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Sample records for dna interacting proteins

  1. Screening for protein-DNA interactions by automatable DNA-protein interaction ELISA.

    Directory of Open Access Journals (Sweden)

    Luise H Brand

    Full Text Available DNA-binding proteins (DBPs, such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs the binding sequence had been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation. To close this gap, we report an automatable DNA-protein-interaction (DPI-ELISA screen of an optimized double-stranded DNA (dsDNA probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. We could show that the DPI-ELISA screen disclosed the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 was used to demonstrate that the automated DPI-ELISA screen revealed the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 were analyzed, which led to a refinement of their known DNA-binding consensi. Finally, we performed a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA-consensus was uncovered and we could show that the GATC-core is compulsory for AtTIFY1 binding. This specific interaction between AtTIFY1 and its DNA-binding motif was confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a

  2. Dynamics of DNA conformations and DNA-protein interaction

    DEFF Research Database (Denmark)

    Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen;

    2005-01-01

    in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent...... findings on the search process of proteins for a specific target on the DNA. © 2006 Materials Research Society....

  3. On the ion-mediated interaction between protein and DNA

    CERN Document Server

    Barbi, Maria

    2013-01-01

    The mechanism allowing a protein to search of a target sequence on DNA is currently described as an intermittent process composed of 3D diffusion in bulk and 1D diffusion along the DNA molecule. Due to the relevant charge of protein and DNA, electrostatic interaction should play a crucial role during this search. In this paper, we explicitly derive the mean field theory allowing for a description of the protein-DNA electrostatics in solution. This approach leads to a unified model of the search process, where 1D and 3D diffusion appear as a natural consequence of the diffusion on an extended interaction energy profile.

  4. REVIEW ARTICLE: DNA protein interactions and bacterial chromosome architecture

    Science.gov (United States)

    Stavans, Joel; Oppenheim, Amos

    2006-12-01

    Bacteria, like eukaryotic organisms, must compact the DNA molecule comprising their genome and form a functional chromosome. Yet, bacteria do it differently. A number of factors contribute to genome compaction and organization in bacteria, including entropic effects, supercoiling and DNA-protein interactions. A gamut of new experimental techniques have allowed new advances in the investigation of these factors, and spurred much interest in the dynamic response of the chromosome to environmental cues, segregation, and architecture, during both exponential and stationary phases. We review these recent developments with emphasis on the multifaceted roles that DNA-protein interactions play.

  5. Multiplex single-molecule interaction profiling of DNA barcoded proteins

    OpenAIRE

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

    2014-01-01

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling le...

  6. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex.

    Science.gov (United States)

    Gell, D; Jackson, S P

    1999-01-01

    In mammalian cells, the Ku and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) proteins are required for the correct and efficient repair of DNA double-strand breaks. Ku comprises two tightly-associated subunits of approximately 69 and approximately 83 kDa, which are termed Ku70 and Ku80 (or Ku86), respectively. Previously, a number of regions of both Ku subunits have been demonstrated to be involved in their interaction, but the molecular mechanism of this interaction remains unknown. We have identified a region in Ku70 (amino acid residues 449-578) and a region in Ku80 (residues 439-592) that participate in Ku subunit interaction. Sequence analysis reveals that these interaction regions share sequence homology and suggests that the Ku subunits are structurally related. On binding to a DNA double-strand break, Ku is able to interact with DNA-PKcs, but how this interaction is mediated has not been defined. We show that the extreme C-terminus of Ku80, specifically the final 12 amino acid residues, mediates a highly specific interaction with DNA-PKcs. Strikingly, these residues appear to be conserved only in Ku80 sequences from vertebrate organisms. These data suggest that Ku has evolved to become part of the DNA-PK holo-enzyme by acquisition of a protein-protein interaction motif at the C-terminus of Ku80. PMID:10446239

  7. Energetics of the protein-DNA-water interaction

    Directory of Open Access Journals (Sweden)

    Marabotti Anna

    2007-01-01

    Full Text Available Abstract Background To understand the energetics of the interaction between protein and DNA we analyzed 39 crystallographically characterized complexes with the HINT (Hydropathic INTeractions computational model. HINT is an empirical free energy force field based on solvent partitioning of small molecules between water and 1-octanol. Our previous studies on protein-ligand complexes demonstrated that free energy predictions were significantly improved by taking into account the energetic contribution of water molecules that form at least one hydrogen bond with each interacting species. Results An initial correlation between the calculated HINT scores and the experimentally determined binding free energies in the protein-DNA system exhibited a relatively poor r2 of 0.21 and standard error of ± 1.71 kcal mol-1. However, the inclusion of 261 waters that bridge protein and DNA improved the HINT score-free energy correlation to an r2 of 0.56 and standard error of ± 1.28 kcal mol-1. Analysis of the water role and energy contributions indicate that 46% of the bridging waters act as linkers between amino acids and nucleotide bases at the protein-DNA interface, while the remaining 54% are largely involved in screening unfavorable electrostatic contacts. Conclusion This study quantifies the key energetic role of bridging waters in protein-DNA associations. In addition, the relevant role of hydrophobic interactions and entropy in driving protein-DNA association is indicated by analyses of interaction character showing that, together, the favorable polar and unfavorable polar/hydrophobic-polar interactions (i.e., desolvation mostly cancel.

  8. An ancient protein-DNA interaction underlying metazoan sex determination.

    Science.gov (United States)

    Murphy, Mark W; Lee, John K; Rojo, Sandra; Gearhart, Micah D; Kurahashi, Kayo; Banerjee, Surajit; Loeuille, Guy-André; Bashamboo, Anu; McElreavey, Kenneth; Zarkower, David; Aihara, Hideki; Bardwell, Vivian J

    2015-06-01

    DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. Here we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are used in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.

  9. The interaction of amino acids, peptides, and proteins with DNA.

    Science.gov (United States)

    Solovyev, Andrey Y; Tarnovskaya, Svetlana I; Chernova, Irina A; Shataeva, Larisa K; Skorik, Yury A

    2015-01-01

    Amino acids that carry charges on their side groups can bind to double stranded DNA (dsDNA) and change the strength of the double helix. Measurement of the DNA melting temperature (Tm) confirmed that acidic amino acids (Glu, Asp) weaken the H-bonds between DNA strands, whereas basic amino acids (Arg, Lys) strengthen the interaction between the strands. A rank correlation exists between the amino acid isoelectric points and the observed changes in Tm. A similar dependence of the hyperchromic effect on the isoelectric point of a protein (pepsin, insulin, cortexin, and protamine) was observed for DNA-protein complexes at room temperature. Short peptides (KE, AEDG, and KEDP) containing a mixture of acidic and basic amino acid residues also affect Tm and the stability of the double helix. A model for binding Glu and Lys to dsDNA was explored by a docking simulation. The model shows that Glu, in an untwisted shape, binds to dsDNA in its major groove and disrupts three H-bonds between the strands, thereby destabilizing the double helix. Lys, in an untwisted shape, binds to the external side of the dsDNA and forms two bonds with O atoms of neighboring phosphodiester groups, thereby strengthening the DNA helix.

  10. Micromechanical study of protein-DNA interactions and chromosomes

    Science.gov (United States)

    Marko, John

    I will discuss micromechanics experiments that our group has used to analyze protein-DNA interactions and chromosome organization. In single-DNA experiments we have found that a feature of protein-DNA complexes is that their dissociation rates can depend strikingly on bulk solution concentrations of other proteins and DNA segments; I will describe experiments which demonstrate this effect, which can involve tens-fold changes in off-rates with submicromolar changes in solution concentrations. Second, I will discuss experiments aimed at analyzing large-scale human chromosome structure; we isolate metaphase chromosomes, which in their native form behave as remarkably elastic networks of chromatin. Exposure to DNA-cutting restriction enzymes completely eliminates this elasticity, indicating that there is not a mechanically contiguous protein ''scaffold'' from which the chromosome gains its stability. I will show results of siRNA experiments indicating that depletion of condensin proteins leads to destabilization of chromosome mechanics, indicating condensin's role as the major chromatin ''cross-linker'' in metaphase chromosomes. Finally I will discuss similar experiments on human G1 nuclei, where we use genetic and chemical modifications to separate the contributions of the nuclear lamina and chromatin to the mechanical stiffness of the nucleus as a whole. Supported by the NSF (DMR-1206868, MCB-1022117) and the NIH (GM105847, CA193419).

  11. DNA Origami Reorganizes upon Interaction with Graphite: Implications for High-Resolution DNA Directed Protein Patterning

    Directory of Open Access Journals (Sweden)

    Masudur Rahman

    2016-10-01

    Full Text Available Although there is a long history of the study of the interaction of DNA with carbon surfaces, limited information exists regarding the interaction of complex DNA-based nanostructures with the important material graphite, which is closely related to graphene. In view of the capacity of DNA to direct the assembly of proteins and optical and electronic nanoparticles, the potential for combining DNA-based materials with graphite, which is an ultra-flat, conductive carbon substrate, requires evaluation. A series of imaging studies utilizing Atomic Force Microscopy has been applied in order to provide a unified picture of this important interaction of structured DNA and graphite. For the test structure examined, we observe a rapid destabilization of the complex DNA origami structure, consistent with a strong interaction of single-stranded DNA with the carbon surface. This destabilizing interaction can be obscured by an intentional or unintentional primary intervening layer of single-stranded DNA. Because the interaction of origami with graphite is not completely dissociative, and because the frustrated, expanded structure is relatively stable over time in solution, it is demonstrated that organized structures of pairs of the model protein streptavidin can be produced on carbon surfaces using DNA origami as the directing material.

  12. New Environment-Sensitive Multichannel DNA Fluorescent Label for Investigation of the Protein-DNA Interactions

    OpenAIRE

    Kuznetsova, Alexandra A.; Kuznetsov, Nikita A.; Vorobjev, Yuri N.; Barthes, Nicolas P. F.; Benoît Y Michel; Alain Burger; Fedorova, Olga S.

    2014-01-01

    Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside...

  13. Effect of DNA methylation on protein-DNA interaction of HL-60 cells

    Institute of Scientific and Technical Information of China (English)

    何忠效; 白坚石; 张昱

    1999-01-01

    HL-60 cells have been induced with differentiation index 16 % by S-adenosyl-L-rnethionine (SAM) as inducer in the presence of optimum conceptration of 10 μmol/L. The methylation level of genorne DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma I, Sal I, XhoI and Hind Ⅲ which are sensitive to 5-methylcytosine were used to cleave the genorne DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.

  14. Nonlinear Model of the Specificity of DNA-Protein Interactions and Its Stability

    Science.gov (United States)

    Dwiputra, D.; Hidayat, W.; Khairani, R.; Zen, F. P.

    2016-08-01

    Specific DNA-protein interactions are fundamental processes of living cells. We propose a new model of DNA-protein interactions to explain the site specificity of the interactions. The hydrogen bonds between DNA base pairs and between DNA-protein peptide groups play a significant role in determination of the specific binding site. We adopt the Morse potential with coupling terms to construct the Hamiltonian of coupled oscillators representing the hydrogen bonds in which the depth of the potentials vary in the DNA chain. In this paper we investigate the stability of the model to determine the conditions satisfying the biological circumstances of the DNA-protein interactions.

  15. New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions.

    Directory of Open Access Journals (Sweden)

    Alexandra A Kuznetsova

    Full Text Available Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu, pyrrolocytosine (Cpy and 1,3-diaza-2-oxophenoxazine (tCO. For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside 5,6-dihydrouridine (DHU, which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.

  16. New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions.

    Science.gov (United States)

    Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Vorobjev, Yuri N; Barthes, Nicolas P F; Michel, Benoît Y; Burger, Alain; Fedorova, Olga S

    2014-01-01

    Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.

  17. Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

    CERN Document Server

    Williams, Mark C

    2011-01-01

    This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

  18. Titantium Dioxide Nanoparticles Assembled by DNA Molecules Hybridization and Loading of DNA Interacting Proteins.

    Science.gov (United States)

    Wu, Aiguo; Paunesku, Tatjana; Brown, Eric M B; Babbo, Angela; Cruz, Cecille; Aslam, Mohamed; Dravid, Vinayak; Woloschak, Gayle E

    2008-02-01

    This work demonstrates the assembly of TiO(2) nanoparticles with attached DNA oligonucleotides into a 3D mesh structure by allowing base pairing between oligonucleotides. A change of the ratio of DNA oligonucleotide molecules and TiO(2) nanoparticles regulates the size of the mesh as characterized by UV-visible light spectra, transmission electron microscopy and atomic force microscopy images. This type of 3D mesh, based on TiO(2)-DNA oligonucleotide nanoconjugates, can be used for studies of nanoparticle assemblies in material science, energy science related to dye-sensitized solar cells, environmental science as well as characterization of DNA interacting proteins in the field of molecular biology. As an example of one such assembly, proliferating cell nuclear antigen protein (PCNA) was cloned, its activity verified, and the protein was purified, loaded onto double strand DNA oligonucleotide-TiO(2) nanoconjugates, and imaged by atomic force microscopy. This type of approach may be used to sample and perhaps quantify and/or extract specific cellular proteins from complex cellular protein mixtures affinity based on their affinity for chosen DNA segments assembled into the 3D matrix.

  19. DBD-Hunter: a knowledge-based method for the prediction of DNA-protein interactions.

    Science.gov (United States)

    Gao, Mu; Skolnick, Jeffrey

    2008-07-01

    The structures of DNA-protein complexes have illuminated the diversity of DNA-protein binding mechanisms shown by different protein families. This lack of generality could pose a great challenge for predicting DNA-protein interactions. To address this issue, we have developed a knowledge-based method, DNA-binding Domain Hunter (DBD-Hunter), for identifying DNA-binding proteins and associated binding sites. The method combines structural comparison and the evaluation of a statistical potential, which we derive to describe interactions between DNA base pairs and protein residues. We demonstrate that DBD-Hunter is an accurate method for predicting DNA-binding function of proteins, and that DNA-binding protein residues can be reliably inferred from the corresponding templates if identified. In benchmark tests on approximately 4000 proteins, our method achieved an accuracy of 98% and a precision of 84%, which significantly outperforms three previous methods. We further validate the method on DNA-binding protein structures determined in DNA-free (apo) state. We show that the accuracy of our method is only slightly affected on apo-structures compared to the performance on holo-structures cocrystallized with DNA. Finally, we apply the method to approximately 1700 structural genomics targets and predict that 37 targets with previously unknown function are likely to be DNA-binding proteins. DBD-Hunter is freely available at http://cssb.biology.gatech.edu/skolnick/webservice/DBD-Hunter/.

  20. Electrophoretic mobility shift assays for the analysis of DNA-protein interactions.

    Science.gov (United States)

    Gaudreault, Manon; Gingras, Marie-Eve; Lessard, Maryse; Leclerc, Steeve; Guérin, Sylvain L

    2009-01-01

    Electromobility shift assay is a simple, efficient, and rapid method for the study of specific DNA-protein interactions. It relies on the reduction in the electrophoretic mobility conferred to a DNA fragment by an interacting protein. The technique is suitable to qualitative, quantitative, and kinetic analyses. It can also be used to analyze conformational changes.

  1. Predicting functionality of protein-DNA interactions by integrating diverse evidence

    DEFF Research Database (Denmark)

    Ucar, Duygu; Beyer, A.; Parthasarathy, S.

    2009-01-01

    Chromatin immunoprecipitation (ChIP-chip) experiments enable capturing physical interactions between regulatory proteins and DNA in vivo. However, measurement of chromatin binding alone is not sufficient to detect regulatory interactions. A detected binding event may not be biologically relevant...

  2. Visualization of interaction between ribosome-inactivating proteins and supercoiled DNA with an atomic force microscope

    Institute of Scientific and Technical Information of China (English)

    吴晓华; 刘望夷; 欧阳振乾; 李民乾

    1997-01-01

    The interaction between ribosome-inactivating proteins (RIPs) and supercoiled DNA was observed with an atomic force microscope (AFM). It was found that RIPs can bind to both supercoiled DNA and the unwound double stranded loop region in supercoiled DNA. The RIPs hound to the supercoils can induce the conformational change of supercoiled DNA. Furthermore, the supercoiled DNA was relaxed and cleaved into nick or linear form by RIPs. It indicated that RIP seemed to be a supercoil-dependent DNA binding protein and exhibited the activity of su-percoil-dependent DNA endonuclease.

  3. Genome-Wide Mapping of in Vivo Protein-DNA Interactions

    OpenAIRE

    Johnson, David S.; Mortazavi, Ali; Myers, Richard M.; Wold, Barbara

    2007-01-01

    In vivo protein-DNA interactions connect each transcription factor with its direct targets to form a gene network scaffold. To map these protein-DNA interactions comprehensively across entire mammalian genomes, we developed a large-scale chromatin immunoprecipitation assay (ChIPSeq) based on direct ultrahigh-throughput DNA sequencing. This sequence census method was then used to map in vivo binding of the neuron-restrictive silencer factor (NRSF; also known as REST, for repressor element–1 si...

  4. Interaction of DNA and Proteins with Single Nanopores

    Science.gov (United States)

    Kasianowicz, J. J.

    2006-03-01

    The bacterial toxins Staphylococcus aureus alpha-hemolysin and Bacillus anthracis protective antigen kill cells in part by forming ion channels in target membranes. We are using electrophysiology, molecular biology/protein biochemistry and computer modeling to study how biopolymers (e.g., single-stranded DNA and proteins) bind to and transport through these nanometer-scale pores. The results provide insight into the mechanism by which these toxins work and are the basis for several potential nanobiotechnology applications including ultra-rapid DNA sequencing, the sensitive and selective detection of a wide range of analytes and high throughput screening of therapeutic agents against several anthrax toxins. In collaboration with V.M. Stanford, M. Misakian, B. Nablo, S.E. Henrickson, NIST, EEEL, Gaithersburg, MD; T. Nguyen, R. Gussio, NCI, Ft. Detrick, MD; and K.M. Halverson, S. Bavari, R.G. Panchal, USAMRIID, Ft. Detrick, MD.

  5. Comparative interactomics for virus-human protein-protein interactions: DNA viruses versus RNA viruses.

    Science.gov (United States)

    Durmuş, Saliha; Ülgen, Kutlu Ö

    2017-01-01

    Viruses are obligatory intracellular pathogens and completely depend on their hosts for survival and reproduction. The strategies adopted by viruses to exploit host cell processes and to evade host immune systems during infections may differ largely with the type of the viral genetic material. An improved understanding of these viral infection mechanisms is only possible through a better understanding of the pathogen-host interactions (PHIs) that enable viruses to enter into the host cells and manipulate the cellular mechanisms to their own advantage. Experimentally-verified protein-protein interaction (PPI) data of pathogen-host systems only became available at large scale within the last decade. In this study, we comparatively analyzed the current PHI networks belonging to DNA and RNA viruses and their human host, to get insights into the infection strategies used by these viral groups. We investigated the functional properties of human proteins in the PHI networks, to observe and compare the attack strategies of DNA and RNA viruses. We observed that DNA viruses are able to attack both human cellular and metabolic processes simultaneously during infections. On the other hand, RNA viruses preferentially interact with human proteins functioning in specific cellular processes as well as in intracellular transport and localization within the cell. Observing virus-targeted human proteins, we propose heterogeneous nuclear ribonucleoproteins and transporter proteins as potential antiviral therapeutic targets. The observed common and specific infection mechanisms in terms of viral strategies to attack human proteins may provide crucial information for further design of broad and specific next-generation antiviral therapeutics.

  6. Circular Dichroism for the Analysis of Protein-DNA Interactions.

    Science.gov (United States)

    Scarlett, Garry; Siligardi, Giuliano; Kneale, Geoffrey G

    2015-01-01

    The aim of this chapter is to provide information on the practical aspects of circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD) in protein-nucleic acids interaction solution studies. The chapter will describe the guidelines appropriate to designing experiments and conducting correct data interpretation, the use of both benchtop and synchrotron CD approaches is discussed and the advantages of SRCD outlined. Further information and a good general review of the field a can be found in Gray (Circular Dichroism of protein-nucleic acid interactions. In: Fasman GD (ed) Circular dichroism and the conformational analysis of biomolecules. Plenum Press, New York. pp 469-500, 1996).

  7. The development of protein microarrays and their applications in DNA-protein and protein-protein interaction analyses of Arabidopsis transcription factors.

    Science.gov (United States)

    Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S P; Snyder, Michael; Harmer, Stacey L; Zhu, Yu-Xian; Deng, Xing Wang

    2008-01-01

    We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale.

  8. Modelling protein-DNA interactions: bend and twist until it fits

    NARCIS (Netherlands)

    van Dijk, M.

    2010-01-01

    DNA-interacting proteins fulfil a vital role in the living cell. They allow the cell to quickly respond to changes in its environment by tuning the level of transcription. Furthermore, sophisticated protein machineries guard the DNA against hazardous influences that may damage it. A disruption of th

  9. Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction.

    Science.gov (United States)

    Talukder, Poulami; Chen, Shengxi; Roy, Basab; Yakovchuk, Petro; Spiering, Michelle M; Alam, Mohammad P; Madathil, Manikandadas M; Bhattacharya, Chandrabali; Benkovic, Stephen J; Hecht, Sidney M

    2015-12-29

    Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2'-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2'-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.

  10. DOMMINO 2.0: integrating structurally resolved protein-, RNA-, and DNA-mediated macromolecular interactions.

    Science.gov (United States)

    Kuang, Xingyan; Dhroso, Andi; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

    2016-01-01

    Macromolecular interactions are formed between proteins, DNA and RNA molecules. Being a principle building block in macromolecular assemblies and pathways, the interactions underlie most of cellular functions. Malfunctioning of macromolecular interactions is also linked to a number of diseases. Structural knowledge of the macromolecular interaction allows one to understand the interaction's mechanism, determine its functional implications and characterize the effects of genetic variations, such as single nucleotide polymorphisms, on the interaction. Unfortunately, until now the interactions mediated by different types of macromolecules, e.g. protein-protein interactions or protein-DNA interactions, are collected into individual and unrelated structural databases. This presents a significant obstacle in the analysis of macromolecular interactions. For instance, the homogeneous structural interaction databases prevent scientists from studying structural interactions of different types but occurring in the same macromolecular complex. Here, we introduce DOMMINO 2.0, a structural Database Of Macro-Molecular INteractiOns. Compared to DOMMINO 1.0, a comprehensive database on protein-protein interactions, DOMMINO 2.0 includes the interactions between all three basic types of macromolecules extracted from PDB files. DOMMINO 2.0 is automatically updated on a weekly basis. It currently includes ∼1,040,000 interactions between two polypeptide subunits (e.g. domains, peptides, termini and interdomain linkers), ∼43,000 RNA-mediated interactions, and ∼12,000 DNA-mediated interactions. All protein structures in the database are annotated using SCOP and SUPERFAMILY family annotation. As a result, protein-mediated interactions involving protein domains, interdomain linkers, C- and N- termini, and peptides are identified. Our database provides an intuitive web interface, allowing one to investigate interactions at three different resolution levels: whole subunit network

  11. The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

    Institute of Scientific and Technical Information of China (English)

    Wei Gong; Kun He; Mike Covington; S.R Dinesh-Kumar; Michael Snyder; Stacey L.Harmer; Yu-Xian Zhu; Xing Wang Deng

    2008-01-01

    We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to constructprotein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and proteinprotein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale.

  12. Fabrication of Unimolecular Double-stranded DNA Microarrays on Solid Surfaces for Probing DNA-Protein/Drug Interactions

    Directory of Open Access Journals (Sweden)

    Zuhong Lu

    2003-01-01

    Full Text Available We present a novel method for fabricating unimole cular double-stranded DNA microarrays on solid surfaces, which were used to probe sequence-specific DNA/protein interactions. For manufacturing the unimolecular double-stranded DNA microarrays, two kinds of special single-stranded oligonucleotides, constant oligonucleotide and target oligonucleotide, were chemically synthesized. The constant oligonucleotides with internal aminated dT were used to capture and immobilize the target oligonucleotides onto the solid surface, and also to provide a primer for later enzymatic extension reactions, while target oligonucleotides took the role of harbouring DNA-binding sites of DNA-binding proteins. The variant target oligonucleotides were annealed and ligated with the constant oligonucleotides to form the new unimolecular oligonucleotides for microspotting. The prepared unimolecular oligonucleotides were microspotted on aldehyde-derivatized glass slides to make partial-dsDNA microarrays. Finally, the partial-dsDNA microarrays were converted into a unimolecular complete-dsDNA microarray by a DNA polymerase extension reaction. The efficiency and accuracy of the polymerase synthesis were demonstrated by the fluorescent-labeled dUTP incorporation in the enzymatic extension reaction and the restriction endonuclease digestion of the fabricated unimolecular complete-dsDNA microarray. The accessibility and specificity of the sequence-specific DNA-binding proteins binding to the immobilized unimolecular dsDNA probes were demonstrated by the binding of Cy3 labeled NF-?B (p50·p50 to the unimolecular dsDNA microarray. This unimolecular dsDNA microarray provides a general technique for high-throughput DNA-protein or DNA-drugs interactions.

  13. Evaluation of small ligand-protein interaction by ligation reaction with DNA-modified ligand.

    Science.gov (United States)

    Sugita, Rie; Mie, Masayasu; Funabashi, Hisakage; Kobatake, Eiry

    2010-01-01

    A method for the evaluation of interactions between protein and ligand using DNA-modified ligands, including signal enhancement of the DNA ligation reactions, is described. For proof of principle, a DNA probe modified by biotin was used. Two DNA probes were prepared with complementary sticky-ends. While one DNA probe was modified at the 5'-end of the sticky-end, the other was not modified. The probes could be ligated together by T4 DNA ligase along the strand without biotin modification. However, in the presence of streptavidin or anti-biotin Fab, the ligation reaction joining the two probes could not occur on either strand.

  14. Toxoplasma gondii: a bradyzoite-specific DnaK-tetratricopeptide repeat (DnaK-TPR) protein interacts with p23 co-chaperone protein.

    Science.gov (United States)

    Ueno, Akio; Dautu, George; Haga, Kaori; Munyaka, Biscah; Carmen, Gabriella; Kobayashi, Yoshiyasu; Igarashi, Makoto

    2011-04-01

    The DnaK-tetratricopeptide repeat (DnaK-TPR) gene (ToxoDB ID, TGME49_002020) is expressed predominantly at the bradyzoite stage. DnaK-TPR protein has a heat shock protein (DnaK) and tetratricopeptide repeat (TPR) domains with amino acid sequence similarity to the counterparts of other organisms (40.2-43.7% to DnaK domain and 41.1-66.0% to TPR domain). These findings allowed us to infer that DnaK-TPR protein is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. An immunofluorescence assay (IFA) revealed that DnaK-TPR protein was expressed in Toxoplasma gondii-encysted and in vitro-induced bradyzoites and distributed in the whole part of parasite cells. We conducted yeast two-hybrid screening to identify proteins interacting with DnaK-TPR protein, and demonstrated that DnaK-TPR protein interacts with p23 co-chaperone protein (Tgp23). It was expected that DnaK-TPR protein would have a function as a molecular chaperon in bradyzoite cells associated with Tgp23. Possible mechanisms for this gene are discussed.

  15. Mechanics of protein-DNA interaction studied with ultra-fast optical tweezers

    Science.gov (United States)

    Monico, Carina; Tempestini, Alessia; Vanzi, Francesco; Pavone, Francesco S.; Capitanio, Marco

    2014-05-01

    The lac operon is a well known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). LacI and other DNA-binding proteins bind their specific target sequences with rates higher than allowed by 3D diffusion alone. Generally accepted models predict a combination of free 3D diffusion and 1D sliding along non-specific DNA. We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with two different DNA constructs: a construct with two copies of the O1 operator separated by 300 bp and a construct containing the native E.coli operator sequences. Our measurements show at least two classes of LacI-DNA interactions: long (in the tens of s range) and short (tens of ms). Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operator sequences (long events) and transient interactions with nonspecific sequences (short events). Moreover, we observe continuous sliding of the protein along DNA, passively driven by the force applied with the optical tweezers.

  16. Direct inference of protein-DNA interactions using compressed sensing methods.

    Science.gov (United States)

    AlQuraishi, Mohammed; McAdams, Harley H

    2011-09-06

    Compressed sensing has revolutionized signal acquisition, by enabling complex signals to be measured with remarkable fidelity using a small number of so-called incoherent sensors. We show that molecular interactions, e.g., protein-DNA interactions, can be analyzed in a directly analogous manner and with similarly remarkable results. Specifically, mesoscopic molecular interactions act as incoherent sensors that measure the energies of microscopic interactions between atoms. We combine concepts from compressed sensing and statistical mechanics to determine the interatomic interaction energies of a molecular system exclusively from experimental measurements, resulting in a "de novo" energy potential. In contrast, conventional methods for estimating energy potentials are based on theoretical models premised on a priori assumptions and extensive domain knowledge. We determine the de novo energy potential for pairwise interactions between protein and DNA atoms from (i) experimental measurements of the binding affinity of protein-DNA complexes and (ii) crystal structures of the complexes. We show that the de novo energy potential can be used to predict the binding specificity of proteins to DNA with approximately 90% accuracy, compared to approximately 60% for the best performing alternative computational methods applied to this fundamental problem. This de novo potential method is directly extendable to other biomolecule interaction domains (enzymes and signaling molecule interactions) and to other classes of molecular interactions.

  17. Delineation of Methyl-DNA Binding Protein Interactions in the Prostate Cancer Genome (PC110091)

    Science.gov (United States)

    2014-03-01

    DNA Binding Protein Interactions in the Prostate Cancer Genome (PC110091) PRINCIPAL INVESTIGATOR: Roderick T Hori, PhD...13. SUPPLEMENTARY NOTES Prostate Cancer, Methylated DNA, Methyl- CpG Binding Domain, Chromatin Immunoprecipitation 14. ABSTRACT The purpose...of this study is to generate a genome-wide association profile of Methyl- CpG Domain-containing (MBD) proteins, such as MeCP2, MBD1, MBD2 and MBD4, in

  18. Learning protein-DNA interaction landscapes by integrating experimental data through computational models.

    Science.gov (United States)

    Zhong, Jianling; Wasson, Todd; Hartemink, Alexander J

    2014-10-15

    Transcriptional regulation is directly enacted by the interactions between DNA and many proteins, including transcription factors (TFs), nucleosomes and polymerases. A critical step in deciphering transcriptional regulation is to infer, and eventually predict, the precise locations of these interactions, along with their strength and frequency. While recent datasets yield great insight into these interactions, individual data sources often provide only partial information regarding one aspect of the complete interaction landscape. For example, chromatin immunoprecipitation (ChIP) reveals the binding positions of a protein, but only for one protein at a time. In contrast, nucleases like MNase and DNase can be used to reveal binding positions for many different proteins at once, but cannot easily determine the identities of those proteins. Currently, few statistical frameworks jointly model these different data sources to reveal an accurate, holistic view of the in vivo protein-DNA interaction landscape. Here, we develop a novel statistical framework that integrates different sources of experimental information within a thermodynamic model of competitive binding to jointly learn a holistic view of the in vivo protein-DNA interaction landscape. We show that our framework learns an interaction landscape with increased accuracy, explaining multiple sets of data in accordance with thermodynamic principles of competitive DNA binding. The resulting model of genomic occupancy provides a precise mechanistic vantage point from which to explore the role of protein-DNA interactions in transcriptional regulation. The C source code for compete and Python source code for MCMC-based inference are available at http://www.cs.duke.edu/∼amink. amink@cs.duke.edu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. 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.

  20. The physics of protein-DNA interaction networks in the control of gene expression

    Science.gov (United States)

    Saiz, Leonor

    2012-05-01

    Protein-DNA interaction networks play a central role in many fundamental cellular processes. In gene regulation, physical interactions and reactions among the molecular components together with the physical properties of DNA control how genes are turned on and off. A key player in all these processes is the inherent flexibility of DNA, which provides an avenue for long-range interactions between distal DNA elements through DNA looping. Such versatility enables multiple interactions and results in additional complexity that is remarkably difficult to address with traditional approaches. This topical review considers recent advances in statistical physics methods to study the assembly of protein-DNA complexes with loops, their effects in the control of gene expression, and their explicit application to the prototypical lac operon genetic system of the E. coli bacterium. In the last decade, it has been shown that the underlying physical properties of DNA looping can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including the balance between robustness and sensitivity of the induction process. These physical properties are largely dependent on the free energy of DNA looping, which accounts for DNA bending and twisting effects. These new physical methods have also been used in reverse to uncover the actual in vivo free energy of looping double-stranded DNA in living cells, which was not possible with existing experimental techniques. The results obtained for DNA looping by the lac repressor inside the E. coli bacterium showed a more malleable DNA than expected as a result of the interplay of the simultaneous presence of two distinct conformations of looped DNA.

  1. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4.

    Science.gov (United States)

    Wu, Peï-Yu; Frit, Philippe; Meesala, SriLakshmi; Dauvillier, Stéphanie; Modesti, Mauro; Andres, Sara N; Huang, Ying; Sekiguchi, JoAnn; Calsou, Patrick; Salles, Bernard; Junop, Murray S

    2009-06-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  2. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    Energy Technology Data Exchange (ETDEWEB)

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  3. Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

    Science.gov (United States)

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

    2014-11-27

    In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

  4. WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

    Energy Technology Data Exchange (ETDEWEB)

    Rangaraj, K.; Cooper, P.K.; Trego, K.S.

    2009-01-01

    The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of

  5. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  6. 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.

  7. Thermodynamics of the interaction between Alzheimer's disease related tau protein and DNA.

    Directory of Open Access Journals (Sweden)

    Sergio Camero

    Full Text Available Tau hyperphosphorylation can be considered as one of the hallmarks of Alzheimer's disease and other tauophaties. Besides its well-known role as a microtubule associated protein, Tau displays a key function as a protector of genomic integrity in stress situations. Phosphorylation has been proven to regulate multiple processes including nuclear translocation of Tau. In this contribution, we are addressing the physicochemical nature of DNA-Tau interaction including the plausible influence of phosphorylation. By means of surface plasmon resonance (SPR we measured the equilibrium constant and the free energy, enthalpy and entropy changes associated to the Tau-DNA complex formation. Our results show that unphosphorylated Tau binding to DNA is reversible. This fact is in agreement with the protective role attributed to nuclear Tau, which stops binding to DNA once the insult is over. According to our thermodynamic data, oscillations in the concentration of dephosphorylated Tau available to DNA must be the variable determining the extent of Tau binding and DNA protection. In addition, thermodynamics of the interaction suggest that hydrophobicity must represent an important contribution to the stability of the Tau-DNA complex. SPR results together with those from Tau expression in HEK cells show that phosphorylation induces changes in Tau protein which prevent it from binding to DNA. The phosphorylation-dependent regulation of DNA binding is analogous to the Tau-microtubules binding inhibition induced by phosphorylation. Our results suggest that hydrophobicity may control Tau location and DNA interaction and that impairment of this Tau-DNA interaction, due to Tau hyperphosphorylation, could contribute to Alzheimer's pathogenesis.

  8. Atomic Force Microscopy of dynamic protein DNA interactions

    NARCIS (Netherlands)

    Noort, van Simon Johannes Theodorus

    1999-01-01

    In this thesis a dedicated Atomic Force Microscopy (AFM) setup is used for imaging biochemical reactions with molecular resolution. The basis for the high resolution of AFM is the combination of a small probe, close proximity to the sample and a short-range interaction between the probe and the samp

  9. Landscape of π-π and sugar-π contacts in DNA-protein interactions.

    Science.gov (United States)

    Wilson, Katie A; Wells, Rachael A; Abendong, Minette N; Anderson, Colin B; Kung, Ryan W; Wetmore, Stacey D

    2016-01-01

    There were 1765 contacts identified between DNA nucleobases or deoxyribose and cyclic (W, H, F, Y) or acyclic (R, E, D) amino acids in 672 X-ray structures of DNA-protein complexes. In this first study to compare π-interactions between the cyclic and acyclic amino acids, visual inspection was used to categorize amino acid interactions as nucleobase π-π (according to biological edge) or deoxyribose sugar-π (according to sugar edge). Overall, 54% of contacts are nucleobase π-π interactions, which involve all amino acids, but are more common for Y, F, and R, and involve all DNA nucleobases with similar frequencies. Among binding arrangements, cyclic amino acids prefer more planar (stacked) π-systems than the acyclic counterparts. Although sugar-π interactions were only previously identified with the cyclic amino acids and were found to be less common (38%) than nucleobase-cyclic amino acid contacts, sugar-π interactions are more common than nucleobase π-π contacts for the acyclic series (61% of contacts). Similar to DNA-protein π-π interactions, sugar-π contacts most frequently involve Y and R, although all amino acids adopt many binding orientations relative to deoxyribose. These DNA-protein π-interactions stabilize biological systems, by up to approximately -40 kJ mol(-1) for neutral nucleobase or sugar-amino acid interactions, but up to approximately -95 kJ mol(-1) for positively or negatively charged contacts. The high frequency and strength, despite variation in structure and composition, of these π-interactions point to an important function in biological systems.

  10. HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.

    Science.gov (United States)

    Guillen-Ahlers, Hector; Rao, Prahlad K; Levenstein, Mark E; Kennedy-Darling, Julia; Perumalla, Danu S; Jadhav, Avinash Y L; Glenn, Jeremy P; Ludwig-Kubinski, Amy; Drigalenko, Eugene; Montoya, Maria J; Göring, Harald H; Anderson, Corianna D; Scalf, Mark; Gildersleeve, Heidi I S; Cole, Regina; Greene, Alexandra M; Oduro, Akua K; Lazarova, Katarina; Cesnik, Anthony J; Barfknecht, Jared; Cirillo, Lisa A; Gasch, Audrey P; Shortreed, Michael R; Smith, Lloyd M; Olivier, Michael

    2016-06-01

    Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of Saccharomyces cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges.

  11. Study of Cytochrome c-DNA Interaction - Evaluation of Binding Sites on the Redox Protein

    Science.gov (United States)

    Wettstein, Christoph; Kyne, Ciara; M. Doolan, Aishling; Möhwald, Helmuth; Crowley, Peter B.; Lisdat, Fred

    2014-10-01

    Artificial assemblies consisting of the cationic cytochrome c (cyt c) and double-stranded DNA are interesting for the field of biohybrid systems because of the high electro-activity of the incorporated redox protein. However, little is known about the interactions between these two biomolecules. Here, the complex of reduced cyt c and a 41 base pair oligonucleotide was characterized in solution as a function of pH and ionic strength. Persistent cyt c-DNA agglomerates were observed by UV-vis and DLS (dynamic light scattering) at pH 5.0 and low ionic strength. The strength of the interaction was attenuated by raising the pH or the ionic strength. At pH 7.0 agglomerates were not formed, allowing interaction analysis by NMR spectroscopy. Using TROSY (transverse relaxation-optimized spectroscopy)-HSQC (heteronuclear single quantum coherence) experiments it was possible to identify the DNA binding site on the cyt c surface. Numerous residues surrounding the exposed heme edge of cyt c were involved in transient binding to DNA under these conditions. This result was supported by SEC (size exclusion chromatography) experiments at pH 7.0 showing that the interaction is sufficient for co-elution of cyt c and DNA.Artificial assemblies consisting of the cationic cytochrome c (cyt c) and double-stranded DNA are interesting for the field of biohybrid systems because of the high electro-activity of the incorporated redox protein. However, little is known about the interactions between these two biomolecules. Here, the complex of reduced cyt c and a 41 base pair oligonucleotide was characterized in solution as a function of pH and ionic strength. Persistent cyt c-DNA agglomerates were observed by UV-vis and DLS (dynamic light scattering) at pH 5.0 and low ionic strength. The strength of the interaction was attenuated by raising the pH or the ionic strength. At pH 7.0 agglomerates were not formed, allowing interaction analysis by NMR spectroscopy. Using TROSY (transverse relaxation

  12. Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication.

    Science.gov (United States)

    Wang, Guili; Ren, Gaowei; Cui, Xin; Lu, Zhitao; Ma, Yanpin; Qi, Ying; Huang, Yujing; Liu, Zhongyang; Sun, Zhengrong; Ruan, Qiang

    2016-06-01

    The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns.Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer inHCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.

  13. Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication

    Indian Academy of Sciences (India)

    Guili Wang; Gaowei Ren; Xin Cui; Yanpin Ma; Ying Qi; Yujing Huang; Zhongyang Liu; Zhengrong Sun; Qiang Ruan

    2016-06-01

    The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns. Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer in HCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.

  14. Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions

    Science.gov (United States)

    Sprengel, Andreas; Lill, Pascal; Stegemann, Pierre; Bravo-Rodriguez, Kenny; Schöneweiß, Elisa-C.; Merdanovic, Melisa; Gudnason, Daniel; Aznauryan, Mikayel; Gamrad, Lisa; Barcikowski, Stephan; Sanchez-Garcia, Elsa; Birkedal, Victoria; Gatsogiannis, Christos; Ehrmann, Michael; Saccà, Barbara

    2017-02-01

    The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.

  15. Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions.

    Science.gov (United States)

    Sprengel, Andreas; Lill, Pascal; Stegemann, Pierre; Bravo-Rodriguez, Kenny; Schöneweiß, Elisa-C; Merdanovic, Melisa; Gudnason, Daniel; Aznauryan, Mikayel; Gamrad, Lisa; Barcikowski, Stephan; Sanchez-Garcia, Elsa; Birkedal, Victoria; Gatsogiannis, Christos; Ehrmann, Michael; Saccà, Barbara

    2017-02-16

    The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.

  16. De novo DNA methyltransferase DNMT3b interacts with NEDD8-modified proteins.

    Science.gov (United States)

    Shamay, Meir; Greenway, Melanie; Liao, Gangling; Ambinder, Richard F; Hayward, S Diane

    2010-11-19

    DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. We found that DNMT3b associates with NEDD8-modified proteins. Whereas DNMT3b interacts directly in vitro with NEDD8, conjugation of NEDD8 to target proteins enhances this interaction in vivo. DNMT3b immunoprecipitated two major bands of endogenously NEDDylated proteins at the size of NEDDylated cullins, and indeed DNMT3b interacted with CUL1, CUL2, CUL3, CUL4A, and CUL5. Moreover, DNMT3b preferentially immunoprecipitated the NEDDylated form of endogenous CUL4A. NEDD8 enhanced DNMT3b-dependent DNA methylation. Chromatin immunoprecipitation assays suggest that DNMT3b recruits CUL4A and NEDD8 to chromatin, whereas deletion of Dnmt3b reduces the association of CUL4A and NEDD8 at a repressed promoter in a cancer cell line.

  17. Study of cytochrome c-DNA interaction--evaluation of binding sites on the redox protein.

    Science.gov (United States)

    Wettstein, Christoph; Kyne, Ciara; Doolan, Aishling M; Möhwald, Helmuth; Crowley, Peter B; Lisdat, Fred

    2014-11-21

    Artificial assemblies consisting of the cationic cytochrome c (cyt c) and double-stranded DNA are interesting for the field of biohybrid systems because of the high electro-activity of the incorporated redox protein. However, little is known about the interactions between these two biomolecules. Here, the complex of reduced cyt c and a 41 base pair oligonucleotide was characterized in solution as a function of pH and ionic strength. Persistent cyt c-DNA agglomerates were observed by UV-vis and DLS (dynamic light scattering) at pH 5.0 and low ionic strength. The strength of the interaction was attenuated by raising the pH or the ionic strength. At pH 7.0 agglomerates were not formed, allowing interaction analysis by NMR spectroscopy. Using TROSY (transverse relaxation-optimized spectroscopy)-HSQC (heteronuclear single quantum coherence) experiments it was possible to identify the DNA binding site on the cyt c surface. Numerous residues surrounding the exposed heme edge of cyt c were involved in transient binding to DNA under these conditions. This result was supported by SEC (size exclusion chromatography) experiments at pH 7.0 showing that the interaction is sufficient for co-elution of cyt c and DNA.

  18. Real-Time Analysis of Specific Protein-DNA Interactions with Surface Plasmon Resonance

    Directory of Open Access Journals (Sweden)

    Markus Ritzefeld

    2012-01-01

    Full Text Available Several proteins, like transcription factors, bind to certain DNA sequences, thereby regulating biochemical pathways that determine the fate of the corresponding cell. Due to these key positions, it is indispensable to analyze protein-DNA interactions and to identify their mode of action. Surface plasmon resonance is a label-free method that facilitates the elucidation of real-time kinetics of biomolecular interactions. In this article, we focus on this biosensor-based method and provide a detailed guide how SPR can be utilized to study binding of proteins to oligonucleotides. After a description of the physical phenomenon and the instrumental realization including fiber-optic-based SPR and SPR imaging, we will continue with a survey of immobilization methods. Subsequently, we will focus on the optimization of the experiment, expose pitfalls, and introduce how data should be analyzed and published. Finally, we summarize several interesting publications of the last decades dealing with protein-DNA and RNA interaction analysis by SPR.

  19. DNA-protein interaction at erythroid important regulatory elements of MEL cells by in vivo footprinting

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using ligation-mediated PCR method to study the status of DNA-protein interaction at hypersensitive site 2 of locus control Region and β maj promoter of MEL cell line before and after induction, MEL cell has been cultured and induced to differentiation by Hemin and DMSO, then the live cells have been treated with dimethyl sulfate. Ligation mediated PCR has been carried out following the chemical cleavage. The results demonstrate that before and after induction, the status of DNA-protein interaction at both hypersensitive site 2 and β maj promoter change significantly, indicating that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of β -globin gene cluster participate in the regulation of developmental specificity.

  20. Probing and quantifying DNA-protein interactions with asymmetrical flow field-flow fractionation.

    Science.gov (United States)

    Ashby, Jonathan; Schachermeyer, Samantha; Duan, Yaokai; Jimenez, Luis A; Zhong, Wenwan

    2014-09-05

    Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Probing DNA-Protein Interactions on Surfaces Using Spectral Self-interference Fluorescence Microscopy

    Science.gov (United States)

    Dogan, Mehmet; Droge, Peter; Swan, Anna K.; Unlu, Selim; Goldberg, Bennett B.

