WorldWideScience

Sample records for b3 dna binding

  1. Evolution of the B3 DNA binding superfamily: new insights into REM family gene diversification.

    Directory of Open Access Journals (Sweden)

    Elisson A C Romanel

    Full Text Available BACKGROUND: The B3 DNA binding domain includes five families: auxin response factor (ARF, abscisic acid-insensitive3 (ABI3, high level expression of sugar inducible (HSI, related to ABI3/VP1 (RAV and reproductive meristem (REM. The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily. METHODOLOGY: In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family. CONCLUSIONS: Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.

  2. ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Fumin; Telesco, Shannon E.; Liu, Yingting; Radhakrishnan, Ravi; Lemmon, Mark A. (UPENN); (UPENN-MED)

    2010-06-21

    ErbB3/HER3 is one of four members of the human epidermal growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase family. ErbB3 binds neuregulins via its extracellular region and signals primarily by heterodimerizing with ErbB2/HER2/Neu. A recently appreciated role for ErbB3 in resistance of tumor cells to EGFR/ErbB2-targeted therapeutics has made it a focus of attention. However, efforts to inactivate ErbB3 therapeutically in parallel with other ErbB receptors are challenging because its intracellular kinase domain is thought to be an inactive pseudokinase that lacks several key conserved (and catalytically important) residues - including the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular region - although it is substantially less active than EGFR and does not phosphorylate exogenous peptides. The ErbB3 kinase domain binds ATP with a K{sub d} of approximately 1.1 {micro}M. We describe a crystal structure of ErbB3 kinase bound to an ATP analogue, which resembles the inactive EGFR and ErbB4 kinase domains (but with a shortened {alpha}C-helix). Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely inactivates ErbB3. Using quantum mechanics/molecular mechanics simulations, we delineate a reaction pathway for ErbB3-catalyzed phosphoryl transfer that does not require the conserved catalytic base and can be catalyzed by the 'inactive-like'configuration observed crystallographically. These findings suggest that ErbB3 kinase activity within receptor dimers may be crucial for signaling and could represent an important therapeutic target.

  3. DNA binding hydroxyl radical probes

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Vicky J.; Konigsfeld, Katie M.; Aguilera, Joe A. [Department of Radiology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0610 (United States); Milligan, Jamie R., E-mail: jmilligan@ucsd.edu [Department of Radiology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0610 (United States)

    2012-01-15

    The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores, which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA. - Highlights: > Examined four aromatic groups as a means to detect hydroxyl radicals by fluorescence. > Coumarin system suffers from the fewest disadvantages. > Characterized its reactivity when linked to a hexa-arginine peptide.

  4. The helical structure of DNA facilitates binding

    Science.gov (United States)

    Berg, Otto G.; Mahmutovic, Anel; Marklund, Emil; Elf, Johan

    2016-09-01

    The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

  5. Opposing roles of the two isoforms of ErbB3 binding protein 1 in human cancer cells.

    Science.gov (United States)

    Ko, Hyo Rim; Chang, Yun Sil; Park, Won Soon; Ahn, Jee-Yin

    2016-09-15

    The different functions of the two isoforms of ErbB3 binding protein 1 (Ebp1), p48 and p42, have recently become the focus of interest as they reveal contradictory roles in cell growth promoting ability. The conformational change that crystal structure of p42 was shown to lack α helices at the amino-terminus present in p48 represents the differential binding partners and protein modifications of two Ebp1 isoforms. N-terminal specific phosphorylation by CDK2 and deregulation of the p53 tumor suppressor through specific interaction with HDM2 and Akt activation is postulated to contribute to p48-mediated tumorigenesis. The short isoform p42 Ebp1, which is actual binding partner of ErbB3 has been implicated as a tumor suppressor with many binding partners such as Rb, HDAC2, Sin3A and the p85 subunit of PI3K with HSP70/CHIP, inhibiting its own antiproliferative activity or inhibiting PI3K activity. The aim of the current review is to provide a summary on distinctive cellular functions of two Ebp1 proteins and their molecular partners that might be responsible for the unique functions of each isoform of Ebp1. PMID:27130196

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

    OpenAIRE

    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 hig...

  7. DNA-Aptamers Binding Aminoglycoside Antibiotics

    OpenAIRE

    Nadia Nikolaus; Beate Strehlitz

    2014-01-01

    Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminog...

  8. DNA Triplexes That Bind Several Cofactor Molecules.

    Science.gov (United States)

    Vollmer, Sven; Richert, Clemens

    2015-12-14

    Cofactors are critical for energy-consuming processes in the cell. Harnessing such processes for practical applications requires control over the concentration of cofactors. We have recently shown that DNA triplex motifs with a designed binding site can be used to capture and release nucleotides with low micromolar dissociation constants. In order to increase the storage capacity of such triplex motifs, we have explored the limits of ligand binding through designed cavities in the oligopurine tract. Oligonucleotides with up to six non-nucleotide bridges between purines were synthesized and their ability to bind ATP, cAMP or FAD was measured. Triplex motifs with several single-nucleotide binding sites were found to bind purines more tightly than triplexes with one large binding site. The optimized triplex consists of 59 residues and four C3-bridges. It can bind up to four equivalents of ligand with apparent Kd values of 52 µM for ATP, 9 µM for FAD, and 2 µM for cAMP. An immobilized version fuels bioluminescence via release of ATP at body temperature. These results show that motifs for high-density capture, storage and release of energy-rich biomolecules can be constructed from synthetic DNA. PMID:26561335

  9. MiR-291b-3p Induces Apoptosis in Liver Cell Line NCTC1469 by Reducing the Level of RNA-binding Protein HuR

    Directory of Open Access Journals (Sweden)

    Jun Guo

    2014-03-01

    Full Text Available Background: There is increasing evidence that miRNAs are involved in cellular apoptosis. However, the specific role of miR-291b-3p in apoptosis has not been elucidated. In the present study, we investigated the effect of miR-291b-3p on NCTC1469 cell growth and apoptosis. Methods: Cell viability and apoptosis were examined in NCTC1469 cells transfected with miR-291b-3p mimics, inhibitor miRNA or negative control. Using computational miRNA target prediction databases, HuR was predicted as a target of miR-291b-3p. Luciferase assay, immunofluorescence and western blot were used to further explore the effects of miR-291b-3p on HuR expression. In addition, the effect of HuR on cell apoptosis was evaluated using a HuR-specific siRNA. Results: TNF-α-induced hepatocyte apoptosis was accompanied by enhanced expression of miR-291b-3p, suggesting that miR-291b-3p might contribute to the apoptotic process. Follow-up experiments showed that upregulation of miR-291b-3p decreased cell viability and induced NCTC1469 cell apoptosis. Additionally, similar to the activity of miR-519, which is another member of the same miRNA family, miR-291b-3p suppressed HuR translation through binding to the HuR coding region (CR. We further showed that the downregulation of HuR expression by miR-291b-3p was accompanied by reduced Bcl-2 expression. Moreover, knockdown of HuR also impaired Bcl-2 expression and increased the ratio of Bax/Bcl-2. More significantly, downregulation of miR-291b-3p failed to increase Bcl2 expression in NCTC1469 cells that were co-transfected with siRNA-HuR. Finally, inhibition of miR-291b-3p led to reduced apoptosis, while knockdown of HuR by siRNA promoted apoptosis, even in NCTC1469 cells that were co-transfected with the miR-291b-3p inhibitor. Conclusion: The current data suggested that miR-291b-3p contributed to NCTC1469 cell apoptosis by regulating the expression of HuR, which in turn increased Bcl-2 stability.

  10. Quantitative radiommunoassay for DNA-binding antibodies

    International Nuclear Information System (INIS)

    A radioimmunoassay (RIA) is described for the measurement of serum immunoglobulins capable of binding to double-standard or single-standard DNA. DNA attached to Sephadex G-50 by ultraviolet radiation was used as a solid- phase immunoabsorbent for DNA-binding proteins from serum. Goat anti-human (GAH) IgG (125I-labeled) were used to detect the human immunoglobulins bound onto the washed DNA-Sephadex. The quantities of immunoglobulins bound were determined by comparison with a standard curve constructed by dilution of a plasma from an systemic lupus erythematosus (SLE) patient containing known amounts of bound, DNA-specific IgM and IgG. Another RIA was employed for measuring levels of IgG and IgM. In combination with measurements of the total serum IgM and IgG, the RIA allowed for the determination of the fraction of the total serum IgM or IgG that was specific for double- or single-standard DNA. For a pool of normal human sera the quantities were as follows: 0.04% of the total IgM and 0.001% of the total IgG bound double-standard DNA; 0.22% of the total IgM and 0.05% of the total IgG bound single-stranded DNA. This capability is important because information regarding the quantitative measurement of antibodies to DNA and their class determination may be of significance in monitoring the status of subjects with SLE

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

    Directory of Open Access Journals (Sweden)

    Nancy A Stearns

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

  12. DNA-Aptamers Binding Aminoglycoside Antibiotics

    Directory of Open Access Journals (Sweden)

    Nadia Nikolaus

    2014-02-01

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

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

    Science.gov (United States)

    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. Computational Design of DNA-Binding Proteins.

    Science.gov (United States)

    Thyme, Summer; Song, Yifan

    2016-01-01

    Predicting the outcome of engineered and naturally occurring sequence perturbations to protein-DNA interfaces requires accurate computational modeling technologies. It has been well established that computational design to accommodate small numbers of DNA target site substitutions is possible. This chapter details the basic method of design used in the Rosetta macromolecular modeling program that has been successfully used to modulate the specificity of DNA-binding proteins. More recently, combining computational design and directed evolution has become a common approach for increasing the success rate of protein engineering projects. The power of such high-throughput screening depends on computational methods producing multiple potential solutions. Therefore, this chapter describes several protocols for increasing the diversity of designed output. Lastly, we describe an approach for building comparative models of protein-DNA complexes in order to utilize information from homologous sequences. These models can be used to explore how nature modulates specificity of protein-DNA interfaces and potentially can even be used as starting templates for further engineering. PMID:27094297

  15. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  16. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

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

  17. AFM studies of nonspecific binding of enzyme on DNA

    Institute of Scientific and Technical Information of China (English)

    张益; 谢恒月; 等

    1996-01-01

    Atomic force microscope(AFM) is used to study restriction endonuclease digestion of plasmid DNA,pWRr plasmid DNA is digested by Hind Ⅲ,and the specific and the nonspecific binding of the restriction endonuclease are imaged,and the biological function of the enzyme binding to nonspecific sites is discussed.In addition,it is found that nonspecific binding of Hind ǚ could not induce the DNA characteristic bending angle.

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

    DEFF Research Database (Denmark)

    Ratilainen, T; Holmén, A; Tuite, E;

    2000-01-01

    For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and seq......For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes...

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

    Science.gov (United States)

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

    2016-01-01

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

  20. Mechanisms of DNA Binding and Regulation of Bacillus anthracis DNA Primase

    OpenAIRE

    Biswas, Subhasis B; Wydra, Eric; Biswas, Esther E.

    2009-01-01

    DNA primases are pivotal enzymes in chromosomal DNA replication in all organisms. In this article, we report unique mechanistic characteristics of recombinant DNA primase from Bacillus anthracis (B. anthracis). The mechanism of action of B. anthracis DNA primase (DnaGBA) may be described in several distinct steps as follows. Its mechanism of action is initiated when it binds to single-stranded DNA (ssDNA) in the form of a trimer. Although DnaGBA binds to different DNA sequences with moderate ...

  1. Control of Glycosylation-Related Genes by DNA Methylation: the Intriguing Case of the B3GALT5 Gene and Its Distinct Promoters.

    Science.gov (United States)

    Trinchera, Marco; Zulueta, Aida; Caretti, Anna; Dall'Olio, Fabio

    2014-08-04

    Glycosylation is a metabolic pathway consisting of the enzymatic modification of proteins and lipids through the stepwise addition of sugars that gives rise to glycoconjugates. To determine the full complement of glycoconjugates that cells produce (the glycome), a variety of genes are involved, many of which are regulated by DNA methylation. The aim of the present review is to briefly describe some relevant examples of glycosylation-related genes whose DNA methylation has been implicated in their regulation and to focus on the intriguing case of a glycosyltransferase gene (B3GALT5). Aberrant promoter methylation is frequently at the basis of their modulation in cancer, but in the case of B3GALT5, at least two promoters are involved in regulation, and a complex interplay is reported to occur between transcription factors, chromatin remodelling and DNA methylation of typical CpG islands or even of other CpG dinucleotides. Transcription of the B3GALT5 gene underwent a particular evolutionary fate, so that promoter hypermethylation, acting on one transcript, and hypomethylation of other sequences, acting on the other, cooperate on one gene to obtain full cancer-associated silencing. The findings may also help in unravelling the complex origin of serum CA19.9 antigen circulating in some patients.

  2. Selective binding of anti-DNA antibodies to native dsDNA fragments of differing sequence.

    Science.gov (United States)

    Uccellini, Melissa B; Busto, Patricia; Debatis, Michelle; Marshak-Rothstein, Ann; Viglianti, Gregory A

    2012-03-30

    Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.

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

    Science.gov (United States)

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

    2013-03-01

    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).

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

    Science.gov (United States)

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

    2013-10-15

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

  5. High-Fidelity DNA Sensing by Protein Binding Fluctuations

    CERN Document Server

    Tlusty, Tsvi; Libchaber, Albert; 10.1103/PhysRevLett.93.258103

    2010-01-01

    One of the major functions of RecA protein in the cell is to bind single-stranded DNA exposed upon damage, thereby triggering the SOS repair response.We present fluorescence anisotropy measurements at the binding onset, showing enhanced DNA length discrimination induced by adenosine triphosphate consumption. Our model explains the observed DNA length sensing as an outcome of out-of equilibrium binding fluctuations, reminiscent of microtubule dynamic instability. The cascade architecture of the binding fluctuations is a generalization of the kinetic proofreading mechanism. Enhancement of precision by an irreversible multistage pathway is a possible design principle in the noisy biological environment.

  6. Binding Parameters of Alkaloids Berberine and Sanguinarine with DNA

    CERN Document Server

    Gumenyuk, V G; Kutovyy, S Yu; Yashchuk, V M; Zaika, L A

    2012-01-01

    We study the interaction of berberine and sanguinarine (plant alkaloids) with DNA in aqueous solutions, by using optical spectroscopy methods (absorption and fluorescence). The dependencies of alkaloid spectral characteristics on the concentration ratio N/c between the DNA base pairs and alkaloid molecules in the solutions are considered, and the manifestations of the alkaloid-DNA binding are revealed. The character of binding is found to depend on N/c. The parameters of the binding of berberine and sanguinarine with DNA are determined, by using the modified Scatchard and McGhee-von Hippel equations

  7. The binding of in vitro synthesized adenovirus DNA binding protein to single-stranded DNA is stimulated by zinc ions

    NARCIS (Netherlands)

    Vos, H.L.; Lee, F.M. van der; Sussenbach, J.S.

    1988-01-01

    We have synthesized wild type DNA binding protein (DBP) of adenovirus type 5 (Ad5) and several truncated forms of this protein by a combination of in vitro transcription and translation. The proteins obtained were tested for binding to a single-stranded DNA-cellulose column. It could be shown that f

  8. High-throughput analysis of protein-DNA binding affinity.

    Science.gov (United States)

    Franco-Zorrilla, José M; Solano, Roberto

    2014-01-01

    Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro. PMID:24057393

  9. TAL Effector DNA-Binding Principles and Specificity.

    Science.gov (United States)

    Richter, Annekatrin; Streubel, Jana; Boch, Jens

    2016-01-01

    Transcription activator-like effectors (TALEs) are proteins with a unique DNA-binding domain that confers both a predictable and programmable specificity. The DNA-binding domain consists typically of 34-amino acid near-identical repeats. The repeats form a right-handed superhelical structure that wraps around the DNA double helix and exposes the variable amino acids at position 13 of each repeat to the sense strand DNA bases. Each repeat binds one base in a highly specific, non-overlapping, and comma-free fashion. Although TALE specificities are encoded in a simple way, sophisticated rules can be taken into account to build highly efficient DNA-binding modules for biotechnological use. PMID:26443210

  10. Tight-binding parameters for charge transfer along DNA

    OpenAIRE

    Hawke, L. G.D.; Kalosakas, G.; Simserides, C.

    2009-01-01

    We systematically examine all the tight-binding parameters pertinent to charge transfer along DNA. The $\\pi$ molecular structure of the four DNA bases (adenine, thymine, cytosine, and guanine) is investigated by using the linear combination of atomic orbitals method with a recently introduced parametrization. The HOMO and LUMO wavefunctions and energies of DNA bases are discussed and then used for calculating the corresponding wavefunctions of the two B-DNA base-pairs (adenine-thymine and gua...

  11. Tight-binding modeling of charge migration in DNA devices

    OpenAIRE

    Cuniberti, G.; Macia, E.; Rodriguez, A.; R.A. Römer

    2007-01-01

    Long range charge transfer experiments in DNA oligomers and the subsequently measured -- and very diverse -- transport response of DNA wires in solid state experiments exemplifies the need for a thorough theoretical understanding of charge migration in DNA-based natural and artificial materials. Here we present a review of tight-binding models for DNA conduction which have the intrinsic merit of containing more structural information than plain rate-equation models while still retaining suffi...

  12. Mechanochemical regulations of RPA's binding to ssDNA

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-20

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

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... spectrometry. The determined molecular masses are often sufficient for identification. If not, the proteins are subjected to mass spectrometric peptide mapping followed by database searches. Apart from protein identification, the protocol also yields information on posttranslational modifications. The protocol...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

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

    Energy Technology Data Exchange (ETDEWEB)

    De Ioannes, Pablo; Escalante, Carlos R.; Aggarwal, Aneel K. (VCU); (Mount Sinai Hospital)

    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.

  17. Thermodynamic characterization of proflavine–DNA binding through microcalorimetric studies

    International Nuclear Information System (INIS)

    Highlights: • Energetics of the interaction of proflavine with DNA has been studied. • The binding reaction was favored by both negative enthalpy and positive entropy. • Enthalpy–entropy compensation phenomenon was observed. • Non-polyelectrolytic forces played a dominant role in the binding process. • Proflavine enhanced the thermal stability of DNA remarkably. - Abstract: The interaction of an important acridine dye, proflavine hydrochloride, with double stranded DNA was investigated using isothermal titration calorimetry and differential scanning calorimetry. The equilibrium constant for the binding reaction was calculated to be (1.60 ± 0.04) · 105 · M−1 at T = 298.15 K. The binding of proflavine hydrochloride to DNA was favored by both negative enthalpy and positive entropy contributions to the Gibbs energy. The equilibrium constant for the binding reaction decreased with increasing temperature. The standard molar enthalpy change became increasingly negative while the standard molar entropy change became less positive with rise in temperature. However, the standard molar Gibbs free energy change varied marginally suggesting the occurrence of enthalpy–entropy compensation phenomenon. The binding reaction was dominated by non-polyelectrolytic forces which remained virtually unchanged at all the salt concentrations studied. The binding also significantly increased the thermal stability of DNA against thermal denaturation

  18. MOLECULAR MODELING STUDY OF THE CONTRIBUTIONS OF SIDE AMINO ACID RESIDUES OF POLYMYXIN B3 TO ITS BINDING WITH E.COLI OUTER MEMBRANE LIPOPOLYSACCHARIDE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2014-12-01

    Full Text Available Last decades, antimicrobial peptides (AMPs are the subject of intense investigations aimed to develop effective drugs against extremely resistant nosocomial bacterial pathogens (especially Gram-negative bacteria. In particular, there has been greatly renewed interest to polymyxins, the representatives of AMPs which are specific and highly potent against Gram-negative bacteria, but have potential nephrotoxic side effect. A prerequisite of purposeful enhancement of therapeutic properties of polymyxins is a detailed knowledge of the molecular mechanisms of their interactions with cell targets. Lipopolysaccharide (LPS, the main component of the outer leaflet of outer membrane of gram-negative bacteria, is a primary cell target of polymyxins. The aim of the paper was to study the peculiarities of molecular interactions of polymyxin В3 with lipopolysaccharide of the outer membrane of gram-negative bacterium. Materials and methods The complexes of polymyxin В3 (PmВ3 and its alaninederivatives with E. coli outer membrane lipopolysaccharide were built and studied by molecular modeling methods (minimization, simulated annealing, docking. Atom coordinates of polymyxin В3 and LPS structures were taken from nuclear magnetic resonance and X-ray crystallography experiments, respectively. The AMBER03 force field was used with a 1.05 nm force cutoff. Longrange electrostatic interactions were treated by the Particle Mesh Ewald method. Results and discussion Alanine scanning of PmВ3 molecule has been carried out and the role of its side amino acid residues in the formation of complex with lipopolysaccharide has been investigated. It has been shown that substitutions of polymyxin’s Dab residues in positions 1, 3, 5, 8 and 9 for alanine markedly reduce the binding energy of PmB3-LPS complex, where as the similar substitutions of residues in positions 2, 6, 7 and 10 leave the binding energy virtually unchanged. Structural aspects of antimicrobial action of

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

    Indian Academy of Sciences (India)

    Sumedha Roy; Shayoni Dutta; Kanika Khanna; Shruti Singla; Durai Sundar

    2012-07-01

    Zinc finger proteins interact via their individual fingers to three base pair subsites on the target DNA. The four key residue positions −1, 2, 3 and 6 on the alpha-helix of the zinc fingers have hydrogen bond interactions with the DNA. Mutating these key residues enables generation of a plethora of combinatorial possibilities that can bind to any DNA stretch of interest. Exploiting the binding specificity and affinity of the interaction between the zinc fingers and the respective DNA can help to generate engineered zinc fingers for therapeutic purposes involving genome targeting. Exploring the structure–function relationships of the existing zinc finger–DNA complexes can aid in predicting the probable zinc fingers that could bind to any target DNA. Computational tools ease the prediction of such engineered zinc fingers by effectively utilizing information from the available experimental data. A study of literature reveals many approaches for predicting DNA-binding specificity in zinc finger proteins. However, an alternative approach that looks into the physico-chemical properties of these complexes would do away with the difficulties of designing unbiased zinc fingers with the desired affinity and specificity. We present a physico-chemical approach that exploits the relative strengths of hydrogen bonding between the target DNA and all combinatorially possible zinc fingers to select the most optimum zinc finger protein candidate.

  20. The specificity of the secondary DNA binding site of RecA protein defines its role in DNA strand exchange.

    OpenAIRE

    Mazin, A V; Kowalczykowski, S C

    1996-01-01

    The RecA protein-single-stranded DNA (ssDNA) filament can bind a second DNA molecule. Binding of ssDNA to this secondary site shows specificity, in that polypyrimidinic DNA binds to the RecA protein-ssDNA filament with higher affinity than polypurinic sequences. The affinity of ssDNA, which is identical in sequence to that bound in the primary site, is not always greater than that of nonhomologous DNA. Moreover, this specificity of DNA binding does not depend on the sequence of the DNA bound ...

  1. The function of the secondary DNA-binding site of RecA protein during DNA strand exchange.

    OpenAIRE

    Mazin, A V; Kowalczykowski, S C

    1998-01-01

    RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product...

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

    Science.gov (United States)

    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 localization of the

  3. Characterization of DNA Binding and Retinoic Acid Binding Properties of Retinoic Acid Receptor

    Science.gov (United States)

    Yang, Na; Schule, Roland; Mangelsdorf, David J.; Evans, Ronald M.

    1991-05-01

    High-level expression of the full-length human retinoic acid receptor (RAR) α and the DNA binding domain of the RAR in Escherichia coli was achieved by using a T7 RNA polymerase-directed expression system. After induction, full-length RAR protein was produced at an estimated level of 20% of the total bacterial proteins. Both intact RAR molecules and the DNA binding domain bind to the cognate DNA response element with high specificity in the absence of retinoic acid. However, this binding is enhanced to a great extent upon the addition of eukaryotic cell extracts. The factor responsible for this enhancement is heat-sensitive and forms a complex with RAR that binds to DNA and exhibits a distinct migration pattern in the gel-mobility-shift assay. The interaction site of the factor with RAR is localized in the 70-amino acid DNA binding region of RAR. The hormone binding ability of the RARα protein was assayed by a charcoal absorption assay and the RAR protein was found to bind to retinoic acid with a K_d of 2.1 x 10-10 M.

  4. Binding Isotherms and Cooperative Effects for Metal-DNA Complexes

    CERN Document Server

    Gelagutashvili, Eteri

    2008-01-01

    The stoichiometric binding constants of Nickel(II), Cobalt(II), Manganese(II), Silver(I), Zinc(II) ions with DNA, from Spirulina platensis were determined from their binding isotherms by equilibrium dialysis and atomic absorption spectroscopy. It was shown, that the nature of these ions interaction with DNA, from S .platensis is different. For Cobalt(II), Zinc(II) ions were observed cooperative effects and existence of two different types of the binding sites. Nickel(II)_, Silver(I) -DNA complexes shows independent and identical binding sites and Manganese(II)_ negative cooperative interaction. The logarithm of binding constants for Cobalt (II)_, Nickel (II)_, Manganese (II)_, Zinc (II)_, Silver (I) - DNA, from S. platensis in 3 mM Na(I) are 5.11; 5.18; 4.77; 5.05; 5.42; respectively. The linear correlation of logarithm of binding constants (for complexes of metal-DNA from S. platensis) and the covalent index of Pauling are observed.

  5. AcEBP1, an ErbB3-Binding Protein (EBP1) from halophyte Atriplex canescens, negatively regulates cell growth and stress responses in Arabidopsis.

    Science.gov (United States)

    Li, Jingtao; Yu, Gang; Sun, Xinhua; Zhang, Xianghui; Liu, Jinliang; Pan, Hongyu

    2016-07-01

    An ErbB-3-binding protein gene AcEBP1, also known as proliferation-associated 2G4 gene (PA2G4s) belonging to the M24 superfamily, was obtained from the saltbush Atriplex canescens. Subcellular localization imaging showed the fusion protein AcEBP1-eGFP was located in the nucleus of epidermal cells in Nicotiana benthamiana. The AcEBP1 gene expression levels were up-regulated under salt, osmotic stress, and hormones treatment as revealed by qRT-PCR. Overexpression of AcEBP1 in Arabidopsis demonstrated that AcEBP1 was involved in root cell growth and stress responses (NaCl, osmotic stress, ABA, low temperature, and drought). These phenotypic data were correlated with the expression patterns of stress responsive genes and PR genes. The AcEBP1 transgenic Arabidopsis plants also displayed increased sensitivity under low temperature and evaluated resistance to drought stress. Together, these results demonstrate that AcEBP1 negatively affects cell growth and is a regulator under stress conditions. PMID:27181948

  6. Structure-based Analysis to Hu-DNA Binding

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. DNA binding of dinuclear iron(II) metallosupramolecular cylinders. DNA unwinding and sequence preference.

    Science.gov (United States)

    Malina, Jaroslav; Hannon, Michael J; Brabec, Viktor

    2008-06-01

    [Fe(2)L(3)](4+) (L = C(25)H(20)N(4)) is a synthetic tetracationic supramolecular cylinder (with a triple helical architecture) that targets the major groove of DNA and can bind to DNA Y-shaped junctions. To explore the DNA-binding mode of [Fe(2)L(3)](4+), we examine herein the interactions of pure enantiomers of this cylinder with DNA by biochemical and molecular biology methods. The results have revealed that, in addition to the previously reported bending of DNA, the enantiomers extensively unwind DNA, with the M enantiomer being the more efficient at unwinding, and exhibit preferential binding to regular alternating purine-pyrimidine sequences, with the M enantiomer showing a greater preference. Also, interestingly, the DNA binding of bulky cylinders [Fe(2)(L-CF(3))(3)](4+) and [Fe(2)(L-Ph)(3)](4+) results in no DNA unwinding and also no sequence preference of their DNA binding was observed. The observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code. PMID:18467423

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

    Science.gov (United States)

    Frégeau, Chantal J; De Moors, Anick

    2012-09-01

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

  9. Binding and Transformation of Extracellular DNA in Soil

    Institute of Scientific and Technical Information of China (English)

    CAI Peng; HUANG Qiao-Yun; ZHANG Xue-Wen; CHEN Hao

    2005-01-01

    DNA is the genetic material of various organisms. Extracellular DNA adsorbed or bound on surface-active particles in soils has been shown to persist for long periods against nucleases degradation and still retain the ability to transform competent cells. This paper reviews some recent advances on the binding and transformation of extracellular DNA in soils,which is fundamental to understanding the nature of the soil, regulating biodiversity, and assessing the risk of releasing genetically engineered microorganisms (GEMs) as well as being helpful for development of the genetic evolutional theory of bacteria. Several influencing factors, such as soil pH, ionic strength, soil surface properties, and characteristics of the DNA polymer, are discussed. To date, the understanding of the type of molecular binding sites and the conformation of adsorbed and bound DNA to soil particles is still in its infancy.

  10. Tight-binding approach to strain-dependent DNA electronics

    Science.gov (United States)

    Malakooti, Sadeq; Hedin, Eric; Joe, Yong

    2013-07-01

    Small mechanical strain perturbations are considered in calculations of the poly(G)-poly(C) DNA molecular electronic structure, using a tight-binding framework in conjunction with the theories of Slater-Koster and linear elasticity. Results reveal a strain-induced band gap for DNA which is linearly dependent on the induced strain. Local density of states calculations expose that the contribution of the guanine-cytosine base pairs in the charge transport mechanism is significantly enhanced relative to the backbones when DNA is compressed. Transport investigations also disclose a strain-induced metal-semiconductor transition for the DNA molecule, which suggests possible potential uses for sensing applications.

  11. DnaT is a PriC-binding protein.

    Science.gov (United States)

    Huang, Chien-Chih; Huang, Cheng-Yang

    2016-09-01

    DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT-PriC changed from red to purple, which indicated the protein-protein interactions due to gold nanoparticle aggregate. In addition, this DnaT-PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10(-8) M. These results constitute a pioneering study of the DnaT-PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC-SSB-DNA tricomplex and regulates the PriC-dependent replication restart. PMID:27387236

  12. DnaT is a PriC-binding protein.

    Science.gov (United States)

    Huang, Chien-Chih; Huang, Cheng-Yang

    2016-09-01

    DnaT and PriC are replication restart primosomal proteins required for re-initiating chromosomal DNA replication. DnaT is a component of the PriA-dependent primosome, while PriC belongs to the PriC-dependent primosome. Whether DnaT can interact with PriC is still unknown. In this study, we define a direct interaction between PriC, a key initiator protein in PriC-mediated DNA replication restart, and DnaT, a DnaB/C complex loader protein, from Klebsiella pneumoniae. In fluorescence titrations, PriC bound to single-stranded DNA with a binding-site size of approximately 9 nt. Gold nanoparticle assay showed that the solution of DnaT-PriC changed from red to purple, which indicated the protein-protein interactions due to gold nanoparticle aggregate. In addition, this DnaT-PriC complex could be co-purified by the heparin HP column. Surface plasmon resonance analysis showed that the Kd value of DnaT bound to PriC was 2.9 × 10(-8) M. These results constitute a pioneering study of the DnaT-PriC interaction and present a putative link between the two independent replication restart pathways, namely, PriA- and PriC-dependent primosome assemblies. Further research can directly focus on determining how DnaT binds to the PriC-SSB-DNA tricomplex and regulates the PriC-dependent replication restart.

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

    DEFF Research Database (Denmark)

    Bentin, T; Nielsen, Peter E.

    1996-01-01

    efficient with supercoiled than with linear DNA. In the presence of 140 mM KCI, the PNA binding rate was reduced but, notably, highly dependent on template topology. Negative supercoiling (mean superhelix density, sigma approximately -0.051) increased the rate of binding by 2 orders of magnitude compared...... to that of relaxed DNA. The pseudo-first-order rate constant [k psi (sigma)] obeys an exponential function, k psi (sigma) = k psi (lin)e-sigma delta, where delta is a constant of 105 and k psi lin is the rate of PNA binding to linear DNA (sigma = 0). The activation energy [Ea(sigma)] was determined as approximately...... 93 and approximately 48 kJ mol-1 for PNA binding to linear and supercoiled DNA, respectively. The results are discussed in relation to the possible future use of PNA as an antigene agent and in the framework of DNA "breathing" dynamics....

  14. enDNA-Prot: Identification of DNA-Binding Proteins by Applying Ensemble Learning

    Directory of Open Access Journals (Sweden)

    Ruifeng Xu

    2014-01-01

    Full Text Available DNA-binding proteins are crucial for various cellular processes, such as recognition of specific nucleotide, regulation of transcription, and regulation of gene expression. Developing an effective model for identifying DNA-binding proteins is an urgent research problem. Up to now, many methods have been proposed, but most of them focus on only one classifier and cannot make full use of the large number of negative samples to improve predicting performance. This study proposed a predictor called enDNA-Prot for DNA-binding protein identification by employing the ensemble learning technique. Experiential results showed that enDNA-Prot was comparable with DNA-Prot and outperformed DNAbinder and iDNA-Prot with performance improvement in the range of 3.97–9.52% in ACC and 0.08–0.19 in MCC. Furthermore, when the benchmark dataset was expanded with negative samples, the performance of enDNA-Prot outperformed the three existing methods by 2.83–16.63% in terms of ACC and 0.02–0.16 in terms of MCC. It indicated that enDNA-Prot is an effective method for DNA-binding protein identification and expanding training dataset with negative samples can improve its performance. For the convenience of the vast majority of experimental scientists, we developed a user-friendly web-server for enDNA-Prot which is freely accessible to the public.

  15. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA.

    Directory of Open Access Journals (Sweden)

    Janet L Smith

    2015-05-01

    Full Text Available DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq. We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.

  16. Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.

    Science.gov (United States)

    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

    2016-07-27

    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

  17. Synthesis, DNA binding and cytotoxic evaluation of aminoquinoline scaffolds

    Indian Academy of Sciences (India)

    Gopal Senthil Kumar; Mohamed Ashraf Ali; Tan Soo Choon; Rajendra Prasad Karnam Jayarampillai

    2016-03-01

    An effortless synthetic route has been developed for the synthesis of a new class of aminoquinoline substituted isoindolin-1,3-diones from regio-isomerical hydrazinylquinolines with phthalic anhydride in presence of Eaton’s reagent. DNA binding studies of selected isomeric compounds showed interaction withDNA via intercalation mode with higher binding affinity of 4-substituted quinolines rather than 2-substituted counterparts. Further, all compounds were screened for cytotoxic activity against three human cancer cell lines,among them compound 2c outranged standard doxorubicin against CCRF-CEM cell line.

  18. DNA binding to SMC ATPases-trapped for release.

    Science.gov (United States)

    Schüler, Herwig; Sjögren, Camilla

    2016-04-01

    The SMC/Rad50/RecN proteins are universal DNA‐associated ABC‐type ATPases with crucial functions in genome maintenance. New insights into Rad50-DNA complex structure and cohesin regulation inspire a speculative look at the entire superfamily. Identification of a continuous DNA binding site across the Rad50 dimer interface (Liu et al, 2016; Seifert et al, 2016) suggests a similar site in cohesin. The localization of this site hints a DNA-activated mechanism for cohesin removal from chromosomes.

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

    Science.gov (United States)

    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. PMID:26188387

  20. Tight-binding parameters for charge transfer along DNA

    CERN Document Server

    Hawke, L G D; Simserides, C

    2009-01-01

    We systematically examine all the tight-binding parameters pertinent to charge transfer along DNA. The $\\pi$ molecular structure of the four DNA bases (adenine, thymine, cytosine, and guanine) is investigated by using the linear combination of atomic orbitals method with a recently introduced parametrization. The HOMO and LUMO wavefunctions and energies of DNA bases are discussed and then used for calculating the corresponding wavefunctions of the two B-DNA base-pairs (adenine-thymine and guanine-cytosine). The obtained HOMO and LUMO energies of the bases are in good agreement with available experimental values. Our results are then used for estimating the complete set of charge transfer parameters between neighboring bases and also between successive base-pairs, considering all possible combinations between them, for both electrons and holes. The calculated microscopic quantities can be used in mesoscopic theoretical models of electron or hole transfer along the DNA double helix, as they provide the necessar...

  1. Extensive mapping of PPAR binding to genomic DNA

    DEFF Research Database (Denmark)

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

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

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by...

  3. RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.

    Science.gov (United States)

    Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael; Korolev, Sergey

    2014-10-17

    Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. PMID:25170075

  4. Binding of DNA with Abf2p Increases Efficiency of DNA Uptake by Isolated Mitochondria.

    Science.gov (United States)

    Samoilova, E O; Krasheninnikov, I A; Vinogradova, E N; Kamenski, P A; Levitskii, S A

    2016-07-01

    Mutations in mitochondrial DNA often lead to severe hereditary diseases that are virtually resistant to symptomatic treatment. During the recent decades, many efforts were made to develop gene therapy approaches for treatment of such diseases using nucleic acid delivery into the organelles. The possibility of DNA import into mitochondria has been shown, but this process has low efficiency. In the present work, we demonstrate that the efficiency of DNA import can be significantly increased by preforming its complex with a mitochondria-targeted protein nonspecifically binding with DNA. As a model protein, we used the yeast protein Abf2p. In addition, we measured the length of the DNA site for binding this protein and the dissociation constant of the corresponding DNA-protein complex. Our data can serve as a basis for development of novel, highly efficient approaches for suppressing mutations in the mitochondrial genome. PMID:27449618

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

    OpenAIRE

    Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan; M. Bompiani, Kristin; Hansen, Majken Nørgaard; Gothelf, Kurt Vesterager; Sullenger, Bruce A; LaBean, Thomas H.

    2012-01-01

    Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by a flexible single-strand linker, have been shown to possess anticoagulant activity. Here, we link multiple aptamers at programmed positions on DNA nanostructures to optimize spacing and orientation o...

  6. Z-DNA binding protein from chicken blood nuclei

    Science.gov (United States)

    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.

  7. DNA binding properties of the small cascade subunit Csa5.

    Directory of Open Access Journals (Sweden)

    Michael Daume

    Full Text Available CRISPR-Cas systems provide immunity against viral attacks in archaeal and bacterial cells. Type I systems employ a Cas protein complex termed Cascade, which utilizes small CRISPR RNAs to detect and degrade the exogenic DNA. A small sequence motif, the PAM, marks the foreign substrates. Previously, a recombinant type I-A Cascade complex from the archaeon Thermoproteus tenax was shown to target and degrade DNA in vitro, dependent on a native PAM sequence. Here, we present the biochemical analysis of the small subunit, Csa5, of this Cascade complex. T. tenax Csa5 preferentially bound ssDNA and mutants that showed decreased ssDNA-binding and reduced Cascade-mediated DNA cleavage were identified. Csa5 oligomerization prevented DNA binding. Specific recognition of the PAM sequence was not observed. Phylogenetic analyses identified Csa5 as a universal member of type I-A systems and revealed three distinct groups. A potential role of Csa5 in R-loop stabilization is discussed.

  8. Structural, vibrational, NMR, quantum chemical, DNA binding and protein docking studies of two flexible imine oximes

    Indian Academy of Sciences (India)

    YUNUS KAYA

    2016-09-01

    Two flexible imine oxime molecules, namely, 3-(pyridin-2-ylmethylimino)-butan-2-one oxime (HL¹) and 3-(pyridin-2-ylmethylimino)-pentan-2-one oxime (HL²) have been synthesized and characterized by elemental analysis, IR and NMR techniques. The conformational behavior was investigated using the density functional theory (DFT) with the B3LYP method combined with the 6-311++G(d,p) basis set. As a result of the conformational studies, three stable molecules and the most stable conformer were determined for the both imine oximes. The spectroscopic properties such as vibrational and NMR were calculated for the most stable conformer of the HL¹ and HL². The calculation results were applied to simulate infrared spectra of the title compounds, which show good agreement with observed spectra. In addition, the stable three molecules of the both imine oximes have been used to carry out DNA binding and protein docking studies with DNA and protein structures (downloaded from Protein Data Bank) using Discovery Studio 3.5 to find the most preferred binding mode of the ligands inside the DNA and protein cavity.

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

    Science.gov (United States)

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

    2011-08-01

    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. PMID:21504984

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-25

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

  11. Roles of RNA-Binding Proteins in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Mihoko Kai

    2016-02-01

    Full Text Available Living cells experience DNA damage as a result of replication errors and oxidative metabolism, exposure to environmental agents (e.g., ultraviolet light, ionizing radiation (IR, and radiation therapies and chemotherapies for cancer treatments. Accumulation of DNA damage can lead to multiple diseases such as neurodegenerative disorders, cancers, immune deficiencies, infertility, and also aging. Cells have evolved elaborate mechanisms to deal with DNA damage. Networks of DNA damage response (DDR pathways are coordinated to detect and repair DNA damage, regulate cell cycle and transcription, and determine the cell fate. Upstream factors of DNA damage checkpoints and repair, “sensor” proteins, detect DNA damage and send the signals to downstream factors in order to maintain genomic integrity. Unexpectedly, we have discovered that an RNA-processing factor is involved in DNA repair processes. We have identified a gene that contributes to glioblastoma multiforme (GBM’s treatment resistance and recurrence. This gene, RBM14, is known to function in transcription and RNA splicing. RBM14 is also required for maintaining the stem-like state of GBM spheres, and it controls the DNA-PK-dependent non-homologous end-joining (NHEJ pathway by interacting with KU80. RBM14 is a RNA-binding protein (RBP with low complexity domains, called intrinsically disordered proteins (IDPs, and it also physically interacts with PARP1. Furthermore, RBM14 is recruited to DNA double-strand breaks (DSBs in a poly(ADP-ribose (PAR-dependent manner (unpublished data. DNA-dependent PARP1 (poly-(ADP ribose polymerase 1 makes key contributions in the DNA damage response (DDR network. RBM14 therefore plays an important role in a PARP-dependent DSB repair process. Most recently, it was shown that the other RBPs with intrinsically disordered domains are recruited to DNA damage sites in a PAR-dependent manner, and that these RBPs form liquid compartments (also known as

  12. In vitro binding of nitracrine to DNA in chromatin.

    Science.gov (United States)

    Wilmańska, D; Szmigiero, L; Gniazdowski, M

    1989-01-01

    In the presence of sulfhydryl compounds nitracrine, an anticancer drug, binds covalently to DNA. The accessibility of DNA in chromatin both to nitracrine and to 8-methoxypsoralen, which was used as a reference compound in this study, when assayed in NaCl concentrations from 0 to 2 M show similar characteristics. The initial decrease reaches a minimum at 0.15 M NaCl above which dissociation of non-histone proteins and histones at higher ionic strengths is demonstrated by an increase in accessible sites. The relative accessibility of DNA in chromatin to nitracrine is, however, lower than that found for 8-methoxypsoralen. Partial dissociation of chromatin with 0.7 M NaCl increases the accessibility of DNA in chromatin when assayed in the absence of NaCl but has no apparent influence when estimated at ionic strength close to physiological conditions. PMID:2742691

  13. Characterization of the target DNA sequence for the DNA-binding domain of zinc finger protein 191

    Institute of Scientific and Technical Information of China (English)

    Haoyue Wang; Ruilin Sun; Guoxiang Liu; Minghui Yao; Jian Fei; Hebai Shen

    2008-01-01

    Studies on the DNA-binding properties of transcription factors are important in searching for the downstream genes regulated by these factors. In the present study, we report on the DNA-binding property of a Cys2His2-type transcription factor, zinc finger protein 191 (Zfp191), which has been newly found to play a significant role in mice.By constructing a fusion protein containing the DNA-binding domain of Zfp191,we characterized target DNA by determining the protein's binding specificity and dependence on zinc.The data showed that the DNA-binding domain of Zfp191can specifically bind to the TCAT repeat motif and that there is a cooperative effect among the target DNA's multiple binding sites.Furthermore,the binding reaction is dependent on zinc.This work provides a foundation for further studies on the role of Zfp191 in gene regulation and development.

  14. Mucosal immunization with high-mobility group box 1 in chitosan enhances DNA vaccine-induced protection against coxsackievirus B3-induced myocarditis.

    Science.gov (United States)

    Wang, Maowei; Yue, Yan; Dong, Chunsheng; Li, Xiaoyun; Xu, Wei; Xiong, Sidong

    2013-11-01

    Coxsackievirus B3 (CVB3), a small single-stranded RNA virus, belongs to the Picornaviridae family. Its infection is the most common cause of myocarditis, with no vaccine available. Gastrointestinal mucosa is the major entry port for CVB3; therefore, the induction of local immunity in mucosal tissues may help control initial viral infections and alleviate subsequent myocardial injury. Here we evaluated the ability of high-mobility group box 1 (HMGB1) encapsulated in chitosan particles to enhance the mucosal immune responses induced by the CVB3-specific mucosal DNA vaccine chitosan-pVP1. Mice were intranasally coimmunized with 4 doses of chitosan-pHMGB1 and chitosan-pVP1 plasmids, at 2-week intervals, and were challenged with CVB3 4 weeks after the last immunization. Compared with chitosan-pVP1 immunization alone, coimmunization with chitosan-pHMGB1 significantly (P loads, decreased myocardial injury, and increased survival rates. Flow cytometric analysis indicated that HMGB1 enhanced dendritic cell (DC) recruitment to mesenteric lymph nodes and promoted DC maturation, which might partly account for its mucosal adjuvant effect. This strategy may represent a promising approach to candidate vaccines against CVB3-induced myocarditis. PMID:24027262

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

    Directory of Open Access Journals (Sweden)

    Yuki Nakayama

    Full Text Available 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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    OpenAIRE

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

    1987-01-01

    A monoclonal antibody (Jel 318) was produced by immunizing mice with poly[d(TmC)].poly[d(GA)].poly[d(mCT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly[d(TG)].poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g. poly[d(TC)].poly[d(GmA)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes was as...

