WorldWideScience

Sample records for adenine-modified functionalized dna

  1. DNA: Structure and function

    Sinden, Richard R.; E. Pearson, Christopher; N. Potaman, Vladimir;

    1998-01-01

    This chapter discusses the structure and function of DNA. DNA occupies a critical role in cells, because it is the source of all intrinsic genetic information. Chemically, DNA is a very stable molecule, a characteristic important for a macromolecule that may have to persist in an intact form for a...... long period of time before its information is accessed by the cell. Although DNA plays a critical role as an informational storage molecule, it is by no means as unexciting as a computer tape or disk drive. The structure of the DNA described by Watson and Crick in 1953 is a right handed helix of two...... individual antiparallel DNA strands. Hydrogen bonds provide specificity that allows pairing between the complementary bases (A.T and G.C) in opposite strands. Base stacking occurs near the center of the DNA helix and provides a great deal of stability to the helix (in addition to hydrogen bonding). The sugar...

  2. Functionalizing Designer DNA Crystals

    Chandrasekaran, Arun Richard

    nucleotides is usually pH dependent (pH TFOs were examined: TFO-1 was unmodified while TFOs 2-4 contained additional stabilizing analogues capable of extending triplex formation to pH 7. In addition, each of the TFOs contained a Cy5 dye at the 5'-end of the oligonucleotide to aid in characterization of TFO binding - crystals were obtained with all four variations of TFOs. Formation of DNA triplex in the motif was characterized by an electrophoretic mobility shift assay (EMSA), UV melting studies and FRET. Crystals containing TFO-1 (unmodified) and TFO-2 (with 2'-amino ethoxy modification) were isolated and flash-frozen in liquid nitrogen for X-ray data collection at beam line NSLS-X25. X-ray data was also collected for crystals of the 3-turn triangle without any TFO bound to it. Difference maps were done between the crystals with TFO against the one without to identify any additional electron density corresponding to the third strand in the triplex binding region. The data from the crystal containing TFO-2 was used to further analyze if the additional density can match the expected position of the TFO on the triangle motif. Since the additional density did not correspond to the entire binding region, 2Fo-Fc, 3Fo-2Fc and 4Fo-3Fc maps were done to check for missing pieces of the electron density. From the resulting 2Fo-Fc map, the asymmetric unit from the 3-turn triangle (31-bp duplex model based on previous structure 3UBI) was inserted into the density as a reference. However, the electron density corresponding to the TFO was still not continuous throughout the 13-nt triplex binding region and allowed only a partial fit of the TFO. The third nucleotide in positions 1, 3, 4, 6, 7 were fit into the density in the major groove of the underlying duplex with proper triplex configuration. The third chapter describes the triplex approach to position a functional group (the UV cross-linking agent psoralen) within a pre-formed DNA motif. Triplex formation and psoralen cross-linking of

  3. Functionalizing Designer DNA Crystals

    Chandrasekaran, Arun Richard

    nucleotides is usually pH dependent (pH < 6) four different TFOs were examined: TFO-1 was unmodified while TFOs 2-4 contained additional stabilizing analogues capable of extending triplex formation to pH 7. In addition, each of the TFOs contained a Cy5 dye at the 5'-end of the oligonucleotide to aid in characterization of TFO binding - crystals were obtained with all four variations of TFOs. Formation of DNA triplex in the motif was characterized by an electrophoretic mobility shift assay (EMSA), UV melting studies and FRET. Crystals containing TFO-1 (unmodified) and TFO-2 (with 2'-amino ethoxy modification) were isolated and flash-frozen in liquid nitrogen for X-ray data collection at beam line NSLS-X25. X-ray data was also collected for crystals of the 3-turn triangle without any TFO bound to it. Difference maps were done between the crystals with TFO against the one without to identify any additional electron density corresponding to the third strand in the triplex binding region. The data from the crystal containing TFO-2 was used to further analyze if the additional density can match the expected position of the TFO on the triangle motif. Since the additional density did not correspond to the entire binding region, 2Fo-Fc, 3Fo-2Fc and 4Fo-3Fc maps were done to check for missing pieces of the electron density. From the resulting 2Fo-Fc map, the asymmetric unit from the 3-turn triangle (31-bp duplex model based on previous structure 3UBI) was inserted into the density as a reference. However, the electron density corresponding to the TFO was still not continuous throughout the 13-nt triplex binding region and allowed only a partial fit of the TFO. The third nucleotide in positions 1, 3, 4, 6, 7 were fit into the density in the major groove of the underlying duplex with proper triplex configuration. The third chapter describes the triplex approach to position a functional group (the UV cross-linking agent psoralen) within a pre-formed DNA motif. Triplex formation and

  4. Structure and function of DNA polymerase μ

    DNA polymerases are enzymes playing the central role in DNA metabolism, including DNA replication, DNA repair and recombination. DNA polymerase μ (pol μ DNA polymerase λ (pol λ) and terminal deoxynucleotidyltransferase (TdT) in X family DNA polymerases function in non-homologous end-joining (NHEJ), which is the predonmiant repair pathway for DNA double-strand breaks (DSBs). NHEJ involves enzymes that capture both ends of the broken DNA strand, bring them together in a synaptic DNA-protein complex, and repair the DSB. Pol μ and pol λ fill in the gaps at the junction to maintain the genomic integrity. TdT synthesizes N region at the junction during V(D)J recombination and promotes diversity of immunoglobulin or T-cell receptor gene. Among these three polymerases, the regulatory mechanisms of pol μ remain rather unclear. We have approached the mechanism of pol μ from both sides of structure and cellular dynamics. Here, we propose some new insights into pol μ and the probable NHEJ model including our findings. (author)

  5. Controlling Function and Structure with DNA

    Tørring, Thomas

    2011-01-01

    that the DNA origami could be massively modified fast and efficiently with versatile groups such as amines, fluorophores and biotin. Applying small dynamic DNA structures to control the function of so-called photosensitizers, was the aim of the third and final research topic presented. Photosensitizers...... are functional molecules that upon irradiation can excite naturally occurring triplet oxygen to the cytotoxic singlet oxygen. This process is interesting in terms of photodynamic therapy, but lack of control is limiting the applications. The excitation can be controlled by installing quenching...... molecules close to the photosensitizer, and we exploited this by tethering the two molecules together with a dynamic DNA nanostructure, known as an i-motif. This allowed us to control the distance between the two molecules, and thereby the excitation of triplet oxygen. Finally, the thesis is concluded with...

  6. DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

    Mossi, R; Ferrari, E.; Hübscher, U

    1998-01-01

    The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by th...

  7. Functional roles of DNA polymerases β and γ

    The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC2.7.7.7)β and γ were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. uv irradiation of neuronal nuclei from 60-day-old rats resulted in a 7- to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase β which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this was probably due to the inability of brain tissue to excise alkylated bases from DNA. The role of DNA polymerase γ was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that under these conditions, DNA replication occurs in miatochondria, and exploiting the fact that DNA polymerase γ is the only DNA polymerase present in mitochondria, evidence was obtained for a role of DNA polymerase γ in mitochondrial DNA replication. Based on these results and on the wealth of literature on DNA polymerase α, we conclude that DNA polymerase α is mainly responsible for DNA replication in nuclei, DNA polymerase β is involved in nuclear DNA repair, and DNA polymerase γ is the mitochondrial replicating enzyme. However, minor roles for DNA polymerase α in DNA repair or for DNA polymerase β in DNA replication cannot be excluded

  8. Dielectric Function and Electronic Excitations of Functionalized DNA Thin Films

    Lee, Hosuk; Lee, Hosun; Lee, Jung Eun; Rha Lee, U.; Choi, Dong Hoon

    2010-06-01

    We measure the dielectric functions of organic-soluble, functionalized DNAs bearing functional moieties in the near-infrared, visible, and ultra-violet spectra by using spectroscopic ellipsometry. Natural double-stranded DNA is dissolved in water and reacted with carbazole-based trimethyl ammonium bromide, cetyltrimethylammonium bromide, and chalcone-terminated trimethyl ammonium bromide. The functional DNA products are all precipitated and filtered for washing and drying. We successfully prepare functionalized DNAs that are insoluble in water but soluble in organic solvents. The thin films are fabricated by using the spin coating technique after preparing solutions in either homogeneous or mixed organic solvents. We measure the ultraviolet-visible absorbance spectra of the films. The absorbance spectra show that the optical energy gaps of the functionalized DNAs change little even though the DNAs are connected to the complex molecules by electrostatic interaction. From the measured ellipsometric angles, we estimate the dielectric functions by using parametric optical constant model and layer model analysis. Depending on the nature of the attached complex molecules, the dielectric functions change, new optical structures develop below and above band gaps arising from the side molecules, and the optical energy gaps of the DNAs are altered slightly by weak coupling to the tethered complex molecules.

  9. Eukaryotic DNA Ligases: Structural and Functional Insights

    Ellenberger, Tom; Tomkinson, Alan E.

    2008-01-01

    DNA ligases are required for DNA replication, repair, and recombination. In eukaryotes, there are three families of ATP-dependent DNA ligases. Members of the DNA ligase I and IV families are found in all eukaryotes, whereas DNA ligase III family members are restricted to vertebrates. These enzymes share a common catalytic region comprising a DNA-binding domain, a nucleotidyltransferase (NTase) domain, and an oligonucleotide/oligosaccharide binding (OB)-fold domain. The catalytic region encirc...

  10. Regulation and function of DNA methylation in plants and animals

    He, Xinjian

    2011-02-15

    DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. © 2011 IBCB, SIBS, CAS All rights reserved.

  11. Functions and applications of polypyridyl complexes in DNA

    2001-01-01

    The syntheses and desymmetration of a series of novel polypyridyl ligands as well as their complexes, and their DNA-binding properties are reported in this review.The stresses are focused on the functions and potential applications of such complexes as DNA structural probe, DNA molecular light switches, anti-cancer drugs, and photoactivated cleavage agents.

  12. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2011-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excisio...

  13. Functional redundancy between DNA ligases I and III in DNA replication in vertebrate cells

    Arakawa, Hiroshi; Bednar, Theresa; Wang, Minli; Paul, Katja; Mladenov, Emil; Bencsik-Theilen, Alena A.; Iliakis, George

    2012-01-01

    In eukaryotes, the three families of ATP-dependent DNA ligases are associated with specific functions in DNA metabolism. DNA ligase I (LigI) catalyzes Okazaki-fragment ligation at the replication fork and nucleotide excision repair (NER). DNA ligase IV (LigIV) mediates repair of DNA double strand breaks (DSB) via the canonical non-homologous end-joining (NHEJ) pathway. The evolutionary younger DNA ligase III (LigIII) is restricted to higher eukaryotes and has been associated with base excision (BER) and single strand break repair (SSBR). Here, using conditional knockout strategies for LIG3 and concomitant inactivation of the LIG1 and LIG4 genes, we show that in DT40 cells LigIII efficiently supports semi-conservative DNA replication. Our observations demonstrate a high functional versatility for the evolutionary new LigIII in DNA replication and mitochondrial metabolism, and suggest the presence of an alternative pathway for Okazaki fragment ligation. PMID:22127868

  14. Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

    Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

  15. Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

    Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription

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

    Mazin, A V; Kowalczykowski, S C

    1998-01-01

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

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

    Tatarinova, Olga N.; Smirnov, Igor P. [Research Institute for Physico-Chemical Medicine of the Federal Medical-Biological Agency of the Russian Federation (Russian Federation); Safenkova, Irina V. [A.N. Bach Institute of Biochemistry (Russian Federation); Varizhuk, Anna M.; Pozmogova, Galina E., E-mail: pozmge@gmail.com [Research Institute for Physico-Chemical Medicine of the Federal Medical-Biological Agency of the Russian Federation (Russian Federation)

    2012-10-15

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

  18. Resurrection of DNA function in vivo from an extinct genome.

    Andrew J Pask

    Full Text Available There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine, obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

  19. Carbon nanotube-DNA nanoarchitectures and electronic functionality.

    Wang, Xu; Liu, Fei; Andavan, G T Senthil; Jing, Xiaoye; Singh, Krishna; Yazdanpanah, Vahid R; Bruque, Nicolas; Pandey, Rajeev R; Lake, Roger; Ozkan, Mihrimah; Wang, Kang L; Ozkan, Cengiz S

    2006-11-01

    Biological molecules such as deoxyribonucleic acid (DNA) possess inherent recognition and self-assembly capabilities, and are attractive templates for constructing functional hierarchical material structures as building blocks for nanoelectronics. Here we report the assembly and electronic functionality of nanoarchitectures based on conjugates of single-walled carbon nanotubes (SWNTs) functionalized with carboxylic groups and single-stranded DNA (ssDNA) sequences possessing terminal amino groups on both ends, hybridized together through amide linkages by adopting a straightforward synthetic route. Morphological and chemical-functional characterization of the nanoarchitectures are investigated using scanning electron microscopy, transmission electron microscopy, atomic force microscopy, energy-dispersive X-ray spectroscopy, Raman spectroscopy, and Fourier-transform infrared spectroscopy. Electrical measurements (I-V characterization) of the nanoarchitectures demonstrate negative differential resistance in the presence of SWNT/ssDNA interfaces, which indicates a biomimetic route to fabricating resonant tunneling diodes. I-V characterization on platinum-metallized SWNT-ssDNA nanoarchitectures via salt reduction indicates modulation of their electrical properties, with effects ranging from those of a resonant tunneling diode to a resistor, depending on the amount of metallization. Electron transport through the nanoarchitectures has been analyzed by density functional theory calculations. Our studies illustrate the great promise of biomimetic assembly of functional nanosystems based on biotemplated materials and present new avenues toward exciting future opportunities in nanoelectronics and nanobiotechnology. PMID:17192987

  20. Structure and Function Study of Phi29 DNA packaging motor

    Fang, Huaming

    molecules were required to bind to one short dsDNA molecule. The inhibitive curve of Walker B mutant gp16 analyzed by binomial distribution model showed that one inactive mutant gp16 in the gp16 ring could block the function of the motor and the stoichiometry of gp16 was six. These findings facilitate our understanding of the molecular mechanism of viral DNA packaging: a novel viral DNA packaging model "push through a one-way valve" was proposed. In this model, the connector functioned as a valve to allow DNA to enter but prevented it from sliding out during DNA packaging; the six subunits in the gp16 ring acted sequentially to push DNA into the connector channel. ATP binding of gp16 induced a conformation change with a high affinity for dsDNA. Then, the ATP was hydrolyzed which resulted in the movement of subdomains in this individual gp16 subunit and DNA was pushed forward, followed by the double helix of dsDNA being brought forward to the adjacent subunit in the gp16 ring. The elucidation of the viral DNA packaging mechanism holds great potential for developing artificial motors for delivering drugs and other molecular cargos.