    2007-03-01

    We are probing the interactions between double-stranded DNA and integration host factor (IHF) proteins [1] on surfaces using Spectral Self-interference Fluorescence Microscopy (SSFM) [2].The probing technique utilizes the spectral fringes produced by interference of direct and reflected emission from fluorescent molecules. The modified spectrum provides a unique signature of the axial position of the fluorophores. Using the SSFM technique, we probe the average location of the fluorescent markers attached to the DNA molecules to study the conformational changes in double-stranded DNA tethered to SiO2 surfaces. In the presence of IHF, a DNA bending protein, we observe reduction in the vertical position of fluorescent molecules suggesting the formation of IHF-DNA complex and IHF-induced DNA bending. We also discuss the results with different IHF strains and different binding conditions. [1] Q. Bao et. al., Gene, Vol.343 pp.99-106 (2004) [2] L.A. Moiseev et. al., Journal of Applied Physics, Vol.96, pp. 5311-5315 (2004)

  2. Interatomic Coulombic Decay Effects in Theoretical DNA Recombination Systems Involving Protein Interaction Sites

    Science.gov (United States)

    Vargas, E. L.; Rivas, D. A.; Duot, A. C.; Hovey, R. T.; Andrianarijaona, V. M.

    2015-03-01

    DNA replication is the basis for all biological reproduction. A strand of DNA will ``unzip'' and bind with a complimentary strand, creating two identical strands. In this study, we are considering how this process is affected by Interatomic Coulombic Decay (ICD), specifically how ICD affects the individual coding proteins' ability to hold together. ICD mainly deals with how the electron returns to its original state after excitation and how this affects its immediate atomic environment, sometimes affecting the connectivity between interaction sites on proteins involved in the DNA coding process. Biological heredity is fundamentally controlled by DNA and its replication therefore it affects every living thing. The small nature of the proteins (within the range of nanometers) makes it a good candidate for research of this scale. Understanding how ICD affects DNA molecules can give us invaluable insight into the human genetic code and the processes behind cell mutations that can lead to cancer. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

  3. Discovering approximate-associated sequence patterns for protein-DNA interactions

    KAUST Repository

    Chan, Tak Ming

    2010-12-30

    Motivation: The bindings between transcription factors (TFs) and transcription factor binding sites (TFBSs) are fundamental protein-DNA interactions in transcriptional regulation. Extensive efforts have been made to better understand the protein-DNA interactions. Recent mining on exact TF-TFBS-associated sequence patterns (rules) has shown great potentials and achieved very promising results. However, exact rules cannot handle variations in real data, resulting in limited informative rules. In this article, we generalize the exact rules to approximate ones for both TFs and TFBSs, which are essential for biological variations. Results: A progressive approach is proposed to address the approximation to alleviate the computational requirements. Firstly, similar TFBSs are grouped from the available TF-TFBS data (TRANSFAC database). Secondly, approximate and highly conserved binding cores are discovered from TF sequences corresponding to each TFBS group. A customized algorithm is developed for the specific objective. We discover the approximate TF-TFBS rules by associating the grouped TFBS consensuses and TF cores. The rules discovered are evaluated by matching (verifying with) the actual protein-DNA binding pairs from Protein Data Bank (PDB) 3D structures. The approximate results exhibit many more verified rules and up to 300% better verification ratios than the exact ones. The customized algorithm achieves over 73% better verification ratios than traditional methods. Approximate rules (64-79%) are shown statistically significant. Detailed variation analysis and conservation verification on NCBI records demonstrate that the approximate rules reveal both the flexible and specific protein-DNA interactions accurately. The approximate TF-TFBS rules discovered show great generalized capability of exploring more informative binding rules. © The Author 2010. Published by Oxford University Press. All rights reserved.

  4. Effective interaction studies for inhibition of DNA ligase protein from Staphylococcus aureus.

    Science.gov (United States)

    Vijayalakshmi, Periyasamy; Daisy, Pitchai

    2015-02-01

    Staphylococcus aureus has been recognized as an important human pathogen for more than 100 years. It is among the most important causative agent of human infections in the twenty-first century. DNA ligase is the main protein responsible for the replication of S. aureus. In order to control the replication mechanism, DNA ligase is a successive drug target, hence we have chosen this protein for this study. We performed virtual screening using ZINC database for identification of potent inhibitor against DNA ligase. Based on the scoring methods, we have selected best five compounds from the ZINC database. In order to improve the accuracy, selected compounds were subjected into Quantum Polarized Ligand Docking (QPLD) docking, for which the results showed high docking score, compared to glide docking score. QPLD is more accurate as it includes charges in the scoring function, which was not available in the glide docking. Binding energy calculation results also indicated that selected compounds have good binding capacity with the target protein. In addition, these compounds on screening have good absorption, distribution, metabolism, excretion and toxicity property. In this study, we identified few compounds that particularly work against DNA ligase protein, having better interaction phenomenon and it would help further the experimental analysis.

  5. DNA fluorescent labeling with naphtho[1,2,3-cd]indol-6(2H)-one for investigation of protein-DNA interactions.

    Science.gov (United States)

    Vasilyeva, Svetlana V; Kuznetsov, Nikita A; Kuznetsova, Anastasya S; Khalyavina, Juliya G; Tropina, Darya A; Lavrikova, Tatyana I; Kargina, Olga I; Gornostaev, Leonid M

    2017-06-01

    Fluorescently labeled DNA to study protein-DNA interactions was synthesized using the Cu(I)-catalysed cycloaddition (CuAAC) reaction. For this purpose, a new azido-containing fluorophore based on the naphtho[1,2,3-cd]indol-6(2H)-one derivative was obtained. The fluorescent properties of naphtho[1,2,3-cd]indol-6(2H)-one derivatives and labeled DNA were studied. The new fluorescent DNA conjugate was shown to be a useful tool to study complex mechanisms of protein-DNA interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Tailored protein encapsulation into a DNA host using geometrically organized supramolecular interactions

    Science.gov (United States)

    Sprengel, Andreas; Lill, Pascal; Stegemann, Pierre; Bravo-Rodriguez, Kenny; Schöneweiß, Elisa-C.; Merdanovic, Melisa; Gudnason, Daniel; Aznauryan, Mikayel; Gamrad, Lisa; Barcikowski, Stephan; Sanchez-Garcia, Elsa; Birkedal, Victoria; Gatsogiannis, Christos; Ehrmann, Michael; Saccà, Barbara

    2017-01-01

    The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA–protein conjugation still limit true emulation of natural host–guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA–protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host–guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging. PMID:28205515

  7. 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.

  8. The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision.

    Science.gov (United States)

    Perreault, Andrea A; Venters, Bryan J

    2016-12-23

    Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

  9. Genome-wide mapping of in vivo protein-DNA interactions.

    Science.gov (United States)

    Johnson, David S; Mortazavi, Ali; Myers, Richard M; Wold, Barbara

    2007-06-08

    In vivo protein-DNA interactions connect each transcription factor with its direct targets to form a gene network scaffold. To map these protein-DNA interactions comprehensively across entire mammalian genomes, we developed a large-scale chromatin immunoprecipitation assay (ChIPSeq) based on direct ultrahigh-throughput DNA sequencing. This sequence census method was then used to map in vivo binding of the neuron-restrictive silencer factor (NRSF; also known as REST, for repressor element-1 silencing transcription factor) to 1946 locations in the human genome. The data display sharp resolution of binding position [+/-50 base pairs (bp)], which facilitated our finding motifs and allowed us to identify noncanonical NRSF-binding motifs. These ChIPSeq data also have high sensitivity and specificity [ROC (receiver operator characteristic) area >/= 0.96] and statistical confidence (P <10(-4)), properties that were important for inferring new candidate interactions. These include key transcription factors in the gene network that regulates pancreatic islet cell development.

  10. Developmental and organ-specific changes in promoter DNA-protein interactions in the tomato rbcS gene family.

    Science.gov (United States)

    Manzara, T; Carrasco, P; Gruissem, W

    1991-12-01

    The five genes encoding ribulose-1,5-bisphosphate carboxylase (rbcS) from tomato are differentially expressed. Transcription of the genes is organ specific and developmentally regulated in fruit and light regulated in cotyledons and leaves. DNase I footprinting assays were used to map multiple sites of DNA-protein interaction in the promoter regions of all five genes and to determine whether the differential transcriptional activity of each gene correlated with developmental or organ-specific changes in DNA-protein interactions. We show organ-specific differences in DNase I protection patterns, suggesting that differential transcription of rbcS genes is controlled at least in part at the level of DNA-protein interactions. In contrast, no changes were detected in the DNase I footprint pattern generated with nuclear extracts from dark-grown cotyledons versus cotyledons exposed to light, implying that light-dependent regulation of rbcS transcription is controlled by protein-protein interactions or modification of DNA binding proteins. During development of tomato fruit, most DNA-protein interactions in the rbcS promoter regions disappear, coincident with the transcriptional inactivation of the rbcS genes. In nuclear extracts from nonphotosynthetic roots and red fruit, the only detectable DNase I protection corresponds to a G-box binding activity. Detection of other DNA binding proteins in extracts from these organs and expression of nonphotosynthetic genes exclude the possibility that roots and red fruit are transcriptionally inactive. The absence of complex promoter protection patterns in these organs suggests either that cooperative interactions between different DNA binding proteins are necessary to form functional transcription complexes or that there is developmental and organ-specific regulation of several rbcS-specific transcription factors in these organs. The DNase I-protected DNA sequences defined in this study are discussed in the context of conserved DNA

  11. Using protein design algorithms to understand the molecular basis of disease caused by protein-DNA interactions: the Pax6 example

    DEFF Research Database (Denmark)

    Alibes, A.; Nadra, A.; De Masi, Federico

    2010-01-01

    Quite often a single or a combination of protein mutations is linked to specific diseases. However, distinguishing from sequence information which mutations have real effects in the protein's function is not trivial. Protein design tools are commonly used to explain mutations that affect protein...... stability, or protein-protein interaction, but not for mutations that could affect protein-DNA binding. Here, we used the protein design algorithm FoldX to model all known missense mutations in the paired box domain of Pax6, a highly conserved transcription factor involved in eye development and in several...... diseases such as aniridia. The validity of FoldX to deal with protein-DNA interactions was demonstrated by showing that high levels of accuracy can be achieved for mutations affecting these interactions. Also we showed that protein-design algorithms can accurately reproduce experimental DNA-binding logos...

  12. In vitro Selection and Interaction Studies of a DNA Aptamer Targeting Protein A.

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    Full Text Available A new DNA aptamer targeting Protein A is presented. The aptamer was selected by use of the FluMag-SELEX procedure. The SELEX technology (Systematic Evolution of Ligands by EXponential enrichment is widely applied as an in vitro selection and amplification method to generate target-specific aptamers and exists in various modified variants. FluMag-SELEX is one of them and is characterized by the use of magnetic beads for target immobilization and fluorescently labeled oligonucleotides for monitoring the aptamer selection progress. Structural investigations and sequence truncation experiments of the selected aptamer for Protein A led to the conclusion, that a stem-loop structure at its 5'-end including the 5'-primer binding site is essential for aptamer-target binding. Extensive interaction analyses between aptamer and Protein A were performed by methods like surface plasmon resonance, MicroScale Thermophoresis and bead-based binding assays using fluorescence measurements. The binding of the aptamer to its target was thus investigated in assays with immobilization of one of the binding partners each, and with both binding partners in solution. Affinity constants were determined in the low micromolar to submicromolar range, increasing to the nanomolar range under the assumption of avidity. Protein A provides more than one binding site for the aptamer, which may overlap with the known binding sites for immunoglobulins. The aptamer binds specifically to both native and recombinant Protein A, but not to other immunoglobulin-binding proteins like Protein G and L. Cross specificity to other proteins was not found. The application of the aptamer is directed to Protein A detection or affinity purification. Moreover, whole cells of Staphylococcus aureus, presenting Protein A on the cell surface, could also be bound by the aptamer.

  13. Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA

    OpenAIRE

    Kapur, Amit; Beck, Jennifer L.; Brown, Susan E.; Dixon, Nicholas E.; Sheil, Margaret M.

    2002-01-01

    Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus–Ter complex by electrospray ionization mass spectrometry (ESI-MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus–Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the io...

  14. Interaction of HMG proteins and H1 with hybrid PNA-DNA junctions.

    Science.gov (United States)

    Totsingan, Filbert; Bell, Anthony J

    2013-11-01

    The objective of this study was to evaluate the effects of inserting peptide nucleic acid (PNA) sequences into the protein-binding surface of an immobilized four-way junction (4WJ). Here we compare the classic immobile DNA junction, J1, with two PNA containing hybrid junctions (4WJ-PNA1 and 4WJ-PNA3 ). The protein interactions of each 4WJ were evaluated using recombinant high mobility group proteins from rat (HMGB1b and HMGB1b/R26A) and human histone H1. In vitro studies show that both HMG and H1 proteins display high binding affinity toward 4WJ's. A 4WJ can access different conformations depending on ionic environment, most simply interpreted by a two-state equilibrium between: (i) an open-x state favored by absence of Mg(2+), low salt, and protein binding, and (ii) a compact stacked-x state favored by Mg(2+). 4WJ-PNA3, like J1, shifts readily from an open to stacked conformation in the presence of Mg(+2), while 4WJ-PNA1 does not. Circular dichroism spectra indicate that HMGB1b recognizes each of the hybrid junctions. H1, however, displays a strong preference for J1 relative to the hybrids. More extensive binding analysis revealed that HMGB1b binds J1 and 4WJ-PNA3 with nearly identical affinity (K(D)s) and 4WJ-PNA1 with two-fold lower affinity. Thus both the sequence/location of the PNA sequence and the protein determine the structural and protein recognition properties of 4WJs.

  15. Inducible DNA - protein interactions of the murine kappa immunoglobulin enhancer in intact cells: comparison with in vitro interactions

    Energy Technology Data Exchange (ETDEWEB)

    Hromas, R.; Pauli, U.; Marcuzzi, A.; Lafrenz, D.; Nick, H.; Stein, J.; Stein, G.; Ness, B.V.

    1988-02-11

    The large intron of the kappa immunoglobulin gene contains a cis-acting enhancer element, which is important in the tissue-specific expressions of the gene. The authors have confirmed the binding activity of a sequence-specific factor present in lymphoid extracts derived from cell lines expressing, or induced to express, the kappa gene. They have extended these studies to show the binding activity is present in normal activated splenic B cells as well as lambda producing cells, and have demonstrated by DNAse footprint analysis full, protection of a sequence containing the 11 bp homology to the SV-40 core enhancer. They have compared these in vitro binding studies with an analysis of protein-DNA interactions in intact murine cell lines using genomic sequencing techniques. They demonstrate significant alterations in DMS reactivity of DNA in the murine 70Z/3 cell line after it is induced to kappa expression. These alterations occur at guanine residues which are part of the 11 bp core sequence, and are identical to those observed in cells constitutively expressing kappa. This provides direct evidence for the induced binding of the tissue specific factor to intact chromatin. In intact chromatin they also observed significant alteration in the reactivity of a guanine, 3' of the core sequence, which is part of a potential secondary DNA structure, and protection of four residues that are part of a region homologous to the heavy chain enhancer.

  16. Inducible DNA-protein interactions of the murine kappa immunoglobulin enhancer in intact cells: comparison with in vitro interactions.

    Science.gov (United States)

    Hromas, R; Pauli, U; Marcuzzi, A; Lafrenz, D; Nick, H; Stein, J; Stein, G; Van Ness, B

    1988-01-01

    The large intron of the kappa immunoglobulin gene contains a cis-acting enhancer element, which is important in the tissue-specific expression of the gene. We have confirmed the binding activity of a sequence-specific factor present in lymphoid extracts derived from cell lines expressing, or induced to express, the kappa gene. We have extended these studies to show the binding activity is present in normal activated splenic B cells as well as lambda producing cells, and have demonstrated by DNAse footprint analysis full protection of a sequence containing the 11 bp homology to the SV-40 core enhancer. We have compared these in vitro binding studies with an analysis of protein-DNA interactions in intact murine cell lines using genomic sequencing techniques. We demonstrate significant alterations in DMS reactivity of DNA in the murine 70Z/3 cell line after it is induced to kappa expression. These alterations occur at guanine residues which are part of the the 11 bp core sequence, and are identical to those observed in cells constitutively expressing kappa. This provides direct evidence for the induced binding of the tissue specific factor to intact chromatin. In intact chromatin we also observed significant alteration in the reactivity of a guanine, 3' of the core sequence, which is part of a potential secondary DNA structure, and protection of four residues that are part of a region homologous to the heavy chain enhancer. Images PMID:2830597

  17. De Novo DNA Methyltransferase DNMT3b Interacts with NEDD8-modified Proteins*

    OpenAIRE

    Shamay, Meir; Greenway, Melanie; Liao, Gangling; AMBINDER, RICHARD F; Hayward, S. Diane

    2010-01-01

    DNA methylation and histone modifications play an important role in transcription regulation. In cancer cells, many promoters become aberrantly methylated through the activity of the de novo DNA methyltransferases DNMT3a and DNMT3b and acquire repressive chromatin marks. NEDD8 is a ubiquitin-like protein modifier that is conjugated to target proteins, such as cullins, to regulate their activity, and cullin 4A (CUL4A) in its NEDD8-modified form is essential for repressive chromatin formation. ...

  18. Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level

    Directory of Open Access Journals (Sweden)

    Gionata Belcastro

    2013-02-01

    Full Text Available The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates, explained in current models by a combination of 3D and 1D diffusion. Here, we present a review of the single-molecule approaches at the forefront of the study of protein-DNA interaction dynamics and target search in vitro and in vivo. Flow stretch, optical and magnetic manipulation, single fluorophore detection and localization as well as combinations of different methods are described and the results obtained with these techniques are discussed in the framework of the current facilitated diffusion model.

  19. Extracting physical chemistry from mechanics: a new approach to investigate DNA interactions with drugs and proteins in single molecule experiments.

    Science.gov (United States)

    Rocha, M S

    2015-09-01

    In this review we focus on the idea of establishing connections between the mechanical properties of DNA-ligand complexes and the physical chemistry of DNA-ligand interactions. This type of connection is interesting because it opens the possibility of performing a robust characterization of such interactions by using only one experimental technique: single molecule stretching. Furthermore, it also opens new possibilities in comparing results obtained by very different approaches, in particular when comparing single molecule techniques to ensemble-averaging techniques. We start the manuscript reviewing important concepts of DNA mechanics, from the basic mechanical properties to the Worm-Like Chain model. Next we review the basic concepts of the physical chemistry of DNA-ligand interactions, revisiting the most important models used to analyze the binding data and discussing their binding isotherms. Then, we discuss the basic features of the single molecule techniques most used to stretch DNA-ligand complexes and to obtain "force × extension" data, from which the mechanical properties of the complexes can be determined. We also discuss the characteristics of the main types of interactions that can occur between DNA and ligands, from covalent binding to simple electrostatic driven interactions. Finally, we present a historical survey of the attempts to connect mechanics to physical chemistry for DNA-ligand systems, emphasizing a recently developed fitting approach useful to connect the persistence length of DNA-ligand complexes to the physicochemical properties of the interaction. Such an approach in principle can be used for any type of ligand, from drugs to proteins, even if multiple binding modes are present.

  20. Generalized theory on the mechanism of site-specific DNA-protein interactions

    Science.gov (United States)

    Niranjani, G.; Murugan, R.

    2016-05-01

    We develop a generalized theoretical framework on the binding of transcription factor proteins (TFs) with specific sites on DNA that takes into account the interplay of various factors regarding overall electrostatic potential at the DNA-protein interface, occurrence of kinetic traps along the DNA sequence, presence of other roadblock protein molecules along DNA and crowded environment, conformational fluctuations in the DNA binding domains (DBDs) of TFs, and the conformational state of the DNA. Starting from a Smolochowski type theoretical framework on site-specific binding of TFs we logically build our model by adding the effects of these factors one by one. Our generalized two-step model suggests that the electrostatic attractive forces present inbetween the positively charged DBDs of TFs and the negatively charged phosphate backbone of DNA, along with the counteracting shielding effects of solvent ions, is the core factor that creates a fluidic type environment at the DNA-protein interface. This in turn facilitates various one-dimensional diffusion (1Dd) processes such as sliding, hopping and intersegmental transfers. These facilitating processes as well as flipping dynamics of conformational states of DBDs of TFs between stationary and mobile states can enhance the 1Dd coefficient on a par with three-dimensional diffusion (3Dd). The random coil conformation of DNA also plays critical roles in enhancing the site-specific association rate. The extent of enhancement over the 3Dd controlled rate seems to be directly proportional to the maximum possible 1Dd length. We show that the overall site-specific binding rate scales with the length of DNA in an asymptotic way. For relaxed DNA, the specific binding rate will be independent of the length of DNA as length increases towards infinity. For condensed DNA as in in vivo conditions, the specific binding rate depends on the length of DNA in a turnover way with a maximum. This maximum rate seems to scale with the

  1. Molecular interactions of UvrB protein and DNA from Helicobacter pylori: Insight into a molecular modeling approach.

    Science.gov (United States)

    Bavi, Rohit; Kumar, Raj; Rampogu, Shailima; Son, Minky; Park, Chanin; Baek, Ayoung; Kim, Hyong-Ha; Suh, Jung-Keun; Park, Seok Ju; Lee, Keun Woo

    2016-08-01

    Helicobacter pylori (H. pylori) persevere in the human stomach, an environment in which they encounter many DNA-damaging conditions, including gastric acidity. The pathogenicity of H. pylori is enhanced by its well-developed DNA repair mechanism, thought of as 'machinery,' such as nucleotide excision repair (NER). NER involves multi-enzymatic excinuclease proteins (UvrABC endonuclease), which repair damaged DNA in a sequential manner. UvrB is the central component in prokaryotic NER, essential for damage recognition. Therefore, molecular modeling studies of UvrB protein from H. pylori are carried out with homology modeling and molecular dynamics (MD) simulations. The results reveal that the predicted structure is bound to a DNA hairpin with 3-bp stem, an 11-nucleotide loop, and 3-nt 3' overhang. In addition, a mutation of the Y96A variant indicates reduction in the binding affinity for DNA. Free-energy calculations demonstrate the stability of the complex and help identify key residues in various interactions based on residue decomposition analysis. Stability comparative studies between wild type and mutant protein-DNA complexes indicate that the former is relatively more stable than the mutant form. This predicted model could also be useful in designing new inhibitors for UvrB protein, as well as preventing the pathogenesis of H. pylori.

  2. The Inner Membrane Protein PilG Interacts with DNA and the Secretin PilQ in Transformation.

    Science.gov (United States)

    Frye, Stephan A; Lång, Emma; Beyene, Getachew Tesfaye; Balasingham, Seetha V; Homberset, Håvard; Rowe, Alexander D; Ambur, Ole Herman; Tønjum, Tone

    2015-01-01

    Expression of type IV pili (Tfp), filamentous appendages emanating from the bacterial surface, is indispensable for efficient neisserial transformation. Tfp pass through the secretin pore consisting of the membrane protein PilQ. PilG is a polytopic membrane protein, conserved in Gram-positive and Gram-negative bacteria, that is required for the biogenesis of neisserial Tfp. PilG null mutants are devoid of pili and non-competent for transformation. Here, recombinant full-length, truncated and mutated variants of meningococcal PilG were overexpressed, purified and characterized. We report that meningococcal PilG directly binds DNA in vitro, detected by both an electromobility shift analysis and a solid phase overlay assay. PilG DNA binding activity was independent of the presence of the consensus DNA uptake sequence. PilG-mediated DNA binding affinity was mapped to the N-terminus and was inactivated by mutation of residues 43 to 45. Notably, reduced meningococcal transformation of DNA in vivo was observed when PilG residues 43 to 45 were substituted by alanine in situ, defining a biologically significant DNA binding domain. N-terminal PilG also interacted with the N-terminal region of PilQ, which previously was shown to bind DNA. Collectively, these data suggest that PilG and PilQ in concert bind DNA during Tfp-mediated transformation.

  3. The Inner Membrane Protein PilG Interacts with DNA and the Secretin PilQ in Transformation.

    Directory of Open Access Journals (Sweden)

    Stephan A Frye

    Full Text Available Expression of type IV pili (Tfp, filamentous appendages emanating from the bacterial surface, is indispensable for efficient neisserial transformation. Tfp pass through the secretin pore consisting of the membrane protein PilQ. PilG is a polytopic membrane protein, conserved in Gram-positive and Gram-negative bacteria, that is required for the biogenesis of neisserial Tfp. PilG null mutants are devoid of pili and non-competent for transformation. Here, recombinant full-length, truncated and mutated variants of meningococcal PilG were overexpressed, purified and characterized. We report that meningococcal PilG directly binds DNA in vitro, detected by both an electromobility shift analysis and a solid phase overlay assay. PilG DNA binding activity was independent of the presence of the consensus DNA uptake sequence. PilG-mediated DNA binding affinity was mapped to the N-terminus and was inactivated by mutation of residues 43 to 45. Notably, reduced meningococcal transformation of DNA in vivo was observed when PilG residues 43 to 45 were substituted by alanine in situ, defining a biologically significant DNA binding domain. N-terminal PilG also interacted with the N-terminal region of PilQ, which previously was shown to bind DNA. Collectively, these data suggest that PilG and PilQ in concert bind DNA during Tfp-mediated transformation.

  4. 14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

    DEFF Research Database (Denmark)

    Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil

    2012-01-01

    Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins...... are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites...... are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding...

  5. Specific interactions between lactose repressor protein and DNA affected by ligand binding: ab initio molecular orbital calculations.

    Science.gov (United States)

    Ohyama, Tatsuya; Hayakawa, Masato; Nishikawa, Shin; Kurita, Noriyuki

    2011-06-01

    Transcription mechanisms of gene information from DNA to mRNA are essentially controlled by regulatory proteins such as a lactose repressor (LacR) protein and ligand molecules. Biochemical experiments elucidated that a ligand binding to LacR drastically changes the mechanism controlled by LacR, although the effect of ligand binding has not been clarified at atomic and electronic levels. We here investigated the effect of ligand binding on the specific interactions between LacR and operator DNA by the molecular simulations combined with classical molecular mechanics and ab initio fragment molecular orbital methods. The results indicate that the binding of anti-inducer ligand strengthens the interaction between LacR and DNA, which is consistent with the fact that the binding of anti-inducer enhances the repression of gene transcription by LacR. It was also elucidated that hydrating water molecules existing between LacR and DNA contribute to the specific interactions between LacR and DNA. Copyright © 2011 Wiley Periodicals, Inc.

  6. Specific interaction of terminase, the DNA packaging enzyme of bacteriophage lambda, with the portal protein of the prohead.

    Science.gov (United States)

    Yeo, A; Feiss, M

    1995-01-13

    Terminase, the bacteriophage lambda DNA packaging protein, is a heteromultimer of two subunits, gpNu1 and gpA, the products of genes Nu1 and A, resp. Phage 21 is a lambdoid phage that produces a terminase similar to that of lambda terminase, the subunits of 21 terminase, gp1 and gp2, have the same domain structures of their lambda analog, gpNu1 and gpA, respectively. The lambda and 21 terminases have different DNA binding and prohead binding specificities. When the C-terminal 32 amino residues of gpA replace the C-terminal 32 residues of gp2, the resulting chimeric terminase specifically uses lambda proheads, indicating that the C-terminal 32 residues of gpA are a specificity domain for prohead binding. A second chimeric terminase, in which the C-terminal six residues of gpA are replaced by the C-terminal six residues of gp2, is unable to utilize lambda proheads, and a lambda phage producing this terminase, lambda Are636, is unable to form plaques. In the present work, a pseudorevertant of lambda Are636 was isolated that contained a mutation Bms8, affecting the prohead. The B gene encodes the portal protein of lambda proheads, which forms the special vertex that is thought to serve as (1) the site of DNA entry into the prohead during packaging, (2) the site for DNA exit during DNA injection, and (3) the site of tail attachment during virion assembly. Bms8 is predicted to change residue 331 of gpB from proline to serine. Burst size measurements and in vitro DNA packaging experiments demonstrated allele-specific interactions between the Are636 terminase and Bms8 proheads. That is, wild-type terminase interacted more efficiently with wild-type proheads than with Bms8 proheads, and Are636 terminase interacted with Bms8 proheads more efficiently than with wild-type proheads. Prohead binding by lambda terminase is stimulated by an assembly catalyst, gpFI. In vitro packaging extracts lacking gpFI were used under conditions in which packaging was gpFI-independent. In the

  7. Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing.

    Science.gov (United States)

    Barfeld, Stefan J; Mills, Ian G

    2016-01-01

    Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.

  8. An interaction between teh DNA repair factor XPA and replication protein A appears essential for nucleotide excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lei; Lu, Xiaoyan; Peterson, C.A.; Legerski, R.J. [Univ. of Texas, Houston, TX (United States)

    1995-10-01

    Replication protein A (RPA) is required for simian virus 40-directed DNA replication in vitro and for nucleotide excision repair (NER). Here we report that RPA and the human repair protein XPA specifically interact both in vitro and in vivo. Mapping of the RPA-interactive domains in XPA revealed that both of the largest subunits of RPA, RPA-70 and RPA-34, interact with XPA at distinct sites. A domain involved in mediating the interaction with RPA-70 was located between XPA residues 153 and 176. Deletion of highly conserved motifs within this region identified two mutants that were deficient in binding RPA in vitro and highly defective in NER both in vitro and in vivo. The second domain mediating the interaction with RPA-34 was identified within the first 58 residues in XPA. Deletion of this region, however, only moderately affects the complementing activity of XPA in vivo. Finally, the XPA-RPA complex is shown to have a greater affinity for damaged DNA than XPA alone. Taken together, these results indicate that the interaction between XPA and RPA is required for NER but that only the interaction with RPA-70 is essential. 52 refs., 7 figs.

  9. Topology of RNA-protein nucleobase-amino acid π-π interactions and comparison to analogous DNA-protein π-π contacts.

    Science.gov (United States)

    Wilson, Katie A; Holland, Devany J; Wetmore, Stacey D

    2016-05-01

    The present work analyzed 120 high-resolution X-ray crystal structures and identified 335 RNA-protein π-interactions (154 nonredundant) between a nucleobase and aromatic (W, H, F, or Y) or acyclic (R, E, or D) π-containing amino acid. Each contact was critically analyzed (including using a visual inspection protocol) to determine the most prevalent composition, structure, and strength of π-interactions at RNA-protein interfaces. These contacts most commonly involve F and U, with U:F interactions comprising one-fifth of the total number of contacts found. Furthermore, the RNA and protein π-systems adopt many different relative orientations, although there is a preference for more parallel (stacked) arrangements. Due to the variation in structure, the strength of the intermolecular forces between the RNA and protein components (as determined from accurate quantum chemical calculations) exhibits a significant range, with most of the contacts providing significant stability to the associated RNA-protein complex (up to -65 kJ mol(-1)). Comparison to the analogous DNA-protein π-interactions emphasizes differences in RNA- and DNA-protein π-interactions at the molecular level, including the greater abundance of RNA contacts and the involvement of different nucleobase/amino acid residues. Overall, our results provide a clearer picture of the molecular basis of nucleic acid-protein binding and underscore the important role of these contacts in biology, including the significant contribution of π-π interactions to the stability of nucleic acid-protein complexes. Nevertheless, more work is still needed in this area in order to further appreciate the properties and roles of RNA nucleobase-amino acid π-interactions in nature.

  10. Functional interaction of CCAAT/enhancer-binding-protein-α basic region mutants with E2F transcription factors and DNA.

    Science.gov (United States)

    Kowenz-Leutz, Elisabeth; Schuetz, Anja; Liu, Qingbin; Knoblich, Maria; Heinemann, Udo; Leutz, Achim

    2016-07-01

    The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα.

  11. Role of the LEXE Motif of Protein-primed DNA Polymerases in the Interaction with the Incoming Nucleotide*

    Science.gov (United States)

    Santos, Eugenia; Lázaro, José M.; Pérez-Arnaiz, Patricia; Salas, Margarita; de Vega, Miguel

    2014-01-01

    The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding φ29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg2+ ions, a reaction highly improved when Mn2+ was used as metal activator. These results, together with previous crystallographic resolution of φ29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg2+. Recovery of both reactions with Mn2+ supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP. PMID:24324256

  12. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers....... The biophysical and structural investigations of PPIs consequently demand hybrid approaches, implementing orthogonal methods and strategies for global data analysis. Currently, impressive developments in hardware and software within several methodologies define a new era for the biostructural community. Data can...

  13. Engineered holliday junctions as single-molecule reporters for protein-DNA interactions with application to a MerR-family regulator.

    Science.gov (United States)

    Sarkar, Susanta K; Andoy, Nesha May; Benítez, Jaime J; Chen, Peng R; Kong, Jason S; He, Chuan; Chen, Peng

    2007-10-17

    Protein-DNA interactions are essential for gene maintenance, replication, and expression. Characterizing how proteins interact with and change the structure of DNA is crucial in elucidating the mechanisms of protein function. Here, we present a novel and generalizable method of using engineered DNA Holliday junctions (HJs) that contain specific protein-recognition sequences to report protein-DNA interactions in single-molecule FRET measurements, utilizing the intrinsic structural dynamics of HJs. Because the effects of protein binding are converted to the changes in the structure and dynamics of HJs, protein-DNA interactions that involve small structural changes of DNA can be studied. We apply this method to investigate how the MerR-family regulator PbrR691 interacts with DNA for transcriptional regulation. Both apo- and holo-PbrR691 bind the stacked conformers of the engineered HJ, change their structures, constrain their conformational distributions, alter the kinetics, and shift the equilibrium of their structural dynamics. The information obtained maps the potential energy surfaces of HJ before and after PbrR691 binding and reveals the protein actions that force DNA structural changes for transcriptional regulation. The ability of PbrR691 to bind both HJ conformers and still allow HJ structural dynamics also informs about its conformational flexibility that may have significance for its regulatory function. This method of using engineered HJs offers quantification of the changes both in structure and in dynamics of DNA upon protein binding and thus provides a new tool to elucidate the correlation of structure, dynamics, and function of DNA-binding proteins.

  14. Interaction of a rhizobial DNA-binding protein with the promoter region of a plant leghemoglobin gene

    Energy Technology Data Exchange (ETDEWEB)

    Welters, P.; Metz, B.; Felix, G.; Palme, K. (Max Planck Insitut fur Zuchtungsforschung, Koeln (Germany)); Szczyglowski, K. (Michigan State Univ., East Lansing, MI (United States)); Bruijn, F.J. de (Max Planck Institut fur Zuchtungsforschung, Koeln (Germany) Michigan State Univ., East Lansing, MI (United States))

    1993-08-01

    A nucleotide sequence was identified approximately 650 bp upstream of the Sesbania rostrata leghemoglobin gene Srglb3 start codon, which interacts specifically with a proteinaceous DNA-binding factor found in nodule extracts but not in extracts from leaves or root. The binding site for this factor was delimited using footprinting techniques. The DNA-binding activity of this factor was found to be heat stable, dependent on divalent cations, and derived from the (infecting) Azorhizobium caulinodans bacteria or bacteroids (A. caulinodans bacterial binding factor 1, AcBBF1). A 9- to 10-kD protein was isolated from a free-living culture of A. caulinodans that co-purifies with the DNA-binding activity (A. caulinodans bacterial binding protein 1, AcBBP1) and interacts specifically with its target (S. rostrata bacterial binding site 1, SrBBS1). The amino acid sequence of the N-terminal 27 residues of AcBBP1 was determined and was found to share significant similarity (46% identity; 68% similarity) with a domain of the herpes simplex virus major DNA-binding protein infected cell protein 8(ICP8). An insertion mutation in the SrBBS1 was found to result in a substantial reduction of the expression of a Srglb3-gus reporter gene fusion in nodules of transgenic Lotus corniculatus plants, suggesting a role for this element in Srglb3 promoter activity. Based on these results, the authors propose that (a) bacterial transacting factor(s) may play a role in infected cell-specific expression of the symbiotically induced plant lb genes. 70 refs., 11 figs.

  15. A feature-based approach to modeling protein-DNA interactions.

    Directory of Open Access Journals (Sweden)

    Eilon Sharon

    Full Text Available Transcription factor (TF binding to its DNA target site is a fundamental regulatory interaction. The most common model used to represent TF binding specificities is a position specific scoring matrix (PSSM, which assumes independence between binding positions. However, in many cases, this simplifying assumption does not hold. Here, we present feature motif models (FMMs, a novel probabilistic method for modeling TF-DNA interactions, based on log-linear models. Our approach uses sequence features to represent TF binding specificities, where each feature may span multiple positions. We develop the mathematical formulation of our model and devise an algorithm for learning its structural features from binding site data. We also developed a discriminative motif finder, which discovers de novo FMMs that are enriched in target sets of sequences compared to background sets. We evaluate our approach on synthetic data and on the widely used TF chromatin immunoprecipitation (ChIP dataset of Harbison et al. We then apply our algorithm to high-throughput TF ChIP data from mouse and human, reveal sequence features that are present in the binding specificities of mouse and human TFs, and show that FMMs explain TF binding significantly better than PSSMs. Our FMM learning and motif finder software are available at http://genie.weizmann.ac.il/.

  16. Identification of the first small-molecule inhibitor of the REV7 DNA repair protein interaction.

    Science.gov (United States)

    Actis, Marcelo L; Ambaye, Nigus D; Evison, Benjamin J; Shao, Youming; Vanarotti, Murugendra; Inoue, Akira; McDonald, Ezelle T; Kikuchi, Sotaro; Heath, Richard; Hara, Kodai; Hashimoto, Hiroshi; Fujii, Naoaki

    2016-09-15

    DNA interstrand crosslink (ICL) repair (ICLR) has been implicated in the resistance of cancer cells to ICL-inducing chemotherapeutic agents. Despite the clinical significance of ICL-inducing chemotherapy, few studies have focused on developing small-molecule inhibitors for ICLR. The mammalian DNA polymerase ζ, which comprises the catalytic subunit REV3L and the non-catalytic subunit REV7, is essential for ICLR. To identify small-molecule compounds that are mechanistically capable of inhibiting ICLR by targeting REV7, high-throughput screening and structure-activity relationship (SAR) analysis were performed. Compound 1 was identified as an inhibitor of the interaction of REV7 with the REV7-binding sequence of REV3L. Compound 7 (an optimized analog of compound 1) bound directly to REV7 in nuclear magnetic resonance analyses, and inhibited the reactivation of a reporter plasmid containing an ICL in between the promoter and reporter regions. The normalized clonogenic survival of HeLa cells treated with cisplatin and compound 7 was lower than that for cells treated with cisplatin only. These findings indicate that a small-molecule inhibitor of the REV7/REV3L interaction can chemosensitize cells by inhibiting ICLR.

  17. JAB1 regulates unphosphorylated STAT3 DNA-binding activity through protein–protein interaction in human colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Nishimoto, Arata, E-mail: anishimo@yamaguchi-u.ac.jp [Department of Surgery and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505 (Japan); Kugimiya, Naruji; Hosoyama, Toru; Enoki, Tadahiko [Department of Surgery and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505 (Japan); Li, Tao-Sheng [Department of Stem Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Hamano, Kimikazu [Department of Surgery and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505 (Japan)

    2013-08-30

    Highlights: •JAB1 interacted with unphosphorylated STAT3 in the nucleus. •JAB1 knockdown tended to increase nuclear STAT3 expression. •JAB1 knockdown significantly decreased unphosphorylated STAT3 DNA-binding activity. •JAB1 knockdown significantly decreased MDR1, NANOG, and VEGF expressions. •Nuclear JAB1, but not nuclear STAT3, correlated with STAT3 DNA-binding activity. -- Abstract: Recent studies have revealed that unphosphorylated STAT3 forms a dimer, translocates to the nucleus, binds to the STAT3 binding site, and activates the transcription of STAT3 target genes, thereby playing an important role in oncogenesis in addition to phosphorylated STAT3. Among signaling steps of unphosphorylated STAT3, nuclear translocation and target DNA-binding are the critical steps for its activation. Therefore, elucidating the regulatory mechanism of these signaling steps of unphosphorylated STAT3 is a potential step in the discovery of a novel cancer drug. However, the mechanism of unphosphorylated STAT3 binding to the promoter of target genes remains unclear. In this study, we focused on Jun activation domain-binding protein 1 (JAB1) as a candidate protein that regulates unphosphorylated STAT3 DNA-binding activity. Initially, we observed that both unphosphorylated STAT3 and JAB1 existed in the nucleus of human colon cancer cell line COLO205 at the basal state (no cytokine stimulation). On the other hand, phosphorylated STAT3 did not exist in the nucleus of COLO205 cells at the basal state. Immunoprecipitation using nuclear extract of COLO205 cells revealed that JAB1 interacted with unphosphorylated STAT3. To investigate the effect of JAB1 on unphosphorylated STAT3 activity, RNAi studies were performed. Although JAB1 knockdown tended to increase nuclear STAT3 expression, it significantly decreased unphosphorylated STAT3 DNA-binding activity. Subsequently, JAB1 knockdown significantly decreased the expression levels of MDR1, NANOG, and VEGF, which are STAT3 target

  18. Human ribosomal protein S3 interacts with DNA base excision repair proteins hAPE/Ref-1 and hOGG1.

    Science.gov (United States)

    Hegde, Vijay; Wang, Mu; Deutsch, Walter A

    2004-11-09

    The human ribosomal protein S3 (hS3) possesses associated activities that suggest alternative roles beyond its participation in protein translation. For example, it is capable of cleaving apurinic/apyrimidinic (AP) DNA via a beta-elimination reaction, an activity that is missing in partially purified extracts of xeroderma pigmentosum group-D fibroblasts. In a recent study, we showed by surface plasmon resonance (SPR) that hS3 also has a very high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) and AP sites in DNA. Using the same SPR technology, it is shown here that hS3 positively interacts with the human base excision repair (BER) enzymes N-glycosylase/AP lyase OGG1 and APE/Ref-1. Using a DNA substrate that allows for the detection of 8-oxoG repair, we also show that hOGG1 N-glycosylase activity becomes increasingly more robust in the presence of hS3. Human S3 was found to co-immunoprecipitate with both hOGG1 and APE/Ref-1, indicating that these proteins physically interact with one another. These results raise the possibility that hS3 not only functions as a ribosomal protein but, in addition, may influence repair activities at sites of DNA damage.

  19. Deformed epidermal autoregulatory factor-1 (DEAF1 interacts with the Ku70 subunit of the DNA-dependent protein kinase complex.

    Directory of Open Access Journals (Sweden)

    Philip J Jensik

    Full Text Available Deformed Epidermal Autoregulatory Factor 1 (DEAF1 is a transcription factor linked to suicide, cancer, autoimmune disorders and neural tube defects. To better understand the role of DEAF1 in protein interaction networks, a GST-DEAF1 fusion protein was used to isolate interacting proteins in mammalian cell lysates, and the XRCC6 (Ku70 and the XRCC5 (Ku80 subunits of DNA dependent protein kinase (DNA-PK complex were identified by mass spectrometry, and the DNA-PK catalytic subunit was identified by immunoblotting. Interaction of DEAF1 with Ku70 and Ku80 was confirmed to occur within cells by co-immunoprecipitation of epitope-tagged proteins, and was mediated through interaction with the Ku70 subunit. Using in vitro GST-pulldowns, interaction between DEAF1 and the Ku70 subunit was mapped to the DEAF1 DNA binding domain and the C-terminal Bax-binding region of Ku70. In transfected cells, DEAF1 and Ku70 colocalized to the nucleus, but Ku70 could not relocalize a mutant cytoplasmic form of DEAF1 to the nucleus. Using an in vitro kinase assay, DEAF1 was phosphorylated by DNA-PK in a DNA-independent manner. Electrophoretic mobility shift assays showed that DEAF1 or Ku70/Ku80 did not interfere with the DNA binding of each other, but DNA containing DEAF1 binding sites inhibited the DEAF1-Ku70 interaction. The data demonstrates that DEAF1 can interact with the DNA-PK complex through interactions of its DNA binding domain with the carboxy-terminal region of Ku70 that contains the Bax binding domain, and that DEAF1 is a potential substrate for DNA-PK.

  20. A novel microfluidic mixer based on dual-hydrodynamic focusing for interrogating the kinetics of DNA-protein interaction.

    Science.gov (United States)

    Li, Ying; Xu, Fei; Liu, Chao; Xu, Youzhi; Feng, Xiaojun; Liu, Bi-Feng

    2013-08-21

    Kinetic measurement of biomacromolecular interaction plays a significant role in revealing the underlying mechanisms of cellular activities. Due to the small diffusion coefficient of biomacromolecules, it is difficult to resolve the rapid kinetic process with traditional analytical methods such as stopped-flow or laminar mixers. Here, we demonstrated a unique continuous-flow laminar mixer based on microfluidic dual-hydrodynamic focusing to characterize the kinetics of DNA-protein interactions. The time window of this mixer for kinetics observation could cover from sub-milliseconds to seconds, which made it possible to capture the folding process with a wide dynamic range. Moreover, the sample consumption was remarkably reduced to <0.55 μL min⁻¹, over 1000-fold saving in comparison to those reported previously. We further interrogated the interaction kinetics of G-quadruplex and the single-stranded DNA binding protein, indicating that this novel micromixer would be a useful approach for analyzing the interaction kinetics of biomacromolecules.

  1. BayesPI - a new model to study protein-DNA interactions: a case study of condition-specific protein binding parameters for Yeast transcription factors

    Directory of Open Access Journals (Sweden)

    Morigen

    2009-10-01

    Full Text Available Abstract Background We have incorporated Bayesian model regularization with biophysical modeling of protein-DNA interactions, and of genome-wide nucleosome positioning to study protein-DNA interactions, using a high-throughput dataset. The newly developed method (BayesPI includes the estimation of a transcription factor (TF binding energy matrices, the computation of binding affinity of a TF target site and the corresponding chemical potential. Results The method was successfully tested on synthetic ChIP-chip datasets, real yeast ChIP-chip experiments. Subsequently, it was used to estimate condition-specific and species-specific protein-DNA interaction for several yeast TFs. Conclusion The results revealed that the modification of the protein binding parameters and the variation of the individual nucleotide affinity in either recognition or flanking sequences occurred under different stresses and in different species. The findings suggest that such modifications may be adaptive and play roles in the formation of the environment-specific binding patterns of yeast TFs and in the divergence of TF binding sites across the related yeast species.