  19. In Vitro Whole Genome DNA Binding Analysis of the Bacterial Replication Initiator and Transcription Factor DnaA

    OpenAIRE

    Smith, Janet L.; Grossman, Alan D.

    2015-01-01

    DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep ...

  20. Genomic DNA binding to ZnO microrods

    Science.gov (United States)

    Guzmán-Embús, D. A.; Cardozo, M. Orrego; Vargas-Hernández, C.

    2015-08-01

    In this work, ZnO microrods were produced by hydrothermal synthesis. DNA was extracted from pork spleen cells by cellular lysis, deproteinization and precipitation. The analysis of the DNA binding to the ZnO was performed using Raman spectroscopy a technique that allowed for the evaluation of the effect that the presence of the ZnO in the complex has on the DNA structure. Vibrational spectral bands from the DNA molecule and hexagonal wurtzite ZnO were observed and classified as E2(M), A1(TO), E2(High), E1(LO) and 2LO. The Raman signals from the vibrational bands corresponding to the phosphodiester bond 5‧-C-O-P-O-C-3‧ and bond stretching of the PO2- group, as well as ring vibrations of the nitrogenous bases of the DNA, were enhanced by the presence of the ZnO microrods. The bands from the modes corresponding to the C-O and Odbnd Psbnd O- molecules of the DNA backbone were observed to exhibit larger spectral shifts due to the compression and tensile stresses generated at the ZnO/DNA interface, respectively. In addition, the relative vibrational mode intensities of the nitrogenous bases increased.

  1. Elasticity of DNA and the effect of Dendrimer Binding

    CERN Document Server

    Mogurampelly, Santosh; Netz, Roland R; Maiti, Prabal K

    2013-01-01

    Negatively charged DNA can be compacted by positively charged dendrimers and the degree of compaction is a delicate balance between the strength of the electrostatic interaction and the elasticity of DNA. We report various elastic properties of short double stranded DNA (dsDNA) and the effect of dendrimer binding using fully atomistic molecular dynamics and numerical simulations. In equilibrium at room temperature, the contour length distribution P(L) and end-to-end distance distribution P(R) are nearly Gaussian, the former gives an estimate of the stretch modulus {\\gamma}_1 of dsDNA in quantitative agreement with the literature value. The bend angle distribution P({\\theta}) of the dsDNA also has a Gaussian form and allows to extract a persistence length, L_p of 43 nm. When the dsDNA is compacted by positively charged dendrimer, the stretch modulus stays invariant but the effective bending rigidity estimated from the end-to-end distance distribution decreases dramatically due to backbone charge neutralization...

  2. Synthesis, DNA binding and topoisomerase inhibition of mononaphthalimide homospermidine derivatives

    Institute of Scientific and Technical Information of China (English)

    Zhi Yong Tian; Hong Xia Ma; Song Qiang Xie; Xue Wang; Jin Zhao; Chao Jie Wang; Wen Yuan Gao

    2008-01-01

    Two novel mononaphthalimide homospermidine derivatives (2a, 2b) with three or four methylene unit as linkages weresynthesized and evaluated for cytotoxicity against human leukemia K562, murine melanoma B 16 and Chinese hamster ovary CHOcell lines. The presence of homospermidine motif could greatly elevate the potency of 1,8-naphthalimide. Conjugate 2b with longerspacer exhibited higher in vitro cytotoxicity than 2a. The DNA binding experiments indicated that conjugates 2b could bind toherring sperm DNA. The topoisomerase Ⅱ poison trials revealed that 2b could inhibit the activity of top. Ⅱ.2008 Chao Jie Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

  3. Effect of DNA binding protein Ssh12 from hyperthermophilic archaeon Sulfolobus shibatae on DNA supercoiling

    Institute of Scientific and Technical Information of China (English)

    楼慧强; 黄力; VietQ.Mai

    1999-01-01

    An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.

  4. Binding of cationic surfactants to DNA, protein and DNA-protein mixtures.

    Science.gov (United States)

    Gani, S A; Chattoraj, D K; Mukherjee, D C

    1999-06-01

    Extent of binding (gamma 2(1)) of cationic surfactants cetyltrimethyl ammonium bromide (CTAB), myristyltrimethyl ammonium bromide (MTAB) and dodecyl trimethyl ammonium bromide (DTAB) to calf-thymus DNA, bovine serum albumin (BSA) and to their binary mixture respectively have been measured as function of bulk concentration of the surfactant by using equilibrium dialysis technique. Binding of CTAB has been studied at different pH, ionic strength (mu), temperature and biopolymer composition and with native and denatured states of the biopolymers. The chain-length of different long chain amines plays a significant role in the extent of binding under identical solution condition. The binding ratios for CTAB to collagen, gelatin, DNA-collagen and DNA-gelatin mixtures respectively have also been determined. The conformational structures of different biopolymers are observed to play significant role in macromolecular interactions between protein and DNA in the presence of CTAB. From the experimental values of the maximum binding ratio (gamma 2m) at the saturation level for each individual biopolymer, ideal values (gamma 2m)id have been theoretically calculated for binary mixtures of biopolymers using additivity rule. The protein-DNA-CTAB interaction in mixture has been explained in terms of the deviation (delta) of (gamma 2m) from (gamma 2m)id in the presence of a surfactant in bulk. The binding of surfactants to biopolymers and to their binary mixtures are compared more precisely in terms of the Gibbs' free energy decrease (-delta G degree) for the saturation of the binding sites in the biopolymers or biopolymer mixtures with the change of the bulk surfactant activity from zero to unity in the rational mole fraction scale. PMID:10650715

  5. Computational redesign of endonuclease DNA binding and cleavage specificity

    Science.gov (United States)

    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.

  6. Cytotoxic, DNA binding, DNA cleavage and antibacterial studies of ruthenium-fluoroquinolone complexes

    Indian Academy of Sciences (India)

    Mohan N Patel; Hardik N Joshi; Chintan R Patel

    2014-05-01

    Six new Ru(II) and Ru(III) complexes have been synthesized and characterized by elemental analysis, LC-MS, electronic spectra, IR spectra and magnetic moment measurements. DNA-binding properties of Ru complexes have been studied by means of absorption spectrophotometry and viscosity measurements as well as their HS DNA cleavage properties by means of agarose gel electrophoresis. The experimental results show that all the complexes can bind to DNA via partial intercalative mode. The b values of complexes were found in the range 2.14 × 104 to 2.70 × 105 M-1. All the complexes show excellent efficiency of cleaving DNA than respective fluoroquinolones. Brine shrimp lethality bioassay has been performed to check the cytotoxic activity. The IC50 values of the complexes are in the range of 6.27 to 16.05 g mL-1.

  7. Molecular basis for oligomeric-DNA binding and episome maintenance by KSHV LANA.

    Directory of Open Access Journals (Sweden)

    John F Domsic

    Full Text Available LANA is the KSHV-encoded terminal repeat binding protein essential for viral replication and episome maintenance during latency. We have determined the X-ray crystal structure of LANA C-terminal DNA binding domain (LANADBD to reveal its capacity to form a decameric ring with an exterior DNA binding surface. The dimeric core is structurally similar to EBV EBNA1 with an N-terminal arm that regulates DNA binding and is required for replication function. The oligomeric interface between LANA dimers is dispensable for single site DNA binding, but is required for cooperative DNA binding, replication function, and episome maintenance. We also identify a basic patch opposite of the DNA binding surface that is responsible for the interaction with BRD proteins and contributes to episome maintenance function. The structural features of LANADBD suggest a novel mechanism of episome maintenance through DNA-binding induced oligomeric assembly.

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

    Science.gov (United States)

    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

  9. A plant DNA-binding protein that recognizes 5-methylcytosine residues.

    OpenAIRE

    Zhang, D. L.; Ehrlich, K C; Supakar, P C; Ehrlich, M

    1989-01-01

    A novel, 5-methylcytosine-specific, DNA-binding protein, DBP-m, has been identified in nuclear extracts of peas. DBP-m specifically recognizes 5-methylcytosine residues in DNA without appreciable DNA sequence specificity, unlike a mammalian DNA-binding protein (MDBP), which recognizes 5-methylcytosine residues but only in a related family of 14-base-pair sequences.

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

    Science.gov (United States)

    Schrader, Anna; Gross, Thomas; Thalhammer, Verena; Längst, Gernot

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Anna Schrader

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

  12. Dynamic binding of replication protein a is required for DNA repair

    Science.gov (United States)

    Chen, Ran; Subramanyam, Shyamal; Elcock, Adrian H.; Spies, Maria; Wold, Marc S.

    2016-01-01

    Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA–DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions. PMID:27131385

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

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Xun; Guanga, Gerald P; Wan, Cheng; Rose, Robert B [Z; (W Elec.); (NCSU)

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

    Science.gov (United States)

    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.

  15. Binding of transcription factor GabR to DNA requires recognition of DNA shape at a location distinct from its cognate binding site

    Science.gov (United States)

    Al-Zyoud, Walid A.; Hynson, Robert MG.; Ganuelas, Lorraine A.; Coster, Adelle CF.; Duff, Anthony P.; Baker, Matthew AB.; Stewart, Alastair G.; Giannoulatou, Eleni; Ho, Joshua WK.; Gaus, Katharina; Liu, Dali; Lee, Lawrence K.; Böcking, Till

    2016-01-01

    Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small-angle X-ray scattering and multi-angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR–DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR–DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location from the cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites. PMID:26681693

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

    Science.gov (United States)

    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.

  17. Prediction of Protein-DNA binding by Monte Carlo method

    Science.gov (United States)

    Deng, Yuefan; Eisenberg, Moises; Korobka, Alex

    1997-08-01

    We present an analysis and prediction of protein-DNA binding specificity based on the hydrogen bonding between DNA, protein, and auxillary clusters of water molecules. Zif268, glucocorticoid receptor, λ-repressor mutant, HIN-recombinase, and tramtrack protein-DNA complexes are studied. Hydrogen bonds are approximated by the Lennard-Jones potential with a cutoff distance between the hydrogen and the acceptor atoms set to 3.2 Åand an angular component based on a dipole-dipole interaction. We use a three-stage docking algorithm: geometric hashing that matches pairs of hydrogen bonding sites; (2) least-squares minimization of pairwise distances to filter out insignificant matches; and (3) Monte Carlo stochastic search to minimize the energy of the system. More information can be obtained from our first paper on this subject [Y.Deng et all, J.Computational Chemistry (1995)]. Results show that the biologically correct base pair is selected preferentially when there are two or more strong hydrogen bonds (with LJ potential lower than -0.20) that bind it to the protein. Predicted sequences are less stable in the case of weaker bonding sites. In general the inclusion of water bridges does increase the number of base pairs for which correct specificity is predicted.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-10-22

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

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

    Science.gov (United States)

    Szmigiero, L; Gniazdowski, M

    1981-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sheik Dawood Shahida Parveen

    2014-01-01

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

  1. Characterization of DNA-binding proteins from pea mitochondria

    DEFF Research Database (Denmark)

    Hatzack, F.A.; Dombrowski, S.; Brennicke, A.;

    1998-01-01

    unknown. Proteins binding to double-stranded oligonucleotides representing different parts of the pea (Pisum sativum) mitochondrial atp9 were analyzed by denaturation-renaturation chromatography and mobility-shift experiments. Two DNA-protein complexes were detected, which appeared to be sequence specific...... in competition experiments. Purification by hydroxyapatite, phosphocellulose, and reversed-phase high-pressure liquid chromatography separated two polypeptides with apparent molecular masses of 32 and 44 kD. Both proteins bound to conserved structures of the pea atp9 and the heterologous Oenothera berteriana atp...

  2. Statistical analysis of structural determinants for protein-DNA-binding specificity.

    Science.gov (United States)

    Corona, Rosario I; Guo, Jun-Tao

    2016-08-01

    DNA-binding proteins play critical roles in biological processes including gene expression, DNA packaging and DNA repair. They bind to DNA target sequences with different degrees of binding specificity, ranging from highly specific (HS) to nonspecific (NS). Alterations of DNA-binding specificity, due to either genetic variation or somatic mutations, can lead to various diseases. In this study, a comparative analysis of protein-DNA complex structures was carried out to investigate the structural features that contribute to binding specificity. Protein-DNA complexes were grouped into three general classes based on degrees of binding specificity: HS, multispecific (MS), and NS. Our results show a clear trend of structural features among the three classes, including amino acid binding propensities, simple and complex hydrogen bonds, major/minor groove and base contacts, and DNA shape. We found that aspartate is enriched in HS DNA binding proteins and predominately binds to a cytosine through a single hydrogen bond or two consecutive cytosines through bidentate hydrogen bonds. Aromatic residues, histidine and tyrosine, are highly enriched in the HS and MS groups and may contribute to specific binding through different mechanisms. To further investigate the role of protein flexibility in specific protein-DNA recognition, we analyzed the conformational changes between the bound and unbound states of DNA-binding proteins and structural variations. The results indicate that HS and MS DNA-binding domains have larger conformational changes upon DNA-binding and larger degree of flexibility in both bound and unbound states. Proteins 2016; 84:1147-1161. © 2016 Wiley Periodicals, Inc.

  3. Interaction of zinc and cobalt with dipeptides and their DNA binding studies

    Indian Academy of Sciences (India)

    P Rabindra Reddy; M Radhika; K Srinivas Rao

    2004-06-01

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

  4. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part I. Importance of hydrophobic interactions in stabilization of β-hairpin structure

    OpenAIRE

    Skwierawska, Agnieszka; Makowska, Joanna; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2009-01-01

    We previously studied a 16-amino acid-residue fragment of the C-terminal β-hairpin (residues 46–61), [IG(46–61)], of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, and found that hydrophobic interactions and the turn region play an important role in stabilizing the structure. Based on these results, we carried out systematic structural studies of peptides derived from the sequence of IG(46–61) by systematically shortening the peptide by one residue at a time from bo...

  5. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part III. Dynamics of long-range hydrophobic interactions

    OpenAIRE

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2010-01-01

    A 20-residue peptide, IG(42–61), derived from the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using CD, NMR spectroscopy at various temperatures and by differential scanning calorimetry. Unlike other related peptides studied so far, this peptide displays two heat capacity peaks in DSC measurements (at a scanning rate of 1.5 deg/min at a peptide concentration of 0.07mM) which suggests a three-state folding/unfolding process. The ...

  6. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part IV. Implication for the mechanism of folding of the parent protein

    OpenAIRE

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2010-01-01

    A 34-residue α/β peptide, [IG(28-61)], derived from the C-terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using CD and NMR spectroscopy at various temperatures, and by differential scanning calorimetry. It was found that the C-terminal part (a 16-residue-long fragment) of this peptide, which corresponds to the sequence of the β-hairpin in the native structure, forms structure similar to the β-hairpin only at T = 313 K, and the structure is...

  7. Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

    Institute of Scientific and Technical Information of China (English)

    Yun Fei BAI; Qin Yu GE; Tong Xiang LI; Jin Ke WANG; Quan Jun LIU; Zu Hong LU

    2005-01-01

    The double-stranded DNA (dsDNA) probe contains two different protein binding sites.One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme.The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.

  8. Endonuclease-rolling circle amplification-based method for sensitive analysis of DNA-binding protein

    Institute of Scientific and Technical Information of China (English)

    Min Li Li; Dong Rui Zhou; Hong Zhao; Jin Ke Wang; Zu Hong Lu

    2009-01-01

    A sensitive approach for the qualitative detection of DNA-binding protein on the microarray was developed. DNA complexes in which a partial duplex region is formed from a biotin-primer and a circle single strand DNA (ssDNA) were spotted on a microarray. The endonuclease recognition site (ERS) and the DNA-binding sites (DBS) were arranged side by side within the duplex region. The working principle of the detection system is described as follows: when the DNA-binding protein capture the DBS, the endonuclease could not attach to the ERS, and the immobilized primer in the DNA complex could be extended along the circle ssDNA by rolling circle amplification (RCA). When no protein protects the DBS, the ERS could be attacked by the endonuclease and subsequently no rolling circle amplification occurs. Thereby we can detect the sequence specific DNA-binding activity with high-sensitivity due to the signal amplification of RCA.

  9. ncDNA and drift drive binding site accumulation

    Directory of Open Access Journals (Sweden)

    Ruths Troy

    2012-08-01

    Full Text Available Abstract Background The amount of transcription factor binding sites (TFBS in an organism’s genome positively correlates with the complexity of the regulatory network of the organism. However, the manner by which TFBS arise and accumulate in genomes and the effects of regulatory network complexity on the organism’s fitness are far from being known. The availability of TFBS data from many organisms provides an opportunity to explore these issues, particularly from an evolutionary perspective. Results We analyzed TFBS data from five model organisms – E. coli K12, S. cerevisiae, C. elegans, D. melanogaster, A. thaliana – and found a positive correlation between the amount of non-coding DNA (ncDNA in the organism’s genome and regulatory complexity. Based on this finding, we hypothesize that the amount of ncDNA, combined with the population size, can explain the patterns of regulatory complexity across organisms. To test this hypothesis, we devised a genome-based regulatory pathway model and subjected it to the forces of evolution through population genetic simulations. The results support our hypothesis, showing neutral evolutionary forces alone can explain TFBS patterns, and that selection on the regulatory network function does not alter this finding. Conclusions The cis-regulome is not a clean functional network crafted by adaptive forces alone, but instead a data source filled with the noise of non-adaptive forces. From a regulatory perspective, this evolutionary noise manifests as complexity on both the binding site and pathway level, which has significant implications on many directions in microbiology, genetics, and synthetic biology.

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

    Science.gov (United States)

    Moskaluk, Christopher; Bastia, Deepak

    1988-03-01

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

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

    Science.gov (United States)

    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.

  12. DNABINDPROT: fluctuation-based predictor of DNA-binding residues within a network of interacting residues

    OpenAIRE

    Ozbek, Pemra; Soner, Seren; Erman, Burak; Haliloglu, Turkan

    2010-01-01

    DNABINDPROT is designed to predict DNA-binding residues, based on the fluctuations of residues in high-frequency modes by the Gaussian network model. The residue pairs that display high mean-square distance fluctuations are analyzed with respect to DNA binding, which are then filtered with their evolutionary conservation profiles and ranked according to their DNA-binding propensities. If the analyses are based on the exact outcome of fluctuations in the highest mode, using a conservation thre...

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

    Science.gov (United States)

    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

  14. Drug binding to higher ordered DNA structures: netropsin complexation with a nucleic acid triple helix.

    OpenAIRE

    Park, Y. W.; Breslauer, K J

    1992-01-01

    We have used a combination of spectroscopic and calorimetric techniques to characterize how netropsin, a ligand that binds in the minor groove of DNA, influences the properties of a DNA triple helix. Specifically, our data allow us to reach the following conclusions: (i) netropsin binds to the triplex without displacing the major-groove-bound third strand; (ii) netropsin binding to the triplex exhibits a lower saturation binding density (7.0 base triplets per netropsin bound) than netropsin b...

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

    International Nuclear Information System (INIS)

    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 32P-end-labeled type I and type III procollagen promoter DNAs in the presence of equimolar amounts of 32P-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

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-09-03

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

  18. Solution measurement of DNA curvature in papillomavirus E2 binding sites

    OpenAIRE

    Zimmerman, Jeff M.; Maher, L. James

    2003-01-01

    ‘Indirect readout’ refers to the proposal that proteins can recognize the intrinsic three-dimensional shape or flexibility of a DNA binding sequence apart from direct protein contact with DNA base pairs. The differing affinities of human papillomavirus (HPV) E2 proteins for different E2 binding sites have been proposed to reflect indirect readout. DNA bending has been observed in X-ray structures of E2 protein–DNA complexes. X-ray structures of three different E2 DNA binding sites revealed di...

  19. DNA structure, binding mechanism and biology functions of polypyridyl complexes in biomedicine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    There is considerable research interest and vigorous debate about the DNA binding of polypyridyl complexes including the electron transfer involving DNA. In this review, based on the fluorescence quenching experiments, it was proposed that DNA might serve as a conductor. From the time-interval CD spectra, the different binding rates of D- and L-enantiomer to calf thymus DNA were observed. The factors influencing the DNA-binding of polypyridyl complexes, and the potential bio-functions of the complexes are also discussed.

  20. 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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

    Directory of Open Access Journals (Sweden)

    TIJANA SAVIC

    2006-02-01

    Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

  3. Agrobacterium tumefaciens virE operon encodes a single-stranded DNA-binding protein.

    Science.gov (United States)

    Das, A

    1988-05-01

    The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmid are essential for transformation of plant cells. Overproduction of a virE-encoded gene product in Escherichia coli was achieved by construction of an operon fusion with the E. coli tryptophan (trp) operon. The virE2 gene product in E. coli partitioned into the insoluble membrane fraction. The protein was solubilized by treatment with 4 M urea at 0 degree C. DNA-protein binding experiments showed that a strong single-stranded (ss) DNA-binding activity was present in protein fractions containing the virE2 gene product. The binding was highly specific with little or no binding observed with either double-stranded DNA or ssRNA. No significant binding to Ti plasmid DNA sequences was observed. Protein blotting studies indicated that the ssDNA-binding activity was associated with the 68-kDa virE2 polypeptide. PMID:2452439

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

    Science.gov (United States)

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

    2015-04-30

    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.

  5. The effect of polyamines on the binding of anti-DNA antibodies from patients with SLE and normal human subjects.

    Science.gov (United States)

    Wang, Xiao; Stearns, Nancy A; Li, Xingfu; Pisetsky, David S

    2014-07-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE). To elucidate specificity further, the effect of polyamines on the binding of anti-DNA antibodies from patients with lupus was tested by ELISA to calf thymus (CT) DNA; we also assessed the binding of plasmas of patients and normal human subjects (NHS) to Micrococcus luteus (MC) DNA. As these studies showed, spermine can dose-dependently inhibit SLE anti-DNA binding to CT DNA and can promote dissociation of preformed immune complexes. With MC DNA as antigen, spermine failed to inhibit the NHS anti-DNA binding. Studies using plasmas adsorbed to a CT DNA cellulose affinity indicated that SLE plasmas are mixtures of anti-DNA that differ in inhibition by spermine and binding to conserved and non-conserved determinants. Together, these studies demonstrate that spermine can influence the binding of anti-DNA autoantibodies and may contribute to the antigenicity of DNA.

  6. Multispectroscopic studies of paeoniflorin binding to calf thymus DNA in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Guowen, E-mail: gwzhang@ncu.edu.cn [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235, Nanjing East Road, Nanchang, Jiangxi 330047 (China); Fu, Peng; Pan, Junhui [State Key Laboratory of Food Science and Technology, Nanchang University, No. 235, Nanjing East Road, Nanchang, Jiangxi 330047 (China)

    2013-02-15

    The mechanism of paeoniflorin binding to calf thymus DNA in physiological buffer (pH 7.4) was investigated by multispectroscopic methods including UV-vis absorption, fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, coupled with viscosity measurements and DNA melting techniques. The results suggested that paeoniflorin molecules could bind to DNA via groove binding mode as evidenced by no significant change in iodide quenching effect, increase in single-stranded DNA (ssDNA) quenching effect, and almost unchanged relative viscosity and melting temperature of DNA. The observed changes in CD signals revealed that DNA remains in the B-conformation. Further, the displacement experiments with Hoechst 33258 probe and the results of FT-IR spectra indicated that paeoniflorin mainly binds in the region of rich A-T base pairs of DNA. The thermodynamic parameters, enthalpy change ({Delta}H Degree-Sign ) and entropy change ({Delta}S Degree-Sign ) were calculated to be -30.09{+-}0.18 kJ mol{sup -1} and -14.07{+-}0.61 J mol{sup -1} K{sup -1} by the van't Hoff equation, suggesting that hydrogen bond and van der Waals forces play a predominant role in the binding of paeoniflorin to DNA. - Highlights: Black-Right-Pointing-Pointer The binding mode of paeoniflorin to calf thymus DNA is the minor groove binding. Black-Right-Pointing-Pointer Paeoniflorin mainly binds in the region of rich A-T base pairs of DNA. Black-Right-Pointing-Pointer The binding does not alter the native B-conformation of DNA. Black-Right-Pointing-Pointer The binding is driven mainly by hydrogen bonds and van der Waals forces.

  7. DNA bending is induced in an enhancer by the DNA-binding domain of the bovine papillomavirus E2 protein.

    OpenAIRE

    Moskaluk, C; Bastia, D

    1988-01-01

    The E2 gene of bovine papillomavirus type 1 has been shown to encode a DNA-binding protein and to trans-activate the viral enhancer. We have localized the DNA-binding domain of the E2 protein to the carboxyl-terminal 126 amino acids of the E2 open reading frame. The DNA-binding domain has been expressed in Escherichia coli and partially purified. Gel retardation and DNase I "footprinting" on the bovine papillomavirus type 1 enhancer identify the sequence motif ACCN6GGT (in which N = any nucle...

  8. Study of MMLV RT- Binding with DNA using Surface Plasmon Resonance Biosensor

    Institute of Scientific and Technical Information of China (English)

    Lei WU; Ming-Hui HUANG; Jian-Long ZHAO; Meng-Su YANG

    2005-01-01

    Surface plasmon resonance biosensor technique was used to study the binding of Moloney murine leukemia virus reverse transcriptase without RNase H domain (MMLV RT-) with DNA in the absence and in the presence of inhibitors. Different DNA substrates, including single-stranded DNA (ssDNA),DNA template-primer (T-P) duplex and gapped DNA, were immobilized on the biosensor chip surface using streptavidin-biotin, and MMLV RT--DNA binding kinetics were analyzed by different models. MMLV RT-could bind with ssDNA and the binding was involved in conformation change. MMLV RT- binding DNA T-P duplex and gapped DNA could be analyzed using the simple 1:1 Langmuir model. The lack of RNase H domain reduced the affinity between MMLV RT- and T-P duplex. The effects of RT inhibitors, including efavirenz, nevirapine and quercetin, on the interaction between MMLV RT- and gapped DNA were analyzed according to recovered kinetics parameters. Efavirenz slightly interfered with the binding between RT and DNA and the affinity constant in the presence of the inhibitor (KA=1.21× 106 M-1) was lower than in the absence of the inhibitor (KA=4.61× 106 M-1). Nevirapine induced relatively tight binding between RT and DNA and the affinity constant in the presence of the inhibsitor (KA=l.47×107 M-1) was approximately three folds higher than without nevirapine, mainly due to rapid association and slow dissociation. Quercetin, a flavonoid originating from plant which has previously shown strong inhibition of the activity of RT, was found to have minimal effect on the RT-DNA binding.

  9. A general approach to visualize protein binding and DNA conformation without protein labelling.

    Science.gov (United States)

    Song, Dan; Graham, Thomas G W; Loparo, Joseph J

    2016-01-01

    Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein-DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein-DNA interactions.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    KAUST Repository

    Li, Lixin

    2013-07-01

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

  12. Sequence-selective DNA binding with cell-permeable oligoguanidinium-peptide conjugates.

    Science.gov (United States)

    Mosquera, Jesús; Sánchez, Mateo I; Valero, Julián; de Mendoza, Javier; Vázquez, M Eugenio; Mascareñas, José L

    2015-03-21

    Conjugation of a short peptide fragment from a bZIP protein to an oligoguanidinium tail results in a DNA-binding miniprotein that selectively interacts with composite sequences containing the peptide-binding site next to an A/T-rich tract. In addition to stabilizing the complex with the target DNA, the oligoguanidinium unit also endows the conjugate with cell internalization properties.

  13. Interindividual variation in binding of benzo[a]pyrene to DNA in cultured human Bronchi

    DEFF Research Database (Denmark)

    Harris, C.C.; Autrup, Herman; Connor, R.;

    1976-01-01

    The binding of benzo[a]pyrene to DNA in cultured human bronchus was measured in specimens from 37 patients. The binding values ranged from 2 to 151 picomoles of benzo[a]pyrene per milligram of DNA with an overall mean +/- standard error of 34.2 +/- 5.2. This 75-fold interindividual variation in t...

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

    Science.gov (United States)

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

    2015-06-14

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

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

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

    Science.gov (United States)

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

    2014-06-01

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

  18. The Caulobacter crescentus chromosome replication origin evolved two classes of weak DnaA binding sites.

    Science.gov (United States)

    Taylor, James A; Ouimet, Marie-Claude; Wargachuk, Richard; Marczynski, Gregory T

    2011-10-01

    The Caulobacter crescentus replication initiator DnaA and essential response regulator CtrA compete to control chromosome replication. The C. crescentus replication origin (Cori) contains five strong CtrA binding sites but only two apparent DnaA boxes, termed G-boxes (with a conserved second position G, TGATCCACA). Since clusters of DnaA boxes typify bacterial replication origins, this discrepancy suggested that C. crescentus DnaA recognizes different DNA sequences or compensates with novel DNA-binding proteins. We searched for novel DNA sites by scanning mutagenesis of the most conserved Cori DNA. Autonomous replication assays showed that G-boxes and novel W-boxes (TCCCCA) are essential for replication. Further analyses showed that C. crescentus DnaA binds G-boxes with moderate and W-boxes with very weak affinities significantly below DnaA's capacity for high-affinity Escherichia coli-boxes (TTATCCACA). Cori has five conserved W-boxes. Increasing W-box affinities increases or decreases autonomous replication depending on their strategic positions between the G-boxes. In vitro, CtrA binding displaces DnaA from proximal G-boxes and from distal W-boxes implying CtrA-DnaA competition and DnaA-DnaA cooperation between G-boxes and W-boxes. Similarly, during cell cycle progression, CtrA proteolysis coincides with DnaA binding to Cori. We also observe highly conserved W-boxes in other replication origins lacking E. coli-boxes. Therefore, strategically weak DnaA binding can be a general means of replication control.

  19. DNA-binding studies of the natural β-carboline eudistomin U.

    Science.gov (United States)

    Giulietti, Jennifer M; Tate, Patrick M; Cai, Ang; Cho, Bongsup; Mulcahy, Seann P

    2016-10-01

    Eudistomin U is a member of the β-carboline class of heterocyclic amine-containing molecules that are capable of binding to DNA. The structure of eudistomin U is unique since it contains an indole ring at the 1-position of the pyridine ring. While simple β-carbolines are reported to intercalate DNA, an examination of the mode of binding of eudistomin U has been lacking. We report preliminary spectroscopic (UV-Vis, thermal denaturation, CD) and calorimetric (DSC) data on the binding of eudistomin U to DNA, which suggest that eudistomin U binds weakly according to a mechanism that is more complicated than other members of its class. PMID:27567367

  20. Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains.

    OpenAIRE

    Vallee, B L; Coleman, J E; Auld, D S

    1991-01-01

    We now recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families of proteins. Those of the DNA-binding domains of the fungal transcription factor GAL4 and the rat glucocorticoid receptor are the first to have be...

  1. DNA binding during expanded bed adsorption and factors affecting adsorbent aggregation

    DEFF Research Database (Denmark)

    Arpanaei, Ayyoob; Mathiasen, N.; Hobley, Timothy John

    2008-01-01

    tolerance of anion exchangers when binding DNA. However, more importantly. with the adsorbents examined here. attempts to reduce bed aggregation by feedstock conditioning with added salt may increase DNA binding leading to a reduction in expanded bed adsorption performance compromising protein capture...... ligand densities to be examined. Very high dynamic binding capacities at 10% breakthrough were found in the absence of added salt. However, the highest binding capacities (similar to 10 and similar to 19mg DNA ml(-1) gel) were found in buffers containing added salt at concentrations of either 0.25 or 0......) even though the dynamic binding capacity was reduced as DNA concentration was increased. The extent of bed contraction during DNA loading was found to be a function of added salt concentration and ligand density of the adsorbent. The results imply that ligand density significantly affects the salt...

  2. A Novel Approach to Predict Core Residues on Cancer-Related DNA-Binding Domains

    OpenAIRE

    Ka-Chun Wong

    2016-01-01

    Protein–DNA interactions are involved in different cancer pathways. In particular, the DNA-binding domains of proteins can determine where and how gene regulatory regions are bound in different cell lines at different stages. Therefore, it is essential to develop a method to predict and locate the core residues on cancer-related DNA-binding domains. In this study, we propose a computational method to predict and locate core residues on DNA-binding domains. In particular, we have selected the ...

  3. High Purity DNA Extraction with a SPE Microfluidic Chip Using KI as the Binding Salt

    Institute of Scientific and Technical Information of China (English)

    Xing CHEN; Da Fu CUI; Chang Chun LIU

    2006-01-01

    Based on solid phase extraction method, a novel silicon-PDMS-glass microchip for high purity DNA extraction has been developed by using KI as the binding salt. The microfluidic chip fabricated by MEMS technology was composed of a silicon substrate with a coiled channel and a compounded PDMS-glass cover. With this microfluidic chip, the wall of the coiled channel was used as solid phase matrix for binding DNA and DNA was extracted by the fluxion of the binding buffer, washing buffer and elution buffer. KI as a substitute for guanidine, was used successfully as binding salt for purification DNA, obtaining higher purity of genomic DNA and about 13.9 ng DNA from 1 μL rat whole blood in 35 minutes.

  4. Stepwise bending of DNA by a single TATA box binding protein

    DEFF Research Database (Denmark)

    Tolic-Nørrelykke, Simon F; Rasmussen, Mette B; Pavone, Francesco S;

    2006-01-01

    bead is reduced compared to that of unbent DNA. We detected individual binding and dissocation events and derived kinetic parameters for the process. Dissociation was induced by increasing the salt concentration or by directly pulling on the tethered bead using optical tweezers. In addition to the well......The TATA-box binding protein (TBP) is required by all three eucaryotic RNA polymerases for the initiation of transcription from most promoters. TBP recognizes, binds to, and bends promoter sequences called "TATA-boxes" in the DNA. We present results from the study of individual Saccharomyces...... cerevisiae TBPs interacting with single DNA molecules containing a TATA-box. Using video microscopy, we observed the Brownian motion of the beads tethered by short surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the DNA changes and the amplitude of Brownian motion of the tehtered...

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

    Science.gov (United States)

    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.

  6. A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae.

    OpenAIRE

    Fox, M E; Feldman, B. J.; Chu, G.

    1994-01-01

    DNA photolyase binds to and repairs cyclobutane pyrimidine dimers induced by UV radiation. Here we demonstrate that in the yeast Saccharomyces cerevisiae, photolyase also binds to DNA damaged by the anticancer drugs cis-diamminedichloroplatinum (cis-DDP) and nitrogen mustard (HN2) and by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Surprisingly, mutations in photolyase were associated with resistance of yeast cells to cis-DDP, MNNG, 4-nitroquinoline oxide (4NQO), and HN2....

  7. Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

    Science.gov (United States)

    Mason, Claire E.; Jergic, Slobodan; Lo, Allen T. Y.; Wang, Yao; Dixon, Nicholas E.; Beck, Jennifer L.

    2013-02-01

    Single-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)70, one molecule of (dT)35, or two molecules of (dT)35] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

  8. Linkage structures strongly influence the binding cooperativity of DNA intercalators conjugated to triplex forming oligonucleotides.

    OpenAIRE

    1994-01-01

    Conjugation of DNA intercalators to triple helix forming oligodeoxynucleotides (ODN's) can enhance ODN binding properties and consequently their potential ability to modulate gene expression. To test the hypothesis that linkage structure could strongly influence the binding enhancement of intercalator conjugation with triplex forming ODN's, we have used a model system to investigate binding avidity of short oligomers conjugated to DNA intercalators through various linkages. Using a dA10.T10 t...

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

    Directory of Open Access Journals (Sweden)

    Marx Kenneth A

    2006-03-01

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

  10. Mycobacterium tuberculosis WhiB3 maintains redox homeostasis by regulating virulence lipid anabolism to modulate macrophage response.

    Directory of Open Access Journals (Sweden)

    Amit Singh

    2009-08-01

    Full Text Available The metabolic events associated with maintaining redox homeostasis in Mycobacterium tuberculosis (Mtb during infection are poorly understood. Here, we discovered a novel redox switching mechanism by which Mtb WhiB3 under defined oxidizing and reducing conditions differentially modulates the assimilation of propionate into the complex virulence polyketides polyacyltrehaloses (PAT, sulfolipids (SL-1, phthiocerol dimycocerosates (PDIM, and the storage lipid triacylglycerol (TAG that is under control of the DosR/S/T dormancy system. We developed an in vivo radio-labeling technique and demonstrated for the first time the lipid profile changes of Mtb residing in macrophages, and identified WhiB3 as a physiological regulator of virulence lipid anabolism. Importantly, MtbDeltawhiB3 shows enhanced growth on medium containing toxic levels of propionate, thereby implicating WhiB3 in detoxifying excess propionate. Strikingly, the accumulation of reducing equivalents in MtbDeltawhiB3 isolated from macrophages suggests that WhiB3 maintains intracellular redox homeostasis upon infection, and that intrabacterial lipid anabolism functions as a reductant sink. MtbDeltawhiB3 infected macrophages produce higher levels of pro- and anti-inflammatory cytokines, indicating that WhiB3-mediated regulation of lipids is required for controlling the innate immune response. Lastly, WhiB3 binds to pks2 and pks3 promoter DNA independent of the presence or redox state of its [4Fe-4S] cluster. Interestingly, reduction of the apo-WhiB3 Cys thiols abolished DNA binding, whereas oxidation stimulated DNA binding. These results confirmed that WhiB3 DNA binding is reversibly regulated by a thiol-disulfide redox switch. These results introduce a new paradigmatic mechanism that describes how WhiB3 facilitates metabolic switching to fatty acids by regulating Mtb lipid anabolism in response to oxido-reductive stress associated with infection, for maintaining redox balance. The link

  11. Analyzing the forces binding a restriction endonuclease to DNA using a synthetic nanopore

    OpenAIRE

    Dorvel, B.; Sigalov, G.; Zhao, Q.; Comer, J.; Dimitrov, V; Mirsaidov, U.; Aksimentiev, A.; Timp, G.

    2009-01-01

    Restriction endonucleases are used prevalently in recombinant DNA technology because they bind so stably to a specific target sequence and, in the presence of cofactors, cleave double-helical DNA specifically at a target sequence at a high rate. Using synthetic nanopores along with molecular dynamics (MD), we have analyzed with atomic resolution how a prototypical restriction endonuclease, EcoRI, binds to the DNA target sequence—GAATTC—in the absence of a Mg2+ ion cofactor. We have previously...

  12. Electrical Detection of TATA Binding Protein at DNA-Modified Microelectrodes

    OpenAIRE

    Gorodetsky, Alon A.; Ebrahim, Ali; Barton, Jacqueline K.

    2008-01-01

    A simple method for the electrochemical detection of TATA-binding protein is demonstrated at DNA-modified microelectrodes. The assay is general and based on the interruption of DNA-mediated charge transport to Nile Blue, a redox-active probe covalently attached to the DNA base pair stack. Nanomolar quantities of TATA binding protein can be detected on the microelectrodes even in the presence of micromolar amounts of bovine serum albumin, EndonucleaseIII, or Bam HI methyltransferase. The schem...

  13. Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.

    Science.gov (United States)

    Luo, G; Lewis, R A

    1992-12-01

    RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.

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

    OpenAIRE

    Olimpo, Jeffrey T.; DeStefano, Jeffrey J

    2010-01-01

    Human immunodeficiency virus reverse transcriptase (HIV-RT) binds more stably in binary complexes with RNA–DNA versus DNA–DNA. Current results indicate that only the -2 and -4 RNA nucleotides (-1 hybridized to the 3′ recessed DNA base) are required for stable binding to RNA–DNA, and even a single RNA nucleotide conferred significantly greater stability than DNA–DNA. Replacing 2′- hydroxyls on pivotal RNA bases with 2′-O-methyls did not affect stability, indicating that interactions between hy...

  15. A Novel Cobalt(Ⅲ) Mixed-polypyridyl Complex: Synthesis,Characterization and DNA Binding

    Institute of Scientific and Technical Information of China (English)

    CHEN,Hui-Li(陈绘丽); YANG,Pin(杨频)

    2002-01-01

    A novel complex[Co(phen)2HPIP]Cl3[phen=phenanethroline,HPIP=2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanethroline]has been synthesized and structurally characterized by elemental analysis,UV,IR and 1H NMR spectroscopies. The interaction of the complex with calf thymus DNA(CT DNA)has been studied using absorption and emission spectroscopy, DNA melting techniques and cyclic voltammetry. The compound shows absorption hypochromicity, fluorescence enhancement and DNA melting temperature increment when binding to CT DNA. CV measurement shows a shift in reduction potential and a change in peak current with addition of DNA.These results prove that the compound inserts into DNA base pairs. The shift of peak potential indicates the ion interaction mode between the complex and DNA. The binding constant of the compound to DNA is 4.37×104. The complex also seems to be an efficient photocleavage reagent.

  16. Specific binding of a dihydropyrimidinone derivative with DNA: Spectroscopic, calorimetric and modeling investigations

    Energy Technology Data Exchange (ETDEWEB)

    Wang Gongke, E-mail: wanggongke@126.com [School of Chemistry and Environmental Science, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China); Yan Changling; Wang Dongchao; Li Dan [School of Chemistry and Environmental Science, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China); Lu Yan, E-mail: yanlu2001@sohu.com [School of Chemistry and Environmental Science, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007 (China)

    2012-07-15

    One of the dihydropyrimidinone derivative 5-(ethoxycarbonyl)-6-methyl-4-(4-methoxyphenyl) -3,4-dihydropyrimidin-2(1H)-one (EMMD) was synthesized, and its binding properties with calf-thymus DNA (ctDNA) were investigated using spectroscopic, viscometric, isothermal titration calorimetric (ITC) and molecular modeling techniques. Fluorescence spectra suggested that the fluorescence enhancement of the binding interaction of EMMD to ctDNA was a static process with ground state complex formation. The binding constant determined with spectroscopic titration and ITC was found to be in the same order of 10{sup 4} M{sup -1}. According to the results of the viscosity analysis, fluorescence competitive binding experiment, fluorescence quenching studies, absorption spectral and ITC investigations, it can be concluded that EMMD is intercalative binding to ctDNA. Furthermore, the results of molecular modeling confirmed those obtained from spectroscopic, viscosimetric and ITC investigations. Additionally, ITC studies also indicated that the binding interaction is predominantly enthalpy driven. - Highlights: Black-Right-Pointing-Pointer Medically important dihydropyrimidinones derivative EMMD is synthesized. Black-Right-Pointing-Pointer EMMD is intercalative binding into ctDNA helix. Black-Right-Pointing-Pointer Hydrogen bonding may play an essential role in the binding of EMCD with ctDNA. Black-Right-Pointing-Pointer This binding interaction is predominantly enthalpy driven.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-15

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

  19. Statistical-mechanical lattice models for protein-DNA binding in chromatin

    CERN Document Server

    Teif, Vladimir B

    2010-01-01

    Statistical-mechanical lattice models for protein-DNA binding are well established as a method to describe complex ligand binding equilibriums measured in vitro with purified DNA and protein components. Recently, a new field of applications has opened up for this approach since it has become possible to experimentally quantify genome-wide protein occupancies in relation to the DNA sequence. In particular, the organization of the eukaryotic genome by histone proteins into a nucleoprotein complex termed chromatin has been recognized as a key parameter that controls the access of transcription factors to the DNA sequence. New approaches have to be developed to derive statistical mechanical lattice descriptions of chromatin-associated protein-DNA interactions. Here, we present the theoretical framework for lattice models of histone-DNA interactions in chromatin and investigate the (competitive) DNA binding of other chromosomal proteins and transcription factors. The results have a number of applications for quant...

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

    Science.gov (United States)

    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

    2015-01-01

    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 × 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. PMID:26068233

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

    Directory of Open Access Journals (Sweden)

    Sinara Mônica Vitalino de Almeida

    2015-06-01

    Full Text Available In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide derivatives (3a–h were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 104 to 1.0 × 106 M−1 and quenching constants from −0.2 × 104 to 2.18 × 104 M−1 indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z-2-(acridin-9-ylmethylene-N- (4-chlorophenyl hydrazinecarbothioamide (3f, while the most active compound in antiproliferative assay was (Z-2-(acridin-9-ylmethylene-N-phenylhydrazinecarbothioamide (3a. There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.

  2. How does a protein reach its binding locus: sliding along DNA chain or not?

    CERN Document Server

    Li, Jingwei

    2016-01-01

    In gene expression, various kinds of proteins need to bind to specific locus of DNA. It is still not clear how these proteins find their target locus. In this study, the mean first-passage time (FPT) of protein binding to its target locus on DNA chain is discussed by a chain-space coupled model. Our results show that the 1-dimensional diffusion constant has a critical value, with which the mean time spent by a protein to find its target locus is almost independent of the binding rate of protein to DNA chain and the detachment rate from DNA chain. Which implies that, the frequency of protein binding to DNA and the sliding time on DNA chain have little influence on the search efficiency, and therefore whether or not the 1-dimensional sliding on DNA chain increases the search efficiency depends on the 1-dimensional diffusion constant of the protein on DNA chain. This study also finds that only protein bindings to DNA loci which are close to the target locus help to increase the search efficiency, while bindings ...