  1. Beyond the dna: a prototype for functional genomics

    Albala, J

    2000-03-02

    A prototype oligonucleotide ''functional chip'' has been developed to screen novel DNA repair proteins for their ability to bind or alter different forms of DNA. This chip has been developed as a functional genomics screen for analysis of protein-DNA interactions for novel proteins identified from the Human Genome Project The process of novel gene identification that has ensued as a consequence of available sequence information is remarkable. The challenge how lies in determining the function of newly identified gene products in a time-and cost-effective high-throughput manner. The functional chip is generated by the robotic application of DNA spotted in a microarray format onto a glass slide. Individual proteins are then analyzed against the different form of DNA bound to the slide. Several prototype functional chips were designed to contain various DNA fragments tethered to a glass slide for analysis of protein-DNA binding or enzymatic activity of known proteins. The technology has been developed to screen novel, putative DNA repair proteins for their ability to bind various types of DNA alone and in concert with protein partners. An additional scheme has been devised to screen putative repair enzymes for their ability to process different types of DNA molecules. Current methods to analyze gene expression primarily utilize either of two technologies. The oligonucleotide chip, pioneered by Fodor and co-workers and Affymetrix, Inc., consists of greater than 64,000 oligonucleotides attached in situ to a glass support. The oligonucleotide chip has been used primarily to identify specific mutations in a given gene by hybridization against a fluorescently-labeled substrate. The second method is the microarray, whereby DNA targets are systematically arranged on a glass slide and then hybridized with fluorescently-labeled complex targets for gene expression analysis (Jordan, 1998). By this technique, a large amount of information can be obtained

  2. Functional Dissection of the DNA Interface of the Nucleotidyltransferase Domain of Chlorella Virus DNA Ligase*

    Samai, Poulami; Shuman, Stewart

    2011-01-01

    Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3′-OH ...

  3. Functionalization of DNA Nanostructures for Cell Signaling Applications

    Pedersen, Ronnie O.

    Transforming growth factor beta (TGF-beta) is an important cytokine responsible for a wide range of different cellular functions including extracellular matrix formation, angiogenesis and epithelial-mesenchymal transition. We have sought to use self-assembling DNA nanostructures to influence TGF-beta signaling. The predictable Watson Crick base pairing allows for designing self-assembling nanoscale structures using oligonucleotides. We have used the method of DNA origami to assemble structures functionalized with multiple peptides that bind TGF-beta receptors outside the ligand binding domain. This allows the nanostructures to cluster TGF-beta receptors and lower the energy barrier of ligand binding thus sensitizing the cells to TGF-beta stimulation. To prove efficacy of our nanostructures we have utilized immunofluorescent staining of Smad2/4 in order to monitor TGF-beta mediated translocation of Smad2/4 to the cell nucleus. We have also utilized Smad2/4 responsive luminescence constructs that allows us to quantify TGF-beta stimulation with and without nanostructures. To functionalize our nanostructures we relied on biotin-streptavidin linkages. This introduces a multivalency that is not necessarily desirable in all designs. Therefore we have investigated alternative means of functionalization. The first approach is based on targeting DNA nanostructure by using zinc finger binding proteins. Efficacy of zinc finger binding proteins was assayed by the use of enzyme-linked immunosorbent (ELISA) assay and atomic force microscopy (AFM). While ELISA indicated a relative specificity of zinc finger proteins for target DNA sequences AFM showed a high degree of non-specific binding and insufficient affinity. The second approach is based on using peptide nucleic acid (PNA) incorporated in the nanostructure through base pairing. PNA is a synthetic DNA analog consisting of a backbone of repeating N-(2-aminoethyl)-glycine units to which purine and pyrimidine bases are linked by

  4. DNA Modifications: Function and Applications in Normal and Disease States

    Vichithra R. B. Liyanage

    2014-10-01

    Full Text Available Epigenetics refers to a variety of processes that have heritable effects on gene expression programs without changes in DNA sequence. Key players in epigenetic control are chemical modifications to DNA, histone, and non-histone chromosomal proteins, which establish a complex regulatory network that controls genome function. Methylation of DNA at the fifth position of cytosine in CpG dinucleotides (5-methylcytosine, 5mC, which is carried out by DNA methyltransferases, is commonly associated with gene silencing. However, high resolution mapping of DNA methylation has revealed that 5mC is enriched in exonic nucleosomes and at intron-exon junctions, suggesting a role of DNA methylation in the relationship between elongation and RNA splicing. Recent studies have increased our knowledge of another modification of DNA, 5-hydroxymethylcytosine (5hmC, which is a product of the ten-eleven translocation (TET proteins converting 5mC to 5hmC. In this review, we will highlight current studies on the role of 5mC and 5hmC in regulating gene expression (using some aspects of brain development as examples. Further the roles of these modifications in detection of pathological states (type 2 diabetes, Rett syndrome, fetal alcohol spectrum disorders and teratogen exposure will be discussed.

  5. Functional DNA: Teaching Infinite Series through Genetic Analogy

    Kowalski, R. Travis

    2011-01-01

    This article presents an extended analogy that connects infinite sequences and series to the science of genetics, by identifying power series as "DNA for a function." This analogy allows standard topics such as convergence tests or Taylor approximations to be recast in a "forensic" light as mathematical analogs of genetic concepts such as DNA…

  6. Function of YY1 in long-distance DNA interactions

    Michael L. Atchison

    2014-02-01

    Full Text Available During B cell development long-distance DNA interactions are needed for V(DJ somatic rearrangement of the immunoglobulin (Ig loci to produce functional Ig genes, and for class switch recombination (CSR needed for antibody maturation. The tissue-specificity and developmental timing of these mechanisms are a subject of active investigation. A small number of factors are implicated in controlling Ig locus long-distance interactions including Pax5, YY1, EZH2, IKAROS, CTCF, cohesin, and condensin proteins. Here we will focus on the role of YY1 in controlling these mechanisms. YY1 is a multifunctional transcription factor involved in transcriptional activation and repression, X chromosome inactivation, Polycomb Group (PcG protein DNA recruitment, and recruitment of proteins required for epigenetic modifications (acetylation, deacetylation, methylation, ubiquitiation, sumoylation etc.. YY1 conditional knock-out indicated that YY1 is required for B cell development, at least in part, by controlling long-distance DNA interactions at the IgH and Igκ loci. Our recent data show that YY1 is also required for CSR. The mechanisms implicated in YY1 control of long-distance DNA interactions include controlling non-coding antisense RNA transcripts, recruitment of PcG proteins to DNA, and interaction with complexes involved in long-distance DNA interactions including the cohesin and condensin complexes. Though common rearrangement mechanisms operate at all Ig loci, their distinct temporal activation along with the ubiquitious nature of YY1 poses challenges for determining the specific mechanisms of YY1 function in these processes, and their regulation at the tissue-specific and B cell stage-specific level. The large numbers of post-translational modifications that control YY1 functions are possible candidates for regulation.

  7. Function of YY1 in Long-Distance DNA Interactions.

    Atchison, Michael L

    2014-01-01

    During B cell development, long-distance DNA interactions are needed for V(D)J somatic rearrangement of the immunoglobulin (Ig) loci to produce functional Ig genes, and for class switch recombination (CSR) needed for antibody maturation. The tissue-specificity and developmental timing of these mechanisms is a subject of active investigation. A small number of factors are implicated in controlling Ig locus long-distance interactions including Pax5, Yin Yang 1 (YY1), EZH2, IKAROS, CTCF, cohesin, and condensin proteins. Here we will focus on the role of YY1 in controlling these mechanisms. YY1 is a multifunctional transcription factor involved in transcriptional activation and repression, X chromosome inactivation, Polycomb Group (PcG) protein DNA recruitment, and recruitment of proteins required for epigenetic modifications (acetylation, deacetylation, methylation, ubiquitination, sumoylation, etc.). YY1 conditional knock-out indicated that YY1 is required for B cell development, at least in part, by controlling long-distance DNA interactions at the immunoglobulin heavy chain and Igκ loci. Our recent data show that YY1 is also required for CSR. The mechanisms implicated in YY1 control of long-distance DNA interactions include controlling non-coding antisense RNA transcripts, recruitment of PcG proteins to DNA, and interaction with complexes involved in long-distance DNA interactions including the cohesin and condensin complexes. Though common rearrangement mechanisms operate at all Ig loci, their distinct temporal activation along with the ubiquitous nature of YY1 poses challenges for determining the specific mechanisms of YY1 function in these processes, and their regulation at the tissue-specific and B cell stage-specific level. The large numbers of post-translational modifications that control YY1 functions are possible candidates for regulation. PMID:24575094

  8. Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes

    Nourisaeid, Elham; Mousavi, Amir; Arpanaei, Ayyoob

    2016-01-01

    In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.

  9. Specificity and function of Archaeal DNA replication initiator proteins

    Samson, Rachel Y.; Xu, Yanqun; Gadelha, Catarina;

    2013-01-01

    Chromosomes with multiple DNA replication origins are a hallmark of Eukaryotes and some Archaea. All eukaryal nuclear replication origins are defined by the origin recognition complex (ORC) that recruits the replicative helicase MCM(2-7) via Cdc6 and Cdt1. We find that the three origins in the...... investigate the role of ATP binding and hydrolysis in initiator function in vivo and in vitro. We find that the ATP-bound form of Orc1-1 is proficient for replication and implicates hydrolysis of ATP in downregulation of origin activity. Finally, we reveal that ATP and DNA binding by Orc1-1 remodels the...... protein's structure rather than that of the DNA template....

  10. What cost mitochondria? The maintenance of functional mitochondrial DNA within and across generations

    Aanen, D.K.; Spelbrink, J.N.; Beekman, M.

    2014-01-01

    The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is r

  11. Androgen receptor function links human sexual dimorphism to DNA methylation.

    Ole Ammerpohl

    Full Text Available Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

  12. Functions that protect Escherichia coli from DNA-protein crosslinks.

    Krasich, Rachel; Wu, Sunny Yang; Kuo, H Kenny; Kreuzer, Kenneth N

    2015-04-01

    Pathways for tolerating and repairing DNA-protein crosslinks (DPCs) are poorly defined. We used transposon mutagenesis and candidate gene approaches to identify DPC-hypersensitive Escherichia coli mutants. DPCs were induced by azacytidine (aza-C) treatment in cells overexpressing cytosine methyltransferase; hypersensitivity was verified to depend on methyltransferase expression. We isolated hypersensitive mutants that were uncovered in previous studies (recA, recBC, recG, and uvrD), hypersensitive mutants that apparently activate phage Mu Gam expression, and novel hypersensitive mutants in genes involved in DNA metabolism, cell division, and tRNA modification (dinG, ftsK, xerD, dnaJ, hflC, miaA, mnmE, mnmG, and ssrA). Inactivation of SbcCD, which can cleave DNA at protein-DNA complexes, did not cause hypersensitivity. We previously showed that tmRNA pathway defects cause aza-C hypersensitivity, implying that DPCs block coupled transcription/translation complexes. Here, we show that mutants in tRNA modification functions miaA, mnmE and mnmG cause defects in aza-C-induced tmRNA tagging, explaining their hypersensitivity. In order for tmRNA to access a stalled ribosome, the mRNA must be cleaved or released from RNA polymerase. Mutational inactivation of functions involved in mRNA processing and RNA polymerase elongation/release (RNase II, RNaseD, RNase PH, RNase LS, Rep, HepA, GreA, GreB) did not cause aza-C hypersensitivity; the mechanism of tmRNA access remains unclear. PMID:25731940

  13. Evidence implying DNA polymerase beta function in excision repair.

    Siedlecki, J A; Szyszko, J.; Pietrzykowska, I; Zmudzka, B

    1980-01-01

    Comparison was made of the ability of calf thymus DNA polymerases alpha and beta to replicate the following templates: native E. coli CR-34 DNA (T-DNA), calf thymus DNA activated by DNase I (act.DNA), BU-DNA (from E. coli CR-34 cells cultured on BUdR-containing medium) with damages resulting from incomplete excision repair, as well as thermally denatured act.DNA and BU-DNA (s.s.act.DNA and s.s.BU-DNA). 3H-TTP incorporation during extensive replication of act.DNA was similar for both enzymes, ...

  14. Functionalized nanopore-embedded electrodes for rapid DNA sequencing

    He, Haiying; Pandey, Ravindra; Rocha, Alexandre Reily; Sanvito, Stefano; Grigoriev, Anton; Ahuja, Rajeev; Karna, Shashi P

    2007-01-01

    The determination of a patient's DNA sequence can, in principle, reveal an increased risk to fall ill with particular diseases [1,2] and help to design "personalized medicine" [3]. Moreover, statistical studies and comparison of genomes [4] of a large number of individuals are crucial for the analysis of mutations [5] and hereditary diseases, paving the way to preventive medicine [6]. DNA sequencing is, however, currently still a vastly time-consuming and very expensive task [4], consisting of pre-processing steps, the actual sequencing using the Sanger method, and post-processing in the form of data analysis [7]. Here we propose a new approach that relies on functionalized nanopore-embedded electrodes to achieve an unambiguous distinction of the four nucleic acid bases in the DNA sequencing process. This represents a significant improvement over previously studied designs [8,9] which cannot reliably distinguish all four bases of DNA. The transport properties of the setup investigated by us, employing state-o...

  15. Density functional calculations of planar DNA base-pairs

    Machado, M V T; Artacho, E; Sánchez-Portál, D; Soler, J M; Machado, Maider; Ordejon, Pablo; Artacho, Emilio; Sanchez-Portal, Daniel; Soler, Jose M.

    1999-01-01

    We present a systematic Density Functional Theory (DFT) study of geometries and energies of the nucleic acid DNA bases (guanine, adenine, cytosine and thymine) and 30 different DNA base-pairs. We use a recently developed linear-scaling DFT scheme, which is specially suited for systems with large numbers of atoms. As a first step towards the study of large DNA systems, in this work: (i) We establish the reliability of the approximations of our method (including pseudopotentials and basis sets) for the description of the hydrogen-bonded base pairs, by comparing our results with those of former calculations. We show that the interaction energies at Hartree-Fock geometries are in very good agreement with those of second order M{ø}ller-Plesset (MP2) perturbation theory (the most accurate technique that can be applied at present for system of the sizes of the base-pairs). (ii) We perform DFT structural optimizations for the 30 different DNA base-pairs, only three of which had been previously studied with DFT. Our ...

  16. Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi.

    Carolina Furtado

    Full Text Available The oxidative lesion 8-oxoguanine (8-oxoG is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1. This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1, the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1(-/- (CD138 to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H(2O(2. Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H(2O(2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER.