  2. Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

    KAUST Repository

    Ghosh, Sharmistha

    2010-04-06

    The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Interaction of the Chlamydia trachomatis histone H1-like protein (Hc1) with DNA and RNA causes repression of transcription and translation in vitro

    DEFF Research Database (Denmark)

    Pedersen, Lotte Bang; Birkelund, S; Christiansen, G

    1994-01-01

    The 18 kDa histone H1-like protein from Chlamydia trachomatis (Hc1) is a DNA-binding protein thought to be involved in condensation of the chlamydial chromosome during late stages in the chlamydial life cycle. Expression of Hc1 in Escherichia coli results in an overall relaxation of DNA and sever......The 18 kDa histone H1-like protein from Chlamydia trachomatis (Hc1) is a DNA-binding protein thought to be involved in condensation of the chlamydial chromosome during late stages in the chlamydial life cycle. Expression of Hc1 in Escherichia coli results in an overall relaxation of DNA...... and severely affects DNA, RNA and protein synthesis. We have analysed the interaction of Hc1 with single-stranded DNA and RNA by Southwestern and Northwestern blotting. Furthermore, we show that purified, recombinant Hc1 dramatically affects transcription and translation in vitro at physiologically relevant...

  4. The Role of Cdkn1A-Interacting Zinc Finger Protein 1 (CIZ1 in DNA Replication and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Qiang Liu

    2016-02-01

    Full Text Available Cdkn1A-interacting zinc finger protein 1 (CIZ1 was first identified in a yeast-2-hybrid system searching for interacting proteins of CDK2 inhibitor p21Cip1/Waf1. Ciz1 also binds to CDK2, cyclin A, cyclin E, CDC6, PCNA, TCF4 and estrogen receptor-α. Recent studies reveal numerous biological functions of CIZ1 in DNA replication, cell proliferation, and differentiation. In addition, splicing variants of CIZ1 mRNA is associated with a variety of cancers and Alzheimer’s disease, and mutations of the CIZ1 gene lead to cervical dystonia. CIZ1 expression is increased in cancers and rheumatoid arthritis. In this review, we will summarize the biological functions and molecular mechanisms of CIZ1 in these physiological and pathological processes.

  5. Interaction of the retinoblastoma protein with Orc1 and its recruitment to human origins of DNA replication.

    Directory of Open Access Journals (Sweden)

    Ramiro Mendoza-Maldonado

    Full Text Available BACKGROUND: The retinoblastoma protein (Rb is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1 specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. CONCLUSIONS/SIGNIFICANCE: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication.

  6. Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    Yan-Ping Huang; Xue-Song Gao; Dong Ji; Shu-Mei Lin; Yan-Wei Zhong; Qing Shao; Shu-Lin Zhang; Jun Cheng; Lin Wang; Jiang Guo; Yan Liu; Yuan Yang; Li-Ying Zhang; Gui-Qin Bai

    2005-01-01

    AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes.METHODS: We constructed p7 protein bait plasmid by doning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (α type) containing liver cDNA library plasmid, pACT2 in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics.RESULTS: Fifty colonies were selected and sequenced.Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens.CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.

  7. A DNA-Centric Protein Interaction Map of Ultraconserved Elements Reveals Contribution of Transcription Factor Binding Hubs to Conservation

    Directory of Open Access Journals (Sweden)

    Tar Viturawong

    2013-10-01

    Full Text Available Ultraconserved elements (UCEs have been the subject of great interest because of their extreme sequence identity and their seemingly cryptic and largely uncharacterized functions. Although in vivo studies of UCE sequences have demonstrated regulatory activity, protein interactors at UCEs have not been systematically identified. Here, we combined high-throughput affinity purification, high-resolution mass spectrometry, and SILAC quantification to map intrinsic protein interactions for 193 UCE sequences. The interactome contains over 400 proteins, including transcription factors with known developmental roles. We demonstrate based on our data that UCEs consist of strongly conserved overlapping binding sites. We also generated a fine-resolution interactome of a UCE, confirming the hub-like nature of the element. The intrinsic interactions mapped here are reflected in open chromatin, as indicated by comparison with existing ChIP data. Our study argues for a strong contribution of protein-DNA interactions to UCE conservation and provides a basis for further functional characterization of UCEs.

  8. A High-Throughput Screening Strategy to Identify Protein-Protein Interaction Inhibitors That Block the Fanconi Anemia DNA Repair Pathway.

    Science.gov (United States)

    Voter, Andrew F; Manthei, Kelly A; Keck, James L

    2016-07-01

    Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol.

  9. Interaction of the Chlamydia trachomatis histone H1-like protein (Hc1) with DNA and RNA causes repression of transcription and translation in vitro

    DEFF Research Database (Denmark)

    Pedersen, Lotte Bang; Birkelund, S; Christiansen, G

    1994-01-01

    and severely affects DNA, RNA and protein synthesis. We have analysed the interaction of Hc1 with single-stranded DNA and RNA by Southwestern and Northwestern blotting. Furthermore, we show that purified, recombinant Hc1 dramatically affects transcription and translation in vitro at physiologically relevant...

  10. Replication protein A: single-stranded DNA's first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair.

    Science.gov (United States)

    Chen, Ran; Wold, Marc S

    2014-12-01

    Replication protein A (RPA), the major single-stranded DNA-binding protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. © 2014 WILEY Periodicals, Inc.

  11. Better estimation of protein-DNA interaction parameters improve prediction of functional sites

    Directory of Open Access Journals (Sweden)

    O'Flanagan Ruadhan A

    2008-12-01

    Full Text Available Abstract Background Characterizing transcription factor binding motifs is a common bioinformatics task. For transcription factors with variable binding sites, we need to get many suboptimal binding sites in our training dataset to get accurate estimates of free energy penalties for deviating from the consensus DNA sequence. One procedure to do that involves a modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment method designed to produce many such sequences. Results We analyzed low stringency SELEX data for E. coli Catabolic Activator Protein (CAP, and we show here that appropriate quantitative analysis improves our ability to predict in vitro affinity. To obtain large number of sequences required for this analysis we used a SELEX SAGE protocol developed by Roulet et al. The sequences obtained from here were subjected to bioinformatic analysis. The resulting bioinformatic model characterizes the sequence specificity of the protein more accurately than those sequence specificities predicted from previous analysis just by using a few known binding sites available in the literature. The consequences of this increase in accuracy for prediction of in vivo binding sites (and especially functional ones in the E. coli genome are also discussed. We measured the dissociation constants of several putative CAP binding sites by EMSA (Electrophoretic Mobility Shift Assay and compared the affinities to the bioinformatics scores provided by methods like the weight matrix method and QPMEME (Quadratic Programming Method of Energy Matrix Estimation trained on known binding sites as well as on the new sites from SELEX SAGE data. We also checked predicted genome sites for conservation in the related species S. typhimurium. We found that bioinformatics scores based on SELEX SAGE data does better in terms of prediction of physical binding energies as well as in detecting functional sites. Conclusion We think that training binding site detection

  12. Synthesis, molecular structure, theoretical calculation, DNA/protein interaction and cytotoxic activity of manganese(III) complex with 8-hydroxyquinoline.

    Science.gov (United States)

    Thamilarasan, V; Sengottuvelan, N; Sudha, A; Srinivasan, P; Siva, A

    2015-01-01

    Manganese(III) complex (1) [Mn(8-hq)3] (where 8-hq=8-hydroxyquinoline) has been synthesized and characterized by elemental, spectral (UV-vis, FT-IR) and thermal analysis. The structure of complex (1) has been determined by single crystal X-ray diffraction studies and the configuration around manganese(III) ion was elongated octahedral coordination geometry. Density functional theory calculations were performed for ligand and its complex. Binding studies of ligand and complex 1 with calf thymus DNA (CT-DNA) was investigated by absorption, fluorescence, circular dichroic (CD) spectroscopy and viscosity measurements. Absorption spectral studies revealed that ligand and complex 1 binds to DNA groove and its intrinsic binding strength has been found to be 2.57×10(4) and 2.91×10(4)M(-1). A molecular docking study confirm that the complex 1 is a minor groove binder and was stabilized through hydrogen bonding interactions. Complex 1 exhibits a good binding propensity to bovine serum albumin (BSA) protein. The in vitro cytotoxicity study of complex 1 on breast cancer cell line (MCF-7) indicate that it has the potential to act as effective anticancer drug, with IC50 values of 3.25μM. The ligand and its complex have been screened for antimicrobial activities and the complex showed better antimicrobial activity than the free ligand.

  13. Integrated Method to Attach DNA Handles and Functionally Select Proteins to Study Folding and Protein-Ligand Interactions with Optical Tweezers.

    Science.gov (United States)

    Hao, Yuxin; Canavan, Clare; Taylor, Susan S; Maillard, Rodrigo A

    2017-09-07

    Optical tweezers has emerged as a powerful tool to study folding, ligand binding, and motor enzymes. The manipulation of proteins with optical tweezers requires attaching molecular handles to the protein of interest. Here, we describe a novel method that integrates the covalent attachment of DNA handles to target proteins with a selection step for functional and properly folded molecules. In addition, this method enables obtaining protein molecules in different liganded states and can be used with handles of different lengths. We apply this method to study the cAMP binding domain A (CBD-A) of Protein kinase A. We find that the functional selection step drastically improves the reproducibility and homogeneity of the single molecule data. In contrast, without a functional selection step, proteins often display misfolded conformations. cAMP binding stabilizes the CBD-A against a denaturing force, and increases the folded state lifetime. Data obtained with handles of 370 and 70 base pairs are indistinguishable, but at low forces short handles provide a higher spatial resolution. Altogether, this method is flexible, selects for properly folded molecules in different liganded states, and can be readily applicable to study protein folding or protein-ligand interactions with force spectroscopy that require molecular handles.

  14. The range of interaction between DNA-bending proteins is controlled by the second-longest correlation length for bending fluctuations

    Science.gov (United States)

    2013-01-01

    When a DNA molecule is stretched, the zero-force correlation length for its bending fluctuations – the persistence length A – bifurcates into two different correlation lengths - the shorter “longitudinal” correlation length ξ‖(f) and the longer “transverse” correlation length ξ⊥(f). In the high-force limit, ξ‖(f)=ξ⊥(f)/2=kBTA/f/2. When DNA-bending proteins bind to the DNA molecule, there is an effective interaction between the protein-generated bends mediated by DNA elasticity and bending fluctuations. Surprisingly, the range of this interaction is not the longest correlation length associated with transverse fluctuations of the tangent vector along the polymer, but instead is the second longest longitudinal correlation length ξ‖ (f, μ). The effect arises from the protein-bend contribution to the Hamiltonian having an axial rotational symmetry which eliminates its coupling to the transverse fluctuations. PMID:23368394

  15. Identification of the replication origins from Cyanothece ATCC 51142 and their interactions with the DnaA protein: from in silico to in vitro studies

    Directory of Open Access Journals (Sweden)

    He eHuang

    2015-12-01

    Full Text Available Based on the complete genome of Cyanothece ATCC 51142, the oriCs of both the circular and linear chromosomes in Cyanothece ATCC 51142 have been predicted by utilizing a web-based system Ori-Finder. Here, we provide the experimental supports for the results of Ori-Finder to identify the origins of replication of Cyanothece ATCC 51142 and their interactions with initiator protein DnaA . The two replication origins are both composed of three characteristically arranged DnaA boxes, and an AT-rich stretch, and the oriC in circular chromosome is followed by the dnaN gene. The dnaA gene is located downstream of the origin of circular chromosome and expresses a typical DnaA protein that follows the division into four domains (I, II, III, IV, as with other members of the DnaA protein family. Here, we report the purification of DnaA (IV and characterize the interaction of the purified protein with the replication origins, in order to offer experimental supports for the prediction. Combined with experimental validation, we identified the oriCs of Cyanothece ATCC 51142. The results of EMSA and DNase I footprint assay demonstrate that the C-terminal domain of Cyanothece ATCC 51142 DnaA protein specifically binds the oriCs of both the circular and linear chromosomes, and DNase I footprint assay demonstrates that DnaA (IV has a hypersensitive affinity interaction with DnaA boxes in both oriCs. The results also confirm the results predicted by Ori-Finder.

  16. Regulation of the nucleosome repeat length in vivo by the DNA sequence, protein concentrations and long-range interactions.

    Directory of Open Access Journals (Sweden)

    Daria A Beshnova

    2014-07-01

    Full Text Available The nucleosome repeat length (NRL is an integral chromatin property important for its biological functions. Recent experiments revealed several conflicting trends of the NRL dependence on the concentrations of histones and other architectural chromatin proteins, both in vitro and in vivo, but a systematic theoretical description of NRL as a function of DNA sequence and epigenetic determinants is currently lacking. To address this problem, we have performed an integrative biophysical and bioinformatics analysis in species ranging from yeast to frog to mouse where NRL was studied as a function of various parameters. We show that in simple eukaryotes such as yeast, a lower limit for the NRL value exists, determined by internucleosome interactions and remodeler action. For higher eukaryotes, also the upper limit exists since NRL is an increasing but saturating function of the linker histone concentration. Counterintuitively, smaller H1 variants or non-histone architectural proteins can initiate larger effects on the NRL due to entropic reasons. Furthermore, we demonstrate that different regimes of the NRL dependence on histone concentrations exist depending on whether DNA sequence-specific effects dominate over boundary effects or vice versa. We consider several classes of genomic regions with apparently different regimes of the NRL variation. As one extreme, our analysis reveals that the period of oscillations of the nucleosome density around bound RNA polymerase coincides with the period of oscillations of positioning sites of the corresponding DNA sequence. At another extreme, we show that although mouse major satellite repeats intrinsically encode well-defined nucleosome preferences, they have no unique nucleosome arrangement and can undergo a switch between two distinct types of nucleosome positioning.

  17. Identification of interacting partners of Human Mpv17-like protein with a mitigating effect of mitochondrial dysfunction through mtDNA damage.

    Science.gov (United States)

    Iida, Reiko; Ueki, Misuzu; Yasuda, Toshihiro

    2015-10-01

    Human Mpv17-like protein (M-LPH) has been suggested to participate in mitochondrial function. In this study, we investigated the proteins that interact with M-LPH, and identified four: H2A histone family, member X (H2AX), ribosomal protein S14 (RPS14), ribosomal protein S3 (RPS3) and B-cell receptor-associated protein 31 (Bap31). Immunofluorescence and subcellular fractionation studies revealed that M-LPH is localized predominantly in the nucleus, to some extent in a subset of mitochondria, and marginally in the cytosol. Mitochondrial M-LPH appeared as punctate foci, and these were co-localized with a subset of mitochondrial transcription factor A (TFAM) and mtDNA, indicating that M-LPH is localized in or in close proximity to mitochondrial nucleoids. RNAi-mediated knockdown of M-LPH resulted in an increase of mtDNA damage and reduced the expression of mtDNA-encoded genes. A ROS inducer, antimycin A, caused an increase in both the number and size of the mitochondrial M-LPH foci, and these foci were co-localized with two enzymes, DNA polymerase γ (POLG) and DNA ligase III (LIG3), both involved in mtDNA repair. Furthermore, knockdown of M-LPH hampered mitochondrial localization of these enzymes. Taken together, these observations suggest that M-LPH is involved in the maintenance of mtDNA and protects cells from mitochondrial dysfunction.

  18. The role of the mycobacterial DNA-binding protein 1 (MDP1 from Mycobacterium bovis BCG in host cell interaction

    Directory of Open Access Journals (Sweden)

    Kunisch Ralph

    2012-08-01

    Full Text Available Abstract Background Mycobacterium tuberculosis differs from most pathogens in its ability to multiply inside monocytes and to persist during long periods of time within granuloma in a status of latency. A class of proteins called mycobacterial histone-like proteins has been associated with regulation of replication and latency, but their precise role in the infection process has yet to be uncovered. Our study aimed at defining the impact of the histone-like protein MDP1 from M. bovis BCG (mycobacterial DNA-binding protein 1, corresponding to Rv2986c from M. tuberculosis on early steps of infection. Results Previously, a BCG (Bacillus Calmette Guérin strain had been generated by antisense-technique exhibiting reduced MDP1 expression. This strain was now used to analyse the impact of reduced amount of MDP1 on the interaction with human blood monocytes, macrophage lines and PBMC (peripheral blood mononuclear cells. MDP1 was revealed to be required for growth at acidic pH and for intracellular replication in human blood monocytes. Down-regulation of MDP1 resulted in reduced secretion of the cytokine IL-1β by infected human PBMC. In addition, a reduction of MDP1 expression had a major impact on the formation of fused multi-nucleated macrophages. In monocyte preparations from human blood as well as in human and mouse macrophage cell lines, both the percentage of multi-nucleated cells and the number of nuclei per cell were much enhanced when the monocytes were infected with BCG expressing less MDP1. Conclusion MDP1 from M. bovis BCG affects the growth at acidic pH and the intracellular replication in human monocytes. It furthermore affects cytokine secretion by host cells, and the formation of fused multi-nucleated macrophages. Our results suggest an important role of MDP1 in persistent infection.

  19. Extracting physical chemistry from mechanics: a new approach to investigate DNA interactions with drugs and proteins in single molecule experiments

    CERN Document Server

    Rocha, M S

    2015-01-01

    In this review we focus on the idea of establishing connections between the mechanical properties of DNAligand complexes and the physical chemistry of DNA-ligand interactions. This type of connection is interesting because it opens the possibility of performing a robust characterization of such interactions by using only one experimental technique: single molecule stretching. Furthermore, it also opens new possibilities in comparing results obtained by very different approaches, in special when comparing single molecule techniques to ensemble-averaging techniques. We start the manuscript reviewing important concepts of the DNA mechanics, from the basic mechanical properties to the Worm-Like Chain model. Next we review the basic concepts of the physical chemistry of DNA-ligand interactions, revisiting the most important models used to analyze the binding data and discussing their binding isotherms. Then, we discuss the basic features of the single molecule techniques most used to stretch the DNA-ligand complex...

  20. Interaction between the Chlamydia trachomatis histone H1-like protein (Hc1) and DNA

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Pedersen, LB; Koehler, JF;

    1993-01-01

    The gene encoding the Chlamydia trachomatis histone H1-like protein (Hc1) from serovar L2 was cloned into Escherichia coli by use of expression vector pET11d. In this vector, transcription of the gene is under the control of a bacteriophage T7 promoter, and T7 RNA polymerase is inducible in the h......The gene encoding the Chlamydia trachomatis histone H1-like protein (Hc1) from serovar L2 was cloned into Escherichia coli by use of expression vector pET11d. In this vector, transcription of the gene is under the control of a bacteriophage T7 promoter, and T7 RNA polymerase is inducible...

  1. Homeodomain-interacting Protein Kinase-2 (HIPK2) Phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and Modulates Its DNA Binding Affinity

    OpenAIRE

    Zhang, Qingchun; Wang, Yinsheng

    2007-01-01

    The chromosomal high-mobility group A (HMGA) proteins, comprising of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation and phosphorylation. Recently, it was found that the homeodomain-interacting pro...

  2. The repeat domain of the type III effector protein PthA shows a TPR-like structure and undergoes conformational changes upon DNA interaction.

    Science.gov (United States)

    Murakami, Mário Tyago; Sforça, Mauricio Luis; Neves, Jorge Luiz; Paiva, Joice Helena; Domingues, Mariane Noronha; Pereira, André Luiz Araujo; Zeri, Ana Carolina de Mattos; Benedetti, Celso Eduardo

    2010-12-01

    Many plant pathogenic bacteria rely on effector proteins to suppress defense and manipulate host cell mechanisms to cause disease. The effector protein PthA modulates the host transcriptome to promote citrus canker. PthA possesses unusual protein architecture with an internal region encompassing variable numbers of near-identical tandem repeats of 34 amino acids termed the repeat domain. This domain mediates protein-protein and protein-DNA interactions, and two polymorphic residues in each repeat unit determine DNA specificity. To gain insights into how the repeat domain promotes protein-protein and protein-DNA contacts, we have solved the structure of a peptide corresponding to 1.5 units of the PthA repeat domain by nuclear magnetic resonance (NMR) and carried out small-angle X-ray scattering (SAXS) and spectroscopic studies on the entire 15.5-repeat domain of PthA2 (RD2). Consistent with secondary structure predictions and circular dichroism data, the NMR structure of the 1.5-repeat peptide reveals three α-helices connected by two turns that fold into a tetratricopeptide repeat (TPR)-like domain. The NMR structure corroborates the theoretical TPR superhelix predicted for RD2, which is also in agreement with the elongated shape of RD2 determined by SAXS. Furthermore, RD2 undergoes conformational changes in a pH-dependent manner and upon DNA interaction, and shows sequence similarities to pentatricopeptide repeat (PPR), a nucleic acid-binding motif structurally related to TPR. The results point to a model in which the RD2 structure changes its compactness as it embraces the DNA with the polymorphic diresidues facing the interior of the superhelix oriented toward the nucleotide bases.

  3. Screening for Novel Binding Proteins Interacting with Human Papillomavirus Type 18 E6 Oncogene in the Hela cDNA Library by Yeast Two-Hybrid System

    Institute of Scientific and Technical Information of China (English)

    Shuang LI; Ping LIU; Ling XI; Xuefeng JIANG; Jianfeng ZHOU; Shixuan WANG; Li MENG; Yunping LU; Ding Ma

    2008-01-01

    To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH109 was transformed with pGBKT7-HPV18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPVl8 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate im- muno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transeriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

  4. Time-Resolved Fluorescence Anisotropy Study of the Interaction Between DNA and a Peptide Truncated from the p53 Protein Core Domain.

    Science.gov (United States)

    Liu, Chengxuan; Liang, Gaiting; Liu, Zhen; Zu, Lily

    2014-03-01

    Time-resolved fluorescence anisotropy spectroscopy was applied to study the interaction between a peptide truncated from the binding site of tumor suppressor p53 protein and the DNAs covalently labeled with 6-carboxyfluorescein (FAM) dye. Fluorescence intensity quenching and changes of anisotropy decay lifetime were monitored when FAM labeled DNA formed complex with the peptide. The results demonstrated that the sequence of DNA could not define the binding specificity between the peptide and DNA. But the anisotropy decay of FAM can be used to examine the binding affinity of the peptide to DNA. The fluorescent dynamics of FAM can also be used to represent the rigidity of the complex formed between the peptide and DNA.

  5. PIC: Protein Interactions Calculator.

    Science.gov (United States)

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside.

  6. A novel type of DNA-binding protein interacts with a conserved sequence in an early nodulin ENOD12 promoter

    DEFF Research Database (Denmark)

    Christiansen, H; Hansen, A C; Vijn, I

    1996-01-01

    in the nodule parenchyma and meristem. The presence of three small overlapping ORFs in the 5'-untranslated region of the ENBP1 cDNA indicates that ENBP1 expression might be regulated at the translational level. The interaction of ENBP1 with a conserved AT-rich element within the ENOD12 promoter and the presence...

  7. Terminal protection of small molecule-linked ssDNA-SWNT nanoassembly for sensitive detection of small molecule and protein interaction

    Institute of Scientific and Technical Information of China (English)

    Yu Wang; Dian-Ming Zhou; Zhan Wu; Li-Juan Tang; Jian-Hui Jiang

    2013-01-01

    The interactions between small molecules and proteins constitute a critical regulatory mechanism in many fundamental biological processes.A novel biosensing strategy has been developed for sensitive and selective detection of small molecule and protein interaction on the basis of terminal protection of small molecule-linked ssDNA-SWNT nanoassembly.The developed strategy is demonstrated using folate and its binding protein folate receptor (FR) as a model case.The results reveal the developed technique displays superb resistance to non-specific binding,very low detection limit as low as subnanomolar,and a wide dynamic range from 100 pmol/L to 500 nmol/L of FR.Thus,it may offer a simple,cost-effective,highly selective and sensitive platform for homogeneous fluorescence detection of small molecule-protein interaction and related biochemical studies.

  8. Significance of the pathogenic mutation T372R in the Yin Yang 1 protein interaction with DNA--thermodynamic studies.

    Science.gov (United States)

    Nieborak, Anna; Górecki, Andrzej

    2016-03-01

    This work focuses on the pathogenic missense mutation in YY1 protein correlated with insulinomas. Based on in vitro studies, we demonstrate that the mutation does not affect the secondary structure of either zinc fingers or the N-terminal fragment (NTF) of the protein. Apart from a slight increase in the protein's compactness, no changes in the tertiary structure were observed. The introduced mutation significantly alters DNA-binding properties, both the affinity and enthalpy-entropy contribution of the process, which are highly dependent on the recognized sequence. Obtained results indicate concerted rather than a modular mode of sequence recognition by YY1 with the significant impact of a disordered NTF.

  9. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    of research are explored. Here we present an overview of the most widely used protein-protein interaction databases and the methods they employ to gather, combine, and predict interactions. We also point out the trade-off between comprehensiveness and accuracy and the main pitfall scientists have to be aware......Years of meticulous curation of scientific literature and increasingly reliable computational predictions have resulted in creation of vast databases of protein interaction data. Over the years, these repositories have become a basic framework in which experiments are analyzed and new directions...

  10. Observation of Transcription Regulation in the Mouse Heart Nuclear DNA Fragments and the Specific-protein Interaction by AFM

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Using atom force microscopy (AFM), in vitro transcription, PAGE and other experimental technologies, it is observed that, in active genes of mice (Balb/c) nuclear DNA fragments of non-transcriptional state, only regulation sequences at both ends are associated with scaffold proteins (indissociable proteins) and some transcriptional factors such as complexes (dissociable proteins) made of gene-coding proteins and specific auxiliary small molecules, while there are no combining proteins in intermediate coding sequences. However, in active genes of transcriptional state, both regulation sequences and intermediate coding sequences are associated with active transcriptional factors by non-covalent bonds.This paper shows the prospective application of AFM observation and in vitro transcription in the research on gene expression and regulation. It also offers some theoretical basis for localization of specific genes in human genomes.

  11. Protein Synthesis--An Interactive Game.

    Science.gov (United States)

    Clements, Lee Ann J.; Jackson, Karen E.

    1998-01-01

    Describes an interactive game designed to help students see and understand the dynamic relationship between DNA, RNA, and proteins. Appropriate for either a class or laboratory setting, following a lecture session about protein synthesis. (DDR)

  12. PprA Protein Is Involved in Chromosome Segregation via Its Physical and Functional Interaction with DNA Gyrase in Irradiated Deinococcus radiodurans Bacteria.

    Science.gov (United States)

    Devigne, Alice; Guérin, Philippe; Lisboa, Johnny; Quevillon-Cheruel, Sophie; Armengaud, Jean; Sommer, Suzanne; Bouthier de la Tour, Claire; Servant, Pascale

    2016-01-01

    PprA, a radiation-induced Deinococcus-specific protein, was previously shown to be required for cell survival and accurate chromosome segregation after exposure to ionizing radiation. Here, we used an in vivo approach to determine, by shotgun proteomics, putative PprA partners coimmunoprecipitating with PprA when cells were exposed to gamma rays. Among them, we found the two subunits of DNA gyrase and, thus, chose to focus our work on characterizing the activities of the deinococcal DNA gyrase in the presence or absence of PprA. Loss of PprA rendered cells hypersensitive to novobiocin, an inhibitor of the B subunit of DNA gyrase. We showed that treatment of bacteria with novobiocin resulted in induction of the radiation desiccation response (RDR) regulon and in defects in chromosome segregation that were aggravated by the absence of PprA. In vitro, the deinococcal DNA gyrase, like other bacterial DNA gyrases, possesses DNA negative supercoiling and decatenation activities. These two activities are inhibited in vitro by novobiocin and nalidixic acid, whereas PprA specifically stimulates the decatenation activity of DNA gyrase. Together, these results suggest that PprA plays a major role in chromosome decatenation via its interaction with the deinococcal DNA gyrase when D. radiodurans cells are recovering from exposure to ionizing radiation. IMPORTANCE D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation that are

  13. Polyomavirus interaction with the DNA damage response

    Institute of Scientific and Technical Information of China (English)

    Joshua; L.Justice; Brandy; Verhalen; Mengxi; Jiang

    2015-01-01

    Viruses are obligate intracellular parasites that subvert cellular metabolism and pathways to mediate their own replication—normally at the expense of the host cell. Polyomaviruses are a group of small DNA viruses, which have long been studied as a model for eukaryotic DNA replication. Polyomaviruses manipulate host replication proteins, as well as proteins involved in DNA maintenance and repair, to serve as essential cofactors for productive infection. Moreover, evidence suggests that polyomavirus infection poses a unique genotoxic threat to the host cell. In response to any source of DNA damage, cells must initiate an effective DNA damage response(DDR) to maintain genomic integrity, wherein two protein kinases, ataxia telangiectasia mutated(ATM) and ATM- and Rad3-related(ATR), are major regulators of DNA damage recognition and repair. Recent investigation suggests that these essential DDR proteins are required for productive polyomavirus infection. This review will focus on polyomaviruses and their interaction with ATMand ATR-mediated DNA damage responses and the effect of this interaction on host genomic stability.

  14. Theory on the mechanism of site-specific DNA-protein interactions in the presence of traps

    Science.gov (United States)

    Niranjani, G.; Murugan, R.

    2016-08-01

    The speed of site-specific binding of transcription factor (TFs) proteins with genomic DNA seems to be strongly retarded by the randomly occurring sequence traps. Traps are those DNA sequences sharing significant similarity with the original specific binding sites (SBSs). It is an intriguing question how the naturally occurring TFs and their SBSs are designed to manage the retarding effects of such randomly occurring traps. We develop a simple random walk model on the site-specific binding of TFs with genomic DNA in the presence of sequence traps. Our dynamical model predicts that (a) the retarding effects of traps will be minimum when the traps are arranged around the SBS such that there is a negative correlation between the binding strength of TFs with traps and the distance of traps from the SBS and (b) the retarding effects of sequence traps can be appeased by the condensed conformational state of DNA. Our computational analysis results on the distribution of sequence traps around the putative binding sites of various TFs in mouse and human genome clearly agree well the theoretical predictions. We propose that the distribution of traps can be used as an additional metric to efficiently identify the SBSs of TFs on genomic DNA.

  15. The Polycomb group protein MEDEA and the DNA methyltransferase MET1 interact to repress autonomous endosperm development in Arabidopsis.

    Science.gov (United States)

    Schmidt, Anja; Wöhrmann, Heike J P; Raissig, Michael T; Arand, Julia; Gheyselinck, Jacqueline; Gagliardini, Valeria; Heichinger, Christian; Walter, Joern; Grossniklaus, Ueli

    2013-03-01

    In flowering plants, double fertilization of the female gametes, the egg and the central cell, initiates seed development to give rise to a diploid embryo and the triploid endosperm. In the absence of fertilization, the FERTILIZATION-INDEPENDENT SEED Polycomb Repressive Complex 2 (FIS-PRC2) represses this developmental process by histone methylation of certain target genes. The FERTILIZATION-INDEPENDENT SEED (FIS) class genes MEDEA (MEA) and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) encode two of the core components of this complex. In addition, DNA methylation establishes and maintains the repression of gene activity, for instance via DNA METHYLTRANSFERASE1 (MET1), which maintains methylation of symmetric CpG residues. Here, we demonstrate that Arabidopsis MET1 interacts with MEA in vitro and in a yeast two-hybrid assay, similar to the previously identified interaction of the mammalian homologues DNMT1 and EZH2. MET1 and MEA share overlapping expression patterns in reproductive tissues before and after fertilization, a prerequisite for an interaction in vivo. Importantly, a much higher percentage of central cells initiate endosperm development in the absence of fertilization in mea-1/MEA; met1-3/MET1 as compared to mea-1/MEA mutant plants. In addition, DNA methylation at the PHERES1 and MEA loci, imprinted target genes of the FIS-PRC2, was affected in the mea-1 mutant compared with wild-type embryos. In conclusion, our data suggest a mechanistic link between two major epigenetic pathways involved in histone and DNA methylation in plants by physical interaction of MET1 with the FIS-PRC2 core component MEA. This concerted action is relevant for the repression of seed development in the absence of fertilization. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  16. Bound or free: interaction of the C-terminal domain of Escherichia coli single-stranded DNA-binding protein (SSB) with the tetrameric core of SSB.

    Science.gov (United States)

    Su, Xun-Cheng; Wang, Yao; Yagi, Hiromasa; Shishmarev, Dmitry; Mason, Claire E; Smith, Paul J; Vandevenne, Marylène; Dixon, Nicholas E; Otting, Gottfried

    2014-04-01

    Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

  17. Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Son, Ora [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Sunghan [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Shin, Yun-jeong [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Woo-Young [College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Koh, Hee-Jong, E-mail: heejkoh@snu.ac.kr [Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Cheon, Choong-Ill, E-mail: ccheon@sookmyung.ac.kr [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2015-09-18

    The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex. - Highlights: • Nucleosome assembly protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1. • Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription.

  18. 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.

  19. Further Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and its Interaction with ICP8, the Major DNA-Binding Protein of Herpes Simplex Virus

    Science.gov (United States)

    1994-01-01

    Diseases Produced by Herpes Simplex Viruses Disease Gingivostomatitis Pha ryngotonsill itis Herpes labialis Genital herpes Primary (P) or Recurrent... herpes simplex labialis : experimental induction of lesions with UV light. J. Clin. Micro. 22:366-368. Stanberry, L.R. 1986. Herpesvirus latency and...UL37 Protein of Herpes Simplex Virus Type 1 and its Interaction with [CPS, the Major DNA~Binding Protein of Herpes Simplex Virus" beyond brief

  20. Analysis of the DNA-binding profile and function of TALE homeoproteins reveals their specialization and specific interactions with Hox genes/proteins.

    Science.gov (United States)

    Penkov, Dmitry; Mateos San Martín, Daniel; Fernandez-Díaz, Luis C; Rosselló, Catalina A; Torroja, Carlos; Sánchez-Cabo, Fátima; Warnatz, H J; Sultan, Marc; Yaspo, Marie L; Gabrieli, Arianna; Tkachuk, Vsevolod; Brendolan, Andrea; Blasi, Francesco; Torres, Miguel

    2013-04-25

    The interactions of Meis, Prep, and Pbx1 TALE homeoproteins with Hox proteins are essential for development and disease. Although Meis and Prep behave similarly in vitro, their in vivo activities remain largely unexplored. We show that Prep and Meis interact with largely independent sets of genomic sites and select different DNA-binding sequences, Prep associating mostly with promoters and housekeeping genes and Meis with promoter-remote regions and developmental genes. Hox target sequences associate strongly with Meis but not with Prep binding sites, while Pbx1 cooperates with both Prep and Meis. Accordingly, Meis1 shows strong genetic interaction with Pbx1 but not with Prep1. Meis1 and Prep1 nonetheless coregulate a subset of genes, predominantly through opposing effects. Notably, the TALE homeoprotein binding profile subdivides Hox clusters into two domains differentially regulated by Meis1 and Prep1. During evolution, Meis and Prep thus specialized their interactions but maintained significant regulatory coordination.

  1. Allostery through protein-induced DNA bubbles.

    Science.gov (United States)

    Traverso, Joseph J; Manoranjan, Valipuram S; Bishop, A R; Rasmussen, Kim Ø; Voulgarakis, Nikolaos K

    2015-01-01

    Allostery through DNA is increasingly recognized as an important modulator of DNA functions. Here, we show that the coalescence of protein-induced DNA bubbles can mediate allosteric interactions that drive protein aggregation. We propose that such allostery may regulate DNA's flexibility and the assembly of the transcription machinery. Mitochondrial transcription factor A (TFAM), a dual-function protein involved in mitochondrial DNA (mtDNA) packaging and transcription initiation, is an ideal candidate to test such a hypothesis owing to its ability to locally unwind the double helix. Numerical simulations demonstrate that the coalescence of TFAM-induced bubbles can explain experimentally observed TFAM oligomerization. The resulting melted DNA segment, approximately 10 base pairs long, around the joints of the oligomers act as flexible hinges, which explains the efficiency of TFAM in compacting DNA. Since mitochondrial polymerase (mitoRNAP) is involved in melting the transcription bubble, TFAM may use the same allosteric interaction to both recruit mitoRNAP and initiate transcription.

  2. Allostery through protein-induced DNA bubbles

    Science.gov (United States)

    Traverso, Joseph J.; Manoranjan, Valipuram S.; Bishop, A. R.; Rasmussen, Kim Ø.; Voulgarakis, Nikolaos K.

    2015-03-01

    Allostery through DNA is increasingly recognized as an important modulator of DNA functions. Here, we show that the coalescence of protein-induced DNA bubbles can mediate allosteric interactions that drive protein aggregation. We propose that such allostery may regulate DNA's flexibility and the assembly of the transcription machinery. Mitochondrial transcription factor A (TFAM), a dual-function protein involved in mitochondrial DNA (mtDNA) packaging and transcription initiation, is an ideal candidate to test such a hypothesis owing to its ability to locally unwind the double helix. Numerical simulations demonstrate that the coalescence of TFAM-induced bubbles can explain experimentally observed TFAM oligomerization. The resulting melted DNA segment, approximately 10 base pairs long, around the joints of the oligomers act as flexible hinges, which explains the efficiency of TFAM in compacting DNA. Since mitochondrial polymerase (mitoRNAP) is involved in melting the transcription bubble, TFAM may use the same allosteric interaction to both recruit mitoRNAP and initiate transcription.

  3. Efficient expression and purification of human replication fork-stabilizing factor, Claspin, from mammalian cells: DNA-binding activity and novel protein interactions.

    Science.gov (United States)

    Uno, Syuzi; Masai, Hisao

    2011-08-01

    Purification of recombinant proteins of a large size often poses problems of instability or low expression in bacterial or insect cells. Here, we established a method for a high-level expression of large-sized recombinant proteins in mammalian cells and subsequent purification of the full-length proteins. We applied this method to express human Claspin and Tim-Tipin complex, which play important roles in replication checkpoint responses as fork-stabilizing factors, and successfully purified them in functional forms in amount sufficient for enzymatic characterization. Purified Claspin behaves as a monomer and binds preferentially to fork-like DNA. Over-expression of tagged Claspin in mammalian cells facilitated the detection of its interacting factors. Claspin interacts with many factors involved in checkpoint regulation and replication fork machinery, including ATR, ATM, Chk1, Tim, MCM4, MCM10, Cdc45, DNA polymerases α, δ, ε and Cdc7 kinase. We will discuss the potential implication of these findings in architecture of replication fork. We will also discuss the advantage of this system for purification and characterization of those proteins that are large and have been difficult to deal with.

  4. A comparative study of cytotoxicity and interaction with DNA/protein of five transition metal complexes with Schiff base ligands.

    Science.gov (United States)

    Niu, Meiju; Hong, Min; Chang, Guoliang; Li, Xiao; Li, Zhen

    2015-07-01

    Five transition metal complexes NiL(1)2 (1), CuL(1)2 (2), ZnL(1)2 (3), [MnL(1)2(N3)]n·nCH2Cl2 (4), CuL(2)2 (5) {HL(1)=3-{[2-(2-hydroxy-ethoxy)-ethylimino]-methyl}-naphthalen-2-ol, HL(2)=2-{[2-(2-hydroxy-ethoxy)-ethylimino]-methyl}-phenol} have been synthesized and fully characterized. In all of the complexes, the ligands coordinated to the metal ion in a negative fashion via O and N donor atoms. The X-ray structures of nickel complex 1 and copper complexes 2 and 5 are four-coordinated monomers and show slightly distorted square-planar geometry in the vicinity of the central metal atom. Zinc complex 3 exhibits a four-coordinated tetrahedral structure. Differently, manganese complex 4 reveals a six-coordinated octahedral structure, one-dimensional chain is linked by azide in the end-to-end mode. In vitro cytotoxicity of these complexes to various tumor cell lines was assayed by the MTT method. The results showed that most of these metal-Schiff base complexes exhibited enhanced cytotoxicity than Schiff base ligands, which clearly implied a positive synergistic effect. Moreover, these complexes appeared to be selectively active against certain cell lines. The interactions of these metal complexes with CT-DNA were investigated by UV-vis, fluorescence and CD spectroscopy, the results indicated that these complexes are metallointercalators and can interact with CT-DNA. The study of interaction between complexes and BSA indicated that all of the complexes could quench the intrinsic fluorescence of BSA in a static quenching process.

  5. Theoretical studies on interactions between low energy electrons and protein-DNA fragments: valence anions of AT-amino acids side chain complexes.

    Science.gov (United States)

    Szyperska, Anna; Gajewicz, Agnieszka; Mazurkiewicz, Kamil; Leszczynski, Jerzy; Rak, Janusz

    2011-11-21

    Electron attachment to trimeric complexes that mimic most frequent hydrogen bonding interactions between an amino acid side chain (AASC) and the Watson-Crick (WC) 9-methyladenine-1-methylthymine (MAMT) base pair has been studied at the B3LYP/6-31++G(d,p) level of theory. Although the neutral trimers will not occur in the gas phase due to unfavorable free energy of stabilization (G(stab)) they should form a protein-DNA complex where entropy changes related to formation of such a complex will more than balance its disadvantageous G(stab). The most stable neutrals possess an identical pattern of hydrogen bonds (HBs). In addition, the proton-acceptor (N7) and proton-donor (N10) atoms of adenine involved in those HBs are located in the main groove of DNA. All neutral structures support the adiabatically stable valence anions in which the excess electron is localized on a π* orbital of thymine. The vertical detachment energies (VDEs) of anions corresponding to the most stable neutrals are substantially smaller than that of the isolated WC MAMT base pair. Hence, electron transfer from the anionic thymine to the phosphate group and as a consequence formation of a single strand break (SSB) should proceed more efficiently in a protein-dsDNA complex than in the naked dsDNA as far as electron attachment to thymine is concerned. This journal is © the Owner Societies 2011

  6. Stacking interactions and DNA intercalation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Dr. Shen [Fred Hutchinson Cancer Research Center; Cooper, Valentino R [ORNL; Thonhauser, Prof. Timo [Wake Forest University, Winston-Salem, NC; Lundqvist, Prof. Bengt I. [Chalmers University of Technology, Sweden; Langreth, David C. [Rutgers University

    2009-01-01

    The relationship between stacking interactions and the intercalation of proflavine and ellipticine within DNA is investigated using a nonempirical van der Waals density functional for the correlation energy. Our results, employing a binary stack model, highlight fundamental, qualitative differences between base-pair base-pair interactions and that of the stacked intercalator base pair system. Most notable result is the paucity of torque which so distinctively defines the Twist of DNA. Surprisingly, this model, when combined with a constraint on the twist of the surrounding base-pair steps to match the observed unwinding of the sugar-phosphate backbone, was sufficient for explaining the experimentally observed proflavine intercalator configuration. Our extensive mapping of the potential energy surface of base-pair intercalator interactions can provide valuable information for future nonempirical studies of DNA intercalation dynamics.

  7. DNMT3B interacts with constitutive centromere protein CENP-C to modulate DNA methylation and the histone code at centromeric regions.

    Science.gov (United States)

    Gopalakrishnan, Suhasni; Sullivan, Beth A; Trazzi, Stefania; Della Valle, Giuliano; Robertson, Keith D

    2009-09-01

    DNA methylation is an epigenetically imposed mark of transcriptional repression that is essential for maintenance of chromatin structure and genomic stability. Genome-wide methylation patterns are mediated by the combined action of three DNA methyltransferases: DNMT1, DNMT3A and DNMT3B. Compelling links exist between DNMT3B and chromosome stability as emphasized by the mitotic defects that are a hallmark of ICF syndrome, a disease arising from germline mutations in DNMT3B. Centromeric and pericentromeric regions are essential for chromosome condensation and the fidelity of segregation. Centromere regions contain distinct epigenetic marks, including dense DNA hypermethylation, yet the mechanisms by which DNA methylation is targeted to these regions remains largely unknown. In the present study, we used a yeast two-hybrid screen and identified a novel interaction between DNMT3B and constitutive centromere protein CENP-C. CENP-C is itself essential for mitosis. We confirm this interaction in mammalian cells and map the domains responsible. Using siRNA knock downs, bisulfite genomic sequencing and ChIP, we demonstrate for the first time that CENP-C recruits DNA methylation and DNMT3B to both centromeric and pericentromeric satellite repeats and that CENP-C and DNMT3B regulate the histone code in these regions, including marks characteristic of centromeric chromatin. Finally, we demonstrate that loss of CENP-C or DNMT3B leads to elevated chromosome misalignment and segregation defects during mitosis and increased transcription of centromeric repeats. Taken together, our data reveal a novel mechanism by which DNA methylation is targeted to discrete regions of the genome and contributes to chromosomal stability.

  8. A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability.

    Science.gov (United States)

    De, Ananya; Campbell, Colin

    2007-02-15

    The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.

  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. Conjugated Polymers/DNA Hybrid Materials for Protein Inactivation.

    Science.gov (United States)

    Zhao, Likun; Zhang, Jiangyan; Xu, Huiming; Geng, Hao; Cheng, Yongqiang

    2016-09-01

    Chromophore-assisted light inactivation (CALI) is a powerful tool for analyzing protein functions due to the high degree of spatial and temporal resolution. In this work, we demonstrate a CALI approach based on conjugated polymers (CPs)/DNA hybrid material for protein inactivation. The target protein is conjugated with single-stranded DNA in advance. Single-stranded DNA can form CPs/DNA hybrid material with cationic CPs via electrostatic and hydrophobic interactions. Through the formation of CPs/DNA hybrid material, the target protein that is conjugated with DNA is brought into close proximity to CPs. Under irradiation, CPs harvest light and generate reactive oxygen species (ROS), resulting in the inactivation of the adjacent target protein. This approach can efficiently inactivate any target protein which is conjugated with DNA and has good specificity and universality, providing a new strategy for studies of protein function and adjustment of protein activity.