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

    Science.gov (United States)

    Agarwal, Shweta; Chadha, Deepti; Mehrotra, Ranjana

    2015-01-01

    Chloroethyl nitrosoureas constitute an important family of cancer chemotherapeutic agents, used in the treatment of various types of cancer. They exert antitumor activity by inducing DNA interstrand cross-links. Semustine, a chloroethyl nitrosourea, is a 4-methyl derivative of lomustine. There exist some interesting reports dealing with DNA-binding properties of chloroethyl nitrosoureas; however, underlying mechanism of cytotoxicity caused by semustine has not been precisely and completely delineated. The present work focuses on understanding semustine-DNA interaction to comprehend its anti-proliferative action at molecular level using various spectroscopic techniques. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy is used to determine the binding site of semustine on DNA. Conformational transition in DNA after semustine complexation is investigated using circular dichroism (CD) spectroscopy. Stability of semustine-DNA complexes is determined using absorption spectroscopy. Results of the present study demonstrate that semustine performs major-groove-directed DNA alkylation at guanine residues in an incubation-time-drug-concentration-dependent manner. CD spectral outcomes suggest partial transition of DNA from native B-conformation to C-form. Calculated binding constants (Ka) for semustine and lomustine interactions with DNA are 1.53 × 10(3) M(-1) and 8.12 × 10(3) M(-1), respectively. Moreover, molecular modeling simulation is performed to predict preferential binding orientation of semustine with DNA that corroborates well with spectral outcomes. Results based on comparative study of DNA-binding properties of semustine and lomustine, presented here, may establish a correlation between molecular structure and cytotoxicity of chloroethyl nitrosoureas that may be instrumental in the designing and synthesis of new nitrosourea therapeutics possessing better efficacy and fewer side effects. PMID:25350567

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

    Science.gov (United States)

    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.

  5. The role of monogamous bivalency and Fc interactions in the binding of anti-DNA antibodies to DNA antigen.

    Science.gov (United States)

    Stearns, Nancy A; Pisetsky, David S

    2016-05-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus. These antibodies can bind DNA avidly by monogamous bivalency, a mechanism which requires the interaction of both Fab combining regions with antigenic determinants on the same polynucleotide. To explore further this mechanism, we tested Fab and F(ab')2 fragments prepared from IgG from patient plasmas in an ELISA with native DNA antigen, detecting antibody with a peroxidase conjugated anti-Fab reagent. These studies showed that Fab fragments, which can only bind monovalently, had negligible activity. Although bivalent F(ab')2 fragments would be predicted to bind DNA, these fragments also showed poor anti-DNA activity. Control studies showed that the fragments retained antibody activity to tetanus toxoid and an EBV antigen preparation. Together, these findings suggest that anti-DNA avidity depends on monogamous bivalency, with the antibody Fc portion also influencing DNA binding, in a mechanism which can be termed Fc-dependent monogamous bivalency.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  8. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    赵文; 姜志胜; 倪菊华; 陈光慧; 刘乃奎; 汤健; 贾弘褆; 唐朝枢

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sar-coplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

  9. Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2+ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2+-ATPase activity in SR and changing of character of Ca2+ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2+ transport of SR.

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

    Science.gov (United States)

    Warfel, Jaycob D; LiCata, Vince J

    2015-07-01

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

  11. Specific and Efficient Binding of XPA to Double-strand/Single-strand DNA Junctions with 3′- and/or 5′-ssDNA Branches

    OpenAIRE

    Yang, Zhengguan; Roginskaya, Marina; Colis, Laureen C.; Basu, Ashis K.; Shell, Steven M.; Liu, Yiyong; Musich, Phillip R.; Harris, Constance M.; Harris, Thomas M.; Zou, Yue

    2006-01-01

    Human XPA is an important DNA damage recognition protein in nucleotide excision repair (NER). We previously observed that XPA binds to DNA lesion as a homodimer (1). Herein we report that XPA recognized undamaged DNA doublestrand/ single-strand (ds-ssDNA) junctions containing ssDNA branches with binding affinity (Kd = 49.1±5.1 nM) much higher than its ability to bind to DNA damage. The recognized DNA junction structures include Y-shape junction (with both 3′- and 5′- ssDNA branches), 3′-overh...

  12. Identification of a DNA binding protein that recognizes the nonamer recombinational signal sequence of immunoglobulin genes.

    Science.gov (United States)

    Halligan, B D; Desiderio, S V

    1987-10-01

    Extracts of nuclei from B- and T-lymphoid cells contain a protein that binds specifically to the conserved nonamer DNA sequence within the recombinational signals of immunoglobulin genes. Complexes with DNA fragments from four kappa light-chain joining (J) segments have the same electrophoretic mobility. Nonamer-containing DNA fragments from heavy-chain and light-chain genes compete for binding. Within the 5'-flanking DNA of the J kappa 4 gene segment, the binding site has been localized to a 27-base-pair interval spanning the nonamer region. The binding activity is recovered as a single peak after ion-exchange chromatography. The site of binding of the protein and its presence in nuclei of lymphoid cells suggest that it may function in the assembly of immunoglobulin genes.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1996-05-01

    The cationic peptide ligands distamycin and netropsin bind noncovalently to the minor groove of DNA. The binding site, orientation, stoichiometry, and qualitative affinity of distamycin binding to several short DNA oligomers were investigated by NMR spectroscopy. The oligomers studied contain A,T-rich or I,C-rich binding sites, where I = 2-desaminodeoxyguanosine. I{center_dot}C base pairs are functional analogs of A{center_dot}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.

  14. The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase.

    Science.gov (United States)

    Yu, Zhong; Genest, Paul-André; ter Riet, Bas; Sweeney, Kate; DiPaolo, Courtney; Kieft, Rudo; Christodoulou, Evangelos; Perrakis, Anastassis; Simmons, Jana M; Hausinger, Robert P; van Luenen, Henri G A M; Rigden, Daniel J; Sabatini, Robert; Borst, Piet

    2007-01-01

    Trypanosomatids contain an unusual DNA base J (beta-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe(2+) and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe(2+) and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase. PMID:17389644

  15. Spectroscopic probe of the competitive binding of ethidium bromide and neomycin to DNA

    Science.gov (United States)

    Pal, Medini Kanta; Ghosh, Jimut Kanti

    1995-03-01

    The three spectroscopic changes of ethidium bromide (EB) on its binding to DNA, namely red-shift of the νmax, enhancement of fluorescence and induced dichroism are utilized to study the competitive binding of neomycin (NMC) and EB to DNA. Reversion of νmax, decrease in fluorescence and reduction of dichroism of DNA-EB on addition of NMC shows that the binding of NMC and EB to DNA is competitive in nature, over a limited concentration of the polymer. The binding constant of EB-DNA falls from 4.00 × 10 6 to 2.27 × 10 4 1 mol -1 in the presence of added NMC.

  16. Study on the Binding Mode of a Co(Ⅱ) Complex with DNA

    Institute of Scientific and Technical Information of China (English)

    ZHOU Qing-Hua; YANG Pin

    2005-01-01

    The mode of binding of CoLCl2, here L=bis(2-benzimidazolylmethyl)amine, with calf thymus DNA has been investigated by fluorescence measurements, equilibrium dialysis, viscosity experiments and gel electrophoresis. The complex was found to bind but weakly to DNA, with binding constant of 1.96× 104 L/mol determind at 20 ℃ in a solution containing 5 mmol/L Tris-HCl (pH 7.1) and 50 mmol/L NaCl. Polyelectrolyte theory was applied to analyse these values. Viscosity experiments show that binding did not alter the relative viscosity of DNA with any complexes to an appreciable extent. Electrophoresis test displayed that the compound could not cleave the DNA.These results show that the complex is essentially electrostatically bound to DNA.

  17. Cooperative binding of Agrobacterium tumefaciens VirE2 protein to single-stranded DNA.

    Science.gov (United States)

    Sen, P; Pazour, G J; Anderson, D; Das, A

    1989-05-01

    The VirE2 protein of Agrobacterium tumefaciens Ti plasmid pTiA6 is a single-stranded-DNA-binding protein. Density gradient centrifugation studies showed that it exists as a tetramer in solution. Monomeric VirE2 active in DNA binding could also be obtained by using a different protein isolation procedure. VirE2 was found to be thermolabile; brief incubation at 37 degrees C abolished its DNA-binding activity. It was insensitive to the sulfhydryl-specific reagent N-ethylmaleimide. Removal of the carboxy-terminal 37 residues of the 533-residue VirE2 polypeptide led to complete loss of DNA-binding activity; however, chimeric fusion proteins containing up to 125 residues of the VirE2 C terminus were inactive in DNA binding. In nuclease protection studies, VirE2 protected single-stranded DNA against degradation by DNase I. Analysis of the DNA-VirE2 complex by electron microscopy demonstrated that VirE2 coats a single-stranded DNA molecule and that the binding of VirE2 to its substrate is cooperative. PMID:2708313

  18. Early diagnosis of systemic lupus erythmatosus using ANN models of dsDNA binding antibody sequence data

    OpenAIRE

    Bahari, Mohamad Hasan; Mahmoudi, Mahmoud; Azemi, Asad; Mirsalehi, Mir Mojtaba; Khademi, Morteza

    2010-01-01

    In this paper a new method based on artificial neural networks (ANN), is introduced for identifying pathogenic antibodies in Systemic Lupus Erythmatosus (SLE). dsDNA binding antibodies have been implicated in the pathogenesis of this autoimmune disease. In order to identify these dsDNA binding antibodies, the protein sequences of 42 dsDNA binding and 608 non-dsDNA binding antibodies were extracted from Kabat database and encoded using a physicochemical property of their amino acids namely Hyd...

  19. Structure of a Thyroid Hormone Receptor DNA-Binding Domain Homodimer Bound to an Inverted Palindrome DNA Response Element

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yi; Young, Matthew A. (Michigan)

    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.

  20. STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

    Science.gov (United States)

    Bhattacharjee, Anukana; Stewart, Jason; Chaiken, Mary; Price, Carolyn M.

    2016-01-01

    Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress. PMID:27690379

  1. Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

    Energy Technology Data Exchange (ETDEWEB)

    Lindner, Scott E.; De Silva, Erandi K.; Keck, James L.; Llinás, Manuel (Princeton); (UW-MED)

    2010-11-05

    Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan Apetala2 (AP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical Apicomplexan AP2 protein, PF14{_}0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14{_}0633 AP2 domain bound to DNA reveals a {beta}-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent {alpha}-helix supports the {beta}-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened or eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14{_}0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the {alpha}-helices of the AP2 domains pack against the {beta}-sheets of the dimer mates. DNA-induced dimerization of PF14{_}0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multisite binding mode, at least two copies of the consensus sequence recognized by PF14{_}0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.

  2. Dynamic Coupling among Protein Binding, Sliding, and DNA Bending Revealed by Molecular Dynamics.

    Science.gov (United States)

    Tan, Cheng; Terakawa, Tsuyoshi; Takada, Shoji

    2016-07-13

    Protein binding to DNA changes the DNA's structure, and altered DNA structure can, in turn, modulate the dynamics of protein binding. This mutual dependency is poorly understood. Here we investigated dynamic couplings among protein binding to DNA, protein sliding on DNA, and DNA bending by applying a coarse-grained simulation method to the bacterial architectural protein HU and 14 other DNA-binding proteins. First, we verified our method by showing that the simulated HU exhibits a weak preference for A/T-rich regions of DNA and a much higher affinity for gapped and nicked DNA, consistent with biochemical experiments. The high affinity was attributed to a local DNA bend, but not the specific chemical moiety of the gap/nick. The long-time dynamic analysis revealed that HU sliding is associated with the movement of the local DNA bending site. Deciphering single sliding steps, we found the coupling between HU sliding and DNA bending is akin to neither induced-fit nor population-shift; instead they moved concomitantly. This is reminiscent of a cation transfer on DNA and can be viewed as a protein version of polaron-like sliding. Interestingly, on shorter time scales, HU paused when the DNA was highly bent at the bound position and escaped from pauses once the DNA spontaneously returned to a less bent structure. The HU sliding is largely regulated by DNA bending dynamics. With 14 other proteins, we explored the generality and versatility of the dynamic coupling and found that 6 of the 15 assayed proteins exhibit the polaron-like sliding. PMID:27309278

  3. Minor Groove Binding between Norfloxacin and DNA Duplexes in Solution: A Molecular Dynamics Study

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Molecular dynamics were used to investigate the interaction between norfloxacin and DNA duplex. The results showed that norfloxacin was situated in the minor groove of DNA,binding to the TCGA region of d [ATATCGATAT] 2. Specific hydrogen bonds were formed between norfloxacin and guanine base of DNA during the 2 ns MD, which may be the reason for the preferentiality of quinolone antibacterial towards the guanine base of DNA duplex.

  4. Quest for the binding mode of tetrabromobisphenol A with Calf thymus DNA

    Science.gov (United States)

    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.

  5. Deciphering the groove binding modes of tau-fluvalinate and flumethrin with calf thymus DNA

    Science.gov (United States)

    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.

  6. Deciphering the groove binding modes of tau-fluvalinate and flumethrin with calf thymus DNA.

    Science.gov (United States)

    Tao, Mo; Zhang, Guowen; Pan, Junhui; Xiong, Chunhong

    2016-02-15

    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 10(4)Lmol(-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.

  7. Prospects of nanoparticle-DNA binding and its implications in medical biotechnology.

    Science.gov (United States)

    An, Hongjie; Jin, Bo

    2012-01-01

    Bio-nanotechnology is a new interdisciplinary R&D area that integrates engineering and physical science with biology through the development of multifunctional devices and systems, focusing biology inspired processes or their applications, in particular in medical biotechnology. DNA based nanotechnology, in many ways, has been one of the most intensively studied fields in recent years that involves the use and the creation of bio-inspired materials and their technologies for highly selective biosensing, nanoarchitecture engineering and nanoelectronics. Increasing researches have been offered to a fundamental understanding how the interactions between the nanoparticles and DNA molecules could alter DNA molecular structure and its biochemical activities. This minor review describes the mechanisms of the nanoparticle-DNA binding and molecular interactions. We present recent discoveries and research progresses how the nanoparticle-DNA binding could vary DNA molecular structure, DNA detection, and gene therapy. We report a few case studies associated with the application of the nanoparticle-DNA binding devices in medical detection and biotechnology. The potential impacts of the nanoparticles via DNA binding on toxicity of the microorganisms are briefly discussed. The nanoparticle-DNA interactions and their impact on molecular and microbial functionalities have only drown attention in recent a few years. The information presented in this review can provide useful references for further studies on biomedical science and technology.

  8. DNA methylation changes are a late event in acute promyelocytic leukemia and coincide with loss of transcription factor binding

    DEFF Research Database (Denmark)

    Schoofs, Till; Rohde, Christian; Hebestreit, Katja;

    2013-01-01

    . Transcription factor-binding sites (eg, the c-myc-binding sites) were associated with low methylation. However, SUZ12- and REST-binding sites identified in embryonic stem cells were preferentially DNA hypermethylated in APL cells. Unexpectedly, PML-RARα-binding sites were also protected from aberrant DNA...

  9. Direct zonal liquid chromatographic method for the kinetic study of actinomycin-DNA binding.

    Science.gov (United States)

    Vidal-Madjar, Claire; Florentina, Cañada-Cañada; Gherghi, Ioanna; Jaulmes, Alain; Pantazaki, Anastasia; Taverna, Myriam

    2004-07-01

    The binding of an anticancer drug (actinomycin D or ACTD) to double-stranded DNA (dsDNA) was studied by means of high-performance liquid chromatography (HPLC). ACTD is an antitumor antibiotic containing one chromophore group and two pentapeptidic lactone cycles that binds dsDNA. Incubations of ACTD with DNA were performed at physiological pH. The complexed and free ligand concentrations of the mixture were quantified at 440 nm from their separation on a size-exclusion chromatographic (SEC) column using the same buffer for the elution and the sample incubation. The DNA and the ACTD-DNA complexes were eluted at the column exclusion volume while the ligand was retained on the support. An apparent binding curve was obtained by plotting the amount emerging at the exclusion column volume against that eluted at free ACTD retention volume. A dissociating effect was evidenced and the binding parameters were significantly different from those obtained at equilibrium by visible absorbance titration. The equilibrium binding parameters determined by absorption spectroscopy were used as starting data in the numerical simulations of the chromatographic process. The results showed a strong dependency of the apparent binding parameters on the reaction kinetics. Finally the comparison of the apparent binding curve obtained from the HPLC experiments and from the numerical simulations permitted an evaluation of the dissociation rate constant (kd = 0.004 s(-1)). PMID:15296384

  10. The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase

    OpenAIRE

    Yu, Zhong; Genest, Paul-André; ter Riet, Bas; Sweeney, Kate; DiPaolo, Courtney; Kieft, Rudo; Christodoulou, Evangelos; Perrakis, Anastassis; Simmons, Jana M.; Hausinger, Robert P.; van Luenen, Henri G.A.M.; Daniel J Rigden; Sabatini, Robert; Borst, Piet

    2007-01-01

    Trypanosomatids contain an unusual DNA base J (β-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe...

  11. DNA minor groove binding of small molecules: Experimental and computational evidence

    Indian Academy of Sciences (India)

    Prateek Pandya; Md Maidul Islam; G Suresh Kumar; B Jayaram; Surat Kumar

    2010-03-01

    Eight indole derivatives were studied for their DNA binding ability using fluorescence quenching and molecular docking methods. These indole compounds have structural moieties similar as in few indole alkaloids. Experimental and theoretical studies have suggested that indole derivatives bind in the minor groove of DNA. Thermodynamic profiles of DNA complexes of indole derivatives were obtained from computational methods. The complexes were largely stabilized by H-bonding and van der Waal’s forces with positive entropy values. Indole derivatives were found to possess some Purine (Pu) - Pyrimidine (Py) specificity with DNA sequences. The results obtained from experimental and computational methods showed good agreement with each other, supported by their correlation constant values.

  12. Assembly of custom TALE-type DNA binding domains by modular cloning.

    Science.gov (United States)

    Morbitzer, Robert; Elsaesser, Janett; Hausner, Jens; Lahaye, Thomas

    2011-07-01

    Transcription activator-like effector (TALE) DNA binding proteins show tremendous potential as molecular tools for targeted binding to any desired DNA sequence. Their DNA binding domain consists of tandem arranged repeats, and due to this repetitive structure it is challenging to generate designer TALEs (dTALEs) with user-defined specificity. We present a cloning approach that facilitates the assembly of multiple repeat-encoding DNA fragments that translate into dTALEs with pre-defined DNA binding specificity. This method makes use of type IIS restriction enzymes in two sequential cut-ligase reactions to build dTALE repeat arrays. We employed this modular approach for generation of a dTALE that differentiates between two highly similar DNA sequences that are both targeted by the Xanthomonas TALE, AvrBs3. These data show that this modular assembly system allows rapid generation of highly specific TALE-type DNA binding domains that target binding sites of predefined length and sequence. This approach enables the rapid and flexible production of dTALEs for gene regulation and genome editing in routine and high-throughput applications.

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

    Science.gov (United States)

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

    2012-11-01

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

  14. From face to interface recognition: a differential geometric approach to distinguish DNA from RNA binding surfaces.

    Science.gov (United States)

    Shazman, Shula; Elber, Gershon; Mandel-Gutfreund, Yael

    2011-09-01

    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.

  15. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

    Science.gov (United States)

    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.

  16. Patchiness of ion-exchanged mica revealed by DNA binding dynamics at short length scales.

    Science.gov (United States)

    Billingsley, D J; Lee, A J; Johansson, N A B; Walton, A; Stanger, L; Crampton, N; Bonass, W A; Thomson, N H

    2014-01-17

    The binding of double-stranded (ds) DNA to mica can be controlled through ion-exchanging the mica with divalent cations. Measurements of the end-to-end distance of linear DNA molecules discriminate whether the binding mechanism occurs through 2D surface equilibration or kinetic trapping. A range of linear dsDNA fragments have been used to investigate length dependences of binding. Mica, ion-exchanged with Ni(II) usually gives rise to kinetically trapped DNA molecules, however, short linear fragments (mica is heterogeneous, and contains patches or domains, separating different ionic species. These results correlate with imaging of dsDNA under aqueous buffer on Ni(II)-mica and indicate that binding domains are of the order of 100 nm in diameter. Shorter DNA fragments behave intermediate to the two extreme cases of 2D equilibration and kinetic trapping. Increasing the incubation time of Ni(II) on mica, from minutes to hours, brings the conformations of the shorter DNA fragments closer to the theoretical value for kinetic trapping, indicating that long timescale kinetics play a role in ion-exchange. X-ray photoelectron spectroscopy (XPS) was used to confirm that the relative abundance of Ni(II) ions on the mica surface increases with time. These findings can be used to enhance spatial control of binding of DNA to inorganic surfaces with a view to patterning high densities arrays.

  17. Dithiocarbamate/piperazine bridged pyrrolobenzodiazepines as DNA-minor groove binders: synthesis, DNA-binding affinity and cytotoxic activity.

    Science.gov (United States)

    Kamal, Ahmed; Sreekanth, Kokkonda; Shankaraiah, Nagula; Sathish, Manda; Nekkanti, Shalini; Srinivasulu, Vunnam

    2015-04-01

    A new series of C8-linked dithiocarbamate/piperazine bridged pyrrolo[2,1-c][1,4]benzodiazepine conjugates (5a-c, 6a,b) have been synthesized and evaluated for their cytotoxic potential and DNA-binding ability. The representative conjugates 5a and 5b have been screened for their cytotoxicity against a panel of 60 human cancer cell lines. Compound 5a has shown promising cytotoxic activity on selected cancer cell lines that display melanoma, leukemia, CNS, ovarian, breast and renal cancer phenotypes. The consequence of further replacement of the 3-cyano-3,3-diphenylpropyl 1-piperazinecarbodithioate in 5b and 5c with 4-methylpiperazine-1-carbodithioate yielded new conjugates 6a and 6b respectively. In addition, the compounds 5c and 6a,b have been evaluated for their in vitro cytotoxicity on some of the selected human cancer cell lines and these conjugates have exhibited significant cytotoxic activity. Further, the DNA-binding ability of these new conjugates has been evaluated by using thermal denaturation (ΔTm) studies. The correlation between structure and DNA-binding ability has been investigated by molecular modeling studies which predicted that 6b exhibits superior DNA-binding ability and these are in agreement with the experimental DNA-binding studies. PMID:25665519

  18. Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein.

    OpenAIRE

    Keeney, S.; Eker, André; Brody, T.; Vermeulen, Wim; Bootsma, Dirk; Hoeijmakers, Jan; Linn, S.(Florida International University, Miami, USA)

    1994-01-01

    textabstractCells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xerode...

  19. Probing the binding of coumarins and cyclothialidines to DNA gyrase

    DEFF Research Database (Denmark)

    Kampranis, S C; Gormley, N A; Tranter, R;

    1999-01-01

    B and coumarin and cyclothialidine drugs and made mutations by site-directed mutagenesis. We used proteolysis as a probe of drug binding to wild-type and mutant proteins. Limited proteolysis of gyrase revealed that binding of these antibiotics is associated with a characteristic proteolytic fingerprint......, suggesting a drug-induced conformational change. The ability of the mutants to bind the drugs was studied by testing their ability to induce the coumarin-associated proteolytic signature and to bind to a novobiocin-affinity column. To analyze further the interaction of the drugs with gyrase, we studied...

  20. Effects of calmodulin on DNA-binding activity of heat shock transcription factor in vitro

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The DNA-binding activity of heat shock transcription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific to CaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF for binding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar results were obtained with wheat and tomato. Our observations provide the first example of the involvement of CaM in regulation of the DNA-binding activity of the HSF.

  1. Interaction of coumarin with calf thymus DNA: deciphering the mode of binding by in vitro studies.

    Science.gov (United States)

    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.

  2. Binding of radiation-induced phenylalanine radicals to DNA

    International Nuclear Information System (INIS)

    When an aqueous solution of double-stranded DNA of bacteriophage PM2 containing phenylalanine and saturated with N2O is irradiated with γ-rays, radiation-induced phenylalanine radicals are bound covalently. Under the conditions used about 25 phenylalanine molecules may be bound per lethal hit. Also for single-stranded PM2 DNA, most of the phenylalanine radicals bound are non-lethal. Evidence is presented that in double-stranded DNA an appreciable fraction of the single-strand breaks is induced by phenylalanine radicals. Radiation products of phenylalanine and the phenylalanine bound to the DNA decrease the sensitivity of the DNA to the induction of single-strand breaks. There are indications that the high efficiency of protection by radiation products of phenylalanine is due to their positive charge, which will result in a relatively high concentration of these compounds in the vicinity of the negatively charged DNA molecules

  3. Transcriptional Regulation in Mammalian Cells by Sequence-Specific DNA Binding Proteins

    Science.gov (United States)

    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.

  4. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction between 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod-odecane. High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover,the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA condensation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  5. Novel cyclen-based linear polymer as a high-affinity binding material for DNA condensation

    Institute of Scientific and Technical Information of China (English)

    XIANG YongZhe; WANG Na; ZHANG Ji; LI Kun; ZHANG ZhongWei; LIN HongHui; YU XiaoQi

    2009-01-01

    A novel cyclen-based linear polyamine (POGEC) was designed and synthesized from the reaction be-tween 1,3-propanediol diglycidyl ether and 1,7-bis(diethoxyphosphory)-1,4,7,10-tetraazacyclod- odecane.High-affinity binding between POGEC and DNA was demonstrated by agarose gel electrophoresis and scanning electron microscopy (SEM). Moreover, the formed POGEC/DNA complex (termed polyplex) could be disassociated to release the free DNA through addition of the physiological concentration of NaCl solution. Fluorescence spectrum was used to measure the high-affinity binding and DNA con-densation capability of POGEC. Circular dichroism (CD) spectrum indicates that the DNA conformation did not change after binding to POEGC.

  6. DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy.

    Science.gov (United States)

    Kim, Sung Hyun; Kim, So Young; Jung, Kwang-Hwan; Kim, Doseok

    2015-01-01

    Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, K(D), of ASRT to 20 bp-long DNA fragments lied in micro-molar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination.

  7. Mechanism of formation of the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. I. Importance of hydrophobic interactions in stabilization of beta-hairpin structure.

    Science.gov (United States)

    Skwierawska, Agnieszka; Makowska, Joanna; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A

    2009-06-01

    We previously studied a 16-amino acid-residue fragment of the C-terminal beta-hairpin of the B3 domain (residues 46-61), [IG(46-61)] of the immunoglobulin binding protein G from Streptoccocus, and found that hydrophobic interactions and the turn region play an important role in stabilizing the structure. Based on these results, we carried out systematic structural studies of peptides derived from the sequence of IG (46-61) by systematically shortening the peptide by one residue at a time from both the C- and the N-terminus. To determine the structure and stability of two resulting 12- and 14-amino acid-residue peptides, IG(48-59) and IG(47-60), respectively, we carried out circular dichroism, NMR, and calorimetric studies of these peptides in pure water. Our results show that IG(48-59) possesses organized three-dimensional structure stabilized by hydrophobic interactions (Tyr50-Phe57 and Trp48-Val59) at T = 283 and 305 K. At T = 313 K, the structure breaks down because of increased chain entropy, but the turn region is preserved in the same position observed for the structure of the whole protein. The breakdown of structure occurs near the melting temperature of this peptide (T(m) = 310 K) measured by differential scanning calorimetry (DSC). The melting temperature of IG(47-60) determined by DSC is T(m) = 330 K and its structure is similar to that of the native beta-hairpin at all (lower) temperatures examined (283-313 K). Both of these truncated sequences are conserved in all known amino acid sequences of the B domains of the immunoglobulin binding protein G from bacteria. Thus, this study contributes to an understanding of the mechanism of folding of this whole family of proteins, and provides information about the mechanism of formation and stabilization of a beta-hairpin structural element. PMID:19089955

  8. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part III. Dynamics of long-range hydrophobic interactions

    Science.gov (United States)

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2010-01-01

    A 20-residue peptide, IG(42–61), derived from the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using CD, NMR spectroscopy at various temperatures and by differential scanning calorimetry. Unlike other related peptides studied so far, this peptide displays two heat capacity peaks in DSC measurements (at a scanning rate of 1.5 deg/min at a peptide concentration of 0.07mM) which suggests a three-state folding/unfolding process. The results from DSC and NMR measurements suggest the formation of a dynamic network of hydrophobic interactions stabilizing the structure, which resembles a β-hairpin shape over a wide range of temperatures (283 – 313 K). Our results show that IG(42–61) possesses a well-organized three-dimensional structure stabilized by long-range hydrophobic interactions (Tyr50 ··· Phe57 and Trp48 ··· Val59) at T = 283 K and (Trp48 ··· Val59) at 305 and 313 K. The mechanism of β-hairpin folding and unfolding, as well as the influence of peptide length on its conformational properties, are also discussed. PMID:19847914

  9. Mechanism of formation of the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. III. Dynamics of long-range hydrophobic interactions.

    Science.gov (United States)

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A

    2010-02-15

    A 20-residue peptide, IG(42-61), derived from the C-terminal beta-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using circular dichroism, nuclear magnetic resonance (NMR) spectroscopy at various temperatures and by differential scanning calorimetry (DSC). Unlike other related peptides studied so far, this peptide displays two heat capacity peaks in DSC measurements (at a scanning rate of 1.5 deg/min at a peptide concentration of 0.07 mM), which suggests a three-state folding/unfolding process. The results from DSC and NMR measurements suggest the formation of a dynamic network of hydrophobic interactions stabilizing the structure, which resembles a beta-hairpin shape over a wide range of temperatures (283-313 K). Our results show that IG (42-61) possesses a well-organized three-dimensional structure stabilized by long-range hydrophobic interactions (Tyr50 ... Phe57 and Trp48 ... Val59) at T = 283 K and (Trp48 ... Val59) at 305 and 313 K. The mechanism of beta-hairpin folding and unfolding, as well as the influence of peptide length on its conformational properties, are also discussed. PMID:19847914

  10. Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding.

    Science.gov (United States)

    Tomlinson, Christopher G; Moye, Aaron L; Holien, Jessica K; Parker, Michael W; Cohen, Scott B; Bryan, Tracy M

    2015-01-15

    The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3' ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3' region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3' end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight. PMID:25365545

  11. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    Science.gov (United States)

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

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

    KAUST Repository

    Lee, Seung-Joo

    2010-03-28

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

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

    NARCIS (Netherlands)

    Wingender, Edgar

    2013-01-01

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

  14. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray

    KAUST Repository

    Wong, Ka-Chun

    2015-06-11

    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=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build 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 using 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.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    The family of NAC (NAM/ATAF1,2/CUC2) transcription factors has been implicated in a wide range of plant processes, but knowledge on the DNA-binding properties of the family is limited. Using a reiterative selection procedure on random oligonucleotides, we have identified consensus binding sites...

  16. Discovery of selective inhibitors of tyrosyl-DNA phosphodiesterase 2 by targeting the enzyme DNA-binding cleft.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2012-02-01

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

  18. Exploiting anthracene photodimerization within peptides: light induced sequence-selective DNA binding.

    Science.gov (United States)

    Bullen, Gemma A; Tucker, James H R; Peacock, Anna F A

    2015-05-11

    The unprecedented use of anthracene photodimerization within a protein or peptide system is explored through its incorporation into a DNA-binding peptide, derived from the GCN4 transcription factor. This study demonstrates an effective and dynamic interplay between a photoreaction and a peptide-DNA assembly, with each process able to exert control over the other.

  19. Stapling monomeric GCN4 peptides allows for DNA binding and enhanced cellular uptake.

    Science.gov (United States)

    Iyer, Abhishek; Van Lysebetten, Dorien; Ruiz García, Yara; Louage, Benoit; De Geest, Bruno G; Madder, Annemieke

    2015-04-01

    The basic DNA recognition region of the GCN4 protein comprising 23 amino acids has been modified to contain two optimally positioned cysteines which have been linked and stapled using cross-linkers of suitable lengths. This results in stapled peptides with a stabilized α-helical conformation which allows for DNA binding and concurrent enhancement of cellular uptake.

  20. Recognition of methylated DNA through methyl-CpG binding domain proteins

    DEFF Research Database (Denmark)

    Zou, Xueqing; Ma, Wen; Solov'yov, Ilia;

    2012-01-01

    DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although...... the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines...

  1. Applying DNA affinity chromatography to specifically screen for sucrose-related DNA-binding transcriptional regulators of Xanthomonas campestris.

    Science.gov (United States)

    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. PMID:27060555

  2. Binding of 2,7-diaminomitosene to DNA: model for the precovalent recognition of DNA by activated mitomycin C.

    Science.gov (United States)

    Kumar, G S; He, Q Y; Behr-Ventura, D; Tomasz, M

    1995-02-28

    Mitomycin C (MC), mitomycin A, porfiromycin, BMY-25067, and BMY-25287, antitumor antibiotics collectively termed "mitosanes", were found to have no appreciable binding affinity to various natural and synthetic DNAs, as tested by UV spectrophotometry and equilibrium dialysis. Further tests of DNA binding applied to MC including thermal melting measurements, displacement of ethidium fluorescence, and unwinding of closed circular DNA were similarly negative. In contrast, 2,7-diaminomitosene (2,7-DAM), a major end product of the reductive activation of MC, binds to the same series of DNAs by all of these criteria. In the presence of DNA its UV absorbance at the 313 nm maximum decreased and underwent a slight red shift. This effect was used for determining DNA binding constants (Kb) by the spectrophotometric titration method. At pH 6.0 the Kbs of three natural DNAs with varying GC content, as well as poly(dA-dT).poly(dA-dT), and poly(dG-dC).poly(dG-dC), were all in the range of (1.2-5.3) x 10(4) (M nucleotide)-1, with no apparent specificity of binding. Poly(dG-m5dC).poly(dG-m5dC) displayed a slightly higher Kb ((7.5-8.4) x 10(4)). Binding of other, closely related mitosenes was tested to calf thymus DNA by equilibrium dialysis. Neither the presence of a 1-OH substituent, removal of the 10-carbamoyl group, nor methylation of the 2-amino group modifies the binding affinity of the mitosenes significantly. The 1-phosphate substituent abolishes binding. The binding of 2,7-DAM to DNA increased with decreasing pH and decreasing ionic strength. It was determined that 2,7-DAM is protonated at the 2-amino group with a pKa = 7.55, and this correlated well with the observed pH dependence of the binding, indicating that the binding affinity has a strong electrostatic component. This was confirmed by the finding that the extrapolated Kb to 1 M Na+ concentration diminishes to only 10% of the value of Kb at 0.01 M Na+ concentration. Viscosity tests showed conclusively that 2,7-DAM

  3. Atomic-resolution STM structure of DNA and localization of the retinoic acid binding site

    International Nuclear Information System (INIS)

    Single-molecule imaging by scanning tunnelling microscopy (STM) yields the atomic-resolution (0.6 A) structure of individual B-type DNA molecules. The strong correlation between these STM structures and those predicted from the known base sequence indicates that sequencing of single DNA molecules using STM may be feasible. There is excellent agreement between the STM and X-ray structures, but subtle differences exist due to radial distortions. We show that the interactions of other molecules with DNA, their binding configurations, and the structure of these complexes can be studied at the single-molecule level. The anti-cancer drug retinoic acid (RA) binds selectively to the minor groove of DNA with up to 6 RA molecules per DNA turn and with the plane of the RA molecule approximately parallel to the DNA symmetry axis. Similar studies for other drug molecules will be valuable in the a priori evaluation of the effectiveness of anti-cancer drugs

  4. Structural Basis for Telomerase Catalytic Subunit TERT Binding to RNA Template and Telomeric DNA

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

    Science.gov (United States)

    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.

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

    OpenAIRE

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

    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 re...

  7. Characterisation of the DNA binding domain of the yeast RAP1 protein

    OpenAIRE

    Henry, Y A; Chambers, A.; Tsang, J S; Kingsman, A J; Kingsman, S M

    1990-01-01

    The 827 amino acid yeast RAP1 protein interacts with DNA to regulate gene expression at numerous unrelated loci in the yeast genome. By a combination of amino, carboxy and internal deletions, we have defined an internal 235 amino acid fragment of the yeast RAP1 protein that can bind efficiently to the RAP1 binding site of the PGK Upstream Activation Sequence (UAS). This domain spans residues 361 to 596 of the full length protein and lacks any homology to the DNA binding 'zinc finger' or 'heli...

  8. DNA-binding activity in the excretory-secretory products of Trichinella pseudospiralis (Nematoda: Trichinelloidea)

    OpenAIRE

    Mak, CH; Ko, RCC

    2001-01-01

    A novel DNA-binding peptide of Mr approximately 30 kDa was documented for the first time in the excretory-secretory (E-S) products of the infective-stage larvae of Trichinella pseudospiralis. Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E-S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of the Mr of protei...

  9. Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA.

    Science.gov (United States)

    Furukohri, Asako; Nishikawa, Yoshito; Akiyama, Masahiro Tatsumi; Maki, Hisaji

    2012-07-01

    DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.

  10. A small molecule inhibitor of Pot1 binding to telomeric DNA.

    Science.gov (United States)

    Altschuler, Sarah E; Croy, Johnny E; Wuttke, Deborah S

    2012-10-01

    Chromosome ends are complex structures, consisting of repetitive DNA sequence terminating in an ssDNA overhang with many associated proteins. Because alteration of the regulation 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 1), 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 Schizosaccharomyces 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 sub-micromolar affinity. Furthermore, this compound, subsequently identified as the bis-azo dye Congo red (CR), was able to competitively inhibit hPOT1 binding to telomeric DNA. Isothermal titration calorimetry 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.

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

    Science.gov (United States)

    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.

  12. Molecular dynamics simulations and binding free energy analysis of DNA minor groove complexes of curcumin.

    Science.gov (United States)

    Koonammackal, Mathew Varghese; Nellipparambil, Unnikrishnan Viswambharan Nair; Sudarsanakumar, Chellappanpillai

    2011-11-01

    Curcumin is a natural phytochemical that exhibits a wide range of pharmacological properties, including antitumor and anticancer activities. The similarity in the shape of curcumin to DNA minor groove binding drugs is the motivation for exploring its binding affinity in the minor grooves of DNA sequences. Interactions of curcumin with DNA have not been extensively examined, while its pharmacological activities have been studied and documented in depth. Curcumin was docked with two DNA duplexes, d(GTATATAC)(2) and d(CGCGATATCGCG)(2), and molecular dynamics simulations of the complexes were performed in explicit solvent to determine the stability of the binding. In all systems, the curcumin is positioned in the minor groove in the A·T region, and was stably bound throughout the simulation, causing only minor modifications to the structural parameters of DNA. Water molecules were found to contribute to the stability of the binding of the ligand. Free energy analyses of the complexes were performed with MM-PBSA, and the binding affinities that were calculated are comparable to the values reported for other similar nucleic acid-ligand systems, indicating that curcumin is a suitable natural molecule for the development of minor groove binding drugs. PMID:21287216

  13. A physiological role for androgen actions in the absence of androgen receptor DNA binding activity.

    Science.gov (United States)

    Pang, Tammy P S; Clarke, Michele V; Ghasem-Zadeh, Ali; Lee, Nicole K L; Davey, Rachel A; MacLean, Helen E

    2012-01-01

    We tested the hypothesis that androgens have physiological actions via non-DNA binding-dependent androgen receptor (AR) signaling pathways in males, using our genetically modified mice that express a mutant AR with deletion of the 2nd zinc finger of the DNA binding domain (AR(ΔZF2)) that cannot bind DNA. In cultured genital skin fibroblasts, the mutant AR(ΔZF2) has normal ligand binding ability, phosphorylates ERK-1/2 in response to 1 min DHT treatment (blocked by the AR antagonist bicalutamide), but has reduced androgen-dependent nuclear localization compared to wildtype (WT). AR(ΔZF2) males have normal baseline ERK-1/2 phosphorylation, with a 1.5-fold increase in Akt phosphorylation in AR(ΔZF2) muscle vs WT. To identify physiological actions of non-DNA binding-dependent AR signaling, AR(ΔZF2) males were treated for 6 weeks with dihydrotestosterone (DHT). Cortical bone growth was suppressed by DHT in AR(ΔZF2) mice (6% decrease in periosteal and 7% decrease in medullary circumference vs untreated AR(ΔZF2) males). In conclusion, these data suggest that non-DNA binding dependent AR actions suppress cortical bone growth, which may provide a mechanism to fine-tune the response to androgens in bone.

  14. Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme.

    Science.gov (United States)

    Viswanathan, Ramya; True, Jason D; Auble, David T

    2016-07-22

    The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short single-stranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.

  15. Binding of an anticancer Rutaceae plant flavonoid glycoside with calf thymus DNA: Biophysical and electrochemical studies

    International Nuclear Information System (INIS)

    In the present work, we report the interaction of a bioactive Rutaceae plant flavonoid glycoside, diosmin (DIO) with calf thymus DNA employing ethidium bromide as a fluorescence probe. The mode of binding between DIO and DNA was investigated by UV absorption, fluorescence, 3D-fluorescence, fluorescence polarization, FT-IR, circular dichroism, melting temperature (Tm) measurements and differential pulse voltammogram studies. The results revealed the intercalative mode of binding between DIO and DNA. Further, the values of thermodynamic parameters, ∆H° (−388.32 kJ mol−1) and ∆S° (−1.22 kJ mol−1 K−1) indicated that the van der Waals forces and hydrogen bond played a major role in the binding of DIO to DNA. The observed negative ∆G° values revealed the spontaneity of interaction process. The binding of DIO to DNA–EB was found to be stronger in the presence of coexisting substances. -- Highlights: • Mechanism of interaction of diosmin with DNA was studied by spectroscopic methods. • Ethidium bromide was used as a fluorescence probe in the present study. • The van der Waals forces and hydrogen bond played a significant role in the interaction. • Intercalative mode of binding was proposed between DIO and DNA

  16. Binding of an anticancer Rutaceae plant flavonoid glycoside with calf thymus DNA: Biophysical and electrochemical studies

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishnan, Sandhya; Jaldappagari, Seetharamappa, E-mail: jseetharam@yahoo.com

    2013-10-15

    In the present work, we report the interaction of a bioactive Rutaceae plant flavonoid glycoside, diosmin (DIO) with calf thymus DNA employing ethidium bromide as a fluorescence probe. The mode of binding between DIO and DNA was investigated by UV absorption, fluorescence, 3D-fluorescence, fluorescence polarization, FT-IR, circular dichroism, melting temperature (T{sub m}) measurements and differential pulse voltammogram studies. The results revealed the intercalative mode of binding between DIO and DNA. Further, the values of thermodynamic parameters, ∆H° (−388.32 kJ mol{sup −1}) and ∆S° (−1.22 kJ mol{sup −1} K{sup −1}) indicated that the van der Waals forces and hydrogen bond played a major role in the binding of DIO to DNA. The observed negative ∆G° values revealed the spontaneity of interaction process. The binding of DIO to DNA–EB was found to be stronger in the presence of coexisting substances. -- Highlights: • Mechanism of interaction of diosmin with DNA was studied by spectroscopic methods. • Ethidium bromide was used as a fluorescence probe in the present study. • The van der Waals forces and hydrogen bond played a significant role in the interaction. • Intercalative mode of binding was proposed between DIO and DNA.

  17. DNA Binding Proteins and Drug Delivery Vehicles: Tales of Elephants and Snakes.

    Science.gov (United States)

    Karpel, Richard L

    2015-01-01

    We compare the DNA-interactive properties of bacteriophage T4 gene 32 protein (gp32) with those of crotamine, a component of the venom of the South American rattlesnake. Gene 32 protein is a classical single-stranded DNA binding protein that has served as a model for this class of proteins. We discuss its biological functions, structure, binding specificities, and how it controls its own expression. In addition, we delineate the roles of the structural domains of gp32 and how they regulate the protein's various activities. Crotamine, a component of the venom of the South American rattlesnake, is probably not a DNA binding protein in nature, but clearly shows significant DNA binding in vitro. Crotamine has been shown to selectively disrupt rapidly dividing cells and this specificity has been demonstrated for crotamine-facilitated delivery of plasmid DNA Thus, crotamine, or a variant of the protein, could have important clinical and/or diagnostic roles. Understanding its DNA binding properties may therefore lead to more effective drug delivery vehicles.

  18. Ephemeral protein binding to DNA shapes stable nuclear bodies and chromatin domains

    CERN Document Server

    Brackley, C A; Michieletto, D; Mouvet, F; Cook, P R; Marenduzzo, D

    2016-01-01

    Fluorescence microscopy reveals that the contents of many (membrane-free) nuclear "bodies" exchange rapidly with the soluble pool whilst the underlying structure persists; such observations await a satisfactory biophysical explanation. To shed light on this, we perform large-scale Brownian dynamics simulations of a chromatin fiber interacting with an ensemble of (multivalent) DNA-binding proteins; these proteins switch between two states -- active (binding) and inactive (non-binding). This system provides a model for any DNA-binding protein that can be modified post-translationally to change its affinity for DNA (e.g., like the phosphorylation of a transcription factor). Due to this out-of-equilibrium process, proteins spontaneously assemble into clusters of self-limiting size, as individual proteins in a cluster exchange with the soluble pool with kinetics like those seen in photo-bleaching experiments. This behavior contrasts sharply with that exhibited by "equilibrium", or non-switching, proteins that exis...

  19. High-throughput prediction of RNA, DNA and protein binding regions mediated by intrinsic disorder.

    Science.gov (United States)

    Peng, Zhenling; Kurgan, Lukasz

    2015-10-15

    Intrinsically disordered proteins and regions (IDPs and IDRs) lack stable 3D structure under physiological conditions in-vitro, are common in eukaryotes, and facilitate interactions with RNA, DNA and proteins. Current methods for prediction of IDPs and IDRs do not provide insights into their functions, except for a handful of methods that address predictions of protein-binding regions. We report first-of-its-kind computational method DisoRDPbind for high-throughput prediction of RNA, DNA and protein binding residues located in IDRs from protein sequences. DisoRDPbind is implemented using a runtime-efficient multi-layered design that utilizes information extracted from physiochemical properties of amino acids, sequence complexity, putative secondary structure and disorder and sequence alignment. Empirical tests demonstrate that it provides accurate predictions that are competitive with other predictors of disorder-mediated protein binding regions and complementary to the methods that predict RNA- and DNA-binding residues annotated based on crystal structures. Application in Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila melanogaster proteomes reveals that RNA- and DNA-binding proteins predicted by DisoRDPbind complement and overlap with the corresponding known binding proteins collected from several sources. Also, the number of the putative protein-binding regions predicted with DisoRDPbind correlates with the promiscuity of proteins in the corresponding protein-protein interaction networks. Webserver: http://biomine.ece.ualberta.ca/DisoRDPbind/.