  17. Functional Characterization of 8-Oxoguanine DNA Glycosylase of Trypanosoma cruzi

    Mendes, Isabela Cecília; de Moura, Michelle Barbi; Campos, Priscila Carneiro; Macedo, Andrea Mara; Franco, Glória Regina; Pena, Sérgio Danilo Junho; Teixeira, Santuza Maria Ribeiro; Van Houten, Bennett; Machado, Carlos Renato

    2012-01-01

    The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1−/− (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H2O2). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H2O2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER. PMID:22876325

  18. Functional demonstration of adaptive immunity in zebrafish using DNA vaccination

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja; Rasmussen, Jesper Skou; Kjær, Torben Egil; Vesely, Thomas

    Due to the well characterized genome, overall highly synteny with the human genome and its suitability for functional genomics studies, the zebrafish is considered to be an ideal animal model for basic studies of mechanisms of diseases and immunity in vertebrates including humans. While several...... studies have documented existence of a classical innate immune response, there is mainly indirect evidence of functional adaptive immunity. To address this aspect, groups of zebrafish were vaccinated with DNA-vaccines against the rhabdoviruses VHSV, IHNV and SVCV. Seven weeks later, the fish were...... challenged with SVCV by immersion. Despite some variability between replicate aquaria, there was a protective effect of the homologous vaccine and no effect of the heterologous vaccines. The results therefore confirm the existence of not only a well developed but also a fully functional adaptive immune...

  19. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

    Yoshimura, Akari, E-mail: akari_yo@stu.musashino-u.ac.jp [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan); Kobayashi, Yume [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan); Tada, Shusuke [Department of Medical Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510 (Japan); Seki, Masayuki [Department of Biochemistry, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai-shi, Miyagi 981-8558 (Japan); Enomoto, Takemi [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan)

    2014-09-12

    Highlights: • The UV sensitivity of POLH{sup −/−} cells was suppressed by disruption of WRNIP1. • In WRNIP1{sup −/−/−}/POLH{sup −/−} cells, mutation frequencies and SCE after irradiation reduced. • WRNIP1 defect recovered rate of fork progression after irradiation in POLH{sup −/−} cells. • WRNIP1 functions upstream of Polη in the translesion DNA synthesis pathway. - Abstract: WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH{sup −/−}) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

  20. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

    Highlights: • The UV sensitivity of POLH−/− cells was suppressed by disruption of WRNIP1. • In WRNIP1−/−/−/POLH−/− cells, mutation frequencies and SCE after irradiation reduced. • WRNIP1 defect recovered rate of fork progression after irradiation in POLH−/− cells. • WRNIP1 functions upstream of Polη in the translesion DNA synthesis pathway. - Abstract: WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH−/−) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation

  1. Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'.

    Ciafrè, S A; Rinaldi, M; Gasparini, P; Seripa, D.; L. Bisceglia; Zelante, L; M.G. Farace; Fazio, V M

    1995-01-01

    Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of t...

  2. Functionalizing designer DNA crystals with a triple-helical veneer.

    Rusling, David A; Chandrasekaran, Arun Richard; Ohayon, Yoel P; Brown, Tom; Fox, Keith R; Sha, Ruojie; Mao, Chengde; Seeman, Nadrian C

    2014-04-01

    DNA is a very useful molecule for the programmed self-assembly of 2D and 3D nanoscale objects.1 The design of these structures exploits Watson-Crick hybridization and strand exchange to stitch linear duplexes into finite assemblies.2-4 The dimensions of these complexes can be increased by over five orders of magnitude through self-assembly of cohesive single-stranded segments (sticky ends).5, 6 Methods that exploit the sequence addressability of DNA nanostructures will enable the programmable positioning of components in 2D and 3D space, offering applications such as the organization of nanoelectronics,7 the direction of biological cascades,8 and the structure determination of periodically positioned molecules by X-ray diffraction.9 To this end we present a macroscopic 3D crystal based on the 3-fold rotationally symmetric tensegrity triangle3, 6 that can be functionalized by a triplex-forming oligonucleotide on each of its helical edges. PMID:24615910

  3. Beyond DNA repair: additional functions of PARP-1 in cancer.

    AliceNWeaver; EddySYang

    2013-01-01

    Poly(ADP-ribose) polymerases (PARPs) are DNA-dependent nuclear enzymes that transfer negatively charged ADP-ribose moieties from cellular nicotinamide-adenine-dinucleotide (NAD+) to a variety of protein substrates, altering protein-protein and protein-DNA interactions. The most studied of these enzymes is PARP-1, which is an excellent therapeutic target in cancer due to its pivotal role in the DNA damage response. Clinical studies have shown susceptibility to PARP inhibitors in DNA repair d...

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

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

    2006-01-01

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

  5. Molecular underpinnings of Aprataxin RNA/DNA deadenylase function and dysfunction in neurological disease.

    Schellenberg, Matthew J; Tumbale, Percy P; Williams, R Scott

    2015-03-01

    Eukaryotic DNA ligases seal DNA breaks in the final step of DNA replication and repair transactions via a three-step reaction mechanism that can abort if DNA ligases encounter modified DNA termini, such as the products and repair intermediates of DNA oxidation, alkylation, or the aberrant incorporation of ribonucleotides into genomic DNA. Such abortive DNA ligation reactions act as molecular checkpoint for DNA damage and create 5'-adenylated nucleic acid termini in the context of DNA and RNA-DNA substrates in DNA single strand break repair (SSBR) and ribonucleotide excision repair (RER). Aprataxin (APTX), a protein altered in the heritable neurological disorder Ataxia with Oculomotor Apraxia 1 (AOA1), acts as a DNA ligase "proofreader" to directly reverse AMP-modified nucleic acid termini in DNA- and RNA-DNA damage responses. Herein, we survey APTX function and the emerging cell biological, structural and biochemical data that has established a molecular foundation for understanding the APTX mediated deadenylation reaction, and is providing insights into the molecular bases of APTX deficiency in AOA1. PMID:25637650

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

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

    2016-07-27

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

  7. Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids

    Kenyon, Lesley; Moraes, Carlos T.

    1997-01-01

    The nuclear and mitochondrial genomes coevolve to optimize approximately 100 different interactions necessary for an efficient ATP-generating system. This coevolution led to a species-specific compatibility between these genomes. We introduced mitochondrial DNA (mtDNA) from different primates into mtDNA-less human cells and selected for growth of cells with a functional oxidative phosphorylation system. mtDNA from common chimpanzee, pigmy chimpanzee, and gorilla were able to restore oxidative...

  8. Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

    Hertoghs, Kirsten M. L.; Ellis, Jonathan H.; Catchpole, Ian R.

    2003-01-01

    The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first ...

  9. Size-selective separation of DNA fragments by using lysine-functionalized silica particles

    Liu, Lingling; Guo, Zilong; Huang, Zhenzhen; Zhuang, Jiaqi; Yang, Wensheng

    2016-02-01

    In this work, a facile and efficient approach has been demonstrated for size-selective separation of DNA fragments by using lysine-functionalized silica particles. At a given pH, the environmental ionic strength can be utilized to alter the electrostatic interactions of lysine-functionalized silica particles with DNA fragments and in turn the DNA fragments on the silica particle surfaces, which exhibits a clear dependence on the DNA fragment sizes. By carefully adjusting the environmental pH and salt concentration, therefore, the use of the lysine-functionalized silica particles allows effective separation of binary and ternary DNA mixtures, for example, two different DNA fragments with sizes of 101 and 1073 bp, 101 and 745 bp, 101 and 408 bp, respectively, and three different DNA fragments with sizes of 101, 408 and 1073 bp.

  10. Beyond DNA Repair: Additional Functions of PARP-1 in Cancer

    Weaver, Alice N.; Eddy S. Yang

    2013-01-01

    Poly(ADP-ribose) polymerases (PARPs) are DNA-dependent nuclear enzymes that transfer negatively charged ADP-ribose moieties from cellular nicotinamide-adenine-dinucleotide (NAD+) to a variety of protein substrates, altering protein–protein and protein-DNA interactions. The most studied of these enzymes is poly(ADP-ribose) polymerase-1 (PARP-1), which is an excellent therapeutic target in cancer due to its pivotal role in the DNA damage response. Clinical studies have shown susceptibility to P...

  11. Injection molded nanofluidic chips: Fabrication method and functional tests using single-molecule DNA experiments

    Utko, Pawel; Persson, Karl Fredrik; Kristensen, Anders;

    2011-01-01

    We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels.......We demonstrate that fabrication of nanofluidic systems can be greatly simplified by injection molding of polymers. We functionally test our devices by single-molecule DNA experiments in nanochannels....

  12. Influence of exogenous DNA on macrophage functional activity in experimental radiotherapy

    A study was made of peritoneal macrophage functional activity in C57BL/6 mice witb transplantable Lewis carcinoma during administration of exogenous DNA and use of local radiotherapy. The results show that the use of exogenous DNA in radiotherapy incroases peritoneal macrophage functional activity in mice with transplantable tumors

  13. A baculovirus photolyase with DNA repair activity and circadian clock regulatory function

    Biernat, M.A.; Eker, A.P.M.; Oers, van M.M.; Vlak, J.M.; Horst, van der G.T.J.; Chaves, I.

    2012-01-01

    Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors,

  14. Structure and function of the translesion DNA polymerases and interactions with damaged DNA

    F. Peter Guengerich

    2015-03-01

    Pre-steady-state kinetic analysis has been used to develop minimum kinetic models with rate constants of (the eight individual reaction steps in the catalytic cycle. The use of single-tryptophan mutants of Sulfolobus solfataricus Dpo4 and human (h pol κ has led to discernment of the steps for the conformation change (associated with dNTP binding and relocation and nucleotidyl transfer. X-ray crystal structures have been obtained for a number of the DNA adduct/DNA polymerase pairs in both binary and ternary complexes. Two isomeric etheno guanine adducts differ considerably in their interactions with DNA polymerases, explaining the base preferences. Further, even when several DNA polymerases cause the same mispairs with a single DNA adduct, the structural bases for this can differ considerably.

  15. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely

    Xiaoyan Guo; Ping Chen; Xiaohu Hou; Wenjuan Xu; Dan Wang; Tian-yan Wang; Liping Zhang; Gang Zheng; Zhi-liang Gao; Cheng-Yi He; Boping Zhou; Zhi-Ying Chen

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C g...

  16. Single and double stranded DNA detection using locked nucleic acid (LNA) functionalized nanoparticles

    McKenzie, Fiona; Stokes, Robert; Faulds, Karen; Graham, Duncan

    2008-08-01

    Gold and silver nanoparticles functionalized with oligonucleotides can be used for the detection of specific sequences of DNA. We show that gold nanoparticles modified with locked nucleic acid (LNA) form stronger duplexes with a single stranded DNA target and offer better discrimination against single base pair mismatches than analogous DNA probes. Our LNA nanoparticle probes have also been used to detect double stranded DNA through triplex formation, whilst still maintaining selectivity for only complementary targets. Nanoparticle conjugates embedded with suitable surface enhanced resonance Raman scattering (SERRS) labels have been synthesized enabling simultaneous detection and identification of multiple DNA targets.

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

    2001-01-01

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

  18. Left-handed Z-DNA: structure and function

    Herbert, A.; Rich, A.

    1999-01-01

    Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

  19. Functional amyloids as inhibitors of plasmid DNA replication.

    Molina-García, Laura; Gasset-Rosa, Fátima; Moreno-Del Álamo, María; Fernández-Tresguerres, M Elena; Moreno-Díaz de la Espina, Susana; Lurz, Rudi; Giraldo, Rafael

    2016-01-01

    DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is 'handcuffing', i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation. PMID:27147472

  20. Glom is a novel mitochondrial DNA packaging protein in Physarum polycephalum and causes intense chromatin condensation without suppressing DNA functions.

    Sasaki, Narie; Kuroiwa, Haruko; Nishitani, Chikako; Takano, Hiroyoshi; Higashiyama, Tetsuya; Kobayashi, Tamaki; Shirai, Yuki; Sakai, Atsushi; Kawano, Shigeyuki; Murakami-Murofushi, Kimiko; Kuroiwa, Tsuneyoshi

    2003-12-01

    Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions. PMID:12960433

  1. DNA based arithmetic function: a half adder based on DNA strand displacement

    Li, Wei; Zhang, Fei; Yan, Hao; Liu, Yan

    2016-02-01

    Biomolecular programming utilizes the reactions and information stored in biological molecules, such as proteins and nucleic acids, for computational purposes. DNA has proven itself an excellent candidate for building logic operating systems due to its highly predictable molecular behavior. In this work we designed and realized an XOR logic gate and an AND logic gate based on DNA strand displacement reactions. These logic gates utilize ssDNA as input and output signals. The XOR gate and the AND gate were used as building blocks for constructing a half adder logic circuit, which is a primary step in constructing a full adder, a basic arithmetic unit in computing. This work provides the field of DNA molecular programming with a potential universal arithmetic tool.Biomolecular programming utilizes the reactions and information stored in biological molecules, such as proteins and nucleic acids, for computational purposes. DNA has proven itself an excellent candidate for building logic operating systems due to its highly predictable molecular behavior. In this work we designed and realized an XOR logic gate and an AND logic gate based on DNA strand displacement reactions. These logic gates utilize ssDNA as input and output signals. The XOR gate and the AND gate were used as building blocks for constructing a half adder logic circuit, which is a primary step in constructing a full adder, a basic arithmetic unit in computing. This work provides the field of DNA molecular programming with a potential universal arithmetic tool. Electronic supplementary information (ESI) available: Detailed descriptions of DNA logic gate design, materials and methods, and additional data analysis. See DOI: 10.1039/c5nr08497k

  2. Imparting the unique properties of DNA into complex material architectures and functions

    Phyllis F. Xu

    2013-07-01

    Full Text Available While the remarkable chemical and biological properties of DNA have been known for decades, these properties have only been imparted into materials with unprecedented function much more recently. The inimitable ability of DNA to form programmable, complex assemblies through stable, specific, and reversible molecular recognition has allowed the creation of new materials through DNA's ability to control a material's architecture and properties. In this review we discuss recent progress in how DNA has brought unmatched function to materials, focusing specifically on new advances in delivery agents, devices, and sensors.

  3. Evaluation of Fluorescent Analogs of Deoxycytidine for Monitoring DNA Transitions from Duplex to Functional Structures

    Yogini P. Bhavsar

    2011-01-01

    Full Text Available Topological variants of single-strand DNA (ssDNA structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC and 1,3-diaza-2-oxophenoxazine (tC∘, can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC∘ is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

  4. DNA based arithmetic function: a half adder based on DNA strand displacement.

    Li, Wei; Zhang, Fei; Yan, Hao; Liu, Yan

    2016-02-14

    Biomolecular programming utilizes the reactions and information stored in biological molecules, such as proteins and nucleic acids, for computational purposes. DNA has proven itself an excellent candidate for building logic operating systems due to its highly predictable molecular behavior. In this work we designed and realized an XOR logic gate and an AND logic gate based on DNA strand displacement reactions. These logic gates utilize ssDNA as input and output signals. The XOR gate and the AND gate were used as building blocks for constructing a half adder logic circuit, which is a primary step in constructing a full adder, a basic arithmetic unit in computing. This work provides the field of DNA molecular programming with a potential universal arithmetic tool. PMID:26814628

  5. RNA-directed DNA methylation: Mechanisms and functions

    Mahfouz, Magdy M.

