific ligands along the DNA minor groove.

  5. Effect of pH on the Structure and DNA Binding of the FOXP2 Forkhead Domain.

    PubMed

    Blane, Ashleigh; Fanucchi, Sylvia

    2015-06-30

    Forkhead box P2 (FOXP2) is a transcription factor expressed in cardiovascular, intestinal, and neural tissues during embryonic development and is implicated in language development. FOXP2 like other FOX proteins contains a DNA binding domain known as the forkhead domain (FHD). The FHD interacts with DNA by inserting helix 3 into the major groove. One of these DNA-protein interactions is a direct hydrogen bond that is formed with His554. FOXP2 is localized in the nuclear compartment that has a pH of 7.5. Histidine contains an imidazole side chain in which the amino group typically has a pKa of ~6.5. It seems possible that pH fluctuations around 6.5 may result in changes in the protonation state of His554 and thus the ability of the FOXP2 FHD to bind DNA. To investigate the effect of pH on the FHD, both the structure and the binding affinity were studied in the pH range of 5-9. This was done in the presence and absence of DNA. The structure was assessed using size exclusion chromatography, far-UV circular dichroism, and intrinsic and extrinsic fluorescence. The results indicated that while pH did not affect the secondary structure in the presence or absence of DNA, the tertiary structure was pH sensitive and the protein was less compact at low pH. Furthermore, the presence of DNA caused the protein to become more compact at low pH and also had the potential to increase the dimerization propensity. Fluorescence anisotropy was used to investigate the effect of pH on the FOXP2 FHD DNA binding affinity. It was found that pH had a direct effect on binding affinity. This was attributed to the altered hydrogen bonding patterns upon protonation or deprotonation of His554. These results could implicate pH as a means of regulating transcription by the FOXP2 FHD, which may also have repercussions for the behavior of this protein in cancer cells.

  6. [Expression and purification of FOXO1 DNA binding domain and its DNA properties].

    PubMed

    Ha, Yinuer; Li, Jun; Chen, Yongheng; Chen, Lin; Chen, Zhuchu

    2017-01-28

    目的:探讨翼螺旋转录因子FOXO1的DNA结合域(FOXO1 DNA binding domain,FOXO1-DBD)的表达、纯化及与DNA的结合特性。方法:采用优化FOXO1-DNA的基因序列和低温诱导的方式实现FOXO1-DBD蛋白的可溶性表达,通过镍亲和层析及阳离子交换层析进行纯化,并经凝胶迁移实验(electrophoretic mobility shift assay,EMSA)验证FOXO1-D