  20. Obesity risk gene TMEM18 encodes a sequence-specific DNA-binding protein.

    Directory of Open Access Journals (Sweden)

    Jaana M Jurvansuu

    Full Text Available Transmembrane protein 18 (TMEM18 has previously been connected to cell migration and obesity. However, the molecular function of the protein has not yet been described. Here we show that TMEM18 localises to the nuclear membrane and binds to DNA in a sequence-specific manner. The protein binds DNA with its positively charged C-terminus that contains also a nuclear localisation signal. Increase in the amount of TMEM18 in cells suppresses expression from a reporter vector with the TMEM18 target sequence. TMEM18 is a small protein of 140 residues and is predicted to be mostly alpha-helical with three transmembrane parts. As a consequence the DNA binding by TMEM18 would bring the chromatin very near to nuclear membrane. We speculate that this closed perinuclear localisation of TMEM18-bound DNA might repress transcription from it.

  1. Studies on a Novel Minor-groove Targeting Artificial Nuclease: Synthesis and DNA Binding Behavior

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nucleases play an important role in molecular biology, for example, in DNA sequencing. Synthetic polyamide conjugates can be considered as a novel tool for the selective inhibition of gene expressions and also as potential drugs in anticancer or antiviral chemotherapy. In this article, the synthesis of a novel minor-groove targeting artificial nuclease, an oligopyrrol-containing compound, has been reported. It was found that this novel compound can bind DNA in AT-rich minor groove with high affinity and site specificity. DNA binding behavior was determined by using UV-Vis and CD. It is indicated that compound 6 can enhance the Tm of DNA from 80. 4 C to 84. 4 ℃ and that it possesses a high binding constant value(Kb = 3.05×104 L/mol).

  2. Relevance of arginines in the mode of binding of H1 histones to DNA.

    Science.gov (United States)

    Piscopo, Marina; Conte, Mariachiara; Di Paola, Flaviano; Conforti, Salvatore; Rana, Gina; De Petrocellis, Luciano; Fucci, Laura; Geraci, Giuseppe

    2010-07-01

    The mode of binding of sperm and somatic H1 histones to DNA has been investigated by analyzing the effect of their addition on the electrophoretic mobility of linear and circular plasmid molecules. Low concentrations of sperm histones do not appear to alter the electrophoretic mobility of DNA, whereas at increasing concentrations, an additional DNA band is observed near the migration origin. This band then becomes the only component at higher values. In contrast, somatic histones cause a gradual retardation in the mobility of the DNA band at low concentrations and aggregated structures are observed only at higher values. Experiments on the H1 globular domain obtained by limited proteolysis indicate that the mode of binding to DNA depends on the H1 globular domain. The arginine residues appear to be relevant for the different effects as indicated by experiments on sperm histone and on protamine with arginines deguanidinated to ornithines. The modified molecules influence DNA mobility like somatic H1s, indicating that the positive guanidino groups of arginines cannot be substituted by the positive amino groups of ornithines. Modifications of the amino groups of lysines show that these residues are necessary for the binding of H1 histones to DNA but they have no influence on the binding mode. PMID:20438368

  3. Generalizing and learning protein-DNA binding sequence representations by an evolutionary algorithm

    KAUST Repository

    Wong, Kachun

    2011-02-05

    Protein-DNA bindings are essential activities. Understanding them forms the basis for further deciphering of biological and genetic systems. In particular, the protein-DNA bindings between transcription factors (TFs) and transcription factor binding sites (TFBSs) play a central role in gene transcription. Comprehensive TF-TFBS binding sequence pairs have been found in a recent study. However, they are in one-to-one mappings which cannot fully reflect the many-to-many mappings within the bindings. An evolutionary algorithm is proposed to learn generalized representations (many-to-many mappings) from the TF-TFBS binding sequence pairs (one-to-one mappings). The generalized pairs are shown to be more meaningful than the original TF-TFBS binding sequence pairs. Some representative examples have been analyzed in this study. In particular, it shows that the TF-TFBS binding sequence pairs are not presumably in one-to-one mappings. They can also exhibit many-to-many mappings. The proposed method can help us extract such many-to-many information from the one-to-one TF-TFBS binding sequence pairs found in the previous study, providing further knowledge in understanding the bindings between TFs and TFBSs. © 2011 Springer-Verlag.

  4. Zinc-induced interaction of the metal-binding domain of amyloid-β peptide with DNA.

    Science.gov (United States)

    Khmeleva, Svetlana A; Mezentsev, Yuri V; Kozin, Sergey A; Tsvetkov, Philipp O; Ivanov, Alexis S; Bodoev, Nikolay V; Makarov, Alexander A; Radko, Sergey P

    2013-01-01

    The interaction of the 16-mer synthetic peptide (Aβ16), which represents the metal-binding domain of the amyloid-β with DNA, was studied employing the surface plasmon resonance technique. It has been shown that Aβ16 binds to the duplex DNA in the presence of zinc ions and thus the metal-binding domain can serve as a zinc-dependent DNA-binding site of the amyloid-β. The interaction of Aβ16 with DNA most probably depends on oligomerization of the peptide and is dominated by interaction with phosphates of the DNA backbone.

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

    Directory of Open Access Journals (Sweden)

    Ioudinkova E. S.

    2009-12-01

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

  6. The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.; Volkert, Michael R.

    2004-02-01

    Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

  7. Both HMG boxes in Hmo1 are essential for DNA binding in vitro and in vivo.

    Science.gov (United States)

    Higashino, Ayako; Shiwa, Yuh; Yoshikawa, Hirofumi; Kokubo, Tetsuro; Kasahara, Koji

    2015-01-01

    Hmo1, a member of the high mobility group B family proteins in Saccharomyces cerevisiae, associates with the promoters of ribosomal protein genes (RPGs) to direct accurate transcriptional initiation. Here, to identify factors involved in the binding of Hmo1 to its targets and the mechanism of Hmo1-dependent transcriptional initiation, we developed a novel reporter system using the promoter of the RPG RPS5. A genetic screen did not identify any factors that influence Hmo1 binding, but did identify a number of mutations in Hmo1 that impair its DNA binding activity in vivo and in vitro. These results suggest that Hmo1 binds to its target promoters autonomously without any aid of additional factors. Furthermore, characterization of Hmo1 mutants showed that the box A domain plays a pivotal role in DNA binding and may be required for the recognition of structural properties of target promoters that occur in native chromatin. PMID:25410521

  8. Differential modes of DNA binding by mismatch uracil DNA glycosylase from Escherichia coli: implications for abasic lesion processing and enzyme communication in the base excision repair pathway

    OpenAIRE

    Grippon, Seden; Zhao, Qiyuan; Robinson, Tom; Marshall, Jacqueline J. T.; O’Neill, Rory J.; Manning, Hugh; Kennedy, Gordon; Dunsby, Christopher; Neil, Mark; Halford, Stephen E.; French, Paul M. W.; Baldwin, Geoff S.

    2010-01-01

    Mismatch uracil DNA glycosylase (Mug) from Escherichia coli is an initiating enzyme in the base-excision repair pathway. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. Since Mug is known to bind its product tightly, inhibiting enzyme turnover, understanding how Mug binds DNA is of significance when considering how Mug interacts with downstream enzymes in the base-excision repair pathway. We have demonstrated differential binding modes o...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-06-01

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

  10. False-positive myeloperoxidase binding activity due to DNA/anti-DNA antibody complexes: a source for analytical error in serologic evaluation of anti-neutrophil cytoplasmic autoantibodies.

    Science.gov (United States)

    Jethwa, H S; Nachman, P H; Falk, R J; Jennette, J C

    2000-09-01

    Anti-myeloperoxidase antibodies (anti-MPO) are a major type of anti-neutrophil cytoplasmic antibody (ANCA). While evaluating anti-MPO monoclonal antibodies from SCG/Kj mice, we observed several hybridomas that appeared to react with both MPO and DNA. Sera from some patients with systemic lupus erythematosus (SLE) also react with MPO and DNA. We hypothesized that the MPO binding activity is a false-positive result due to the binding of DNA, contained within the antigen binding site of anti-DNA antibodies, to the cationic MPO. Antibodies from tissue culture supernatants from 'dual reactive' hybridomas were purified under high-salt conditions (3 M NaCl) to remove any antigen bound to antibody. The MPO and DNA binding activity were measured by ELISA. The MPO binding activity was completely abrogated while the DNA binding activity remained. The MPO binding activity was restored, in a dose-dependent manner, by the addition of increasing amount of calf-thymus DNA (CT-DNA) to the purified antibody. Sera from six patients with SLE that reacted with both MPO and DNA were treated with DNase and showed a decrease in MPO binding activity compared with untreated samples. MPO binding activity was observed when CT-DNA was added to sera from SLE patients that initially reacted with DNA but not with MPO. These results suggest that the DNA contained within the antigen binding site of anti-DNA antibodies could bind to the highly cationic MPO used as substrate antigen in immunoassays, resulting in a false-positive test.

  11. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hah, S S; Stivers, K M; Vere White, R; Henderson, P T

    2005-12-29

    Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.

  12. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part IV. Implication for the mechanism of folding of the parent protein

    Science.gov (United States)

    Lewandowska, Agnieszka; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2010-01-01

    A 34-residue α/β peptide, [IG(28-61)], derived from the C-terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus was studied using CD and NMR spectroscopy at various temperatures, and by differential scanning calorimetry. It was found that the C-terminal part (a 16-residue-long fragment) of this peptide, which corresponds to the sequence of the β-hairpin in the native structure, forms structure similar to the β-hairpin only at T = 313 K, and the structure is stabilized by non-native long-range hydrophobic interactions (Val47 – Val59). On the other hand, the N-terminal part of IG(28-61), which corresponds to the middle α-helix in the native structure, is unstructured at low temperature (283 K), and forms an α-helix-like structure at 305 K and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305 and 313 K), we do not observe any long-range connectivities which would have supported packing between the C-terminal (β-hairpin) and the N-terminal (α-helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Koliński [Kmiecik, S.; Kolinski, A. Biophys J 2008, 94, 726-736], based on Monte Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. PMID:20049918

  13. Mechanism of formation of the C-terminal beta-hairpin of the B3 domain of the immunoglobulin-binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein.

    Science.gov (United States)

    Lewandowska, Agnieszka; Ołdziej, Stanislaw; Liwo, Adam; Scheraga, Harold A

    2010-05-01

    A 34-residue alpha/beta peptide [IG(28-61)], derived from the C-terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C-terminal part (a 16-residue-long fragment) of this peptide, which corresponds to the sequence of the beta-hairpin in the native structure, forms structure similar to the beta-hairpin only at T = 313 K, and the structure is stabilized by non-native long-range hydrophobic interactions (Val47-Val59). On the other hand, the N-terminal part of IG(28-61), which corresponds to the middle alpha-helix in the native structure, is unstructured at low temperature (283 K) and forms an alpha-helix-like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long-range connectivities which would have supported packing between the C-terminal (beta-hairpin) and the N-terminal (alpha-helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726-736), based on Monte-Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. PMID:20049918

  14. Structural and dynamic properties of linker histone H1 binding to DNA

    CERN Document Server

    Dootz, Rolf; Pfohl, Thomas

    2010-01-01

    Found in all eukaryotic cells, linker histones H1 are known to bind to and rearrange nucleosomal linker DNA. In vitro, the fundamental nature of H1/DNA interactions has attracted wide interest among research communities - for biologists from a chromatin organization deciphering point of view, and for physicists from the study of polyelectrolyte interactions point of view. Hence, H1/DNA binding processes, structural and dynamical information about these self-assemblies is of broad importance. Targeting a quantitative understanding of H1 induced DNA compaction mechanisms our strategy is based on using small angle X-ray microdiffraction in combination with microfluidics. The usage of microfluidic hydrodynamic focusing devices facilitate a microscale control of these self-assembly processes. In addition, the method enables time-resolved access to structure formation in situ, in particular to transient intermediate states. The observed time dependent structure evolution shows that the interaction of H1 with DNA ca...

  15. Mitochondrial transcription termination factor 2 binds to entire mitochondrial DNA and negatively regulates mitochondrial gene expression

    Institute of Scientific and Technical Information of China (English)

    Weiwei Huang; Min Yu; Yang Jiao; Jie Ma; Mingxing Ma; Zehua Wang; Hong Wu; Deyong Tan

    2011-01-01

    Mitochondrial transcription termination factor 2 (mTERF2) is a mitochondriai matrix protein that binds to the mitochondriai DNA.Previous studies have shown that overexpression of mTERF2 can inhibit cell proliferation, but the mechanism has not been well defined so far.This study aimed to present the binding pattern of mTERF2 to the mitochondrial DNA (mtDNA) in vivo, and investigated the biological function of mTERF2 on the replication of mtDNA, mRNA transcription, and protein translation.The mTERF2 binding to entire mtDNA was identified via the chromatin immunoprecipitation analysis.The mtDNA replication efficiency and expression levels of mitochondria genes were significantly inhibited when the mTERF2 was overexpressed in HeLa cells.The inhibition level of mtDNA content was the same with the decreased levels of mRNA and mitochondrial protein expression.Overall, the mTERF2 might be a cell growth inhibitor based on its negative effect on mtDNA replication, which eventually own-regulated all of the oxidative phosphorylation components in the mitochondria that were essential for the cell's energy metabolism.

  16. Kinetics and binding of the thymine-DNA mismatch glycosylase, Mig-Mth, with mismatch-containing DNA substrates.

    Science.gov (United States)

    Begley, Thomas J; Haas, Brian J; Morales, Juan C; Kool, Eric T; Cunningham, Richard P

    2003-01-01

    We have examined the removal of thymine residues from T-G mismatches in DNA by the thymine-DNA mismatch glycosylase from Methanobacterium thermoautrophicum (Mig-Mth), within the context of the base excision repair (BER) pathway, to investigate why this glycosylase has such low activity in vitro. Using single-turnover kinetics and steady-state kinetics, we calculated the catalytic and product dissociation rate constants for Mig-Mth, and determined that Mig-Mth is inhibited by product apyrimidinic (AP) sites in DNA. Electrophoretic mobility shift assays (EMSA) provide evidence that the specificity of product binding is dependent upon the base opposite the AP site. The binding of Mig-Mth to DNA containing the non-cleavable substrate analogue difluorotoluene (F) was also analyzed to determine the effect of the opposite base on Mig-Mth binding specificity for substrate-like duplex DNA. The results of these experiments support the idea that opposite strand interactions play roles in determining substrate specificity. Endonuclease IV, which cleaves AP sites in the next step of the BER pathway, was used to analyze the effect of product removal on the overall rate of thymine hydrolysis by Mig-Mth. Our results support the hypothesis that endonuclease IV increases the apparent activity of Mig-Mth significantly under steady-state conditions by preventing reassociation of enzyme to product. PMID:12509271

  17. Surface shapes and surrounding environment analysis of single- and double-stranded DNA-binding proteins in protein-DNA interface.

    Science.gov (United States)

    Wang, Wei; Liu, Juan; Sun, Lin

    2016-07-01

    Protein-DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double-stranded DNA-binding proteins (DSBs) and single-stranded DNA-binding proteins (SSBs) in protein-DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single-stranded DNA) or dsDNA (double-stranded DNA). Proteins 2016; 84:979-989. © 2016 Wiley Periodicals, Inc.

  18. Bayesian Unsupervised Learning of DNA Regulatory Binding Regions

    Directory of Open Access Journals (Sweden)

    Jukka Corander

    2009-01-01

    positions within a set of DNA sequences are very rare in the literature. Here we show how such a learning problem can be formulated using a Bayesian model that targets to simultaneously maximize the marginal likelihood of sequence data arising under multiple motif types as well as under the background DNA model, which equals a variable length Markov chain. It is demonstrated how the adopted Bayesian modelling strategy combined with recently introduced nonstandard stochastic computation tools yields a more tractable learning procedure than is possible with the standard Monte Carlo approaches. Improvements and extensions of the proposed approach are also discussed.

  19. Role of DNA binding sites and slow unbinding kinetics in titration-based oscillators.

    Science.gov (United States)

    Karapetyan, Sargis; Buchler, Nicolas E

    2015-12-01

    Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ultrasensitivity that is specific for repression. We then use stochastic simulation to show that multiple binding sites increase the coherence of oscillations by mitigating the binary noise. Slow values of DNA unbinding rate are also effective in alleviating molecular noise due to the increased distance from the bifurcation point. Our work demonstrates how the number of DNA binding sites and slow unbinding kinetics, which are often omitted in biophysical models of gene circuits, can have a significant impact on the temporal and stochastic dynamics of genetic oscillators.

  20. Role of DNA binding sites and slow unbinding kinetics in titration-based oscillators

    Science.gov (United States)

    Karapetyan, Sargis; Buchler, Nicolas E.

    2015-12-01

    Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ultrasensitivity that is specific for repression. We then use stochastic simulation to show that multiple binding sites increase the coherence of oscillations by mitigating the binary noise. Slow values of DNA unbinding rate are also effective in alleviating molecular noise due to the increased distance from the bifurcation point. Our work demonstrates how the number of DNA binding sites and slow unbinding kinetics, which are often omitted in biophysical models of gene circuits, can have a significant impact on the temporal and stochastic dynamics of genetic oscillators.

  1. Evaluation of DNA binding, DNA cleavage, protein binding, radical scavenging and in vitro cytotoxic activities of ruthenium(II) complexes containing 2,4-dihydroxy benzylidene ligands.

    Science.gov (United States)

    Mohanraj, Maruthachalam; Ayyannan, Ganesan; Raja, Gunasekaran; Jayabalakrishnan, Chinnasamy

    2016-12-01

    The new ruthenium(II) complexes with hydrazone ligands, 4-Methyl-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(1)), 4-Methoxy-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(2)), 4-Bromo-benzoic acid (2,4-dihydroxy-benzylidene)-hydrazide (HL(3)), were synthesized and characterized by various spectro analytical techniques. The molecular structures of the ligands were confirmed by single crystal X-ray diffraction technique. The DNA binding studies of the ligands and complexes were examined by absorption, fluorescence, viscosity and cyclic voltammetry methods. The results indicated that the ligands and complexes could interact with calf thymus DNA (CT-DNA) through intercalation. The DNA cleavage activity of the complexes was evaluated by gel electrophoresis assay, which revealed that the complexes are good DNA cleaving agents. The binding interaction of the ligands and complexes with bovine serum albumin (BSA) was investigated using fluorescence spectroscopic method. Antioxidant studies showed that the complexes have a strong radical scavenging properties. Further, the cytotoxic effect of the complexes examined on cancerous cell lines showed that the complexes exhibit significant anticancer activity. PMID:27612830

  2. DNA-MATRIX: a tool for constructing transcription factor binding sites Weight matrix

    Directory of Open Access Journals (Sweden)

    Chandra Prakash Singh,

    2009-12-01

    Full Text Available Despite considerable effort to date, DNA transcription factor binding sites prediction in whole genome remains a challenge for the researchers. Currently the genome wide transcription factor binding sites prediction tools required either direct pattern sequence or weight matrix. Although there are known transcription factor binding sites pattern databases and tools for genome level prediction but no tool for weight matrix construction. Considering this, we developed a DNA-MATRIX tool for searching putative transcription factor binding sites in genomic sequences. DNA-MATRIX uses the simple heuristic approach for weight matrix construction, which can be transformed into different formats as per the requirement of researcher’s for further genome wide prediction and therefore provides the possibility to identify the conserved known DNA binding sites in the coregulated genes and also to search for a great variety of different regulatory binding patterns. The user may construct and save specific weight or frequency matrices in different formats derived through user selected set of known motif sequences.

  3. Thermodynamics of the DNA binding of biogenic polyamines: Calorimetric and spectroscopic investigations

    International Nuclear Information System (INIS)

    Highlights: ► DNA binding of spermine > spermidine >> putrescine > cadaverine. ► Binding of the polyamines stabilized the DNA melting temperature by 6–17 K. ► Binding was entropy driven with small enthalpy contribution at all temperatures. ► Electrostatic interaction is the major contributing force to the Gibbs energy term. ► Small but negative heat capacity changes indicate the role of hydrophobic forces. - Abstract: The thermodynamics of the reaction of biogenic polyamines spermine, spermidine, putrescine and cadaverine with calf thymus DNA was studied by thermal melting, differential scanning calorimetry and isothermal titration calorimetry experiments. These results were supplemented by ethidium bromide displacement and circular dichroism experiments. Melting studies show enhanced stabilization of DNA melting temperature by spermine (17 K) > spermidine (11 K) > putrescine (7 K) > cadaverine (6 K). The binding affinity of the polyamines to DNA as determined from calorimetry experiments was in the order, spermine > spermidine > putrescine > cadaverine with KiSPM = 6.20 × 105 M−1, KiSPD = 3.10 × 105 M−1, KiPUT = 1.80 × 104 M−1, and KiCAD = 1.60 × 104 M−1 at T = 293.15 K, which correlated with their increasing number of positive charges. The trend in the binding affinity was also in agreement with the IC50 values of ethidium bromide displacement ability and circular dichroism perturbations. Absorbance and circular dichroism studies showed perturbation of DNA conformation within the B-form by spermine to be the highest and that by cadaverine to be the least. The binding of all the four polyamines was entropy driven with small enthalpy contributions that were unfavourable. Electrostatic interaction is judged to be the major contributing force to the Gibbs energy term. The heat capacity values denote some extent of hydrophobic interaction between the polyamines and DNA.

  4. gDNA-Prot: Predict DNA-binding proteins by employing support vector machine and a novel numerical characterization of protein sequence.

    Science.gov (United States)

    Zhang, Yan-Ping; Wuyunqiqige; Zheng, Wei; Liu, Shuyi; Zhao, Chunguang

    2016-10-01

    DNA-binding proteins are the functional proteins in cells, which play an important role in various essential biological activities. An effective and fast computational method gDNA-Prot is proposed to predict DNA-binding proteins in this paper, which is a DNA-binding predictor that combines the support vector machine classifier and a novel kind of feature called graphical representation. The DNA-binding protein sequence information was described with the 20 probabilities of amino acids and the 23 new numerical graphical representation features of a protein sequence, based on 23 physicochemical properties of 20 amino acids. The Principal Components Analysis (PCA) was employed as feature selection method for removing the irrelevant features and reducing redundant features. The Sigmod function and Min-max normalization methods for PCA were applied to accelerate the training speed and obtain higher accuracy. Experiments demonstrated that the Principal Components Analysis with Sigmod function generated the best performance. The gDNA-Prot method was also compared with the DNAbinder, iDNA-Prot and DNA-Prot. The results suggested that gDNA-Prot outperformed the DNAbinder and iDNA-Prot. Although the DNA-Prot outperformed gDNA-Prot, gDNA-Prot was faster and convenient to predict the DNA-binding proteins. Additionally, the proposed gNDA-Prot method is available at http://sourceforge.net/projects/gdnaprot.

  5. gDNA-Prot: Predict DNA-binding proteins by employing support vector machine and a novel numerical characterization of protein sequence.

    Science.gov (United States)

    Zhang, Yan-Ping; Wuyunqiqige; Zheng, Wei; Liu, Shuyi; Zhao, Chunguang

    2016-10-01

    DNA-binding proteins are the functional proteins in cells, which play an important role in various essential biological activities. An effective and fast computational method gDNA-Prot is proposed to predict DNA-binding proteins in this paper, which is a DNA-binding predictor that combines the support vector machine classifier and a novel kind of feature called graphical representation. The DNA-binding protein sequence information was described with the 20 probabilities of amino acids and the 23 new numerical graphical representation features of a protein sequence, based on 23 physicochemical properties of 20 amino acids. The Principal Components Analysis (PCA) was employed as feature selection method for removing the irrelevant features and reducing redundant features. The Sigmod function and Min-max normalization methods for PCA were applied to accelerate the training speed and obtain higher accuracy. Experiments demonstrated that the Principal Components Analysis with Sigmod function generated the best performance. The gDNA-Prot method was also compared with the DNAbinder, iDNA-Prot and DNA-Prot. The results suggested that gDNA-Prot outperformed the DNAbinder and iDNA-Prot. Although the DNA-Prot outperformed gDNA-Prot, gDNA-Prot was faster and convenient to predict the DNA-binding proteins. Additionally, the proposed gNDA-Prot method is available at http://sourceforge.net/projects/gdnaprot. PMID:27378005

  6. Binding and entry of DNA in bacterial transformation

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1976-01-01

    Bacterial transformation in relation to DNA transport and competence in Streptococcus pneumoniae (also called Diplococcus pneumoniae) is discussed. This species will serve as a model with which to compare transformation in other bacterial species, particularly Bacillus subtilis and Haemophilus influenzae, with emphasis on the many similarities as well as differences.

  7. Nuc-ErbB3 regulates H3K27me3 levels and HMT activity to establish epigenetic repression during peripheral myelination.

    Science.gov (United States)

    Ness, Jennifer K; Skiles, Amanda A; Yap, Eng-Hui; Fajardo, Eduardo J; Fiser, Andras; Tapinos, Nikos

    2016-06-01

    Nuc-ErbB3 an alternative transcript from the ErbB3 locus binds to a specific DNA motif and associates with Schwann cell chromatin. Here we generated a nuc-ErbB3 knockin mouse that lacks nuc-ErbB3 expression in the nucleus without affecting the neuregulin-ErbB3 receptor signaling. Nuc-ErbB3 knockin mice exhibit hypermyelination and aberrant myelination at the paranodal region. This phenotype is attributed to de-repression of myelination associated gene transcription following loss of nuc-ErbB3 and histone H3K27me3 promoter occupancy. Nuc-ErbB3 knockin mice exhibit reduced association of H3K27me3 with myelination-associated gene promoters and increased RNA Pol-II rate of transcription of these genes. In addition, nuc-ErbB3 directly regulates levels of H3K27me3 in Schwann cells. Nuc-ErbB3 knockin mice exhibit significant decrease of histone H3K27me3 methyltransferase (HMT) activity and reduced levels of H3K27me3. Collectively, nuc-ErbB3 is a master transcriptional repressor, which regulates HMT activity to establish a repressive chromatin landscape on promoters of genes during peripheral myelination.

  8. Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome.

    Science.gov (United States)

    Yue, Hongjun; Fang, He; Wei, Sijie; Hayes, Jeffrey J; Lee, Tae-Hee

    2016-04-12

    Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.

  9. Stepwise bending of DNA by a single TATA-box Binding Protein

    CERN Document Server

    Tolic-Norrelykke, S F; Pavone, F S; Berg-Sørensen, K; Oddershede, L B; Tolic-Norrelykke, Simon F.; Rasmusssen, Mette B.; Pavone, Francesco S.; Berg-Sorensen, Kirstine; Oddershede, Lene B.

    2006-01-01

    The TATA-box Binding Protein (TBP) is required by all three eukaryotic RNA polymerases for the initiation of transcription from most promoters. TBP recognizes, binds to, and bends promoter sequences called ``TATA-boxes'' in the DNA. We present results from the study of individual Saccharomyces cerevisia TBPs interacting with single DNA molecules containing a TATA-box. Using video microscopy, we observed the Brownian motion of beads tethered by short surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the DNA changes and the amplitude of Brownian motion of the tethered bead is reduced compared to that of unbent DNA. We detected individual binding and dissociation events and derived kinetic parameters for the process. Dissociation was induced by increasing the salt concentration or by directly pulling on the tethered bead using optical tweezers. In addition to the well-defined free and bound classes of Brownian motion, we observed another two classes of motion. These extra classes were i...

  10. Studies of the binding mode of TXNHCH2COOH with calf thymus DNA by spectroscopic methods.

    Science.gov (United States)

    Ataci, Nese; Arsu, Nergis

    2016-12-01

    In this study, a thioxanthone derivative named 2-(9-oxo-9H-thioxanthen-2ylamino) acetic acid (TX-NHCH2COOH) was used to investigate small molecule and DNA binding interactions. Absorption and fluorescence emission spectroscopy were used and melting studies were used to explain the binding mode of TXNHCH2COOH-DNA. Intrinsic binding constant Kb TXNHCH2COOH was found 6×10(5)M(-1)from UV-Vis absorption spectroscopy. Fluorescence emmision intensity increased by adding ct-DNA to the TXNHCH2COOH and KI quenching experiments resulted with low Ksv value. Additionally, 3.7°C increase for Tm was observed. The observed quenching of EB and ct-DNA complex and increase viscosity values of ct-DNA by addition of TXNHCH2COOH was determined. All those results indicate that TXNHCH2COOH can intercalate into DNA base pairs. Fluorescence microscopy helped to display imaging of the TXNHCH2COOH-DNA solution. PMID:27367618

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

    Directory of Open Access Journals (Sweden)

    Anne De Cian

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

  12. The role of DNA binding sites and slow unbinding kinetics in titration-based oscillators

    CERN Document Server

    Karapetyan, Sargis

    2015-01-01

    Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ul...

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  14. A type IV pilus mediates DNA binding during natural transformation in Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Raphaël Laurenceau

    Full Text Available Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.

  15. A Review of Protein-DNA Binding Motif using Association Rule Mining

    Directory of Open Access Journals (Sweden)

    Virendra Kumar Tripathi,

    2013-04-01

    Full Text Available Thesurvival of gene regulation and lifemechanisms is pre-request of finding unknownpattern oftranscription factor binding sites. Thediscovery motif of gene regulation inbioinformaticsis challenging jobs for getting relation betweentranscription factors and transcription factorbinding sites. The increasing size and length ofstring pattern of motif is issued a problem related tomodeling and optimization of gene selectionprocess. In this paper we give a survey of protein-DNA binding using association rule mining.Association rule mining well knowndata miningtechnique for pattern analysis. The capability ofnegative and positive pattern generation help fullfordiscoveringof new pattern in DNA bindingbioinformatics data. The other data miningapproach such as clustering and classification alsoapplied the process of gene selection grouping forknown and unknown pattern. But faced a problemof valid string of DNA data, the rule miningprinciple find a better relation between transcriptionfactors and transcription factor binding sites.

  16. Differential effect of glucocorticoid receptor antagonists on glucocorticoid receptor nuclear translocation and DNA binding

    Science.gov (United States)

    Spiga, Francesca; Knight, David M; Droste, Susanne K; Conway-Campbell, Becky; Kershaw, Yvonne; MacSweeney, Cliona P; Thomson, Fiona J; Craighead, Mark; Peeters, Bernard WMM; Lightman, Stafford L

    2016-01-01

    The effects of RU486 and S-P, a more selective glucocorticoid receptor antagonist from Schering-Plough, were investigated on glucocorticoid receptor nuclear translocation and DNA binding. In the in vitro study, AtT20 cells were treated with vehicle or with RU486, S-P or corticosterone (3–300 nM) or co-treated with vehicle or glucocorticoid receptor antagonists (3–300 nM) and 30 nM corticosterone. Both glucocorticoid receptor antagonists induced glucocorticoid receptor nuclear translocation but only RU486 induced DNA binding. RU486 potentiated the effect of corticosterone on glucocorticoid receptor nuclear translocation and DNA binding, S-P inhibited corticosterone-induced glucocorticoid receptor nuclear translocation, but not glucocorticoid receptor-DNA binding. In the in vivo study, adrenalectomized rats were treated with vehicle, RU486 (20 mg/kg) and S-P (50 mg/kg) alone or in combination with corticosterone (3 mg/kg). RU486 induced glucocorticoid receptor nuclear translocation in the pituitary, hippocampus and prefrontal cortex and glucocorticoid receptor-DNA binding in the hippocampus, whereas no effect of S-P on glucocorticoid receptor nuclear translocation or DNA binding was observed in any of the areas analysed. These findings reveal differential effects of RU486 and S-P on areas involved in regulation of hypothalamic–pituitary–adrenal axis activity in vivo and they are important in light of the potential use of this class of compounds in the treatment of disorders associated with hyperactivity of the hypothalamic–pituitary–adrenal axis. PMID:20093322

  17. RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

    OpenAIRE

    Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

    2015-01-01

    APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G d...

  18. An expanded binding model for Cys2His2 zinc finger protein–DNA interfaces

    International Nuclear Information System (INIS)

    Cys2His2 zinc finger (C2H2-ZF) proteins comprise the largest class of eukaryotic transcription factors. The 'canonical model' for C2H2-ZF protein–DNA interaction consists of only four amino acid–nucleotide contacts per zinc finger domain, and this model has been the basis for several efforts for computationally predicting and experimentally designing protein–DNA interfaces. Here, we perform a systematic analysis of structural and experimental binding data and find that, in addition to the canonical contacts, several other amino acid and base pair combinations frequently play a role in C2H2-ZF protein–DNA binding. We suggest an expansion of the canonical C2H2-ZF model to include one to three additional contacts, and show that computational approaches including these additional contacts improve predictions of DNA targets of zinc finger proteins

  19. Binding of a new bisphenol analogue, bisphenol S to bovine serum albumin and calf thymus DNA.

    Science.gov (United States)

    Wang, Yan-Qing; Zhang, Hong-Mei; Cao, Jian; Tang, Bo-Ping

    2014-09-01

    Interactions of bisphenol S, a new bisphenol analogue with bovine serum albumin and calf thymus DNA were investigated using different spectroscopic methods and molecular modeling calculation. According to the analysis of experimental and theoretical data, we concluded that hydrophobic interactions and hydrogen bonding primarily mediated the binding processes of bisphenol S with bovine serum albumin and DNA. In addition, the electrostatic force should not be excluded. Molecular modeling studies indicated that the binding site of bisphenol S to bovine serum albumin located in the subdomain IB, while bisphenol S was a groove binder of DNA. In addition, BPS did not obviously induce second structural changes of bovine serum albumin, but it induced a conformational change of calf thymus DNA.

  20. DNA-Binding, Photocleavage, and Photodynamic Anti-cancer Activities of Pyridyl Corroles.

    Science.gov (United States)

    Liang, Zhen-Hua; Liu, Hai-Yang; Zhou, Rong; Zhang, Zao; Ali, Atif; Han, Bing-Jie; Liu, Yun-Jun; Xiao, Xin-Yan

    2016-08-01

    The DNA-binding, photocleavage, and antitumor activity of three free base pyridyl corroles 1, 2, and 3 have been investigated. The binding affinity toward CT-DNA decreases with increasing number of pentafluorophenyl, whereas the photocleavage activity toward pBR322 DNA becomes more efficient. Singlet oxygen was demonstrated as active species responsible for DNA cleavage. These corroles exhibited high cytotoxicity against three tested cancer cells (Hela, HapG2, and A549) and the cytotoxicity could be further enhanced under irradiation. Intracellular reactive oxygen species level was also monitored using HeLa Cells upon the combined treatment of corroles and light. These corroles could be absorbed by HeLa cells at low concentration. They can induce the decrease of mitochondrial membrane potential and apoptosis of tumor cells under irradiation. PMID:26895317

  1. Human TFDP3, a novel DP protein, inhibits DNA binding and transactivation by E2F

    DEFF Research Database (Denmark)

    Qiao, Huan; Di Stefano, Luisa; Tian, Chan;

    2006-01-01

    The two known DP proteins, TFDP1 and -2, bind E2Fs to form heterodimers essential for high affinity DNA binding and efficient transcriptional activation/repression. Here we report the identification of a new member of the DP family, human TFDP3. Despite the high degree of sequence similarity, TFDP3...... a new DP protein and a novel mechanism whereby E2F function is regulated....

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

    OpenAIRE

    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 transcri...

  3. DNA-binding activity of TNF-α inducing protein from Helicobacter pylori

    International Nuclear Information System (INIS)

    Tumor necrosis factor-α (TNF-α) inducing protein (Tipα) is a carcinogenic factor secreted from Helicobacter pylori (H. pylori), mediated through both enhanced expression of TNF-α and chemokine genes and activation of nuclear factor-κB. Since Tipα enters gastric cancer cells, the Tipα binding molecules in the cells should be investigated. The direct DNA-binding activity of Tipα was observed by pull down assay using single- and double-stranded genomic DNA cellulose. The surface plasmon resonance assay, indicating an association between Tipα and DNA, revealed that the affinity of Tipα for (dGdC)10 is 2400 times stronger than that of del-Tipα, an inactive Tipα. This suggests a strong correlation between DNA-binding activity and carcinogenic activity of Tipα. And the DNA-binding activity of Tipα was first demonstrated with a molecule secreted from H. pylori

  4. Studies on the Synthesis, Characterization, DNA Binding, Cytotoxicity and Antioxidant activity of 2-methyl-4-nitrophenylferrocene

    International Nuclear Information System (INIS)

    We report herein the synthesis, structural characterization, DNA binding, BamH1 digestion, cytotoxicity and antioxidant activity of 2-methyl-4-nitrophenylferrocene. Structural characterization is based on multinuclear (1H and 13C) NMR, FT-IR spectroscopy and elemental analysis. Interaction of 2-methyl-4-nitrophenylferrocene with pBR322 plasmid DNA shows noncovalent interactions however these noncovalent interactions reveal the prevention of BamH1 restriction site (g/ggtcc). In the voltammogram, a negative shift in peak potential has been observed on addition of increasing concentration of CT-DNA, which shows electrostatic interaction for 2-methyl-4-nitrophenylferro with negatively charged phosphate of DNA backbone. The binding ratio, binding constant, binding free energy and diffusion coefficient of free and bound drug were calculated to understand the mechanism. The high negative value of -delta G signifies the spontaneity and high conformational stability of 2-methyl-4-nitrophenylferro with CT-DNA. The compound has the ability to scavenge free radicals as have been revealed by DPPH findings. (author)

  5. Synthesis, characterization; DNA binding and antitumor activity of ruthenium(II) polypyridyl complexes.

    Science.gov (United States)

    Srishailam, A; Gabra, Nazar Mohammed; Kumar, Yata Praveen; Reddy, Kotha Laxma; Devi, C Shobha; Anil Kumar, D; Singh, Surya S; Satyanarayana, S

    2014-12-01

    Three new ruthenium(II) polypyridyl complexes [Ru(phen)2BrIPC](2+) (1), [Ru(bpy)2 BrIPC](2+) (2) and [Ru(dmb)2BrIPC](2+) (3) where, BrIPC = (6-bromo-3-(1H-imidazo[4,5-f] [1,10]-phenanthroline, phen = 1,10-phenanthroline, bpy = 2,2' bipyridine, dmb = 4,4'-dimethyl 2,2' bipyridine, were synthesised and characterised. DNA-binding nature was investigated by spectroscopic titrations and mode of binding was assessed by viscosity measurements. The DNA-binding constants Kb of complexes 1, 2 and 3 were determined to be in the order of 10(5). Experimental results showed that these complexes interact with CT-DNA by intercalative mode. Photocleavage and antimicrobial activities were complex concentration dependent, at high concentration, high activity and vice versa. MTT assay was performed on HeLa cell lines, IC50 values of complexes in the order of 3 > 2 > 1 > cisplatin. From comet assay, cellular uptake studies, we observed that complexes could enter into the cell membrane and accumulate inside the nucleus. Molecular docking studies support the DNA binding affinity with hydrogen bonding and van der Waals attractions between base pairs and phosphate backbone of DNA with metal complexes. PMID:25318017

  6. The modified base J is the target for a novel DNA-binding protein in kinetoplastid protozoans.

    OpenAIRE

    Cross, M.; Kieft, R.; Sabatini, R.; Wilm, M; Kort, M. de; van der Marel, G A; Boom, J.H. van; Leeuwen, F. van; Borst, P

    1999-01-01

    DNA from Kinetoplastida contains the unusual modified base beta-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. We have now identified a protein in nuclear extracts of bloodstream stage T.brucei that binds specifically to J-containing duplex DNA. J-specific DNA binding was also observed with extracts from the kinetoplastids Crithidia fascic...

  7. Preliminary Crystallographic Study of Streptomyces coelicolor Single-stranded DNA-binding Protein

    OpenAIRE

    Štefanić, Zoran; Vujaklija, Dušica; Andrišić, Luka; Mikleušević, Goran; Andrejašič, Miha; Turk, Dušan; Luić, Marija

    2007-01-01

    Single-stranded DNA-binding proteins (SSBs) play a crucial role in DNA processing such as replication, repair and recombination in all organisms, from bacteria to human. Streptomyces coelicolor ssb gene was overexpressed in a heterologous host, Escherichia coli NM522. 15 mg of purified protein from 1 dm(3) of culture was obtained in one-step procedure applying Ni2+ chelating chromatography. Among bacterial SSBs with the solved crystal structure, the S. coelicolor SSB displayed significant seq...

  8. Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

    Science.gov (United States)

    Smidt, M P; Russchen, B; Snippe, L; Wijnholds, J; Ab, G

    1995-07-11

    Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed. PMID:7630716

  9. The DNA Binding Activity of p53 Displays Reaction-Diffusion Kinetics

    Science.gov (United States)

    Hinow, Peter; Rogers, Carl E.; Barbieri, Christopher E.; Pietenpol, Jennifer A.; Kenworthy, Anne K.; DiBenedetto, Emmanuele

    2006-01-01

    The tumor suppressor protein p53 plays a key role in maintaining the genomic stability of mammalian cells and preventing malignant transformation. In this study, we investigated the intracellular diffusion of a p53-GFP fusion protein using confocal fluorescence recovery after photobleaching. We show that the diffusion of p53-GFP within the nucleus is well described by a mathematical model for diffusion of particles that bind temporarily to a spatially homogeneous immobile structure with binding and release rates k1 and k2, respectively. The diffusion constant of p53-GFP was estimated to be Dp53-GFP = 15.4 μm2 s−1, significantly slower than that of GFP alone, DGFP = 41.6 μm2 s−1. The reaction rates of the binding and unbinding of p53-GFP were estimated as k1 = 0.3 s−1 and k2 = 0.4 s−1, respectively, values suggestive of nonspecific binding. Consistent with this finding, the diffusional mobilities of tumor-derived sequence-specific DNA binding mutants of p53 were indistinguishable from that of the wild-type protein. These data are consistent with a model in which, under steady-state conditions, p53 is latent and continuously scans DNA, requiring activation for sequence-specific DNA binding. PMID:16603489

  10. Nucleotide excision repair is impaired by binding of transcription factors to DNA.

    Science.gov (United States)

    Sabarinathan, Radhakrishnan; Mularoni, Loris; Deu-Pons, Jordi; Gonzalez-Perez, Abel; López-Bigas, Núria

    2016-04-14

    Somatic mutations are the driving force of cancer genome evolution. The rate of somatic mutations appears to be greatly variable across the genome due to variations in chromatin organization, DNA accessibility and replication timing. However, other variables that may influence the mutation rate locally are unknown, such as a role for DNA-binding proteins, for example. Here we demonstrate that the rate of somatic mutations in melanomas is highly increased at active transcription factor binding sites and nucleosome embedded DNA, compared to their flanking regions. Using recently available excision-repair sequencing (XR-seq) data, we show that the higher mutation rate at these sites is caused by a decrease of the levels of nucleotide excision repair (NER) activity. Our work demonstrates that DNA-bound proteins interfere with the NER machinery, which results in an increased rate of DNA mutations at the protein binding sites. This finding has important implications for our understanding of mutational and DNA repair processes and in the identification of cancer driver mutations.

  11. Cationic polymers for DNA origami coating - examining their binding efficiency and tuning the enzymatic reaction rates.

    Science.gov (United States)

    Kiviaho, Jenny K; Linko, Veikko; Ora, Ari; Tiainen, Tony; Järvihaavisto, Erika; Mikkilä, Joona; Tenhu, Heikki; Nonappa; Kostiainen, Mauri A

    2016-06-01

    DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The effect of the polymer structure on the binding was investigated and the toxicity of the polymer-origami complexes evaluated. The study shows that all of the analyzed polymers had a suitable binding efficiency irrespective of the block structure. It was also observed that the toxicity of polymer-origami complexes was insignificant at the biologically relevant concentration levels. Besides brick-like DNA origamis, tubular origami carriers equipped with enzymes were also coated with the polymers. By adjusting the amount of cationic polymers that cover the DNA structures, we showed that it is possible to control the enzyme kinetics of the complexes. This work gives a starting point for further development of biocompatible and effective polycation-based block copolymers that can be used in coating different DNA origami nanostructures for various bioapplications. PMID:27219684

  12. Atomic-Scale Molecular Dynamics Simulations of DNA-Polycation Complexes: Two Distinct Binding Patterns.

    Science.gov (United States)

    Kondinskaia, Diana A; Kostritskii, Andrei Yu; Nesterenko, Alexey M; Antipina, Alexandra Yu; Gurtovenko, Andrey A

    2016-07-14

    Synthetic cationic polymers represent a promising class of delivery vectors for gene therapy. Here, we employ atomistic molecular dynamics simulations to gain insight into the structure and properties of complexes of DNA with four linear polycations: polyethylenimine (PEI), poly-l-lysine (PLL), polyvinylamine (PVA), and polyallylamine (PAA). These polycations differ in their polymer geometries, protonation states, and hydrophobicities of their backbone chains. Overall, our results demonstrate for the first time the existence of two distinct patterns of binding of DNA with polycations. For PEI, PLL, and PAA, the complex is stabilized by the electrostatic attraction between protonated amine groups of the polycation and phosphate groups of DNA. In contrast, PVA demonstrates an alternative binding pattern as it gets embedded into the DNA major groove. It is likely that both the polymer topology and affinity of the backbone chain of PVA to the DNA groove are responsible for such behavior. The differences in binding patterns can have important biomedical implications: embedding PVA into a DNA groove makes it less sensitive to changes in the aqueous environment (pH level, ionic strength, etc.) and could therefore hinder the intracellular release of genetic material from a delivery vector, leading to lower transfection activity. PMID:27280954

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

    Science.gov (United States)

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

    2009-04-01

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

  14. Binding of histone H1 to DNA is differentially modulated by redox state of HMGB1.

    Directory of Open Access Journals (Sweden)

    Eva Polanská

    Full Text Available HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.