    2010-07-01

    Epigenetic RNA based gene silencing mechanisms play a major role in genome stability and control of gene expression. Transcriptional gene silencing via RNA-directed DNA methylation (RdDM) guides the epigenetic regulation of the genome in response to disease states, growth, developmental and stress signals. RdDM machinery is composed of proteins that produce and modify 24-nt- long siRNAs, recruit the RdDM complex to genomic targets, methylate DNA and remodel chromatin. The final DNA methylation pattern is determined by either DNA methyltransferase alone or by the combined action of DNA methyltransferases and demethylases. The dynamic interaction between RdDM and demethylases may render the plant epigenome plastic to growth, developmental, and environmental cues. The epigenome plasticity may allow the plant genome to assume many epigenomes and to have the right epigenome at the right time in response to intracellular or extracellular stimuli. This review discusses recent advances in RdDM research and considers future perspectives.

  6. DNA-Templated Polymerization of Side-Chain-Functionalized Peptide Nucleic Acid Aldehydes

    Kleiner, Ralph E.; Brudno, Yevgeny; Birnbaum, Michael E.; Liu, David R.

    2008-01-01

    The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We obs...

  7. Mitochondrial DNA damage and vascular function in patients with diabetes mellitus and atherosclerotic cardiovascular disease

    Fetterman, Jessica L.; Holbrook, Monica; Westbrook, David G.; Brown, Jamelle A.; Kyle P. Feeley; Bretón-Romero, Rosa; Linder, Erika A.; Berk, Brittany D.; Weisbrod, Robert M.; Widlansky, Michael E.; Gokce, Noyan; Ballinger, Scott W.; Hamburg, Naomi M.

    2016-01-01

    Objective Prior studies demonstrate mitochondrial dysfunction with increased reactive oxygen species generation in peripheral blood mononuclear cells in diabetes mellitus. Oxidative stress-mediated damage to mitochondrial DNA promotes atherosclerosis in animal models. Thus, we evaluated the relation of mitochondrial DNA damage in peripheral blood mononuclear cells s with vascular function in patients with diabetes mellitus and with atherosclerotic cardiovascular disease. Approach and results ...

  8. DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection.

    Hao, Rong-Zhang; Song, Hong-Bin; Zuo, Guo-Min; Yang, Rui-Fu; Wei, Hong-Ping; Wang, Dian-Bing; Cui, Zong-Qiang; Zhang, ZhiPing; Cheng, Zhen-Xing; Zhang, Xian-En

    2011-04-15

    The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis. PMID:21315574

  9. Optimized DNA-targeting using triplex forming C5-alkynyl functionalized LNA.

    Sau, Sujay P; Kumar, Pawan; Anderson, Brooke A; Østergaard, Michael E; Deobald, Lee; Paszczynski, Andrzej; Sharma, Pawan K; Hrdlicka, Patrick J

    2009-11-28

    Triplex forming oligonucleotides (TFOs) modified with C5-alkynyl functionalized LNA (locked nucleic acid) monomers display extraordinary thermal affinity toward double stranded DNA targets, excellent discrimination of Hoogsteen-mismatched targets, and high stability against 3?-exonucleases. PMID:19885469

  10. Optimized DNA-targeting using triplex forming C5-alkynyl functionalized LNA†

    Sau, Sujay P.; Kumar, Pawan; Anderson, Brooke A.; Østergaard, Michael E.; Deobald, Lee; Paszczynski, Andrzej; Sharma, Pawan K.; Hrdlicka, Patrick J.

    2009-01-01

    Triplex forming oligonucleotides (TFOs) modified with C5-alkynyl functionalized LNA (locked nucleic acid) monomers display extraordinary thermal affinity toward double stranded DNA targets, excellent discrimination of Hoogsteen-mismatched targets, and high stability against 3′-exonucleases.

  11. DNA damage-dependent regulation of MyoD function

    Simonatto, Marta

    1980-01-01

    Durante la rigenerazione muscolare, le cellule progenitrici sono esposte ad una serie di stimoli extracellulari che coordinano la loro attivazione, proliferazione e differenziamento. Al tempo stesso, tuttavia, l’ambiente rigenerativo, stimolando la sintesi del DNA e aumentando lo stato di ossidazione intracellulare, costituisce una fonte di stress genotossico, come mostriamo in vivo in questo lavoro. Uno studio svolto in precedenza nel nostro laboratorio ha identificato e ca...

  12. Recombinant DNA data management at the restriction and functional site level.

    Shalloway, D; Deering, N R

    1984-01-01

    We have created a system to manage in a unified manner the restriction and functional site information required for design and analysis of recombinant DNA experiments. Primary source DNA data and recombinant clone specifications are used to generate recombinant restriction maps and restriction fragment lists. Sequence data, restriction-site data, and functional-site data may be combined in the data base. Interaction and output is user-friendly and versatile.

  13. A one-pot functionalization strategy for immobilizing proteins onto linear dsDNA scaffolds

    Functional DNA scaffolds can be defined as DNA-based structures comprising chemical moieties facilitating and guiding the immobilization of additional nanocomponents. Due to the limited reactivity of DNA there is currently a need to develop rapid routes to expand its chemical repertoire and increase its versatility as a nanostructuring scaffold. We report a simple synthetic strategy for generating linear and stable double-stranded DNA scaffolds functionalized with multiple sites reactive towards free thiols, and the utility of this approach is demonstrated by immobilizing a model protein containing an accessible free thiol. This procedure is very versatile and could be easily expanded to other types of chemistries. This approach could also potentially be employed for the specific, oriented immobilization of various biomolecules and nanoparticles on predefined DNA architectures.

  14. Association of DNA repair polymorphisms with DNA repair functional outcomes in healthy human subjects

    Vodička, Pavel; Štětina, R.; Poláková, Veronika; Tulupová, Elena; Naccarati, Alessio; Vodičková, Ludmila; Kumar, R.; Hánová, Monika; Pardini, Barbara; Slyšková, Jana; Musak, L.; De Palma, G.; Souček, P.; Hemminki, K.

    2007-01-01

    Roč. 28, č. 3 (2007), s. 657-664. ISSN 0143-3334 R&D Projects: GA MZd NR8563; GA ČR GA310/05/2626 Institutional research plan: CEZ:AV0Z50390512 Keywords : Base excision DNA * Single-strand breaks * Peripheral blood lymphocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.406, year: 2007

  15. Epigenetic features in the oyster Crassostrea gigas suggestive of functionally relevant promoter DNA methylation in invertebrates.

    GuillaumeRiviere

    2014-04-01

    Full Text Available DNA methylation is evolutionarily conserved. Vertebrates exhibit high, widespread DNA methylation whereas invertebrate genomes are less methylated, predominantly within gene bodies. DNA methylation in invertebrates is associated with transcription level, alternative splicing and genome evolution, but functional outcomes of DNA methylation remain poorly described in lophotrochozoans. Recent genome-wide approaches improve understanding in distant taxa such as molluscs, where the phylogenetic position and life traits of Crassostrea gigas make this bivalve an ideal model to study the physiological and evolutionary implications of DNA methylation. We review the literature about DNA methylation in invertebrates and focus on DNA methylation features in the oyster. Indeed, though our MeDIP-seq results confirm predominant intragenic methylation, the profiles depend on the oyster’s developmental and reproductive stage. We discuss the perspective that oyster DNA methylation could be biased toward the 5’-end of some genes, depending on physiological status, suggesting important functional outcomes of putative promoter methylation from cell differentiation during early development to sustained adaptation of the species to the environment.

  16. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely

    Guo, Xiaoyan; Chen, Ping; Hou, Xiaohu; Xu, Wenjuan; Wang, Dan; Wang, Tian-yan; Zhang, Liping; Zheng, Gang; Gao, Zhi-liang; He, Cheng-Yi; Zhou, Boping; Chen, Zhi-Ying

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus. PMID:27174254

  17. The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.

    Guo, Xiaoyan; Chen, Ping; Hou, Xiaohu; Xu, Wenjuan; Wang, Dan; Wang, Tian-Yan; Zhang, Liping; Zheng, Gang; Gao, Zhi-Liang; He, Cheng-Yi; Zhou, Boping; Chen, Zhi-Ying

    2016-01-01

    HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus. PMID:27174254

  18. Construction of DNA damage response gene pprI function-deficient and function-complementary mutants in Deinococcus radiodurans

    GAO Guanjun; LU Huiming; HUANG Lifen; HUA Yuejin

    2005-01-01

    PprI, a DNA damage response factor from the extraordinary radioresistant bacterium Deinococcus radiodurans, plays a central regulatory role in multiple DNA damage repair. In this study, a fusion DNA fragment carrying kanamycin resistance gene with the D. Radiodurans groEL promoter was cloned by PCR amplification and reversely inserted into the pprI locus in the genome of the wild-type strain R1. The resulting pprI-deficient strain, designated YR1, was very sensitive to ionizing radiation. Meanwhile, the re- combinant DNA fragment was cloned into the shuttle vector pRADZ3, and resulted in plasmid pRADK with kanamycin resistance in D. Radiodurans. The fragments containing complete pprI gene and 3'-terminal deletion pprI△ were cloned into plasmid pRADK. The resulted plasmids designated pRADKpprI and pRADKpprI△ were then transformed to YR1. Results show that YR1 carrying pRADKpprI was able to fully restore the extreme radioresistance to the same level as the wild-type D. Raiodurans R1, whereas YR1 pRADKpprI△ failed to do so. Construction of DNA repair switch PprI function-deficient and function-complementary mutants in D. Radiodurans is not only useful to elucidating the relationship between domains and functions of PprI protein, but also opens the door to the further studies of the biological functions of PprI protein in vivo.

  19. DNA-based stable isotope probing: a link between community structure and function

    DNA-based molecular techniques permit the comprehensive determination of microbial diversity but generally do not reveal the relationship between the identity and the function of microorganisms. The first direct molecular technique to enable the linkage of phylogeny with function is DNA-based stable isotope probing (DNA-SIP). Applying this method first helped describe the utilization of simple compounds, such as methane, methanol or glucose and has since been used to detect microbial communities active in the utilization of a wide variety of compounds, including various xenobiotics. The principle of the method lies in providing 13C-labeled substrate to a microbial community and subsequent analyses of the 13C-DNA isolated from the community. Isopycnic centrifugation permits separating 13C-labeled DNA of organisms that utilized the substrate from 12C-DNA of the inactive majority. As the whole metagenome of active populations is isolated, its follow-up analysis provides successful taxonomic identification as well as the potential for functional gene analyses. Because of its power, DNA-SIP has become one of the leading techniques of microbial ecology research. But from other point of view, it is a labor-intensive method that requires careful attention to detail during each experimental step in order to avoid misinterpretation of results.

  20. The function analysis of full-length cDNA sequence from IRM-2 mouse cDNA library

    Objective: To identify the function of full-length cDNA sequence from IRM-2 mouse cDNA library. Methods: Full-length cDNA products were amplified by PCR from IRM-2 mouse cDNA library according to twenty-one pieces of expressed sequence tag. The expression of full-length cDNAs were detected after mouse embryonic fibroblasts were exposed to 6.5 Gy γ-ray radiation. And the effect on the growth of radiosensitivity cells AT5B1VA transfected with full-length cDNAs was investigated. Results: The expression of No.4, 5 and 2 full-length cDNAs from IRM-2 mouse were higher than that of parental ICR and 615 mouse after mouse embryonic fibroblasts irradiated with γ-ray radiation. And the survival rate of AT5B1VA cells transfected with No.4, 5 and 2 full-length cDNAs was high. Conclusion: No.4, 5 and 2 full-length cDNAs of IRM-2 mouse are of high radioresistance. (authors)

  1. Dispersion stability and exothermic properties of DNA-functionalized single-walled carbon nanotubes

    Kawaguchi M

    2011-04-01

    Full Text Available Minoru Kawaguchi1,2, Jun Ohno2,3, Akihito Irie4, Tadao Fukushima2,5, Jun Yamazaki2,6, Naotoshi Nakashima2,71Department of Dental Engineering, Biomaterials Section, Fukuoka Dental College, Sawara-ku, Fukuoka, Japan; 2Japan Science and Technology Agency (JST, Core Research of Evolutional Science and Technology (CREST, Chiyoda-ku, Tokyo, Japan; 3Department of Morphological Biology, Pathology Section, Fukuoka Dental College, Sawara-ku, Fukuoka, Japan; 4Department of Oral Rehabilitation, Section of Oral Implantology, Fukuoka Dental College, Sawara-ku, Fukuoka, Japan; 5Department of Dental Engineering, Bioengineering Section, Fukuoka Dental College, Sawara-ku, Fukuoka, Japan; 6Department of Physiological Science and Molecular Biology, Section of cellular and Molecular Regulation, Fukuoka Dental College, Sawara-ku, Fukuoka, Japan; 7Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Nishi-ku, Fukuoka, JapanAbstract: Carbon nanotubes act as a photon antenna that serves as an effective “molecular heater” around the near-infrared (NIR region. This exothermic generation can be used as a possible heating source for hyperthermia therapy. The current study reports the dispersible and exothermic properties with NIR irradiation for single-walled carbon nanotubes (SWNTs treated with a strong acid (acid-treated SWNTs, and the SWNTs further functionalized with double-stranded DNA (DNA-functionalized SWNTs: DNA-SWNTs. DNA-SWNTs significantly improved the dispersibility of SWNTs when compared with the acid-treated SWNTs. The binding ratio of the acid-treated SWNT and DNA was calculated to be 3.1 (DNA/SWNTs from the phosphorous content in the DNA-SWNT. This interaction of the SWNTs and DNA would contribute to the stable dispersion of the DNA-SWNTs in a culture medium. With NIR irradiation by a halogen lamp light source, the acid-treated SWNTs and the DNA-SWNTs showed strong heat evolution in vitro (in a culture medium and in vivo

  2. Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms.