  11. The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination.

    Directory of Open Access Journals (Sweden)

    Rebecca C Burgess

    Full Text Available Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR. Although its activity has been implicated in several steps of HR, its exact role(s at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA. This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box of Rad54 (Rad54-AA. Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

  12. C. elegans ring finger protein RNF-113 is involved in interstrand DNA crosslink repair and interacts with a RAD51C homolog.

    Directory of Open Access Journals (Sweden)

    Hyojin Lee

    Full Text Available The Fanconi anemia (FA pathway recognizes interstrand DNA crosslinks (ICLs and contributes to their conversion into double-strand DNA breaks, which can be repaired by homologous recombination. Seven orthologs of the 15 proteins associated with Fanconi anemia are functionally conserved in the model organism C. elegans. Here we report that RNF-113, a ubiquitin ligase, is required for RAD-51 focus formation after inducing ICLs in C. elegans. However, the formation of foci of RPA-1 or FCD-2/FANCD2 in the FA pathway was not affected by depletion of RNF-113. Nevertheless, the RPA-1 foci formed did not disappear with time in the depleted worms, implying serious defects in ICL repair. As a result, RNF-113 depletion increased embryonic lethality after ICL treatment in wild-type worms, but it did not increase the ICL-induced lethality of rfs-1/rad51C mutants. In addition, the persistence of RPA-1 foci was suppressed in doubly-deficient rnf-113;rfs-1 worms, suggesting that there is an epistatic interaction between the two genes. These results lead us to suggest that RNF-113 and RFS-1 interact to promote the displacement of RPA-1 by RAD-51 on single-stranded DNA derived from ICLs.

  13. Interaction of a C-terminal Truncated Hepatitis C Virus Core Protein with Plasmid DNA Vaccine Leads to in vitro Assembly of Heterogeneous Virus-like Particles

    Directory of Open Access Journals (Sweden)

    Nelson Acosta-Rivero

    2005-01-01

    Full Text Available Recently, it has been shown that HCV core proteins (HCcAg with C-terminal deletions assemble in vitro into virus-like particles (VLPs in the presence of structured RNA molecules. Results presented in this work showed that a truncated HCcAg variant covering the first 120 aa (HCcAg.120 with a 32 aa N-terminal fusion peptide (6xHistag-XpressTMepitope interacts with plasmid DNA vaccine. Interestingly, the buoyant density of VLPs containing HCcAg.120 in CsCl gradients changed from 1.15-1,17 g mLˉ1 to 1.30-1.34 g mLˉ1 after addition of plasmid DNA to assembly reactions. In addition, a delay in electrophoretic mobility of HCcAg.120-plasmid samples on agarose gels was observed indicating a direct interaction between VLPs and nucleic acids. Remarkably, addition of either plasmid DNA or tRNA to assembly reactions leaded to heterogeneous and larger VLPs formation than those observed in HCcAg.120 assembly reactions. VLPs containing HCcAg.120 induced a specific IgG antibodies in mice that reacted with hepatocytes from HCV-infected patients. VLPs obtained in this work would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure. Besides, the capacity of particles containing HCcAg.120 to interact with nucleic acids could be used in the development of DNA vaccines and viral vectors based on these particles.

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

    Science.gov (United States)

    Ishibashi, Yu; Oura, Shusuke; Umemura, Kazuo

    2017-09-01

    We examined the adsorption of DNA binding proteins on functionalized, single-walled carbon nanotubes (SWNTs). When SWNTs were functionalized with polyethylene glycol (PEG-SWNT), moderate adsorption of protein molecules was observed. In contrast, nanotubes functionalized with CONH2 groups (CONH2-SWNT) exhibited very strong interactions between the CONH2-SWNT and DNA binding proteins. Instead, when these SWNT surfaces were wrapped with DNA molecules (thymine 30-mers), protein binding was a little decreased. Our results revealed that DNA wrapped PEG-SWNT was one of the most promising candidates to realize DNA nanodevices involving protein reactions on DNA-SWNT surfaces. In addition, the DNA binding protein RecA was more adhesive than single-stranded DNA binding proteins to the functionalized SWNT surfaces.

  15. 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.

  16. Screening for compounds that affect the interaction between bacterial two-component signal transduction response regulator protein and cognate promoter DNA.

    Science.gov (United States)

    Erickson, Matthew G; Ulijasz, Andrew T; Weisblum, Bernard

    2008-01-01

    Bacterial signal transduction systems can be used as drug targets. The signal transduction targets fall into two groups--sensor kinases and response regulators. Previously reported studies describe hits that were thought to inactivate sensor kinases but on closer examination were found to act elsewhere instead; a possible reason for this is that full-length sensor kinases are integral membrane proteins whose activity might reflect interaction with the cell membrane or with membrane components. We describe a model system that instead is based on the interaction between a test compound and a response regulator in a homogeneous phase reaction. In this system, response regulator-DNA complex formation and its inhibition by a test compound are measured by fluorescence polarization. The model system should be readily adaptable to drug discovery based on other bacterial two-component s transduction systems.

  17. Analysis of damaged DNA / proteins interactions: Methodological optimizations and applications to DNA lesions induced by platinum anticancer drugs; Analyse des interactions ADN lese / proteines: Optimisations methodologiques et applications aux dommages de l'ADN engendres par les derives du platine

    Energy Technology Data Exchange (ETDEWEB)

    Bounaix Morand du Puch, Ch

    2010-10-15

    DNA lesions contribute to the alteration of DNA structure, thereby inhibiting essential cellular processes. Such alterations may be beneficial for chemotherapies, for example in the case of platinum anticancer agents. They generate bulky adducts that, if not repaired, ultimately cause apoptosis. A better understanding of the biological response to such molecules can be obtained through the study of proteins that directly interact with the damages. These proteins constitute the DNA lesions interactome. This thesis presents the development of tools aiming at increasing the list of platinum adduct-associated proteins. Firstly, we designed a ligand fishing system made of damaged plasmids immobilized onto magnetic beads. Three platinum drugs were selected for our study: cisplatin, oxali-platin and satra-platin. Following exposure of the trap to nuclear extracts from HeLa cancer cells and identification of retained proteins by proteomics, we obtained already known candidates (HMGB1, hUBF, FACT complex) but also 29 new members of the platinated-DNA interactome. Among them, we noted the presence of PNUTS, TOX4 and WDR82, which associate to form the recently-discovered PTW/PP complex. Their capture was then confirmed with a second model, namely breast cancer cell line MDA MB 231, and the biological consequences of such an interaction now need to be elucidated. Secondly, we adapted a SPRi bio-chip to the study of platinum-damaged DNA/proteins interactions. Affinity of HMGB1 and newly characterized TOX4 for adducts generated by our three platinum drugs could be validated thanks to the bio-chip. Finally, we used our tools, as well as analytical chemistry and biochemistry methods, to evaluate the role of DDB2 (a factor involved in the recognition of UV-induced lesions) in the repair of cisplatin adducts. Our experiments using MDA MB 231 cells differentially expressing DDB2 showed that this protein is not responsible for the repair of platinum damages. Instead, it appears to act

  18. Interactive protein manipulation

    Energy Technology Data Exchange (ETDEWEB)

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  19. Identification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and Demonstration that it Interacts with ICP8, the Major DNA Binding Protein of Herpes Simplex Virus

    Science.gov (United States)

    1992-10-20

    simplex. J.A.M.A. 117:999-1005 . Segal, A.L. t A.H. Katcher, V.J. Brightman, and M. F . Miller. 1974. Recurrent herpes labialis , recurrent aphthous...AII’I fORCE MEDICAL C(NTEIt Title of Dissertation : "Ideatification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and...Demonstration that It Interacts with reps. the Major DNA Binding Protein of Herpes Simplex Virus" Name of Candidate: Lisa Shelton Doctor of

  20. Cardiolipin Interactions with Proteins.

    Science.gov (United States)

    Planas-Iglesias, Joan; Dwarakanath, Himal; Mohammadyani, Dariush; Yanamala, Naveena; Kagan, Valerian E; Klein-Seetharaman, Judith

    2015-09-15

    Cardiolipins (CL) represent unique phospholipids of bacteria and eukaryotic mitochondria with four acyl chains and two phosphate groups that have been implicated in numerous functions from energy metabolism to apoptosis. Many proteins are known to interact with CL, and several cocrystal structures of protein-CL complexes exist. In this work, we describe the collection of the first systematic and, to the best of our knowledge, the comprehensive gold standard data set of all known CL-binding proteins. There are 62 proteins in this data set, 21 of which have nonredundant crystal structures with bound CL molecules available. Using binding patch analysis of amino acid frequencies, secondary structures and loop supersecondary structures considering phosphate and acyl chain binding regions together and separately, we gained a detailed understanding of the general structural and dynamic features involved in CL binding to proteins. Exhaustive docking of CL to all known structures of proteins experimentally shown to interact with CL demonstrated the validity of the docking approach, and provides a rich source of information for experimentalists who may wish to validate predictions.

  1. 6H-Indolo[2,3-b]quinoxalines: DNA and protein interacting scaffold for pharmacological activities.

    Science.gov (United States)

    Moorthy, N S Hari Narayana; Manivannan, E; Karthikeyan, C; Trivedi, Piyush

    2013-08-01

    6H-Indolo[2,3-b]quinoxaline, a planar fused heterocyclic compound exhibits a wide variety of pharmacological activities. The mechanism of pharmacological action exerted by these compounds is predominantly DNA intercalation. The thermal stability of the intercalated complex (DNA and 6H-indolo[2,3-b]quinoxaline derivatives) is an important parameter for the elucidation of anticancer, antiviral and other activities. This thermal stability of the 6H-indolo[2,3- b]quinoxaline-DNA complex depends on the type of substituents and side chains attached to the 6H-indolo[2,3- b]quinoxaline nucleus and also the orientation of the side chain towards the GC rich minor groove of the DNA. Highly active 6H-indolo[2,3-b]quinoxaline derivatives such as NCA0424, B-220 and 9-OH-B-220 have shown good binding affinity to DNA as evident from high thermal stability of compound-DNA complex. Interestingly, these compounds possessed poor inhibitory activity on topoisomerase II enzyme but have significant MDR modulating activity. This review establishes '6H-indolo[2,3-b]quinoxaline' as a valuable template for design and development of novel molecules with different biological activities.

  2. Identification of conserved amino acids in the herpes simplex virus type 1 UL8 protein required for DNA synthesis and UL52 primase interaction in the virus replisome.

    Science.gov (United States)

    Muylaert, Isabella; Zhao, Zhiyuan; Andersson, Torbjörn; Elias, Per

    2012-09-28

    We have used oriS-dependent transient replication assays to search for species-specific interactions within the herpes simplex virus replisome. Hybrid replisomes derived from herpes simplex virus type 1 (HSV-1) and equine herpesvirus type 1 (EHV-1) failed to support DNA replication in cells. Moreover, the replisomes showed a preference for their cognate origin of replication. The results demonstrate that the herpesvirus replisome behaves as a molecular machine relying on functionally important interactions. We then searched for functional interactions in the replisome context by subjecting HSV-1 UL8 protein to extensive mutagenesis. 52 mutants were made by replacing single or clustered charged amino acids with alanines. Four mutants showed severe replication defects. Mutant A23 exhibited a lethal phenotype, and mutants A49, A52 and A53 had temperature-sensitive phenotypes. Mutants A49 and A53 did not interact with UL52 primase as determined by co-immunoprecipitation experiments. Using GFP-tagged UL8, we demonstrate that all mutants were unable to support formation of ICP8-containing nuclear replication foci. Extended mutagenesis suggested that a highly conserved motif corresponding to mutant A49 serves an important role for establishing a physical contact between UL8 and UL52. The replication-defective mutations affected conserved amino acids, and similar phenotypes were observed when the corresponding mutations were introduced into EHV-1 UL8.

  3. Analysis of the DNA-Binding Profile and Function of TALE Homeoproteins Reveals Their Specialization and Specific Interactions with Hox Genes/Proteins

    Directory of Open Access Journals (Sweden)

    Dmitry Penkov

    2013-04-01

    Full Text Available The interactions of Meis, Prep, and Pbx1 TALE homeoproteins with Hox proteins are essential for development and disease. Although Meis and Prep behave similarly in vitro, their in vivo activities remain largely unexplored. We show that Prep and Meis interact with largely independent sets of genomic sites and select different DNA-binding sequences, Prep associating mostly with promoters and housekeeping genes and Meis with promoter-remote regions and developmental genes. Hox target sequences associate strongly with Meis but not with Prep binding sites, while Pbx1 cooperates with both Prep and Meis. Accordingly, Meis1 shows strong genetic interaction with Pbx1 but not with Prep1. Meis1 and Prep1 nonetheless coregulate a subset of genes, predominantly through opposing effects. Notably, the TALE homeoprotein binding profile subdivides Hox clusters into two domains differentially regulated by Meis1 and Prep1. During evolution, Meis and Prep thus specialized their interactions but maintained significant regulatory coordination.

  4. Dynamic protein assemblies in homologous recombination with single DNA molecules

    NARCIS (Netherlands)

    van der Heijden, A.H.

    2007-01-01

    What happens when your DNA breaks? This thesis describes experimental work on the single-molecule level focusing on the interaction between DNA and DNA-repair proteins, in particular bacterial RecA and human Rad51, involved in homologous recombination. Homologous recombination and its central event

  5. Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase α

    Institute of Scientific and Technical Information of China (English)

    Jia Sun; Neal F Lue; Ming Lei; Yuting Yang; Ke Wan; Ninghui Mao; Tai-Yuan Yu; Yi-Chien Lin; Diane C DeZwaan; Brian C Freeman; Jing-Jer Lin

    2011-01-01

    Budding yeast Cdc13-Stnl-Tenl (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB ' fold and the catalytic subunit of DNA polymerase a (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.

  6. Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase [alpha

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Jia; Yang, Yuting; Wan, Ke; Mao, Ninghui; Yu, Tai-Yuan; Lin, Yi-Chien; DeZwaan, Diane C.; Freeman, Brian C.; Lin, Jing-Jer; Lue, Neal F.; Lei, Ming

    2011-08-24

    Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase {alpha} (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.

  7. Cobalt(III) complexes as potential anticancer agents: Physicochemical, structural, cytotoxic activity and DNA/protein interactions.

    Science.gov (United States)

    Thamilarasan, V; Sengottuvelan, N; Sudha, A; Srinivasan, P; Chakkaravarthi, G

    2016-09-01

    Cobalt(III) complexes (1-3) such as [Co(acac)(bpy)(N3)2·H2O] 1, [Co(acac)(en)(N3)2] 2, and [Co(acac)(2-pic)(N3)2] 3 (where, acac=acetylacetone, bpy=2.2'-bipyridine, en=ethylenediamine, 2-pic=2-picolylamine and NaN3=sodium azide) were synthesized and characterized. The structure of complexes (1-3) has been determined by single crystal X-ray diffraction studies and the configuration around cobalt(III) ion was distorted octahedral coordination geometry. Density functional theory calculations were performed to examine the molecular geometry and frontier molecular orbital properties of complexes (1-3). DNA binding properties of the cobalt(III) complexes with calf thymus DNA (CT-DNA) were investigated by UV-visible absorption, fluorescence, circular dichroism spectroscopy and viscosity measurements. The docking studies showed the preferred orientation of sterically acceptable Co(III) complexes (1, 2) inside the DNA through the mode of intercalation, whereas complex 3 exhibited minor groove binding modes. The intrinsic binding constants Kb of complexes (1-3) with CT-DNA were in the following order 1>3>2. Complexes (1-3) exhibit a good binding propensity to bovine serum albumin (BSA) and gel electrophoresis assay demonstrated that the complexes (1-3) promote the cleavage of the pBR322 DNA in the presence of 3-mercaptopropionic acid (MPA) and cleavage process was found to proceed by singlet oxygen cleavage mechanism. Further, the in vitro cytotoxicity studies of complexes (1-3) were tested on human breast cancer cell line (MCF-7). Copyright © 2016. Published by Elsevier B.V.

  8. Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC

    NARCIS (Netherlands)

    Luijsterburg, Martijn S.; Goedhart, Joachim; Moser, Jill; Kool, Hanneke; Geverts, Bart; Houtsmuller, Adriaan B.; Mullenders, Leon H. F.; Vermeulen, Wim; van Driel, Roel

    2007-01-01

    Damage DNA binding protein 2 ( DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and

  9. Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC.

    NARCIS (Netherlands)

    Luijsterburg, M.S.; Goedhart, J.; Moser, J.; Kool, H.; Geverts, B.; Houtsmuller, A.B.; Mullenders, L.H.; Vermeulen, W.; van Driel, R.

    2007-01-01

    Damage DNA binding protein 2 (DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and CUL4A

  10. Computational design of co-assembling protein-DNA nanowires

    Science.gov (United States)

    Mou, Yun; Yu, Jiun-Yann; Wannier, Timothy M.; Guo, Chin-Lin; Mayo, Stephen L.

    2015-09-01

    Biomolecular self-assemblies are of great interest to nanotechnologists because of their functional versatility and their biocompatibility. Over the past decade, sophisticated single-component nanostructures composed exclusively of nucleic acids, peptides and proteins have been reported, and these nanostructures have been used in a wide range of applications, from drug delivery to molecular computing. Despite these successes, the development of hybrid co-assemblies of nucleic acids and proteins has remained elusive. Here we use computational protein design to create a protein-DNA co-assembling nanomaterial whose assembly is driven via non-covalent interactions. To achieve this, a homodimerization interface is engineered onto the Drosophila Engrailed homeodomain (ENH), allowing the dimerized protein complex to bind to two double-stranded DNA (dsDNA) molecules. By varying the arrangement of protein-binding sites on the dsDNA, an irregular bulk nanoparticle or a nanowire with single-molecule width can be spontaneously formed by mixing the protein and dsDNA building blocks. We characterize the protein-DNA nanowire using fluorescence microscopy, atomic force microscopy and X-ray crystallography, confirming that the nanowire is formed via the proposed mechanism. This work lays the foundation for the development of new classes of protein-DNA hybrid materials. Further applications can be explored by incorporating DNA origami, DNA aptamers and/or peptide epitopes into the protein-DNA framework presented here.

  11. Scaffolds for blocking protein-protein interactions.

    Science.gov (United States)

    Hershberger, Stefan J; Lee, Song-Gil; Chmielewski, Jean

    2007-01-01

    Due to the pivotal roles that protein-protein interactions play in a plethora of biological processes, the design of therapeutic agents targeting these interactions has become an attractive and important area of research. The development of such agents is faced with a variety of challenges. Nevertheless, considerable progress has been made in the design of proteomimetics capable of disrupting protein-protein interactions. Those inhibitors based on molecular scaffold designs hold considerable interest because of the ease of variation in regard to their displayed functionality. In particular, protein surface mimetics, alpha-helical mimetics, beta-sheet/beta-strand mimetics, as well as beta-turn mimetics have successfully modulated protein-protein interactions involved in such diseases as cancer and HIV. In this review, current progress in the development of molecular scaffolds designed for the disruption of protein-protein interactions will be discussed with an emphasis on those active against biological targets.

  12. Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus: probing a human leukocyte cDNA library using the yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-kang; ZHAO Long-feng; CHENG Jun; GUO Jiang; LUN Yong-zhi; HONG Yuan

    2006-01-01

    Background The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS 1TP5 protein.Methods The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS 1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization.After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH 109 with Y 187 containing a leukocyte cDNA library plasmid.The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.Results Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes

  13. Protein-protein interaction assays: eliminating false positive interactions

    OpenAIRE

    Nguyen, Tuan N.; Goodrich, James A.

    2006-01-01

    Many methods commonly used to identify and characterize interactions between two or more proteins are variations of the immobilized protein-protein interaction assay (for example, glutathione S-transferase (GST) pulldown and coimmunoprecipitation). A potential, and often overlooked, problem with these assays is the possibility that an observed interaction is mediated not by direct contact between proteins, but instead by nucleic acid contaminating the protein preparations. As a negatively cha...

  14. Controllability in protein interaction networks.

    Science.gov (United States)

    Wuchty, Stefan

    2014-05-13

    Recently, the focus of network research shifted to network controllability, prompting us to determine proteins that are important for the control of the underlying interaction webs. In particular, we determined minimum dominating sets of proteins (MDSets) in human and yeast protein interaction networks. Such groups of proteins were defined as optimized subsets where each non-MDSet protein can be reached by an interaction from an MDSet protein. Notably, we found that MDSet proteins were enriched with essential, cancer-related, and virus-targeted genes. Their central position allowed MDSet proteins to connect protein complexes and to have a higher impact on network resilience than hub proteins. As for their involvement in regulatory functions, MDSet proteins were enriched with transcription factors and protein kinases and were significantly involved in bottleneck interactions, regulatory links, phosphorylation events, and genetic interactions.

  15. A novel type of DNA-binding protein interacts with a conserved sequence in an early nodulin ENOD12 promoter

    DEFF Research Database (Denmark)

    Christiansen, H; Hansen, A C; Vijn, I

    1996-01-01

    of the ENBP1 transcript in cells expressing ENOD12 strongly suggest that ENBP1 is a transcription factor involved in the regulation of ENOD12. Finally, the C-terminal region of ENBP1 shows strong homology to a protein from rat that is specifically expressed in testis tissue. Udgivelsesdato: 1996-Dec...

  16. Replication of adenovirus DNA-protein complex with purified proteins.

    OpenAIRE

    Ikeda, J E; Enomoto, T.; Hurwitz, J

    1981-01-01

    A protein fraction isolated from the cytosol of adenovirus-infected HeLa cells, which contained DNA polymerase alpha, catalyzed adenoviral DNA replication in the presence of adenovirus DNA binding protein, eukaryotic DNA polymerase beta, ATP, all four dNTPs, and MgCl2. DNA replication started at either end of exogenously added adenoviral DNA and was totally dependent on the presence of terminal 55,000-dalton proteins on the DNA template. The replicaton of adenovirus DNA in the system was sens...

  17. 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.

  18. A Raman spectroscopic study of the interaction between nucleotides and the DNA binding protein gp32 of bacteriophage T4

    NARCIS (Netherlands)

    Otto, Cornelis; de Mul, F.F.M.; Greve, Jan

    1987-01-01

    Raman spectra of the bacteriophage T4 denaturing protein gp32, its complex with the polynucleotides poly(rA), poly(dA), poly(dT), poly(rU), and poly(rC), and with the oligonucleotides (dA)8 and (dA)2, were recorded and interpreted. According to an analysis of the gp32 spectra with the reference

  19. The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Sebesta, Marek; Sisakova, Alexandra

    2013-01-01

    Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54...... and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage...

  20. Protein-DNA complexes: specificity and DNA readout mechanisms

    Directory of Open Access Journals (Sweden)

    Shestopalova A. V.

    2011-02-01

    Full Text Available Protein-nucleic acid recognition is essential in a number of cellular processes, in particular, gene regulation, DNA replication and compaction. Studies on the recognition mechanisms show that DNA sequence carries information which is read out by proteins that selectively bind to specific DNA sites. The review is focused on the processes taking place during formation of specific and nonspecific complexes of proteins and DNA. Special attention is paid to direct and indirect mechanisms of sequence-specific recognition. Several examples of protein-nucleic acid complexes are given to illustrate the variety of recognition mechanisms

  1. Cloning of a DNA-binding protein that interacts with the ethylene-responsive enhancer element of the carnation GST1 gene.

    Science.gov (United States)

    Maxson, J M; Woodson, W R

    1996-07-01

    Ethylene transcriptionally activates a glutathione S-transferase gene (GST1) at the onset of the senescence program in carnation (Dianthus caryophyllus L.) flower petals. A 126 bp region of the GST1 promoter sequence has been identified as an ethylene-responsive enhancer element (ERE). In this paper, we demonstrate the ability of nuclear proteins from senescing petals to recognize a 22 bp sequence within the ERE (ERE oligonucleotide). Mutation of the ERE oligonucleotide sequence significantly alters the strength of this nuclear protein-DNA association. The wild-type ERE oligonucleotide sequence was used to isolate a cDNA clone encoding a sequence-specific DNA binding protein. Nucleotide sequencing and deduced amino acid sequence analysis of this cDNA predicted a 32 kDa protein which we have designated carnation ethylene-responsive element-binding protein-1 (CEBP-1). The mRNA expression pattern of CEBP-1 suggests that it is not transcriptionally regulated by ethylene. The amino acid sequence homology of CEBP-1 with other plant nucleic acid binding proteins indicates a conserved nucleic acid binding domain. Within this domain are two highly conserved RNA-binding motifs, RNP-1 and RNP-2. An acidic region and a putative nuclear localization signal are also identified.

  2. Conductometric monitoring of protein-protein interactions.

    Science.gov (United States)

    Spera, Rosanna; Festa, Fernanda; Bragazzi, Nicola L; Pechkova, Eugenia; LaBaer, Joshua; Nicolini, Claudio

    2013-12-06

    Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.

  3. Dynamics of nucleosome invasion by DNA binding proteins.

    Science.gov (United States)

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

    2011-08-12

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

  4. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage phi29

    Science.gov (United States)

    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J.; Smith, Douglas E.

    2016-01-01

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine3+ causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation. PMID:24996111

  5. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage Phi29.

    Science.gov (United States)

    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J; Smith, Douglas E

    2014-06-20

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine(3+) causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interactions facilitate packaging despite increasing the energy of the theoretical optimum spooled DNA conformation.

  6. The Mre11 protein interacts with both Rad50 and the HerA bipolar helicase and is recruited to DNA following gamma irradiation in the archaeon Sulfolobus acidocaldarius

    Directory of Open Access Journals (Sweden)

    Forterre Patrick

    2008-02-01

    Full Text Available Abstract Background The ubiquitous Rad50 and Mre11 proteins play a key role in many processes involved in the maintenance of genome integrity in Bacteria and Eucarya, but their function in the Archaea is presently unknown. We showed previously that in most hyperthermophilic archaea, rad50-mre11 genes are linked to nurA encoding both a single-strand endonuclease and a 5' to 3' exonuclease, and herA, encoding a bipolar DNA helicase which suggests the involvement of the four proteins in common molecular pathway(s. Since genetic tools for hyperthermophilic archaea are just emerging, we utilized immuno-detection approaches to get the first in vivo data on the role(s of these proteins in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius. Results We first showed that S. acidocaldarius can repair DNA damage induced by high doses of gamma rays, and we performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA along with the RadA recombinase in both control and irradiated cells. We found that during the exponential phase, all proteins are synthesized and display constant levels, but that all of them exhibit a different sub-cellular partitioning. Following gamma irradiation, both Mre11 and RadA are immediately recruited to DNA and remain DNA-bound in the course of DNA repair. Furthermore, we show by immuno-precipitation assays that Rad50, Mre11 and the HerA helicase interact altogether. Conclusion Our analyses strongly support that in Sulfolobus acidocaldarius, the Mre11 protein and the RadA recombinase might play an active role in the repair of DNA damage introduced by gamma rays and/or may act as DNA damage sensors. Moreover, our results demonstrate the functional interaction between Mre11, Rad50 and the HerA helicase and suggest that each protein play different roles when acting on its own or in association with its partners. This report provides the first in vivo evidence supporting the

  7. Studying protein-protein interactions using peptide arrays

    NARCIS (Netherlands)

    Katz, C.; Levy-Beladev, L.; Rotem-Bamberger, S.; Rito, T.; Rudiger, S.G.D.; Friedler, A.

    2010-01-01

    Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins

  8. Differential protein-DNA interactions at the promoter and enhancer regions of developmentally regulated U4 snRNA genes.

    Science.gov (United States)

    Miyake, J H; Botros, I W; Stumph, W E

    1992-01-01

    In the chicken genome there are two closely-linked genes, U4B and U4X, that code for different sequence variants of U4 small nuclear RNA (snRNA). Both genes are expressed with nearly equal efficiency in the early embryo, but U4X gene expression is specifically down-regulated relative to U4B as development proceeds. At the present time, little is known about the mechanisms that regulate differential expression of snRNA genes. We have now identified a novel chicken factor, PPBF, that binds sequence-specifically in vitro to the proximal regulatory region of the U4X gene, but not to the proximal region of the U4B gene. PPBF is itself regulated during development and may therefore be a key factor involved in differentially regulating U4X gene transcription relative to U4B. The U4X and U4B enhancers contain distinct sequence variants of two essential motifs (octamer and SPH). The Oct-1 transcription factor binds with similar affinities to both the U4X and U4B octamer motifs. However, a second essential snRNA enhancer-binding protein, SBF, has a 20- to 30-fold lower affinity for the SPH motif in the U4X enhancer than for the homologous SPH motif in the U4B enhancer. A potential role therefore exists for SBF, as well as PPBF, in the preferential down-regulation of the U4X RNA gene during chicken development.

  9. INHIBITION OF THE DNA-BINDING ACTIVITY OF DROSOPHILA SUPPRESSOR OF HAIRLESS AND OF ITS HUMAN HOMOLOG, KBF2/RBP-J-KAPPA, BY DIRECT PROTEIN-PROTEIN INTERACTION WITH DROSOPHILA HAIRLESS

    NARCIS (Netherlands)

    BROU, C; LOGEAT, F; LECOURTOIS, M; VANDEKERCKHOVE, Joël; KOURILSKY, P; SCHWEISGUTH, F; ISRAEL, A

    1994-01-01

    We have purified the sequence-specific DNA-binding protein KBF2 and cloned the corresponding cDNA, which is derived from the previously described RBP-J kappa gene, the human homolog of the Drosophila Suppressor of Hairless [Su(H)] gene. Deletion studies of the RBP-J kappa and Su(H) proteins allowed

  10. DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4-ones as Highly Potent and Monoselective Receptor Interacting Protein 1 Kinase Inhibitors.

    Science.gov (United States)

    Harris, Philip A; King, Bryan W; Bandyopadhyay, Deepak; Berger, Scott B; Campobasso, Nino; Capriotti, Carol A; Cox, Julie A; Dare, Lauren; Dong, Xiaoyang; Finger, Joshua N; Grady, LaShadric C; Hoffman, Sandra J; Jeong, Jae U; Kang, James; Kasparcova, Viera; Lakdawala, Ami S; Lehr, Ruth; McNulty, Dean E; Nagilla, Rakesh; Ouellette, Michael T; Pao, Christina S; Rendina, Alan R; Schaeffer, Michelle C; Summerfield, Jennifer D; Swift, Barbara A; Totoritis, Rachel D; Ward, Paris; Zhang, Aming; Zhang, Daohua; Marquis, Robert W; Bertin, John; Gough, Peter J

    2016-03-10

    The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

  11. Investigation by two-photon fluorescence correlation spectroscopy of the interaction of the nucleocapsid protein of HIV-1 with hairpin loop DNA sequences

    Science.gov (United States)

    Mely, Yves; Azoulay, Joel; Beltz, Herve; Clamme, Jean-Pierre; Bernacchi, Serena; Ficheux, Damien; Roques, Bernard P.; Darlix, Jean-Luc

    2004-09-01

    The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two strand transfer reactions required during reverse transcription. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response element, TAR sequence, at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3" terminus of the early product of reverse transcription. To characterize NCp7-mediated nucleic acid destabilization, we investigated by steady-state and time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly-labelled cTAR sequence with NCp7. The conformational fluctuations observed in the absence of NCp7 were associated with the rapid opening and closing (fraying) of the double stranded terminal segment of cTAR. NCp7 destabilizes cTAR mainly through a large increase of the opening rate constant. Additionally, the various destabilizing structures (bulges, internal loop, mismatches) spread all over cTAR secondary structure were found to be critical for NCp7 chaperone activity. Taken together, our data enabled us to propose a molecular mechanism for the destabilizing activity of NCp7 on cTAR which is crucial for the formation of the cTAR-TAR complex during the first strand transfer reaction.

  12. TFinDit: transcription factor-DNA interaction data depository

    Directory of Open Access Journals (Sweden)

    Turner Daniel

    2012-09-01

    Full Text Available Abstract Background One of the crucial steps in regulation of gene expression is the binding of transcription factor(s to specific DNA sequences. Knowledge of the binding affinity and specificity at a structural level between transcription factors and their target sites has important implications in our understanding of the mechanism of gene regulation. Due to their unique functions and binding specificity, there is a need for a transcription factor-specific, structure-based database and corresponding web service to facilitate structural bioinformatics studies of transcription factor-DNA interactions, such as development of knowledge-based interaction potential, transcription factor-DNA docking, binding induced conformational changes, and the thermodynamics of protein-DNA interactions. Description TFinDit is a relational database and a web search tool for studying transcription factor-DNA interactions. The database contains annotated transcription factor-DNA complex structures and related data, such as unbound protein structures, thermodynamic data, and binding sequences for the corresponding transcription factors in the complex structures. TFinDit also provides a user-friendly interface and allows users to either query individual entries or generate datasets through culling the database based on one or more search criteria. Conclusions TFinDit is a specialized structural database with annotated transcription factor-DNA complex structures and other preprocessed data. We believe that this database/web service can facilitate the development and testing of TF-DNA interaction potentials and TF-DNA docking algorithms, and the study of protein-DNA recognition mechanisms.

  13. Synthesis,DNA/protein Interaction of a Copper(II)Complex with Schiff▬base Ligand%席夫碱铜配合物合成及与DNA/蛋白质作用的研究

    Institute of Scientific and Technical Information of China (English)

    李梅; 黄书娟; 叶诚; 黄珊珊

    2014-01-01

    A novel copper( II)complex was prepared by pyridine▬2▬aldehyde and N,N▬dimethylethylenediamine,and characterized by single▬crystal X▬ray diffraction,elemental analysis,and ESI▬MS• Single▬crystal analysis revealed that the complex shows a distorted pseudo▬square▬pyramidal geometry• The binding properties of the complex with calf thymus DNA( CT▬DNA)and HSA were investiga▬ted by UV▬visible( UV▬vis),uorescence and circular dichroism( CD)spectra• The complex binds signicantly to CT▬DNA by intercala▬tion modes,and the complex binding to protein HSA belongs to static quenching mechanism• The interaction between the complex and HSA involve hydrogen bonding and weak van der Waals•%利用2-吡啶甲醛、N,N▬二甲基乙二胺制备了一种新的席夫碱铜配合物,并通过X▬射线单晶衍射、元素分析和ESI▬MS对其结构进行分析•晶体结构分析表明,配合物呈现扭曲的四方锥构型•用紫外▬可见吸收光谱、荧光光谱和圆二色光谱研究了配合物与小牛胸腺DNA( CT▬DNA)和人血清白蛋白( HSA)的结合性质•结果表明:配合物以插入的方式与DNA相互作用,且配合物与HSA的相互作用为静态猝灭机制,分子间作用力为氢键和范德华力•

  14. Functional interactions of DNA topoisomerases with a human replication origin.

    Science.gov (United States)

    Abdurashidova, Gulnara; Radulescu, Sorina; Sandoval, Oscar; Zahariev, Sotir; Danailov, Miltcho B; Demidovich, Alexander; Santamaria, Laura; Biamonti, Giuseppe; Riva, Silvano; Falaschi, Arturo

    2007-02-21

    The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.

  15. DNA-Protein Crosslink Proteolysis Repair.

    Science.gov (United States)

    Vaz, Bruno; Popovic, Marta; Ramadan, Kristijan

    2017-06-01

    Proteins that are covalently bound to DNA constitute a specific type of DNA lesion known as DNA-protein crosslinks (DPCs). DPCs represent physical obstacles to the progression of DNA replication. If not repaired, DPCs cause stalling of DNA replication forks that consequently leads to DNA double-strand breaks, the most cytotoxic DNA lesion. Although DPCs are common DNA lesions, the mechanism of DPC repair was unclear until now. Recent work unveiled that DPC repair is orchestrated by proteolysis performed by two distinct metalloproteases, SPARTAN in metazoans and Wss1 in yeast. This review summarizes recent discoveries on two proteases in DNA replication-coupled DPC repair and establishes DPC proteolysis repair as a separate DNA repair pathway for genome stability and protection from accelerated aging and cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Theoretical studies of protein-protein and protein-DNA binding rates

    Science.gov (United States)

    Alsallaq, Ramzi A.

    Proteins are folded chains of amino acids. Some of the amino acids (e.g. Lys, Arg, His, Asp, and Glu) carry charges under physiological conditions. Proteins almost always function through binding to other proteins or ligands, for example barnase is a ribonuclease protein, found in the bacterium Bacillus amyloliquefaceus. Barnase degrades RNA by hydrolysis. For the bacterium to inhibit the potentially lethal action of Barnase within its own cell it co-produces another protein called barstar which binds quickly, and tightly, to barnase. The biological function of this binding is to block the active site of barnase. The speeds (rates) at which proteins associate are vital to many biological processes. They span a wide range (from less than 103 to 108 M-1s-1 ). Rates greater than ˜ 106 M -1s-1 are typically found to be manifestations of enhancements by long-range electrostatic interactions between the associating proteins. A different paradigm appears in the case of protein binding to DNA. The rate in this case is enhanced through attractive surface potential that effectively reduces the dimensionality of the available search space for the diffusing protein. This thesis presents computational and theoretical models on the rate of association of ligands/proteins to other proteins or DNA. For protein-protein association we present a general strategy for computing protein-protein rates of association. The main achievements of this strategy is the ability to obtain a stringent reaction criteria based on the landscape of short-range interactions between the associating proteins, and the ability to compute the effect of the electrostatic interactions on the rates of association accurately using the best known solvers for Poisson-Boltzmann equation presently available. For protein-DNA association we present a mathematical model for proteins targeting specific sites on a circular DNA topology. The main achievements are the realization that a linear DNA with reflecting ends

  17. A bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor σ32 of Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Elsa Perrody

    Full Text Available The universally conserved J-domain proteins (JDPs are obligate cochaperone partners of the Hsp70 (DnaK chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ(32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ(32 facilitates RB43 bacteriophage proliferation is discussed.

  18. Yeast Interacting Proteins Database: YNL273W, YMR048W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ing gap repair of damaged DNA; interacts with the MCM helicase Rows with this bait as bait (1) Rows with thi...s bait as prey (0) YMR048W CSM3 Protein required for accurate chromosome segregation during meiosis Row...s with this prey as prey (1) Rows with this prey as bait (0) 4 3 2 2 0 0 0 0 0 - - - -...rk to promote sister chromatid cohesion after DNA damage, facilitating gap repair of damaged DNA; interacts with the MCM helicase Row...s with this bait as bait Rows with this bait as bait (1) Rows with this bait as prey Row

  19. Defining interactions between DNA-PK and ligase IV/XRCC4

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Hsin-Ling; Yannone, Steven M.; Chen, David J.

    2001-04-10

    Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct physical interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. No direct interactions are observed between ligase IV and DNA-PKcs or between XRCC4 and Ku. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks.

  20. Recognition of Local DNA Structures by p53 Protein.

    Science.gov (United States)

    Brázda, Václav; Coufal, Jan

    2017-02-10

    p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells.

  1. Synthesis, characterization, DNA/protein interaction and cytotoxicity studies of Cu(II) and Co(II) complexes derived from dipyridyl triazole ligands

    Science.gov (United States)

    Zhang, Wei; Yao, Di; Wei, Yi; Tang, Jie; Bian, He-Dong; Huang, Fu-Ping; Liang, Hong

    2016-06-01

    Four different transition metal complexes containing dipyridyl triazole ligands, namely [Cu(abpt)2Cl2]·2H2O (1), [Cu(abpt)2(ClO4)2] (2), [Co2(abpt)2(H2O)2Cl2]·Cl2·4H2O (3) and [Co2(Hbpt)2(CH3OH)2(NO3)2] (4) have been designed, synthesized and further structurally characterized by X-ray crystallography, ESI-MS, elemental analysis, IR and Raman spectroscopy. In these complexes, the both ligands act as bidentate ligands with N, N donors. DNA binding interactions with calf thymus DNA (ct-DNA) of the ligand and its complexes 1 ~ 4 were investigated via electronic absorption, fluorescence quenching, circular dichroism and viscosity measurements as well as confocal Laser Raman spectroscopy. The results show these complexes are able to bind to DNA via the non-covalent mode i.e. intercalation and groove binding or electrostatic interactions. The interactions with bovine serum albumin (BSA) were also studied using UV-Vis and fluorescence spectroscopic methods which indicated that fluorescence quenching of BSA by these compounds was the presence of both static and dynamic quenching. Moreover, the in vitro cytotoxic effects of the complexes against four cell lines SK-OV-3, HL-7702, BEL7404 and NCI-H460 showed the necessity of the coordination action on the biological properties on the respective complex and that all four complexes exhibited substantial cytotoxic activity.

  2. Synthesis, characterization, DNA/protein interaction and cytotoxicity studies of Cu(II) and Co(II) complexes derived from dipyridyl triazole ligands.

    Science.gov (United States)

    Zhang, Wei; Yao, Di; Wei, Yi; Tang, Jie; Bian, He-Dong; Huang, Fu-Ping; Liang, Hong

    2016-06-15

    Four different transition metal complexes containing dipyridyl triazole ligands, namely [Cu(abpt)2Cl2]·2H2O (1), [Cu(abpt)2(ClO4)2] (2), [Co2(abpt)2(H2O)2Cl2]·Cl2·4H2O (3) and [Co2(Hbpt)2(CH3OH)2(NO3)2] (4) have been designed, synthesized and further structurally characterized by X-ray crystallography, ESI-MS, elemental analysis, IR and Raman spectroscopy. In these complexes, the both ligands act as bidentate ligands with N, N donors. DNA binding interactions with calf thymus DNA (ct-DNA) of the ligand and its complexes 1~4 were investigated via electronic absorption, fluorescence quenching, circular dichroism and viscosity measurements as well as confocal Laser Raman spectroscopy. The results show these complexes are able to bind to DNA via the non-covalent mode i.e. intercalation and groove binding or electrostatic interactions. The interactions with bovine serum albumin (BSA) were also studied using UV-Vis and fluorescence spectroscopic methods which indicated that fluorescence quenching of BSA by these compounds was the presence of both static and dynamic quenching. Moreover, the in vitro cytotoxic effects of the complexes against four cell lines SK-OV-3, HL-7702, BEL7404 and NCI-H460 showed the necessity of the coordination action on the biological properties on the respective complex and that all four complexes exhibited substantial cytotoxic activity.

  3. 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.

  4. DNA2—An Important Player in DNA Damage Response or Just Another DNA Maintenance Protein?

    Directory of Open Access Journals (Sweden)

    Elzbieta Pawłowska

    2017-07-01

    Full Text Available The human DNA2 (DNA replication helicase/nuclease 2 protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER, interacting with the replication protein A (RPA and the flap endonuclease 1 (FEN1. DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN and Bloom syndrome protein (BLM. In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes.

  5. Probing DNA-DNA Interactions with a Combination of Quadruple-Trap Optical Tweezers and Microfluidics.

    Science.gov (United States)

    Brouwer, Ineke; King, Graeme A; Heller, Iddo; Biebricher, Andreas S; Peterman, Erwin J G; Wuite, Gijs J L

    2017-01-01

    DNA metabolism and DNA compaction in vivo involve frequent interactions of remote DNA segments, mediated by proteins. In order to gain insight into such interactions, quadruple-trap optical tweezers have been developed. This technique provides an unprecedented degree of control through the ability to independently manipulate two DNA molecules in three dimensions. In this way, discrete regions of different DNA molecules can be brought into contact with one another, with a well-defined spatial configuration. At the same time, the tension and extension of the DNA molecules can be monitored. Furthermore, combining quadruple-trap optical tweezers with microfluidics makes fast buffer exchange possible, which is important for in situ generation of the dual DNA-protein constructs needed for these kinds of experiments. In this way, processes such as protein-mediated inter-DNA bridging can be studied with unprecedented control. This chapter provides a step-by-step description of how to perform a dual DNA manipulation experiment using combined quadruple-trap optical tweezers and microfluidics.

  6. DNA condensation by high-affinity interaction with avidin.

    Science.gov (United States)

    Morpurgo, Margherita; Radu, Aurelian; Bayer, Edward A; Wilchek, Meir

    2004-01-01

    Avidin, the basic biotin-binding glycoprotein from chicken egg white, is known to interact with DNA, whereas streptavidin, its neutral non-glycosylated bacterial analog, does not. In the present study we investigated the DNA-binding properties of avidin. Its affinity for DNA in the presence and absence of biotin was compared with that of other positively charged molecules, namely the protein lysozyme, the cationic polymers polylysine and polyarginine and an avidin derivative with higher isoelectric point (pI approximately 11) in which most of the lysine residues were converted to homoarginines. Gel-shift assays, transmission electron microscopy and dynamic light scattering experiments demonstrated an unexpectedly strong interaction between avidin and DNA. The most pronounced gel-shift retardation occurred with the avidin-biotin complex, followed by avidin alone and then guanidylated avidin. Furthermore, ultrastructural and light-scattering studies showed that avidin assembles on the DNA molecule in an organized manner. The assembly leads to the formation of nanoparticles that are about 50-100 nm in size (DNA approximately 5 kb) and have a rod-like or toroidal shape. In these particles the DNA is highly condensed and one avidin is bound to each 18 +/- 4 DNA base pairs. The complexes are very stable even at high dilution ([DNA] =10 pM) and are not disrupted in the presence of buffers or salt (up to 200 mM NaCl). The other positively charged molecules also condense DNA and form particles with a globular shape. However, in this case, these particles disassemble by dilution or in the presence of low salt concentration. The results indicate that the interaction of avidin with DNA may also occur under physiological conditions, further enhanced by the presence of biotin. This DNA-binding property of avidin may thus shed light on a potentially new physiological role for the protein in its natural environment.

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

    Science.gov (United States)

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

    2015-04-20

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

  8. 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.

  9. Domain Structure of the Redβ Single-Strand Annealing Protein: the C-terminal Domain is Required for Fine-Tuning DNA-binding Properties, Interaction with the Exonuclease Partner, and Recombination in vivo.