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

    Science.gov (United States)

    Zhou, Donghua; Moktan, Hem; Pezza, Roberto

    2014-03-01

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

  16. DNA-Binding Kinetics Determines the Mechanism of Noise-Induced Switching in Gene Networks.

    Science.gov (United States)

    Tse, Margaret J; Chu, Brian K; Roy, Mahua; Read, Elizabeth L

    2015-10-20

    Gene regulatory networks are multistable dynamical systems in which attractor states represent cell phenotypes. Spontaneous, noise-induced transitions between these states are thought to underlie critical cellular processes, including cell developmental fate decisions, phenotypic plasticity in fluctuating environments, and carcinogenesis. As such, there is increasing interest in the development of theoretical and computational approaches that can shed light on the dynamics of these stochastic state transitions in multistable gene networks. We applied a numerical rare-event sampling algorithm to study transition paths of spontaneous noise-induced switching for a ubiquitous gene regulatory network motif, the bistable toggle switch, in which two mutually repressive genes compete for dominant expression. We find that the method can efficiently uncover detailed switching mechanisms that involve fluctuations both in occupancies of DNA regulatory sites and copy numbers of protein products. In addition, we show that the rate parameters governing binding and unbinding of regulatory proteins to DNA strongly influence the switching mechanism. In a regime of slow DNA-binding/unbinding kinetics, spontaneous switching occurs relatively frequently and is driven primarily by fluctuations in DNA-site occupancies. In contrast, in a regime of fast DNA-binding/unbinding kinetics, switching occurs rarely and is driven by fluctuations in levels of expressed protein. Our results demonstrate how spontaneous cell phenotype transitions involve collective behavior of both regulatory proteins and DNA. Computational approaches capable of simulating dynamics over many system variables are thus well suited to exploring dynamic mechanisms in gene networks.

  17. Theoretical Study of Sequence Selectivity and Preferred Binding Mode of Psoralen with DNA

    Directory of Open Access Journals (Sweden)

    Patricia Saenz-Méndez

    2007-01-01

    Full Text Available Psoralen interaction with two models of DNA was investigated using molecular mechanics and molecular dynamics methods. Calculated energies of minor groove binding and intercalation were compared in order to define a preferred binding mode for the ligand. We found that both binding modes are possible, explaining the low efficiency for monoadduct formation from intercalated ligands. A comparison between the interaction energy for intercalation between different base pairs suggests that the observed sequence selectivity is due to favorable intercalation in 5′-TpA in (ATn sequences.

  18. Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

    OpenAIRE

    Nakagama, H; Heinrich, G.; Pelletier, J; Housman, D E

    1995-01-01

    The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, settin...

  19. In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

    OpenAIRE

    Moens, U; Seternes, O M; Hey, A W; Silsand, Y; Traavik, T.; Johansen, B.; Rekvig, O P

    1995-01-01

    Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render ...

  20. DDB2 (Damaged DNA binding protein 2) in nucleotide excision repair and DNA damage response

    OpenAIRE

    Stoyanova, Tanya; Roy, Nilotpal; Kopanja, Dragana; Raychaudhuri, Pradip; Bagchi, Srilata

    2009-01-01

    DDB2 was identified as a protein involved in the Nucleotide Excision Repair (NER), a major DNA repair mechanism that repairs UV damage to prevent accumulation of mutations and tumorigenesis. However, recent studies indicated additional functions of DDB2 in the DNA damage response pathway. Herein, we discuss the proposed mechanisms by which DDB2 activates NER and programmed cell death upon DNA damage through its E3 ligase activity.

  1. Mechanism of formation of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. Part II. Interplay of local backbone conformational dynamics and long-range hydrophobic interactions in hairpin formation

    OpenAIRE

    Skwierawska, Agnieszka; Żmudzińska, Wioletta; Ołdziej, Stanisław; Liwo, Adam; Scheraga, Harold A.

    2009-01-01

    Two peptides, corresponding to the turn region of the C-terminal β-hairpin of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, consisting of residues 51–56 [IG(51–56)] and 50–57 [IG(50–57)], respectively, were studied by CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. Our results show that the part of the sequence corresponding to the β-turn in the native structure (DDATKT) of the B3 domain forms bent conformations similar to ...

  2. Synthesis, photochemical properties and DNA binding studies of dna cleaving agents based on chiral dipyridine dihydrodioxins salts

    Science.gov (United States)

    Shamaev, Alexei

    Control of chemical reactions becomes especially challenging when chemical processes have to work within the complexity of biological environments. This is one of the reasons why the ability to design "caged" molecules with structure, reactivity, and biological activity that can be activated externally by light continues to draw significant attention, from both the practical and fundamental points of view. Possible applications of such molecules include design of molecular machines and switches, logic gate mimics, optical sensors, drug delivery systems, etc. Since "caged" molecules are of particular use for processes that occur in biochemical systems and in the environment, interesting light-sensitive systems, anti-cancer drugs, have been developed recently to control DNA cleavage. Caged molecules may interact with or bind with DNA and can be classified by their mechanism of action. Each of these classes of molecules has a different structure and interacts with DNA in a different way, but some molecules can combine several functionalities. The preponderance of caged molecules, anti-cancer drugs, capable of DNA cleavage or their metabolites incorporate Electron Transfer (ET) functionalities, which play important roles in physiological responses. These main groups include quinones (or phenolic precursors), metal complexes, aromatic nitro compounds (or reduced derivatives), and conjugated imines (or iminium species). Redox cycling with oxygen can occur giving rise to Oxidation Stress (OS) through generation of Reactive Oxygen Species (ROS) which can contribute to drug efficacy or can lead to undesirable toxicity. In some cases, ET results in interference with normal electron transport chains. In this work a series of caged molecules-chiral Pyrene Dihydridioxins (PDHD)-DNA chiral DNA intecalators and PDHD-metal complexes bearing masked o-quinone functionality activated through intramolecular ET were synthesized. The o-quinone release and intramolecular ET can be easily

  3. Evolutionary conservation and DNA binding properties of the Ssh7 proteins from Sulfolobus shibatae

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DNA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. Shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the cis-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments with a binding size of ~ 6.6 base pairs and an apparent dissociation constant of (0.7-1.0)×10-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled DNA than to linear or relaxed DNA.

  4. DNA damaging, cell cytotoxicity and serum albumin binding efficacy of the rutin-Cu(ii) complex.

    Science.gov (United States)

    Roy, Atanu Singha; Tripathy, Debi Ranjan; Samanta, Sintu; Ghosh, Sudip K; Dasgupta, Swagata

    2016-04-26

    Flavonoids are widely used as anti-oxidants, anti-cancer agents and possess metal ion chelation properties. In this report we have investigated the DNA binding (and damaging), cell cytotoxicity and serum albumin (SA) binding efficacy of the rutin-Cu(ii) complex using differential spectroscopic methods. The rutin-Cu(ii) complex was able to intercalate into calf thymus DNA (ct-DNA) at lower concentrations and its DNA damaging properties were also confirmed from the agarose gel based assay, fluorescence and UV-vis studies. The copper complex was found to be effective against the growth of HeLa cells in vivo. The binding constants (Kb) of the rutin-Cu(ii) complex towards HSA and BSA were found to be (0.98 ± 0.03) and (1.05 ± 0.02) × 10(5) M(-1), respectively, at 299 K and observed to increase with the increase in temperature. Site selectivity studies revealed that the rutin-Cu(ii) complex binds near site 1 (subdomain IIA) of SAs. Thermodynamic parameters indicated that the mode of interaction of rutin and its copper complex with SAs are different from each other. Both ΔH° and ΔS° were observed to be positive for the interaction of the rutin-Cu(ii) complex with SAs, indicating the presence of hydrophobic association in binding. The values of ΔH° were estimated to be negative (-42.07 ± 2.92 and -23.29 ± 2.33 kJ mol(-1) for HSA and BSA respectively) in the binding of rutin with SAs. It implies that after chelation with Cu(ii) ion, rutin alters its binding mode which could have varying applications to its other physicochemical activities. PMID:27035097

  5. Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

    Science.gov (United States)

    Dombek, P; Ream, W

    1997-02-01

    The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends. A. tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex. The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection. Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei. Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2. We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A. tumefaciens. Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity. Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A. tumefaciens. In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation. This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2. PMID:9023198

  6. Altered Specificity of DNA-Binding Proteins with Transition Metal Dimerization Domains

    Science.gov (United States)

    Cuenoud, Bernard; Schepartz, Alanna

    1993-01-01

    The bZIP motif is characterized by a leucine zipper domain that mediates dimerization and a basic domain that contacts DNA. A series of transition metal dimerization domains were used to alter systematically the relative orientation of basic domain peptides. Both the affinity and the specificity of the peptide-DNA interaction depend on domain orientation. These results indicate that the precise configuration linking the domains is important; dimerization is not always sufficient for DNA binding. This approach to studying the effect of orientation on protein function complements mutagenesis and could be used in many systems.

  7. Label-free detection of biomolecular interaction — DNA — Antimicrobial peptide binding

    DEFF Research Database (Denmark)

    Fojan, Peter; Jensen, Kasper Risgaard; Gurevich, Leonid

    2011-01-01

    of plasmon based biosensors to the study of the interaction of Antimicrobial peptide IL4 and DNA. Our results indicate high affinity binding between IL4 and DNA thereby preventing DNA replication and eventually killing the affected cell. We speculate that this is common for a large class of Antimicrobial...... an interest in Antimicrobial peptides that are active against broad range of infections including bacteria, fungi and viruses and were shown to be capable of treating multi-resistant infection either alone or in combination with the conventional antibiotics. In this paper , we demonstrate an application...... peptides and can be a key point explaining their broad range of activity against various pathogens....

  8. Human single-stranded DNA binding proteins: guardians of genome stability

    Institute of Scientific and Technical Information of China (English)

    Yuanzhong Wu; Jinping Lu; Tiebang Kang

    2016-01-01

    Single-stranded DNA-binding proteins (SSBs) are essential for maintaining the integrity of the genome in all organisms.All processes related to DNA,such as replication,excision,repair,and recombination,require the participation of SSBs whose oligonucleotideaoligosaccharide-binding (OB)-fold domain is responsible for the interaction with single-stranded DNA (ssDNA).For a long time,the heterotrimeric replication protein A (RPA) complex was believed to be the only nuclear SSB in eukanyotes to participate in ssDNA processing,while mitochondrial SSBs that are consewed with prokaryotic SSBs were shown to be essential for maintaining genome stability in eukaryotic mitochondria.In recent years,two new proteins,hSSB1 and hSSB2 (human SSBs 1/2),were identified and have better sequence similarity to bacterial and archaeal SSBs than RPA.This review summarizes the current understanding of these human SSBs in DNA damage repair and in cell-cycle checkpoint activation following DNA damage,as well as their relationships with cancer.

  9. One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

    Directory of Open Access Journals (Sweden)

    Stöcklein Walter

    2007-08-01

    Full Text Available Abstract Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

  10. Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

    International Nuclear Information System (INIS)

    Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres

  11. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

    2015-06-01

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  12. A Conserved Myc Protein Domain, MBIV, Regulates DNA Binding, Apoptosis, Transformation, and G2 Arrest†

    Science.gov (United States)

    Cowling, Victoria H.; Chandriani, Sanjay; Whitfield, Michael L.; Cole, Michael D.

    2006-01-01

    The myc family of oncogenes is well conserved throughout evolution. Here we present the characterization of a domain conserved in c-, N-, and L-Myc from fish to humans, N-Myc317-337, designated Myc box IV (MBIV). A deletion of this domain leads to a defect in Myc-induced apoptosis and in some transformation assays but not in cell proliferation. Unlike other Myc mutants, MycΔMBIV is not a simple loss-of-function mutant because it is hyperactive for G2 arrest in primary cells. Microarray analysis of genes regulated by N-MycΔMBIV reveals that it is weakened for transactivation and repression but not nearly as defective as N-MycΔMBII. Although the mutated region is not part of the previously defined DNA binding domain, we find that N-MycΔMBIV has a significantly lower affinity for DNA than the wild-type protein in vitro. Furthermore, chromatin immunoprecipitation shows reduced binding of N-MycΔMBIV to some target genes in vivo, which correlates with the defect in transactivation. Thus, this conserved domain has an unexpected role in Myc DNA binding activity. These data also provide a novel separation of Myc functions linked to the modulation of DNA binding activity. PMID:16705173

  13. Identification of the proteins responsible for SAR DNA binding in nuclear matrix of ''Cucurbita pepo''

    International Nuclear Information System (INIS)

    The nuclear matrices from White bush (''Cucurbita pepo var. patisonina'') cell nuclei have been isolated using three methods: I, standard procedure involving extraction of cell nuclei with 2 M NaCl and 1% Triton X-100; II, the same with pre-treatment of cell nuclei with 0.5 mM CuSO4 (stabilisation step); and III, method with extraction by lithium diiodosalicylate (LIS), and compared the polypeptide pattern. The isolated matrices specifically bind SAR DNA derived from human β-interferon gene in the exogenous SAR binding assay and in the gel mobility shift assay. Using IgG against the 32 kDa endonuclease we have found in the DNA-protein blot assay that this protein is one of the proteins binding SAR DNA. We have identified three proteins with molecular mass of 65 kDa, 60 kDa and 32 kDa which are responsible for SAR DNA binding in the gel mobility shift assay experiments. (author). 21 refs, 3 figs

  14. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B;

    2006-01-01

    by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

  15. Synthesis, DNA Binding and Topoisomerase I Inhibition Activity of Thiazacridine and Imidazacridine Derivatives

    Directory of Open Access Journals (Sweden)

    Elizabeth Almeida Lafayette

    2013-12-01

    Full Text Available Thiazacridine and imidazacridine derivatives have shown promising results as tumors suppressors in some cancer cell lines. For a better understanding of the mechanism of action of these compounds, binding studies of 5-acridin-9-ylmethylidene-3-amino-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-thiazolidin-4-one, 5-acridin-9-ylmethylidene-2-thioxo-imidazolidin-4-one and 3-acridin-9-ylmethyl-thiazolidin-2,4-dione with calf thymus DNA (ctDNA by electronic absorption and fluorescence spectroscopy and circular dichroism spectroscopy were performed. The binding constants ranged from 1.46 × 104 to 6.01 × 104 M−1. UV-Vis, fluorescence and circular dichroism measurements indicated that the compounds interact effectively with ctDNA, both by intercalation or external binding. They demonstrated inhibitory activities to human topoisomerase I, except for 5-acridin-9-ylmethylidene-2-thioxo-1,3-thiazolidin-4-one. These results provide insight into the DNA binding mechanism of imidazacridines and thiazacridines.

  16. A Review of Protein-DNA Binding Motif using Association Rule Mining

    Directory of Open Access Journals (Sweden)

    Virendra Kumar Tripathi

    2013-03-01

    Full Text Available The survival of gene regulation and life mechanisms is pre-request of finding unknown pattern of transcription factor binding sites. The discovery motif of gene regulation in bioinformatics is challenging jobs for getting relation between transcription factors and transcription factor binding sites. The increasing size and length of string pattern of motif is issued a problem related to modeling and optimization of gene selection process. In this paper we give a survey of protein-DNA binding using association rule mining. Association rule mining well known data mining technique for pattern analysis. The capability of negative and positive pattern generation help full for discovering of new pattern in DNA binding bioinformatics data. The other data mining approach such as clustering and classification also applied the process of gene selection grouping for known and unknown pattern. But faced a problem of valid string of DNA data, the rule mining principle find a better relation between transcription factors and transcription factor binding sites.

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

    Science.gov (United States)

    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. ANTI-NUCLEOSOME ANTIBODIES COMPLEXED TO NUCLEOSOMAL ANTIGENS SHOW ANTI-DNA REACTIVITY AND BIND TO RAT GLOMERULAR-BASEMENT-MEMBRANE IN-VIVO

    NARCIS (Netherlands)

    KRAMERS, C; HYLKEMA, MN; VANBRUGGEN, MCJ; VANDELAGEMAAT, R; DIJKMAN, HBPM; ASSMANN, KJM; SMEENK, RJT; BERDEN, JHM; Hylkema, Machteld

    1994-01-01

    Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). Zn ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM

  19. Cytotoxicity and DNA cleavage with core-shell nanocomposites functionalized by a KH domain DNA binding peptide

    Science.gov (United States)

    Bazak, Remon; Ressl, Jan; Raha, Sumita; Doty, Caroline; Liu, William; Wanzer, Beau; Salam, Seddik Abdel; Elwany, Samy; Paunesku, Tatjana; Woloschak, Gayle E.

    2013-11-01

    A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells.A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA

  20. Selective binding of specific mouse genomic DNA fragments by mouse vimentin filaments in vitro.

    Science.gov (United States)

    Wang, X; Tolstonog, G; Shoeman, R L; Traub, P

    1996-03-01

    Mouse vimentin intermediate filaments (IFs) reconstituted in vitro were analyzed for their capacity to select certain DNA sequences from a mixture of about 500-bp-long fragments of total mouse genomic DNA. The fragments preferentially bound by the IFs and enriched by several cycles of affinity binding and polymerase chain reaction (PCR) amplification were cloned and sequenced. In general, they were G-rich and highly repetitive in that they often contained Gn, (GT)n, and (GA)n repeat elements. Other, more complex repeat sequences were identified as well. Apart from the capacity to adopt a Z-DNA and triple helix configuration under superhelical tension, many fragments were potentially able to form cruciform structures and contained consensus binding sites for various transcription factors. All of these sequence elements are known to occur in introns and 5'/3'-flanking regions of genes and to play roles in DNA transcription, recombination and replication. A FASTA search of the EMBL data bank indeed revealed that sequences homologous to the mouse repetitive DNA fragments are commonly associated with gene-regulatory elements. Unexpectedly, vimentin IFs also bound a large number of apparently overlapping, AT-rich DNA fragments that could be aligned into a composite sequence highly homologous to the 234-bp consensus centromere repeat sequence of gamma-satellite DNA. Previous experiments have shown a high affinity of vimentin for G-rich, repetitive telomere DNA sequences, superhelical DNA, and core histones. Taken together, these data support the hypothesis that, after penetration of the double nuclear membrane via an as yet unidentified mechanism, vimentin IFs cooperatively fix repetitive DNA sequence elements in a differentiation-specific manner in the nuclear periphery subjacent to the nuclear lamina and thus participate in the organization of chromatin and in the control of transcription, replication, and recombination processes. This includes aspects of global

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-07-01

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

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

    International Nuclear Information System (INIS)

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

  3. pH modulates the binding of early growth response protein 1 transcription factor to DNA.

    Science.gov (United States)

    Mikles, David C; Bhat, Vikas; Schuchardt, Brett J; Deegan, Brian J; Seldeen, Kenneth L; McDonald, Caleb B; Farooq, Amjad

    2013-08-01

    The transcription factor early growth response protein (EGR)1 orchestrates a plethora of signaling cascades involved in cellular homeostasis, and its downregulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with an increase in pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as His382 by virtue of the fact that its replacement by nonionizable residues abolishes the pH dependence of the binding of EGR1 to DNA. Notably, His382 inserts into the major groove of DNA, and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, His382 is mainly conserved across other members of the EGR family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating the protein-DNA interactions that are central to this family of transcription factors. Collectively, our findings reveal an unexpected but a key step in the molecular recognition of the EGR family of transcription factors, and suggest that they may act as sensors of pH within the intracellular environment. PMID:23718776

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

    Science.gov (United States)

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

    2016-06-01

    A novel isoflavone, 5,6,7-trimethoxy-3-(3',4',5'-trimethoxyphenyl)-4H-chromen-4-one (1) along with a known pyranocoumarin, Seselin (2) have been isolated from the ethanolic extract of the leaves of Cassia siamea (Family: Fabaceae). Compound 1 has been reported for the first time from any natural source and has not been synthesized so far. Their structures were elucidated on the basis of chemical and physical evidences viz. elemental analysis, UV, FT-IR, (1)H-NMR, (13)C-NMR and mass spectral analysis. Structure of compound (1) was further authenticated by single-crystal X-ray analysis and density functional theory (DFT) calculations. A multi-technique approach employing UV-Visible spectroscopy, fluorescence, KI quenching studies, competitive displacement assay, circular dichroism and viscosity studies have been utilized to probe the extent of interaction and possible binding modes of isolated compounds (1-2) with calf thymus DNA (CT-DNA). Both the compounds were found to interact with DNA via non-intercalative binding mode with moderate proficiencies. Groove binding was the major interaction mode in the case of compound 2 while compound 1 probably interacts with DNA through electrostatic interactions. These studies provide deeper insight in understanding of DNA-drug (natural products) interaction which could be helpful to improve their bioavailability for therapeutic purposes. PMID:27085054

  5. The new generation drug candidate molecules: Spectral, electrochemical, DNA-binding and anticancer activity properties

    Science.gov (United States)

    Gölcü, Ayşegül; Muslu, Harun; Kılıçaslan, Derya; Çeşme, Mustafa; Eren, Özge; Ataş, Fatma; Demirtaş, İbrahim

    2016-09-01

    The new generation drug candidate molecules [Cu(5-Fu)2Cl2H2O] (NGDCM1) and [Zn(5-Fu)2(CH3COO)2] (NGDCM2) were obtained from the reaction of copper(II) and zinc(II) salts with the anticancer drug 5-fluoracil (5-Fu). These compounds have been characterized by spectroscopic and analytical techniques. Thermal behavior of the compounds were also investigated. The electrochemical properties of the compounds have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the NGDCM1 and NGDCM2 has been evaluated by examining their ability to bind to fish sperm double strand DNA (FSdsDNA) with UV spectroscopy. UV studies of the interaction of the 5-Fu and metal derivatives with FSdsDNA have shown that these compounds can bind to FSdsDNA. The binding constants of the compounds with FSdsDNA have also been calculated. Thermal decomposition of the compounds lead to the formation of CuO and ZnO as final products. The effect of proliferation 5-Fu, NGDCM1 and NGDCM2 were examined on the HeLa cells using real-time cell analyzer with three different concentrations.

  6. Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

    Science.gov (United States)

    Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

    2016-02-01

    A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.

  7. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    Science.gov (United States)

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury. PMID:26345909

  8. Biological characterization of liver fatty acid binding gene from miniature pig liver cDNA library.

    Science.gov (United States)

    Gao, Y H; Wang, K F; Zhang, S; Fan, Y N; Guan, W J; Ma, Y H

    2015-01-01

    Liver fatty acid binding proteins (L-FABP) are a family of small, highly conserved, cytoplasmic proteins that bind to long-chain fatty acids and other hydrophobic ligands. In this study, a full-length enriched cDNA library was successfully constructed from Wuzhishan miniature pig, and then the L-FABP gene was cloned from this cDNA library and an expression vector (pEGFP-N3-L-FABP) was constructed in vitro. This vector was transfected into hepatocytes to test its function. The results of western blotting analysis demonstrated that the L-FABP gene from our full-length enriched cDNA library regulated downstream genes, including the peroxisome proliferator-activated receptor family in hepatocytes. This study provides a theoretical basis and experimental evidence for the application of L-FABP for the treatment of liver injury.

  9. Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1

    Science.gov (United States)

    Schwartz, T.; Lowenhaupt, K.; Kim, Y. G.; Li, L.; Brown, B. A. 2nd; Herbert, A.; Rich, A.

    1999-01-01

    Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

  10. HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding

    Institute of Scientific and Technical Information of China (English)

    Guo-Zhong Gong; Yong-Fang Jiang; Yan He; Li-Ying Lai; Ying-Hua Zhu; Xian-Shi Su

    2004-01-01

    AIM: To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function.METHODS: p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding ability was observed with the use of electrophoretic mobility-shift assay (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cell lines to observe whether HCV NS5A could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter.Western blot experiment was used for detection of HCV NS5A and p53 proteins expression.RESULTS: Relative luciferase activity driven by p21 promoter increased significantly in the presence of endogenous p53 protein. Compared to the control group, exogenous p53 protein also stimulated p21 promoter driven luciferase gene expression in a dose-dependent way. HCV NS5A protein gradually inhibited both endogenous and exogenous p53 transactivation on p21 promoter with increase of the dose of HCV NS5A expression plasmid. By the experiment of EMSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV NS5A expression vector, the p53 binding affinity to its DNA gradually decreased and finally disappeared. Between the Huh 7 cells transfected with p53 expression vector alone or co-transfected with HCV NS5A expression vector, there was no difference in the p53 protein expression.CONCLUSION: HCV NS5A inhibits p53 transactivation on p21 promoter through abrogating p53 binding affinity to its specific DNA sequence. It does not affect p53 protein expression.

  11. RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.

    Science.gov (United States)

    Bernacchi, Serena; Henriet, Simon; Dumas, Philippe; Paillart, Jean-Christophe; Marquet, Roland

    2007-09-01

    The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors. PMID:17609216

  12. Synthesis and spectroscopic studies of the aminoglycoside (neomycin)--perylene conjugate binding to human telomeric DNA.

    Science.gov (United States)

    Xue, Liang; Ranjan, Nihar; Arya, Dev P

    2011-04-12

    Synthesis of a novel perylene-neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5'-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA-RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV-vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand-quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4.

  13. Early diagnosis of systemic lupus erythmatosus using ANN models of dsDNA binding antibody sequence data.

    Science.gov (United States)

    Bahari, Mohamad Hasan; Mahmoudi, Mahmoud; Azemi, Asad; Mirsalehi, Mir Mojtaba; Khademi, Morteza

    2010-01-01

    In this paper a new method based on artificial neural networks (ANN), is introduced for identifying pathogenic antibodies in Systemic Lupus Erythmatosus (SLE). dsDNA binding antibodies have been implicated in the pathogenesis of this autoimmune disease. In order to identify these dsDNA binding antibodies, the protein sequences of 42 dsDNA binding and 608 non-dsDNA binding antibodies were extracted from Kabat database and encoded using a physicochemical property of their amino acids namely Hydrophilicity. Encoded antibodies were used as the training patterns of a general regression neural network (GRNN). Simulation results show that the accuracy of proposed method in recognizing dsDNA binding antibodies is 83.2%. We have also investigated the roles of the light and heavy chains of anti-dsDNA antibodies in binding to DNA. Simulation results concur with the published experimental findings that in binding to DNA, the heavy chain of anti-dsDNA is more important than their light chain. PMID:21346864

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

    Science.gov (United States)

    Barrilleaux, Bonnie L; Burow, Dana; Lockwood, Sarah H; Yu, Abigail; Segal, David J; Knoepfler, Paul S

    2014-01-01

    The transcription factor Miz-1 can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein. The genomic binding of Miz-1 includes both core promoters and more distal sites, but the preferred DNA binding motif of Miz-1 has been unclear. We used a high-throughput in vitro technique, Bind-n-Seq, to identify two Miz-1 consensus DNA binding motif sequences--ATCGGTAATC and ATCGAT (Mizm1 and Mizm2)--bound by full-length Miz-1 and its zinc finger domain, respectively. We validated these sequences directly as high affinity Miz-1 binding motifs. Competition assays using mutant probes indicated that the binding affinity of Miz-1 for Mizm1 and Mizm2 is highly sequence-specific. Miz-1 strongly activates gene expression through the motifs in a Myc-independent manner. MEME-ChIP analysis of Miz-1 ChIP-seq data in two different cell types reveals a long motif with a central core sequence highly similar to the Mizm1 motif identified by Bind-n-Seq, validating the in vivo relevance of the findings. Miz-1 ChIP-seq peaks containing the long motif are predominantly located outside of proximal promoter regions, in contrast to peaks without the motif, which are highly concentrated within 1.5 kb of the nearest transcription start site. Overall, our results indicate that Miz-1 may be directed in vivo to the novel motif sequences we have identified, where it can recruit its specific binding partners to control gene expression and ultimately regulate cell fate. PMID:24983942

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

    Directory of Open Access Journals (Sweden)

    Bonnie L Barrilleaux

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

  16. Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae

    Science.gov (United States)

    Zhang, S.; Lockshin, C.; Herbert, A.; Winter, E.; Rich, A.

    1992-01-01

    A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

  17. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; Glover, J. N. Mark

    2009-01-01

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC282–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

  18. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold.

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J J; Glover, J N Mark

    2009-06-16

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer. PMID:19482939

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

    Science.gov (United States)

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

    2016-03-01

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

  20. Anti-DNA autoantibodies initiate experimental lupus nephritis by binding directly to the glomerular basement membrane in mice.

    Science.gov (United States)

    Krishnan, Meera R; Wang, Congmiao; Marion, Tony N

    2012-07-01

    The strongest serological correlate for lupus nephritis is antibody to double-stranded DNA, although the mechanism by which anti-DNA antibodies initiate lupus nephritis is unresolved. Most recent reports indicate that anti-DNA must bind chromatin in the glomerular basement membrane or mesangial matrix to form glomerular deposits. Here we determined whether direct binding of anti-DNA antibody to glomerular basement membrane is critical to initiate glomerular binding of anti-DNA in experimental lupus nephritis. Mice were co-injected with IgG monoclonal antibodies or hybridomas with similar specificity for DNA and chromatin but different IgG subclass and different relative affinity for basement membrane. Only anti-DNA antibodies that bound basement membrane bound to glomeruli, activated complement, and induced proteinuria whether injected alone or co-injected with a non-basement-membrane-binding anti-DNA antibody. Basement membrane-binding anti-DNA antibodies co-localized with heparan sulfate proteoglycan in glomerular basement membrane and mesangial matrix but not with chromatin. Thus, direct binding of anti-DNA antibody to antigens in the glomerular basement membrane or mesangial matrix may be critical to initiate glomerular inflammation. This may accelerate and exacerbate glomerular immune complex formation in human and murine lupus nephritis.

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

    Science.gov (United States)

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

    2013-09-01

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

  2. NPM/ALK binds and phosphorylates the RNA/DNA-binding protein PSF in anaplastic large-cell lymphoma.

    Science.gov (United States)

    Galietta, Annamaria; Gunby, Rosalind H; Redaelli, Sara; Stano, Paola; Carniti, Cristiana; Bachi, Angela; Tucker, Philip W; Tartari, Carmen J; Huang, Ching-Jung; Colombo, Emanuela; Pulford, Karen; Puttini, Miriam; Piazza, Rocco G; Ruchatz, Holger; Villa, Antonello; Donella-Deana, Arianna; Marin, Oriano; Perrotti, Danilo; Gambacorti-Passerini, Carlo

    2007-10-01

    The oncogenic fusion tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) induces cellular transformation in anaplastic large-cell lymphomas (ALCLs) carrying the t(2;5) chromosomal translocation. Protein-protein interactions involving NPM/ALK are important for the activation of downstream signaling pathways. This study was aimed at identifying novel NPM/ALK-binding proteins that might contribute to its oncogenic transformation. Using a proteomic approach, several RNA/DNA-binding proteins were found to coimmunoprecipitate with NPM/ALK, including the multifunctional polypyrimidine tract binding proteinassociated splicing factor (PSF). The interaction between NPM/ALK and PSF was dependent on an active ALK kinase domain and PSF was found to be tyrosine-phosphorylated in NPM/ALK-expressing cell lines and in primary ALK(+) ALCL samples. Furthermore, PSF was shown to be a direct substrate of purified ALK kinase domain in vitro, and PSF Tyr293 was identified as the site of phosphorylation. Y293F PSF was not phosphorylated by NPM/ALK and was not delocalized in NPM/ALK(+) cells. The expression of ALK fusion proteins induced delocalization of PSF from the nucleus to the cytoplasm and forced overexpression of PSF-inhibited proliferation and induced apoptosis in cells expressing NPM/ALK. PSF phosphorylation also increased its binding to RNA and decreased the PSF-mediated suppression of GAGE6 expression. These results identify PSF as a novel NPM/ALK-binding protein and substrate, and suggest that PSF function may be perturbed in NPM/ALK-transformed cells.

  3. The DNA intercalators ethidium bromide and propidium iodide also bind to core histones

    Directory of Open Access Journals (Sweden)

    Amrita Banerjee

    2014-01-01

    Full Text Available Eukaryotic DNA is compacted in the form of chromatin, in a complex with histones and other non-histone proteins. The intimate association of DNA and histones in chromatin raises the possibility that DNA-interactive small molecules may bind to chromatin-associated proteins such as histones. Employing biophysical and biochemical techniques we have characterized the interaction of a classical intercalator, ethidium bromide (EB and its structural analogue propidium iodide (PI with hierarchical genomic components: long chromatin, chromatosome, core octamer and chromosomal DNA. Our studies show that EB and PI affect both chromatin structure and function, inducing chromatin compaction and disruption of the integrity of the chromatosome. Calorimetric studies and fluorescence measurements of the ligands demonstrated and characterized the association of these ligands with core histones and the intact octamer in absence of DNA. The ligands affect acetylation of histone H3 at lysine 9 and acetylation of histone H4 at lysine 5 and lysine 8 ex vivo. PI alters the post-translational modifications to a greater extent than EB. This is the first report showing the dual binding (chromosomal DNA and core histones property of a classical intercalator, EB, and its longer analogue, PI, in the context of chromatin.

  4. Arrest of rolling circle amplification by protein-binding DNA aptamers.

    Science.gov (United States)

    Wang, Lida; Tram, Kha; Ali, Monsur M; Salena, Bruno J; Li, Jinghong; Li, Yingfu

    2014-02-24

    Certain DNA polymerases, such as ϕ29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super-long single-stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand-displacement ability of these polymerases. In this work, the ability of ϕ29DNAP to carry out RCA over circular templates containing a protein-binding DNA aptamer sequence was investigated. It was found that protein-aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein-binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.

  5. Synthesis, structure, DNA binding and cleavage activity of a new copper(Ⅱ) complex of bispyridylpyrrolide

    Institute of Scientific and Technical Information of China (English)

    MIN Rui; HU Xiao-hui; YI Xiao-yi; ZHANG Shou-chun

    2015-01-01

    A copper-bispyridylpyrrolide complex [Cu(PDPH)Cl] (PDPH = 2,5-bis(2′-pyridyl)pyrrole) was synthesized and characterized. The complex crystallizes in the orthorhombic system with space groupPccn,a = 0.9016(3) nm,b = 1.0931(4) nm,c = 2.5319(8) nm, andV = 2.4951(15) nm3. The copper center is situated in a square planar geometry. The interaction of the copper(Ⅱ) complexwith calf thymus DNA (CT-DNA) was investigated by electronic absorption, circular dichroism (CD) and fluorescence spectra. It is proposed that the complex binds to CT-DNA through groove binding mode. Nuclease activity of the complex was also studied by gel electrophoresis method. The complex can efficiently cleave supercoiled pBR322 DNA in the presence of ascorbate (H2A) via oxidative pathway. The preliminary mechanism of DNA cleavage by the complex with different inhibiting reagents indicates that the hydroxyl radicals were involved as the active species in the DNA cleavage process.

  6. SSB protein limits RecOR binding onto single-stranded DNA.

    Science.gov (United States)

    Hobbs, Michael D; Sakai, Akiko; Cox, Michael M

    2007-04-13

    The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action. PMID:17272275

  7. DNA binding studies of 3, 5, 6-trichloro-2-pyridinol pesticide metabolite.

    Science.gov (United States)

    Kashanian, Soheila; Shariati, Zohreh; Roshanfekr, Hamideh; Ghobadi, Sirous

    2012-07-01

    3, 5, 6-Trichloro-2-pyridinol (TCP) is a stable metabolite of two major pesticides, Chlopyrifos insecticide and Triclopyr herbicide, which are widely used in the world. The potential health hazard associated with TCP is identified due to its high affinity to the DNA molecule. Therefore, in this study, the interaction of native calf thymus DNA with TCP has been investigated using spectrophotometric, circular dichroism (CD), spectrofluorometric, viscometric and voltametric techniques. It was found that TCP molecules could interact with DNA via a groove-binding mode, as evidenced by hyperchromism, with no red shift in the UV absorption band of TCP, no changes in K(b) values in the presence of salt, no significant changes in the specific viscosity and CD spectra of DNA, and a decrease in peak currents with no shift in the voltamogram. In addition, TCP is able to release Hoechst 33258, a strong groove binder, in the DNA solutions. The results are indicative of the groove-binding mode of TCP to DNA. PMID:22519761

  8. Quantitative radiommunoassay for DNA-binding antibodies. [Iodine 131, Iodine 125

    Energy Technology Data Exchange (ETDEWEB)

    Smith, L.H.; Guyer, R.L.; Minami, R.M.; Teplitz, R.L.

    1981-09-01

    A radioimmunoassay (RIA) is described for the measurement of serum immunoglobulins capable of binding to double-standard or single-standard DNA. DNA attached to Sephadex G-50 by ultraviolet radiation was used as a solid- phase immunoabsorbent for DNA-binding proteins from serum. Goat anti-human (GAH) IgG (/sup 125/I-labeled) were used to detect the human immunoglobulins bound onto the washed DNA-Sephadex. The quantities of immunoglobulins bound were determined by comparison with a standard curve constructed by dilution of a plasma from an systemic lupus erythematosus (SLE) patient containing known amounts of bound, DNA-specific IgM and IgG. Another RIA was employed for measuring levels of IgG and IgM. In combination with measurements of the total serum IgM and IgG, the RIA allowed for the determination of the fraction of the total serum IgM or IgG that was specific for double- or single-standard DNA. For a pool of normal human sera the quantities were as follows: 0.04% of the total IgM and 0.001% of the total IgG bound double-standard DNA; 0.22% of the total IgM and 0.05% of the total IgG bound single-stranded DNA. This capability is important because information regarding the quantitative measurement of antibodies to DNA and their class determination may be of significance in monitoring the status of subjects with SLE.

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

    Directory of Open Access Journals (Sweden)

    Marcin Olszewski

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

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

    Science.gov (United States)

    Olszewski, Marcin; Balsewicz, Jan; Nowak, Marta; Maciejewska, Natalia; Cyranka-Czaja, Anna; Zalewska-Piątek, Beata; Piątek, Rafał; Kur, Józef

    2015-01-01

    Background SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis. Results This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7±1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100°C and melting temperature (Tm) is 100.2°C as shown by differential scanning calorimetry (DSC) analysis. Conclusion NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids. PMID:25973760

  11. Troxerutin, a natural flavonoid binds to DNA minor groove and enhances cancer cell killing in response to radiation.

    Science.gov (United States)

    Panat, Niranjan A; Singh, Beena G; Maurya, Dharmendra K; Sandur, Santosh K; Ghaskadbi, Saroj S

    2016-05-01

    Troxerutin, a flavonoid best known for its radioprotective and antioxidant properties is of considerable interest of study due to its broad pharmacological activities. The present study on troxerutin highlights its abilities to bind DNA and enhance cancer cell killing in response to radiation. Troxerutin showed strong binding with calf thymus DNA in vitro. Troxerutin-DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing troxerutin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. DAPI fluorescence was drastically reduced with linear increase in troxerutin concentration suggesting possible binding of troxerutin to DNA minor groove. Further, computational studies of docking of troxerutin molecule on mammalian DNA also indicated possible troxerutin-DNA interaction at minor groove of DNA. Troxerutin was found to mainly localize in the nucleus of prostate cancer cells. It induced cytotoxicity in radioresistant (DU145) and sensitive (PC3) prostate cancer cells. When troxerutin pre-treated DU145 and PC3 cells were exposed to γ-radiation, cytotoxicity as estimated by MTT assay, was found to be further enhanced. In addition, the % subG1 population detected by propidium iodide staining also showed similar response when combined with radiation. A similar trend was observed in terms of ROS generation and DNA damage in DU145 cells when troxerutin and radiation were combined. DNA binding at minor groove by troxerutin may have contributed to strand breaks leading to increased radiation induced cell death.

  12. Dynamic Local Polymorphisms in the Gbx1 Homeodomain Induced by DNA Binding.

    Science.gov (United States)

    Proudfoot, Andrew; Geralt, Michael; Elsliger, Marc-Andre; Wilson, Ian A; Wüthrich, Kurt; Serrano, Pedro

    2016-08-01

    The Gastrulation Brain Homeobox 1 (Gbx1) gene encodes the Gbx1 homeodomain that targets TAATTA motifs in double-stranded DNA (dsDNA). Residues Glu17 and Arg52 in Gbx1 form a salt bridge, which is preserved in crystal structures and molecular dynamics simulations of homologous homeodomain-DNA complexes. In contrast, our nuclear magnetic resonance (NMR) studies show that DNA binding to Gbx1 induces dynamic local polymorphisms, which include breaking of the Glu17-Arg52 salt bridge. To study this interaction, we produced a variant with Glu17Arg and Arg52Glu mutations, which exhibited the same fold as the wild-type protein, but a 2-fold reduction in affinity for dsDNA. Analysis of the NMR structures of the Gbx1 homeodomain in the free form, the Gbx1[E17R,R52E] variant, and a Gbx1 homeodomain-DNA complex showed that stabilizing interactions of the Arg52 side chain with the DNA backbone are facilitated by transient breakage of the Glu17-Arg52 salt bridge in the DNA-bound Gbx1. PMID:27396829

  13. Cationic polymers for DNA origami coating - examining their binding efficiency and tuning the enzymatic reaction rates

    Science.gov (United States)

    Kiviaho, Jenny K.; Linko, Veikko; Ora, Ari; Tiainen, Tony; Järvihaavisto, Erika; Mikkilä, Joona; Tenhu, Heikki; Nonappa, Affc; Kostiainen, Mauri A.

    2016-06-01

    DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The effect of the polymer structure on the binding was investigated and the toxicity of the polymer-origami complexes evaluated. The study shows that all of the analyzed polymers had a suitable binding efficiency irrespective of the block structure. It was also observed that the toxicity of polymer-origami complexes was insignificant at the biologically relevant concentration levels. Besides brick-like DNA origamis, tubular origami carriers equipped with enzymes were also coated with the polymers. By adjusting the amount of cationic polymers that cover the DNA structures, we showed that it is possible to control the enzyme kinetics of the complexes. This work gives a starting point for further development of biocompatible and effective polycation-based block copolymers that can be used in coating different DNA origami nanostructures for various bioapplications.DNA origamis are fully tailored, programmable, biocompatible and readily functionalizable nanostructures that provide an excellent foundation for the development of sophisticated drug-delivery systems. However, the DNA origami objects suffer from certain drawbacks such as low cell-transfection rates and low stability. A great deal of studies on polymer-based transfection agents, mainly focusing on polyplex formation and toxicity, exists. In this study, the electrostatic binding between a brick-like DNA origami and cationic block-copolymers was explored. The

  14. The wing in yeast heat shock transcription factor (HSF) DNA-binding domain is required for full activity

    OpenAIRE

    Cicero, Marco P.; T. Hubl, Susan; Harrison, Celia J.; Littlefield, Otis; Hardy, Jeanne A.; Nelson, Hillary C. M.

    2001-01-01

    The yeast heat shock transcription factor (HSF) belongs to the winged helix family of proteins. HSF binds DNA as a trimer, and additional trimers can bind DNA co-operatively. Unlike other winged helix–turn–helix proteins, HSF’s wing does not appear to contact DNA, as based on a previously solved crystal structure. Instead, the structure implies that the wing is involved in protein–protein interactions, possibly within a trimer or between adjacent trimers. To unders...

  15. Antiproliferative activity of bicyclic benzimidazole nucleosides: synthesis, DNA-binding and cell cycle analysis.

    Science.gov (United States)

    Sontakke, Vyankat A; Lawande, Pravin P; Kate, Anup N; Khan, Ayesha; Joshi, Rakesh; Kumbhar, Anupa A; Shinde, Vaishali S

    2016-04-26

    An efficient route was developed for synthesis of bicyclic benzimidazole nucleosides from readily available d-glucose. The key reactions were Vörbruggen glycosylation and ring closing metathesis (RCM). Primarily, to understand the mode of DNA binding, we performed a molecular docking study and the binding was found to be in the minor groove region. Based on the proposed binding model, UV-visible and fluorescence spectroscopic techniques using calf thymus DNA (CT-DNA) demonstrated a non-intercalative mode of binding. Antiproliferative activity of nucleosides was tested against MCF-7 and MDA-MB-231 breast cancer cell lines and found to be active at low micromolar concentrations. Compounds and displayed significant antiproliferative activity as compared to and with the reference anticancer drug, doxorubicin. Cell cycle analysis showed that nucleoside induced cell cycle arrest at the S-phase. Confocal microscopy has been performed to validate the induction of cellular apoptosis. Based on these findings, such modified bicyclic benzimidazole nucleosides will make a significant contribution to the development of anticancer drugs. PMID:27074628

  16. A competition assay for DNA binding using the fluorescent probe ANS.

    Science.gov (United States)

    Taylor, Ian A; Kneale, G Geoff

    2009-01-01

    Fluorescence spectroscopy is a technique frequently employed to study protein-nucleic acid interactions. Often, the intrinsic fluorescence emission spectrum of tryptophan residues in a nucleic-acid-binding protein is strongly perturbed upon interaction with a target DNA or RNA. These spectral changes can then be exploited in order to construct binding isotherms and the extract equilibrium association constant together with the stoichiometry of an interaction. However, when a protein contains many tryptophan residues that are not located in the proximity of the nucleic-acid-binding site, changes in the fluorescence emission spectrum may not be apparent or the magnitude too small to be useful. Here, we make use of an extrinsic fluorescence probe, the environmentally sensitive fluorophore 1-anilinonaphthalene-8-sulphonic acid (1,8-ANS). Displacement by DNA of 1,8-ANS molecules from the nucleic-acid-binding site of the Type I modification methylase EcoR124I results in red shifting and an intensity decrease of the 1,8-ANS fluorescence emission spectrum. These spectral changes have been used to investigate the interaction of EcoR124I with DNA target recognition sequences.