    Schwartz, Kelly; Ganesan, Mahesh; Payne, David E; Solomon, Michael J; Boles, Blaise R

    2016-01-01

    Persistent staphylococcal infections often involve surface-associated communities called biofilms. Staphylococcus aureus biofilm development is mediated by the co-ordinated production of the biofilm matrix, which can be composed of polysaccharides, extracellular DNA (eDNA) and proteins including amyloid fibers. The nature of the interactions between matrix components, and how these interactions contribute to the formation of matrix, remain unclear. Here we show that the presence of eDNA in S. aureus biofilms promotes the formation of amyloid fibers. Conditions or mutants that do not generate eDNA result in lack of amyloids during biofilm growth despite the amyloidogeneic subunits, phenol soluble modulin peptides, being produced. In vitro studies revealed that the presence of DNA promotes amyloid formation by PSM peptides. Thus, this work exposes a previously unacknowledged interaction between biofilm matrix components that furthers our understanding of functional amyloid formation and S. aureus biofilm biology. PMID:26365835

  3. Genetic manipulation in Sulfolobus islandicus and functional analysis of DNA repair genes

    Zhang, Changyi; Tian, Bin; Li, Suming;

    2013-01-01

    enzymes already impaired cell growth, highlighting their important roles in archaeal DNA repair. Systematically characterizing these mutants and generating mutants lacking two or more DNA repair genes will yield further insights into the genetic mechanisms of DNA repair in this model organism.......Recently, a novel gene-deletion method was developed for the crenarchaeal model Sulfolobus islandicus, which is a suitable tool for addressing gene essentiality in depth. Using this technique, we have investigated functions of putative DNA repair genes by constructing deletion mutants and studying...... their phenotype. We found that this archaeon may not encode a eukarya-type of NER (nucleotide excision repair) pathway because depleting each of the eukaryal NER homologues XPD, XPB and XPF did not impair the DNA repair capacity in their mutants. However, among seven homologous recombination proteins...

  4. Genetic Organization of the Vibrio harveyi dnaA Gene Region and Analysis of the Function of the V. harveyi DnaA Protein in Escherichia coli

    Berenstein, Dvora; Olesen, Kirsten; Speck, Christian; Skovgaard, Ole

    2002-01-01

    The Vibrionaceae family is distantly related to Enterobacteriaceae within the group of bacteria possessing the Dam methylase system. We have cloned, sequenced, and analyzed the dnaA gene region of Vibrio harveyi and found that although the organization of the V. harveyi dnaA region differs from that of Escherichia coli, the expression of both genes is autoregulated and ATP-DnaA binds cooperatively to ATP-DnaA boxes in the dnaA promoter region. The DnaA proteins of V. harveyi and E. coli are interchangeable and function nearly identically in controlling dnaA transcription and the initiation of chromosomal DNA replication despite the evolutionary distance between these bacteria. PMID:11948168

  5. New functional sites in MutS affect DNA mismatch repair

    2010-01-01

    The MutS protein plays an important role in the DNA mismatch repair system. Mutations in the mutS gene can lead to genome instability and ultimately cell malfunction. Here we have established a method for identifying functional defective mutants of MutS by random mutation and rifampicin screening. Some novel functional sites in MutS were identified. The MutS mutant strains were analyzed using surface plasmon resonance, gel filtration and far-western methods to determine the molecular mechanisms behind the DNA mismatch repair function of MutS.

  6. Altered Mitochondrial Function, Mitochondrial DNA and Reduced Metabolic Flexibility in Patients With Diabetic Nephropathy

    Anna Czajka

    2015-06-01

    Full Text Available The purpose of this study was to determine if mitochondrial dysfunction plays a role in diabetic nephropathy (DN, a kidney disease which affects >100 million people worldwide and is a leading cause of renal failure despite therapy. A cross-sectional study comparing DN with diabetes patients without kidney disease (DC and healthy controls (HCs; and renal mesangial cells (HMCs grown in normal and high glucose, was carried out. Patients with diabetes (DC had increased circulating mitochondrial DNA (MtDNA, and HMCs increased their MtDNA within 24 h of hyperglycaemia. The increased MtDNA content in DCs and HMCs was not functional as transcription was unaltered/down-regulated, and MtDNA damage was present. MtDNA was increased in DC compared to HC, conversely, patients with DN had lower MtDNA than DC. Hyperglycaemic HMCs had fragmented mitochondria and TLR9 pathway activation, and in diabetic patients, mitophagy was reduced. Despite MtDNA content and integrity changing within 4 days, hyperglycaemic HMCs had a normal bio-energetic profile until 8 days, after which mitochondrial metabolism was progressively impaired. Peripheral blood mononuclear cells (PBMCs from DN patients had reduced reserve capacity and maximal respiration, loss of metabolic flexibility and reduced Bioenergetic Health Index (BHI compared to DC. Our data show that MtDNA changes precede bioenergetic dysfunction and that patients with DN have impaired mitochondrial metabolism compared to DC, leading us to propose that systemic mitochondrial dysfunction initiated by glucose induced MtDNA damage may be involved in the development of DN. Longitudinal studies are needed to define a potential cause–effect relationship between changes in MtDNA and bioenergetics in DN.

  7. Complex ABCC8 DNA variations in congenital hyperinsulinism: lessons from functional studies

    Muzyamba, Morris; Farzaneh, Tabasum; Behe, Phillip;

    2007-01-01

    singly or in combination led to intracellular retention of the channel complex and loss of function. By contrast, V1572I is trafficked appropriately and is functional, consistent with a mechanism of reduction to hemizygosity of paternal ABCC8 in focal disease. V1572I is likely to be a benign DNA variant...

  8. RICD: A rice indica cDNA database resource for rice functional genomics

    Zhang Qifa

    2008-11-01

    Full Text Available Abstract Background The Oryza sativa L. indica subspecies is the most widely cultivated rice. During the last few years, we have collected over 20,000 putative full-length cDNAs and over 40,000 ESTs isolated from various cDNA libraries of two indica varieties Guangluai 4 and Minghui 63. A database of the rice indica cDNAs was therefore built to provide a comprehensive web data source for searching and retrieving the indica cDNA clones. Results Rice Indica cDNA Database (RICD is an online MySQL-PHP driven database with a user-friendly web interface. It allows investigators to query the cDNA clones by keyword, genome position, nucleotide or protein sequence, and putative function. It also provides a series of information, including sequences, protein domain annotations, similarity search results, SNPs and InDels information, and hyperlinks to gene annotation in both The Rice Annotation Project Database (RAP-DB and The TIGR Rice Genome Annotation Resource, expression atlas in RiceGE and variation report in Gramene of each cDNA. Conclusion The online rice indica cDNA database provides cDNA resource with comprehensive information to researchers for functional analysis of indica subspecies and for comparative genomics. The RICD database is available through our website http://www.ncgr.ac.cn/ricd.

  9. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism

    Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMSsensitive mutants were killed by an HO-induced double-strand break atMAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages. (author)

  10. DNA methylation status of nuclear-encoded mitochondrial genes underlies the tissue-dependent mitochondrial functions

    Takasugi Masaki

    2010-08-01

    Full Text Available Abstract Background Mitochondria are semi-autonomous, semi-self-replicating organelles harboring their own DNA (mitochondrial DNA, mtDNA, and their dysregulation is involved in the development of various diseases. While mtDNA does not generally undergo epigenetic modifications, almost all mitochondrial proteins are encoded by nuclear DNA. However, the epigenetic regulation of nuclear-encoded mitochondrial genes (nuclear mt genes has not been comprehensively analyzed. Results We analyzed the DNA methylation status of 899 nuclear mt genes in the liver, brain, and heart tissues of mouse, and identified 636 nuclear mt genes carrying tissue-dependent and differentially methylated regions (T-DMRs. These nuclar mt genes are involved in various mitochondrial functions and they also include genes related to human diseases. T-DMRs regulate the expression of nuclear mt genes. Nuclear mt genes with tissue-specific hypomethylated T-DMRs were characterized by enrichment of the target genes of specific transcription factors such as FOXA2 in the liver, and CEBPA and STAT1 in the brain. Conclusions A substantial proportion of nuclear mt genes contained T-DMRs, and the DNA methylation status of numerous T-DMRs should underlie tissue-dependent mitochondrial functions.

  11. DNA biosensors implemented on PNA-functionalized microstructured optical fibers Bragg gratings

    Candiani, A.; Giannetti, S.; Cucinotta, A.; Bertucci, A.; Manicardi, A.; Konstantaki, M.; Margulis, W.; Pissadakis, S.; Corradini, R.; Selleri, S.

    2013-05-01

    A novel DNA sensing platform based on a Peptide Nucleic Acid - functionalized Microstructured Optical Fibers gratings has been demonstrated. The inner surface of different MOFs has been functionalized using PNA probes, OligoNucleotides mimic that are well suited for specific DNA target sequences detection. The hybrid sensing systems were tested for optical DNA detection of targets of relevance in biomedical application, using the cystic fibrosis gene mutation, and food-analysis, using the genomic DNA from genetic modified organism soy flour. After the solutions of DNA molecules has been infiltrated inside the fibers capillaries and hybridization has occurred, oligonucleotidefunctionalized gold nanoparticles were infiltrated and used to form a sandwich-like system to achieve signal amplification. Spectral measurements of the reflected signal reveal a clear wavelength shift of the reflected modes when the infiltrated complementary DNA matches with the PNA probes placed on the inner fiber surface. Measurements have also been made using the mismatched DNA solution for the c, containing a single nucleotide polymorphism, showing no significant changes in the reflected spectrum. Several experiments have been carried out demonstrating the reproducibility of the results and the high selectivity of the sensors, showing the simplicity and the potential of this approach.

  12. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-Km, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus

  13. [Non-homologous DNA end joining--new proteins, new functions, new mechanisms].

    Popławski, Tomasz; Stoczyńska, Ewelina; Błasiak, Janusz

    2009-01-01

    Humans use primarily nonhomologous end joining (NHEJ) to repair DNA double strand breaks (DSBs), which are the most serious DNA damage, resulting in cell death if non-repaired or missrepaired. NHEJ directly joins together DNA ends resulted from DSBs. This pathway plays a key role in the development of vertebrate immune system through its involvement in the V(D)J recombination. Classical NHEJ in vertebrates involves a heterodimer of Ku proteins, the catalytic subunits of DNA-dependent protein kinase (DNA-PKCS), Artemis, Cernunnos-XLF and XRCC4/ligase DNA IV complex. This classical pathway may be assisted by DNA polymerases mu and lambda. Last 2 years brought new information on the mechanisms, proteins and functions of this DNA repair pathway. In 2006 Cernunnos-XLF was discovered, a protein playing a key role in NHEJ. Some alternative NHEJ pathways were also identified, lacking some of the main proteins of classical NHEJ, but involving other factors, including BRCA1, 53BP1, hPNK, WRN or MDC1. The results obtained so far suggest that not all key components and basic mechanisms of NHEJ have been identified. Future aspects of NHEJ research should include the determination of its role in cancer, aging, immune system development and basic nuclear metabolism. PMID:19514464

  14. Crystal Structure of CRN-4: Implications for Domain Function in Apoptotic DNA Degradation▿

    Hsiao, Yu-Yuan; Nakagawa, Akihisa; Shi, Zhonghao; Mitani, Shohei; Xue, Ding; Yuan, Hanna S.

    2008-01-01

    Cell death related nuclease 4 (CRN-4) is one of the apoptotic nucleases involved in DNA degradation in Caenorhabditis elegans. To understand how CRN-4 is involved in apoptotic DNA fragmentation, we analyzed CRN-4's biochemical properties, in vivo cell functions, and the crystal structures of CRN-4 in apo-form, Mn2+-bound active form, and Er3+-bound inactive form. CRN-4 is a dimeric nuclease with the optimal enzyme activity in cleaving double-stranded DNA in apoptotic salt conditions. Both mut...

  15. A novel function of adenomatous polyposis coli (APC) in regulating DNA repair

    Jaiswal, Aruna S.; Narayan, Satya

    2008-01-01

    Prevailing literature suggests diversified cellular functions for the adenomatous polyposis coli (APC) gene. Among them a recently discovered unique role of APC is in DNA repair. The APC gene can modulate the base excision repair (BER) pathway through an interaction with DNA polymerase β (Pol-β) and flap endonuclease 1 (Fen-1). Taken together with the transcriptional activation of APC gene by alkylating agents and modulation of BER activity, APC may play an important role in carcinogenesis an...

  16. DNA barcoding of life: a classification of uses according to function and scale after ten years of development

    Nancai Pei; Bufeng Chen

    2013-01-01

    DNA barcoding technology provides molecular information, standard dataset platforms, and universal technical regulations for modern biological research. We briefly review the history of DNA barcoding between 2003 and 2012, and classify DNA barcoding into three types of biological function: basic function (e.g., storing data, and identifying species), extending function (e.g., building phylogenies, serving specific subjects, and compiling biological atlas) and potential function (e.g., reveali...

  17. Multiplexed aptasensors and amplified DNA sensors using functionalized graphene oxide: application for logic gate operations.

    Liu, Xiaoqing; Aizen, Ruth; Freeman, Ronit; Yehezkeli, Omer; Willner, Itamar

    2012-04-24

    Graphene oxide (GO) is implemented as a functional matrix for developing fluorescent sensors for the amplified multiplexed detection of DNA, aptamer-substrate complexes, and for the integration of predesigned DNA constructs that activate logic gate operations. Fluorophore-labeled DNA strands acting as probes for two different DNA targets are adsorbed onto GO, leading to the quenching of the luminescence of the fluorophores. Desorption of the probes from the GO, through hybridization with the target DNAs, leads to the fluorescence of the respective label. By coupling exonuclease III, Exo III, to the system, the recycling of the target DNAs is demonstrated, and this leads to the amplified detection of the DNA targets (detection limit 5 × 10(-12) M). Similarly, adsorption of fluorophore-functionalized aptamers against thrombin or ATP onto the GO leads to the desorption of the aptamer-substrate complexes from GO and to the triggering of the luminescence corresponding to the respective fluorophore, thus, allowing the multiplexed analysis of the aptamer-substrate complexes. By designing functional fluorophore-labeled DNA constructs and their interaction with GO, in the presence (or absence) of nucleic acids, or two different substrates for aptamers, as inputs, the activation of the "OR" and "AND" logic gates is demonstrated. PMID:22404375

  18. G-quadruplex functionalized nano mesoporous silica for assay of the DNA methyltransferase activity.

    Liu, Pei; Pang, Junling; Yin, Huanshun; Ai, Shiyun

    2015-06-16

    The abnormal level of DNA methyltransferase (MTase) may cause the aberrant DNA methylation, which has been found being associated with a growing number of human diseases, so it is necessary to create a sensitive and selective method to detect DNA MTase activity. In this paper, a new type of DNA functionalized nano mesoporous silica (MSNs) was creatively introduced to the detection of DNA MTase activity with G-quadruplex as a lock for signal molecule to release. The method was carried out by designing a particular DNA which could fold into G-quadruplex and complement with probe DNA. Next, MSNs was prepared before blocking methylene blue (MB) by G-quadruplex. Probe DNA was then fixed on gold nanoparticles modified glass carbon electrode, and the material was able to be transferred to the surface of electrode by DNA hybridization. After methylation of DNA MTase and the cutting of restriction endonuclease, the electrode was transferred to phosphate buffer solution (pH 9.0) for the releasing of MB. The response of differential pulse voltammetry was obtained from the release of MB. Consequently, the difference of signals with or without methylation could prove the assay of M. SssI MTase activity. The results showed that the responses from MB increased linearly with the increasing of the M. SssI MTase concentrations from 0.28 to 50UmL(-1). The limit of detection was 0.28UmL(-1). In addition, Zebularine, a nucleoside analog of cytidine, was utilized for studying the inhibition activity of M. SssI MTase. PMID:26002474