    Science.gov (United States)

    Smith, Christopher E; Bell, Charles E

    2016-02-13

    Redβ is a component of the Red recombination system of bacteriophage λ that promotes a single strand annealing (SSA) reaction to generate end-to-end concatemers of the phage genome for packaging. Redβ interacts with λ exonuclease (λexo), the other component of the Red system, to form a "synaptosome" complex that somehow integrates the end resection and annealing steps of the reaction. Previous work using limited proteolysis and chemical modification revealed that Redβ consists of an N-terminal DNA binding domain, residues 1-177, and a flexible C-terminal "tail", residues 178-261. Here, we quantitatively compare the binding of the full-length protein (Redβ(FL)) and the N-terminal domain (Redβ(177)) to different lengths of ssDNA substrate and annealed duplex product. We find that in general, Redβ(FL) binds more tightly to annealed duplex product than to ssDNA substrate, while Redβ(177) binds more tightly to ssDNA. In addition, the C-terminal region of Redβ corresponding to residues 182-261 was purified and found to fold into an α-helical domain that is required for the interaction with λexo to form the synaptosome complex. Deletion analysis of Redβ revealed that removal of just eleven residues from the C-terminus disrupts the interaction with λexo as well as ssDNA and dsDNA recombination in vivo. By contrast, the determinants for self-oligomerization of Redβ appear to reside solely within the N-terminal domain. The subtle but significant differences in the relative binding of Redβ(FL) and Redβ(177) to ssDNA substrate and annealed duplex product may be important for Redβ to function as a SSA protein in vivo.

  10. Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

    Science.gov (United States)

    Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

    2002-07-01

    The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

  11. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-03-01

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

  13. UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein–DNA interactions

    Science.gov (United States)

    Hume, Maxwell A.; Barrera, Luis A.; Gisselbrecht, Stephen S.; Bulyk, Martha L.

    2015-01-01

    The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k (‘k-mers’). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org. PMID:25378322

  14. Repulsive DNA-DNA interactions accelerate viral DNA packaging in phage phi29

    OpenAIRE

    Keller, Nicholas; delToro, Damian; Grimes, Shelley; Jardine, Paul J.; Smith, Douglas E.

    2014-01-01

    We use optical tweezers to study the effect of attractive versus repulsive DNA-DNA interactions on motor-driven viral packaging. Screening of repulsive interactions accelerates packaging, but induction of attractive interactions by spermidine3+ causes heterogeneous dynamics. Acceleration is observed in a fraction of complexes, but most exhibit slowing and stalling, suggesting that attractive interactions promote nonequilibrium DNA conformations that impede the motor. Thus, repulsive interacti...

  15. Discovering functional interaction patterns in protein-protein interaction networks

    Directory of Open Access Journals (Sweden)

    Can Tolga

    2008-06-01

    Full Text Available Abstract Background In recent years, a considerable amount of research effort has been directed to the analysis of biological networks with the availability of genome-scale networks of genes and/or proteins of an increasing number of organisms. A protein-protein interaction (PPI network is a particular biological network which represents physical interactions between pairs of proteins of an organism. Major research on PPI networks has focused on understanding the topological organization of PPI networks, evolution of PPI networks and identification of conserved subnetworks across different species, discovery of modules of interaction, use of PPI networks for functional annotation of uncharacterized proteins, and improvement of the accuracy of currently available networks. Results In this article, we map known functional annotations of proteins onto a PPI network in order to identify frequently occurring interaction patterns in the functional space. We propose a new frequent pattern identification technique, PPISpan, adapted specifically for PPI networks from a well-known frequent subgraph identification method, gSpan. Existing module discovery techniques either look for specific clique-like highly interacting protein clusters or linear paths of interaction. However, our goal is different; instead of single clusters or pathways, we look for recurring functional interaction patterns in arbitrary topologies. We have applied PPISpan on PPI networks of Saccharomyces cerevisiae and identified a number of frequently occurring functional interaction patterns. Conclusion With the help of PPISpan, recurring functional interaction patterns in an organism's PPI network can be identified. Such an analysis offers a new perspective on the modular organization of PPI networks. The complete list of identified functional interaction patterns is available at http://bioserver.ceng.metu.edu.tr/PPISpan/.

  16. UV-laser crosslinking of proteins to DNA.

    Science.gov (United States)

    Moss, T; Dimitrov, S I; Houde, D

    1997-02-01

    Photochemical crosslinking is now a powerful method for studying protein-nucleic acid interactions. UV light is a zero-length crosslinking agent that predominantly or exclusively crosslinks proteins to nucleic acids at their contact points. It can therefore provide strong evidence for close protein-nucleic acid interactions. However, to achieve an acceptable degree of crosslinking with conventional UV light sources, exposure times ranging from minutes to several hours are necessary. Such prolonged irradiation allows for the artifactual redistribution of proteins and precludes kinetic studies. The use of UV lasers overcomes these difficulties since the number of photons required for the crosslinking may be delivered in time intervals on the order of nano- or even picoseconds. We described detailed procedures for UV laser-induced protein-DNA crosslinking both in vivo and in vitro. Technical aspects, including the choice of UV laser for irradiation, the isolation of covalently crosslinked protein-DNA complexes, immunochemical techniques for both the identification and isolation of specific protein-DNA complexes and the identification of the crosslinked DNA sequences, are reviewed in detail. The application of UV laser crosslinking in kinetic studies is illustrated by the example of the TATA-binding protein (TBP) interaction with the adenovirus E4 promoter.

  17. A novel Schiff base derived from the gabapentin drug and copper (II) complex: Synthesis, characterization, interaction with DNA/protein and cytotoxic activity.

    Science.gov (United States)

    Shokohi-Pour, Zahra; Chiniforoshan, Hossein; Momtazi-Borojeni, Amir Abbas; Notash, Behrouz

    2016-09-01

    A novel Schiff base [C20H23NO3], has been prepared and characterized using FT-IR, UV-vis, (1)H NMR spectroscopy, elemental analysis and X-ray crystallography. A copper (II) complex [Cu(C20H22NO3)2]·H2O has also been synthesized and characterized. The new ligand and complex thus obtained were investigated by their interaction with calf thymus DNA and BSA using electronic absorption spectroscopy, fluorescence spectroscopy, and thermal denaturation. The intrinsic binding constants Kb of the ligand and Cu (II) complex, with CT-DNA obtained from UV-vis absorption studies were 1.53×10(4)M(-1) and 3.71×10(5)M(-1), respectively. Moreover the addition of the two compounds to CT-DNA (1:2) led to an increase of the melting temperature of DNA up to around 2.61°C for the ligand and 3.99°C for the Cu (II) complex. The ligand and Cu (II) complex bind to CT-DNA via a partial intercalative, as shown by the experimental data. In addition, the albumin interactions of the two compounds were studied by fluorescence quenching spectra, the results indicating that the binding mechanism is a static quenching process. The in vitro cytotoxicity of the two compounds on three different cancer cell lines was evaluated by MTT assay. The results showed that the copper complex exerted enhanced cytotoxicity compared with the Schiff base ligand; thereby, this complex clearly implies a positive synergistic effect. Furthermore, the copper complex showed a high, selective, and dose-dependent cytotoxicity against cancer cell lines.

  18. DNA Interaction Studies of Selected Polyamine Conjugates

    Directory of Open Access Journals (Sweden)

    Marta Szumilak

    2016-09-01

    Full Text Available The interaction of polyamine conjugates with DNA double helix has been studied. Binding properties were examined by ethidium bromide (EtBr displacement and DNA unwinding/topoisomerase I/II (Topo I/II activity assays, as well as dsDNA thermal stability studies and circular dichroism spectroscopy. Genotoxicity of the compounds was estimated by a comet assay. It has been shown that only compound 2a can interact with dsDNA via an intercalative binding mode as it displaced EtBr from the dsDNA-dye complex, with Kapp = 4.26 × 106 M−1; caused an increase in melting temperature; changed the circular dichroism spectrum of dsDNA; converted relaxed plasmid DNA into a supercoiled molecule in the presence of Topo I and reduced the amount of short oligonucleotide fragments in the comet tail. Furthermore, preliminary theoretical study has shown that interaction of the discussed compounds with dsDNA depends on molecule linker length and charge distribution over terminal aromatic chromophores.

  19. DNA Interaction Studies of Selected Polyamine Conjugates

    Science.gov (United States)

    Szumilak, Marta; Merecz, Anna; Strek, Malgorzata; Stanczak, Andrzej; Inglot, Tadeusz W.; Karwowski, Boleslaw T.

    2016-01-01

    The interaction of polyamine conjugates with DNA double helix has been studied. Binding properties were examined by ethidium bromide (EtBr) displacement and DNA unwinding/topoisomerase I/II (Topo I/II) activity assays, as well as dsDNA thermal stability studies and circular dichroism spectroscopy. Genotoxicity of the compounds was estimated by a comet assay. It has been shown that only compound 2a can interact with dsDNA via an intercalative binding mode as it displaced EtBr from the dsDNA-dye complex, with Kapp = 4.26 × 106 M−1; caused an increase in melting temperature; changed the circular dichroism spectrum of dsDNA; converted relaxed plasmid DNA into a supercoiled molecule in the presence of Topo I and reduced the amount of short oligonucleotide fragments in the comet tail. Furthermore, preliminary theoretical study has shown that interaction of the discussed compounds with dsDNA depends on molecule linker length and charge distribution over terminal aromatic chromophores. PMID:27657041

  20. Protopia: a protein-protein interaction tool

    Science.gov (United States)

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  1. 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

  2. Detecting mutually exclusive interactions in protein-protein interaction maps.

    Directory of Open Access Journals (Sweden)

    Carmen Sánchez Claros

    Full Text Available Comprehensive protein interaction maps can complement genetic and biochemical experiments and allow the formulation of new hypotheses to be tested in the system of interest. The computational analysis of the maps may help to focus on interesting cases and thereby to appropriately prioritize the validation experiments. We show here that, by automatically comparing and analyzing structurally similar regions of proteins of known structure interacting with a common partner, it is possible to identify mutually exclusive interactions present in the maps with a sensitivity of 70% and a specificity higher than 85% and that, in about three fourth of the correctly identified complexes, we also correctly recognize at least one residue (five on average belonging to the interaction interface. Given the present and continuously increasing number of proteins of known structure, the requirement of the knowledge of the structure of the interacting proteins does not substantially impact on the coverage of our strategy that can be estimated to be around 25%. We also introduce here the Estrella server that embodies this strategy, is designed for users interested in validating specific hypotheses about the functional role of a protein-protein interaction and it also allows access to pre-computed data for seven organisms.

  3. Detecting mutually exclusive interactions in protein-protein interaction maps.

    KAUST Repository

    Sánchez Claros, Carmen

    2012-06-08

    Comprehensive protein interaction maps can complement genetic and biochemical experiments and allow the formulation of new hypotheses to be tested in the system of interest. The computational analysis of the maps may help to focus on interesting cases and thereby to appropriately prioritize the validation experiments. We show here that, by automatically comparing and analyzing structurally similar regions of proteins of known structure interacting with a common partner, it is possible to identify mutually exclusive interactions present in the maps with a sensitivity of 70% and a specificity higher than 85% and that, in about three fourth of the correctly identified complexes, we also correctly recognize at least one residue (five on average) belonging to the interaction interface. Given the present and continuously increasing number of proteins of known structure, the requirement of the knowledge of the structure of the interacting proteins does not substantially impact on the coverage of our strategy that can be estimated to be around 25%. We also introduce here the Estrella server that embodies this strategy, is designed for users interested in validating specific hypotheses about the functional role of a protein-protein interaction and it also allows access to pre-computed data for seven organisms.

  4. Protein interactions in genome maintenance as novel antibacterial targets.

    Directory of Open Access Journals (Sweden)

    Aimee H Marceau

    Full Text Available Antibacterial compounds typically act by directly inhibiting essential bacterial enzyme activities. Although this general mechanism of action has fueled traditional antibiotic discovery efforts for decades, new antibiotic development has not kept pace with the emergence of drug resistant bacterial strains. These limitations have severely restricted the therapeutic tools available for treating bacterial infections. Here we test an alternative antibacterial lead-compound identification strategy in which essential protein-protein interactions are targeted rather than enzymatic activities. Bacterial single-stranded DNA-binding proteins (SSBs form conserved protein interaction "hubs" that are essential for recruiting many DNA replication, recombination, and repair proteins to SSB/DNA nucleoprotein substrates. Three small molecules that block SSB/protein interactions are shown to have antibacterial activity against diverse bacterial species. Consistent with a model in which the compounds target multiple SSB/protein interactions, treatment of Bacillus subtilis cultures with the compounds leads to rapid inhibition of DNA replication and recombination, and ultimately to cell death. The compounds also have unanticipated effects on protein synthesis that could be due to a previously unknown role for SSB/protein interactions in translation or to off-target effects. Our results highlight the potential of targeting protein-protein interactions, particularly those that mediate genome maintenance, as a powerful approach for identifying new antibacterial compounds.

  5. Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

    Science.gov (United States)

    Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

    2014-10-01

    To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome.

  6. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  7. Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

    Directory of Open Access Journals (Sweden)

    Françoise Paquet

    Full Text Available In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1 from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

  8. Database of Interacting Proteins (DIP)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The DIP database catalogs experimentally determined interactions between proteins. It combines information from a variety of sources to create a single, consistent...

  9. Deciphering protein-protein interactions. Part II. Computational methods to predict protein and domain interaction partners

    National Research Council Canada - National Science Library

    Shoemaker, Benjamin A; Panchenko, Anna R

    2007-01-01

    .... In this review we describe different approaches to predict protein interaction partners as well as highlight recent achievements in the prediction of specific domains mediating protein-protein interactions...

  10. Equilibrium and kinetic aspects of protein-DNA recognition.

    Science.gov (United States)

    Livshitz, M A; Gursky, G V; Zasedatelev, A S; Volkenstein, M V

    1979-01-01

    The specificity of regulatory protein binding to DNA is due to a complementarity between the sequence of reaction centres on the protein and the base pair sequence in the specific DNA site allowing the formation of a number of specific noncovalent bonds between the interacting entities. In the present communication the thermodynamic and kinetic aspects of these interactions are considered. The extent of binding specificity is shown to increase with an increase of the bond stability constants and with an increase in the number of ligand reaction centres. Kinetic analysis is carried out assuming that association process is very fast and that dissociation of nonspecific complexes is a rate-limiting step in the recognition of a specific binding site on DNA. The calculations show that a ligand can recognize its specific binding site on DNA within a reasonably limited time interval if the number of its reaction centres and the corresponding stability constants are strongly limited.

  11. Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

    Science.gov (United States)

    Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

    2015-12-10

    D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression.

  12. Characterization of Staphylococcus aureus Primosomal DnaD Protein: Highly Conserved C-Terminal Region Is Crucial for ssDNA and PriA Helicase Binding but Not for DnaA Protein-Binding and Self-Tetramerization.

    Directory of Open Access Journals (Sweden)

    Yen-Hua Huang

    Full Text Available The role of DnaD in the recruitment of replicative helicase has been identified. However, knowledge of the DNA, PriA, and DnaA binding mechanism of this protein for the DnaA- and PriA-directed replication primosome assemblies is limited. We characterized the DNA-binding properties of DnaD from Staphylococcus aureus (SaDnaD and analyzed its interactions with SaPriA and SaDnaA. The gel filtration chromatography analysis of purified SaDnaD and its deletion mutant proteins (SaDnaD1-195, SaDnaD1-200 and SaDnaD1-204 showed a stable tetramer in solution. This finding indicates that the C-terminal region aa 196-228 is not crucial for SaDnaD oligomerization. SaDnaD forms distinct complexes with ssDNA of different lengths. In fluorescence titrations, SaDnaD bound to ssDNA with a binding-site size of approximately 32 nt. A stable complex of SaDnaD1-195, SaDnaD1-200, and SaDnaD1-204 with ssDNA dT40 was undetectable, indicating that the C-terminal region of SaDnaD (particularly aa 205-228 is crucial for ssDNA binding. The SPR results revealed that SaDnaD1-195 can interact with SaDnaA but not with SaPriA, which may indicate that DnaD has different binding sites for PriA and DnaA. Both SaDnaD and SaDnaDY176A mutant proteins, but not SaDnaD1-195, can significantly stimulate the ATPase activity of SaPriA. Hence, the stimulation effect mainly resulted from direct contact within the protein-protein interaction, not via the DNA-protein interaction. Kinetic studies revealed that the SaDnaD-SaPriA interaction increases the Vmax of the SaPriA ATPase fivefold without significantly affecting the Km. These results indicate that the conserved C-terminal region is crucial for ssDNA and PriA helicase binding, but not for DnaA protein-binding and self-tetramerization.

  13. Re-visiting protein-centric two-tier classification of existing DNA-protein complexes

    Directory of Open Access Journals (Sweden)

    Malhotra Sony

    2012-07-01

    Full Text Available Abstract Background Precise DNA-protein interactions play most important and vital role in maintaining the normal physiological functioning of the cell, as it controls many high fidelity cellular processes. Detailed study of the nature of these interactions has paved the way for understanding the mechanisms behind the biological processes in which they are involved. Earlier in 2000, a systematic classification of DNA-protein complexes based on the structural analysis of the proteins was proposed at two tiers, namely groups and families. With the advancement in the number and resolution of structures of DNA-protein complexes deposited in the Protein Data Bank, it is important to revisit the existing classification. Results On the basis of the sequence analysis of DNA binding proteins, we have built upon the protein centric, two-tier classification of DNA-protein complexes by adding new members to existing families and making new families and groups. While classifying the new complexes, we also realised the emergence of new groups and families. The new group observed was where β-propeller was seen to interact with DNA. There were 34 SCOP folds which were observed to be present in the complexes of both old and new classifications, whereas 28 folds are present exclusively in the new complexes. Some new families noticed were NarL transcription factor, Z-α DNA binding proteins, Forkhead transcription factor, AP2 protein, Methyl CpG binding protein etc. Conclusions Our results suggest that with the increasing number of availability of DNA-protein complexes in Protein Data Bank, the number of families in the classification increased by approximately three fold. The folds present exclusively in newly classified complexes is suggestive of inclusion of proteins with new function in new classification, the most populated of which are the folds responsible for DNA damage repair. The proposed re-visited classification can be used to perform genome

  14. Protein mixtures: interactions and gelation

    OpenAIRE

    Ersch, C.

    2015-01-01

    Gelation is a ubiquitous process in the preparation of foods. As most foods are multi constituent mixtures, understanding gelation in mixtures is an important goal in food science. Here we presented a systematic investigation on the influence of molecular interactions on the gelation in protein mixtures. Gelatin gels with added globular protein and globular protein gels with added gelatin were analyzed for their gel microstructure and rheological properties. Mixed gels with altered microstruc...

  15. Regulation of PCNA-protein interactions for genome stability

    DEFF Research Database (Denmark)

    Mailand, Niels; Gibbs-Seymour, Ian; Bekker-Jensen, Simon

    2013-01-01

    Proliferating cell nuclear antigen (PCNA) has a central role in promoting faithful DNA replication, providing a molecular platform that facilitates the myriad protein-protein and protein-DNA interactions that occur at the replication fork. Numerous PCNA-associated proteins compete for binding...... to a common surface on PCNA; hence these interactions need to be tightly regulated and coordinated to ensure proper chromosome replication and integrity. Control of PCNA-protein interactions is multilayered and involves post-translational modifications, in particular ubiquitylation, accessory factors...... and regulated degradation of PCNA-associated proteins. This regulatory framework allows cells to maintain a fine-tuned balance between replication fidelity and processivity in response to DNA damage....

  16. Cobalt (II) complex with novel unsymmetrical tetradentate Schiff base (ON) ligand: in vitro cytotoxicity studies of complex, interaction with DNA/protein, molecular docking studies, and antibacterial activity.

    Science.gov (United States)

    Shokohi-Pour, Zahra; Chiniforoshan, Hossein; Sabzalian, Mohammad R; Esmaeili, Seyed-Alireza; Momtazi-Borojeni, Amir Abbas

    2017-03-08

    [C20H17N3O2] and cobalt (II) complex [Co(L(2))(MeOH)2].ClO4, (L(2) = 4-((E)-1-((2-(((E)-pyridin-2-ylmethylene) amino) phenyl) imino) ethyl) benzene-1, 3-diol) novel Schiff base has been synthesiszed and chracterized by Fourier transform infrared, UV-vis, (1)H-NMR spectroscopy, and elemental analysis techniques. The interaction of Co(II) complex with DNA and BSA was investigated by electronic absorption spectroscopy, fluorescence spectroscopy, circular dichroism, and thermal denaturation studies. Our experiments indicate that this complex could strongly bind to CT-DNA via minor groove mechanism. In addition, fluorescence spectrometry of BSA with the complex showed that the fluorescence quenching mechanism of BSA was of static type. The complex exhibited significant in vitro cytotoxicity against three human cancer cell lines (JURKAT, SKOV3, and U87). The molecular docking experiment effectively proved the binding of complex to DNA and BSA. Finally, antibacterial assay over gram-positive and gram-negative pathogenic bacterial strains was studied.

  17. Insights on protein-DNA recognition by coarse grain modelling

    Science.gov (United States)

    Poulain, Pierre; Saladin, Adrien; Hartmann, Brigitte; Prévost, Chantal

    2008-01-01

    Coarse grain modelling of macromolecules is a new approach potentially well adapted to answer numerous issues, ranging from physics to biology. We propose here an original DNA coarse grain model specifically dedicated to protein–DNA docking, a crucial, but still largely unresolved, question in molecular biology. Using a representative set of protein–DNA complexes, we first show that our model is able to predict the interaction surface between the macromolecular partners taken in their bound form. In a second part, the impact of the DNA sequence and electrostatics, together with the DNA and protein conformations on docking is investigated. Our results strongly suggest that the overall DNA structure mainly contributes in discriminating the interaction site on cognate proteins. Direct electrostatic interactions between phosphate groups and amino acids side chains strengthen the binding. Overall, this work demonstrates that coarse grain modelling can reveal itself a precious auxiliary for a general and complete description and understanding of protein–DNA association mechanisms. PMID:18478582

  18. Effective interactions of DNA-stars

    Science.gov (United States)

    Abaurrea Velasco, Clara; Likos, Christos N.; Kahl, Gerhard

    2015-09-01

    We put forward a model that allows the calculation of the effective potential of two interacting DNA-stars, i.e., three-armed, Y-shaped, charged macromolecules, built up by three intertwined single-stranded DNAs. These particles are assumed to float on a flat interface separating two media with different dielectric properties. As the only input, our model requires the charge density along the branches and the interaction between two infinitesimally short segments, along two interacting rods. With this effective interaction at hand, a detailed investigations of the self-assembly scenarios of these molecules either via computer simulations or via theoretical frameworks comes within reach.

  19. A simple motif for protein recognition in DNA secondary structures.

    Science.gov (United States)

    Landt, Stephen G; Ramirez, Alejandro; Daugherty, Matthew D; Frankel, Alan D

    2005-09-02

    DNA in a single-stranded form (ssDNA) exists transiently within the cell and comprises the telomeres of linear chromosomes and the genomes of some DNA viruses. As with RNA, in the single-stranded state, some DNA sequences are able to fold into complex secondary and tertiary structures that may be recognized by proteins and participate in gene regulation. To better understand how such DNA elements might fold and interact with proteins, and to compare recognition features to those of a structured RNA, we used in vitro selection to identify ssDNAs that bind an RNA-binding peptide from the HIV Rev protein with high affinity and specificity. The large majority of selected binders contain a non-Watson-Crick G.T base-pair and an adjacent C:G base-pair and both are essential for binding. This GT motif can be presented in different DNA contexts, including a nearly perfect duplex and a branched three-helix structure, and appears to be recognized in large part by arginine residues separated by one turn of an alpha-helix. Interestingly, a very similar GT motif is necessary also for protein binding and function of a well-characterized model ssDNA regulatory element from the proenkephalin promoter.

  20. DNA detection using recombination proteins.

    Directory of Open Access Journals (Sweden)

    Olaf Piepenburg

    2006-07-01

    Full Text Available DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA, couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid-based tests.

  1. 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

  2. Protein-protein interactions as drug targets.

    Science.gov (United States)

    Skwarczynska, Malgorzata; Ottmann, Christian

    2015-01-01

    Modulation of protein-protein interactions (PPIs) is becoming increasingly important in drug discovery and chemical biology. While a few years ago this 'target class' was deemed to be largely undruggable an impressing number of publications and success stories now show that targeting PPIs with small, drug-like molecules indeed is a feasible approach. Here, we summarize the current state of small-molecule inhibition and stabilization of PPIs and review the active molecules from a structural and medicinal chemistry angle, especially focusing on the key examples of iNOS, LFA-1 and 14-3-3.

  3. Multiple DNA Interactions Contribute to the Initiation of Telomerase Elongation.

    Science.gov (United States)

    Karademir Andersson, Ahu; Gustafsson, Cecilia; Krishnankutty, Roopesh; Cohn, Marita

    2017-07-07

    Telomerase maintains telomere length and chromosome integrity by adding short tandem repeats of single-stranded DNA to the 3' ends, via reverse transcription of a defined template region of its RNA subunit. To further understand the telomerase elongation mechanism, we studied the primer utilization and extension activity of the telomerase from the budding yeast Naumovozyma castellii (Saccharomyces castellii), which displays a processive nucleotide and repeat addition polymerization. For the efficient initiation of canonical elongation, telomerase required 4-nt primer 3' end complementarity to the template RNA. This DNA-RNA hybrid formation was highly important for the stabilization of an initiation-competent telomerase-DNA complex. Anchor site interactions with the DNA provided additional stabilization to the complex. Our studies indicate three additional separate interactions along the length of the DNA primer, each providing different and distinct contributions to the initiation event. A sequence-independent anchor site interaction acts immediately adjacent to the base-pairing 3' end, indicating a protein anchor site positioned very close to the catalytic site. Two additional anchor regions further 5' on the DNA provide sequence-specific contributions to the initiation of elongation. Remarkably, a non-telomeric sequence in the distal 25- to 32-nt region negatively influences the initiation of telomerase elongation, suggesting an anchor site with a regulatory role in the telomerase elongation decision. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Screening and identification of proteins interacting with nucleostemin

    Institute of Scientific and Technical Information of China (English)

    Hai-Xia Yang; Geng-Lin Jin; Ling Meng; Jian-Zhi Zhang; Wen-Bin Liu; Cheng-Chao Shou

    2005-01-01

    AIM: To identify the proteins interacting with nucleostemin (NS), thereby gaining an insight into the function of NS.METHODS: Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and β-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing.To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively.Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially.RESULTS: Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B (B56) (PPP2R5A) and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A.CONCLUSION: NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.

  5. 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...

  6. Formaldehyde-mediated Hyperphosphorylation Disturbs The Interaction Between Tau Protein and DNA%甲醛诱导的磷酸化减弱Tau蛋白与DNA相互作用

    Institute of Scientific and Technical Information of China (English)

    卢静; 苗君叶; 潘荣; 赫荣乔

    2011-01-01

    Hyperphosphorylation of Tau protein and related neuron death is one of the most important characteristics of Alzheimer's disease. Our laboratory has shown that Tau protein is able to bind and protect DNA. It is still unknown whether phosphorylation affects the binding of Tau to DNA. Therefore, it is of importance to investigate the effect of phosphorylation on the interaction of Tau with DNA in cells. In this work, we treated N2a cells with formaldehyde and found that Tau protein was hyperphosphorylated in the cells under the experimental conditions. Phosphorylation was remarkably observed at both T181 and S396 of Tau protein in the cells in the presence of formaldehyde compared with those in the absence of formaldehyde. Cytoimmunofluorescence hardly showed that most of the nuclear phosphorylated Tau protein was co-localized with DNA, while Tau protein was partially co-localized with DNA in the absence of formaldehyde as control. Electrophoretic mobility shift assay (EMSA) showed that phosphorylated Tau catalyzed by GSK-3(3 reduced the interaction between Tau protein and DNA in vitro. These findings reveal that hyperphosphorylation declines Tau protein to protect DNA, and may thereafter lead to damage of DNA and even cell death, giving a novel viewpoint to the pathology of Alzheimer's disease.%异常磷酸化Tau蛋白是神经纤维缠结的主要成分,也是老年痴呆的典型病理特征之一.本实验室前期报道了Tau蛋白具有保护DNA的作用,但磷酸化对Tau与DNA相互作用的影响需要进行探索,这对于揭示Tau蛋白异常磷酸化与神经细胞死亡之间的关系具有一定的参考价值.本文采用甲醛孵育N2a细胞,引起了细胞内Tau蛋白的过度磷酸化.实验结果进一步显示,甲醛孵育组的细胞核内磷酸化Tau蛋白与DNA非共定位存在,而对照组细胞核内Tau蛋白与DNA存在一定程度的共定位现象.电泳迁移率实验检测GSK-33催化的磷酸化Tau蛋白与DNA的结合情况,可以观

  7. Synthesis and Structural Investigation of New Bio-Relevant Complexes of Lanthanides with 5-Hydroxyflavone: DNA Binding and Protein Interaction Studies

    Directory of Open Access Journals (Sweden)

    Alexandra-Cristina Munteanu

    2016-12-01

    Full Text Available In the present work, we attempted to develop new metal coordination complexes of the natural flavonoid 5-hydroxyflavone with Sm(III, Eu(III, Gd(III, Tb(III. The resultant hydroxo complexes have been characterized by a variety of spectroscopic techniques, including fluorescence, FT-IR, UV-Vis, EPR and mass spectral studies. The general chemical formula of the complexes is [Ln(C15H9O33(OH2(H2Ox]·nH2O, where Ln is the lanthanide cation and x = 0 for Sm(III, x = 1 for Eu(III, Gd(III, Tb(III and n = 0 for Sm(III, Gd(III, Tb(III, n = 1 for Eu(III, respectively. The proposed structures of the complexes were optimized by DFT calculations. Theoretical calculations and experimental determinations sustain the proposed structures of the hydroxo complexes, with two molecules of 5-hydroxyflavone acting as monoanionic bidentate chelate ligands. The interaction of the complexes with calf thymus DNA has been explored by fluorescence titration and UV-Vis absorption binding studies, and revealed that the synthesized complexes interact with DNA with binding constants (Kb ~ 104. Human serum albumin (HSA and transferrin (Tf binding studies have also been performed by fluorescence titration techniques (fluorescence quenching studies, synchronous fluorescence spectra. The apparent association constants (Ka and thermodynamic parameters have been calculated from the fluorescence quenching experiment at 299 K, 308 K, and 318 K. The quenching curves indicate that the complexes bind to HSA with smaller affinity than the ligand, but to Tf with higher binding affinities than the ligand.

  8. Yeast Interacting Proteins Database: YJL199C, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available d in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies...cies; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey (4) Ro...n; not conserved in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies... species; protein detected in large-scale protein-protein interaction studies Rows with this prey as prey Ro

  9. 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.

  10. Automatic Extraction of Protein Interaction in Literature

    OpenAIRE

    Liu, Peilei; Wang, Ting

    2014-01-01

    Protein-protein interaction extraction is the key precondition of the construction of protein knowledge network, and it is very important for the research in the biomedicine. This paper extracted directional protein-protein interaction from the biological text, using the SVM-based method. Experiments were evaluated on the LLL05 corpus with good results. The results show that dependency features are import for the protein-protein interaction extraction and features related to the interaction w...

  11. Prediction of Protein-Protein Interactions Using Protein Signature Profiling

    Institute of Scientific and Technical Information of China (English)

    Mahmood A. Mahdavi; Yen-Han Lin

    2007-01-01

    Protein domains are conserved and functionally independent structures that play an important role in interactions among related proteins. Domain-domain inter- actions have been recently used to predict protein-protein interactions (PPI). In general, the interaction probability of a pair of domains is scored using a trained scoring function. Satisfying a threshold, the protein pairs carrying those domains are regarded as "interacting". In this study, the signature contents of proteins were utilized to predict PPI pairs in Saccharomyces cerevisiae, Caenorhabditis ele- gans, and Homo sapiens. Similarity between protein signature patterns was scored and PPI predictions were drawn based on the binary similarity scoring function. Results show that the true positive rate of prediction by the proposed approach is approximately 32% higher than that using the maximum likelihood estimation method when compared with a test set, resulting in 22% increase in the area un- der the receiver operating characteristic (ROC) curve. When proteins containing one or two signatures were removed, the sensitivity of the predicted PPI pairs in- creased significantly. The predicted PPI pairs are on average 11 times more likely to interact than the random selection at a confidence level of 0.95, and on aver- age 4 times better than those predicted by either phylogenetic profiling or gene expression profiling.

  12. Unique and universal features of Epsilonproteobacterial origins of chromosome replication and DnaA-DnaA box interactions

    Directory of Open Access Journals (Sweden)

    Pawel Jaworski

    2016-09-01

    Full Text Available In bacteria, chromosome replication is initiated by the interaction of the initiator protein DnaA with a defined region of a chromosome at which DNA replication starts (oriC. While DnaA proteins share significant homology regardless of phylogeny, oriC regions exhibit more variable structures. The general architecture of oriCs is universal, i.e. they are composed of a cluster of DnaA binding sites, a DNA-unwinding element, and sequences that bind regulatory proteins. However, detailed structures of oriCs are shared by related species while being significantly different in unrelated bacteria. In this work, we characterised Epsilonproteobacterial oriC regions. Helicobacter pylori was the only species of the class for which oriC was characterised. A few unique features were found such as bipartite oriC structure, not encountered in any other Gram-negative species, and topology-sensitive DnaA-DNA interactions, which have not been found in any other bacterium. These unusual H. pylori oriC features raised questions of whether oriC structure and DnaA-DNA interactions are unique to this bacterium or they are common to related species. By in silico and in vitro analyses we identified putative oriCs in three Epsilonproteobacterial species: pathogenic Arcobacter butzleri, symbiotic Wolinella succinogenes and free-living Sulfurimonas denitrificans. We propose that oriCs typically co-localize with ruvC-dnaA-dnaN in Epsilonproteobacteria, with the exception of Helicobacteriaceae species. The clusters of DnaA boxes localize upstream (oriC1 and downstream (oriC2 of dnaA, and they likely constitute bipartite origins. In all cases, DNA unwinding was shown to occur in oriC2. Unlike the DnaA box pattern, which is not conserved in Epsilonproteobacterial oriCs, the consensus DnaA box sequences and the mode of DnaA-DnaA box interactions are common to the class. We propose that the typical Epsilonproteobacterial DnaA box consists of the core nucleotide sequence 5

  13. Unique and Universal Features of Epsilonproteobacterial Origins of Chromosome Replication and DnaA-DnaA Box Interactions

    Science.gov (United States)

    Jaworski, Pawel; Donczew, Rafal; Mielke, Thorsten; Thiel, Marcel; Oldziej, Stanislaw; Weigel, Christoph; Zawilak-Pawlik, Anna

    2016-01-01

    In bacteria, chromosome replication is initiated by the interaction of the initiator protein DnaA with a defined region of a chromosome at which DNA replication starts (oriC). While DnaA proteins share significant homology regardless of phylogeny, oriC regions exhibit more variable structures. The general architecture of oriCs is universal, i.e., they are composed of a cluster of DnaA binding sites, a DNA-unwinding element, and sequences that bind regulatory proteins. However, detailed structures of oriCs are shared by related species while being significantly different in unrelated bacteria. In this work, we characterized Epsilonproteobacterial oriC regions. Helicobacter pylori was the only species of the class for which oriC was characterized. A few unique features were found such as bipartite oriC structure, not encountered in any other Gram-negative species, and topology-sensitive DnaA-DNA interactions, which have not been found in any other bacterium. These unusual H. pylori oriC features raised questions of whether oriC structure and DnaA-DNA interactions are unique to this bacterium or whether they are common to related species. By in silico and in vitro analyses we identified putative oriCs in three Epsilonproteobacterial species: pathogenic Arcobacter butzleri, symbiotic Wolinella succinogenes, and free-living Sulfurimonas denitrificans. We propose that oriCs typically co-localize with ruvC-dnaA-dnaN in Epsilonproteobacteria, with the exception of Helicobacteriaceae species. The clusters of DnaA boxes localize upstream (oriC1) and downstream (oriC2) of dnaA, and they likely constitute bipartite origins. In all cases, DNA unwinding was shown to occur in oriC2. Unlike the DnaA box pattern, which is not conserved in Epsilonproteobacterial oriCs, the consensus DnaA box sequences and the mode of DnaA-DnaA box interactions are common to the class. We propose that the typical Epsilonproteobacterial DnaA box consists of the core nucleotide sequence 5′-TTCAC-3

  14. Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing.

    Science.gov (United States)

    Marshall, Owen J; Southall, Tony D; Cheetham, Seth W; Brand, Andrea H

    2016-09-01

    This protocol is an extension to: Nat. Protoc. 2, 1467-1478 (2007); doi:10.1038/nprot.2007.148; published online 7 June 2007The ability to profile transcription and chromatin binding in a cell-type-specific manner is a powerful aid to understanding cell-fate specification and cellular function in multicellular organisms. We recently developed targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling of DNA- and chromatin-binding proteins in vivo without cell isolation. As a protocol extension, this article describes substantial modifications to an existing protocol, and it offers additional applications. TaDa builds upon DamID, a technique for detecting genome-wide DNA-binding profiles of proteins, by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution. TaDa ensures that Dam-fusion proteins are expressed at very low levels, thus avoiding toxicity and potential artifacts from overexpression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to next-generation sequencing technology. TaDa is robust, reproducible and highly sensitive. Compared with other methods for cell-type-specific profiling, the technique requires no cell-sorting, cross-linking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II (Pol II), TaDa can also identify transcribed genes in a cell-type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next-generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure-from collecting tissue samples to generating sequencing libraries-can be accomplished within 5 d.

  15. Protein patterning by a DNA origami framework.

    Science.gov (United States)

    Aslan, Hüsnü; Krissanaprasit, Abhichart; Besenbacher, Flemming; Gothelf, Kurt V; Dong, Mingdong

    2016-08-18

    A spatial arrangement of proteins provides structural and functional advantages in vast technological applications as well as fundamental research. Most protein patterning procedures employ complicated, time consuming and very costly nanofabrication techniques. As an alternative route, we developed a fully biomolecular self-assembly method using DNA Origami Frames (DOF) as a template for both small and large scale protein patterning. We employed a triangular DOF (tDOF) to arrange the Bovine Serum Albumin (BSA) protein. Our in situ protein patterning strategy provides a novel, fully organic platform using a fast and low-cost surface approach with possible utilization in fundamental science and technological applications.

  16. Hepatitis B virus X protein interacts with β5 subunit of heterotrimeric guanine nucleotide binding protein

    Directory of Open Access Journals (Sweden)

    Chen Wei

    2005-08-01

    Full Text Available Abstract Background To isolate cellular proteins interacting with hepatitis B virus X protein (HBX, from HepG2 cells infected with hepatitis B virus (HBV. Results HBV particles were produced in culture medium of HepG2 cells transfected with the mammalian expression vector containing the linear HBV genome, as assessed by commercially available ELISA assay. A cDNA library was made from these cells exposed to HBV. From yeast two hybrid screening with HBX as bait, human guanine nucleotide binding protein β subunit 5L (GNβ5 was isolated from the cDNA library constructed in this study as a new HBX-interacting protein. The HBX-GNβ5 interaction was further supported by mammalian two hybrid assay. Conclusion The use of a cDNA library constructed from HBV-transfected HepG2 cells has resulted in the isolation of new cellular proteins interacting with HBX.

  17. Protein mixtures: interactions and gelation

    NARCIS (Netherlands)

    Ersch, C.

    2015-01-01

    Gelation is a ubiquitous process in the preparation of foods. As most foods are multi constituent mixtures, understanding gelation in mixtures is an important goal in food science. Here we presented a systematic investigation on the influence of molecular interactions on the gelation in protein mixt

  18. Protein mixtures: interactions and gelation

    NARCIS (Netherlands)

    Ersch, C.

    2015-01-01

    Gelation is a ubiquitous process in the preparation of foods. As most foods are multi constituent mixtures, understanding gelation in mixtures is an important goal in food science. Here we presented a systematic investigation on the influence of molecular interactions on the gelation in protein mixt

  19. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  20. The role of DNA dependent protein kinase in synapsis of DNA ends

    NARCIS (Netherlands)

    E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); J.H.J. Hoeijmakers (Jan); D.C. van Gent (Dik)

    2003-01-01

    textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

  1. The role of DNA dependent protein kinase in synapsis of DNA ends

    NARCIS (Netherlands)

    E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); D.C. van Gent (Dik); J.H.J. Hoeijmakers (Jan)

    2003-01-01

    textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

  2. New Compound Classes: Protein-Protein Interactions.

    Science.gov (United States)

    Ottmann, C

    2016-01-01

    "Protein-protein interactions (PPIs) are one of the most promising new targets in drug discovery. With estimates between 300,000 and 650,000 in human physiology, targeted modulation of PPIs would tremendously extend the "druggable" genome. In fact, in every disease a wealth of potentially addressable PPIs can be found making pharmacological intervention based on PPI modulators in principle a generally applicable technology. An impressing number of success stories in small-molecule PPI inhibition and natural-product PPI stabilization increasingly encourage academia and industry to invest in PPI modulation. In this chapter examples of both inhibition as well as stabilization of PPIs are reviewed including some of the technologies which has been used for their identification."

  3. Prediction of Protein-protein Interactions on the Basis of Evolutionary Conservation of Protein Functions

    Directory of Open Access Journals (Sweden)

    Ekaterina Kotelnikova

    2007-01-01

    Full Text Available Motivation: Although a great deal of progress is being made in the development of fast and reliable experimental techniques to extract genome-wide networks of protein-protein and protein-DNA interactions, the sequencing of new genomes proceeds at an even faster rate. That is why there is a considerable need for reliable methods of in-silico prediction of protein interaction based solely on sequence similarity information and known interactions from well-studied organisms. This problem can be solved if a dependency exists between sequence similarity and the conservation of the proteins’ functions.Results: In this paper, we introduce a novel probabilistic method for prediction of protein-protein interactions using a new empirical probabilistic formula describing the loss of interactions between homologous proteins during the course of evolution. This formula describes an evolutional process quite similar to the process of the Earth’s population growth. In addition, our method favors predictions confi rmed by several interacting pairs over predictions coming from a single interacting pair. Our approach is useful in working with “noisy” data such as those coming from high-throughput experiments. We have generated predictions for fi ve “model” organisms: H. sapiens, D. melanogaster, C. elegans, A. thaliana, and S. cerevisiae and evaluated the quality of these predictions.

  4. Interaction of maize chromatin-associated HMG proteins with mononucleosomes

    DEFF Research Database (Denmark)

    Lichota, J.; Grasser, Klaus D.

    2003-01-01

    maize HMGA and five different HMGB proteins with mononucleosomes (containing approx. 165 bp of DNA) purified from micrococcal nuclease-digested maize chromatin. The HMGB proteins interacted with the nucleosomes independent of the presence of the linker histone H1, while the binding of HMGA...

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

    Science.gov (United States)

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

    2013-12-01

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

  6. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  7. Predicting DNA-binding sites of proteins based on sequential and 3D structural information.

    Science.gov (United States)

    Li, Bi-Qing; Feng, Kai-Yan; Ding, Juan; Cai, Yu-Dong

    2014-06-01

    Protein-DNA interactions play important roles in many biological processes. To understand the molecular mechanisms of protein-DNA interaction, it is necessary to identify the DNA-binding sites in DNA-binding proteins. In the last decade, computational approaches have been developed to predict protein-DNA-binding sites based solely on protein sequences. In this study, we developed a novel predictor based on support vector machine algorithm coupled with the maximum relevance minimum redundancy method followed by incremental feature selection. We incorporated not only features of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure, solvent accessibility, but also five three-dimensional (3D) structural features calculated from PDB data to predict the protein-DNA interaction sites. Feature analysis showed that 3D structural features indeed contributed to the prediction of DNA-binding site and it was demonstrated that the prediction performance was better with 3D structural features than without them. It was also shown via analysis of features from each site that the features of DNA-binding site itself contribute the most to the prediction. Our prediction method may become a useful tool for identifying the DNA-binding sites and the feature analysis described in this paper may provide useful insights for in-depth investigations into the mechanisms of protein-DNA interaction.

  8. Interaction of sulforaphane with DNA and RNA.

    Directory of Open Access Journals (Sweden)

    Farzaneh Abassi Joozdani

    Full Text Available Sulforaphane (SFN is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN-DNA and -RNA complexes by Fourier transform infrared (FTIR and UV-Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2, while RNA binding is through G, U, A bases with some degree of SFN-phosphate (PO2 interaction. Overall binding constants were estimated to be K(SFN-DNA=3.01 (± 0.035×10(4 M(-1 and K(SFN-RNA= 6.63 (±0.042×10(3 M(-1. At high SFN concentration (SFN/RNA = 1/1, DNA conformation changed from B to A occurred, while RNA remained in A-family structure.

  9. Interaction of Sulforaphane with DNA and RNA

    Science.gov (United States)

    Abassi Joozdani, Farzaneh; Yari, Faramarz; Abassi Joozdani, Parvaneh; Nafisi, Shohreh

    2015-01-01

    Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO2) interaction. Overall binding constants were estimated to be K(SFN–DNA)=3.01 (± 0.035)×104 M-1 and K(SFN–RNA)= 6.63 (±0.042)×103 M-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure. PMID:26030290

  10. Preliminary Study on Cordycepin-DNA Interaction by Raman Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    Jian Ya LING; Qin Zheng YANG; Shan Shan LUO; Yan LI; Chang Kai ZHANG

    2005-01-01

    The interaction of cordycepin with calf thymus DNA was investigated at physiological pH with drug/DNA molar ratio of 8. The Raman spectroscopy results indicated that the intercalation of high concentration cordycepin and the interaction of cordycepin with PO2 group led to a major reduction of B-form DNA structure in favor of A-form DNA.

  11. The synergy between protein positioning and DNA elasticity: energy minimization of protein-decorated DNA minicircles

    CERN Document Server

    Clauvelin, Nicolas

    2014-01-01

    The binding of proteins onto DNA contributes to the shaping and packaging of genome as well as to the expression of specific genetic messages. With a view to understanding the interplay between the presence of proteins and the deformation of DNA involved in such processes, we developed a new method to minimize the elastic energy of DNA fragments at the mesoscale level. Our method makes it possible to obtain the optimal pathways of protein-decorated DNA molecules for which the terminal base pairs are spatially constrained. We focus in this work on the deformations induced by selected architectural proteins on circular DNA. We report the energy landscapes of DNA minicircles subjected to different levels of torsional stress and containing one or two proteins as functions of the chain length and spacing between the proteins. Our results reveal cooperation between the elasticity of the double helix and the structural distortions of DNA induced by bound proteins. We find that the imposed mechanical stress influence...