  17. PDNAsite: Identification of DNA-binding Site from Protein Sequence by Incorporating Spatial and Sequence Context.

    Science.gov (United States)

    Zhou, Jiyun; Xu, Ruifeng; He, Yulan; Lu, Qin; Wang, Hongpeng; Kong, Bing

    2016-01-01

    Protein-DNA interactions are involved in many fundamental biological processes essential for cellular function. Most of the existing computational approaches employed only the sequence context of the target residue for its prediction. In the present study, for each target residue, we applied both the spatial context and the sequence context to construct the feature space. Subsequently, Latent Semantic Analysis (LSA) was applied to remove the redundancies in the feature space. Finally, a predictor (PDNAsite) was developed through the integration of the support vector machines (SVM) classifier and ensemble learning. Results on the PDNA-62 and the PDNA-224 datasets demonstrate that features extracted from spatial context provide more information than those from sequence context and the combination of them gives more performance gain. An analysis of the number of binding sites in the spatial context of the target site indicates that the interactions between binding sites next to each other are important for protein-DNA recognition and their binding ability. The comparison between our proposed PDNAsite method and the existing methods indicate that PDNAsite outperforms most of the existing methods and is a useful tool for DNA-binding site identification. A web-server of our predictor (http://hlt.hitsz.edu.cn:8080/PDNAsite/) is made available for free public accessible to the biological research community. PMID:27282833

  18. Cdc13 N-Terminal Dimerization DNA Binding and Telomere Length Regulation

    Energy Technology Data Exchange (ETDEWEB)

    M Mitchell; J Smith; M Mason; S Harper; D Speicher; F Johnson; E Skordalakes

    2011-12-31

    The essential yeast protein Cdc13 facilitates chromosome end replication by recruiting telomerase to telomeres, and together with its interacting partners Stn1 and Ten1, it protects chromosome ends from nucleolytic attack, thus contributing to genome integrity. Although Cdc13 has been studied extensively, the precise role of its N-terminal domain (Cdc13N) in telomere length regulation remains unclear. Here we present a structural, biochemical, and functional characterization of Cdc13N. The structure reveals that this domain comprises an oligonucleotide/oligosaccharide binding (OB) fold and is involved in Cdc13 dimerization. Biochemical data show that Cdc13N weakly binds long, single-stranded, telomeric DNA in a fashion that is directly dependent on domain oligomerization. When introduced into full-length Cdc13 in vivo, point mutations that prevented Cdc13N dimerization or DNA binding caused telomere shortening or lengthening, respectively. The multiple DNA binding domains and dimeric nature of Cdc13 offer unique insights into how it coordinates the recruitment and regulation of telomerase access to the telomeres.

  19. Synthesis, photochemistry, DNA cleavage/binding and cytotoxic properties of fluorescent quinoxaline and quinoline hydroperoxides.

    Science.gov (United States)

    Chowdhury, Nilanjana; Gangopadhyay, Moumita; Karthik, S; Pradeep Singh, N D; Baidya, Mithu; Ghosh, S K

    2014-01-01

    Novel fluorescent quinoxaline and quinoline hydroperoxides were shown to perform dual role as both fluorophores for cell imaging and photoinduced DNA cleaving agents. Photophysical studies of newly synthesized quinoxaline and quinoline hydroperoxides showed that they all exhibited moderate to good fluorescence. Photolysis of quinoxaline and quinoline hydroperoxides in acetonitrile using UV light above 350nm resulted in the formation of corresponding ester compounds via γ-hydrogen abstraction by excited carbonyl chromophore. Single strand DNA cleavage was achieved on irradiation of newly synthesized hydroperoxides by UV light (⩾350nm). Both hydroxyl radicals and singlet oxygen were identified as reactive oxygen species (ROS) responsible for the DNA cleavage. Further, we showed quinoline hydroperoxide binds to ct-DNA via intercalative mode. In vitro biological studies revealed that quinoline hydroperoxide has good biocompatibility, cellular uptake property and cell imaging ability. Finally, we showed that quinoline hydroperoxide can permeate into cells efficiently and may cause cytotoxicity upon irradiation by UV light.

  20. Sequence specificity of formaldehyde-mediated covalent binding of anthracycline derivatives to DNA.

    Science.gov (United States)

    Szulawska, Agata; Gniazdowski, Marek; Czyz, Malgorzata

    2005-01-01

    Daunorubicin (DRB) and doxorubicin (DOX) in the presence of formaldehyde (CH2O) form covalent adducts with DNA. A G-specific adduct is formed by producing an aminal bridge between the C-3' of daunosamine and the C-2 of guanine. New derivatives of DRB, DOX and epidoxorubicin (EDOX) with an amidine group bonded to the C-3' of the daunosamine moiety, with either a morpholine or hexamethyleneimine ring attached to the amidine group, were studied in this paper. DNase I footprinting and analyses with restriction endonucleases were applied to compare the specificity of adduct formed by the amidine derivatives and their parent compounds. These approaches provide consistent results, proving that a GC pair is required for covalent binding of anthracycline derivatives to DNA and that different flanking sequences are able to modify the sequence preference of the drugs. The 5'-GC-3', 5'-CG-3' and 5'-TC-3' sequences were protected most efficiently by the parent compounds and their morpholine derivatives and some increased protection of 5'-TC-3' sequence was observed for morpholine analogues. Hexamethyleneimine derivatives bind to DNA with much lower efficiency. Finally, the sequence specificity of anthracycline derivatives was correlated with their ability to inhibit binding of transcription factors Sp1 and AP-1 to their DNA recognition sequences. The anthracycline derivatives were more potent in inhibiting Sp1 binding to its cognate GC box than in preventing AP-1 from binding to its mixed A.T and G.C site. Overall, the results indicate that the amidine derivatives of anthracyclines show similar, but not identical sequence specificity as parent compounds, though they exert their effect at a higher concentration. PMID:15588709

  1. CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins

    Science.gov (United States)

    Savic, Daniel; Partridge, E. Christopher; Newberry, Kimberly M.; Smith, Sophia B.; Meadows, Sarah K.; Roberts, Brian S.; Mackiewicz, Mark; Mendenhall, Eric M.; Myers, Richard M.

    2015-01-01

    Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable data sets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIP-seq through epitope tagging of endogenous TFs. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR epitope tagging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several DNA-binding proteins spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIP-quality antibodies are not available. PMID:26355004

  2. Cross-talk between the ligand- and DNA-binding domains of estrogen receptor.

    Science.gov (United States)

    Huang, Wei; Greene, Geoffrey L; Ravikumar, Krishnakumar M; Yang, Sichun

    2013-11-01

    Estrogen receptor alpha (ERα) is a hormone-responsive transcription factor that contains several discrete functional domains, including a ligand-binding domain (LBD) and a DNA-binding domain (DBD). Despite a wealth of knowledge about the behaviors of individual domains, the molecular mechanisms of cross-talk between LBD and DBD during signal transduction from hormone to DNA-binding of ERα remain elusive. Here, we apply a multiscale approach combining coarse-grained (CG) and atomistically detailed simulations to characterize this cross-talk mechanism via an investigation of the ERα conformational landscape. First, a CG model of ERα is built based on crystal structures of individual LBDs and DBDs, with more emphasis on their interdomain interactions. Second, molecular dynamics simulations are implemented and enhanced sampling is achieved via the "push-pull-release" strategy in the search for different LBD-DBD orientations. Third, multiple energetically stable ERα conformations are identified on the landscape. A key finding is that estradiol-bound LBDs utilize the well-described activation helix H12 to pack and stabilize LBD-DBD interactions. Our results suggest that the estradiol-bound LBDs can serve as a scaffold to position and stabilize the DBD-DNA complex, consistent with experimental observations of enhanced DNA binding with the LBD. Final assessment using atomic-level simulations shows that these CG-predicted models are significantly stable within a 15-ns simulation window and that specific pairs of lysine residues in close proximity at the domain interfaces could serve as candidate sites for chemical cross-linking studies. Together, these simulation results provide a molecular view of the role of ERα domain interactions in response to hormone binding.

  3. CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins.

    Science.gov (United States)

    Savic, Daniel; Partridge, E Christopher; Newberry, Kimberly M; Smith, Sophia B; Meadows, Sarah K; Roberts, Brian S; Mackiewicz, Mark; Mendenhall, Eric M; Myers, Richard M

    2015-10-01

    Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable data sets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIP-seq through epitope tagging of endogenous TFs. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR epitope tagging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several DNA-binding proteins spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIP-quality antibodies are not available.

  4. DNA binding, DNA cleavage, antioxidant and cytotoxicity studies on ruthenium(II) complexes of benzaldehyde 4-methyl-3-thiosemicarbazones

    Science.gov (United States)

    Sampath, Krishnan; Sathiyaraj, Subbaiyan; Jayabalakrishnan, Chinnasamy

    2013-03-01

    Four new ruthenium(II) complexes with N(4)-methyl thiosemicarbazone ligands, (E)-2-(2-chlorobenzylidene)-N-methylhydrazinecarbothioamide (HL1) and (E)-N-methyl-2-(2-nitrobenzylidene)hydrazinecarbothioamide (HL2), were prepared and fully characterized by various spectro-analytical techniques. The Schiff bases act as bidentate, monobasic chelating ligands with S and N as the donor sites and are preferably found in the thiol form in all the complexes studied. The molecular structure of HL1 and HL2 were determined by single crystal X-ray diffraction method. DNA binding of the compounds was investigated by absorption spectroscopy which indicated that the complexes bind to DNA via intercalation. The oxidative cleavage of the complexes with CT-DNA inferred that the effects of cleavage are dose dependent. Antioxidant studies of the ligands and complexes showed the significant antioxidant activity against DPPH radical. In addition, the in vitro cytotoxicity of the ligands and complexes against MCF-7 cell line was assayed which showed higher cytotoxic activity with the lower IC50 values indicating their efficiency in killing the cancer cells even at low concentrations.

  5. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon–helix–helix DNA-binding fold

    OpenAIRE

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J. J.; Glover, J. N. Mark

    2009-01-01

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-Å X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC282–202, which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DN...

  6. Binding of PFOS to serum albumin and DNA: insight into the molecular toxicity of perfluorochemicals

    Directory of Open Access Journals (Sweden)

    Ma Yin-Sheng

    2009-02-01

    Full Text Available Abstract Background Health risk from exposure of perfluorochemicals (PFCs to wildlife and human has been a subject of great interest for understanding their molecular mechanism of toxicity. Although much work has been done, the toxigenicity of PFCs remains largely unknown. In this work, the non-covalent interactions between perfluorooctane sulfonate (PFOS and serum albumin (SA and DNA were investigated under normal physiological conditions, aiming to elucidate the toxigenicity of PFCs. Results In equilibrium dialysis assay, the bindings of PFOS to SA correspond to the Langmuir isothermal model with two-step sequence model. The saturation binding number of PFOS was 45 per molecule of SA and 1 per three base-pairs of DNA, respectively. ITC results showed that all the interactions were spontaneous driven by entropy change. Static quenching of the fluorescence of SA was observed when interacting with PFOS, indicating PFOS bound Trp residue of SA. CD spectra of SA and DNA changed obviously in the presence of PFOS. At normal physiological conditions, 1.2 mmol/l PFOS reduces the binding ratio of Vitamin B2 to SA by more than 30%. Conclusion The ion bond, van der Waals force and hydrophobic interaction contributed to PFOS binding to peptide chain of SA and to the groove bases of DNA duplex. The non-covalent interactions of PFOS with SA and DNA alter their secondary conformations, with the physiological function of SA to transport Vitamin B2 being inhibited consequently. This work provides a useful experimental method for further studying the toxigenicity of PFCs.

  7. Synthesis, characterization, molecular docking, DNA binding, cytotoxicity and DFT studies of 1-(4-methoxyphenyl)-3-(pyridine-3-ylmethyl)thiourea

    Science.gov (United States)

    Mushtaque, Md; Jahan, Meriyam; Ali, Murtaza; Khan, Md Shahzad; Khan, Mohd Shahid; Sahay, Preeti; Kesarwani, Ashwani

    2016-10-01

    A new compound 1-(4-methoxyphenyl)-3-(pyridine-3-ylmethyl)thiourea was synthesized and structure of compound (3) was elucidated by FT-IR, 1H-NMR, and mass spectrophotometer. The computational quantum chemical studies of compound (3) like, IR, UV, NBO analysis were performed by DFT with B3LYP exchange-correlation functional in combination with 6-311++G(d, p) basis sets. The compound (3) adopted syn-anti-configuration around sulphur atom, possessing stablization relative energy -740715 kcal/mol. The chemical potential of compound (3) is -3.37 eV and chemical hardness is -2.33 eV. However, ionization and electron affinity of compound (3) are -5.70 eV and -1.04 eV. The compound (3) was docked with B-DNA (1BNA) and the binding energy was found to be -7.41 kcal/mol. The nitrogen atom of thiourea of compound (3) binds with O3 and O4 of cytosine of A strand of DNA having bond lengths (1.92 Å) and (1.74 Å) respectively Furthermore, DNA binding constant was performed by UV-visible spectrophotometer. The binding constant was found 3.71 × 106 Lmol-1. In order to assess cytotoxic nature of the lead compound, MTT-assay was performed against MCF-7 cell line and IC50 value of compound (3) was observed at 160.97 μ M. Theoretical studies revealed that they are good agreement with experimental results.

  8. Genetic recombination in Bacillus subtilis: a division of labor between two single-strand DNA-binding proteins.

    Science.gov (United States)

    Yadav, Tribhuwan; Carrasco, Begoña; Myers, Angela R; George, Nicholas P; Keck, James L; Alonso, Juan C

    2012-07-01

    We have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) DNA-binding proteins from Bacillus subtilis, SsbA and SsbB. During transformation, SsbB localizes at the DNA entry pole where it binds and protects internalized ssDNA. The 2.8-Å resolution structure of SsbB bound to ssDNA reveals a similar overall protein architecture and ssDNA-binding surface to that of Escherichia coli SSB. SsbA, which binds ssDNA with higher affinity than SsbB, co-assembles onto SsbB-coated ssDNA and the two proteins inhibit ssDNA binding by the recombinase RecA. During chromosomal transformation, the RecA mediators RecO and DprA provide RecA access to ssDNA. Interestingly, RecO interaction with ssDNA-bound SsbA helps to dislodge both SsbA and SsbB from the DNA more efficiently than if the DNA is coated only with SsbA. Once RecA is nucleated onto the ssDNA, RecA filament elongation displaces SsbA and SsbB and enables RecA-mediated DNA strand exchange. During plasmid transformation, RecO localizes to the entry pole and catalyzes annealing of SsbA- or SsbA/SsbB-coated complementary ssDNAs to form duplex DNA with ssDNA tails. Our results provide a mechanistic framework for rationalizing the coordinated events modulated by SsbA, SsbB and RecO that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation. PMID:22373918

  9. Preferential DNA Cleavage under Anaerobic Conditions by a DNA Binding Ruthenium Dimer

    OpenAIRE

    Janaratne, Thamara K.; Ongeri, Fiona; Yadav, Abishek; MacDonnell, Frederick M.

    2007-01-01

    In the absence of O2, the cationic complex, [(phen)2Ru(tatpp)Ru(phen)2]4+ (P4+), undergoes in situ reduction by glutathione (GSH) to form a species that induces DNA cleavage. Exposure to air strongly attenuates the cleavage activity, even in the presence of a large excess of reducing agent (e.g., 40 equiv GSH per P4+) suggesting the complex may be useful in targeting cells with a low oxygen microenvironment (hypoxia) for destruction via DNA cleavage. The active species is identified as the do...

  10. Binding hot-spots in an antibody-ssDNA interface: a molecular dynamics study.

    Science.gov (United States)

    Wang, Yeng-Tseng; Lee, Wen-Jay

    2012-10-30

    Simulating antigen-antibody interactions is essential for elucidating antigen-antibody mechanics. Proteins interactions are vital for elucidating antibody-ssDNA associations in immunology. Therefore, this study investigated the dissociation of the human systemic lupus erythematosus antibody-ssDNA complex structure. Dissociation (i.e. the distance between the center of mass of the ssDNA and the antibody) is also studied using the potential of mean force calculations based on molecular dynamics and the explicit water model. The MM-PBSA method is also used to prove our dissociation simulations. With 605 nanosecond molecular dynamics simulations, the results indicate that the 8 residues (i.e. Gly44 (HCDR2), Asn54 (HCDR2), Arg98 (HCDR3), Tyr100 (HCDR3), Asp101 (HCDR3), Tyr32 (LCDR1), Tyr49 (LCDR2) and Asn50 (LCDR2)), and the five inter-protein molecular hydrogen bonds may profoundly impact the antibody-ssDNA interaction, a finding which may be useful for protein engineering of this antibody-ssDNA structure. Experimental binding affinity of this antibody-ssDNA complex equals 7.00 kcal mol(-1). Our dissociation binding affinity is 7.96 ± 0.33 kcal mol(-1) and MM-PBSA binding affinity is 9.12 ± 1.65 kcal mol(-1), which is close to the experimental value. Additionally, the 8 residues Gly44 (HCDR2), Asn54 (HCDR2), Arg98 (HCDR3), Tyr100 (HCDR3), Asp101 (HCDR3), Tyr32 (LCDR1), Tyr49 (LCDR2) and Asn50 (LCDR2) may play a more significant role in developing bioactive antibody analogues. PMID:23079742

  11. Structural Insight into the DNA-Binding Mode of the Primosomal Proteins PriA, PriB, and DnaT

    Directory of Open Access Journals (Sweden)

    Yen-Hua Huang

    2014-01-01

    Full Text Available Replication restart primosome is a complex dynamic system that is essential for bacterial survival. This system uses various proteins to reinitiate chromosomal DNA replication to maintain genetic integrity after DNA damage. The replication restart primosome in Escherichia coli is composed of PriA helicase, PriB, PriC, DnaT, DnaC, DnaB helicase, and DnaG primase. The assembly of the protein complexes within the forked DNA responsible for reloading the replicative DnaB helicase anywhere on the chromosome for genome duplication requires the coordination of transient biomolecular interactions. Over the last decade, investigations on the structure and mechanism of these nucleoproteins have provided considerable insight into primosome assembly. In this review, we summarize and discuss our current knowledge and recent advances on the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.

  12. Specific binding of PapI to Lrp-pap DNA complexes.

    OpenAIRE

    Kaltenbach, L S; Braaten, B A; Low, D A

    1995-01-01

    Expression of pyelonephritis-associated pili (Pap) varies between transcriptionally active (ON) and inactive (OFF) phase states. Pap phase variation is controlled by the binding of leucine-responsive regulatory protein (Lrp) to two pap regulatory DNA regions, each containing a deoxyadenosine methylase site and designated GATC-I and GATC-II. Methylation of these GATC sites modulates binding of Lrp and plays an essential role in phase variation. PapI, an 8.8-kDa pap-encoded regulatory protein, ...

  13. Inflammatory properties of inhibitor of DNA binding 1 secreted by synovial fibroblasts in rheumatoid arthritis

    OpenAIRE

    Edhayan, Gautam; Ohara, Ray A.; Stinson, W. Alex; Amin, M. Asif; Isozaki, Takeo; Ha, Christine M; Haines, G. Kenneth; Morgan, Rachel; Campbell, Phillip L.; Arbab, Ali S; Friday, Sean C.; Fox, David A; Ruth, Jeffrey H.

    2016-01-01

    Background Inhibitor of DNA binding 1 (Id1) is a nuclear protein containing a basic helix-loop-helix (bHLH) domain that regulates cell growth by selective binding and prevention of gene transcription. Sources of Id1 production in rheumatoid arthritis synovial tissue (RA ST) and its range of functional effects in RA remain to be clarified. Methods We analyzed Id1 produced from synovial fibroblasts and endothelial cells (ECs) with histology and real-time polymerase chain reaction (RT-PCR). Fibr...

  14. Isoquinoline alkaloids and their binding with DNA: calorimetry and thermal analysis applications.

    Science.gov (United States)

    Bhadra, Kakali; Kumar, Gopinatha Suresh

    2010-11-01

    Alkaloids are a group of natural products with unmatched chemical diversity and biological relevance forming potential quality pools in drug screening. The molecular aspects of their interaction with many cellular macromolecules like DNA, RNA and proteins are being currently investigated in order to evolve the structure activity relationship. Isoquinolines constitute an important group of alkaloids. They have extensive utility in cancer therapy and a large volume of data is now emerging in the literature on their mode, mechanism and specificity of binding to DNA. Thermodynamic characterization of the binding of these alkaloids to DNA may offer key insights into the molecular aspects that drive complex formation and these data can provide valuable information about the balance of driving forces. Various thermal techniques have been conveniently used for this purpose and modern calorimetric instrumentation provides direct and quick estimation of thermodynamic parameters. Thermal melting studies and calorimetric techniques like isothermal titration calorimetry and differential scanning calorimetry have further advanced the field by providing authentic, reliable and sensitive data on various aspects of temperature dependent structural analysis of the interaction. In this review we present the application of various thermal techniques, viz. isothermal titration calorimetry, differential scanning calorimetry and optical melting studies in the characterization of drug-DNA interactions with particular emphasis on isoquinoline alkaloid-DNA interaction.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    NAC (NAM/ATAF/CUC) plant transcription factors regulate essential processes in development, stress responses and nutrient distribution in important crop and model plants (rice, Populus, Arabidopsis), which makes them highly relevant in the context of crop optimization and bioenergy production...... transcription factors and the mammalian GCM (Glial cell missing) transcription factors, which all use a ß-strand motif for DNA-binding. The structure shows that the NAC domain inserts the edge of its core ß-sheet into the major groove, while leaving the DNA largely undistorted. The structure of the NAC...

  16. NMR characterization of the DNA binding properties of a novel Hoechst 33258 analogue peptide building block

    DEFF Research Database (Denmark)

    Bunkenborg, Jakob; Behrens, Carsten; Jacobsen, Jens Peter

    2002-01-01

    A novel aryl-bis-benzimidazole amino acid analogue of the DNA-binding compound Hoechst 33258 has recently been designed for incorporation in peptide combinatorial libraries by replacing the N-methylpiperazine group with a carboxyl group and the hydroxy group with an amino-methyl group. The DNA...... preference with the bis-benzimidazole moiety displaced toward the 3'-end from the center of the duplex. Two families of models of the complexes with A(5) and A(3)T(3) were derived with restrained molecular dynamics based on a large set of 70 and 61, respectively, intermolecular ligand NOEs. Both models give...

  17. Single-stranded DNA-binding proteins: multiple domains for multiple functions.

    Science.gov (United States)

    Dickey, Thayne H; Altschuler, Sarah E; Wuttke, Deborah S

    2013-07-01

    The recognition of single-stranded DNA (ssDNA) is integral to myriad cellular functions. In eukaryotes, ssDNA is present stably at the ends of chromosomes and at some promoter elements. Furthermore, it is formed transiently by several cellular processes including telomere synthesis, transcription, and DNA replication, recombination, and repair. To coordinate these diverse activities, a variety of proteins have evolved to bind ssDNA in a manner specific to their function. Here, we review the recognition of ssDNA through the analysis of high-resolution structures of proteins in complex with ssDNA. This functionally diverse set of proteins arises from a limited set of structural motifs that can be modified and arranged to achieve distinct activities, including a range of ligand specificities. We also investigate the ways in which these domains interact in the context of large multidomain proteins/complexes. These comparisons reveal the structural features that define the range of functions exhibited by these proteins.

  18. Microbial interactions chapter: binding and entry of DNA in bacterial transformation

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.A.

    1977-01-01

    Genetic transformation of bacteria by DNA released from cells of a related strain is discussed. The mechanism by which the giant information-bearing molecules of DNA are transported into the bacterial cell was investigated. It was concluded that the overall process of DNA uptake consists of two main steps, binding of donor DNA to the outside of the cell and entry of the bound DNA into the cell. Each step is discussed in detail. Inasmuch as these phenomena occur at the cell surface, they are related to structures and functions of the cell wall and membrane. In addition, the development of competence, that is the formation of cell surface structures allowing DNA uptake, is examined from both a physiological and evolutionary point of view. Genetic transfer mediated by free DNA is an obvious and important form of cellular interaction. The development of competence involves another, quite distinct system of interaction between bacterial cells. Streptococcus pneumoniae, Bacillus subtilis, and Hemophilus influenzae were used as the test organisms. 259 references.

  19. Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence

    OpenAIRE

    Breslin, Mary B; Zhu, Min; Notkins, Abner L.; Lan, Michael S.

    2002-01-01

    A novel cDNA, insulinoma-associated antigen-1 (IA-1), containing five zinc-finger DNA-binding motifs, was isolated from a human insulinoma subtraction library. IA-1 expression is restricted to fetal but not adult pancreatic and brain tissues as well as tumors of neuroendocrine origin. Using various GAL4 DNA binding domain (DBD)/IA-1 fusion protein constructs, we demonstrated that IA-1 functions as a transcriptional repressor and that the region between amino acids 168 and 263 contains the maj...

  20. Mixed-ligand complexes of ruthenium(II) incorporating a diazo ligand: Synthesis, characterization and DNA binding

    Indian Academy of Sciences (India)

    Megha S Deshpande; Avinash S Kumbhar

    2005-03-01

    Mixed-ligand complexes of the type [Ru(N-N)2(dzdf)]Cl2, where N-N is 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen) and 9-diazo-4,5-diazafluorene (dzdf), have been synthesized and characterized by elemental analysis, UV-Vis, IR and NMR spectroscopy. Binding of these complexes with calf thymus DNA (CT-DNA) has been investigated by absorption spectroscopy, steady-state emission spectroscopy and viscosity measurements. The experimental results indicate that the size and shape of the intercalating ligands have marked effect on the binding affinity of the complexes to CT-DNA. The complex [Ru(phen)2(dzdf)]Cl2 binds with CT-DNA through an intercalative binding mode, while the complex [Ru(bpy)2(dzdf)]Cl2 binds electrostatically.

  1. Cu(II) complexes of glyco-imino-aromatic conjugates in DNA binding, plasmid cleavage and cell cytotoxicity

    Indian Academy of Sciences (India)

    Amit Kumar; Atanu Mitra; Amrendra Kumar Ajay; Manoj Kumar Bhat; Chebrolu P Rao

    2012-11-01

    Binding of metal complexes of C2-glucosyl conjugates with DNA has been established by absorption and fluorescence studies. Conformational changes occurred in DNA upon binding have been studied by circular dichroism. All these studies are suggestive that the metal complexes bind to DNA through intercalation. Binding of di-nuclear copper complex 5 was found to be stronger when compared to the other complexes studied. Copper complexes were found to cleave the plasmid DNA in the absence of oxidizing or reducing agent, whereas, zinc complexes do not cleave. Metal complexes have shown toxicity to the HeLa and MCF-7 cell lines.Morphological studies, western blot and FACS analysis are suggestive of apoptotic cell death induced by the metal complexes. Di-nuclear copper complexes were found to be better as compared to the mononuclear ones in binding, plasmid cleavage and also in causing more cell death.

  2. The role of the Zn(II binding domain in the mechanism of E. coli DNA topoisomerase I

    Directory of Open Access Journals (Sweden)

    Tse-Dinh Yuk-Ching

    2002-05-01

    Full Text Available Abstract Background Escherichia coli DNA topoisomerase I binds three Zn(II with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain. The 67 kDa N-terminal domain (Top67 has the active site tyrosine for DNA cleavage but cannot relax negatively supercoiled DNA. We analyzed the role of the ZD domain in the enzyme mechanism. Results Addition of purified ZD domain to Top67 partially restored the relaxation activity, demonstrating that covalent linkage between the two domains is not necessary for removal of negative supercoils from DNA. The two domains had similar affinities to ssDNA. However, only Top67 could bind dsDNA with high affinity. DNA cleavage assays showed that the Top67 had the same sequence and structure selectivity for DNA cleavage as the intact enzyme. DNA rejoining also did not require the presence of the ZD domain. Conclusions We propose that during relaxation of negatively supercoiled DNA, Top67 by itself can position the active site tyrosine near the junction of double-stranded and single-stranded DNA for cleavage. However, the interaction of the ZD domain with the passing single-strand of DNA, coupled with enzyme conformational change, is needed for removal of negative supercoils.

  3. Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex.

    Science.gov (United States)

    Routledge, M N; Allan, J M; Garner, R C

    1997-07-01

    To investigate the use of UvrB-binding to detect DNA damage, mobility shift gel electrophoresis was used to detect binding of UvrB protein to a 136 bp DNA fragment that was randomly adducted with aflatoxin B1 8,9-epoxide and end-labelled with 32P. After polyacrylamide gel electrophoresis, the shifted band that contained DNA bound by UvrB was quantified as a percentage of total radioactive substrate DNA. This method was applied to analyse plasmid DNA that was adducted with various DNA modifying agents in vitro. These adducts competed for UvrB-binding to the labelled substrate. By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + visible light, did not compete for UvrB-binding, even though the presence of UvrABC sensitive sites were confirmed on this DNA by a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which contained an internal 32P-label and either a single apurinic site, aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or non-adducted guanine, were also used as substrates for UvrA- and UvrB-binding to examine the stability of UvrB-DNA complexes with specific adducts. Under similar conditions used for the competition assay, significant UvrB-binding was seen only for the aflatoxin adducted substrate. These results suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts. It was also found that rat liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA in the competition assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.

  4. The role of antigen specificity in the binding of murine monoclonal anti-DNA antibodies to microparticles from apoptotic cells.

    Science.gov (United States)

    Ullal, Anirudh J; Marion, Tony N; Pisetsky, David S

    2014-10-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus and markers of underlying immune system disturbances. These antibodies bind to both single-stranded and double-stranded DNA, mediating pathogenesis by forming immune complexes. As shown recently, DNA in blood exists in both free and particulate forms, with DNA representing an important component of microparticles. Microparticles are membrane-bound vesicles containing nuclear molecules, released by membrane blebbing during cell death and activation. A panel of monoclonal NZB/NZW F1 anti-DNA antibodies was tested for binding to microparticles generated from apoptotic THP-1 and Jurkat cells. These studies showed that only certain anti-DNA antibodies in the panel, specific for double-stranded DNA, bound to microparticles. Binding to particles was reduced by soluble DNA or DNase treatment. Together, these results indicate that particle binding is a feature of only certain anti-DNA antibodies, reflecting immunochemical properties of the antibodies and the nature of the exposed DNA antigens.

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

    Directory of Open Access Journals (Sweden)

    Samta Jain

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

  6. Methylated DNA binding proteins%DNA甲基化结合蛋白

    Institute of Scientific and Technical Information of China (English)

    邹丹丹; 王晓利; 汪海林

    2014-01-01

    DNA methylation is one of the most important epigenetic modifications in mammalian cells, approximately 70%-80% CpG dinucleotides are methylated. Dysregulation of DNA methylation occurs in many cancer cells, in which hypermethylation of promoter CpG islands may inactivate some tumor suppressor genes. The methylated DNA signal is read out by methylated DNA binding proteins (MBPs). They can specifically recognize and bind to methylated CpG sites and further recruit co-repressors, such as histone deacetylases (HDAC), to establish silent chromatin, thus providing a link between DNA methylation and gene silencing. In mammals, three structurally distinct types of MBPs have been identified so far:MBD( Methyl-CpG-Binding Domain) , Kaiso and SRA( Set and Ring finger-associated) family proteins. Their structure, function, binding properties ( focused on MBD protein ) to methylated DNA and contribution to human disease process are reviewed and discussed in this article.%DNA甲基化是哺乳动物细胞中最重要的表观遗传学修饰之一,大约70%-80%的CpG发生这种甲基化修饰。异常的甲基化在许多癌症中频发,启动子CpG岛的高甲基化作为普遍的失活机制介导抑癌基因沉默。甲基化信号由甲基化结合蛋白来转译,它们能够特异性识别并结合至甲基化位点通过募集辅阻遏复合物例如组蛋白去乙酰化酶( Histone Deacetylase, HDAC)等建立沉默的染色质,从而在DNA甲基化和基因沉默中起桥梁作用。目前,哺乳动物中已鉴定出的甲基化结合蛋白有三类,分别是:MBD ( Methyl-CpG-Binding Domain)、Kaiso以及SRA( Set and Ring finger-associated)家族。本文就这三大家族(以MBD为主)各自的结构、功能、结合甲基化DNA的特性以及它们在某些疾病发生中的作用做一综述。

  7. Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein

    DEFF Research Database (Denmark)

    Bendtsen, Jannick Dyrløv; Nilsson, A.S.; Lehnherr, H.

    2002-01-01

    Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium Escherichia coli. A phylogenetic analysis indicated that the P1 ssb gene coexists with its E. coli counterpart as an independent unit...... phase. These results reconciled the observed evolutionary conservation with the seemingly redundant presence of ssb genes in many bacteriophages and conjugative plasmids....

  8. Binding of synthetic double-stranded DNA by serum from patients with systemic lupus erythematosus: correlation with renal histology.

    Science.gov (United States)

    Steinman, C R; Grishman, E; Spiera, H; Deesomochok, U

    1977-03-01

    Detection of antibody to double-stranded DNA by direct binding assays has proved useful in clinical management of patients with systemic lupus erythematosus (SLE). Recent confusion regarding specificity of these antibodies for SLE appears to be due, at least in part, to contamination of natural DNA preparations with nondouble-stranded DNA antigens. Measurement of binding of a synthetic, self-complementary DNA copolymer (dAT) rather than of natural DNA (KB) has been shown to obviate some of these difficulties, apparently because of freedom of dAT from nondouble-stranded DNA antigens. Among the advantages found in this way was a higher degree of specificity of antibodies to double-stranded DNA for clinically-judged active lupus nephritis than had been suspected. Since activity of nephritis is difficult to assess clinically, histologic data were sought to confirm these observations. Thirty-two kidney specimens were examined by light and/or electron microscopy. The degree of histologic activity and the amount and location of glomerular electron-dense deposits were semiquantitated blindly. The binding of both dAT and KB DNA was measured by the ammonium sulfate method. Correlation with the amount of electron-defense deposits was highly significant for dAT binding and somewhat less so for KB DNA binding as determined by both parametric and nonparametric statistical methods. Significant correlation with histologic activity was found for dAT but not KB DNA binding. These results are consistent with previous data and suggest that dAT binding may provide a useful, noninvasive means of clinically assessing both nephritis activity and the intensity of glomerular immune-complex deposition as reflected by the amount of electron-dense deposits. If it can be confirmed that the latter provides long-term prognostic information, then dAT binding (and perhaps its reponse to therapy) may also prove of value in this regard.

  9. The process of displacing the single-stranded DNA-binding protein from single-stranded DNA by RecO and RecR proteins.

    Science.gov (United States)

    Inoue, Jin; Honda, Masayoshi; Ikawa, Shukuko; Shibata, Takehiko; Mikawa, Tsutomu

    2008-01-01

    The regions of single-stranded (ss) DNA that result from DNA damage are immediately coated by the ssDNA-binding protein (SSB). RecF pathway proteins facilitate the displacement of SSB from ssDNA, allowing the RecA protein to form protein filaments on the ssDNA region, which facilitates the process of recombinational DNA repair. In this study, we examined the mechanism of SSB displacement from ssDNA using purified Thermus thermophilus RecF pathway proteins. To date, RecO and RecR are thought to act as the RecOR complex. However, our results indicate that RecO and RecR have distinct functions. We found that RecR binds both RecF and RecO, and that RecO binds RecR, SSB and ssDNA. The electron microscopic studies indicated that SSB is displaced from ssDNA by RecO. In addition, pull-down assays indicated that the displaced SSB still remains indirectly attached to ssDNA through its interaction with RecO in the RecO-ssDNA complex. In the presence of both SSB and RecO, the ssDNA-dependent ATPase activity of RecA was inhibited, but was restored by the addition of RecR. Interestingly, the interaction of RecR with RecO affected the ssDNA-binding properties of RecO. These results suggest a model of SSB displacement from the ssDNA by RecF pathway proteins. PMID:18000001

  10. Molecular docking study investigating the possible mode of binding of C.I. Acid Red 73 with DNA.

    Science.gov (United States)

    Guo, Yumei; Yue, Qinyan; Gao, Baoyu

    2011-07-01

    C.I. Acid Red 73 is a reactive azo dye with a variable potential carcinogenicity. The mechanism mediating interactions that occur between the dye and DNA have not been completely understood thus far. In this study, molecular docking techniques were applied to describe the most probable mode of DNA binding as well as the sequence selectivity of the C.I. Acid Red 73 dye. These docking experiments revealed that the dye is capable of interacting with the minor groove of the DNA on the basis of its curved shape, which fits well with the topology of double-stranded DNA. In addition, the dye can bind selectively to the minor groove of the DNA by applying CGT sequence selectivity. Further, the minor groove can be recognized although DNA targets present intercalation gaps. However, intercalative binding can also occur when the DNA target possesses an appropriate intercalation gap. Compared with the other eight DNA sequences that were studied, the DNA dodecamer d(CGCGATATCGCG)(2) (PDB ID: 1DNE) presents a very favorable target for the binding of C.I. Acid Red 73 to the minor groove, with the lowest binding free energy -9.19 kcal/mol. Results reported from this study are expected to provide useful information for research involving further simulations of molecular dynamics and toxicology investigations of the dye.

  11. Insights into the mechanism of DNA recognition by the methylated LINE binding protein EhMLBP of Entamoeba histolytica.

    Science.gov (United States)

    Lavi, Tal; Siman-Tov, Rama; Ankri, Serge

    2009-08-01

    EhMLBP is an essential Entamoeba histolytica protein that binds preferentially to methylated long interspersed nuclear elements and rDNA. In an effort to identify more EhMLBP DNA substrates, we developed an affinity-based technique in which the C-terminal DNA binding domain of EhMLBP (GST-CterEhMLBP) was used as the ligand. Bioinformatic analysis of the DNA sequences that were isolated by this affinity method revealed the presence of a 29-nucleotide consensus motif that includes a stretch of ten adenines. Gel retardation analysis showed that EhMLBP binds to the consensus motif with a preference for its methylated form. Four DNA sequences, namely those that encoded either dihydrouridine synthetase, RAP GTPase activating protein, serine/threonine protein kinase or leucine-rich repeat containing protein (LRPP) were then selected for further analysis. In vivo binding of EhMLBP to these genes was confirmed by chromatin immunoprecipitation. The presence of methylated cytosines was detected in DNA encoding LRPP and to a lower extent in the other genes. EhMLBP binds preferentially to the methylated forms of these DNA targets. The ability of the consensus motif to compete with EhMLBP binding to its DNA substrates indicates that the adenine stretch is involved in the mechanism of DNA recognition. The results of this investigation extend our existing knowledge on the number of DNA sequences that are recognized by EhMLBP and reinforce the notion that this protein is an innate methylated DNA binding protein in E. histolytica.

  12. Molecular crowding effect on dynamics of DNA-binding proteins search for their targets

    Science.gov (United States)

    Liu, Lin; Luo, Kaifu

    2014-12-01

    DNA-binding proteins locate and bind their target sequences positioned on DNA in crowded environments, but the molecular crowding effect on this search process is not clear. Using analytical techniques and Langevin dynamics simulations in two dimensions (2D), we find that the essential physics for facilitated diffusion in 2D search and 3D search is the same. We observe that the average search times have minima at the same optimal nonspecific binding energy for the cases with and without the crowding particle. Moreover, the molecular crowding increases the search time by increasing the average search rounds and the one-dimensional (1D) sliding time of a round, but almost not changing the average 2D diffusion time of a round. In addition, the fraction of 1D sliding time out of the total search time increases with increasing the concentration of crowders. For 2D diffusion, the molecular crowding decreases the jumping length and narrows its distribution due to the cage effect from crowders. These results shed light on the role of facilitated diffusion in DNA targeting kinetics in living cells.

  13. How Aromatic Compounds Block DNA Binding of HcaR Catabolite Regulator.

    Science.gov (United States)

    Kim, Youngchang; Joachimiak, Grazyna; Bigelow, Lance; Babnigg, Gyorgy; Joachimiak, Andrzej

    2016-06-17

    Bacterial catabolism of aromatic compounds from various sources including phenylpropanoids and flavonoids that are abundant in soil plays an important role in the recycling of carbon in the ecosystem. We have determined the crystal structures of apo-HcaR from Acinetobacter sp. ADP1, a MarR/SlyA transcription factor, in complexes with hydroxycinnamates and a specific DNA operator. The protein regulates the expression of the hca catabolic operon in Acinetobacter and related bacterial strains, allowing utilization of hydroxycinnamates as sole sources of carbon. HcaR binds multiple ligands, and as a result the transcription of genes encoding several catabolic enzymes is increased. The 1.9-2.4 Å resolution structures presented here explain how HcaR recognizes four ligands (ferulate, 3,4-dihydroxybenzoate, p-coumarate, and vanillin) using the same binding site. The ligand promiscuity appears to be an adaptation to match a broad specificity of hydroxycinnamate catabolic enzymes while responding to toxic thioester intermediates. Structures of apo-HcaR and in complex with a specific DNA hca operator when combined with binding studies of hydroxycinnamates show how aromatic ligands render HcaR unproductive in recognizing a specific DNA target. The current study contributes to a better understanding of the hca catabolic operon regulation mechanism by the transcription factor HcaR. PMID:27129205

  14. Calculating the contribution of different binding modes to Quinacrine - DNA complex formation from polarized fluorescence data

    CERN Document Server

    Voloshin, Igor; Karachevtsev, Victor; Zozulya, Victor

    2013-01-01

    Binding of acridine derivative quinacrine (QA) to chicken erythrocyte DNA was studied by methods of absorption and polarized fluorescent spectroscopy. Measurements were carried out in aqueous buffered solutions (pH 6.9) of different dye concentrations (QA concentration range from $10^{-6}$ till $10^{-4}$ M) and ionic strengths ($Na^{+}$ concentration rang from $10^{-3}$ till 0.15 M) in a wide range of phosphate-to-dye molar ratios ($P/D$). It is established that the minimum of fluorescent titration curve plotted as relative fluorescence intensity $vs$ $P/D$ is conditioned by the competition between the two types of QA binding to DNA which posses by different emission parameters: (i) intercalative one dominating under high $P/D$ values, and (ii) outside electrostatic binding dominating under low $P/D$ values, which is accompanied by the formation of non-fluorescent dye associates on the DNA backbone. Absorption and fluorescent characteristics of complexes formed were determined. The method of calculation of di...

  15. Functional importance of the DNA binding activity of Candida albicans Czf1p.

    Directory of Open Access Journals (Sweden)

    Ivana Petrovska

    Full Text Available The human opportunistic pathogen Candida albicans undergoes a reversible morphological transition between the yeast and hyphal states in response to a variety of signals. One such environmental trigger is growth within a semisolid matrix such as agar medium. This growth condition is of interest because it may mimic the growth of C. albicans in contact with host tissue during infection. During growth within a semisolid matrix, hyphal growth is positively regulated by the transcriptional regulator Czf1p and negatively by a second key transcriptional regulator, Efg1p. Genetic studies indicate that Czf1p, a member of the zinc-cluster family of transcriptional regulators, exerts its function by opposing the inhibitory influence of Efg1p on matrix-induced filamentous growth. We examined the importance of the two known activities of Czf1p, DNA-binding and interaction with Efg1p. We found that the two activities were separable by mutation allowing us to demonstrate that the DNA-binding activity of Czf1p was essential for its role as a positive regulator of morphogenesis. Surprisingly, however, interactions with Efg1p appeared to be largely dispensable. Our studies provide the first evidence of a key role for the DNA-binding activity of Czf1p in the morphological yeast-to-hyphal transition triggered by matrix-embedded growth.

  16. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    International Nuclear Information System (INIS)

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer

  17. CLONING AND CHARACTERIZATION OF HUMAN UBIQUITIN BINDING ENZYME 2 cDNA

    Institute of Scientific and Technical Information of China (English)

    李光涛; 吕鸿雁; 周严; 金鉴; 蒋科艺; 彭小忠; 袁建刚; 强伯勤

    2002-01-01

    Objective.To clone and identify the gene encoding human ubiquitin binding enzyme 2 and study its expression pattern. Methods.According to the sequence of human EST,which is highly homologous to the mouse ubiquitin binding/conjugating enzyme (E2),primers were synthesized to screen the human fetal brain cDNA library.The gene was analyzed by bioinformatics technique and its expression pattern was studied by using multiple tissue Northern blot. Results.Two cDNA clones encoding human ubiquitin conjugating enzyme have been isolated and identified.Both containing the ubiquitin conjugating domain,the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence.They belong to a highly conserved and widely expressed E2 enzyme family.Northern blot shows that they are expressed exclusively in adult human heart,placenta,and pancreas but no transcripts can be detected in brain,lung,liver,skeletal muscle or kidney.Conclusions.The gene encoding human ubiquitin binding enzyme is expressed under temporal control.As a key enzyme in the degradation of proteins,ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post translation.

  18. Structure-function analysis indicates that sumoylation modulates DNA-binding activity of STAT1

    Directory of Open Access Journals (Sweden)

    Grönholm Juha

    2012-10-01

    Full Text Available Abstract Background STAT1 is an essential transcription factor for interferon-γ-mediated gene responses. A distinct sumoylation consensus site (ψKxE 702IKTE705 is localized in the C-terminal region of STAT1, where Lys703 is a target for PIAS-induced SUMO modification. Several studies indicate that sumoylation has an inhibitory role on STAT1-mediated gene expression but the molecular mechanisms are not fully understood. Results Here, we have performed a structural and functional analysis of sumoylation in STAT1. We show that deconjugation of SUMO by SENP1 enhances the transcriptional activity of STAT1, confirming a negative regulatory effect of sumoylation on STAT1 activity. Inspection of molecular model indicated that consensus site is well exposed to SUMO-conjugation in STAT1 homodimer and that the conjugated SUMO moiety is directed towards DNA, thus able to form a sterical hindrance affecting promoter binding of dimeric STAT1. In addition, oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant has higher DNA-binding activity on STAT1 responsive gene promoters than wild-type STAT1. Furthermore, sumoylation deficient STAT1 E705Q mutant displayed enhanced histone H4 acetylation on interferon-γ-responsive promoter compared to wild-type STAT1. Conclusions Our results suggest that sumoylation participates in regulation of STAT1 responses by modulating DNA-binding properties of STAT1.