  19. Inhibition of the mitochondrial respiratory chain function abrogates quartz induced DNA damage in lung epithelial cells

    Respirable quartz dust has been classified as a human carcinogen by the International Agency for Research on Cancer. The aim of our study was to investigate the mechanisms of DNA damage by DQ12 quartz in RLE-6TN rat lung epithelial type II cells (RLE). Transmission electron microscopy and flow-cytometry analysis showed a rapid particle uptake (30 min to 4 h) of quartz by the RLE cells, but particles were not found within the cell nuclei. This suggests that DNA strand breakage and induction of 8-hydroxydeoxyguanosine - as also observed in these cells during these treatment intervals - did not result from direct physical interactions between particles and DNA, or from short-lived particle surface-derived reactive oxygen species. DNA damage by quartz was significantly reduced in the presence of the mitochondrial inhibitors rotenone and antimycin-A. In the absence of quartz, these inhibitors did not affect DNA damage, but they reduced cellular oxygen consumption. No signs of apoptosis were observed by quartz. Flow-cytometry analysis indicated that the reduced DNA damage by rotenone was not due to a possible mitochondria-mediated reduction of particle uptake by the RLE cells. Further proof of concept for the role of mitochondria was shown by the failure of quartz to elicit DNA damage in mitochondria-depleted 143B (rho-0) osteosarcoma cells, at concentrations where it elicited DNA damage in the parental 143B cell line. In conclusion, our data show that respirable quartz particles can elicit oxidative DNA damage in vitro without entering the nuclei of type II cells, which are considered to be important target cells in quartz carcinogenesis. Furthermore, our observations indicate that such indirect DNA damage involves the mitochondrial electron transport chain function, by an as-yet-to-be elucidated mechanism

  20. Increasing the stability of DNA-functionalized gold nanoparticles using mercaptoalkanes

    Stakenborg, T., E-mail: tim.stakenborg@imec.b [Veterinary Research Institute, Department of Bacteriology and Immunology (Belgium); Peeters, S.; Reekmans, G.; Laureyn, W.; Jans, H.; Borghs, G. [IMEC vzw, Nano Engineered Component Science (Belgium); Imberechts, H. [Veterinary Research Institute, Department of Bacteriology and Immunology (Belgium)

    2008-12-15

    In this work, the stability of DNA functionalized gold nanoparticles was examined in relation to their size, temperature, as well as the presence of mono- and bivalent ions. Furthermore, we report on the stabilizing effect of an additional post-functionalization with mercaptoalkanes, optionally bearing triethylene glycol (TEG) units. Although such so-called backfilling molecules are commonly used for planar gold surfaces, they have rarely been reported in combination with DNA-functionalized nanoparticles. Our results show that, conform the DLVO theory, smaller citrate-capped gold nanoparticles were more stable towards higher concentrations of salt. Citrate nanoparticles of 30 nm in size were only stable in sodium chloride concentrations up to {approx}0.05 M and up to 45 {sup o}C. The stability of these uncoated nanoparticles was even lower when bivalent salts were used (i.e. <2 x 10{sup -4} M). Immobilization of DNA on these nanoparticles, on the other hand, improved the stability in salt solutions with at least one order of magnitude. The additional use of backfilling molecules stabilized the gold nanoparticles even further, without negatively affecting the DNA hybridization efficiency. DNA functionalization also had a positive impact on the thermal stability of the nanoparticles. Unfortunately, this beneficial effect was not observed after a subsequent backfilling step.

  1. Increasing the stability of DNA-functionalized gold nanoparticles using mercaptoalkanes

    In this work, the stability of DNA functionalized gold nanoparticles was examined in relation to their size, temperature, as well as the presence of mono- and bivalent ions. Furthermore, we report on the stabilizing effect of an additional post-functionalization with mercaptoalkanes, optionally bearing triethylene glycol (TEG) units. Although such so-called backfilling molecules are commonly used for planar gold surfaces, they have rarely been reported in combination with DNA-functionalized nanoparticles. Our results show that, conform the DLVO theory, smaller citrate-capped gold nanoparticles were more stable towards higher concentrations of salt. Citrate nanoparticles of 30 nm in size were only stable in sodium chloride concentrations up to ∼0.05 M and up to 45 oC. The stability of these uncoated nanoparticles was even lower when bivalent salts were used (i.e. -4 M). Immobilization of DNA on these nanoparticles, on the other hand, improved the stability in salt solutions with at least one order of magnitude. The additional use of backfilling molecules stabilized the gold nanoparticles even further, without negatively affecting the DNA hybridization efficiency. DNA functionalization also had a positive impact on the thermal stability of the nanoparticles. Unfortunately, this beneficial effect was not observed after a subsequent backfilling step.

  2. Overexpression of mtDNA-associated AtWhy2 compromises mitochondrial function

    Abou-Rached Charbel

    2008-04-01

    Full Text Available Abstract Background StWhy1, a member of the plant-specific Whirly single-stranded DNA-binding protein family, was first characterized as a transcription factor involved in the activation of the nuclear PR-10a gene following defense-related stress in potato. In Arabidopsis thaliana, Whirlies have recently been shown to be primarily localized in organelles. Two representatives of the family, AtWhy1 and AtWhy3 are imported into plastids while AtWhy2 localizes to mitochondria. Their function in organelles is currently unknown. Results To understand the role of mitochondrial Whirlies in higher plants, we produced A. thaliana lines with altered expression of the atwhy2 gene. Organellar DNA immunoprecipitation experiments demonstrated that AtWhy2 binds to mitochondrial DNA. Overexpression of atwhy2 in plants perturbs mitochondrial function by causing a diminution in transcript levels and mtDNA content which translates into a low activity level of respiratory chain complexes containing mtDNA-encoded subunits. This lowered activity of mitochondria yielded plants that were reduced in size and had distorted leaves that exhibited accelerated senescence. Overexpression of atwhy2 also led to early accumulation of senescence marker transcripts in mature leaves. Inactivation of the atwhy2 gene did not affect plant development and had no detectable effect on mitochondrial morphology, activity of respiratory chain complexes, transcription or the amount of mtDNA present. This lack of phenotype upon abrogation of atwhy2 expression suggests the presence of functional homologues of the Whirlies or the activation of compensating mechanisms in mitochondria. Conclusion AtWhy2 is associated with mtDNA and its overexpression results in the production of dysfunctional mitochondria. This report constitutes the first evidence of a function for the Whirlies in organelles. We propose that they could play a role in the regulation of the gene expression machinery of organelles.

  3. The role of active DNA demethylation and Tet enzyme function in memory formation and cocaine action.

    Alaghband, Yasaman; Bredy, Timothy W; Wood, Marcelo A

    2016-06-20

    Active DNA modification is a major epigenetic mechanism that regulates gene expression in an experience-dependent manner, which is thought to establish stable changes in neuronal function and behavior. Recent discoveries regarding the Ten eleven translocation (Tet1-3) family of DNA hydroxylases have provided a new avenue for the study of active DNA demethylation, and may thus help to advance our understanding of how dynamic DNA modifications lead to long-lasting changes in brain regions underlying learning and memory, as well as drug-seeking and propensity for relapse following abstinence. Drug addiction is a complex, relapsing disorder in which compulsive drug-seeking behavior can persist despite aversive consequences. Therefore, understanding the molecular mechanisms that underlie the onset and persistence of drug addiction, as well as the pronounced propensity for relapse observed in addicts, is necessary for the development of selective treatments and therapies. In this mini-review, we provide an overview of the involvement of active DNA demethylation with an emphasis on the Tet family of enzymes and 5-hydroxymethylcytosine (5-hmC) in learning and memory, as well as in drug-seeking behavior. Memory and addiction share overlapping molecular, cellular, and circuit functions allowing research in one area to inform the other. Current discrepancies and directions for future studies focusing on the dynamic interplay between DNA methylation and demethylation, and how they orchestrate gene expression required for neuronal plasticity underlying memory formation, are discussed. PMID:26806038

  4. A van der Waals density functional mapping of attraction in DNA dimers

    Londero, Elisa; Hyldgaard, Per; Schroder, Elsebeth

    2013-01-01

    The dispersion interaction between a pair of parallel DNA double-helix structures is investigated by means of the van der Waals density functional (vdW-DF) method. Each double-helix structure consists of an infinite repetition of one B-DNA coil with 10 base pairs. This parameter-free density functional theory (DFT) study illustrates the initial step in a proposed vdW-DF computational strategy for large biomolecular problems. The strategy is to first perform a survey of interaction geometries,...

  5. Fluorescent Structural DNA Nanoballs Functionalized With Phosphate-Linked Nucleotide Triphosphates

    Anderson, Jon P.; Reynolds, Bambi L.; Baum, Kristin; Williams, John G.

    2010-01-01

    Highly labeled DNA nanoballs functionalized with phosphate-linked nucleotide triphosphates (dNTPs) were developed as a source of dNTPs for DNA polymerase. The particles were prepared by strand-displacement polymerization from a self-complementary circular template. Imaged by atomic force microscopy, these functionalized particles appear as condensed fuzzy balls with diameters between 50–150 nm. They emit a bright fluorescent signal, detected in 2 msec exposures with a signal-to-noise of 25 when imaged using a TIR fluorescence microscope. PMID:20158249

  6. Identification of Peptides That Inhibit the DNA Binding, trans-Activator, and DNA Replication Functions of the Human Papillomavirus Type 11 E2 Protein

    Deng, Su-Jun; Pearce, Kenneth H.; Dixon, Eric P.; Hartley, Kelly A.; Stanley, Thomas B.; Lobe, David C.; Garvey, Edward P.; Kost, Thomas A.; Petty, Regina L.; Rocque, Warren J.; Alexander, Kenneth A.; Underwood, Mark R.

    2004-01-01

    Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-...

  7. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  8. Nucleolar organization, ribosomal DNA array stability, and acrocentric chromosome integrity are linked to telomere function.

    Kaitlin M Stimpson

    Full Text Available The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA. The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2 causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.

  9. Diamondoid-functionalized gold nanogaps as sensors for natural, mutated, and epigenetically modified DNA nucleotides

    Sivaraman, Ganesh; Amorim, Rodrigo G.; Scheicher, Ralph H.; Fyta, Maria

    2016-05-01

    Modified tiny hydrogen-terminated diamond structures, known as diamondoids, show a high efficiency in sensing DNA molecules. These diamond cages, as recently proposed, could offer functionalization possibilities for gold junction electrodes. In this investigation, we report on diamondoid-functionalized electrodes, showing that such a device would have a high potential in sensing and sequencing DNA. The smallest diamondoid including an amine modification was chosen for the functionalization. Here, we report on the quantum tunneling signals across diamondoid-functionalized Au(111) electrodes. Our work is based on quantum-transport calculations and predicts the expected signals arising from different DNA units within the break junctions. Different gating voltages are proposed in order to tune the sensitivity of the functionalized electrodes with respect to specific nucleotides. The relation of this sensitivity to the coupling or decoupling of the electrodes is discussed. Our results also shed light on the sensing capability of such a device in distinguishing the DNA nucleotides, in their natural and mutated forms.

  10. Construction of heteroduplex DNA and in vitro model for functional analysis of mismatch repair

    WANG Yi; Clark Alan; WANG Jiaxun; SUN Menghong; SHI Daren

    2004-01-01

    Functional deficiency of mismatch repair (MMR) system is one of the mechanisms of tumorigenesis. With the development of the investigation and the requirement from the clinical diagnosis and treatment it is necessary to build up a method to evaluate the functional status of the whole MMR system in the concerned tumors. The original ssDNA and dsDNA from wild type (wt) bacteriophage M13mp2 and its three derivates with mutation points in the lacZα Gene have been used to construct two kinds of heteroduplex DNA molecules. One named del(2) has two bases deleted in the negative strand, the other has a G·G mismatch base pair in the negative strand too. Introducing this heteroduplex DNA into E. Coli NR9162 (mutS-) without the MMR ability on the indicator plate with x-gal and IPTG, there are three kinds of plaques, mixture plaque as the characteristic phenotype of heteroduplex DNA, blue and clear plaques. If the cell extract is mismatch repair competent the percentage of the mixture plaque will decrease after incubation with these heteroduplex DNA, the repair efficiency is expressed in percentage as 100× (1 minus the ratio of percentages of mixture plaque obtained from the extract-treated sample and untreated samples), which can imply the functional status of MMR system of certain samples. After large T-antigen-dependent SV-40 DNA replication assay cell extract from TK6, a human lymphoblastoid B-cell lymphoma cell line with MMR ability, and Lovo, a human colonic carcinoma cell line with MMR deficiency have incubated with these heteroduplex DNA. The repair efficiency of TK6 to del(2) is more than 60%, to G·G is more than 50%. The Lovo efficiency to del(2) is less than 10%, to G·G is less than 20%. Therefore, in this in vitro model used for functional analysis of mismatch repair of heteroduplex DNA as the repair target, TK6 can serve as the control for MMR proficiency and Lovo as the control for MMR deficiency. Using this model the tumor tissue from a case of hereditary

  11. Bio-functionalized hollow core photonic crystal fibers for label-free DNA detection

    Candiani, A.; Salloom, Hussein T.; Coscelli, E.; Sozzi, M.; Manicardi, A.; Ahmad, Ahmad K.; Al-Janabi, A. Hadi; Corradini, R.; Picchi, G.; Cucinotta, A.; Selleri, S.

    2014-02-01

    Bio-functionalization of inner surfaces of all silica Hollow Core-Photonic Crystal Fibers (HC-PCF) has been investigated. The approach is based on layer-by-layer self-assembly Peptide Nucleic Acid (PNA) probes, which is an oligonucleotide mimic that is well suited for specific DNA target recognition. Two kinds of HC-PCFs have been considered: a photonic Bragg fiber and a hollow core (HC-1060) fiber. After spectral characterization and internal surface functionalization by using PNA probes, genomic DNA solutions from soy flour were infiltrated into the fibers. The experimental results indicate that hybridization of the complementary strand of target DNA increases the thickness of the silica layer and leads up to the generation of surface modes, resulting in a significant modulation of the transmission spectra. Numerical analysis confirms such behavior, suggesting the possibility to realize biological sensing.