  12. Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication, SirA, with domain I of DnaA.

    Science.gov (United States)

    Jameson, Katie H; Rostami, Nadia; Fogg, Mark J; Turkenburg, Johan P; Grahl, Anne; Murray, Heath; Wilkinson, Anthony J

    2014-09-01

    Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA-interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N-terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α-helical with predominantly apolar side-chains packing in a hydrophobic interface. Site-directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP-SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA-interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor.

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

    Science.gov (United States)

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

    2007-06-01

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

  14. JavaScript DNA translator: DNA-aligned protein translations.

    Science.gov (United States)

    Perry, William L

    2002-12-01

    There are many instances in molecular biology when it is necessary to identify ORFs in a DNA sequence. While programs exist for displaying protein translations in multiple ORFs in alignment with a DNA sequence, they are often expensive, exist as add-ons to software that must be purchased, or are only compatible with a particular operating system. JavaScript DNA Translator is a shareware application written in JavaScript, a scripting language interpreted by the Netscape Communicator and Internet Explorer Web browsers, which makes it compatible with several different operating systems. While the program uses a familiar Web page interface, it requires no connection to the Internet since calculations are performed on the user's own computer. The program analyzes one or multiple DNA sequences and generates translations in up to six reading frames aligned to a DNA sequence, in addition to displaying translations as separate sequences in FASTA format. ORFs within a reading frame can also be displayed as separate sequences. Flexible formatting options are provided, including the ability to hide ORFs below a minimum size specified by the user. The program is available free of charge at the BioTechniques Software Library (www.Biotechniques.com).

  15. Daunomycin, an antitumor DNA intercalator, influences histone-DNA interactions.

    Science.gov (United States)

    Wójcik, Krzysztof; Zarębski, Mirosław; Cossarizza, Andrea; Dobrucki, Jurek W

    2013-09-01

    Although daunomycin and adriamycin are considered effective antitumor drugs and have been used in the clinic for over 40 years, their mechanism of action is still a matter of debate. We investigated the influence of daunomycin on interaction between linker or core histones and DNA in live HeLa cells in vitro, using image and flow cytometry. Exposure to daunomycin at clinically relevant concentrations (25-250 nM) caused dissociation of wild-type H1.1 as well as 4 H1 point mutants from DNA, followed by their accumulation in nucleoli and aggregation of chromatin. A detectable dissociation of H2B core histones occurred only at much higher concentrations of the drug (500 nM). Replication of DNA and synthesis of RNA were not halted by daunomycin (up to 2500 nM); however the characteristic subnuclear distribution of sites of transcription and replication was lost. Dissociation of the H1.1 linker histones and subsequent loss of higher order chromatin structures may constitute an important component of the mechanism of cytotoxicity of daunomycin.

  16. Conjugation of fluorescent proteins with DNA oligonucleotides.

    Science.gov (United States)

    Lapiene, Vidmantas; Kukolka, Florian; Kiko, Kathrin; Arndt, Andreas; Niemeyer, Christof M

    2010-05-19

    This work describes the synthesis of covalent ssDNA conjugates of six fluorescent proteins, ECFP, EGFP, E(2)GFP, mDsRed, Dronpa, and mCherry, which were cloned with an accessible C-terminal cystein residue to enable site-selective coupling using a heterobispecific cross-linker. The resulting conjugates revealed similar fluorescence emission intensity to the unconjugated proteins, and the functionality of the tethered oligonucleotide was proven by specific Watson-Crick base pairing to cDNA-modified gold nanoparticles. Fluorescence spectroscopy analysis indicated that the fluorescence of the FP is quenched by the gold particle, and the extent of quenching varied with the intrinsic spectroscopic properties of FP as well as with the configuration of surface attachment. Since this study demonstrates that biological fluorophores can be selectively incorporated into and optically coupled with nanoparticle-based devices, applications in DNA-based nanofabrication can be foreseen.

  17. Coevolution of gene expression among interacting proteins

    OpenAIRE

    2004-01-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically inter...

  18. The Mitochondrial DNA (mtDNA)-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination

    KAUST Repository

    Blomme, Jonas

    2017-04-19

    In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana. Gainand loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.

  19. Interplay between human high mobility group protein 1 and replication protein A on psoralen-cross-linked DNA

    DEFF Research Database (Denmark)

    Reddy, Madhava C; Christensen, Jesper; Vasquez, Karen M

    2005-01-01

    Human high mobility group box (HMGB) 1 and -2 proteins are highly conserved and abundant chromosomal proteins that regulate chromatin structure and DNA metabolism. HMGB proteins bind preferentially to DNA that is bent or underwound and to DNA damaged by agents such as cisplatin, UVC radiation......, and benzo[a]pyrenediol epoxide (BPDE). Binding of HMGB1 to DNA adducts is thought to inhibit nucleotide excision repair (NER), leading to cell death, but the biological roles of these proteins remain obscure. We have used psoralen-modified triplex-forming oligonucleotides (TFOs) to direct a psoralen-DNA...... interstrand cross-link (ICL) to a specific site to determine the effect of HMGB proteins on recognition of these lesions. Our results reveal that human HMGB1 (but not HMGB2) binds with high affinity and specificity to psoralen ICLs, and interacts with the essential NER protein, replication protein A (RPA...

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

    Science.gov (United States)

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

    2009-08-15

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

  1. A facile method for reversibly linking a recombinant protein to DNA.

    Science.gov (United States)

    Goodman, Russell P; Erben, Christoph M; Malo, Jonathan; Ho, Wei M; McKee, Mireya L; Kapanidis, Achillefs N; Turberfield, Andrew J

    2009-06-15

    We present a facile method for linking recombinant proteins to DNA. It is based on the nickel-mediated interaction between a hexahistidine tag (His(6)-tag) and DNA functionalized with three nitrilotriacetic acid (NTA) groups. The resulting DNA-protein linkage is site-specific. It can be broken quickly and controllably by the addition of a chelating agent that binds nickel. We have used this new linker to bind proteins to a variety of DNA motifs commonly used in the fabrication of nanostructures by DNA self-assembly.

  2. Photosensitive surfactants: micellization and interaction with DNA.

    Science.gov (United States)

    Zakrevskyy, Yuriy; Roxlau, Julian; Brezesinski, Gerald; Lomadze, Nino; Santer, Svetlana

    2014-01-28

    Recently, photosensitive surfactants have re-attracted considerable attention. It has been shown that their association with oppositely charged biologically important polyelectrolytes, such as DNA or microgels, can be efficiently manipulated simply by light exposure. In this article, we investigate the self-assembly of photosensitive surfactants as well as their interactions with DNA by calorimetric and spectroscopic methods. Critical micelle concentration (CMC), standard micellization enthalpy, entropy, and Gibbs energy were determined in different conditions (ionic strengths and temperatures) for a series of cationic surfactants with an azobenzene group in their tail. It is shown, that aggregation forces of photosensitive units play an important role in the micellization giving the major contribution to the micellization enthalpy. The onset of the aggregation can be traced from shift of the absorption peak position in the UV-visible spectrum. Titration UV-visible spectroscopy is used as an alternative, simple, and sensitive approach to estimate CMC. The titration UV-visible spectroscopy was also employed to investigate interactions (CAC: critical aggregation concentration, precipitation, and colloidal stabilization) in the DNA-surfactant complex.

  3. Photosensitive surfactants: Micellization and interaction with DNA

    Science.gov (United States)

    Zakrevskyy, Yuriy; Roxlau, Julian; Brezesinski, Gerald; Lomadze, Nino; Santer, Svetlana

    2014-01-01

    Recently, photosensitive surfactants have re-attracted considerable attention. It has been shown that their association with oppositely charged biologically important polyelectrolytes, such as DNA or microgels, can be efficiently manipulated simply by light exposure. In this article, we investigate the self-assembly of photosensitive surfactants as well as their interactions with DNA by calorimetric and spectroscopic methods. Critical micelle concentration (CMC), standard micellization enthalpy, entropy, and Gibbs energy were determined in different conditions (ionic strengths and temperatures) for a series of cationic surfactants with an azobenzene group in their tail. It is shown, that aggregation forces of photosensitive units play an important role in the micellization giving the major contribution to the micellization enthalpy. The onset of the aggregation can be traced from shift of the absorption peak position in the UV-visible spectrum. Titration UV-visible spectroscopy is used as an alternative, simple, and sensitive approach to estimate CMC. The titration UV-visible spectroscopy was also employed to investigate interactions (CAC: critical aggregation concentration, precipitation, and colloidal stabilization) in the DNA-surfactant complex.

  4. NMR studies of a new family of DNA binding proteins: the THAP proteins

    Energy Technology Data Exchange (ETDEWEB)

    Gervais, Virginie, E-mail: virginie.gervais@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France); Campagne, Sebastien [ETH Zurich (Switzerland); Durand, Jade; Muller, Isabelle; Milon, Alain, E-mail: alain.milon@ipbs.fr [IPBS (Institut de Pharmacologie et de Biologie Structurale), CNRS (France)

    2013-05-15

    The THAP (THanatos-Associated Protein) domain is an evolutionary conserved C2CH zinc-coordinating domain shared with a large family of cellular factors (THAP proteins). Many members of the THAP family act as transcription factors that control cell proliferation, cell cycle progression, angiogenesis, apoptosis and epigenetic gene silencing. They recognize specific DNA sequences in the promoters of target genes and subsequently recruit effector proteins. Recent structural and functional studies have allowed getting better insight into the nuclear and cellular functions of some THAP members and the molecular mechanisms by which they recognize DNA. The present article reviews recent advances in the knowledge of the THAP domains structures and their interaction with DNA, with a particular focus on NMR. It provides the solution structure of the THAP domain of THAP11, a recently characterized human THAP protein with important functions in transcription and cell growth in colon cancer.

  5. Mapping DNA-Lac repressor interaction with ultra-fast optical tweezers

    Science.gov (United States)

    Monico, Carina; Tempestini, Alessia; Vanzi, Francesco; Pavone, Francesco S.; Capitanio, Marco

    2015-03-01

    The lac operon is a well-known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with different DNA constructs. Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operator sequences and transient interactions with nonspecific sequences.

  6. Interactions within the mammalian DNA methyltransferase family

    Directory of Open Access Journals (Sweden)

    Ehrenhofer-Murray Ann E

    2003-05-01

    Full Text Available Abstract Background In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity. Results The role played by the N-terminal domain in this regulation has been investigated using the yeast two-hybrid system. We show here the presence of an intra-molecular interaction in Dnmt1 but not in Dnmt3a or Dnmt3b. This interaction was confirmed by immunoprecipitation and was localized by deletion mapping. Furthermore, a systematic analysis of interactions among the Dnmt family members has revealed that DNMT3L interacts with the C-terminal domain of Dnmt3a and Dnmt3b. Conclusions The lack of methylating ability of the isolated C-terminal domain of Dnmt1 could be explained in part by a physical interaction between N- and C-terminal domains that apparently is required for activation of the catalytic domain. Our deletion analysis suggests that the tertiary structure of Dnmt1 is important in this process rather than a particular sequence motif. Furthermore, the interaction between DNMT3L and the C-terminal domains of Dnmt3a and Dnmt3b suggests a mechanism whereby the enzymatically inactive DNMT3L brings about the methylation of its substrate by recruiting an active methylase.

  7. DNA-Templated Introduction of an Aldehyde Handle in Proteins

    DEFF Research Database (Denmark)

    Kodal, Anne Louise Bank; Rosen, Christian Bech; Mortensen, Michael Rosholm;

    2016-01-01

    -templated reductive amination we create DNA-protein conjugates with control over labeling stoichiometry without genetic engineering. A guiding DNA strand with a metal-binding functionality facilitates site-selectivity by directing coupling of a second reactive DNA strand to the vicinity of a protein metal......-binding site. Here, we demonstrate DNA-templated reductive amination for His6-tagged proteins and native metal-binding proteins, including IgG1 antibodies. We also use a cleavable linker between the DNA and the protein to remove the DNA and introduce a single aldehyde to proteins. This functions as a handle...

  8. Pursuing DNA catalysts for protein modification.

    Science.gov (United States)

    Silverman, Scott K

    2015-05-19

    Catalysis is a fundamental chemical concept, and many kinds of catalysts have considerable practical value. Developing entirely new catalysts is an exciting challenge. Rational design and screening have provided many new small-molecule catalysts, and directed evolution has been used to optimize or redefine the function of many protein enzymes. However, these approaches have inherent limitations that prompt the pursuit of different kinds of catalysts using other experimental methods. Nature evolved RNA enzymes, or ribozymes, for key catalytic roles that in modern biology are limited to phosphodiester cleavage/ligation and amide bond formation. Artificial DNA enzymes, or deoxyribozymes, have great promise for a broad range of catalytic activities. They can be identified from unbiased (random) sequence populations as long as the appropriate in vitro selection strategies can be implemented for their identification. Notably, in vitro selection is different in key conceptual and practical ways from rational design, screening, and directed evolution. This Account describes the development by in vitro selection of DNA catalysts for many different kinds of covalent modification reactions of peptide and protein substrates, inspired in part by our earlier work with DNA-catalyzed RNA ligation reactions. In one set of studies, we have sought DNA-catalyzed peptide backbone cleavage, with the long-term goal of artificial DNA-based proteases. We originally anticipated that amide hydrolysis should be readily achieved, but in vitro selection instead surprisingly led to deoxyribozymes for DNA phosphodiester hydrolysis; this was unexpected because uncatalyzed amide bond hydrolysis is 10(5)-fold faster. After developing a suitable selection approach that actively avoids DNA hydrolysis, we were able to identify deoxyribozymes for hydrolysis of esters and aromatic amides (anilides). Aliphatic amide cleavage remains an ongoing focus, including via inclusion of chemically modified DNA

  9. Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

    Directory of Open Access Journals (Sweden)

    Marijn T J van Loenhout

    Full Text Available The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

  10. Scanning a DNA molecule for bound proteins using hybrid magnetic and optical tweezers.

    Science.gov (United States)

    van Loenhout, Marijn T J; De Vlaminck, Iwijn; Flebus, Benedetta; den Blanken, Johan F; Zweifel, Ludovit P; Hooning, Koen M; Kerssemakers, Jacob W J; Dekker, Cees

    2013-01-01

    The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ~17 nm spatial resolution. An offset of 33 ± 5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/[Formula: see text]. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions.

  11. Yeast Interacting Proteins Database: YDR425W, YGL161C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available icles; computational analysis of large-scale protein-protein interaction data sug...olgi vesicles; computational analysis of large-scale protein-protein interaction data suggests a possible ro

  12. Yeast Interacting Proteins Database: YPL070W, YOR155C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available utational analysis of large-scale protein-protein interaction data suggests a possible role in transcription...9 domain; computational analysis of large-scale protein-protein interaction data suggests a possible role in

  13. Yeast Interacting Proteins Database: YPL070W, YLR245C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available utational analysis of large-scale protein-protein interaction data suggests a possible role in transcription...Vps9 domain; computational analysis of large-scale protein-protein interaction data suggests a possible role

  14. Yeast Interacting Proteins Database: YPL070W, YPR193C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available utational analysis of large-scale protein-protein interaction data suggests a possible role in transcription...in; computational analysis of large-scale protein-protein interaction data suggests a possible role in trans

  15. Yeast Interacting Proteins Database: YGL161C, YDR084C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available les; computational analysis of large-scale protein-protein interaction data suggests a possible role in vesi...GTPases, localized to late Golgi vesicles; computational analysis of large-scale protein-protein interaction

  16. Yeast Interacting Proteins Database: YDL226C, YGL198W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available omputational analysis of large-scale protein-protein interaction data suggests a ... computational analysis of large-scale protein-protein interaction data suggests a possible role in vesicle-

  17. Probing protein-sugar interactions.

    Science.gov (United States)

    Ebel, C; Eisenberg, H; Ghirlando, R

    2000-01-01

    We have investigated the partial specific volumes (2) (ml/g), hydration, and cosolvent interactions of rabbit muscle aldolase by equilibrium sedimentation in the analytical ultracentrifuge and by direct density increment (partial differential/partial differentialc(2))(mu) measurements over a range of sugar concentrations and temperature. In a series of sugars increasing in size, glucose, sucrose, raffinose, and alpha-cyclodextrin, (partial differential/ partial differentialc(2))(mu) decreases linearly with the solvent density rho(0). These sugar cosolvents do not interact with the protein; however, the interaction parameter B(1) (g water/g protein) mildly increases with increasing sugar size. The experimental B(1) values are smaller than values calculated by excluded volume (rolling ball) considerations. B(1) relates to hydration in this and in other instances studied. It decreases with increasing temperature, leading to an increase in (2) due to reduced water of hydration electrostriction. The density increments (partial differential/ partial differentialc(2))(mu), however, decrease in concave up form in the case of glycerol and in concave down form for trehalose, leading to more complex behavior in the case of carbohydrates playing a biological role as osmolytes and antifreeze agents. A critical discussion, based on the thermodynamics of multicomponent solutions, is presented.

  18. Modelling of DNA-protein recognition

    Science.gov (United States)

    Rein, R.; Garduno, R.; Colombano, S.; Nir, S.; Haydock, K.; Macelroy, R. D.

    1980-01-01

    Computer model-building procedures using stereochemical principles together with theoretical energy calculations appear to be, at this stage, the most promising route toward the elucidation of DNA-protein binding schemes and recognition principles. A review of models and bonding principles is conducted and approaches to modeling are considered, taking into account possible di-hydrogen-bonding schemes between a peptide and a base (or a base pair) of a double-stranded nucleic acid in the major groove, aspects of computer graphic modeling, and a search for isogeometric helices. The energetics of recognition complexes is discussed and several models for peptide DNA recognition are presented.

  19. Inferring protein-protein interaction complexes from immunoprecipitation data

    NARCIS (Netherlands)

    Kutzera, J.; Hoefsloot, H.C.J.; Malovannaya, A.; Smit, A.B.; Van Mechelen, I.; Smilde, A.K.

    2013-01-01

    BACKGROUND: Protein inverted question markprotein interactions in cells are widely explored using small inverted question markscale experiments. However, the search for protein complexes and their interactions in data from high throughput experiments such as immunoprecipitation is still a challenge.

  20. Inferring protein-protein interaction complexes from immunoprecipitation data

    NARCIS (Netherlands)

    Kutzera, J.; Hoefsloot, H.C.J.; Malovannaya, A.; Smit, A.B.; Van Mechelen, I.; Smilde, A.K.

    2013-01-01

    BACKGROUND: Protein inverted question markprotein interactions in cells are widely explored using small inverted question markscale experiments. However, the search for protein complexes and their interactions in data from high throughput experiments such as immunoprecipitation is still a challenge.

  1. Human cytomegalovirus IE2 protein interacts with transcription activating factors

    Institute of Scientific and Technical Information of China (English)

    XU; Jinping(徐进平); YE; Linbai(叶林柏)

    2002-01-01

    The human cytomegalovirus (HCMV) IE86 Cdna was cloned into Pgex-2T and fusion protein GST-IE86 was expressed in E. Coli. SDS-PAGE and Western blot assay indicated that fusion protein GST-IE86 with molecular weight of 92 ku is soluble in the supernatant of cell lysate. Protein GST and fusion protein GST-IE86 were purified by affinity chromatography. The technology of co-separation and specific affinity chromatography was used to study the interactions of HCMV IE86 protein with some transcriptional regulatory proteins and transcriptional factors. The results indicated that IE86 interacts separately with transcriptional factor TFIIB and promoter DNA binding transcription trans-activating factors SP1, AP1 and AP2 to form a heterogenous protein complex. These transcriptional trans-activating factors, transcriptional factor and IE86 protein were adsorbed and retained in the affinity chromatography simultaneously. But IE86 protein could not interact with NF-Кb, suggesting that the function of IE86 protein that can interact with transcriptional factor and transcriptional trans-activating factors has no relevance to protein glycosylation. IE86 protein probably has two domains responsible for binding transcriptional trans-activating regulatory proteins and transcriptional factors respectively, thus activating the transcription of many genes. The interactions accelerated the assembly of the transcriptional initiation complexes.

  2. Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage

    Directory of Open Access Journals (Sweden)

    Olsen Birgitte B

    2012-03-01

    Full Text Available Abstract Background The DNA-dependent protein kinase (DNA-PK is a nuclear complex composed of a large catalytic subunit (DNA-PKcs and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. Results In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. Conclusions Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  4. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-06-25

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  5. Pharmacogenomic study using bio- and nanobioelectrochemistry: Drug-DNA interaction.

    Science.gov (United States)

    Hasanzadeh, Mohammad; Shadjou, Nasrin

    2016-04-01

    Small molecules that bind genomic DNA have proven that they can be effective anticancer, antibiotic and antiviral therapeutic agents that affect the well-being of millions of people worldwide. Drug-DNA interaction affects DNA replication and division; causes strand breaks, and mutations. Therefore, the investigation of drug-DNA interaction is needed to understand the mechanism of drug action as well as in designing DNA-targeted drugs. On the other hand, the interaction between DNA and drugs can cause chemical and conformational modifications and, thus, variation of the electrochemical properties of nucleobases. For this purpose, electrochemical methods/biosensors can be used toward detection of drug-DNA interactions. The present paper reviews the drug-DNA interactions, their types and applications of electrochemical techniques used to study interactions between DNA and drugs or small ligand molecules that are potentially of pharmaceutical interest. The results are used to determine drug binding sites and sequence preference, as well as conformational changes due to drug-DNA interactions. Also, the intention of this review is to give an overview of the present state of the drug-DNA interaction cognition. The applications of electrochemical techniques for investigation of drug-DNA interaction were reviewed and we have discussed the type of qualitative or quantitative information that can be obtained from the use of each technique.

  6. Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing

    Energy Technology Data Exchange (ETDEWEB)

    Liang Liang; Dou Peng; Dong Mingming; Ke Xiaokang; Bian Ningsheng [School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China); Liu Zhen, E-mail: zhenliu@nju.edu.cn [School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China)

    2009-09-14

    Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05 {+-} 0.83 x 10{sup -3} s{sup -1}. The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.

  7. The escherichia coli chromosome replication initiator protein, DnaA

    DEFF Research Database (Denmark)

    Nyborg, Malene

    The experimental work presented in this thesis involve mutational analysis of the DNA binding domain of the DnaA protein and analysis of the A184V substitution in the ATP area of domain III and other amino acid substitutions found in the DnaA5 and DnaA4G proteins....

  8. Discovery of a Potent Inhibitor of Replication Protein A Protein-Protein Interactions Using a Fragment-Linking Approach

    Energy Technology Data Exchange (ETDEWEB)

    Frank, Andreas O.; Feldkamp, Michael D.; Kennedy, J. Phillip; Waterson, Alex G.; Pelz, Nicholas F.; Patrone, James D.; Vangamudi, Bhavatarini; Camper, DeMarco V.; Rossanese, Olivia W.; Chazin, Walter J.; Fesik, Stephen W. [Vanderbilt; (Vanderbilt-MED)

    2013-10-22

    Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA)-binding protein, is involved in nearly all cellular DNA transactions. The RPA N-terminal domain (RPA70N) is a recruitment site for proteins involved in DNA-damage response and repair. Selective inhibition of these protein–protein interactions has the potential to inhibit the DNA-damage response and to sensitize cancer cells to DNA-damaging agents without affecting other functions of RPA. To discover a potent, selective inhibitor of the RPA70N protein–protein interactions to test this hypothesis, we used NMR spectroscopy to identify fragment hits that bind to two adjacent sites in the basic cleft of RPA70N. High-resolution X-ray crystal structures of RPA70N–ligand complexes revealed how these fragments bind to RPA and guided the design of linked compounds that simultaneously occupy both sites. We have synthesized linked molecules that bind to RPA70N with submicromolar affinity and minimal disruption of RPA’s interaction with ssDNA.

  9. A stochastic reaction-diffusion model for protein aggregation on DNA

    Science.gov (United States)

    Voulgarakis, Nikolaos K.

    Vital functions of DNA, such as transcription and packaging, depend on the proper clustering of proteins on the double strand. The present study investigates how the interplay between DNA allostery and electrostatic interactions affects protein clustering. The statistical analysis of a simple but transparent computational model reveals two major consequences of this interplay. First, depending on the protein and salt concentration, protein filaments exhibit a bimodal DNA stiffening and softening behavior. Second, within a certain domain of the control parameters, electrostatic interactions can cause energetic frustration that forces proteins to assemble in rigid spiral configurations. Such spiral filaments might trigger both positive and negative supercoiling, which can ultimately promote gene compaction and regulate the promoter. It has been experimentally shown that bacterial histone-like proteins assemble in similar spiral patterns and/or exhibit the same bimodal behavior. The proposed model can, thus, provide computational insights into the physical mechanisms used by proteins to control the mechanical properties of the DNA.

  10. Searching target sites on DNA by proteins: Role of DNA dynamics under confinement.

    Science.gov (United States)

    Mondal, Anupam; Bhattacherjee, Arnab

    2015-10-30

    DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Analytical Ultracentrifugation as a Tool to Study Nonspecific Protein–DNA Interactions

    Science.gov (United States)

    Yang, Teng-Chieh; Catalano, Carlos Enrique; Maluf, Nasib Karl

    2016-01-01

    Analytical ultracentrifugation (AUC) is a powerful tool that can provide thermodynamic information on associating systems. Here, we discuss how to use the two fundamental AUC applications, sedimentation velocity (SV), and sedimentation equilibrium (SE), to study nonspecific protein–nucleic acid interactions, with a special emphasis on how to analyze the experimental data to extract thermodynamic information. We discuss three specific applications of this approach: (i) determination of nonspecific binding stoichiometry of E. coli integration host factor protein to dsDNA, (ii) characterization of nonspecific binding properties of Adenoviral IVa2 protein to dsDNA using SE-AUC, and (iii) analysis of the competition between specific and nonspecific DNA-binding interactions observed for E. coli integration host factor protein assembly on dsDNA. These approaches provide powerful tools that allow thermodynamic interrogation and thus a mechanistic understanding of how proteins bind nucleic acids by both specific and nonspecific interactions. PMID:26412658

  12. New approach for predicting protein-protein interactions

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Protein-protein interactions (PPIs) are of vital importance for virtually all processes of a living cell. The study of these associations of protein molecules could improve people's understanding of diseases and provide basis for therapeutic approaches.

  13. Protein-DNA electrostatics: toward a new paradigm for protein sliding.

    Science.gov (United States)

    Barbi, Maria; Paillusson, Fabien

    2013-01-01

    Gene expression and regulation rely on an apparently finely tuned set of reactions between some proteins and DNA. Such DNA-binding proteins have to find specific sequences on very long DNA molecules and they mostly do so in the absence of any active process. It has been rapidly recognized that, to achieve this task, these proteins should be efficient at both searching (i.e., sampling fast relevant parts of DNA) and finding (i.e., recognizing the specific site). A two-mode search and variants of it have been suggested since the 1970s to explain either a fast search or an efficient recognition. Combining these two properties at a phenomenological level is, however, more difficult as they appear to have antagonist roles. To overcome this difficulty, one may simply need to drop the dichotomic view inherent to the two-mode search and look more thoroughly at the set of interactions between DNA-binding proteins and a given DNA segment either specific or nonspecific. This chapter demonstrates that, in doing so in a very generic way, one may indeed find a potential reconciliation between a fast search and an efficient recognition. Although a lot remains to be done, this could be the time for a change of paradigm.

  14. Protein-DNA electrostatics: toward a new paradigm for protein sliding

    CERN Document Server

    Barbi, Maria

    2013-01-01

    Gene expression and regulation rely on an apparently finely tuned set of reactions between some proteins and DNA. Such DNA-binding proteins have to find specific sequences on very long DNA molecules and they mostly do so in absence of any active process. It has been rapidly recognized that to achieve this task these proteins should be efficient at both searching (i.e. sampling fast relevant parts of DNA) and finding (i.e. recognizing the specific site). A two-mode search and variants of it have been suggested since the 70s to explain either a fast search or an efficient recognition. Combining these two properties at a phenomenological level is however more difficult as they appear to have antagonist roles. To overcome this difficulty, one may simply need to drop the dihcotomic view inherent to the two-mode search and look more thoroughly at the set of interactions between DNA-binding proteins and a given DNA segment either specific or non-specific. This chapter demonstrates that, in doing so in a very generic...

  15. A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response

    DEFF Research Database (Denmark)

    Blasius, Melanie; Wagner, Sebastian A; Choudhary, Chuna Ram

    2014-01-01

    RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome...

  16. A Polycomb complex remains bound through DNA replication in the absence of other eukaryotic proteins

    KAUST Repository

    Lengsfeld, Bettina M.

    2012-09-17

    Propagation of chromatin states through DNA replication is central to epigenetic regulation and can involve recruitment of chromatin proteins to replicating chromatin through interactions with replication fork components. Here we show using a fully reconstituted T7 bacteriophage system that eukaryotic proteins are not required to tether the Polycomb complex PRC1 to templates during DNA replication. Instead, DNA binding by PRC1 can withstand passage of a simple replication fork.

  17. 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...... and by strengthening the interaction between mDNA and MBD proteins. Free-energy perturbation calculations also show that methylation yields favorable contribution to the binding free energy for MBD-mDNA complex....

  18. A Global Protein Kinase and Phosphatase Interaction Network in Yeast

    Science.gov (United States)

    Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

    2011-01-01

    The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

  19. The conserved Est1 protein stimulates telomerase DNA extension activity

    Science.gov (United States)

    DeZwaan, Diane C.; Freeman, Brian C.

    2009-01-01

    The first telomerase cofactor identified was the budding yeast protein Est1, which is conserved through humans. While it is evident that Est1 is required for telomere DNA maintenance, understanding its mechanistic contributions to telomerase regulation has been limited. In vitro, the primary effect of Est1 is to activate telomerase-mediated DNA extension. Although Est1 displayed specific DNA and RNA binding, neither activity contributed significantly to telomerase stimulation. Rather Est1 mediated telomerase upregulation through direct contacts with the reverse transcriptase subunit. In addition to intrinsic Est1 functions, we found that Est1 cooperatively activated telomerase in conjunction with Cdc13 and that the combinatorial effect was dependent upon a known salt-bridge interaction between Est1 (K444) and Cdc13 (E252). Our studies provide insights into the molecular events used to control the enzymatic activity of the telomerase holoenzyme. PMID:19805136

  20. Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit.

    Directory of Open Access Journals (Sweden)

    Derek S Woods

    Full Text Available DNA double strand breaks (DSBs can be generated by endogenous cellular processes or exogenous agents in mammalian cells. These breaks are highly variable with respect to DNA sequence and structure and all are recognized in some context by the DNA-dependent protein kinase (DNA-PK. DNA-PK is a critical component necessary for the recognition and repair of DSBs via non-homologous end joining (NHEJ. Previously studies have shown that DNA-PK responds differentially to variations in DSB structure, but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanisms by which DNA-PK is activated by various DNA substrates and provide evidence that the DNA-PK is differentially activated by DNA structural variations as a function of the C-terminal region of Ku80. Discrimination based on terminal DNA sequence variations, on the other hand, is independent of the Ku80 C-terminal interactions and likely results exclusively from DNA-dependent protein kinase catalytic subunit interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. These results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately the efficiency of DSB repair.

  1. Yeast Interacting Proteins Database: YEL043W, YOR164C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available on quantitative analysis of protein-protein interaction maps; may interact with ribosomes, based on co-purification studies...ing based on quantitative analysis of protein-protein interaction maps; may interact with ribosomes, based on co-purification studies

  2. The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA.

    NARCIS (Netherlands)

    D. Ristic (Dejan); C. Wyman (Claire); C. Paulusma (Coen); R. Kanaar (Roland)

    2001-01-01

    textabstractProper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gai

  3. Towards Inferring Protein Interactions: Challenges and Solutions

    Directory of Open Access Journals (Sweden)

    Ji Xiang

    2006-01-01

    Full Text Available Discovering interacting proteins has been an essential part of functional genomics. However, existing experimental techniques only uncover a small portion of any interactome. Furthermore, these data often have a very high false rate. By conceptualizing the interactions at domain level, we provide a more abstract representation of interactome, which also facilitates the discovery of unobserved protein-protein interactions. Although several domain-based approaches have been proposed to predict protein-protein interactions, they usually assume that domain interactions are independent on each other for the convenience of computational modeling. A new framework to predict protein interactions is proposed in this paper, where no assumption is made about domain interactions. Protein interactions may be the result of multiple domain interactions which are dependent on each other. A conjunctive norm form representation is used to capture the relationships between protein interactions and domain interactions. The problem of interaction inference is then modeled as a constraint satisfiability problem and solved via linear programing. Experimental results on a combined yeast data set have demonstrated the robustness and the accuracy of the proposed algorithm. Moreover, we also map some predicted interacting domains to three-dimensional structures of protein complexes to show the validity of our predictions.

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

    Science.gov (United States)

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

    2015-01-01

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

  5. MITOMI: a microfluidic platform for in vitro characterization of transcription factor-DNA interaction.

    Science.gov (United States)

    Rockel, Sylvie; Geertz, Marcel; Maerkl, Sebastian J

    2012-01-01

    Gene regulatory networks (GRNs) consist of transcription factors (TFs) that determine the level of gene expression by binding to specific DNA sequences. Mapping all TF-DNA interactions and elucidating their dynamics is a major goal to generate comprehensive models of GRNs. Measuring quantitative binding affinities of large sets of TF-DNA interactions requires the application of novel tools and methods. These tools need to cope with the difficulties related to the facts that TFs tend to be expressed at low levels in vivo, and often form only transient interactions with both DNA and their protein partners. Our approach describes a high-throughput microfluidic platform with a novel detection principle based on the mechanically induced trapping of molecular interactions (MITOMI). MITOMI allows the detection of transient and low-affinity TF-DNA interactions in high-throughput.

  6. Protein-Protein Interaction Network and Gene Ontology

    Science.gov (United States)

    Choi, Yunkyu; Kim, Seok; Yi, Gwan-Su; Park, Jinah

    Evolution of computer technologies makes it possible to access a large amount and various kinds of biological data via internet such as DNA sequences, proteomics data and information discovered about them. It is expected that the combination of various data could help researchers find further knowledge about them. Roles of a visualization system are to invoke human abilities to integrate information and to recognize certain patterns in the data. Thus, when the various kinds of data are examined and analyzed manually, an effective visualization system is an essential part. One instance of these integrated visualizations can be combination of protein-protein interaction (PPI) data and Gene Ontology (GO) which could help enhance the analysis of PPI network. We introduce a simple but comprehensive visualization system that integrates GO and PPI data where GO and PPI graphs are visualized side-by-side and supports quick reference functions between them. Furthermore, the proposed system provides several interactive visualization methods for efficiently analyzing the PPI network and GO directedacyclic- graph such as context-based browsing and common ancestors finding.

  7. Coarse-grain modelling of protein-protein interactions

    NARCIS (Netherlands)

    Baaden, Marc; Marrink, Siewert J.

    2013-01-01

    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are separate

  8. Yeast Interacting Proteins Database: YPL095C, YGL198W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available d to late Golgi vesicles; computational analysis of large-scale protein-protein interaction data suggests a ...gene name YIP4 Prey description Protein that interacts with Rab GTPases, localized to late Golgi vesicles; computation

  9. Coevolution of gene expression among interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  10. Stretching and immobilization of DNA for studies of protein–DNA interactions at the single-molecule level

    Directory of Open Access Journals (Sweden)

    Dukkipati VenkatRam

    2007-01-01

    Full Text Available AbstractSingle-molecule studies of the interactions of DNA and proteins are important in a variety of biological or biotechnology processes ranging from the protein’s search for its DNA target site, DNA replication, transcription, or repair, and genome sequencing. A critical requirement for single-molecule studies is the stretching and immobilization of otherwise randomly coiled DNA molecules. Several methods for doing so have been developed over the last two decades, including the use of forces derived from light, magnetic and electric fields, and hydrodynamic flow. Here we review the immobilization and stretching mechanisms for several of these techniques along with examples of single-molecule DNA–protein interaction assays that can be performed with each of them.

  11. Bioinformatic Prediction of WSSV-Host Protein-Protein Interaction

    Directory of Open Access Journals (Sweden)

    Zheng Sun

    2014-01-01

    Full Text Available WSSV is one of the most dangerous pathogens in shrimp aquaculture. However, the molecular mechanism of how WSSV interacts with shrimp is still not very clear. In the present study, bioinformatic approaches were used to predict interactions between proteins from WSSV and shrimp. The genome data of WSSV (NC_003225.1 and the constructed transcriptome data of F. chinensis were used to screen potentially interacting proteins by searching in protein interaction databases, including STRING, Reactome, and DIP. Forty-four pairs of proteins were suggested to have interactions between WSSV and the shrimp. Gene ontology analysis revealed that 6 pairs of these interacting proteins were classified into “extracellular region” or “receptor complex” GO-terms. KEGG pathway analysis showed that they were involved in the “ECM-receptor interaction pathway.” In the 6 pairs of interacting proteins, an envelope protein called “collagen-like protein” (WSSV-CLP encoded by an early virus gene “wsv001” in WSSV interacted with 6 deduced proteins from the shrimp, including three integrin alpha (ITGA, two integrin beta (ITGB, and one syndecan (SDC. Sequence analysis on WSSV-CLP, ITGA, ITGB, and SDC revealed that they possessed the sequence features for protein-protein interactions. This study might provide new insights into the interaction mechanisms between WSSV and shrimp.

  12. PSAIA – Protein Structure and Interaction Analyzer

    Directory of Open Access Journals (Sweden)

    Vlahoviček Kristian

    2008-04-01

    Full Text Available Abstract Background PSAIA (Protein Structure and Interaction Analyzer was developed to compute geometric parameters for large sets of protein structures in order to predict and investigate protein-protein interaction sites. Results In addition to most relevant established algorithms, PSAIA offers a new method PIADA (Protein Interaction Atom Distance Algorithm for the determination of residue interaction pairs. We found that PIADA produced more satisfactory results than comparable algorithms implemented in PSAIA. Particular advantages of PSAIA include its capacity to combine different methods to detect the locations and types of interactions between residues and its ability, without any further automation steps, to handle large numbers of protein structures and complexes. Generally, the integration of a variety of methods enables PSAIA to offer easier automation of analysis and greater reliability of results. PSAIA can be used either via a graphical user interface or from the command-line. Results are generated in either tabular or XML format. Conclusion In a straightforward fashion and for large sets of protein structures, PSAIA enables the calculation of protein geometric parameters and the determination of location and type for protein-protein interaction sites. XML formatted output enables easy conversion of results to various formats suitable for statistic analysis. Results from smaller data sets demonstrated the influence of geometry on protein interaction sites. Comprehensive analysis of properties of large data sets lead to new information useful in the prediction of protein-protein interaction sites.

  13. The role of the adenovirus DNA binding protein in DNA replication and recombination

    NARCIS (Netherlands)

    Breukelen, B. van

    2003-01-01

    Replication of adenovirus DNA in infected cells is an efficient process that, compared to cellular replication, has the use of a protein primer as a hallmark. The mechanism of this DNA replication process and especially the role of one of the replication proteins, the DNA binding protein DBP, is the

  14. Synergy of DNA-bending nucleoid proteins and macromolecular crowd-condensing DNA

    NARCIS (Netherlands)

    Bessa Ramos, E.; Wintraecken, C.H.J.M.; Geerling, A.C.M.; Vries, de R.J.

    2007-01-01

    Many prokaryotic nucleoid proteins bend DNA and form extended helical protein-DNA fibers rather than condensed structures. On the other hand, it is known that such proteins (such as bacterial HU) strongly promote DNA condensation by macromolecular crowding. Using theoretical arguments, we show that

  15. DNA-HMGB1 interaction: The nuclear aggregates of polyamine mediation.

    Science.gov (United States)

    Iacomino, Giuseppe; Picariello, Gianluca; Sbrana, Francesca; Raiteri, Roberto; D'Agostino, Luciano

    2016-10-01

    Nuclear aggregates of polyamines (NAPs) are supramolecular compounds generated by the self-assembly of protonated nuclear polyamines (spermine, spermidine and putrescine) and phosphate ions. In the presence of genomic DNA, the hierarchical process of self-structuring ultimately produces nanotube-like polymers that envelop the double helix. Because of their modular nature and their aggregation-disaggregation dynamics, NAPs confer plasticity and flexibility to DNA. Through the disposition of charges, NAPs also enable a bidirectional stream of information between the genome and interacting moieties. High mobility group (HMG) B1 is a non-histone chromosomal protein that binds to DNA and that influences multiple nuclear processes. Because genomic DNA binds to either NAPs or HMGB1 protein, we explored the ability of in vitro self-assembled NAPs (ivNAPs) to mediate the DNA-HMGB1 interaction. To this end, we structured DNA-NAPs-HMGB1 and DNA-HMGB1-NAPs ternary complexes in vitro through opportune sequential incubations. Mobility shift electrophoresis and atomic force microscopy showed that the DNA-ivNAPs-HGMB1 complex had conformational assets supposedly more suitable those of the DNA-HGMB1-ivNAPs to comply with the physiological and functional requirements of DNA. Our findings indicated that ivNAPs act as mediators of the DNA-HMGB1 interaction.

  16. 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.

  17. Stochastic Ratchet Mechanisms for Replacement of Proteins Bound to DNA

    Science.gov (United States)

    Cocco, S.; Marko, J.F.; Monasson, R.

    2014-01-01

    Experiments indicate that unbinding rates of proteins from DNA can depend on the concentration of proteins in nearby solution. Here we present a theory of multi-step replacement of DNA-bound proteins by solution-phase proteins. For four different kinetic scenarios we calculate the dependence of protein unbinding and replacement rates on solution protein concentration. We find (1) strong effects of progressive ‘rezipping’ of the solution-phase protein onto DNA sites liberated by ‘unzipping’ of the originally bound protein; (2) that a model in which solution-phase proteins bind non-specifically to DNA can describe experiments on exchanges between the non specific DNA-binding proteins Fis-Fis and Fis-HU; (3) that a binding specific model describes experiments on the exchange of CueR proteins on specific binding sites. PMID:24972228

  18. Monothiol glutaredoxin Grx5 interacts with Fe-S scaffold proteins Isa1 and Isa2 and supports Fe-S assembly and DNA integrity in mitochondria of fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyoung-Dong; Chung, Woo-Hyun; Kim, Hyo-Jin; Lee, Kyung-Chang [Laboratory of Molecular Microbiology, School of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of); Roe, Jung-Hye, E-mail: jhroe@snu.ac.kr [Laboratory of Molecular Microbiology, School of Biological Sciences, Institute of Microbiology, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2010-02-12

    Mitochondrial monothiol glutaredoxins that bind Fe-S cluster are known to participate in Fe-S cluster assembly. However, their precise role has not been well understood. Among three monothiol glutaredoxins (Grx3, 4, and 5) in Schizosaccharomyces pombe only Grx5 resides in mitochondria. The {Delta}grx5 mutant requires cysteine on minimal media, and does not grow on non-fermentable carbon source such as glycerol. We found that the mutant is low in the activity of Fe-S enzymes in mitochondria as well as in the cytoplasm. Screening of multi-copy suppressor of growth defects of the mutant identified isa1{sup +} gene encoding a putative A-type Fe-S scaffold, in addition to mas5{sup +} and hsc1{sup +} genes encoding putative chaperones for Fe-S assembly process. Examination of other scaffold and chaperone genes revealed that isa2{sup +}, but not isu1{sup +} and ssc1{sup +}, complemented the growth phenotype of {Delta}grx5 mutant as isa1{sup +} did, partly through restoration of Fe-S enzyme activities. The mutant also showed a significant decrease in the amount of mitochondrial DNA. We demonstrated that Grx5 interacts in vivo with Isa1 and Isa2 proteins in mitochondria by observing bimolecular fluorescence complementation. These results indicate that Grx5 plays a central role in Fe-S assembly process through interaction with A-type Fe-S scaffold proteins Isa1 and Isa2, each of which is an essential protein in S. pombe, and supports mitochondrial genome integrity as well as Fe-S assembly.

  19. Yeast Interacting Proteins Database: YNL189W, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available tein; not conserved in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies...myces species; protein detected in large-scale protein-protein interaction studies Rows with this prey as pr

  20. Inferring interaction partners from protein sequences

    CERN Document Server

    Bitbol, Anne-Florence; Colwell, Lucy J; Wingreen, Ned S

    2016-01-01

    Specific protein-protein interactions are crucial in the cell, both to ensure the formation and stability of multi-protein complexes, and to enable signal transduction in various pathways. Functional interactions between proteins result in coevolution between the interaction partners. Hence, the sequences of interacting partners are correlated. Here we exploit these correlations to accurately identify which proteins are specific interaction partners from sequence data alone. Our general approach, which employs a pairwise maximum entropy model to infer direct couplings between residues, has been successfully used to predict the three-dimensional structures of proteins from sequences. Building on this approach, we introduce an iterative algorithm to predict specific interaction partners from among the members of two protein families. We assess the algorithm's performance on histidine kinases and response regulators from bacterial two-component signaling systems. The algorithm proves successful without any a pri...

  1. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea.

    Science.gov (United States)

    Petitjean, Céline; Moreira, David; López-García, Purificación; Brochier-Armanet, Céline

    2012-11-26

    In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer) domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants). Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs) to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

  2. Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea

    Directory of Open Access Journals (Sweden)

    Petitjean Céline

    2012-11-01

    Full Text Available Abstract Background In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants. Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Results Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. Conclusions We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

  3. Affinity purification of sequence-specific DNA binding proteins.

    OpenAIRE

    1986-01-01

    We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed t...