  19. DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

    Energy Technology Data Exchange (ETDEWEB)

    Lafaye, Céline; Barbier, Ewa; Miscioscia, Audrey; Saint-Pierre, Christine [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Kraut, Alexandra; Couté, Yohann [Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S_1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France); Plo, Isabelle [INSERM, U1009, Institut Gustave Roussy, Université Paris 11, 114 rue Edouard Vaillant, Villejuif F-94805 (France); Gasparutto, Didier; Ravanat, Jean-Luc [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Breton, Jean, E-mail: jean.breton@cea.fr [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France)

    2014-03-28

    Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.

  20. Discovery of widespread GTP-binding motifs in genomic DNA and RNA.

    Science.gov (United States)

    Curtis, Edward A; Liu, David R

    2013-04-18

    Biological RNAs that bind small molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genome-encoded RNA fragments for naturally occurring GTP aptamers. Several aptamer classes were identified, including one (the "G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ~75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (~300 μM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.

  1. DNA fragments binding CTCF in vitro and in vivo are capable of blocking enhancer activity

    Directory of Open Access Journals (Sweden)

    Didych Dmitry A

    2012-04-01

    Full Text Available Abstract Background Earlier we identified ten 100-300-bp long CTCF-binding DNA fragments selected earlier from a 1-Mb human chromosome 19 region. Here the positive-negative selection technique was used to check the ability of CTCF-binding human genomic fragments to block enhancer-promoter interaction when inserted into the genome. Results Ten CTCF-binding DNA fragments were inserted between the CMV enhancer and CMV minimal promoter driving the herpes simplex virus thymidine kinase (HSV-tk gene in a vector expressing also the neoR gene under a separate promoter. The constructs were then integrated into the genome of CHO cells, and the cells resistant to neomycin and ganciclovir (positive-negative selection were picked up, and their DNAs were PCR analyzed to confirm the presence of the fragments between the enhancer and promoter in both orientations. Conclusions We demonstrated that all sequences identified by their CTCF binding both in vitro and in vivo had enhancer-blocking activity when inserted between the CMV minimal promoter and enhancer in stably transfected CHO cells.

  2. Specific binding of DNA to aggregated forms of Alzheimer's disease amyloid peptides.

    Science.gov (United States)

    Camero, Sergio; Ayuso, Jose M; Barrantes, Alejandro; Benítez, María J; Jiménez, Juan S

    2013-04-01

    Anomalous protein aggregation is closely associated to age-related mental illness. Extraneuronal plaques, mainly composed of aggregated amyloid peptides, are considered as hallmarks of Alzheimer's disease. According to the amyloid cascade hypothesis, this disease starts as a consequence of an abnormal processing of the amyloid precursor protein resulting in an excess of amyloid peptides. Nuclear localization of amyloid peptide aggregates together with amyloid-DNA interaction, have been repeatedly reported. In this paper we have used surface plasmon resonance and electron microscopy to study the structure and behavior of different peptides and proteins, including β-lactoglobulin, bovine serum albumin, myoglobin, histone, casein and the amyloid-β peptides related to Alzheimer's disease Aβ25-35 and Aβ1-40. The main purpose of this study is to investigate whether proneness to DNA interaction is a general property displayed by aggregated forms of proteins, or it is an interaction specifically related to the aggregated forms of those particular proteins and peptides related to neurodegenerative diseases. Our results reveal that those aggregates formed by amyloid peptides show a particular proneness to interact with DNA. They are the only aggregated structures capable of binding DNA, and show more affinity for DNA than for other polyanions like heparin and polyglutamic acid, therefore strengthening the hypothesis that amyloid peptides may, by means of interaction with nuclear DNA, contribute to the onset of Alzheimer's disease.

  3. A rapid screening method using DNA binding dyes to determine whether hair follicles have sufficient DNA for successful profiling.

    Science.gov (United States)

    Haines, Alicia M; Linacre, Adrian

    2016-05-01

    We report a simple screening method to assess the viability of successful DNA profiling from single hair follicles. A total of 48 hair samples (shed and plucked) were collected from male and female donors and the root tips (0.5cm) were stained using one of three DNA binding dyes (EvaGreen™, Diamond™ Nucleic Acid Dye and RedSafe™) at 20× concentration. The hairs were subsequently viewed under a Nikon Optiphot fluorescent microscope to count the approximate number of nuclei in one plane of view. The hairs were then processed using either (1) a DNA extraction kit (QIAmp(®) Mini Kit) and then amplified using the AmpFLSTR(®) NGM™ kit, which amplifies 15 short tandem repeat (STR) loci plus the gender marker amelogenin, or (2) by direct PCR amplification using the same DNA profiling kit. Diamond™ dye had the lowest background signal and plucked hairs treated with this dye produced full DNA profiles when amplified directly and was chosen to screen a further 150 mixed hair samples. These hairs were separated into one of five categories (1, >100 nuclei; 1.5, 50-99 nuclei; 2, 1-49 nuclei; 2.5, no nuclei but high fluorescent signal; 3, no nuclei and very low fluorescent signal) from which 60 of the hairs were chosen to undergo direct amplification using the NGM™ kit. It was found that there was a direct correlation to the category designation and the ability to obtain a DNA profile up-loadable to the Australian DNA Database. Approximately 91% of category 1 hairs resulted in either a full or high partial (12-29 alleles) profile by direct PCR whereas about 78% of category 3 hairs exhibited no amplification. The results show that this method can be used to predict successful STR amplification from single hair follicles. It is a rapid, sensitive, cheap, non-destructive and easy to perform methodology applicable for screening multiple hairs in order to aid forensic investigators in predicting hairs that will yield DNA results. PMID:27038658

  4. Ambient UV-B exposure reduces the binding of ofloxacin with bacterial DNA gyrase and induces DNA damage mediated apoptosis.

    Science.gov (United States)

    Singh, Jyoti; Dwivedi, Ashish; Mujtaba, Syed Faiz; Singh, Krishna P; Pal, Manish Kumar; Chopra, Deepti; Goyal, Shruti; Srivastav, Ajeet K; Dubey, Divya; Gupta, Shailendra K; Haldar, Chandana; Ray, Ratan Singh

    2016-04-01

    Ofloxacin (OFLX) is a broad spectrum antibiotic, which generates photo-products under sunlight exposure. Previous studies have failed to explain the attenuated anti-bacterial activity of OFLX. The study was extended to explore the unknown molecular mechanism of photogenotoxicity on human skin cell line (HaCaT) under environmental UV-B irradiation. Photochemically OFLX generates ROS and caused 2'-dGuO photodegradation. We have addressed the binding affinity of OFLX and its photo-products against DNA gyrase. Significant free radical generation such as (1)O2, O2(•-) and (•)OH reduces antioxidants and demonstrated the ROS mediated OFLX phototoxicity. However, the formation of micronuclei and CPDs showed photogenotoxic potential of OFLX. OFLX induced cell cycle arrest in sub-G1 peak. OFLX triggers apoptosis via permeabilization of mitochondrial membrane with the downregulation of anti-apoptotic Bcl-2 and caspase-3 whereas, upregulation of pro-apoptotic Bax and Cyto-C proteins. Our study illustrated that binding affinity of OFLX photo-products with DNA gyrase was mainly responsible for the attenuated antimicrobial activity. It was proved through molecular docking study. Thus, study suggests that sunlight exposure should avoid by drug users especially during peak hours for their safety from photosensitivity. Clinicians may guide patients regarding the safer use of photosensitive drugs during treatment. PMID:26812543

  5. Binding Studies of Natural Product Berberine with DNA G-Quadruplex

    Directory of Open Access Journals (Sweden)

    Nagendra K. Sharma

    2011-01-01

    Full Text Available Problem statement: The ends of chromosome had highly repetitive short G and C-rich sequences of DNA. These sequences were known to form stable tetraplex type of secondary structures which help to maintain gene integratity after cell divison. Approach: Any reagent which controls the random cell division would be useful to design anticancer drugs. Therefore a many natural and synthesized molecules which stabilized tetraplex structures are targeted as anticancer drug entities. Results: Among them, Berberine hydrochloride natural product and its analogues are well studies as G-quadruplex stabilizing agent. In this report, DNA sequence 5’-G3-C5-G3-3’ has been designed which has probability to form i-motif and G-qua druplex types of secondary structures. Herein we studied the interaction between this DNA strands and Berberine hydrochloride by 1H-NMR techniques and UV in two different PH (4.7 and 7.4 conditions. Conclusion/Recommendations: Our preliminary results showed that Berberine bind with this DNA strand in both pH conditions which is further supported by UV melting experiments. In future this sequence can be used as probe to screen out tetraplex binding natural products which help to generate new anticancer drugs.

  6. Theoretical estimates of exposure timescales of protein binding sites on DNA regulated by nucleosome kinetics.

    Science.gov (United States)

    Parmar, Jyotsana J; Das, Dibyendu; Padinhateeri, Ranjith

    2016-02-29

    It is being increasingly realized that nucleosome organization on DNA crucially regulates DNA-protein interactions and the resulting gene expression. While the spatial character of the nucleosome positioning on DNA has been experimentally and theoretically studied extensively, the temporal character is poorly understood. Accounting for ATPase activity and DNA-sequence effects on nucleosome kinetics, we develop a theoretical method to estimate the time of continuous exposure of binding sites of non-histone proteins (e.g. transcription factors and TATA binding proteins) along any genome. Applying the method to Saccharomyces cerevisiae, we show that the exposure timescales are determined by cooperative dynamics of multiple nucleosomes, and their behavior is often different from expectations based on static nucleosome occupancy. Examining exposure times in the promoters of GAL1 and PHO5, we show that our theoretical predictions are consistent with known experiments. We apply our method genome-wide and discover huge gene-to-gene variability of mean exposure times of TATA boxes and patches adjacent to TSS (+1 nucleosome region); the resulting timescale distributions have non-exponential tails.

  7. Using DNA duplex stability information for transcription factor binding site discovery.

    Science.gov (United States)

    Gordân, Raluca; Hartemink, Alexander J

    2008-01-01

    Transcription factor (TF) binding site discovery is an important step in understanding transcriptional regulation. Many computational tools have already been developed, but their success in detecting TF motifs is still limited. We believe one of the main reasons for the low accuracy of current methods is that they do not take into account the structural aspects of TF-DNA interaction. We have previously shown that knowledge about the structural class of the TF and information about nucleosome occupancy can be used to improve motif discovery. Here, we demonstrate the benefits of using information about the DNA double-helical stability for motif discovery. We notice that, in general, the energy needed to destabilize the DNA double helix is higher at TF binding sites than at random DNA sites. We use this information to derive informative positional priors that we incorporate into a motif finding algorithm. When applied to yeast ChIP-chip data, the new informative priors improve the performance of the motif finder significantly when compared to priors that do not use the energetic stability information.

  8. Whole Genome and Transcriptome Sequencing of a B3 Thymoma

    OpenAIRE

    Iacopo Petrini; Arun Rajan; Trung Pham; Donna Voeller; Sean Davis; James Gao; Yisong Wang; Giuseppe Giaccone

    2013-01-01

    Molecular pathology of thymomas is poorly understood. Genomic aberrations are frequently identified in tumors but no extensive sequencing has been reported in thymomas. Here we present the first comprehensive view of a B3 thymoma at whole genome and transcriptome levels. A 55-year-old Caucasian female underwent complete resection of a stage IVA B3 thymoma. RNA and DNA were extracted from a snap frozen tumor sample with a fraction of cancer cells over 80%. We performed array comparative genomi...

  9. ATPase-dependent auto-phosphorylation of the open condensin hinge diminishes DNA binding.

    Science.gov (United States)

    Akai, Yuko; Kanai, Ryuta; Nakazawa, Norihiko; Ebe, Masahiro; Toyoshima, Chikashi; Yanagida, Mitsuhiro

    2014-12-01

    Condensin, which contains two structural maintenance of chromosome (SMC) subunits and three regulatory non-SMC subunits, is essential for many chromosomal functions, including mitotic chromosome condensation and segregation. The ATPase domain of the SMC subunit comprises two termini connected by a long helical domain that is interrupted by a central hinge. The role of the ATPase domain has remained elusive. Here we report that the condensin SMC subunit of the fission yeast Schizosaccharomyces pombe is phosphorylated in a manner that requires the presence of the intact SMC ATPase Walker motif. Principal phosphorylation sites reside in the conserved, glycine-rich stretch at the hinge interface surrounded by the highly basic DNA-binding patch. Phosphorylation reduces affinity for DNA. Consistently, phosphomimetic mutants produce severe mitotic phenotypes. Structural evidence suggests that prior opening (though slight) of the hinge is necessary for phosphorylation, which is implicated in condensin's dissociation from and its progression along DNA.

  10. Oxidation of p53 through DNA charge transport involves a network of disulfides within the DNA-binding domain.

    Science.gov (United States)

    Schaefer, Kathryn N; Geil, Wendy M; Sweredoski, Michael J; Moradian, Annie; Hess, Sonja; Barton, Jacqueline K

    2015-01-27

    Transcription factor p53 plays a critical role in the cellular response to stress stimuli. We have seen that p53 dissociates selectively from various promoter sites as a result of oxidation at long-range through DNA-mediated charge transport (CT). Here, we examine this chemical oxidation and determine the residues in p53 that are essential for oxidative dissociation, focusing on the network of cysteine residues adjacent to the DNA-binding site. Of the eight mutants studied, only the C275S mutation shows decreased affinity for the Gadd45 promoter site. However, both mutations C275S and C277S result in substantial attenuation of oxidative dissociation, with C275S causing the most severe attenuation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide-labeled, whereas oxidized cysteines participating in disulfide bonds were (13)C2D2-iodoacetamide-labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed by mass spectrometry. A distinct shift in peptide labeling toward (13)C2D2-iodoacetamide-labeled cysteines is observed in oxidized samples, confirming that chemical oxidation of p53 occurs at long range. All observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds among the cysteine network. On the basis of these data, it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA. PMID:25584637

  11. Characterization and directed evolution of a methyl-binding domain protein for high-sensitivity DNA methylation analysis.

    Science.gov (United States)

    Heimer, Brandon W; Tam, Brooke E; Sikes, Hadley D

    2015-12-01

    Methyl-binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them useful for DNA methylation analysis. We displayed three of the core members MBD1, MBD2 and MBD4 on the surface of Saccharomyces cerevisiae cells. Using the yeast display platform, we determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9 ± 1.3 nM for binding to singly methylated DNA. The measured affinity for DNA with two methylated sites varied with the distance between the sites. We further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wild-type protein to 3.1 ± 1.0 nM. The most prevalent of these mutations, K161R, occurs away from the DNA-binding site and bridges the N- and C-termini of the protein by forming a new hydrogen bond. The F208Y and L170R mutations added new non-covalent interactions with the bound DNA strand. We finally concatenated the high-affinity MBD variant and expressed it in Escherichia coli as a green fluorescent protein fusion. Concatenating the protein from 1× to 3× improved binding 6-fold for an interfacial binding application. PMID:26384511

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

    Directory of Open Access Journals (Sweden)

    Nicolas Siaud

    2011-12-01

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

  13. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    Science.gov (United States)

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-01

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

  14. Communication routes in ARID domains between distal residues in helix 5 and the DNA-binding loops.

    Directory of Open Access Journals (Sweden)

    Gaetano Invernizzi

    2014-09-01

    Full Text Available ARID is a DNA-binding domain involved in several transcriptional regulatory processes, including cell-cycle regulation and embryonic development. ARID domains are also targets of the Human Cancer Protein Interaction Network. Little is known about the molecular mechanisms related to conformational changes in the family of ARID domains. Thus, we have examined their structural dynamics to enrich the knowledge on this important family of regulatory proteins. In particular, we used an approach that integrates atomistic simulations and methods inspired by graph theory. To relate these properties to protein function we studied both the free and DNA-bound forms. The interaction with DNA not only stabilizes the conformations of the DNA-binding loops, but also strengthens pre-existing paths in the native ARID ensemble for long-range communication to those loops. Residues in helix 5 are identified as critical mediators for intramolecular communication to the DNA-binding regions. In particular, we identified a distal tyrosine that plays a key role in long-range communication to the DNA-binding loops and that is experimentally known to impair DNA-binding. Mutations at this tyrosine and in other residues of helix 5 are also demonstrated, by our approach, to affect the paths of communication to the DNA-binding loops and alter their native dynamics. Overall, our results are in agreement with a scenario in which ARID domains exist as an ensemble of substates, which are shifted by external perturbation, such as the interaction with DNA. Conformational changes at the DNA-binding loops are transmitted long-range by intramolecular paths, which have their heart in helix 5.

  15. Communication routes in ARID domains between distal residues in helix 5 and the DNA-binding loops.

    Science.gov (United States)

    Invernizzi, Gaetano; Tiberti, Matteo; Lambrughi, Matteo; Lindorff-Larsen, Kresten; Papaleo, Elena

    2014-09-01

    ARID is a DNA-binding domain involved in several transcriptional regulatory processes, including cell-cycle regulation and embryonic development. ARID domains are also targets of the Human Cancer Protein Interaction Network. Little is known about the molecular mechanisms related to conformational changes in the family of ARID domains. Thus, we have examined their structural dynamics to enrich the knowledge on this important family of regulatory proteins. In particular, we used an approach that integrates atomistic simulations and methods inspired by graph theory. To relate these properties to protein function we studied both the free and DNA-bound forms. The interaction with DNA not only stabilizes the conformations of the DNA-binding loops, but also strengthens pre-existing paths in the native ARID ensemble for long-range communication to those loops. Residues in helix 5 are identified as critical mediators for intramolecular communication to the DNA-binding regions. In particular, we identified a distal tyrosine that plays a key role in long-range communication to the DNA-binding loops and that is experimentally known to impair DNA-binding. Mutations at this tyrosine and in other residues of helix 5 are also demonstrated, by our approach, to affect the paths of communication to the DNA-binding loops and alter their native dynamics. Overall, our results are in agreement with a scenario in which ARID domains exist as an ensemble of substates, which are shifted by external perturbation, such as the interaction with DNA. Conformational changes at the DNA-binding loops are transmitted long-range by intramolecular paths, which have their heart in helix 5.

  16. APE1/Ref-1 enhances DNA binding activity of mutant p53 in a redox-dependent manner.

    Science.gov (United States)

    Cun, Yanping; Dai, Nan; Li, Mengxia; Xiong, Chengjie; Zhang, Qinhong; Sui, Jiangdong; Qian, Chengyuan; Wang, Dong

    2014-02-01

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a dual function protein; in addition to its DNA repair activity, it can stimulate DNA binding activity of numerous transcription factors as a reduction-oxidation (redox) factor. APE1/Ref-1 has been found to be a potent activator of wild-type p53 (wtp53) DNA binding in vitro and in vivo. Although p53 is mutated in most types of human cancer including hepatocellular carcinoma (HCC), little is known about whether APE1/Ref-1 can regulate mutant p53 (mutp53). Herein, we reported the increased APE1/Ref-1 protein and accumulation of mutp53 in HCC by immunohistochemistry. Of note, it was observed that APE1/Ref-1 high-expression and mutp53 expression were associated with carcinogenesis and progression of HCC. To determine whether APE1/Ref-1 regulates DNA binding of mutp53, we performed electromobility shift assays (EMSAs) and quantitative chromatin immunoprecipitation (ChIP) assays in HCC cell lines. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, reduced mutp53 efficiently bound to nonlinear DNA, but not to linear DNA. Notably, overexpression of APE1/Ref-1 resulted in increased DNA binding activity of mutp53, while downregulation of APE1/Ref-1 caused a marked decrease of mutp53 DNA binding. In addition, APE1/Ref-1 could not potentiate the accumulation of p21 mRNA and protein in mutp53 cells. These data indicate that APE1/Ref-1 can stimulate mutp53 DNA binding in a redox-dependent manner.

  17. Chiroptical properties, binding affinity, and photostability of a conjugated zinc porphyrin dimer complexed with left-handed Z-DNA and right-handed B-DNA.

    Science.gov (United States)

    Choi, Jung Kyu; Reed, Aisha; Balaz, Milan

    2014-01-14

    We have studied the UV-vis absorption and chiroptical properties, binding affinity and photostability of a conjugated positively charged butadiyne-linked Zn(ii) porphyrin dimer bound to DNA sequence poly(dG-dC)2. Right-handed B-DNA, spermine-induced Z-DNA and Co(iii)-induced Z-DNA have been explored. Resonance light scattering (RLS) spectra showed formation of porphyrin aggregates in the presence of all DNA forms with the largest aggregates formed with B-DNA. The porphyrin dimer gave rise to induced bisignate circular dichroism (CD) signals in the presence of the left-handed Z-DNA conformations. On the other hand, the dimer stayed nearly chiroptically silent when complexed with the B-form of poly(dG-dC)2. Our results indicated that the conjugated Zn(ii) porphyrin dimer can be used as a sensor for the chiroptical detection of Z-DNA in the visible (400-500 nm) and near-infrared region of the electromagnetic spectrum (700-800 nm). The helicity of DNA had little effect on the dimer binding affinities. The photostability of the porphyrin dimer complexed with any form of DNA was higher than that of the free molecule. The porphyrin dimer bound to Z-DNA exhibited slower photobleaching than the B-DNA dimer complex.

  18. Microwave-Assisted Synthesis of Arene Ru(II Complexes Induce Tumor Cell Apoptosis Through Selectively Binding and Stabilizing bcl-2 G-Quadruplex DNA

    Directory of Open Access Journals (Sweden)

    Yanhua Chen

    2016-05-01

    Full Text Available A series of arene Ru(II complexes coordinated with phenanthroimidazole derivatives, [(η6-C6H6Ru(lCl]Cl(1b L = p-ClPIP = 2-(4-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 2b L = m-ClPIP = 2-(3-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 3b L = p-NPIP = 2-(4-Nitrophenylimidazole[4,5f] 1,10-phenanthroline; 4b L = m-NPIP = 2-(3-Nitrophenyl imidazole [4,5f] 1,10-phenanthroline were synthesized in yields of 89.9%–92.7% under conditions of microwave irradiation heating for 30 min to liberate four arene Ru(II complexes (1b, 2b, 3b, 4b. The anti-tumor activity of 1b against various tumor cells was evaluated by MTT assay. The results indicated that this complex blocked the growth of human lung adenocarcinoma A549 cells with an IC50 of 16.59 μM. Flow cytometric analysis showed that apoptosis of A549 cells was observed following treatment with 1b. Furthermore, the in vitro DNA-binding behaviors that were confirmed by spectroscopy indicated that 1b could selectively bind and stabilize bcl-2 G-quadruplex DNA to induce apoptosis of A549 cells. Therefore, the synthesized 1b has impressive bcl-2 G-quadruplex DNA-binding and stabilizing activities with potential applications in cancer chemotherapy.

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

    Directory of Open Access Journals (Sweden)

    Caselle Michele

    2007-09-01

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

  20. Macrocyclic nickel(II) complexes: Synthesis, characterization, superoxide scavenging activity and DNA-binding

    Science.gov (United States)

    Ramadan, Abd El-Motaleb M.

    2012-05-01

    A new series of nickel(II) complexes with the tetraaza macrocyclic ligand have been synthesized as possible functional models for nickel-superoxide dismutase enzyme. The reaction of 5-amino-3-methyl-1-phenylpyrazole-4-carbaldehyde (AMPC) with itself in the presence of nickel(II) ion yields, the new macrocyclic cationic complex, [NiL(NO3)2], containing a ligand composed of the self-condensed AMPC (4 mol) bound to a single nickel(II) ion. A series of metathetical reactions have led to the isolation of a number of newly complexes of the types [NiL]X2; X = ClO4 and BF4, [NiLX2], X = Cl and Br (Scheme 1). Structures and characterizations of these complexes were achieved by several physicochemical methods namely, elemental analysis, magnetic moment, conductivity, and spectral (IR and UV-Vis) measurements. The electrochemical properties and thermal behaviors of these chelates were investigated by using cyclic voltammetry and thermogravimetric analysis (TGA and DTG) techniques. A distorted octahedral stereochemistry has been proposed for the six-coordinate nitrato, and halogeno complexes. For the four-coordinate, perchlorate and fluoroborate, complex species a square-planar geometry is proposed. The measured superoxide dismutase mimetic activities of the complexes indicated that they are potent NiSOD mimics and their activities are compared with those obtained previously for nickel(II) complexes. The probable mechanistic implications of the catalytic dismutation of O2rad - by the synthesized nickel(II) complexes are discussed. The DNA-binding properties of representative complexes [NiLCl2] and [NiL](PF4)2 have been investigated by the electronic absorption and fluorescence measurements. The results obtained suggest that these complexes bind to DNA via an intercalation binding mode and the binding affinity for DNA follows the order: [NiLCl2] □ [NiL](PF4)2.

  1. Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Aimee; Higgins, Darren E.; Panne, Daniel; (Harvard-Med); (EMBL)

    2009-07-22

    The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

  2. The single-stranded DNA-binding protein of Deinococcus radiodurans

    Directory of Open Access Journals (Sweden)

    Wood Elizabeth

    2004-01-01

    Full Text Available Abstract Background Deinococcus radiodurans R1 is one of the most radiation-resistant organisms known and is able to repair an unusually large amount of DNA damage without induced mutation. Single-stranded DNA-binding (SSB protein is an essential protein in all organisms and is involved in DNA replication, recombination and repair. The published genomic sequence from Deinococcus radiodurans includes a putative single-stranded DNA-binding protein gene (ssb; DR0100 requiring a translational frameshift for synthesis of a complete SSB protein. The apparently tripartite gene has inspired considerable speculation in the literature about potentially novel frameshifting or RNA editing mechanisms. Immediately upstream of the ssb gene is another gene (DR0099 given an ssb-like annotation, but left unexplored. Results A segment of the Deinococcus radiodurans strain R1 genome encompassing the ssb gene has been re-sequenced, and two errors involving omitted guanine nucleotides have been documented. The corrected sequence incorporates both of the open reading frames designated DR0099 and DR0100 into one contiguous ssb open reading frame (ORF. The corrected gene requires no translational frameshifts and contains two predicted oligonucleotide/oligosaccharide-binding (OB folds. The protein has been purified and its sequence is closely related to the Thermus thermophilus and Thermus aquaticus SSB proteins. Like the Thermus SSB proteins, the SSBDr functions as a homodimer. The Deinococcus radiodurans SSB homodimer stimulates Deinococcus radiodurans RecA protein and Escherichia coli RecA protein-promoted DNA three-strand exchange reactions with at least the same efficiency as the Escherichia coli SSB homotetramer. Conclusions The correct Deinococcus radiodurans ssb gene is a contiguous open reading frame that codes for the largest bacterial SSB monomer identified to date. The Deinococcus radiodurans SSB protein includes two OB folds per monomer and functions as a

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

    Directory of Open Access Journals (Sweden)

    Toshitsugu Fujita

    2015-09-01

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

  4. Structures of the Ets Protein DNA-binding Domains of Transcription Factors Etv1, Etv4, Etv5, and Fev: DETERMINANTS OF DNA BINDING AND REDOX REGULATION BY DISULFIDE BOND FORMATION.

    Science.gov (United States)

    Cooper, Christopher D O; Newman, Joseph A; Aitkenhead, Hazel; Allerston, Charles K; Gileadi, Opher

    2015-05-29

    Ets transcription factors, which share the conserved Ets DNA-binding domain, number nearly 30 members in humans and are particularly involved in developmental processes. Their deregulation following changes in expression, transcriptional activity, or by chromosomal translocation plays a critical role in carcinogenesis. Ets DNA binding, selectivity, and regulation have been extensively studied; however, questions still arise regarding binding specificity outside the core GGA recognition sequence and the mode of action of Ets post-translational modifications. Here, we report the crystal structures of Etv1, Etv4, Etv5, and Fev, alone and in complex with DNA. We identify previously unrecognized features of the protein-DNA interface. Interactions with the DNA backbone account for most of the binding affinity. We describe a highly coordinated network of water molecules acting in base selection upstream of the GGAA core and the structural features that may account for discrimination against methylated cytidine residues. Unexpectedly, all proteins crystallized as disulfide-linked dimers, exhibiting a novel interface (distant to the DNA recognition helix). Homodimers of Etv1, Etv4, and Etv5 could be reduced to monomers, leading to a 40-200-fold increase in DNA binding affinity. Hence, we present the first indication of a redox-dependent regulatory mechanism that may control the activity of this subset of oncogenic Ets transcription factors.

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

    Directory of Open Access Journals (Sweden)

    Chaban Christina

    2010-11-01

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

  6. Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing.

    Science.gov (United States)

    Bidlingmaier, Scott; Ha, Kevin; Lee, Nam-Kyung; Su, Yang; Liu, Bin

    2016-04-01

    Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands. PMID

  7. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    Directory of Open Access Journals (Sweden)

    Tatsuaki Tsuruyama

    Full Text Available It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1 recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  8. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    Science.gov (United States)

    Tsuruyama, Tatsuaki; Nakai, Tonau; Ohmori, Rei; Ozeki, Munetaka; Tamaki, Keiji; Yoshikawa, Kenichi

    2013-01-01

    It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  9. Ligand binding mode to duplex and triplex DNA assessed by combining electrospray tandem mass spectrometry and molecular modeling

    OpenAIRE

    Rosu, Frédéric; Nguyen, Chi-Hung; De Pauw, Edwin; Gabelica, Valérie

    2007-01-01

    In this paper, we report the analysis of seven benzopyridoindole and benzopyridoquinoxaline drugs binding to different duplex DNA and triple helical DNA, using an approach combining electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS/MS), and molecular modeling. The ligands were ranked according to the collision energy (CE(50)) necessary to dissociate 50% of the complex with the duplex or the triplex in tandem MS. To determine the probable ligand binding site and ...

  10. The HMG-box mitochondrial transcription factor xl-mtTFA binds DNA as a tetramer to activate bidirectional transcription.

    OpenAIRE

    Antoshechkin, I; Bogenhagen, D F; Mastrangelo, I A

    1997-01-01

    The mitochondrial HMG-box transcription factor xl-mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl-mtTFA binding to this site. First, co-immunoprecipitation of differentially tagged recombinant mtTFA derivatives established that the protein exists as a multimer. Second, in vitro chemical cross-linking experiments provided e...

  11. High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK

    OpenAIRE

    Schloot Nanette C; Fingberg Waltraud; Alloza Iraide; Vandenbroeck Koen; Blasius Elias; Siegenthaler Rahel K; Burkart Volker; Christen Philipp; Kolb Hubert

    2010-01-01

    Abstract Background Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (pre)proinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK. Results Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further regi...

  12. Structural and functional studies of a large winged Z-DNA-binding domain of Danio rerio protein kinase PKZ.

    Science.gov (United States)

    Subramani, Vinod Kumar; Kim, Doyoun; Yun, Kyunghee; Kim, Kyeong Kyu

    2016-07-01

    The Z-DNA-binding domain of PKZ from zebrafish (Danio rerio; drZαPKZ ) contains the largest β-wing among known Z-DNA-binding domains. To elucidate the functional implication of the β-wing, we solved the crystal structure of apo-drZαPKZ . Structural comparison with its Z-DNA-bound form revealed a large conformational change within the β-wing during Z-DNA binding. Biochemical studies of protein mutants revealed that two basic residues in the β-wing are responsible for Z-DNA recognition as well as fast B-Z transition. Therefore, the extra basic residues in the β-wing of drZαPKZ are necessary for the fast B-Z transition activity. PMID:27265117

  13. Conjugation of benzylvanillin and benzimidazole structure improves DNA binding with enhanced antileukemic properties.

    Directory of Open Access Journals (Sweden)

    Zena A Al-Mudaris

    Full Text Available Benzyl-o-vanillin and benzimidazole nucleus serve as important pharmacophore in drug discovery. The benzyl vanillin (2-(benzyloxy-3-methoxybenzaldehyde compound shows anti-proliferative activity in HL60 leukemia cancer cells and can effect cell cycle progression at G2/M phase. Its apoptosis activity was due to disruption of mitochondrial functioning. In this study, we have studied a series of compounds consisting of benzyl vanillin and benzimidazole structures. We hypothesize that by fusing these two structures we can produce compounds that have better anticancer activity with improved specificity particularly towards the leukemia cell line. Here we explored the anticancer activity of three compounds namely 2-(2-benzyloxy-3-methoxyphenyl-1H-benzimidazole, 2MP, N-1-(2-benzyloxy-3-methoxybenzyl-2-(2-benzyloxy-3-methoxyphenyl-1H-benzimidazole, 2XP, and (R and (S-1-(2-benzyloxy-3-methoxyphenyl-2, 2, 2-trichloroethyl benzenesulfonate, 3BS and compared their activity to 2-benzyloxy-3-methoxybenzaldehyde, (Bn1, the parent compound. 2XP and 3BS induces cell death of U937 leukemic cell line through DNA fragmentation that lead to the intrinsic caspase 9 activation. DNA binding study primarily by the equilibrium binding titration assay followed by the Viscosity study reveal the DNA binding through groove region with intrinsic binding constant 7.39 µM/bp and 6.86 µM/bp for 3BS and 2XP respectively. 2XP and 3BS showed strong DNA binding activity by the UV titration method with the computational drug modeling showed that both 2XP and 3BS failed to form any electrostatic linkages except via hydrophobic interaction through the minor groove region of the nucleic acid. The benzylvanillin alone (Bn1 has weak anticancer activity even after it was combined with the benzimidazole (2MP, but after addition of another benzylvanillin structure (2XP, stronger activity was observed. Also, the combination of benzylvanillin with benzenesulfonate (3BS significantly improved

  14. Binding of the antitumor drug nogalamycin and its derivatives to DNA: Structural comparison

    International Nuclear Information System (INIS)

    The three-dimensional molecular structures of the complexes between a novel antitumor drug nogalamycin and its derivative U-58872 with a modified DNA hexamer d[m5CGT(pS)Am5CG] have been determined at 1.7- and 1.8-angstrom resolution, respectively, by X-ray diffraction analyses. Both structures (in space group P61) have been refined with constrained refinement procedure to final R factors of 0.208 (3386 reflections) and 0.196 (2143 reflections). In both complexes, two nogalamycins bind to the DNA hexamer double helix in a 2:1 ratio with the elongated aglycon chromophore intercalated between the CpG steps at both ends of the helix. The aglycon chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. Most of the sugars remain in the C2'-endo pucker family, except three deoxycytidine residues (terminal C1, C7, and internal C5). All nucleotides are in the anti conformation. Specific hydrogen bonds are found in the complex between the drug and guanine-cytosine bases in both grooves of the helix. One hydroxyl group of the aminoglucose donates a hydrogen bond to the N7 of guanine, while the other receives a hydrogen bond from the N4 amino group of cytosine. The orientation of these two hydrogen bonds suggests that nogalamycin prefers a GC base pair with its aglycon chromophore intercalating at the 5'-side of a guanine (between NpG), or at the 3'-side of a cytosine (between CpN) with the sugars pointing toward the GC base pair. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through, suggesting that nogalamycin prefers GC sequences embedded in a stretch of AT sequences

  15. DNA-binding protein prediction using plant specific support vector machines: validation and application of a new genome annotation tool.

    Science.gov (United States)

    Motion, Graham B; Howden, Andrew J M; Huitema, Edgar; Jones, Susan

    2015-12-15

    There are currently 151 plants with draft genomes available but levels of functional annotation for putative protein products are low. Therefore, accurate computational predictions are essential to annotate genomes in the first instance, and to provide focus for the more costly and time consuming functional assays that follow. DNA-binding proteins are an important class of proteins that require annotation, but current computational methods are not applicable for genome wide predictions in plant species. Here, we explore the use of species and lineage specific models for the prediction of DNA-binding proteins in plants. We show that a species specific support vector machine model based on Arabidopsis sequence data is more accurate (accuracy 81%) than a generic model (74%), and based on this we develop a plant specific model for predicting DNA-binding proteins. We apply this model to the tomato proteome and demonstrate its ability to perform accurate high-throughput prediction of DNA-binding proteins. In doing so, we have annotated 36 currently uncharacterised proteins by assigning a putative DNA-binding function. Our model is publically available and we propose it be used in combination with existing tools to help increase annotation levels of DNA-binding proteins encoded in plant genomes. PMID:26304539

  16. Towards the classification of DYT6 dystonia mutants in the DNA-binding domain of THAP1.

    Science.gov (United States)

    Campagne, Sébastien; Muller, Isabelle; Milon, Alain; Gervais, Virginie

    2012-10-01

    The transcription factor THAP1 (THanatos Associated Protein 1) has emerged recently as the cause of DYT6 primary dystonia, a type of rare, familial and mostly early-onset syndrome that leads to involuntary muscle contractions. Many of the mutations described in the DYT6 patients fall within the sequence-specific DNA-binding domain (THAP domain) of THAP1 and are believed to negatively affect DNA binding. Here, we have used an integrated approach combining spectroscopic (NMR, fluorescence, DSF) and calorimetric (ITC) methods to evaluate the effect of missense mutations, within the THAP domain, on the structure, stability and DNA binding. Our study demonstrates that none of the mutations investigated failed to bind DNA and some of them even bind DNA stronger than the wild-type protein. However, some mutations could alter DNA-binding specificity. Furthermore, the most striking effect is the decrease of stability observed for mutations at positions affecting the zinc coordination, the hydrophobic core or the C-terminal AVPTIF motif, with unfolding temperatures ranging from 46°C for the wild-type to below 37°C for two mutations. These findings suggest that reduction in population of folded protein under physiological conditions could also account for the disease. PMID:22844099

  17. Experimental and molecular docking studies on DNA binding interaction of adefovir dipivoxil: Advances toward treatment of hepatitis B virus infections

    Science.gov (United States)

    Shahabadi, Nahid; Falsafi, Monireh

    The toxic interaction of adefovir dipivoxil with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated drug interacted with CT-DNA in a groove binding mode. The binding constant of UV-visible and the number of binding sites were 3.33 ± 0.2 × 104 L mol-1and 0.99, respectively. The fluorimetric studies showed that the reaction between the drug and CT-DNA is exothermic (ΔH = 34.4 kJ mol-1; ΔS = 184.32 J mol-1 K-1). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of CT-DNA in the presence of adefovir dipivoxil, which verified the groove binding mode. Furthermore, the drug induces detectable changes in its viscosity. The molecular modeling results illustrated that adefovir strongly binds to groove of DNA by relative binding energy of docked structure -16.83 kJ mol-1. This combination of multiple spectroscopic techniques and molecular modeling methods can be widely used in the investigation on the toxic interaction of small molecular pollutants and drugs with bio macromolecules, which contributes to clarify the molecular mechanism of toxicity or side effect in vivo.

  18. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  19. Discovery, optimization and validation of an optimal DNA-binding sequence for the Six1 homeodomain transcription factor.

    Science.gov (United States)

    Liu, Yubing; Nandi, Soumyadeep; Martel, André; Antoun, Alen; Ioshikhes, Ilya; Blais, Alexandre

    2012-09-01

    The Six1 transcription factor is a homeodomain protein involved in controlling gene expression during embryonic development. Six1 establishes gene expression profiles that enable skeletal myogenesis and nephrogenesis, among others. While several homeodomain factors have been extensively characterized with regards to their DNA-binding properties, relatively little is known of the properties of Six1. We have used the genomic binding profile of Six1 during the myogenic differentiation of myoblasts to obtain a better understanding of its preferences for recognizing certain DNA sequences. DNA sequence analyses on our genomic binding dataset, combined with biochemical characterization using binding assays, reveal that Six1 has a much broader DNA-binding sequence spectrum than had been previously determined. Moreover, using a position weight matrix optimization algorithm, we generated a highly sensitive and specific matrix that can be used to predict novel Six1-binding sites with highest accuracy. Furthermore, our results support the idea of a mode of DNA recognition by this factor where Six1 itself is sufficient for sequence discrimination, and where Six1 domains outside of its homeodomain contribute to binding site selection. Together, our results provide new light on the properties of this important transcription factor, and will enable more accurate modeling of Six1 function in bioinformatic studies.

  20. Theory on the mechanisms of combinatorial binding of transcription factors with DNA

    CERN Document Server

    Murugan, R

    2016-01-01

    We develop a theoretical framework on the mechanism of combinatorial binding of transcription factors (TFs) with their specific binding sites on DNA. We consider three possible mechanisms viz. monomer, hetero-oligomer and coordinated recruitment pathways. In the monomer pathway, combinatorial TFs search for their targets in an independent manner and the protein-protein interactions among them will be insignificant. The protein-protein interactions are very strong so that the hetero-oligomer complex of TFs as a whole searches for the cognate sites in case of hetero-oligomer pathway. The TF which arrived first will recruit the adjacent TFs in a sequential manner in the recruitment pathway. The free energy released from the protein-protein interactions among TFs will be in turn utilized to stabilize the TFs-DNA complex. Such coordinated binding of TFs in fact emerges as the cooperative effect. Monomer and hetero-oligomer pathways are efficient only when few TFs are involved in the combinatorial regulation. Detai...

  1. Mesoscopic model and free energy landscape for protein-DNA binding sites: analysis of cyanobacterial promoters.

    Directory of Open Access Journals (Sweden)

    Rafael Tapia-Rojo

    2014-10-01

    Full Text Available The identification of protein binding sites in promoter sequences is a key problem to understand and control regulation in biochemistry and biotechnological processes. We use a computational method to analyze promoters from a given genome. Our approach is based on a physical model at the mesoscopic level of protein-DNA interaction based on the influence of DNA local conformation on the dynamics of a general particle along the chain. Following the proposed model, the joined dynamics of the protein particle and the DNA portion of interest, only characterized by its base pair sequence, is simulated. The simulation output is analyzed by generating and analyzing the Free Energy Landscape of the system. In order to prove the capacity of prediction of our computational method we have analyzed nine promoters of Anabaena PCC 7120. We are able to identify the transcription starting site of each of the promoters as the most populated macrostate in the dynamics. The developed procedure allows also to characterize promoter macrostates in terms of thermo-statistical magnitudes (free energy and entropy, with valuable biological implications. Our results agree with independent previous experimental results. Thus, our methods appear as a powerful complementary tool for identifying protein binding sites in promoter sequences.

  2. Differential immunoadsorption coupled with rate nephelometry for estimation of DNA-binding immunoglobulins

    Energy Technology Data Exchange (ETDEWEB)

    DeBari, V.A.; Nicotra, J.; Blaney, J.F.; Schultz, E.F.; Needle, M.A.

    1984-07-01

    The authors describe a technique for estimating the mass of anti-DNA antibodies by immunonephelometry of serum immunoglobulins (IgG, IgA, IgM) before and after adsorption onto DNA bound to agarose-polylysine columns. Sixteen patients with systemic lupus erythematosus and 16 age- and sex-matched controls were studied. Precision was determined for high-value (in 10 patients) and low-value (in nine controls) ranges for each of the immunoglobulins. They found anti-DNA antibody concentrations (mean +/- SD) in systemic lupus erythematosus of 1.981 +/- 1.015 g/L for IgG 0.257 +/- 0.215 g/L for IgA and 0.282 +/- 0.234 g/L for IgM. Sensitivity and linearity are such that fivefold dilutions of patients' serum with either a buffered albumin solution or control serum yielded values close to the expected values for IgG. Similarly diluted sera gave inordinately high values in the radiometric binding assay. Neither parametric (linear regression) nor nonparametric correlation methods (Spearman's rank and Kendall's tau) show a significant correlation between patients' data obtained by the present technique and that by a radiometric binding assay, although combined data from patients and controls demonstrate a significant nonparametric correlation.

  3. Conformational Dynamics and the Binding of Specific and Nonspecific DNA by the Autoinhibited Transcription Factor Ets-1.

    Science.gov (United States)

    Desjardins, Geneviève; Okon, Mark; Graves, Barbara J; McIntosh, Lawrence P

    2016-07-26

    The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix-turn-helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA.

  4. Two glutamic acid residues in the DNA-binding domain are engaged in the release of STAT1 dimers from DNA

    Directory of Open Access Journals (Sweden)

    Koch Verena

    2012-08-01

    Full Text Available Abstract Background In interferon-γ-stimulated cells, the dimeric transcription factor STAT1 (signal transducer and activator of transcription 1 recognizes semi-palindromic motifs in the promoter regions of cytokine-driven target genes termed GAS (gamma-activated sites. However, the molecular steps that facilitate GAS binding and the subsequent liberation of STAT1 homodimers from these promoter elements are not well understood. Results Using a mutational approach, we identified two critical glutamyl residues within the DNA-binding domain adjacent to the phosphodiester backbone of DNA which efficiently release phospho-STAT1 from DNA. The release of STAT1 dimers from DNA enhances transcriptional activity on both interferon-driven reporter and endogenous target genes. A substitution of either of the two glutamic acid residues broadens the repertoire of putative binding sites on DNA and enhances binding affinity to GAS sites. However, despite elevated levels of tyrosine phosphorylation and a prolonged nuclear accumulation period, the STAT1 DNA-binding mutants show a significantly reduced transcriptional activity upon stimulation of cells with interferon-γ. This reduced transcriptional response may be explained by the deposition of oligomerized STAT1 molecules outside GAS sites. Conclusions Thus, two negatively charged amino acid residues in the DNA-binding domain are engaged in the liberation of STAT1 from DNA, resulting in a high dissociation rate from non-GAS sites as a key feature of STAT1 signal transduction, which positively regulates cytokine-dependent gene expression probably by preventing retention at transcriptionally inert sites.