  12. Synthetic Polymer Hybridization with DNA and RNA Directs Nanoparticle Loading, Silencing Delivery, and Aptamer Function.

    Zhou, Zhun; Xia, Xin; Bong, Dennis

    2015-07-22

    We report herein discrete triplex hybridization of DNA and RNA with polyacrylates. Length-monodisperse triazine-derivatized polymers were prepared on gram-scale by reversible addition-fragmentation chain-transfer polymerization. Despite stereoregio backbone heterogeneity, the triazine polymers bind T/U-rich DNA or RNA with nanomolar affinity upon mixing in a 1:1 ratio, as judged by thermal melts, circular dichroism, gel-shift assays, and fluorescence quenching. We call these polyacrylates "bifacial polymer nucleic acids" (bPoNAs). Nucleic acid hybridization with bPoNA enables DNA loading onto polymer nanoparticles, siRNA silencing delivery, and can further serve as an allosteric trigger of RNA aptamer function. Thus, bPoNAs can serve as tools for both non-covalent bioconjugation and structure-function nucleation. It is anticipated that bPoNAs will have utility in both bio- and nanotechnology. PMID:26138550

  13. Multi-scale coding of genomic information: From DNA sequence to genome structure and function

    Understanding how chromatin is spatially and dynamically organized in the nucleus of eukaryotic cells and how this affects genome functions is one of the main challenges of cell biology. Since the different orders of packaging in the hierarchical organization of DNA condition the accessibility of DNA sequence elements to trans-acting factors that control the transcription and replication processes, there is actually a wealth of structural and dynamical information to learn in the primary DNA sequence. In this review, we show that when using concepts, methodologies, numerical and experimental techniques coming from statistical mechanics and nonlinear physics combined with wavelet-based multi-scale signal processing, we are able to decipher the multi-scale sequence encoding of chromatin condensation-decondensation mechanisms that play a fundamental role in regulating many molecular processes involved in nuclear functions.

  14. Photoligation of self-assembled DNA constructs containing anthracene-functionalized 2'-amino-LNA monomers

    Pasternak, Karol; Pasternak, Anna; Gupta, Pankaj;

    2011-01-01

    Efficient synthesis of a novel anthracene-functionalized 2'-amino-LNA phosphoramidite derivative is described together with its incorporation into oligodeoxynucleotides. Two DNA strands with the novel 2'-N-anthracenylmethyl-2'-amino-LNA monomers can be effectively cross-linked by photoligation at...

  15. DNA-sensors based on functionalized conducting polymers and quantum dots

    Kjällman, Tanja; Peng, Hui; Travas-Sejdic, Jadranka; Soeller, Christian

    2007-12-01

    The availability of rapid and specific biosensors is of great importance for many areas of biomedical research and modern biotechnology. This includes a need for DNA sensors where the progress of molecular biology demands routine detection of minute concentrations of specific gene fragments. A promising alternative approach to traditional DNA essays utilizes novel smart materials, including conducting polymers and nanostructured materials such as quantum dots. We have constructed a number of DNA sensors based on smart materials that allow rapid one-step detection of unlabeled DNA fragments with high specificity. These sensors are based on functionalized conducting polymers derived from polypyrrole (PPy) and poly(p-phenylenevinylene) (PPV). PPy based sensors provide intrinsic electrical readout via cyclic voltammetry and electrochemical impedance spectroscopy. The performance of these sensors is compared to a novel self-assembled monolayer-PNA construct on a gold electrode. Characterization of the novel PNA based sensor shows that it has comparable performance to the PPy based sensors and can also be read out effectively using AC cyclic voltammetry. Complementary to such solid substrate sensors we have developed a novel optical DNA essay based on a new PPV derived cationic conducting polymer. DNA detection in this essay results from sample dependent fluorescence resonance energy transfer changes between the cationic conducting polymer and Cy3 labeled probe oligonucleotides. As an alternative to such fluorochrome based sensors we discuss the use of inorganic nanocrystals ('quantum dots') and present data from water soluble CdTe quantum dots synthesized in an aqueous environment.

  16. Tuning the geometry of shape-restricted DNA molecules on the functionalized Si(1 1 1)

    Designing a well-defined and stable interface between biomolecules and semiconductor surfaces is of great importance for current and future biosensing and bioelectronic applications. The well-characterized chemistry, stability, and easily tunable electronic properties of silicon substrate make it a practical platform for this type of interface. It has been established in our previous work that a robust, covalent attachment between thiol-DNA molecules of a pre-designed geometrical shape and a modified silicon surface can be achieved. This work focuses on using this binding model and altering the distance between the DNA molecules and silicon surface by strategically placing thiol linkers within the pre-determined geometric design of the rectangularly shaped DNA. The statistical analysis of the height profiles of DNA molecules attached to the surface, as determined by AFM, provides specific insight into how the construction of the DNA molecules affects the binding distance. A comparison between two thiol-DNA molecules with different numbers of thiol groups placed either within the rectangular shape or anchored to the free loop of the same geometric design suggest that the average distance of these molecules to the functionalized silicon surface can be changed by approximately 0.5 nm.

  17. Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic 'mini-genes'.

    Ciafrè, S A; Rinaldi, M; Gasparini, P; Seripa, D; Bisceglia, L; Zelante, L; Farace, M G; Fazio, V M

    1995-01-01

    Several gene transfer techniques that employ 'naked DNA' molecules have recently been developed and numerous gene therapy protocols that make use of 'naked-DNA' have been proposed. We studied the possibility of enhancing the stability of 'naked DNA vectors' and thus also gene transfer and expression efficiencies, by constructing phosphorothioate (PS-) double strand DNA molecules and functional transcription units. We first synthesized short PS-double strand DNA molecules by the annealing of two complementary, 35 nt long, oligonucleotides. The accessibility of DNA modifying enzymes to this molecule was significantly decreased: T4-ligase and kinase activity were respectively reduced up to 1/2 and to 1/6, as compared to the normal phosphodiester molecule. Nucleolytic stability was increased either to purified enzymes (DNase I and Bal31) or to incubations in fresh serum, cell culture medium or in muscle protein extract. Phosphorothioate end-capped complete eukaryotic transcription units (obtained by Taq polymerase amplification with PS-primers) were not significantly protected from nucleolytic attack. On the contrary, synthetic transcription units, 'mini genes', obtained by Taq amplification with 1, 2 or 3 PS-dNTP substitutions, were resistant to DNase I and Bal31 nucleolytic activity. Transcription efficiency, driven by the T7 promoter, was 96.5, 95 and 33.5% (respectively with 1, 2 or 3 substitutions), as compared to the normal phosphodiester molecules. Images PMID:7479077

  18. A Functional Bacterium-to-Plant DNA Transfer Machinery of Rhizobium etli.

    Lacroix, Benoît; Citovsky, Vitaly

    2016-03-01

    Different strains and species of the soil phytopathogen Agrobacterium possess the ability to transfer and integrate a segment of DNA (T-DNA) into the genome of their eukaryotic hosts, which is mainly mediated by a set of virulence (vir) genes located on the bacterial Ti-plasmid that also contains the T-DNA. To date, Agrobacterium is considered to be unique in its capacity to mediate genetic transformation of eukaryotes. However, close homologs of the vir genes are encoded by the p42a plasmid of Rhizobium etli; this microorganism is related to Agrobacterium, but known only as a symbiotic bacterium that forms nitrogen-fixing nodules in several species of beans. Here, we show that R. etli can mediate functional DNA transfer and stable genetic transformation of plant cells, when provided with a plasmid containing a T-DNA segment. Thus, R. etli represents another bacterial species, besides Agrobacterium, that encodes a protein machinery for DNA transfer to eukaryotic cells and their subsequent genetic modification. PMID:26968003

  19. Microscopic insight into the DNA condensation process of a zwitterion-functionalized polycation.

    Sun, Hui; Zhou, Li; Chen, Xiaolu; Han, Xia; Wang, Rui; Liu, Honglai

    2016-11-01

    Zwitterion-functionalized polycations are ideal gene carriers with long circulation, high cellular uptaking and low cell viability. However, the trade-off between the DNA condensation efficiency and the cell viability must be addressed. The purpose of this study is to provide a microscopic insight into the DNA condensation process and to explore the effect of a zwitterionic block of zwitterion-functionalized polycation, which is of great significance in designing novel gene delivery systems. Poly[2-(dimethylamino)ethyl methacrylate-b-(sulfobetaine methacrylate)] (PDMAEMA-b-PSBMA) copolymers were synthesized and used as the model systems. Different from the conventional concept that the PSBMA zwitterionic block act only as the "stealthy" groups, the subtle differences in physical and colloidal characteristics between the polycation/DNA polyplexes show that the PSBMA segment is capable of wrapping DNA attributed to the quaternary ammonium cations, without compromising the DNA condensation capability. On the other hand, the incorporation of PSBMA block reduces the surface charge of the polyplexes, which substantially result in the inefficient transfection and the reduced cytotoxicity. PMID:27404763

  20. Nanoparticles and DNA - a powerful and growing functional combination in bionanotechnology

    Samanta, Anirban; Medintz, Igor L.

    2016-04-01

    Functionally integrating DNA and other nucleic acids with nanoparticles in all their different physicochemical forms has produced a rich variety of composite nanomaterials which, in many cases, display unique or augmented properties due to the synergistic activity of both components. These capabilities, in turn, are attracting greater attention from various research communities in search of new nanoscale tools for diverse applications that include (bio)sensing, labeling, targeted imaging, cellular delivery, diagnostics, therapeutics, theranostics, bioelectronics, and biocomputing to name just a few amongst many others. Here, we review this vibrant and growing research area from the perspective of the materials themselves and their unique capabilities. Inorganic nanocrystals such as quantum dots or those made from gold or other (noble) metals along with metal oxides and carbon allotropes are desired as participants in these hybrid materials since they can provide distinctive optical, physical, magnetic, and electrochemical properties. Beyond this, synthetic polymer-based and proteinaceous or viral nanoparticulate materials are also useful in the same role since they can provide a predefined and biocompatible cargo-carrying and targeting capability. The DNA component typically provides sequence-based addressability for probes along with, more recently, unique architectural properties that directly originate from the burgeoning structural DNA field. Additionally, DNA aptamers can also provide specific recognition capabilities against many diverse non-nucleic acid targets across a range of size scales from ions to full protein and cells. In addition to appending DNA to inorganic or polymeric nanoparticles, purely DNA-based nanoparticles have recently surfaced as an excellent assembly platform and have started finding application in areas like sensing, imaging and immunotherapy. We focus on selected and representative nanoparticle-DNA materials and highlight their

  1. Nanoparticles and DNA - a powerful and growing functional combination in bionanotechnology.

    Samanta, Anirban; Medintz, Igor L

    2016-04-28

    Functionally integrating DNA and other nucleic acids with nanoparticles in all their different physicochemical forms has produced a rich variety of composite nanomaterials which, in many cases, display unique or augmented properties due to the synergistic activity of both components. These capabilities, in turn, are attracting greater attention from various research communities in search of new nanoscale tools for diverse applications that include (bio)sensing, labeling, targeted imaging, cellular delivery, diagnostics, therapeutics, theranostics, bioelectronics, and biocomputing to name just a few amongst many others. Here, we review this vibrant and growing research area from the perspective of the materials themselves and their unique capabilities. Inorganic nanocrystals such as quantum dots or those made from gold or other (noble) metals along with metal oxides and carbon allotropes are desired as participants in these hybrid materials since they can provide distinctive optical, physical, magnetic, and electrochemical properties. Beyond this, synthetic polymer-based and proteinaceous or viral nanoparticulate materials are also useful in the same role since they can provide a predefined and biocompatible cargo-carrying and targeting capability. The DNA component typically provides sequence-based addressability for probes along with, more recently, unique architectural properties that directly originate from the burgeoning structural DNA field. Additionally, DNA aptamers can also provide specific recognition capabilities against many diverse non-nucleic acid targets across a range of size scales from ions to full protein and cells. In addition to appending DNA to inorganic or polymeric nanoparticles, purely DNA-based nanoparticles have recently surfaced as an excellent assembly platform and have started finding application in areas like sensing, imaging and immunotherapy. We focus on selected and representative nanoparticle-DNA materials and highlight their

  2. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

    Jana eSlyskova

    2014-05-01

    Full Text Available Thousands of DNA lesions are estimated to occur in each cell every day and almost all are recognized and repaired. DNA repair is an essential system that prevents accumulation of mutations which can lead to serious cellular malfunctions. Phenotypic evaluation of DNA repair activity of individuals is a relatively new approach. Methods to assess base and nucleotide excision repair pathways (BER and NER in peripheral blood cells based on modified comet assay protocols have been widely applied in human epidemiological studies. These provided some interesting observations of individual DNA repair activity being suppressed among cancer patients. However, extension of these results to cancer target tissues requires a different approach. Here we describe the evaluation of BER and NER activities in extracts from deep-frozen colon biopsies using an upgraded version of the in vitro comet-based DNA repair assay in which twelve reactions on one microscope slide can be performed. The aim of this report is to provide a detailed, easy-to-follow protocol together with results of optimization experiments. Additionally, results obtained by functional assays were analysed in the context of other cellular biomarkers, namely single nucleotide polymorphisms and gene expressions. We have shown that measuring DNA repair activity is not easily replaceable by genomic or transcriptomic approaches, but should be applied with the latter techniques in a complementary manner. The ability to measure DNA repair directly in cancer target tissues might finally answer questions about the tissue-specificity of DNA repair processes and their real involvement in the process of carcinogenesis.

  3. Functionalized 2'-amino-alpha-L-LNA: directed positioning of intercalators for DNA targeting.

    Kumar, T Santhosh; Madsen, Andreas S; Østergaard, Michael E; Sau, Sujay P; Wengel, Jesper; Hrdlicka, Patrick J

    2009-02-01

    Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (locked nucleic acid) and its diastereomer alpha-L-LNA are two promising examples thereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization, and molecular modeling of N2'-functionalized 2'-amino-alpha-L-LNA is described. Chemoselective N2'-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2'-N-(pyren-1-yl)carbonyl-2'-amino-alpha-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 degrees C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small nonaromatic N2'-functionalities such as 2'-N-acetyl-2'-amino-alpha-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as -16.5 degrees C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases in circular dichroism signal intensity, and molecular modeling studies suggest that pyrene-functionalized 2'-amino-alpha-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development of probes for emerging therapeutic and diagnostic applications focusing on DNA targeting. PMID:19108636

  4. The Caenorhabditis elegans Werner syndrome protein functions upstream of ATR and ATM in response to DNA replication inhibition and double-strand DNA breaks.