  4. Sliding Clamp–DNA Interactions Are Required for Viability and Contribute to DNA Polymerase Management in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Heltzel, J.; Scouten Ponticelli, S; Sanders, L; Duzen, J; Cody, V; Pace, J; Snell, E; Sutton, M

    2009-01-01

    Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the Escherichia coli {beta} clamp interact physically with the DNA that it topologically encircles. We utilized mutant {beta} clamp proteins bearing G66E and G174A substitutions ({beta}159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 ({beta}148-152), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the X-ray crystal structure of {beta}148-152, which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both {beta}159 and {beta}148-152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of {beta}148-152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired {beta}148-152-DNA interactions. Once loaded, {beta}148-152 was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, {beta}148-152 was severely impaired for Pol II and Pol IV replication and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, {beta}148-152 was unable to support viability of E. coli. Nevertheless, physiological levels of {beta}148-152 expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing {beta}159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp-DNA

  5. Interaction of Murine BiP/GRP78 with the DnaJ Homologue MTJ1*

    OpenAIRE

    Chevalier, Mathieu; Rhee, Hong; Elguindi, Ebrahim C.; Blond, Sylvie Y.

    2000-01-01

    The activity of Hsp70 proteins is regulated by accessory proteins, among which the most studied are the members of the DnaJ-like protein family. BiP/GRP78 chaperones the translocation and maturation of secreted and membrane proteins in the endoplasmic reticulum. No DnaJ-like partner has been described so far to regulate the function of mammalian BiP/GRP78. We show here that murine BiP/GRP78 interacts with the lumenal J domain of the murine transmembrane protein MTJ1 (J-MTJ1). J-MTJ1 stimulate...

  6. In vivo bacterial morphogenetic protein interactions

    NARCIS (Netherlands)

    van der Ploeg, R.; den Blaauwen, T.; Meghea, A.

    2012-01-01

    This chapter will discuss none-invasive techniques that are widely used to study protein-protein interactions. As an example, their application in exploring interactions between proteins involved in bacterial cell division will be evaluated. First, bacterial morphology and cell division of the rod-s

  7. Ontology integration to identify protein complex in protein interaction networks

    Directory of Open Access Journals (Sweden)

    Yang Zhihao

    2011-10-01

    Full Text Available Abstract Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches.

  8. Neurodegenerative diseases: quantitative predictions of protein-RNA interactions.

    Science.gov (United States)

    Cirillo, Davide; Agostini, Federico; Klus, Petr; Marchese, Domenica; Rodriguez, Silvia; Bolognesi, Benedetta; Tartaglia, Gian Gaetano

    2013-02-01

    Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimer's, and Parkinson's diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and α-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction.

  9. Analysis of Protein-Membrane Interactions

    DEFF Research Database (Denmark)

    Kemmer, Gerdi Christine

    Cellular membranes are complex structures, consisting of hundreds of different lipids and proteins. These membranes act as barriers between distinct environments, constituting hot spots for many essential functions of the cell, including signaling, energy conversion, and transport. These functions...... are implemented by soluble proteins reversibly binding to, as well as by integral membrane proteins embedded in, cellular membranes. The activity and interaction of these proteins is furthermore modulated by the lipids of the membrane. Here, liposomes were used as model membrane systems to investigate...... interactions between proteins and lipids. First, interactions of soluble proteins with membranes and specific lipids were studied, using two proteins: Annexin V and Tma1. The protein was first subjected to a lipid/protein overlay assay to identify candidate interaction partners in a fast and efficient way...

  10. DNA-Damage Response RNA-Binding Proteins (DDRBPs): Perspectives from a New Class of Proteins and Their RNA Targets.

    Science.gov (United States)

    Dutertre, Martin; Vagner, Stéphan

    2016-09-29

    Upon DNA damage, cells trigger an early DNA-damage response (DDR) involving DNA repair and cell cycle checkpoints, and late responses involving gene expression regulation that determine cell fate. Screens for genes involved in the DDR have found many RNA-binding proteins (RBPs), while screens for novel RBPs have identified DDR proteins. An increasing number of RBPs are involved in early and/or late DDR. We propose to call this new class of actors of the DDR, which contain an RNA-binding activity, DNA-damage response RNA-binding proteins (DDRBPs). We then discuss how DDRBPs contribute not only to gene expression regulation in the late DDR but also to early DDR signaling, DNA repair, and chromatin modifications at DNA-damage sites through interactions with both long and short noncoding RNAs.

  11. Glutamate promotes SSB protein-protein Interactions via intrinsically disordered regions.

    Science.gov (United States)

    Kozlov, Alexander G; Shinn, Min Kyung; Weiland, Elizabeth A; Lohman, Timothy M

    2017-09-01

    E. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds to ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes that differ in occluded site size and cooperativity. High "unlimited" cooperativity is associated with the 35 site size ((SSB)35) mode at low [NaCl], whereas the 65 site size ((SSB)65) mode formed at higher [NaCl] (> 200mM), where ssDNA wraps completely around the tetramer, displays "limited" cooperativity forming dimers of tetramers. It was previously thought that high cooperativity was associated only with the (SSB)35 binding mode. However, we show here that highly cooperative binding also occurs in the (SSB)65/(SSB)56 binding modes at physiological salt concentrations containing either glutamate or acetate. Highly cooperative binding requires the 56 amino acid intrinsically disordered C-terminal linker (IDL) that connects the DNA binding domain with the 9 amino acid C-terminal acidic tip that is involved in SSB binding to other proteins involved in genome maintenance. These results suggest that high cooperativity involves interactions between IDL regions from different SSB tetramers. Glutamate, which is preferentially excluded from protein surfaces, may generally promote interactions between intrinsically disordered regions of proteins. Since glutamate is the major monovalent anion in E. coli, these results suggest that SSB likely binds to ssDNA with high cooperativity in vivo. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Assessing protein-protein interactions based on the semantic similarity of interacting proteins.

    Science.gov (United States)

    Cui, Guangyu; Kim, Byungmin; Alguwaizani, Saud; Han, Kyungsook

    2015-01-01

    The Gene Ontology (GO) has been used in estimating the semantic similarity of proteins since it has the largest and reliable vocabulary of gene products and characteristics. We developed a new method which can assess Protein-Protein Interactions (PPI) using the branching factor and information content of the common ancestor of interacting proteins in the GO hierarchy. We performed a comparative evaluation of the measure with other GO-based similarity measures and evaluation results showed that our method outperformed others in most GO domains.

  13. Yeast Interacting Proteins Database: YGL198W, YDR084C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available les; computational analysis of large-scale protein-protein interaction data suggests a possible role in vesi... GTPases, localized to late Golgi vesicles; computational analysis of large-scale protein-protein interactio

  14. Yeast Interacting Proteins Database: YNL189W, YOR284W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ait as prey (0) YOR284W HUA2 Cytoplasmic protein of unknown function; computational analysis of large-scal...protein of unknown function; computational analysis of large-scale protein-protein interaction data suggests

  15. Extreme dryness and DNA-protein cross-links

    Science.gov (United States)

    Bieger-Dose, A.; Dose, K.; Meffert, R.; Mehler, M.; Risi, S.

    Exposure of fungal conidia (Aspergillus ochraceus) or spores of Bacillus subtilis to extreme dryness or vacuum induces DNA lesions, including strand breaks and the formation of DNA-protein cross-links. In wet cells only a small amount of protein is bound to DNA, but exposure to conditions of lowered water activity results in an increasing number of cross-links between DNA and proteins. In fungal conidia these cross-links are detected after selective iodination (125J) of the DNA-bound proteins followed by gel electrophoresis and subsequent autoradiography. Another approach is the labelling of DNA with 32p by means of nick translation and the detection of differences in the electrophoretic mobility of DNA before and after digestion with proteinase K of proteins bound to DNA.

  16. DNA Nanoparticles for Improved Protein Synthesis In Vitro.

    Science.gov (United States)

    Galinis, Robertas; Stonyte, Greta; Kiseliovas, Vaidotas; Zilionis, Rapolas; Studer, Sabine; Hilvert, Donald; Janulaitis, Arvydas; Mazutis, Linas

    2016-02-24

    The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 10(4)  clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription-translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.

  17. A conserved mammalian protein interaction network.

    Directory of Open Access Journals (Sweden)

    Åsa Pérez-Bercoff

    Full Text Available Physical interactions between proteins mediate a variety of biological functions, including signal transduction, physical structuring of the cell and regulation. While extensive catalogs of such interactions are known from model organisms, their evolutionary histories are difficult to study given the lack of interaction data from phylogenetic outgroups. Using phylogenomic approaches, we infer a upper bound on the time of origin for a large set of human protein-protein interactions, showing that most such interactions appear relatively ancient, dating no later than the radiation of placental mammals. By analyzing paired alignments of orthologous and putatively interacting protein-coding genes from eight mammals, we find evidence for weak but significant co-evolution, as measured by relative selective constraint, between pairs of genes with interacting proteins. However, we find no strong evidence for shared instances of directional selection within an interacting pair. Finally, we use a network approach to show that the distribution of selective constraint across the protein interaction network is non-random, with a clear tendency for interacting proteins to share similar selective constraints. Collectively, the results suggest that, on the whole, protein interactions in mammals are under selective constraint, presumably due to their functional roles.

  18. The Foundations of Protein-Ligand Interaction

    Science.gov (United States)

    Klebe, Gerhard

    For the specific design of a drug we must first answer the question: How does a drug achieve its activity? An active ingredient must, in order to develop its action, bind to a particular target molecule in the body. Usually this is a protein, but also nucleic acids in the form of RNA and DNA can be target structures for active agents. The most important condition for binding is at first that the active agent exhibits the correct size and shape in order to optimally fit into a cavity exposed to the surface of the protein, the "bindingpocket". It is further necessary for the surface properties of the ligand and protein to be mutually compatible to form specific interactions. In 1894 Emil Fischer compared the exact fit of a substrate for the catalytic centre of an enzyme with the picture of a "lock-and-key". Paul Ehrlich coined in 1913 "Corpora non agunt nisi fixata", literally "bodies do not work when they are not bound". He wanted to imply that active agents that are meant to kill bacteria or parasites must be "fixed" by them, i.e. linked to their structures. Both concepts form the starting point for any rational concept in the development of active pharmaceutical ingredients. In many respects they still apply today. A drug must, after being administered, reach its target and interact with a biological macromolecule. Specific agents have a large affinity and sufficient selectivity to bind to the macromolecule's active site. This is the only way they can develop the desired biological activity without side-effects.

  19. The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Sebesta, Marek; Sisakova, Alexandra;

    2013-01-01

    Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and t...

  20. Hemoglobin interacting proteins and implications of spectrin hemoglobin interaction.

    Science.gov (United States)

    Basu, Avik; Chakrabarti, Abhijit

    2015-10-14

    In this report we have analyzed interacting partners of hemoglobin inside erythrocyte and sought possible implications of hemoglobin-spectrin interaction. Our list of identified cytosolic hemoglobin interacting proteins includes redox regulators like peroxiredoxin-2, Cu-Zn superoxide dismutase, catalase, aldehyde dehydrogenase-1, flavin reductase and chaperones like HSP70, α-hemoglobin stabilizing protein. Others include metabolic enzymes like carbonic anhydrase-1, selenium binding protein-1, purine nucleoside phosphorylase and nucleoside diphosphate kinase. Additionally, various membrane proteins like α and β spectrin, ankyrin, band3, protein4.1, actin and glyceraldehyde 3 phosphate dehydrogenase have been shown to interact with hemoglobin. Our result indicates that major membrane skeleton protein spectrin, that also has a chaperone like activity, helps to fold the unstable alpha-globin chains in vitro. Taken together our results could provide insight into a protein network evolved around hemoglobin molecule inside erythrocyte that may add a new perspective in understanding the hemoglobin function and homeostasis.

  1. Site-Selective Conjugation of Native Proteins with DNA

    DEFF Research Database (Denmark)

    Trads, Julie Brender; Tørring, Thomas; Gothelf, Kurt Vesterager

    2017-01-01

    . In recent years, a number of chemical methods that target conjugation to specific sites at native proteins have become available, and an overview of these methods is provided in this Account. Our laboratory has investigated DNA-templated protein conjugation (DTPC), which offers an alternative approach...... to site-selective conjugation of DNA to proteins. The method is inspired by the concept of DNA-templated synthesis where functional groups conjugated to DNA strands are preorganized by DNA hybridization to dramatically increase the reaction rate. In DPTC, we target metal binding sites in proteins...

  2. Three enhancements to the inference of statistical protein-DNA potentials.

    Science.gov (United States)

    AlQuraishi, Mohammed; McAdams, Harley H

    2013-03-01

    The energetics of protein-DNA interactions are often modeled using so-called statistical potentials, that is, energy models derived from the atomic structures of protein-DNA complexes. Many statistical protein-DNA potentials based on differing theoretical assumptions have been investigated, but little attention has been paid to the types of data and the parameter estimation process used in deriving the statistical potentials. We describe three enhancements to statistical potential inference that significantly improve the accuracy of predicted protein-DNA interactions: (i) incorporation of binding energy data of protein-DNA complexes, in conjunction with their X-ray crystal structures, (ii) use of spatially-aware parameter fitting, and (iii) use of ensemble-based parameter fitting. We apply these enhancements to three widely-used statistical potentials and use the resulting enhanced potentials in a structure-based prediction of the DNA binding sites of proteins. These enhancements are directly applicable to all statistical potentials used in protein-DNA modeling, and we show that they can improve the accuracy of predicted DNA binding sites by up to 21%.

  3. Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

    DEFF Research Database (Denmark)

    Liberti, Sascha E; Andersen, Sofie Dabros; Wang, Jing

    2011-01-01

    Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S......-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein...... (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues...

  4. An Investigation on the Fundamental Interaction between Abeta Peptides and the AT-Rich DNA.

    Science.gov (United States)

    Zhao, Li Na; Zheng, Jie; Chew, Lock Yue; Mu, Yuguang

    2015-07-01

    DNA damage is ubiquitous in all mammalian cells with the occurrence of more than 60,000 times per day per cell. In particular, DNA damage in neurons is found to accumulate with age and has been suggested to interfere with the synthesis of functional proteins. Moreover, recent studies have found through transgenic mice that human amyloid precursor protein causes an increase in DNA double-strand breaks (DSBs) with the effect of a prolongation in DNA repair. It is surmised that amyloid β (Aβ) exacerbates the DNA DSBs in neurons, possibly engendering neuronal dysfunction as a result. However, a good understanding on the holistic interaction mechanisms and the manner in which Aβ intertwines with DNA damage is still in its infancy. In our study, we found that DNA with an AT-rich sequence has a very low binding affinity toward Aβ by means of molecular dynamics simulation. While we have pursued a particular sequence of DNA in this study, other DNA sequences are expected to affect the interaction and binding affinity between DNA and Aβ, and will be pursued in our further research. Nonetheless, we have uncovered favorable interaction between the positively charged side chain of Aβ and the two ends of DNA. The latest experiment reveals that many of the double-stranded breaks in neurons can be fixed via DNA repair mechanisms but not in the case that Aβs are present. It is found that the increased numbers of DSBs prevail in active neurons. Here, on the basis of the favorable interaction between Aβ and the two ends of DNA, we propose the possibility that Aβ prevents DNA repair via binding directly to the break ends of the DNA, which further exacerbates DNA damage. Moreover, we have found that the base pair oxygen of the DNA has a greater preference to form hydrogen bonds than the backbone oxygen with Aβ at the two ends. Thus, we postulate that Aβ could serve to prevent the repair of AT-rich DNA, and it is unlikely to cause its breakage or affect its binding toward

  5. Yeast Interacting Proteins Database: YDL089W, YML008C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available rDNA repeat stability; null mutant causes increase in unequal sister-chromatid exchange; GFP-fusion protein...peat stability; null mutant causes increase in unequal sister-chromatid exchange; GFP-fusion protein localiz

  6. Yeast Interacting Proteins Database: YPL204W, YOL149W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YPL204W HRR25 Protein kinase involved in regulating diverse events including vesicu...tion Protein kinase involved in regulating diverse events including vesicular trafficking, DNA repair, and c

  7. Nedd4 family interacting protein 1 (Ndfip1) is required for ubiquitination and nuclear trafficking of BRCA1-associated ATM activator 1 (BRAT1) during the DNA damage response.

    Science.gov (United States)

    Low, Ley-Hian; Chow, Yuh-Lit; Li, Yijia; Goh, Choo-Peng; Putz, Ulrich; Silke, John; Ouchi, Toru; Howitt, Jason; Tan, Seong-Seng

    2015-03-13

    During injury, cells are vulnerable to apoptosis from a variety of stress conditions including DNA damage causing double-stranded breaks. Without repair, these breaks lead to aberrations in DNA replication and transcription, leading to apoptosis. A major response to DNA damage is provided by the protein kinase ATM (ataxia telangiectasia mutated) that is capable of commanding a plethora of signaling networks for DNA repair, cell cycle arrest, and even apoptosis. A key element in the DNA damage response is the mobilization of activating proteins into the cell nucleus to repair damaged DNA. BRAT1 is one of these proteins, and it functions as an activator of ATM by maintaining its phosphorylated status while also keeping other phosphatases at bay. However, it is unknown how BRAT1 is trafficked into the cell nucleus to maintain ATM phosphorylation. Here we demonstrate that Ndfip1-mediated ubiquitination of BRAT1 leads to BRAT1 trafficking into the cell nucleus. Without Ndfip1, BRAT1 failed to translocate to the nucleus. Under genotoxic stress, cells showed increased expression of both Ndfip1 and phosphorylated ATM. Following brain injury, neurons show increased expression of Ndfip1 and nuclear translocation of BRAT1. These results point to Ndfip1 as a sensor protein during cell injury and Ndfip1 up-regulation as a cue for BRAT1 ubiquitination by Nedd4 E3 ligases, followed by nuclear translocation of BRAT1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Integral UBL domain proteins: a family of proteasome interacting proteins

    DEFF Research Database (Denmark)

    Hartmann-Petersen, Rasmus; Gordon, Colin

    2004-01-01

    The family of ubiquitin-like (UBL) domain proteins (UDPs) comprises a conserved group of proteins involved in a multitude of different cellular activities. However, recent studies on UBL-domain proteins indicate that these proteins appear to share a common property in their ability to interact wi...

  9. Protein-Protein Interactions in Virus-Host Systems.

    Science.gov (United States)

    Brito, Anderson F; Pinney, John W

    2017-01-01

    To study virus-host protein interactions, knowledge about viral and host protein architectures and repertoires, their particular evolutionary mechanisms, and information on relevant sources of biological data is essential. The purpose of this review article is to provide a thorough overview about these aspects. Protein domains are basic units defining protein interactions, and the uniqueness of viral domain repertoires, their mode of evolution, and their roles during viral infection make viruses interesting models of study. Mutations at protein interfaces can reduce or increase their binding affinities by changing protein electrostatics and structural properties. During the course of a viral infection, both pathogen and cellular proteins are constantly competing for binding partners. Endogenous interfaces mediating intraspecific interactions-viral-viral or host-host interactions-are constantly targeted and inhibited by exogenous interfaces mediating viral-host interactions. From a biomedical perspective, blocking such interactions is the main mechanism underlying antiviral therapies. Some proteins are able to bind multiple partners, and their modes of interaction define how fast these "hub proteins" evolve. "Party hubs" have multiple interfaces; they establish simultaneous/stable (domain-domain) interactions, and tend to evolve slowly. On the other hand, "date hubs" have few interfaces; they establish transient/weak (domain-motif) interactions by means of short linear peptides (15 or fewer residues), and can evolve faster. Viral infections are mediated by several protein-protein interactions (PPIs), which can be represented as networks (protein interaction networks, PINs), with proteins being depicted as nodes, and their interactions as edges. It has been suggested that viral proteins tend to establish interactions with more central and highly connected host proteins. In an evolutionary arms race, viral and host proteins are constantly changing their interface

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  11. Yeast Interacting Proteins Database: YOR124C, YGR268C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available that of Type I J-proteins; computational analysis of large-scale protein-protein interaction data suggests a...tational analysis of large-scale protein-protein interaction data suggests a possible role in actin patch as

  12. Yeast Interacting Proteins Database: YLR291C, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ved in closely related Saccharomyces species; protein detected in large-scale protein-protein interaction studies...in large-scale protein-protein interaction studies Rows with this prey as prey Rows with this prey as prey (

  13. Yeast Interacting Proteins Database: YML064C, YJL199C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available y related Saccharomyces species; protein detected in large-scale protein-protein interaction studies Rows wi...in-protein interaction studies Rows with this prey as prey (4) Rows with this prey as bait (1) 28 6 3 4 0 0 ...d in closely related Saccharomyces species; protein detected in large-scale prote

  14. Genome-wide protein-protein interactions and protein function exploration in cyanobacteria.

    Science.gov (United States)

    Lv, Qi; Ma, Weimin; Liu, Hui; Li, Jiang; Wang, Huan; Lu, Fang; Zhao, Chen; Shi, Tieliu

    2015-10-22

    Genome-wide network analysis is well implemented to study proteins of unknown function. Here, we effectively explored protein functions and the biological mechanism based on inferred high confident protein-protein interaction (PPI) network in cyanobacteria. We integrated data from seven different sources and predicted 1,997 PPIs, which were evaluated by experiments in molecular mechanism, text mining of literatures in proved direct/indirect evidences, and "interologs" in conservation. Combined the predicted PPIs with known PPIs, we obtained 4,715 no-redundant PPIs (involving 3,231 proteins covering over 90% of genome) to generate the PPI network. Based on the PPI network, terms in Gene ontology (GO) were assigned to function-unknown proteins. Functional modules were identified by dissecting the PPI network into sub-networks and analyzing pathway enrichment, with which we investigated novel function of underlying proteins in protein complexes and pathways. Examples of photosynthesis and DNA repair indicate that the network approach is a powerful tool in protein function analysis. Overall, this systems biology approach provides a new insight into posterior functional analysis of PPIs in cyanobacteria.

  15. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  16. Adding protein context to the human protein-protein interaction network to reveal meaningful interactions.

    Directory of Open Access Journals (Sweden)

    Martin H Schaefer

    Full Text Available Interactions of proteins regulate signaling, catalysis, gene expression and many other cellular functions. Therefore, characterizing the entire human interactome is a key effort in current proteomics research. This challenge is complicated by the dynamic nature of protein-protein interactions (PPIs, which are conditional on the cellular context: both interacting proteins must be expressed in the same cell and localized in the same organelle to meet. Additionally, interactions underlie a delicate control of signaling pathways, e.g. by post-translational modifications of the protein partners - hence, many diseases are caused by the perturbation of these mechanisms. Despite the high degree of cell-state specificity of PPIs, many interactions are measured under artificial conditions (e.g. yeast cells are transfected with human genes in yeast two-hybrid assays or even if detected in a physiological context, this information is missing from the common PPI databases. To overcome these problems, we developed a method that assigns context information to PPIs inferred from various attributes of the interacting proteins: gene expression, functional and disease annotations, and inferred pathways. We demonstrate that context consistency correlates with the experimental reliability of PPIs, which allows us to generate high-confidence tissue- and function-specific subnetworks. We illustrate how these context-filtered networks are enriched in bona fide pathways and disease proteins to prove the ability of context-filters to highlight meaningful interactions with respect to various biological questions. We use this approach to study the lung-specific pathways used by the influenza virus, pointing to IRAK1, BHLHE40 and TOLLIP as potential regulators of influenza virus pathogenicity, and to study the signalling pathways that play a role in Alzheimer's disease, identifying a pathway involving the altered phosphorylation of the Tau protein. Finally, we provide the

  17. Mass spectrometric analysis of protein interactions

    DEFF Research Database (Denmark)

    Borch, Jonas; Jørgensen, Thomas J. D.; Roepstorff, Peter

    2005-01-01

    Mass spectrometry is a powerful tool for identification of interaction partners and structural characterization of protein interactions because of its high sensitivity, mass accuracy and tolerance towards sample heterogeneity. Several tools that allow studies of protein interaction are now...... available and recent developments that increase the confidence of studies of protein interaction by mass spectrometry include quantification of affinity-purified proteins by stable isotope labeling and reagents for surface topology studies that can be identified by mass-contributing reporters (e.g. isotope...... labels, cleavable cross-linkers or fragment ions. The use of mass spectrometers to study protein interactions using deuterium exchange and for analysis of intact protein complexes recently has progressed considerably....

  18. Quantitative analysis of protein-protein interactions by split firefly luciferase complementation in plant protoplasts.

    Science.gov (United States)

    Li, Jian-Feng; Zhang, Dandan

    2014-07-01

    This unit describes the split firefly luciferase complementation (SFLC) assay, a high-throughput quantitative method that can be used to investigate protein-protein interactions (PPIs) in plant mesophyll protoplasts. In SFLC, the two proteins to be tested for interaction are expressed as chimeric proteins, each fused to a different half of firefly luciferase. If the proteins interact, a functional luciferase can be transitorily reconstituted, and is detected using the cell-permeable substrate D-luciferin. An advantage of the SFLC assay is that dynamic changes in PPIs in a cell can be detected in a near real-time manner. Another advantage is the unusually high DNA co-transfection and protein expression efficiencies that can be achieved in plant protoplasts, thereby enhancing the throughput of the method.

  19. Using the clustered circular layout as an informative method for visualizing protein-protein interaction networks.

    Science.gov (United States)

    Fung, David C Y; Wilkins, Marc R; Hart, David; Hong, Seok-Hee

    2010-07-01

    The force-directed layout is commonly used in computer-generated visualizations of protein-protein interaction networks. While it is good for providing a visual outline of the protein complexes and their interactions, it has two limitations when used as a visual analysis method. The first is poor reproducibility. Repeated running of the algorithm does not necessarily generate the same layout, therefore, demanding cognitive readaptation on the investigator's part. The second limitation is that it does not explicitly display complementary biological information, e.g. Gene Ontology, other than the protein names or gene symbols. Here, we present an alternative layout called the clustered circular layout. Using the human DNA replication protein-protein interaction network as a case study, we compared the two network layouts for their merits and limitations in supporting visual analysis.

  20. Protein found to promote DNA repair, prevent cancer

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    @@ An abundant chromosomal protein that binds to damaged DNA prevents cancer development by enhancing DNA repair, researchers at University of Texas reported on-line in the Proceedings of the National Academies of Science.

  1. High mobility group A-interacting proteins in cancer: focus on chromobox protein homolog 7, homeodomain interacting protein kinase 2 and PATZ

    Directory of Open Access Journals (Sweden)

    Monica Fedele

    2012-03-01

    Full Text Available The High Mobility Group A (HMGA proteins, a family of DNA architectural factors, by interacting with different proteins play crucial roles in neoplastic transformation of a wide range of tissues. Therefore, the search for HMGA-interacting partners was carried out by several laboratories in order to investigate the mechanisms underlying HMGA-dependent tumorigenesis. Three of the several HMGA-binding proteins are discussed in this review. These are the Chromobox family protein (chromobox protein homolog 7, CBX7, the homeodomain interacting protein kinase 2 (HIPK2 and the POZ/domain and Kruppel zinc finger family member, PATZ. All of them play a critical role in tumorigenesis, and may also be independent markers of cancer. Their activities are linked to cell cycle, apoptosis and senescence. In this review, we discuss the properties of each protein, including their effect on HMGA1 functions, and propose a model accounting for how their activities might be coordinated.

  2. Influence of mobile DNA-protein-DNA bridges on DNA configurations: Coarse-grained Monte-Carlo simulations

    NARCIS (Netherlands)

    Vries, de R.

    2011-01-01

    A large literature exists on modeling the influence of sequence-specific DNA-binding proteins on the shape of the DNA double helix in terms of one or a few fixed constraints. This approach is inadequate for the many proteins that bind DNA sequence independently, and that are present in very large qu

  3. Influence of mobile DNA-protein-DNA bridges on DNA configurations: Coarse-grained Monte-Carlo simulations

    NARCIS (Netherlands)

    Vries, de R.

    2011-01-01

    A large literature exists on modeling the influence of sequence-specific DNA-binding proteins on the shape of the DNA double helix in terms of one or a few fixed constraints. This approach is inadequate for the many proteins that bind DNA sequence independently, and that are present in very large

  4. Identification of DNA-dependent protein kinase catalytic subunit (DNA-PKcs as a novel target of bisphenol A.

    Directory of Open Access Journals (Sweden)

    Yuki Ito

    Full Text Available Bisphenol A (BPA forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80, is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM, high doses of BPA were required before cellular effects were observed (100-300 μM. The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient. Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

  5. DNA-templated nickel nanostructures and protein assemblies.

    Science.gov (United States)

    Becerril, Hector A; Ludtke, Paul; Willardson, Barry M; Woolley, Adam T

    2006-11-21

    We report a straightforward method for the fabrication of DNA-templated nickel nanostructures on surfaces. These nickel nanomaterials have potential to be applied as nanowires, as templated catalyst lines, as nanoscale magnetic domains, or in directed protein localization. Indeed, we show here that histidine-tagged phosducin-like protein (His-PhLP) binds with high selectivity to both Ni2+-treated surface DNA and DNA-templated nickel metal to create linear protein assemblies on surfaces. The association of His-PhLP with DNA-templated nickel ions or metal is reversible under appropriate rinsing conditions. Nanoscale DNA-templated protein assemblies might be useful in the construction of high-density protein lines for proteomic analysis, for example. Importantly, these nanofabrication procedures are not limited to linear DNA and can be applied readily to other self-assembled DNA topologies.

  6. Yeast Interacting Proteins Database: YDL089W, YDR233C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available rDNA repeat stability; null mutant causes increase in unequal sister-chromatid exchange; GFP-fusion protein...DNA repeat stability; null mutant causes increase in unequal sister-chromatid exchange; GFP-fusion protein l

  7. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    Science.gov (United States)

    He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

    2012-07-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

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

    DEFF Research Database (Denmark)

    Asklund, Marlene; Atlung, Tove

    2004-01-01

    an extensive mutational analysis of the DNA-binding domain of the Escherichia coli DnaA protein using mutagenic PCR. We analyzed mutants exhibiting more or less partial activity by selecting for complementation of a dnaA(Ts) mutant strain at different expression levels of the new mutant proteins. The selection...

  9. The Escherichia coli Hfq protein: an unattended DNA-transactions regulator

    Directory of Open Access Journals (Sweden)

    Grzegorz M Cech

    2016-07-01

    Full Text Available The Hfq protein was discovered in Escherichia coli as a host factor for bacteriophage Qβ RNA replication. Subsequent studies indicated that Hfq is a pleiotropic regulator of bacterial gene expression. The regulatory role of Hfq is ascribed mainly to its function as an RNA-chaperone, facilitating interactions between bacterial noncoding RNA and its mRNA target. Thus, it modulates mRNA translation and stability. Nevertheless, Hfq is able to interact with DNA as well. Its role in the regulation of DNA-related processes has been demonstrated. In this mini-review, it is discussed how Hfq interacts with DNA and what is the role of this protein in regulation of DNA transactions. Particularly, Hfq has been demonstrated to be involved in the control of ColE1 plasmid DNA replication, transposition, and possibly also transcription. Possible mechanisms of these Hfq-mediated regulations are described and discussed.

  10. PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

    Science.gov (United States)

    Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

    2013-06-14

    Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

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

    Science.gov (United States)

    Papoian, Garegin

    2012-02-01

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

  12. DNA-Conjugated Organic Chromophores in DNA Stacking Interactions

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V.; Pedersen, Erik Bjerregaard

    2009-01-01

    Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic...... review presents those efforts in the design of intercalators/organic chromophores as oligonucleotide conjugates that form a foundation for the generation of novel nucleic acid architectures...

  13. A gateway-based system for fast evaluation of protein-protein interactions in bacteria.

    Directory of Open Access Journals (Sweden)

    Thorsten Wille

    Full Text Available Protein-protein interactions are important layers of regulation in all kingdoms of life. Identification and characterization of these interactions is one challenging task of the post-genomic era and crucial for understanding of molecular processes within a cell. Several methods have been successfully employed during the past decades to identify protein-protein interactions in bacteria, but most of them include tedious and time-consuming manipulations of DNA. In contrast, the MultiSite Gateway system is a fast tool for transfer of multiple DNA fragments between plasmids enabling simultaneous and site directed cloning of up to four fragments into one construct. Here we developed a new set of Gateway vectors including custom made entry vectors and modular Destination vectors for studying protein-protein interactions via Fluorescence Resonance Energy Transfer (FRET, Bacterial two Hybrid (B2H and split Gaussia luciferase (Gluc, as well as for fusions with SNAP-tag and HaloTag for dual-color super-resolution microscopy. As proof of principle, we characterized the interaction between the Salmonella effector SipA and its chaperone InvB via split Gluc and B2H approach. The suitability for FRET analysis as well as functionality of fusions with SNAP- and HaloTag could be demonstrated by studying the transient interaction between chemotaxis response regulator CheY and its phosphatase CheZ.

  14. Yeast Interacting Proteins Database: YPR040W, YDL188C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YPR040W TIP41 Protein that interacts physically and genetically with Tap42p, which ...ait ORF YPR040W Bait gene name TIP41 Bait description Protein that interacts physically and genetically

  15. Yeast Interacting Proteins Database: YPR040W, YDL134C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YPR040W TIP41 Protein that interacts physically and genetically with Tap42p, which ...Bait ORF YPR040W Bait gene name TIP41 Bait description Protein that interacts physically and genetically

  16. Yeast Interacting Proteins Database: YPR103W, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available tein involved in control of glucose-regulated gene expression; interacts with protein kinase Snf1p, glucose sensors...gulated gene expression; interacts with protein kinase Snf1p, glucose sensors Snf

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

    Science.gov (United States)

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

    2014-06-01

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

  18. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    Energy Technology Data Exchange (ETDEWEB)

    Galvão, C.W. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Souza, E.M. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Etto, R.M. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Schumacher, J.; Buck, M. [Department of Life Sciences, Imperial College London, London (United Kingdom); Steffens, M.B.R. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

    2012-10-15

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX{sub Hs}) can interact with the H. seropedicae RecA protein (RecA{sub Hs}) and that RecA{sub Hs} possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX{sub Hs} inhibited 90% of the RecA{sub Hs} DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA{sub Hs}. RecA{sub Hs} ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX{sub Hs} was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX{sub Hs} protein negatively modulates the RecA{sub Hs} activities by protein-protein interactions and also by DNA-protein interactions.

  19. Yeast Interacting Proteins Database: YGR268C, YER125W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available larity to that of Type I J-proteins; computational analysis of large-scale protein-protein interaction data ...equence similarity to that of Type I J-proteins; computational analysis of large-scale protein-protein inter

  20. 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.

  1. Detecting single DNA molecule interactions with optical microcavities (Presentation Recording)

    Science.gov (United States)

    Vollmer, Frank

    2015-09-01

    as the detection of less than 1 kDa intercalating small molecules[1]. [1] M. D. Baaske, M. R. Foreman, and F. Vollmer, "Single molecule nucleic acid interactions monitored on a label-free microcavity biosensing platform," Nature Nanotechnology, vol. 9, pp. 933-939, 2014. [2] Y. Wu, D. Y. Zhang, P. Yin, and F. Vollmer, "Ultraspecific and Highly Sensitive Nucleic Acid Detection by Integrating a DNA Catalytic Network with a Label-Free Microcavity," Small, vol. 10, pp. 2067-2076, 2014. [3] M. R. Foreman, W.-L. Jin, and F. Vollmer, "Optimizing Detection Limits in Whispering Gallery Mode Biosensing," Optics Express, vol. 22, pp. 5491-5511, 2014. [4] M. A. Santiago-Cordoba, S. V. Boriskina, F. Vollmer, and M. C. Demirel, "Nanoparticle-based protein detection by optical shift of a resonant microcavity," Applied Physics Letters, vol. 99, Aug 2011. [5] M. R. Foreman and F. Vollmer, "Theory of resonance shifts of whispering gallery modes by arbitrary plasmonic nanoparticles," New Journal of Physics, vol. 15, p. 083006, Aug 2013. [6] M. R. Foreman and F. Vollmer "Level repulsion in hybrid photonic-plasmonic microresonators for enhanced biodetection" Phys. Rev. A 88, 023831 (2013).

  2. DNA & Protein detection based on microbead agglutination

    KAUST Repository

    Kodzius, Rimantas

    2012-06-06

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microparticles in the presence of a specific analyte thus enabling the macroscopic observation. Agglutination-based tests are most often used to explore the antibody-antigen reactions. Agglutination has been used for mode protein assays using a biotin/streptavidin two-component system, as well as a hybridization based two-component assay; however, as our work shows, two-component systems are prone to self-termination of the linking analyte and thus have a lower sensitivity. Three component systems have also been used with DNA hybridization, as in our work; however, their assay requires 48 hours for incubation, while our assay is performed in 5 minutes making it a real candidate for POC testing. We demonstrate three assays: a two-component biotin/streptavidin assay, a three-component hybridization assay using single stranded DNA (ssDNA) molecules and a stepped three-component hybridization assay. The comparison of these three assays shows our simple stepped three-component agglutination assay to be rapid at room temperature and more sensitive than the two-component version by an order of magnitude. An agglutination assay was also performed in a PDMS microfluidic chip where agglutinated beads were trapped by filter columns for easy observation. We developed a rapid (5 minute) room temperature assay, which is based on microbead agglutination. Our three-component assay solves the linker self-termination issue allowing an order of magnitude increase in sensitivity over two–component assays. Our stepped version of the three-component assay solves the issue with probe site saturation thus enabling a wider range of detection. Detection of the agglutinated beads with the naked eye by trapping in microfluidic channels has been shown.

  3. Mapping interactions of Chikungunya virus nonstructural proteins.

    Science.gov (United States)

    Sreejith, R; Rana, Jyoti; Dudha, Namrata; Kumar, Kapila; Gabrani, Reema; Sharma, Sanjeev K; Gupta, Amita; Vrati, Sudhanshu; Chaudhary, Vijay K; Gupta, Sanjay

    2012-10-01

    The four nonstructural proteins (nsPs1-4) of Chikungunya virus (CHIKV) play important roles involving enzymatic activities and specific interactions with both viral and host components, during different stages of viral pathogenesis. Elucidation of the presence and/or absence of interactions among nsPs in a systematic manner is thus of scientific interest. In the current study, each pair-wise combination among the four nonstructural proteins of CHIKV was systematically analyzed for possible interactions. Six novel protein interactions were identified for CHIKV, using systems such as yeast two-hybrid, GST pull down and ELISA, three of which have not been previously reported for the genus Alphavirus. These interactions form a network of organized associations that suggest the spatial arrangement of nonstructural proteins in the late replicase complex. The study identified novel interactions as well as concurred with previously described associations in related alphaviruses.

  4. 与人巨细胞病毒UL128两种不同结构蛋白相互作用蛋白的筛选与分析%Screening of protein interacting with the transcript of UL128 gene showed two protein patterns by yeast two-hybrid from human fetus brain cDNA library

    Institute of Scientific and Technical Information of China (English)

    任高伟; 崔鑫; 马艳萍; 齐莹; 阮强; 孙峥嵘

    2010-01-01

    Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.%目的 利用酵母双杂交系统从人胎脑cDNA文库中筛选与两种不同转录结构的人巨细胞病毒(HCMV)UL128编码蛋白相互作用的蛋白,比较两者相互作用蛋白之间的异同点.方法 通过3'RACE和5'RACE技术扩增出两种HCMV UL128片段,其大小分别为519 bp和642 bp,并将其成功构建到酵母诱饵表达载体pGBKT7中.将以上两种酵母表达载体分别转化到酵母菌AH109中,再将文库DNA转化到已含有酵母表达载体的AH109中,筛选与两种片段大小不同的UL128编码蛋白相互作用的人胎脑蛋白,并对筛选得到的阳性克隆进行测序和生物信息学分析.结果 筛出EFEMP2与UL128-519 bp编码蛋白相互作用,THY-1

  5. Spectroscopic Electrochemical Studies of Interaction Between Fuchsin Basic DNA

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Visible spectroscopic and electrochemical methods were used to study the interactions between DNA and fuchsin basic(FB). FB has an irreversible electro-oxidation peak in 5 mmol/L Tris-HCl buffer solution at pH = 7.4 on a glassy carbon electrode(GCE). After adding certain concentration of dsDNA, the oxidation peak current of FB decreases, but the peak potential hardly changs. The visible absorption spectroscopic study shows that the binding mode of FB to dsDNA is intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is smaller than 0. 2, and a new substance, which produces a new absorption peak, is obtained via a covalent binding between dsDNA and FB apart from intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is larger than 0. 2. The visible absorption spectra varies no longer when the ratio of the concentration of dsDNA to FB is larger than 1.5. A mean binding ratio of dsDNA to FB was determined to be 1.4: 1,suggesting that two complexes FB-dsDNA and FB-2dsDNA be formed. The interaction between FB and ssDNA was only electrostatic binding. The more powerful interaction of FB with dsDNA than with ssDNA may be applied for the recognition of dsDNA and ssDNA, and in DNA biosensor as hybridization indicator.

  6. 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.

  7. In vivo bacterial morphogenetic protein interactions

    OpenAIRE

    van der Ploeg, R.; den Blaauwen, T.; Meghea, A.

    2012-01-01

    This chapter will discuss none-invasive techniques that are widely used to study protein-protein interactions. As an example, their application in exploring interactions between proteins involved in bacterial cell division will be evaluated. First, bacterial morphology and cell division of the rod-shaped bacterium Escherichia coli will be introduced. Next, three bacterial two-hybrid methods and three Förster resonance energy transfer detection methods that are frequently applied to detect int...

  8. Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

    Science.gov (United States)

    Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

    2015-01-01

    TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

  9. A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes.

    Science.gov (United States)

    Song, Wei; Levin, David S; Varkey, Johnson; Post, Sean; Bermudez, Vladimir P; Hurwitz, Jerard; Tomkinson, Alan E

    2007-08-03

    DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.

  10. An Interactive Introduction to Protein Structure

    Science.gov (United States)

    Lee, W. Theodore

    2004-01-01

    To improve student understanding of protein structure and the significance of noncovalent interactions in protein structure and function, students are assigned a project to write a paper complemented with computer-generated images. The assignment provides an opportunity for students to select a protein structure that is of interest and detail…

  11. Yeast Interacting Proteins Database: YML064C, YOR284W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available th this bait as prey (0) YOR284W HUA2 Cytoplasmic protein of unknown function; computational analysis of large-scale... unknown function; computational analysis of large-scale protein-protein interact

  12. Yeast Interacting Proteins Database: YJR091C, YKL076C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpre...NA-binding proteins, interacts with mRNAs encoding membrane-associated proteins; involved in localizing the

  13. Yeast Interacting Proteins Database: YJR091C, YNR048W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpre...y of RNA-binding proteins, interacts with mRNAs encoding membrane-associated proteins; involved in localizing

  14. Yeast Interacting Proteins Database: YJR091C, YML015C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available encoding membrane-associated proteins; involved in localizing the Arp2/3 complex to mitochondria; overexpre...y of RNA-binding proteins, interacts with mRNAs encoding membrane-associated proteins; involved in localizing

  15. Yeast Interacting Proteins Database: YDL239C, YPL070W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available Vps9 domain; computational analysis of large-scale protein-protein interaction data suggests a possible role...ey description Cytoplasmic protein of unknown function containing a Vps9 domain; computational analysis of large-scale

  16. Inferring interaction partners from protein sequences

    Science.gov (United States)

    Bitbol, Anne-Florence; Dwyer, Robert S.; Colwell, Lucy J.; Wingreen, Ned S.

    2016-01-01

    Specific protein−protein interactions are crucial in the cell, both to ensure the formation and stability of multiprotein complexes and to enable signal transduction in various pathways. Functional interactions between proteins result in coevolution between the interaction partners, causing their sequences to be correlated. Here we exploit these correlations to accurately identify, from sequence data alone, which proteins are specific interaction partners. Our general approach, which employs a pairwise maximum entropy model to infer couplings between residues, has been successfully used to predict the 3D structures of proteins from sequences. Thus inspired, we introduce an iterative algorithm to predict specific interaction partners from two protein families whose members are known to interact. We first assess the algorithm’s performance on histidine kinases and response regulators from bacterial two-component signaling systems. We obtain a striking 0.93 true positive fraction on our complete dataset without any a priori knowledge of interaction partners, and we uncover the origin of this success. We then apply the algorithm to proteins from ATP-binding cassette (ABC) transporter complexes, and obtain accurate predictions in these systems as well. Finally, we present two metrics that accurately distinguish interacting protein families from noninteracting ones, using only sequence data. PMID:27663738

  17. Studies of interaction between two alkaloids and double helix DNA

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yantao [Key Laboratory of Preparation and Applications of Environmentally Friendly Materials (Jilin Normal University), Ministry of Education China, Siping 136000 (China); College of Chemistry, Jilin Normal University, Siping 136000 (China); Peng, Tingting [College of Chemistry, Jilin Normal University, Siping 136000 (China); College of Chemistry, Baicheng Normal University, Baicheng, 130032 (China); Zhao, Lei [Siping Institute for Food and Drug Control, Siping 136000 (China); Jiang, Dayu [Key Laboratory of Preparation and Applications of Environmentally Friendly Materials (Jilin Normal University), Ministry of Education China, Siping 136000 (China); College of Chemistry, Jilin Normal University, Siping 136000 (China); Cui, Yuncheng, E-mail: 1979yanzi@163.com [Key Laboratory of Preparation and Applications of Environmentally Friendly Materials (Jilin Normal University), Ministry of Education China, Siping 136000 (China); College of Chemistry, Jilin Normal University, Siping 136000 (China)

    2014-12-15

    This article presents the study on the interaction of two alkaloids (matrine and evodiamine) and hs-DNA by absorption, fluorescence, circular dichroism (CD), DNA melting and viscosity experiments. The spectroscopic studies suggested that two alkaloids can bind to DNA through an intercalative mode. The viscosity measurement and thermal denaturation also indicated that two alkaloids can intercalate to DNA. The binding constants (K{sub A}) and the number of binding sites (n) were determined. At the same time, some significant thermodynamic parameters of the binding of the alkaloids to DNA were obtained. Competitive binding studies revealed that alkaloids had an effect on ethidium bromide (EB) bound DNA. In addition, it was also proved that the fluorescence quenching was influenced by ionic strength. - Highlights: • Interaction between two alkaloids and DNA is studied by spectral methods. • The binding constant and the binding sites between two alkaloids and DNA are obtained. • There are a classical intercalative mode between alkaloids and DNA. • The binding of matrine with DNA is weaker than that of evodiamine. • It is important for us to understand the alkaloids–DNA interactions at a molecular level.