  5. SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation and gene repression

    DEFF Research Database (Denmark)

    Gjerstorff, Morten; Greve, Katrine Buch Vidén; Møller, Jesper Bonnet;

    2-mediated derepression of the PcG target gene ATF3 is associated with widespread binding of SSX2 to this gene and a reduction in BMI1 and histone H3K27me3 at the proximal promoter. SSX2 binds double-stranded DNA in a sequence non-specific manner, suggesting that it modulates PcG activity through...

  6. Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

    Directory of Open Access Journals (Sweden)

    Metsis Madis

    2008-06-01

    Full Text Available Abstract Background In a traditional electrophoresis mobility shift assay (EMSA a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA ( Results We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (AluI, BsuRI, TruI, etc, separation of low and high molecular weight fractions of resultant DNA fragments, 32P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside

  7. Mixed-ligand copper(ii) Schiff base complexes: the role of the co-ligand in DNA binding, DNA cleavage, protein binding and cytotoxicity.

    Science.gov (United States)

    Lian, Wen-Jing; Wang, Xin-Tian; Xie, Cheng-Zhi; Tian, He; Song, Xue-Qing; Pan, He-Ting; Qiao, Xin; Xu, Jing-Yuan

    2016-05-31

    Four novel mononuclear Schiff base copper(ii) complexes, namely, [Cu(L)(OAc)]·H2O (), [Cu(HL)(C2O4)(EtOH)]·EtOH (), [Cu(L)(Bza)] () and [Cu(L)(Sal)] () (HL = 1-(((2-((2-hydroxypropyl)amino)ethyl)imino)methyl)naphthalene-2-ol), Bza = benzoic acid, Sal = salicylic acid), were synthesized and characterized by X-ray crystallography, elemental analysis and infrared spectroscopy. Single-crystal diffraction analysis revealed that all the complexes were mononuclear molecules, in which the Schiff base ligand exhibited different coordination modes and conformations. The N-HO and O-HO inter- and intramolecular hydrogen bonding interactions linked these molecules into multidimensional networks. Their interactions with calf thymus DNA (CT-DNA) were investigated by UV-visible and fluorescence spectrometry, as well as by viscosity measurements. The magnitude of the Kapp values of the four complexes was 10(5), indicating a moderate intercalative binding mode between the complexes and DNA. Electrophoresis results showed that all these complexes induced double strand breaks of pUC19 plasmid DNA in the presence of H2O2 through an oxidative pathway. In addition, the fluorescence spectrum of human serum albumin (HSA) with the complexes suggested that the quenching mechanism of HSA by the complexes was a static process. Moreover, the antiproliferative activity of the four complexes against HeLa (human cervical carcinoma) and HepG-2 (human liver hepatocellular carcinoma) cells evaluated by colorimetric cell proliferation assay and clonogenic assay revealed that all four complexes had improved cytotoxicity against cancer cells. Inspiringly, complex , with salicylic acid as the auxiliary ligand, displayed a stronger anticancer activity, suggesting that a synergistic effect of the Schiff base complex and the nonsteroidal anti-inflammatory drug may be involved in the cell killing process. The biological features of mixed-ligand copper(ii) Schiff base complexes and how acetic auxiliary

  8. Non-covalent DNA groove-binding by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

    OpenAIRE

    Marsch, G A; Ward, R. L.; Colvin, M.; Turteltaub, K W

    1994-01-01

    The cooked meat mutagen 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) is metabolized in vivo to electrophilic intermediates that covalently bind to DNA guanines. Here we address the mechanism of PhIP's non-covalent interaction with DNA by using spectroscopic and computational methodologies. NMR methodologies indicated that upon addition of DNA, PhIP aromatic protons underwent a small, 0.11-0.12 p.p.m. upfield shift. DNA phosphorus resonances of non-covalent PhIP-DNA complexes broade...

  9. Preferential binding of Escherichia coli RecF protein to gapped DNA in the presence of adenosine (gamma-thio) triphosphate.

    OpenAIRE

    Hegde, S P; Rajagopalan, M; Madiraju, M V

    1996-01-01

    Escherichia coli RecF protein binds, but does not hydrolyze, ATP. To determine the role that ATP binding to RecF plays in RecF protein-mediated DNA binding, we have determined the interaction between RecF protein and single-stranded (ss)DNA, double-stranded (ds)DNA, and dsDNA containing ssDNA regions (gapped [g]DNA) either alone or in various combinations both in the presence and in the absence of adenosine (gamma-thio) triphosphate, gamma-S-ATP, a nonhydrolyzable ATP analog. Protein-DNA comp...

  10. Sensing Conformational Changes in DNA upon Ligand Binding Using QCM-D. Polyamine Condensation and Rad51 Extension of DNA Layers

    KAUST Repository

    Sun, Lu

    2014-10-16

    © 2014 American Chemical Society. Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigating DNA interactions. Here, we investigate if conformational changes induced in surface-attached DNA molecules upon ligand binding can be monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. DNA duplexes containing 59-184 base pairs were formed on QCM-D crystals by stepwise assembly of synthetic oligonucleotides of designed base sequences. The DNA films were exposed to the cationic polyamines spermidine and spermine, known to condense DNA molecules in bulk experiments, or to the recombination protein Rad51, known to extend the DNA helix. The binding and dissociation of the ligands to the DNA films were monitored in real time by measurements of the shifts in resonance frequency (Δf) and in dissipation (ΔD). The QCM-D data were analyzed using a Voigt-based model for the viscoelastic properties of polymer films in order to evaluate how the ligands affect thickness and shear viscosity of the DNA layer. Binding of spermine shrinks all DNA layers and increases their viscosity in a reversible fashion, and so does spermidine, but to a smaller extent, in agreement with its lower positive charge. SPR was used to measure the amount of bound polyamines, and when combined with QCM-D, the data indicate that the layer condensation leads to a small release of water from the highly hydrated DNA films. The binding of Rad51 increases the effective layer thickness of a 59bp film, more than expected from the know 50% DNA helix extension. The combined results provide guidelines for a QCM-D biosensor based on ligand-induced structural changes in DNA films. The QCM-D approach provides high discrimination between ligands affecting the thickness and the structural properties of the DNA layer differently. The reversibility of the film

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

    Science.gov (United States)

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

    2010-03-19

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

  12. Inhibitory Effect of Bridged Nucleosides on Thermus aquaticus DNA Polymerase and Insight into the Binding Interactions.

    Directory of Open Access Journals (Sweden)

    Sung-Kun Kim

    Full Text Available Modified nucleosides have the potential to inhibit DNA polymerases for the treatment of viral infections and cancer. With the hope of developing potent drug candidates by the modification of the 2',4'-position of the ribose with the inclusion of a bridge, efforts were focused on the inhibition of Taq DNA polymerase using quantitative real time PCR, and the results revealed the significant inhibitory effects of 2',4'-bridged thymidine nucleoside on the polymerase. Study on the mode of inhibition revealed the competitive mechanism with which the 2',4'-bridged thymidine operates. With a Ki value of 9.7 ± 1.1 μM, the 2',4'-bridged thymidine proved to be a very promising inhibitor. Additionally, docking analysis showed that all the nucleosides including 2',4'-bridged thymidine were able to dock in the active site, indicating that the substrate analogs reflect a structural complementarity to the enzyme active site. The analysis also provided evidence that Asp610 was a key binding site for 2',4'-bridged thymidine. Molecular dynamics (MD simulations were performed to further understand the conformational variations of the binding. The root-mean-square deviation (RMSD values for the peptide backbone of the enzyme and the nitrogenous base of the inhibitor stabilized within 0.8 and 0.2 ns, respectively. Furthermore, the MD analysis indicates substantial conformational change in the ligand (inhibitor as the nitrogenous base rotated anticlockwise with respect to the sugar moiety, complemented by the formation of several new hydrogen bonds where Arg587 served as a pivot axis for binding formation. In conclusion, the active site inhibition of Taq DNA polymerase by 2',4'-bridged thymidine suggests the potential of bridged nucleosides as drug candidates.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Konstantin Tretyakov

    Full Text Available 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/.

  15. Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication.

    Directory of Open Access Journals (Sweden)

    Greicy H Goto

    2015-08-01

    Full Text Available Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.

  16. DNA binding and condensation properties of the herpes simplex virus type 1 triplex protein VP19C.

    Directory of Open Access Journals (Sweden)

    Alakesh Bera

    Full Text Available Herpesvirus capsids are regular icosahedrons with a diameter of a 125 nm and are made up of 162 capsomeres arranged on a T = 16 lattice. The capsomeres (VP5 interact with the triplex structure, which is a unique structural feature of herpesvirus capsid shells. The triplex is a heterotrimeric complex; one molecule of VP19C and two of VP23 form a three-pronged structure that acts to stabilize the capsid shell through interactions with adjacent capsomeres. VP19C interacts with VP23 and with the major capsid protein VP5 and is required for the nuclear localization of VP23. Mutation of VP19C results in the abrogation of capsid shell synthesis. Analysis of the sequence of VP19C showed the N-terminus of VP19C is very basic and glycine rich. It was hypothesized that this domain could potentially bind to DNA. In this study an electrophoretic mobility shift assay (EMSA and a DNA condensation assay were performed to demonstrate that VP19C can bind DNA. Purified VP19C was able to bind to both a DNA fragment of HSV-1 origin as well as a bacterial plasmid sequence indicating that this activity is non-specific. Ultra-structural imaging of the nucleo-protein complexes revealed that VP19C condensed the DNA and forms toroidal DNA structures. Both the DNA binding and condensing properties of VP19C were mapped to the N-terminal 72 amino acids of the protein. Mutational studies revealed that the positively charged arginine residues in this N-terminal domain are required for this binding. This DNA binding activity, which resides in a non-conserved region of the protein could be required for stabilization of HSV-1 DNA association in the capsid shell.

  17. Sequence-specific binding and cleavage of duplex DNA by a radioiodinated, intercalator-linked, triplex-forming oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Orson, Frank M.; McShan, W. Michael; Kinsey, Berma M

    1996-05-01

    Applications of oligodeoxynucleotides to modulate gene expression have been the subject of much recent research. We have sought to develop a method to permanently inactivate a gene, or potentially kill cells containing abnormal genes. In this report, we show that a DNA intercalator conjugated to a triplex-forming oligonucleotide can be labeled with an Auger electron emitting radioisotope, can cleave its duplex DNA target, and can specifically bind the target sequence contained in a total of 10 kilobases of irrelevant DNA.

  18. Sequence-specific binding and cleavage of duplex DNA by a radioiodinated, intercalator-linked, triplex-forming oligonucleotide

    International Nuclear Information System (INIS)

    Applications of oligodeoxynucleotides to modulate gene expression have been the subject of much recent research. We have sought to develop a method to permanently inactivate a gene, or potentially kill cells containing abnormal genes. In this report, we show that a DNA intercalator conjugated to a triplex-forming oligonucleotide can be labeled with an Auger electron emitting radioisotope, can cleave its duplex DNA target, and can specifically bind the target sequence contained in a total of 10 kilobases of irrelevant DNA

  19. TALEored Epigenetics: A DNA-Binding Scaffold for Programmable Epigenome Editing and Analysis.

    Science.gov (United States)

    Kubik, Grzegorz; Summerer, Daniel

    2016-06-01

    Epigenetic modification of the cytosine 5-position is an important regulator of gene expression with essential roles in genome stability, development, and disease. In addition to 5-methylcytosine (mC), the oxidized mC derivatives 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine (hmC, fC, and caC) have recently been discovered. These are intermediates of an active demethylation pathway but might also represent new epigenetic marks with individual biological roles. This increase in chemical complexity of DNA-encoded information has created a pressing need for new approaches that allow reading and editing of this information. Transcription-activator-like effectors (TALEs) are DNA-binding domains with programmable sequence selectivity that enable the direct reading of epigenetic cytosine modifications but can also guide enzymatic editing domains to genomic loci of choice. Here, we review recent advances in employing TALEs for these applications. PMID:26972580

  20. Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A

    OpenAIRE

    Gangelhoff, Todd A.; Mungalachetty, Purnima S.; Nix, Jay C.; Mair E A Churchill

    2009-01-01

    The mitochondrial transcription factor A (mtTFA) is central to assembly and initiation of the mitochondrial transcription complex. Human mtTFA (h-mtTFA) is a dual high mobility group box (HMGB) protein that binds site-specifically to the mitochondrial genome and demarcates the promoters for recruitment of h-mtTFB1, h-mtTFB2 and the mitochondrial RNA polymerase. The stoichiometry of h-mtTFA was found to be a monomer in the absence of DNA, whereas it formed a dimer in the complex with the light...

  1. A triplex DNA-binding protein from human cells: purification and characterization.

    OpenAIRE

    Kiyama, R; Camerini-Otero, R.D.

    1991-01-01

    A protein that binds to an oligonucleotide triplex, (dT)34.(dA)34.(dT)34 (TAT triplex), was purified from HeLa cells by a combination of conventional column chromatography and triplex DNA affinity chromatography. The protein has an apparent molecular mass of 55 kDa on sodium dodecyl sulfate/polyacrylamide gels. Although the protein has an affinity for AT duplex and TAT triplex, a higher affinity for TAT triplex was demonstrated by comparing the elution profiles from an AT duplex and a TAT tri...

  2. Asparagine deamidation reduces DNA-binding affinity of the Drosophila melanogaster Scr homeodomain.

    Science.gov (United States)

    O'Connell, Nichole E; Lelli, Katherine; Mann, Richard S; Palmer, Arthur G

    2015-10-24

    Spontaneous deamidation of asparagine is a non-enzymatic post-translational modification of proteins. Residue Asn 321 is the main site of deamidation of the Drosophila melanogaster Hox transcription factor Sex Combs Reduced (Scr). Formation of iso-aspartate, the major deamidation product, is detected by HNCACB triple-resonance NMR spectroscopy. The rate of deamidation is quantified by fitting the decay of Asn NH2 side-chain signals in a time-series of (15)N-(1)H HSQC NMR spectra. The deamidated form of Scr binds to specific DNA target sequences with reduced affinity as determined by an electrophoretic mobility shift assay.

  3. Synthesis, characterization and DNA binding of the complexes of rare earth with phenanthroline and demethylcantharate

    Institute of Scientific and Technical Information of China (English)

    ZHANG Fan; ZHU Wenzhong; LIN Qiuyue; GUO Weidong; ZHANG Lingling; LI Shikun

    2011-01-01

    Three novel rare earth complexes, [Ln2(DCA)2(phen)2](NO3)2·6H2O (Ln(Ⅲ)=Sm(Ⅲ)(1), Er(Ⅲ)(2), Yb(Ⅲ)(3); DCA2-=demethylcantharate, 7-oxabicyclo[2.2.1] heptane-2,3-dicarboxylate, C8H8O52-; phen=1,10-phenanthroline, C12H8N2) were synthesized. The structures were characterized by elemental analysis, molar conductance, IR and TGA. The results suggested that the structural features of the complexes were: in each DCA2-, one carboxylate group, as bidentate bridging group, connected two rare earth ions; the other carboxylate group, as bidentate chelate group took part in the coordination with rare earth ion. And cyclic ether oxygen of DCA2- and nitrogen atoms of phen took part in the coordination. The probable coordination number was seven. The interaction of the complexes with DNA was studied by UV-spectra, fluorescence spectra and viscosity measurements. Following increasing the concentration of DNA, the UV absorption bands nearby 265 mn of the three complexes appeared hypochromism and red-shift phenomena. And the values of binding constants Kb were 1.89× 105 L/mol (1), 3.54× 104 L/mol (2) and 3.83× 104 L/mol (3). The complexes could quench the fluorescence of EB-DNA system, and the values of equilibrium constants Ksq were 1.72(1), 0.56(2) and 1.09(3). The relative viscosity of DNA steadily decreased with increasing theconcentration of complexes. So, we could infer that the complexes may partially insert into DNA. The study of agarose gel electrophoresis showed that the complexes could cleave plasmid DNA, and the process of the reaction was through unclassical redox mechanism.

  4. Triazolopyridyl ketones as a novel class of antileishmanial agents. DNA binding and BSA interaction.

    Science.gov (United States)

    Adam, Rosa; Bilbao-Ramos, Pablo; López-Molina, Sonia; Abarca, Belén; Ballesteros, Rafael; González-Rosende, M Eugenia; Dea-Ayuela, M Auxiliadora; Alzuet-Piña, Gloria

    2014-08-01

    A new series of triazolopyridyl pyridyl ketones has been synthetized by regioselective lithiation of the corresponding [1,2,3]triazolo[1,5-a]pyridine at 7 position followed by reaction with different electrophiles. The in vitro antileishmanial activity of these compounds was evaluated against Leishmaniainfantum, Leishmaniabraziliensis, Leishmaniaguyanensis and Leishmaniaamazonensis. Compounds 6 and 7 were found to be the most active leishmanicidal agents. Both of them showed activities at micromolar concentration against cultured promastigotes of Leishmania spp. (IC₅₀=99.8-26.8 μM), without cytotoxicity on J774 macrophage cells. These two compounds were also tested in vivo in a murine model of acute infection by L. infantum. The triazolopyridine derivative 6 was effective against both spleen and liver parasites forms, while 7 was inactive against liver parasites. Mechanistic aspects of the antileishmanial activity were investigated by means of DNA binding studies (UV-titration and viscosimetry). Results have revealed that these active ligands are able to interact strongly with DNA [Kb=1.14 × 10(5)M(-1) (6) and 3.26 × 10(5)M(-1) (7)]. Moreover, a DNA groove binding has been proposed for both 6 and 7. To provide more insight on the mode of action of compounds 6 and 7 under biological conditions, their interaction with bovine serum albumin (BSA) was monitored by fluorescence titrations and UV-visible spectroscopy. The quenching constants and binding parameters were determined. Triazolopyridine ketones 6 and 7 have exhibited significant affinity towards BSA [Kb=2.5 × 10(4)M(-1) (6) and 1.9 × 10(4)M(-1) (7)]. Finally, to identify the binding location of compounds 6 and 7 on the BSA, competitive binding experiments were carried out, using warfarin, a characteristic marker for site I, and ibuprofen as one for site II. Results derived from these studies have indicated that both compounds interact at BSA site I and, to a lesser extent, at site II.

  5. Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

    Science.gov (United States)

    Kitange, Gaspar J.; Mladek, Ann C.; Schroeder, Mark A.; Pokorny, Jenny C.; Carlson, Brett L.; Zhang, Yuji; Nair, Asha A.; Lee, Jeong-Heon; Yan, Huihuang; Decker, Paul A.; Zhang, Zhiguo; Sarkaria, Jann N.

    2016-01-01

    Summary Here we provide evidence that RBBP4 modulates temozolomide (TMZ) sensitivity through coordinate regulation of 2 key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT) and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51 and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac) and increased H3K9 tri-methylation (H3K9me3). Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin modifying complex that binds within the promoter of MGMT, RAD51 and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. RBBP4/CBP/p300 complex may provide an interesting target for developing therapy sensitizing strategies for GBM and other tumors. PMID:26972001

  6. Retinoblastoma Binding Protein 4 Modulates Temozolomide Sensitivity in Glioblastoma by Regulating DNA Repair Proteins

    Directory of Open Access Journals (Sweden)

    Gaspar J. Kitange

    2016-03-01

    Full Text Available Here we provide evidence that RBBP4 modulates temozolomide (TMZ sensitivity through coordinate regulation of two key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition, and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51, and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac and increased H3K9 tri-methylation (H3K9me3. Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin-modifying complex that binds within the promoter of MGMT, RAD51, and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. The RBBP4/CBP/p300 complex may provide an interesting target for developing therapy-sensitizing strategies for GBM and other tumors.

  7. Distinct DNA binding sites contribute to the TCF transcriptional switch in C. elegans and Drosophila.

    Directory of Open Access Journals (Sweden)

    Chandan Bhambhani

    2014-02-01

    Full Text Available Regulation of gene expression by signaling pathways often occurs through a transcriptional switch, where the transcription factor responsible for signal-dependent gene activation represses the same targets in the absence of signaling. T-cell factors (TCFs are transcription factors in the Wnt/ß-catenin pathway, which control numerous cell fate specification events in metazoans. The TCF transcriptional switch is mediated by many co-regulators that contribute to repression or activation of Wnt target genes. It is typically assumed that DNA recognition by TCFs is important for target gene location, but plays no role in the actual switch. TCF/Pangolin (the fly TCF and some vertebrate TCF isoforms bind DNA through two distinct domains, a High Mobility Group (HMG domain and a C-clamp, which recognize DNA motifs known as HMG and Helper sites, respectively. Here, we demonstrate that POP-1 (the C. elegans TCF also activates target genes through HMG and Helper site interactions. Helper sites enhanced the ability of a synthetic enhancer to detect Wnt/ß-catenin signaling in several tissues and revealed an unsuspected role for POP-1 in regulating the C. elegans defecation cycle. Searching for HMG-Helper site clusters allowed the identification of a new POP-1 target gene active in the head muscles and gut. While Helper sites and the C-clamp are essential for activation of worm and fly Wnt targets, they are dispensable for TCF-dependent repression of targets in the absence of Wnt signaling. These data suggest that a fundamental change in TCF-DNA binding contributes to the transcriptional switch that occurs upon Wnt stimulation.

  8. Rational design, synthesis, and DNA binding properties of novel sequence-selective peptidyl congeners of ametantrone.

    Science.gov (United States)

    Gianoncelli, Alessandra; Basili, Serena; Scalabrin, Matteo; Sosic, Alice; Moro, Stefano; Zagotto, Giuseppe; Palumbo, Manlio; Gresh, Nohad; Gatto, Barbara

    2010-07-01

    Natural and synthetic compounds characterized by an anthraquinone nucleus represent an important class of anti-neoplastic agents, the mechanism of action of which is related to intercalation into DNA. Ametantrone (AM) is a synthetic 9,10-anthracenedione bearing two (hydroxyethylamino)ethylamino residues at positions 1 and 4; along with other anthraquinones and anthracyclines, it shares a polycyclic intercalating moiety and charged side chains that stabilize DNA binding. All these drugs elicit adverse side effects, which represent a challenge for antitumor chemotherapy. In the present work the structure of AM was augmented with appropriate groups that target well-defined base pairs in the major groove. These should endow AM with DNA sequence selectivity. We describe the rationale for the synthesis and the evaluation of activity of a new series of compounds in which the planar anthraquinone is conjugated at positions 1 and 4 through the side chains of AM or other bioisosteric linkers to appropriate dipeptides. The designed novel AM derivatives were shown to selectively stabilize two oligonucleotide duplexes that both have a palindromic GC-rich hexanucleotide core, but their stabilizing effects on a random DNA sequence was negligible. In the case of the most effective compound, the 1,4-bis-[Gly-(L-Lys)] derivative of AM, the experimental results confirm the predictions of earlier theoretical computations. In contrast, AM had equal stabilizing effects on all three sequences and showed no preferential binding. This novel peptide derivative can be classified as a strong binder regarding the sequences that it selectively targets, possibly opening the exploitation of less cytotoxic conjugates of AM to the targeted treatment of oncological and viral diseases. PMID:20458714

  9. High-resolution detection of DNA binding sites of the global transcriptional regulator GlxR in Corynebacterium glutamicum

    DEFF Research Database (Denmark)

    Jungwirth, Britta; Sala, Claudia; Kohl, Thomas A;

    2013-01-01

    mapping of these data on the genome sequence of C. glutamicum, 107 enriched DNA fragments were detected from cells grown with glucose as carbon source. GlxR binding sites were identified in the sequence of 79 enriched DNA fragments, of which 21 sites were not previously reported. Electrophoretic mobility...... of the 6C non-coding RNA gene and to non-canonical DNA binding sites within protein-coding regions. The present study underlines the dynamics within the GlxR regulon by identifying in vivo targets during growth on glucose and contributes to the expansion of knowledge of this important transcriptional...

  10. Peptidyl anthraquinones as potential antineoplastic drugs: synthesis, DNA binding, redox cycling, and biological activity.

    Science.gov (United States)

    Gatto, B; Zagotto, G; Sissi, C; Cera, C; Uriarte, E; Palù, G; Capranico, G; Palumbo, M

    1996-08-01

    A series of new compounds containing a 9,10-anthracenedione moiety and one or two peptide chains at position 1 and/or 4 have been synthesized. The amino acid residues introduced are glycine (Gly), lysine (Lys), and tryptophan (Trp), the latter two in both the L- and D-configurations. The peptidyl anthraquinones maintain the ability of intercalating efficiently into DNA, even though the orientation within the base-pair pocket may change somewhat with reference to the parent drugs mitoxantrone (MX) and ametantrone (AM). The interaction constants of the mono-, di-, and triglycyl derivatives are well comparable to those found for AM but 5-10 times lower than the value reported for MX. On the other hand, the glycyl-lysyl compounds bind DNA to the same extent as (L-isomer) or even better than (D-isomer) MX. As for the parent drugs without peptidyl chains, the new compounds prefer alternating CG binding sites, although to different extents. The bis-Gly-Lys derivatives are the least sensitive to base composition, which may be due to extensive aspecific charged interactions with the polynucleotide backbone. As far as redox properties are concerned, all peptidyl anthraquinones show a reduction potential very close to that of AM and 60-80 mV less negative than that of MX; hence, they can produce free-radical-damaging species to an extent similar to the parent drugs. The biological activity has been tested in human tumor and murine leukemia cell lines. Most of the test anthraquinones exhibit cytotoxic properties close to those of AM and considerably lower than those of MX. Stimulation of topoisomerase-mediated DNA cleavage is moderately present in representatives of the glycylanthraquinone family, whereas inhibition of the background cleavage occurs when Lys is present in the peptide chain. For most of the test anthraquinones, the toxicity data are in line with the DNA affinity scale and the topoisomerase II stimulation activity. However, in the lysyl derivatives, for which

  11. Agrobacterium rhizogenes GALLS protein substitutes for Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2.

    Science.gov (United States)

    Hodges, Larry D; Cuperus, Josh; Ream, Walt

    2004-05-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2. PMID:15126468

  12. The incorporation of radiolabelled sulphur from captan into protein and its impact on a DNA binding study.

    Science.gov (United States)

    Provan, W M; Eyton-Jones, H; Lappin, G; Pritchard, D; Moore, R B; Green, T

    1995-05-19

    Repeated administration of high doses of captan is known to produce tumours specifically in the duodenum of mice. Captan is not carcinogenic in the rat. In this study, DNA purified from the liver, stomach, duodenum and jejenum of mice dosed with 35S radiolabelled captan was found to contain radioactivity equivalent to Covalent Binding Indices in the range 38-91; that from the bone marrow had a CBI of 2.8. The distribution of radioactivity between the various tissues did not reflect the target organ specificity of captan. Attempts to further purify the DNA samples using caesium chloride gradients resulted in partial separation of the radioactivity from the DNA suggesting that covalent binding to the DNA may not have occurred. A study of the chemical breakdown of captan showed that captan is unstable, producing a variety of potentially reactive species containing sulphur. Evidence was further obtained to show that the sulphur of captan is incorporated into endogenous amino acids and protein. Hepatic DNA from mice dosed with 35S radiolabelled N-acetylcysteine, and two thiazolidine derivatives which are analogous to known metabolites of captan, was radiolabelled to a similar extent to that from captan treated mice. Furthermore, the DNA from each of these treatments had similar properties on caesium chloride gradients. It was concluded that the radioactivity associated with DNA in the captan DNA binding study was present in the low levels of protein which are always associated with purified DNA samples.

  13. Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

    Science.gov (United States)

    Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

    2015-07-27

    VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. PMID:26044711

  14. Effects of mono- and divalent metal ions on DNA binding and catalysis of human apurinic/apyrimidinic endonuclease 1.

    Science.gov (United States)

    Miroshnikova, Anastasia D; Kuznetsova, Alexandra A; Vorobjev, Yuri N; Kuznetsov, Nikita A; Fedorova, Olga S

    2016-05-26

    Here, we used stopped-flow fluorescence techniques to conduct a comparative kinetic analysis of the conformational transitions in human apurinic/apyrimidinic endonuclease 1 (APE1) and in DNA containing an abasic site in the course of their interaction. Effects of monovalent (K(+)) and divalent (Mg(2+), Mn(2+), Ca(2+), Zn(2+), Cu(2+), and Ni(2+)) metal ions on DNA binding and catalytic stages were studied. It was shown that the first step of substrate binding (corresponding to formation of a primary enzyme-substrate complex) does not depend on the concentration (0.05-5.0 mM) or the nature of divalent metal ions. In contrast, the initial DNA binding efficiency significantly decreased at a high concentration (5-250 mM) of monovalent K(+) ions, indicating the involvement of electrostatic interactions in this stage. It was also shown that Cu(2+) ions abrogated the DNA binding ability of APE1, possibly, due to a strong interaction with DNA bases and the sugar-phosphate backbone. In the case of Ca(2+) ions, the catalytic activity of APE1 was lost completely with retention of binding potential. Thus, the enzymatic activity of APE1 is increased in the order Zn(2+) < Ni(2+) < Mn(2+) < Mg(2+). Circular dichroism spectra and calculation of the contact area between APE1 and DNA reveal that Mg(2+) ions stabilize the protein structure and the enzyme-substrate complex. PMID:27063150

  15. A mechanism for single-stranded DNA-binding protein (SSB) displacement from single-stranded DNA upon SSB-RecO interaction.

    Science.gov (United States)

    Inoue, Jin; Nagae, Takayuki; Mishima, Masaki; Ito, Yutaka; Shibata, Takehiko; Mikawa, Tsutomu

    2011-02-25

    Displacement of single-stranded DNA (ssDNA)-binding protein (SSB) from ssDNA is necessary for filament formation of RecA on ssDNA to initiate homologous recombination. The interaction between RecO and SSB is considered to be important for SSB displacement; however, the interaction has not been characterized at the atomic level. In this study, to clarify the mechanism underlying SSB displacement from ssDNA upon RecO binding, we examined the interaction between Thermus thermophilus RecO and cognate SSB by NMR analysis. We found that SSB interacts with the C-terminal positively charged region of RecO. Based on this result, we constructed some RecO mutants. The R127A mutant had considerably decreased binding affinity for SSB and could not anneal SSB-coated ssDNAs. Further, the mutant in the RecOR complex prevented the recovery of ssDNA-dependent ATPase activity of RecA from inhibition by SSB. These results indicated that the region surrounding Arg-127 is the binding site of SSB. We also performed NMR analysis using the C-terminal peptide of SSB and found that the acidic region of SSB is involved in the interaction with RecO, as seen in other protein-SSB interactions. Taken together with the findings of previous studies, we propose a model for SSB displacement from ssDNA where the acidic C-terminal region of SSB weakens the ssDNA binding affinity of SSB when the dynamics of the C-terminal region are suppressed by interactions with other proteins, including RecO. PMID:21169364

  16. Binding of 12-s-12 dimeric surfactants to calf thymus DNA: Evaluation of the spacer length influence.

    Science.gov (United States)

    Sarrión, Beatriz; Bernal, Eva; Martín, Victoria Isabel; López-López, Manuel; López-Cornejo, Pilar; García-Calderón, Margarita; Moyá, María Luisa

    2016-08-01

    Several cationic dimeric surfactants have shown high affinity towards DNA. Bis-quaternary ammonium salts (m-s-m) have been the most common type of dimeric surfactants investigated and it is generally admitted that those that posses a short spacer (s≤3) show better efficiency to bind or compact DNA. However, experimental results in this work show that 12-s-12 surfactants with long spacers make the surfactant/ctDNA complexation more favorable than those with short spacers. A larger contribution of the hydrophobic interactions, which control the binding Gibbs energy, as well as a higher average charge of the surfactant molecules bound to the nucleic acid, which favors the electrostatic attractions, could explain the experimental observations. Dimeric surfactants with intermediate spacer length seem to be the less efficient for DNA binding. PMID:27108208

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

    Directory of Open Access Journals (Sweden)

    Brandon L Jutras

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

  18. Sequence-specific DNA binding of individual cut repeats of the human CCAAT displacement/cut homeodomain protein.

    Science.gov (United States)

    Aufiero, B; Neufeld, E J; Orkin, S H

    1994-08-01

    CCAAT displacement protein (CDP), a nuclear protein of 180-190 kDa, contains a triplicated motif, the cut domain, similar (80-90% conserved) to three repeats of 60-65 amino acids first identified in Drosophila cut, a homeo-domain protein involved in cell-fate decisions in development. Cut repeats bind DNA and exhibit subtle differences in target-site recognition. DNA sequences specifically bound by cut repeats were isolated by PCR-mediated DNA target-site selection. Sequences selected for cut repeat 2 and 3 (CR2 and CR3) binding are A+T-rich and favor an ATA motif with similar, but not identical, flanking base preferences. CR2 and CR3 discriminate among similar target sequences. CR1, which is more divergent from CR2 and CR3, displays the most restricted pattern of DNA sequence recognition. Methylation interference analysis demonstrates different protein-DNA contacts for CR1 and CR3 binding to a target sequence. Thus, CDP/cut is a complex protein whose DNA-binding properties reflect the combinatorial interaction of four domains (three cut repeats and one homeodomain) with target DNA sequences. PMID:7914370

  19. The Electronic Behavior of Zinc-Finger Protein Binding Sites in the Context of the DNA Extended Ladder Model

    Science.gov (United States)

    Oiwa, Nestor; Cordeiro, Claudette; Heermann, Dieter

    2016-05-01

    Instead of ATCG letter alignments, typically used in bioinformatics, we propose a new alignment method using the probability distribution function of the bottom of the occupied molecular orbital (BOMO), highest occupied molecular orbital (HOMO) and lowest unoccupied orbital (LUMO). We apply the technique to transcription factors with Cys2His2 zinc fingers. These transcription factors search for binding sites, probing for the electronic patterns at the minor and major DNA groves. The eukaryotic Cys2His2 zinc finger proteins bind to DNA ubiquitously at highly conserved domains. They are responsible for gene regulation and the spatial organization of DNA. To study and understand these zinc finger DNA-protein interactions, we use the extended ladder in the DNA model proposed by Zhu, Rasmussen, Balatsky & Bishop (2007) te{Zhu-2007}. Considering one single spinless electron in each nucleotide π-orbital along a double DNA chain (dDNA), we find a typical pattern for the bottom of BOMO, HOMO and LUMO along the binding sites. We specifically looked at two members of zinc finger protein family: specificity protein 1 (SP1) and early grown response 1 transcription factors (EGR1). When the valence band is filled, we find electrons in the purines along the nucleotide sequence, compatible with the electric charges of the binding amino acids in SP1 and EGR1 zinc finger.

  20. The Structure of DdrB from Deinococcus: a New Fold for Single-stranded DNA Binding Proteins

    Energy Technology Data Exchange (ETDEWEB)

    Sugiman-Marangos, S.; Junop, M

    2010-01-01

    Deinococcus spp. are renowned for their amazing ability to recover rapidly from severe genomic fragmentation as a result of exposure to extreme levels of ionizing radiation or desiccation. Despite having been originally characterized over 50 years ago, the mechanism underlying this remarkable repair process is still poorly understood. Here, we report the 2.8 {angstrom} structure of DdrB, a single-stranded DNA (ssDNA) binding protein unique to Deinococcus spp. that is crucial for recovery following DNA damage. DdrB forms a pentameric ring capable of binding single-stranded but not double-stranded DNA. Unexpectedly, the crystal structure reveals that DdrB comprises a novel fold that is structurally and topologically distinct from all other single-stranded binding (SSB) proteins characterized to date. The need for a unique ssDNA binding function in response to severe damage, suggests a distinct role for DdrB which may encompass not only standard SSB protein function in protection of ssDNA, but also more specialized roles in protein recruitment or DNA architecture maintenance. Possible mechanisms of DdrB action in damage recovery are discussed.

  1. Sm and DNA Binding by dual reactive B cells requires distinct V{sub H}, V{sub {kappa}}, and V{sub H} CDR3 structures

    Energy Technology Data Exchange (ETDEWEB)

    Retter, M.W.; Eisenberg, R.A.; Cohen, P.L. [Univ. of North Carolina, Chapel Hill, NC (United States)] [and others

    1995-08-15

    We have previously demonstrated an overlap of the anti-Sm and anti-DNA responses in MRL/Mp-Ipr/Ipr mice. The Ab produced by many anti-Sm hybridomas bind DNA and are encoded by Ig V genes used by anti-DNA hybridomas. In addition, some anti-Sm Ab that bind DNA have acquired mutations that improve DNA binding, indicating that DNA is a selecting Ag in the anti-Sm response. To gain insight into the basis for the dual binding ability of these Ab, we coexpressed the H chain from the anti-Sm hybridoma 2-12 with nine different L chains. Hybridoma 2-12 binds Sm but not DNA, yet expresses the same J558 V{sub H} gene as three anti-Sm hybridomas that bind ssDNA and at least one anti-DNA hybridoma that does not bind Sm. We found that most of the transfectoma Ab bind Sm, but their avidities vary over more than 3 orders of magnitude. Five of the nine transfectoma Ab bind ssDNA, and none bind dsDNA. In general, the ability to bind each Ag follows the binding ability of the hybridoma from which the L chain is derived. H Chain swapping experiments indicate that the H chain, V{sub H} CDR3 in particular contributes to the binding of both Sm and DNA. We conclude that Sm and DNA select for distinct features of V{sub H}, V{sub {kappa}}, and V{sub H} CDR3, suggesting selection by both Ag in the anti-Sm response.

  2. Peeling single-stranded DNA from graphite surface to determine oligonucleotide binding energy by force spectroscopy.

    Science.gov (United States)

    Manohar, Suresh; Mantz, Amber R; Bancroft, Kevin E; Hui, Chung-Yuen; Jagota, Anand; Vezenov, Dmitri V

    2008-12-01

    We measured the force required to peel single-stranded DNA molecules from single-crystal graphite using chemical force microscopy. Force traces during retraction of a tip chemically modified with oligonucleotides displayed characteristic plateaus with abrupt force jumps, which we interpreted as a steady state peeling process punctuated by complete detachment of one or more molecules. We were able to differentiate between bases in pyrimidine homopolymers; peeling forces were 85.3 - 4.7 pN for polythymine and 60.8 +/- 5.5 pN for polycytosine, substantially independent of salt concentration and the rate of detachment. We developed a model for peeling a freely jointed chain from the graphite surface and estimated the average binding energy per monomer to be 11.5 +/- 0.6 k(B)T and 8.3 +/- 0.7 k(B)T in the cases of thymine and cytosine nucleotides, respectively. The equilibrium free-energy profile simulated using molecular dynamics had a potential well of 18.9 k(B)T for thymidine, showing that nonelectrostatic interactions dominate the binding. The discrepancy between the experiment and theory indicates that not all bases are adsorbed on the surface or that there is a population of conformations in which they adsorb. Force spectroscopy using oligonucleotides covalently linked to AFM tips provides a flexible and unambiguous means to quantify the strength of interactions between DNA and a number of substrates, potentially including nanomaterials such as carbon nanotubes. PMID:19368004

  3. Analysis of the DNA-binding and dimerization activities of Neurospora crassa transcription factor NUC-1.

    Science.gov (United States)

    Peleg, Y; Metzenberg, R L

    1994-12-01

    NUC-1, a positive regulatory protein of Neurospora crassa, controls the expression of several unlinked target genes involved in phosphorus acquisition. The carboxy-terminal end of the NUC-1 protein has sequence similarity to the helix-loop-helix family of transcription factors. Bacterially expressed and in vitro-synthesized proteins, which consist of the carboxy-terminal portion of NUC-1, bind specifically to upstream sequences of two of its target genes, pho2+ and pho-4+. These upstream sequences contain the core sequence, CACGTG, a target for many helix-loop-helix proteins. A large loop region (47 amino acids) separates the helix I and helix II domains. Mutations and deletion within the loop region did not interfere with the in vitro or in vivo functions of the protein. Immediately carboxy-proximal to the helix II domain, the NUC-1 protein contains an atypical zipper domain which is essential for function. This domain consists of a heptad repeat of alanine and methionine rather than leucine residues. Analysis of mutant NUC-1 proteins suggests that the helix II and the zipper domains are essential for the protein dimerization, whereas the basic and the helix I domains are involved in DNA binding. The helix I domain, even though likely to participate in dimer formation while NUC-1 is bound to DNA, is not essential for in vitro dimerization.

  4. ACETYLATION INCREASES EWS-FLI1 DNA BINDING AND TRANSCRIPTIONAL ACTIVITY

    Directory of Open Access Journals (Sweden)

    Silke eSchlottmann

    2012-09-01

    Full Text Available Ewing Sarcoma (ES is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant posttranslational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors, and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with histone deacetylase inhibitors (HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in COS7 cells. However, our data that evaluates the acetylation of ful-length EWS-FLI1 remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

  5. Hormone- and DNA-binding mechanisms of the recombinant human estrogen receptor.

    Science.gov (United States)

    Obourn, J D; Koszewski, N J; Notides, A C

    1993-06-22

    We have investigated the hormone- and DNA-binding mechanisms of the wild-type human estrogen receptor (hER) overproduced in insect cells using a baculovirus expression system. The recombinant hER was indistinguishable in size (67 kDa) and immunogenically from the native human estrogen receptor in MCF-7 breast carcinoma cells. The recombinant hER was purified to 70-80% homogeneity with a two-step procedure that included ammonium sulfate precipitation and oligonucleotide affinity chromatography using a unique Teflon affinity matrix. The recombinant hER bound estradiol with a positively cooperative mechanism. At hER concentrations in excess of 13 nM the Hill coefficient reached a maximal value of 1.6, whereas, at lower hER concentrations, the Hill coefficient approached 1.0, suggesting that the hER was dissociated to the monomeric species and site-site interactions were diminished. The hER specifically bound an estrogen responsive element (ERE) from chicken vitellogenin II gene as measured by the gel mobility assay, ethylation, and thymine interference footprinting. Specific interference patterns suggest a two-fold symmetry of the hER binding to the ERE with each monomer of the hER bound in the major groove of the DNA. These data indicate that the recombinant hER is valuable to define the biochemical and structural properties of the native estrogen receptor. PMID:8512933

  6. Probing the functional impact of sequence variation on p53-DNA interactions using a novel microsphere assay for protein-DNA binding with human cell extracts.

    Directory of Open Access Journals (Sweden)

    Maher A Noureddine

    2009-05-01

    Full Text Available The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs. Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation -- including polymorphisms -- and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks.

  7. Structure-function studies of DNA binding domain of response regulator KdpE reveals equal affinity interactions at DNA half-sites.

    Directory of Open Access Journals (Sweden)

    Anoop Narayanan

    Full Text Available Expression of KdpFABC, a K(+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABC(BS via the winged helix-turn-helix type DNA binding domain (KdpE(DBD. Exploration of E. coli KdpE(DBD and kdpFABC(BS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpE(DBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpE(DBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpE(DBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins.

  8. A structural insight into major groove directed binding of nitrosourea derivative nimustine with DNA: a spectroscopic study.

    Directory of Open Access Journals (Sweden)

    Shweta Agarwal

    Full Text Available Nitrosourea therapeutics occupies a definite place in cancer therapy but its exact mechanism of action has yet to be established. Nimustine, a chloroethyl nitrosourea derivative, is used to treat various types of malignancy including gliomas. The present work focuses on the understanding of nimustine interaction with DNA to delineate its mechanism at molecular level. Attenuated total reflection-Fourier transform infrared (ATR-FTIR has been used to determine the binding sites of nimustine on DNA. Circular dichroism (CD spectroscopy has been used to confirm conformational variations in DNA molecule upon nimustine-DNA interaction. Thermodynamic parameters of nimustine-DNA reaction have been calculated by isothermal titration calorimetry. Results of the present study demonstrate that nimustine is not a simple alkylating agent rather it causes major grove-directed-alkylation. Spectroscopic data suggest binding of nimustine with nitrogenous bases guanine (C6 = O6 and thymine (C4 = O4 in DNA major groove. CD spectra of nimustine-DNA complexes point toward the perturbation of native B-conformation of DNA and its partial transition into C-form. Thermodynamically, nimustine-DNA interaction is an entropy driven endothermic reaction, which suggests hydrophobic interaction of nimustine in DNA-major groove pocket. Spectral results suggest base binding and local conformational changes in DNA upon nimustine interaction. Investigation of drug-DNA interaction is an essential part of rational drug designing that also provides information about the drug's action at molecular level. Results, demonstrated here, may contribute in the development of new nitrosourea therapeutics with better efficacy and fewer side effects.

  9. Modification of DNA dynamics by platinum drug binding: a time-dependent fluorescence depolarization study of the interaction of cis- and trans-diamminedichloroplatinum(II) with DNA.

    Science.gov (United States)

    Millar, D P; Ho, K M; Aroney, M J

    1988-11-15

    The interaction of calf thymus DNA with the antitumor drug cis-diamminedichloroplatinum(II), and with the clinically ineffective trans isomer, is studied by time-dependent fluorescence depolarization spectroscopy of intercalated ethidium. The effect of the platinum compounds on the rapid torsional motions of DNA in solution is observed via depolarization of the ethidium fluorescence. The depolarization data are successfully analyzed with an elastic model of DNA dynamics and yield a value for the product of the torsional rigidity of the DNA and the friction factor for DNA twisting. The dependence of this quantity on the degree of platination of the DNA is determined for each isomer. At low levels of platination, the cis isomer increases the solute-solvent friction acting on the DNA torsional motions, which we attribute to local kinking of the helix axis at the sites of platination. At high levels of platination, the cis isomer decreases the torsional rigidity of the DNA, indicating that disruption of DNA duplex structure occurs under these conditions. The binding of the trans isomer to DNA has no effect on the torsional rigidity or the friction. The present results are compared with other findings on the interaction of these platinum compounds with DNA.

  10. Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA

    International Nuclear Information System (INIS)

    Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent. Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid. MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown. Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance. Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells. Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity. Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication. We examined Mgm101p's role in mtDNA repair. As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment. Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome. (author)