  18. 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/.

  19. Noninvasive imaging of protein-protein interactions in living animals

    Science.gov (United States)

    Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

    2002-05-01

    Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

  20. TALE proteins search DNA using a rotationally decoupled mechanism.

    Science.gov (United States)

    Cuculis, Luke; Abil, Zhanar; Zhao, Huimin; Schroeder, Charles M

    2016-10-01

    Transcription activator-like effector (TALE) proteins are a class of programmable DNA-binding proteins used extensively for gene editing. Despite recent progress, however, little is known about their sequence search mechanism. Here, we use single-molecule experiments to study TALE search along DNA. Our results show that TALEs utilize a rotationally decoupled mechanism for nonspecific search, despite remaining associated with DNA templates during the search process. Our results suggest that the protein helical structure enables TALEs to adopt a loosely wrapped conformation around DNA templates during nonspecific search, facilitating rapid one-dimensional (1D) diffusion under a range of solution conditions. Furthermore, this model is consistent with a previously reported two-state mechanism for TALE search that allows these proteins to overcome the search speed-stability paradox. Taken together, our results suggest that TALE search is unique among the broad class of sequence-specific DNA-binding proteins and supports efficient 1D search along DNA.

  1. Polyamine-DNA interactions and development of gene delivery vehicles.

    Science.gov (United States)

    Thomas, T J; Tajmir-Riahi, H A; Thomas, Thresia

    2016-10-01

    Polyamines are positively charged organic cations under physiologic ionic and pH conditions and hence they interact with negatively charged macromolecules such as DNA and RNA. Although electrostatic interaction is the predominant mode of polyamine-nucleic acid interactions, site- and structure-specific binding has also been recognized. A major consequence of polyamine-DNA interaction is the collapse of DNA to nanoparticles of approximately 100 nm diameter. Electron and atomic force microscopic studies have shown that these nanoparticles are spheroids, toroids and rods. DNA transport to cells for gene therapy applications requires the condensation of DNA to nanoparticles and hence the study of polyamines and related compounds with nucleic acids has received technological importance. In addition to natural and synthetic polyamines, several amine-terminated or polyamine-substituted agents are under intense investigation for non-viral gene delivery vehicles.

  2. Protein-protein interaction based on pairwise similarity

    Directory of Open Access Journals (Sweden)

    Zaki Nazar

    2009-05-01

    Full Text Available Abstract Background Protein-protein interaction (PPI is essential to most biological processes. Abnormal interactions may have implications in a number of neurological syndromes. Given that the association and dissociation of protein molecules is crucial, computational tools capable of effectively identifying PPI are desirable. In this paper, we propose a simple yet effective method to detect PPI based on pairwise similarity and using only the primary structure of the protein. The PPI based on Pairwise Similarity (PPI-PS method consists of a representation of each protein sequence by a vector of pairwise similarities against large subsequences of amino acids created by a shifting window which passes over concatenated protein training sequences. Each coordinate of this vector is typically the E-value of the Smith-Waterman score. These vectors are then used to compute the kernel matrix which will be exploited in conjunction with support vector machines. Results To assess the ability of the proposed method to recognize the difference between "interacted" and "non-interacted" proteins pairs, we applied it on different datasets from the available yeast saccharomyces cerevisiae protein interaction. The proposed method achieved reasonable improvement over the existing state-of-the-art methods for PPI prediction. Conclusion Pairwise similarity score provides a relevant measure of similarity between protein sequences. This similarity incorporates biological knowledge about proteins and it is extremely powerful when combined with support vector machine to predict PPI.

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

    Science.gov (United States)

    Yang, Shu-Wei; Nash, Howard A.

    1994-12-01

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

  4. Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks.

    Science.gov (United States)

    Ramakrishnan, Saminathan; Subramaniam, Sivaraman; Stewart, A Francis; Grundmeier, Guido; Keller, Adrian

    2016-11-16

    DNA origami has become a widely used method for synthesizing well-defined nanostructures with promising applications in various areas of nanotechnology, biophysics, and medicine. Recently, the possibility to transfer the shape of single DNA origami nanostructures into different materials via molecular lithography approaches has received growing interest due to the great structural control provided by the DNA origami technique. Here, we use ordered monolayers of DNA origami nanostructures with internal cavities on mica surfaces as molecular lithography masks for the fabrication of regular protein patterns over large surface areas. Exposure of the masked sample surface to negatively charged proteins results in the directed adsorption of the proteins onto the exposed surface areas in the holes of the mask. By controlling the buffer and adsorption conditions, the protein coverage of the exposed areas can be varied from single proteins to densely packed monolayers. To demonstrate the versatility of this approach, regular nanopatterns of four different proteins are fabricated: the single-strand annealing proteins Redβ and Sak, the iron-storage protein ferritin, and the blood protein bovine serum albumin (BSA). We furthermore demonstrate the desorption of the DNA origami mask after directed protein adsorption, which may enable the fabrication of hierarchical patterns composed of different protein species. Because selectivity in adsorption is achieved by electrostatic interactions between the proteins and the exposed surface areas, this approach may enable also the large-scale patterning of other charged molecular species or even nanoparticles.

  5. Characterization of protein hubs by inferring interacting motifs from protein interactions.

    Directory of Open Access Journals (Sweden)

    Ramon Aragues

    2007-09-01

    Full Text Available The characterization of protein interactions is essential for understanding biological systems. While genome-scale methods are available for identifying interacting proteins, they do not pinpoint the interacting motifs (e.g., a domain, sequence segments, a binding site, or a set of residues. Here, we develop and apply a method for delineating the interacting motifs of hub proteins (i.e., highly connected proteins. The method relies on the observation that proteins with common interaction partners tend to interact with these partners through a common interacting motif. The sole input for the method are binary protein interactions; neither sequence nor structure information is needed. The approach is evaluated by comparing the inferred interacting motifs with domain families defined for 368 proteins in the Structural Classification of Proteins (SCOP. The positive predictive value of the method for detecting proteins with common SCOP families is 75% at sensitivity of 10%. Most of the inferred interacting motifs were significantly associated with sequence patterns, which could be responsible for the common interactions. We find that yeast hubs with multiple interacting motifs are more likely to be essential than hubs with one or two interacting motifs, thus rationalizing the previously observed correlation between essentiality and the number of interacting partners of a protein. We also find that yeast hubs with multiple interacting motifs evolve slower than the average protein, contrary to the hubs with one or two interacting motifs. The proposed method will help us discover unknown interacting motifs and provide biological insights about protein hubs and their roles in interaction networks.

  6. The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions.

    Directory of Open Access Journals (Sweden)

    Vladislav Kljashtorny

    Full Text Available The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD. Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP, albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

  7. Information assessment on predicting protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Gerstein Mark

    2004-10-01

    Full Text Available Abstract Background Identifying protein-protein interactions is fundamental for understanding the molecular machinery of the cell. Proteome-wide studies of protein-protein interactions are of significant value, but the high-throughput experimental technologies suffer from high rates of both false positive and false negative predictions. In addition to high-throughput experimental data, many diverse types of genomic data can help predict protein-protein interactions, such as mRNA expression, localization, essentiality, and functional annotation. Evaluations of the information contributions from different evidences help to establish more parsimonious models with comparable or better prediction accuracy, and to obtain biological insights of the relationships between protein-protein interactions and other genomic information. Results Our assessment is based on the genomic features used in a Bayesian network approach to predict protein-protein interactions genome-wide in yeast. In the special case, when one does not have any missing information about any of the features, our analysis shows that there is a larger information contribution from the functional-classification than from expression correlations or essentiality. We also show that in this case alternative models, such as logistic regression and random forest, may be more effective than Bayesian networks for predicting interactions. Conclusions In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology (GO functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed by Jansen et al. Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. We also found that the data discretizations used in the

  8. An expanded binding model for Cys2His2 zinc finger protein-DNA interfaces

    Science.gov (United States)

    Persikov, Anton V.; Singh, Mona

    2011-06-01

    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.

  9. Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends

    Directory of Open Access Journals (Sweden)

    Kosova A. A.

    2014-01-01

    Full Text Available Aim. The identification of a protein from human cell extract which specifically interacts with the apurinic/apyrimidinic (AP site in the partial DNA duplex containing 5'and 3'-dangling ends (DDE-AP DNA and mimicking clustered DNA damage. Methods. The Schiff base-dependent cross-linking of a protein to AP DNA (borohydride trapping, MALDI-TOF-MS, chromatography, and gel electrophoresis. Results. A human cell extract protein which forms a major covalent adduct with the AP DNA duplex with dangling ends was identified as the Ku80 subunit of Ku antigen by peptide mass mapping based on MALDI-TOF-MS data. The Ku antigen purified from the HeLa cell extract was shown to form the covalent adducts with the same mobility as observed in cell extracts. Conclusions. The Ku80 subunit of Ku antigen can specifically interact with AP DNA forming the Schiff base-mediated adducts which electrophoretic mobility depends on the structure of DNA ends. The difference in electrophoretic mobility can be caused by the cross-linking of AP DNA to distinct target amino acids that appears to reflect unequal positioning of AP DNAs in the complex with Ku antigen.

  10. Structural similarity of genetically interacting proteins

    Directory of Open Access Journals (Sweden)

    Nussinov Ruth

    2008-07-01

    Full Text Available Abstract Background The study of gene mutants and their interactions is fundamental to understanding gene function and backup mechanisms within the cell. The recent availability of large scale genetic interaction networks in yeast and worm allows the investigation of the biological mechanisms underlying these interactions at a global scale. To date, less than 2% of the known genetic interactions in yeast or worm can be accounted for by sequence similarity. Results Here, we perform a genome-scale structural comparison among protein pairs in the two species. We show that significant fractions of genetic interactions involve structurally similar proteins, spanning 7–10% and 14% of all known interactions in yeast and worm, respectively. We identify several structural features that are predictive of genetic interactions and show their superiority over sequence-based features. Conclusion Structural similarity is an important property that can explain and predict genetic interactions. According to the available data, the most abundant mechanism for genetic interactions among structurally similar proteins is a common interacting partner shared by two genetically interacting proteins.

  11. Protein-protein interaction predictions using text mining methods.

    Science.gov (United States)

    Papanikolaou, Nikolas; Pavlopoulos, Georgios A; Theodosiou, Theodosios; Iliopoulos, Ioannis

    2015-03-01

    It is beyond any doubt that proteins and their interactions play an essential role in most complex biological processes. The understanding of their function individually, but also in the form of protein complexes is of a great importance. Nowadays, despite the plethora of various high-throughput experimental approaches for detecting protein-protein interactions, many computational methods aiming to predict new interactions have appeared and gained interest. In this review, we focus on text-mining based computational methodologies, aiming to extract information for proteins and their interactions from public repositories such as literature and various biological databases. We discuss their strengths, their weaknesses and how they complement existing experimental techniques by simultaneously commenting on the biological databases which hold such information and the benchmark datasets that can be used for evaluating new tools.

  12. Dynamic fluctuations of protein-carbohydrate interactions promote protein aggregation.

    Directory of Open Access Journals (Sweden)

    Vladimir Voynov

    Full Text Available Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation.

  13. Dynamic Conformational Change Regulates the Protein-DNA Recognition: An Investigation on Binding of a Y-Family Polymerase to Its Target DNA

    Science.gov (United States)

    Chu, Xiakun; Liu, Fei; Maxwell, Brian A.; Wang, Yong; Suo, Zucai; Wang, Haijun; Han, Wei; Wang, Jin

    2014-01-01

    Protein-DNA recognition is a central biological process that governs the life of cells. A protein will often undergo a conformational transition to form the functional complex with its target DNA. The protein conformational dynamics are expected to contribute to the stability and specificity of DNA recognition and therefore may control the functional activity of the protein-DNA complex. Understanding how the conformational dynamics influences the protein-DNA recognition is still challenging. Here, we developed a two-basin structure-based model to explore functional dynamics in Sulfolobus solfataricus DNA Y-family polymerase IV (DPO4) during its binding to DNA. With explicit consideration of non-specific and specific interactions between DPO4 and DNA, we found that DPO4-DNA recognition is comprised of first 3D diffusion, then a short-range adjustment sliding on DNA and finally specific binding. Interestingly, we found that DPO4 is under a conformational equilibrium between multiple states during the binding process and the distributions of the conformations vary at different binding stages. By modulating the strength of the electrostatic interactions, the flexibility of the linker, and the conformational dynamics in DPO4, we drew a clear picture on how DPO4 dynamically regulates the DNA recognition. We argue that the unique features of flexibility and conformational dynamics in DPO4-DNA recognition have direct implications for low-fidelity translesion DNA synthesis, most of which is found to be accomplished by the Y-family DNA polymerases. Our results help complete the description of the DNA synthesis process for the Y-family polymerases. Furthermore, the methods developed here can be widely applied for future investigations on how various proteins recognize and bind specific DNA substrates. PMID:25188490

  14. 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

  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. 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.

  17. Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation.

    Science.gov (United States)

    Taylor, James A; Pastrana, Cesar L; Butterer, Annika; Pernstich, Christian; Gwynn, Emma J; Sobott, Frank; Moreno-Herrero, Fernando; Dillingham, Mark S

    2015-01-01

    The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed.

  18. Detection of regional DNA methylation using DNA-graphene affinity interactions.

    Science.gov (United States)

    Haque, Md Hakimul; Gopalan, Vinod; Yadav, Sharda; Islam, Md Nazmul; Eftekhari, Ehsan; Li, Qin; Carrascosa, Laura G; Nguyen, Nam-Trung; Lam, Alfred K; Shiddiky, Muhammad J A

    2017-01-15

    We report a new method for the detection of regional DNA methylation using base-dependent affinity interaction (i.e., adsorption) of DNA with graphene. Due to the strongest adsorption affinity of guanine bases towards graphene, bisulfite-treated guanine-enriched methylated DNA leads to a larger amount of the adsorbed DNA on the graphene-modified electrodes in comparison to the adenine-enriched unmethylated DNA. The level of the methylation is quantified by monitoring the differential pulse voltammetric current as a function of the adsorbed DNA. The assay is sensitive to distinguish methylated and unmethylated DNA sequences at single CpG resolution by differentiating changes in DNA methylation as low as 5%. Furthermore, this method has been used to detect methylation levels in a collection of DNA samples taken from oesophageal cancer tissues.

  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. Enhanced Binding Affinity for an i-Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids.

    Science.gov (United States)

    Bai, Xiaoguang; Talukder, Poulami; Daskalova, Sasha M; Roy, Basab; Chen, Shengxi; Li, Zhongxian; Dedkova, Larisa M; Hecht, Sidney M

    2017-03-17

    Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the BCL2 i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase 2 into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson-Crick interaction of 2 and G14. In comparison, the introduction of uracil nucleobase 3 had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing 1 exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing 4 bound less tightly to the DNA substrate. Finally, the modified protein containing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing 1 both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA-protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1-DNA interaction, as well as the ability of nucleobase amino acid 1 to stabilize RRM1-DNA interaction.

  1. Interaction between plate make and protein in protein crystallisation screening.

    Directory of Open Access Journals (Sweden)

    Gordon J King

    Full Text Available BACKGROUND: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. METHODOLOGY/PRINCIPAL FINDINGS: We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. CONCLUSIONS/SIGNIFICANCE: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallize, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make.

  2. Cooperative binding interaction of ethidium with allosteric DNA.

    Science.gov (United States)

    Suh, D

    1999-09-30

    The specific association of drugs with deoxyoligonucleotides, containing a B-Z junction between left-handed Z-DNA and right-handed B-DNA, was examined by fluorescence and circular dichroism (CD) technique. Ethidium was chosen for a simple DNA binding compound because it binds to right-handed DNA and hybrid B-Z forms containing a B-Z junction in a highly cooperative manner. The binding isotherms were analyzed by an allosteric model in order to describe the cooperativity of association. Binding of ethidium to the DNA that are initially in the hybrid B-Z forms showed over an order of magnitude higher affinity than other DNA which were entirely in the B-form. The conformational transitions of deoxyoligonucleotides containing a B-Z junction as a result of ethidium binding were monitored by CD and the influence of NaCl on the complex formation was also determined by the CD spectra. The singular value decomposition (SVD) analysis was used to characterize a family of CD spectra of the species in binding equilibria. The results of SVD analysis showed a strikingly complex thermodynamic equilibria of cooperative binding of drugs to the allosterically converted DNA forms. The results also showed that these DNA forms in low- and high-salt were different in the absence or presence of drug. These results demonstrate that DNA-binding-drugs can preferentially interact with specific DNA structures and that these interactions are accompanied by allosteric changes of DNA conformations.

  3. Yeast Interacting Proteins Database: YGL198W, YGL161C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YGL198W YIP4 Protein that interacts with Rab GTPases, localized to late Golgi vesicles; comput...that interacts with Rab GTPases, localized to late Golgi vesicles; computational ...eracts with Rab GTPases, localized to late Golgi vesicles; computational analysis of large-scale protein-pro...ized to late Golgi vesicles; computational analysis of large-scale protein-protein interaction data suggests

  4. Yeast Interacting Proteins Database: YGL161C, YGL198W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YGL161C YIP5 Protein that interacts with Rab GTPases, localized to late Golgi vesicles; comput...that interacts with Rab GTPases, localized to late Golgi vesicles; computational ...eracts with Rab GTPases, localized to late Golgi vesicles; computational analysis of large-scale protein-pro...ized to late Golgi vesicles; computational analysis of large-scale protein-protein interaction data suggests

  5. Non-covalent Interaction of Perfluorooctanoic Acid with DNA

    Institute of Scientific and Technical Information of China (English)

    ZHANG, Xian; SHEN, Rong; CHEN, Ling

    2009-01-01

    Health risk from exposure of perfluorochemicals to wildlife and human has been a subject of concern in many fields such as environmental ecology, toxicology, pathology and life sciences. The interactions of perfluorooctanoic acid (PFOA) with DNA were investigated by equilibrium dialysis, circular dichroism and isothermal titration calorimetry techniques under normal physiological conditions in vitro. The binding of PFOA to DNA was a non-covalent interaction and corresponded to the Langmuir adsorption isotherm in a two-step binding model, in which PFOA climbed along the backbones of DNA and then interacted with the homolateral bases via hydrophobic interactions. The saturation number of PFOA was calculated to be 0.64 per base-pair of DNA. Such an interaction caused the enhancement of circular dichroism spectra of DNA at both 245 and 275 nm, indicating the change of DNA conformation. The acidic media, low electrolyte and temperature ≤35 ℃ are comparatively more favorable for PFOA binding to DNA. This work provides a useful experimental strategy for studying the interactions of perfluorochemicals with biomacromolecules, aiming at a better understanding of the gene toxicity mechanism of perfluorochemicals.

  6. Data management of protein interaction networks

    CERN Document Server

    Cannataro, Mario

    2012-01-01

    Interactomics: a complete survey from data generation to knowledge extraction With the increasing use of high-throughput experimental assays, more and more protein interaction databases are becoming available. As a result, computational analysis of protein-to-protein interaction (PPI) data and networks, now known as interactomics, has become an essential tool to determine functionally associated proteins. From wet lab technologies to data management to knowledge extraction, this timely book guides readers through the new science of interactomics, giving them the tools needed to: Generate

  7. Protein-phospholipid interactions in blood clotting.

    Science.gov (United States)

    Morrissey, James H; Davis-Harrison, Rebecca L; Tavoosi, Narjes; Ke, Ke; Pureza, Vincent; Boettcher, John M; Clay, Mary C; Rienstra, Chad M; Ohkubo, Y Zenmei; Pogorelov, Taras V; Tajkhorshid, Emad

    2010-04-01

    Most steps of the blood clotting cascade require the assembly of a serine protease with its specific regulatory protein on a suitable phospholipid bilayer. Unfortunately, the molecular details of how blood clotting proteins bind to membrane surfaces remain poorly understood, owing to a dearth of techniques for studying protein-membrane interactions at high resolution. Our laboratories are tackling this question using a combination of approaches, including nanoscale membrane bilayers, solid-state NMR, and large-scale molecular dynamics simulations. These studies are now providing structural insights at atomic resolution into clotting protein-membrane interactions. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  8. Laplacian Spectrum and Protein-Protein Interaction Networks

    CERN Document Server

    Banerjee, Anirban

    2007-01-01

    From the spectral plot of the (normalized) graph Laplacian, the essential qualitative properties of a network can be simultaneously deduced. Given a class of empirical networks, reconstruction schemes for elucidating the evolutionary dynamics leading to those particular data can then be developed. This method is exemplified for protein-protein interaction networks. Traces of their evolutionary history of duplication and divergence processes are identified. In particular, we can identify typical specific features that robustly distinguish protein-protein interaction networks from other classes of networks, in spite of possible statistical fluctuations of the underlying data.

  9. Interaction of DNA with Bis(diiminosuccinonitrilo)platinum(Ⅱ)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Zhi-Gang; SUN Yuan-Yuan; JIANG Xiao-Ming

    2008-01-01

    Interaction of DNA with bis(diiminosuccinonitrilo)platinum(Ⅱ) has been studied by UV-visible absorbance spectra, fluorescence spectra and viscosity measurements. The UV-visible absorption spectra of the metal complex exhibit hypochromism with a small blue shift on interaction with DNA. Scatchard plot analyses indicate that the binding sites of the metal complex on DNA are different from those of ethidium bromide. Viscosity experiments reveal that the binding of the metal complex decreases the relative viscosity of DNA. These results suggest that the platinum diimine complex interact with DNA by surface binding. These studies are helpful for us to understand the action mechanism of bis(diiminosuccinonitrilo)platinum(Ⅱ) as a potential photodynamic therapeutic agent, and further to develop it.

  10. Cockayne Syndrome group B protein stimulates NEIL2 DNA glycosylase activity

    DEFF Research Database (Denmark)

    Aamann, Maria Diget; Hvitby, Christina Poulsen; Popuri, Venkateswarlu

    2014-01-01

    Cockayne Syndrome is a segmental premature aging syndrome, which can be caused by loss of function of the CSB protein. CSB is essential for genome maintenance and has numerous interaction partners with established roles in different DNA repair pathways including transcription coupled nucleotide...... excision repair and base excision repair. Here, we describe a new interaction partner for CSB, the DNA glycosylase NEIL2. Using both cell extracts and recombinant proteins, CSB and NEIL2 were found to physically interact independently of DNA. We further found that CSB is able to stimulate NEIL2 glycosylase...... in a DNA bubble structure using whole cell extracts. Taken together, our data supports a biological role for CSB and NEIL2 in transcription associated base excision repair....

  11. Intraviral protein interactions of Chandipura virus.

    Science.gov (United States)

    Kumar, Kapila; Rana, Jyoti; Sreejith, R; Gabrani, Reema; Sharma, Sanjeev K; Gupta, Amita; Chaudhary, Vijay K; Gupta, Sanjay

    2012-10-01

    Chandipura virus (CHPV) is an emerging rhabdovirus responsible for several outbreaks of fatal encephalitis among children in India. The characteristic structure of the virus is a result of extensive and specific interplay among its five encoded proteins. The revelation of interactions among CHPV proteins can help in gaining insight into viral architecture and pathogenesis. In the current study, we carried out comprehensive yeast two-hybrid (Y2H) analysis to elucidate intraviral protein-protein interactions. All of the interactions identified by Y2H were assessed for reliability by GST pull-down and ELISA. A total of eight interactions were identified among four viral proteins. Five of these interactions are being reported for the first time for CHPV. Among these, the glycoprotein (G)-nucleocapsid (N) interaction could be considered novel, as this has not been reported for any members of the family Rhabdoviridae. This study provides a framework within which the roles of the identified protein interactions can be explored further for understanding the biology of this virus at the molecular level.

  12. NOXclass: prediction of protein-protein interaction types

    Directory of Open Access Journals (Sweden)

    Sommer Ingolf

    2006-01-01

    Full Text Available Abstract Background Structural models determined by X-ray crystallography play a central role in understanding protein-protein interactions at the molecular level. Interpretation of these models requires the distinction between non-specific crystal packing contacts and biologically relevant interactions. This has been investigated previously and classification approaches have been proposed. However, less attention has been devoted to distinguishing different types of biological interactions. These interactions are classified as obligate and non-obligate according to the effect of the complex formation on the stability of the protomers. So far no automatic classification methods for distinguishing obligate, non-obligate and crystal packing interactions have been made available. Results Six interface properties have been investigated on a dataset of 243 protein interactions. The six properties have been combined using a support vector machine algorithm, resulting in NOXclass, a classifier for distinguishing obligate, non-obligate and crystal packing interactions. We achieve an accuracy of 91.8% for the classification of these three types of interactions using a leave-one-out cross-validation procedure. Conclusion NOXclass allows the interpretation and analysis of protein quaternary structures. In particular, it generates testable hypotheses regarding the nature of protein-protein interactions, when experimental results are not available. We expect this server will benefit the users of protein structural models, as well as protein crystallographers and NMR spectroscopists. A web server based on the method and the datasets used in this study are available at http://noxclass.bioinf.mpi-inf.mpg.de/.

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

    Science.gov (United States)

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

    2011-07-18

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

  14. Characterization of protein-protein interactions by isothermal titration calorimetry.

    Science.gov (United States)

    Velazquez-Campoy, Adrian; Leavitt, Stephanie A; Freire, Ernesto

    2015-01-01

    The analysis of protein-protein interactions has attracted the attention of many researchers from both a fundamental point of view and a practical point of view. From a fundamental point of view, the development of an understanding of the signaling events triggered by the interaction of two or more proteins provides key information to elucidate the functioning of many cell processes. From a practical point of view, understanding protein-protein interactions at a quantitative level provides the foundation for the development of antagonists or agonists of those interactions. Isothermal Titration Calorimetry (ITC) is the only technique with the capability of measuring not only binding affinity but the enthalpic and entropic components that define affinity. Over the years, isothermal titration calorimeters have evolved in sensitivity and accuracy. Today, TA Instruments and MicroCal market instruments with the performance required to evaluate protein-protein interactions. In this methods paper, we describe general procedures to analyze heterodimeric (porcine pancreatic trypsin binding to soybean trypsin inhibitor) and homodimeric (bovine pancreatic α-chymotrypsin) protein associations by ITC.

  15. Analysis of correlations between protein complex and protein-protein interaction and mRNA expression

    Institute of Scientific and Technical Information of China (English)

    CAI Lun; XUE Hong; LU Hongchao; ZHAO Yi; ZHU Xiaopeng; BU Dongbo; LING Lunjiang; CHEN Runsheng

    2003-01-01

    Protein-protein interaction is a physical interaction of two proteins in living cells. In budding yeast Saccharomyces cerevisiae, large-scale protein-protein interaction data have been obtained through high-throughput yeast two-hybrid systems (Y2H) and protein complex purification techniques based on mass-spectrometry. Here, we collect 11855 interactions between total 2617 proteins. Through seriate genome-wide mRNA expression data, similarity between two genes could be measured. Protein complex data can also be obtained publicly and can be translated to pair relationship that any two proteins can only exist in the same complex or not. Analysis of protein complex data, protein-protein interaction data and mRNA expression data can elucidate correlations between them. The results show that proteins that have interactions or similar expression patterns have a higher possibility to be in the same protein complex than randomized selected proteins, and proteins which have interactions and similar expression patterns are even more possible to exist in the same protein complex. The work indicates that comprehensive integration and analysis of public large-scale bioinformatical data, such as protein complex data, protein-protein interaction data and mRNA expression data, may help to uncover their relationships and common biological information underlying these data. The strategies described here may help to integrate and analyze other functional genomic and proteomic data, such as gene expression profiling, protein-localization mapping and large-scale phenotypic data, both in yeast and in other organisms.

  16. Protein-protein interaction network of celiac disease.

    Science.gov (United States)

    Zamanian Azodi, Mona; Peyvandi, Hassan; Rostami-Nejad, Mohammad; Safaei, Akram; Rostami, Kamran; Vafaee, Reza; Heidari, Mohammadhossein; Hosseini, Mostafa; Zali, Mohammad Reza

    2016-01-01

    The aim of this study is to investigate the Protein-Protein Interaction Network of Celiac Disease. Celiac disease (CD) is an autoimmune disease with susceptibility of individuals to gluten of wheat, rye and barley. Understanding the molecular mechanisms and involved pathway may lead to the development of drug target discovery. The protein interaction network is one of the supportive fields to discover the pathogenesis biomarkers for celiac disease. In the present study, we collected the articles that focused on the proteomic data in celiac disease. According to the gene expression investigations of these articles, 31 candidate proteins were selected for this study. The networks of related differentially expressed protein were explored using Cytoscape 3.3 and the PPI analysis methods such as MCODE and ClueGO. According to the network analysis Ubiquitin C, Heat shock protein 90kDa alpha (cytosolic and Grp94); class A, B and 1 member, Heat shock 70kDa protein, and protein 5 (glucose-regulated protein, 78kDa), T-complex, Chaperon in containing TCP1; subunit 7 (beta) and subunit 4 (delta) and subunit 2 (beta), have been introduced as hub-bottlnecks proteins. HSP90AA1, MKKS, EZR, HSPA14, APOB and CAD have been determined as seed proteins. Chaperons have a bold presentation in curtail area in network therefore these key proteins beside the other hub-bottlneck proteins may be a suitable candidates biomarker panel for diagnosis, prognosis and treatment processes in celiac disease.

  17. The DNA damage response pathways: at the crossroad of protein modifications

    Institute of Scientific and Technical Information of China (English)

    Michael SY Huen; Junjie Chen

    2008-01-01

    Post-translational modifications play a crucial role in coordinating cellular response to DNA damage. Recent evidence suggests an interplay between multiple protein modifications, including phosphorylation, ubiquitylation, acetylation and sumoylation, that combine to propagate the DNA damage signal to elicit cell cycle arrest, DNA repair, apoptosis and senescence. Utility of specific post-translational modifiers allows temporal and spatial control over protein relo-calization and interactions, and may represent a means for trans-regulatory activation of protein activities. The abil-ity to recognize these specific modifiers also underscores the capacity for signal amplification, a crucial step for the maintenance of genomic stability and tumor prevention. Here we have summarized recent findings that highlight the complexity of post-translational modifications in coordinating the DNA damage response, with emphasis on the DNA damage signaling cascade.

  18. Yeast Interacting Proteins Database: YDR020C, YDR020C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available a screen for mutants with increased levels of rDNA transcription; weak similarity with uridine kinases and ...e protein of unknown function; non-essential gene identified in a screen for mutants with increased levels...n of unknown function; non-essential gene identified in a screen for mutants with increased levels of rDNA t...sential gene identified in a screen for mutants with increased levels of rDNA transcription; weak similarity

  19. Evolvability of yeast protein-protein interaction interfaces.

    Science.gov (United States)

    Talavera, David; Williams, Simon G; Norris, Matthew G S; Robertson, David L; Lovell, Simon C

    2012-06-22

    The functional importance of protein-protein interactions indicates that there should be strong evolutionary constraint on their interaction interfaces. However, binding interfaces are frequently affected by amino acid replacements. Change due to coevolution within interfaces can contribute to variability but is not ubiquitous. An alternative explanation for the ability of surfaces to accept replacements may be that many residues can be changed without affecting the interaction. Candidates for these types of residues are those that make interchain interaction only through the protein main chain, β-carbon, or associated hydrogen atoms. Since almost all residues have these atoms, we hypothesize that this subset of interface residues may be more easily substituted than those that make interactions through other atoms. We term such interactions "residue type independent." Investigating this hypothesis, we find that nearly a quarter of residues in protein interaction interfaces make exclusively interchain residue-type-independent contacts. These residues are less structurally constrained and less conserved than residues making residue-type-specific interactions. We propose that residue-type-independent interactions allow substitutions in binding interfaces while the specificity of binding is maintained.

  20. 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...

  1. Yeast Interacting Proteins Database: YDL089W, YCR086W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available rDNA repeat stability; null mutant causes increase in unequal sister-chromatid exchange; GFP-fusion protein...p, Lrs4p; required for rDNA repeat stability; null mutant causes increase in unequal sister-chromatid exchan

  2. Alpha shape and Delaunay triangulation in studies of protein-related interactions.

    Science.gov (United States)

    Zhou, Weiqiang; Yan, Hong

    2014-01-01

    In recent years, more 3D protein structures have become available, which has made the analysis of large molecular structures much easier. There is a strong demand for geometric models for the study of protein-related interactions. Alpha shape and Delaunay triangulation are powerful tools to represent protein structures and have advantages in characterizing the surface curvature and atom contacts. This review presents state-of-the-art applications of alpha shape and Delaunay triangulation in the studies on protein-DNA, protein-protein, protein-ligand interactions and protein structure analysis.

  3. A General System for Studying Protein-Protein Interactions in Gram-Negative Bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Pelletier, Dale A [ORNL; Auberry, Deanna L [ORNL; Buchanan, Michelle V [ORNL; Cannon, Bill [Pacific Northwest National Laboratory (PNNL); Daly, Don S. [Pacific Northwest National Laboratory (PNNL); Doktycz, Mitchel John [ORNL; Foote, Linda J [ORNL; Hervey, IV, William Judson [ORNL; Hooker, Brian [Pacific Northwest National Laboratory (PNNL); Hurst, Gregory {Greg} B [ORNL; Kennel, Steve J [ORNL; Lankford, Patricia K [ORNL; Larimer, Frank W [ORNL; Lu, Tse-Yuan S [ORNL; McDonald, W Hayes [ORNL; McKeown, Catherine K [ORNL; Morrell-Falvey, Jennifer L [ORNL; Owens, Elizabeth T [ORNL; Schmoyer, Denise D [ORNL; Shah, Manesh B [ORNL; Wiley, Steven [Pacific Northwest National Laboratory (PNNL); Wang, Yisong [ORNL; Gilmore, Jason [Pacific Northwest National Laboratory (PNNL)

    2008-01-01

    Abstract One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged bait proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.

  4. Treponema denticola interactions with host proteins

    Directory of Open Access Journals (Sweden)

    J. Christopher Fenno

    2012-02-01

    Full Text Available Oral Treponema species, most notably T. denticola, are implicated in the destructive effects of human periodontal disease. Progress in the molecular analysis of interactions between T. denticola and host proteins is reviewed here, with particular emphasis on the characterization of surface-expressed and secreted proteins of T. denticola involved in interactions with host cells, extracellular matrix components, and components of the innate immune system.

  5. Modes of Escherichia coli Dps Interaction with DNA as Revealed by Atomic Force Microscopy.

    Directory of Open Access Journals (Sweden)

    Vladislav V Melekhov

    Full Text Available Multifunctional protein Dps plays an important role in iron assimilation and a crucial role in bacterial genome packaging. Its monomers form dodecameric spherical particles accumulating ~400 molecules of oxidized iron ions within the protein cavity and applying a flexible N-terminal ends of each subunit for interaction with DNA. Deposition of iron is a well-studied process by which cells remove toxic Fe2+ ions from the genetic material and store them in an easily accessible form. However, the mode of interaction with linear DNA remained mysterious and binary complexes with Dps have not been characterized so far. It is widely believed that Dps binds DNA without any sequence or structural preferences but several lines of evidence have demonstrated its ability to differentiate gene expression, which assumes certain specificity. Here we show that Dps has a different affinity for the two DNA fragments taken from the dps gene regulatory region. We found by atomic force microscopy that Dps predominantly occupies thermodynamically unstable ends of linear double-stranded DNA fragments and has high affinity to the central part of the branched DNA molecule self-assembled from three single-stranded oligonucleotides. It was proposed that Dps prefers binding to those regions in DNA that provide more contact pads for the triad of its DNA-binding bundle associated with one vertex of the protein globule. To our knowledge, this is the first study revealed the nucleoid protein with an affinity to branched DNA typical for genomic regions with direct and inverted repeats. As a ubiquitous feature of bacterial and eukaryotic genomes, such structural elements should be of particular care, but the protein system evolutionarily adapted for this function is not yet known, and we suggest Dps as a putative component of this system.

  6. Moonlighting proteins in sperm-egg interactions.

    Science.gov (United States)

    Petit, François M; Serres, Catherine; Auer, Jana

    2014-12-01

    Sperm-egg interaction is a highly species-specific step during the fertilization process. The first steps consist of recognition between proteins on the sperm head and zona pellucida (ZP) glycoproteins, the acellular coat that protects the oocyte. We aimed to determine which sperm head proteins interact with ZP2, ZP3 and ZP4 in humans. Two approaches were combined to identify these proteins: immunoblotting human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far-Western blotting of human sperm proteins overlaid by each of the human recombinant ZP (hrZP) proteins. We used a proteomic approach with 2D electrophoretic separation of sperm protein revealed using either ASAs eluted from infertile patients or recombinant human ZP glycoproteins expressed in Chinese-hamster ovary (CHO) cells. Only spots highlighted by both methods were analysed by MALDI-MS/MS for identification. We identified proteins already described in human spermatozoa, but implicated in different metabolic pathways such as glycolytic enzymes [phosphokinase type 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and triose phosphate isomerase (TPI)], detoxification enzymes [GST Mu (GSTM) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) 4], ion channels [voltage-dependent anion channel 2 (VDAC2)] or structural proteins (outer dense fibre 2). Several proteins were localized on the sperm head by indirect immunofluorescence, and their interaction with ZP proteins was confirmed by co-precipitation experiments. These results confirm the complexity of the sperm-ZP recognition process in humans with the implication of different proteins interacting with the main three ZP glycoproteins. The multiple roles of these proteins suggest that they are multifaceted or moonlighting proteins.

  7. Interface-resolved network of protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Margaret E Johnson

    Full Text Available We define an interface-interaction network (IIN to capture the specificity and competition between protein-protein interactions (PPI. This new type of network represents interactions between individual interfaces used in functional protein binding and thereby contains the detail necessary to describe the competition and cooperation between any pair of binding partners. Here we establish a general framework for the construction of IINs that merges computational structure-based interface assignment with careful curation of available literature. To complement limited structural data, the inclusion of biochemical data is critical for achieving the accuracy and completeness necessary to analyze the specificity and competition between the protein interactions. Firstly, this procedure provides a means to clarify the information content of existing data on purported protein interactions and to remove indirect and spurious interactions. Secondly, the IIN we have constructed here for proteins involved in clathrin-mediated endocytosis (CME exhibits distinctive topological properties. In contrast to PPI networks with their global and relatively dense connectivity, the fragmentation of the IIN into distinctive network modules suggests that different functional pressures act on the evolution of its topology. Large modules in the IIN are formed by interfaces sharing specificity for certain domain types, such as SH3 domains distributed across different proteins. The shared and distinct specificity of an interface is necessary for effective negative and positive design of highly selective binding targets. Lastly, the organization of detailed structural data in a network format allows one to identify pathways of specific binding interactions and thereby predict effects of mutations at specific surfaces on a protein and of specific binding inhibitors, as we explore in several examples. Overall, the endocytosis IIN is remarkably complex and rich in features masked

  8. Yeast Interacting Proteins Database: YGL237C, YOR047C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available ene expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding prote... expression; interacts with protein kinase Snf1p, glucose sensors Snf3p and Rgt2p, and TATA-binding protein

  9. Mining minimal motif pair sets maximally covering interactions in a protein-protein interaction network

    NARCIS (Netherlands)

    Boyen, P.; Neven, F.; Valentim, F.L.; Dijk, van A.D.J.

    2013-01-01

    Correlated motif covering (CMC) is the problem of finding a set of motif pairs, i.e., pairs of patterns, in the sequences of proteins from a protein-protein interaction network (PPI-network) that describe the interactions in the network as concisely as possible. In other words, a perfect solution fo

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

    Directory of Open Access Journals (Sweden)

    Huiying Zhao

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

  11. A simple dependence between protein evolution rate and the number of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Hirsh Aaron E

    2003-05-01

    Full Text Available Abstract Background It has been shown for an evolutionarily distant genomic comparison that the number of protein-protein interactions a protein has correlates negatively with their rates of evolution. However, the generality of this observation has recently been challenged. Here we examine the problem using protein-protein interaction data from the yeast Saccharomyces cerevisiae and genome sequences from two other yeast species. Results In contrast to a previous study that used an incomplete set of protein-protein interactions, we observed a highly significant correlation between number of interactions and evolutionary distance to either Candida albicans or Schizosaccharomyces pombe. This study differs from the previous one in that it includes all known protein interactions from S. cerevisiae, and a larger set of protein evolutionary rates. In both evolutionary comparisons, a simple monotonic relationship was found across the entire range of the number of protein-protein interactions. In agreement with our earlier findings, this relationship cannot be explained by the fact that proteins with many interactions tend to be important to yeast. The generality of these correlations in other kingdoms of life unfortunately cannot be addressed at this time, due to the incompleteness of protein-protein interaction data from organisms other than S. cerevisiae. Conclusions Protein-protein interactions tend to slow the rate at which proteins evolve. This may be due to structural constraints that must be met to maintain interactions, but more work is needed to definitively establish the mechanism(s behind the correlations we have observed.

  12. DNA methyltransferase DNMT3A associates with viral proteins and impacts HSV-1 infection.

    Science.gov (United States)

    Rowles, Daniell L; Tsai, Yuan-Chin; Greco, Todd M; Lin, Aaron E; Li, Minghao; Yeh, Justin; Cristea, Ileana M

    2015-06-01

    Viral infections can alter the cellular epigenetic landscape, through modulation of either DNA methylation profiles or chromatin remodeling enzymes and histone modifications. These changes can act to promote viral replication or host defense. Herpes simplex virus type 1 (HSV-1) is a prominent human pathogen, which relies on interactions with host factors for efficient replication and spread. Nevertheless, the knowledge regarding its modulation of epigenetic factors remains limited. Here, we used fluorescently-labeled viruses in conjunction with immunoaffinity purification and MS to study virus-virus and virus-host protein interactions during HSV-1 infection in primary human fibroblasts. We identified interactions among viral capsid and tegument proteins, detecting phosphorylation of the capsid protein VP26 at sites within its UL37-binding domain, and an acetylation within the major capsid protein VP5. Interestingly, we found a nuclear association between viral capsid proteins and the de novo DNA methyltransferase DNA (cytosine-5)-methyltransferase 3A (DNMT3A), which we confirmed by reciprocal isolations and microscopy. We show that drug-induced inhibition of DNA methyltransferase activity, as well as siRNA- and shRNA-mediated DNMT3A knockdowns trigger reductions in virus titers. Altogether, our results highlight a functional association of viral proteins with the mammalian DNA methyltransferase machinery, pointing to DNMT3A as a host factor required for effective HSV-1 infection.

  13. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    Science.gov (United States)

    Jette, Nicholas; Lees-Miller, Susan P.

    2015-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

  14. Understanding Protein-Protein Interactions Using Local Structural Features

    DEFF Research Database (Denmark)

    Planas-Iglesias, Joan; Bonet, Jaume; García-García, Javier;

    2013-01-01

    Protein-protein interactions (PPIs) play a relevant role among the different functions of a cell. Identifying the PPI network of a given organism (interactome) is useful to shed light on the key molecular mechanisms within a biological system. In this work, we show the role of structural features...... (loops and domains) to comprehend the molecular mechanisms of PPIs. A paradox in protein-protein binding is to explain how the unbound proteins of a binary complex recognize each other among a large population within a cell and how they find their best docking interface in a short timescale. We use...

  15. 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.

  16. Molecular principles of protein stability and protein-protein interactions

    OpenAIRE

    Lendel, Christofer

    2005-01-01

    Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z ...

  17. HCVpro: Hepatitis C virus protein interaction database

    KAUST Repository

    Kwofie, Samuel K.

    2011-12-01

    It is essential to catalog characterized hepatitis C virus (HCV) protein-protein interaction (PPI) data and the associated plethora of vital functional information to augment the search for therapies, vaccines and diagnostic biomarkers. In furtherance of these goals, we have developed the hepatitis C virus protein interaction database (HCVpro) by integrating manually verified hepatitis C virus-virus and virus-human protein interactions curated from literature and databases. HCVpro is a comprehensive and integrated HCV-specific knowledgebase housing consolidated information on PPIs, functional genomics and molecular data obtained from a variety of virus databases (VirHostNet, VirusMint, HCVdb and euHCVdb), and from BIND and other relevant biology repositories. HCVpro is further populated with information on hepatocellular carcinoma (HCC) related genes that are mapped onto their encoded cellular proteins. Incorporated proteins have been mapped onto Gene Ontologies, canonical pathways, Online Mendelian Inheritance in Man (OMIM) and extensively cross-referenced to other essential annotations. The database is enriched with exhaustive reviews on structure and functions of HCV proteins, current state of drug and vaccine development and links to recommended journal articles. Users can query the database using specific protein identifiers (IDs), chromosomal locations of a gene, interaction detection methods, indexed PubMed sources as well as HCVpro, BIND and VirusMint IDs. The use of HCVpro is free and the resource can be accessed via http://apps.sanbi.ac.za/hcvpro/ or http://cbrc.kaust.edu.sa/hcvpro/. © 2011 Elsevier B.V.

  18. Length, protein–protein interactions, and complexity

    NARCIS (Netherlands)

    Tan, T.; Frenkel, D.; Gupta, V.; Deem, M.W.

    2005-01-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein–protein interactions has driven the selection of longer proteins, as they ar

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

    Science.gov (United States)

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

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

  20. Microwave Interaction with DNA in Solution.

    Science.gov (United States)

    1987-10-10

    DNA fibers. In this contract, we found that the interpretation of the EDSS experimental resonances required a rather small but not unimaginably small...challanged by others who have been unable to reproduce the EDSS results. The resonances may not ultimately stand the test of history. Our analysis shows

  1. Website on Protein Interaction and Protein Structure Related Work

    Science.gov (United States)

    Samanta, Manoj; Liang, Shoudan; Biegel, Bryan (Technical Monitor)

    2003-01-01

    In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2)