    Se-Jin Lee

    2010-01-01

    Full Text Available WRN-1 is the Caenorhabditis elegans homolog of the human Werner syndrome protein, a RecQ helicase, mutations of which are associated with premature aging and increased genome instability. Relatively little is known as to how WRN-1 functions in DNA repair and DNA damage signaling. Here, we take advantage of the genetic and cytological approaches in C. elegans to dissect the epistatic relationship of WRN-1 in various DNA damage checkpoint pathways. We found that WRN-1 is required for CHK1 phosphorylation induced by DNA replication inhibition, but not by UV radiation. Furthermore, WRN-1 influences the RPA-1 focus formation, suggesting that WRN-1 functions in the same step or upstream of RPA-1 in the DNA replication checkpoint pathway. In response to ionizing radiation, RPA-1 focus formation and nuclear localization of ATM depend on WRN-1 and MRE-11. We conclude that C. elegans WRN-1 participates in the initial stages of checkpoint activation induced by DNA replication inhibition and ionizing radiation. These functions of WRN-1 in upstream DNA damage signaling are likely to be conserved, but might be cryptic in human systems due to functional redundancy.

  5. The impact of arginine-modified chitosan-DNA nanoparticles on the function of macrophages

    It has been demonstrated that incorporation of arginine moieties into chitosan significantly elevates the transgenic efficacy of the chitosan. However, little is known about the impact of arginine-modified chitosan on the function of macrophages, which play a vitally important role in the inflammatory response of the body to foreign substances, especially particulate substances. This study was designed to investigate the impact of arginine-modified chitosan/DNA nanoparticles on the function of the murine macrophage through observation of phagocytic activity and production of pro-inflammatory cytokines (IL-1β, IL-6, IL-10, IL-12, and TNF-α). Results showed that both chitosan/DNA nanoparticles and arginine-modified chitosan/DNA nanoparticles, containing 20 μg/mL DNA, were internalized by almost all the macrophages in contact. This led to no significant changes, compared to the non-exposure group, in production of cytokines and phagocytic activity of the macrophages 24 h post co-incubation, whereas exposure to LPS induced obviously elevated cytokine production and phagocytic activity, suggesting that incorporation of arginine moieties into chitosan does not have a negative impact on the function of the macrophages.

  6. Effect of base mismatch on the electronic properties of DNA-DNA and LNA-DNA double strands: Density-functional theoretical calculations

    Natsume, Takayuki; Ishikawa, Yasuyuki; Dedachi, Kenichi; Tsukamoto, Takayuki; Kurita, Noriyuki

    2007-01-01

    The electronic properties of double-stranded octametric DNA-DNA and LNA-DNA with a single-base mismatch were compared with those having fully complementary base pairs to quantify the effect of the base mismatch on hybridization energies (HE). A single T-G mismatch in the LNA-DNA gives rise to a significant reduction in HE, which is consistent with a significant lowering of the melting temperature for mismatched LNA-DNA. By contrast, the hybridization strength of the mismatched DNA-DNA depends...

  7. Synthesis of a drug delivery vehicle for cancer treatment utilizing DNA-functionalized gold nanoparticles

    Brann, Tyler

    The treatment of cancer with chemotherapeutic agents has made great strides in the last few decades but still introduces major systemic side effects. The potent drugs needed to kill cancer cells often cause irreparable damage to otherwise healthy organs leading to further morbidity and mortality. A therapy with intrinsic selective properties and/or an inducible activation has the potential to change the way cancer can be treated. Gold nanoparticles (GNPs) are biocompatible and chemically versatile tools that can be readily functionalized to serve as molecular vehicles. The ability of these particles to strongly absorb light with wavelengths in the therapeutic window combined with the heating effect of surface plasmon resonance makes them uniquely suited for noninvasive heating in biologic applications. Specially designed DNA aptamers have shown their ability to serve as drug carriers through intercalation as well as directly acting as therapeutic agents. By combining these separate molecules a multifaceted drug delivery vehicle can be created with great potential as a selective and controllable treatment for cancer. Oligonucleotide-coated GNPs have been created using spherical GNPs but little work has been reported using gold nanoplates in this way. Using the Diasynth method gold nanoplates were produced to absorb strongly in the therapeutic near infrared (nIR) window. These particles were functionalized with two DNA oligonucleotides: one serving as an intercalation site for doxorubicin, and another, AS1411, serving directly as an anticancer targeting/therapeutic agent. These functional particles were fully synthesized and processed along with confirmation of DNA functionalization and doxorubicin intercalation. Doxorubicin is released via denaturation of the DNA structure into which doxorubicin is intercalated upon the heating of the gold nanoplate well above the DNA melting temperature. This temperature increase, due to light stimulation of surface plasmon

  8. Functions of replication factor C and proliferating-cell nuclear antigen: Functional similarity of DNA polymerase accessory proteins from human cells and bacteriophage T4

    The proliferating-cell nuclear antigen (PCNA) and the replication factors A and C (RF-A and RF-C) are cellular proteins essential for complete elongation of DNA during synthesis from the simian virus 40 origin of DNA replication in vitro. All three cooperate to stimulate processive DNA synthesis by DNA polymerase δ on a primed single-stranded M13 template DNA and as such can be categorized as DNA polymerase accessory proteins. Biochemical analyses with highly purified RF-C and PCNA have demonstrated functions that are completely analogous to the functions of bacteriophage T4 DNA polymerase accessory proteins. A primer-template-specific DNA binding activity and a DNA-dependent ATPase activity copurified with the multisubunit protein RF-C and are similar to the functions of the phage T4 gene 44/62 protein complex. Furthermore, PCNA stimulated the RF-C ATPase activity and is, therefore, analogous to the phage T4 gene 45 protein, which stimulates the ATPase function of the gene 44/62 protein complex. Indeed, some primary sequence similarities between human PCNA and the phage T4 gene 45 protein could be detected. These results demonstrate a striking conservation of the DNA replication apparatus in human cells and bacteriophage T4

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

    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......, or a known regulatory interaction might not be observed under the growth conditions tested so far. To correctly identify physical interactions between transcription factors (TFs) and genes and to determine their regulatory implications under various experimental conditions, we integrated Ch......IP-chip data with motif binding sites, nucleosome occupancy and mRNA expression datasets within a probabilistic framework. This framework was specifically tailored for the identification of functional and nonfunctional DNA binding events. Using this, we estimate that only 50% of condition-specific protein-DNA...

  10. Lipid nanocapsules functionalized with polyethyleneimine for plasmid DNA and drug co-delivery and cell imaging

    Skandrani, Nadia; Barras, Alexandre; Legrand, Dominique; Gharbi, Tijani; Boulahdour, Hatem; Boukherroub, Rabah

    2014-06-01

    The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene delivery systems. The cationic LNCs were produced by a phase inversion process with a nominal size of 25 nm and subsequently modified with PEI chains using a transacylation reaction. The functionalization process allowed good control over the nanoscale particle size (26.2 +/- 3.9 nm) with monodisperse size characteristics (PI stability with no cytotoxicity due to the anchored PEI polymers on the surface of LNCs. Finally, the transfection efficiency of the LNC25-T/pDNA complexes was studied and evaluated on HEK cell lines in comparison with free PEI/pDNA polyplexes. The combination of cationic LNCs with pDNA exhibited more than a 2.8-fold increase in transfection efficiency compared to the standard free PEI/pDNA polyplexes at the same PEI concentrations. Moreover, we have demonstrated that LNC25-T/pDNA loaded with a hydrophobic drug, paclitaxel, showed high drug efficacy. The high transfection efficiency combined with the potential of simultaneous co-delivery of hydrophobic drugs, relatively small size of LNC25-T/pDNA complexes, and fluorescence imaging can be crucial for gene therapy, as small particle sizes may be more favorable for in vivo studies.The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene delivery systems. The cationic LNCs were produced by a phase inversion process with a nominal size of 25 nm and subsequently modified with PEI chains using a transacylation reaction. The functionalization process allowed good control over the nanoscale particle size (26.2 +/- 3.9 nm) with monodisperse size characteristics (PI stability with no cytotoxicity due to the anchored PEI polymers on the surface of LNCs. Finally, the transfection efficiency of the LNC25-T/pDNA complexes was studied and

  11. Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device.

    Woolley, A T; Hadley, D; Landre, P; deMello, A J; Mathies, R A; Northrup, M A

    1996-12-01

    Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 degrees C/s heating, 2.5 degrees C/s cooling) with the high-speed (Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the beta-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 x 10(7) and 4 x 10(5) copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems. PMID:8946790

  12. Highly Sensitive Functionalized Conducting Copolypyrrole Film for DNA Sensing and Protein-resistant%Highly Sensitive Functionalized Conducting Copolypyrrole Film for DNA Sensing and Protein-resistant

    Zhang, Zhihong; Li, Guijuan; Yan, Fufeng; Zhang, Zhonghou; Fang, Shaoming

    2012-01-01

    In order to exploit the applications ofpolypyrrole (PPy) derivatives in biosensors and bioelectronics, the different immobilization mechanisms of biomolecules onto differently functionalized conducting PPy films are investigated. Pyrrole and pyrrole derivatives with carboxyl and amino groups were copolymerized with ω-(N-pyrrolyl)-octylthiol self-assembled on Au surface by the method of the chemical polymerization to form a layer of the copolymer film, i.e., poly[pyrrole-co-(N-pyrrolyl)-caproic acid] (poly(Py-co-PyCA)) and poly[pyrrole-co-(N-pyrrolyl)-hexylamine] (poly(Py-co-PyHA)), in which the carboxyl groups in poly(Py-co-PyCA) were activated to the ester groups. Based on the structure characteristics, the immobilization/hybridization of DNA molecules on PPy, poly(Py-co-PyCA) and poly(Py-co-PyHA) were surveyed by cyclic voltammograms measurements. For differently functionalized copolymers, the immobilization mechanisms of DNA are various. Besides the electrochemical properties of the composite electrodes of PPy and its copolymers being detected before and after bovine serum albumin (BSA) adsorption, the kinetic process of protein binding was determined by surface plasmon resonance of spectroscopy. Since few BSA molecules could anchor onto the PPy and its copolymers surfaces, it suggests this kind of conducting polymers can be applied as the protein-resistant material.

  13. Emerging Molecular and Biological Functions of MBD2, a Reader of DNA Methylation

    Kathleen H Wood

    2016-05-01

    Full Text Available DNA methylation is an epigenetic mark that is essential for many biological processes and is linked to diseases such as cancer. Methylation is usually associated with transcriptional silencing, but new research has challenged this model. Both transcriptional activation and repression have recently been found to be associated with DNA methylation in a context-specific manner. How DNA methylation patterns are interpreted into different functional output remains poorly understood. One mechanism involves the protein ‘readers’ of methylation, which includes the methyl-CpG binding domain (MBD family of proteins. This review examines the molecular and biological functions of MBD2, which binds to CpG methylation and is an integral part of the nucleosome remodeling and histone deacetylation (NuRD complex. MBD2 has been linked to immune system function and tumorigenesis, yet little is known about its functions in vivo. Recent studies have found the MBD2 protein is ubiquitously expressed, with relatively high levels in the lung, liver and colon. Mbd2 null mice surprisingly show relatively mild phenotypes compared to mice with loss of function of other MBD proteins. This evidence has previously been interpreted as functional redundancy between the MBD proteins. Here we examine and contextualize research that suggests MBD2 has unique properties and functions among the MBD proteins. These functions translate to recently described roles in the development and differentiation of multiple cell lineages, including pluripotent stem cells and various cell types of the immune system, as well as in tumorigenesis. We also consider possible models for the dynamic interactions between MBD2 and NuRD in different tissues in vivo. The functions of MBD2 may have direct therapeutic implications for several areas of human disease, including autoimmune conditions and cancer, in addition to providing insights into the actions of NuRD and chromatin regulation.

  14. A novel assay revealed that ribonucleotide reductase is functionally important for interstrand DNA crosslink repair.

    Fujii, Naoaki; Evison, Benjamin J; Actis, Marcelo L; Inoue, Akira

    2015-11-01

    Cells have evolved complex biochemical pathways for DNA interstrand crosslink (ICL) removal. Despite the chemotherapeutic importance of ICL repair, there have been few attempts to identify which mechanistic DNA repair inhibitor actually inhibits ICL repair. To identify such compounds, a new and robust ICL repair assay was developed using a novel plasmid that contains synthetic ICLs between a CMV promoter region that drives transcription and a luciferase reporter gene, and an SV40 origin of replication and the large T antigen (LgT) gene that enables self-replication in mammalian cells. In a screen against compounds that are classified as inhibitors of DNA repair or synthesis, the reporter generation was exquisitely sensitive to ribonucleotide reductase (RNR) inhibitors such as gemcitabine and clofarabine, but not to inhibitors of PARP, ATR, ATM, Chk1, and others. The effect was observed also by siRNA downregulation of RNR. Moreover, the reporter generation was also particularly sensitive to 3-AP, a non-nucleoside RNR inhibitor, but not significantly sensitive to DNA replication stressors, suggesting that the involvement of RNR in ICL repair is independent of incorporation of a nucleotide RNR inhibitor into DNA to induce replication stress. The reporter generation from a modified version of the plasmid that lacks the LgT-SV40ori motif was also adversely affected by RNR inhibitors, further indicating a role for RNR in ICL repair that is independent of DNA replication. Intriguingly, unhooking of cisplatin-ICL from nuclear DNA was significantly inhibited by low doses of gemcitabine, suggesting an unidentified functional role for RNR in the process of ICL unhooking. The assay approach could identify other molecules essential for ICLR in quantitative and flexible manner. PMID:26462050

  15. Z-DNA and its biological function%Z-DNA及其生物学功能

    汤雅男; 杨攀; 胡成钰

    2009-01-01

    Z-DNA是一种处于高能状态、不稳定的DNA分子构象.形成Z-DNA的原因有很多:首先,转录过程中,移动的RNA聚合酶在模板DNA的5'端产生负超螺旋扭曲力,导致Z-DNA的形成;其次,含有d(GC)_n序列的核酸分子在高浓度的NaCl、[Co(NH_3)_6]~(2+)盐溶液中也能够形成Z-DNA;最后,化学修饰也可以使DNA产生稳定的Z-DNA.Z-DNA是在体外首先发现的,但随着研究的不断深入,发现Z-DNA在体内也广泛存在并可能具有功能的多样性,包括参与基因表达调控、染色体断裂、基因重组、抗病毒、病毒发生等生物学过程.%Z-DNA is an instable and high energy DNA conformation in vivo and it can be formed in many conditions.Firstly,when RNA polymerase is moving on a DNA template,transcription can result in negative supercoiling behind the polymerase,thus facilitating Z-DNA conformation at permissive regions.Secondly,the Z-DNA-conformation which is formed by d(GC)_n sequence can also be stabled in high-salt solution of NaCl or [Co(NH_3)_6]~(2+) and lastly,the Z-DNA can be formed by covalent modifications.Though Z-DNA was firstly discovered in vitro,recently many evidences revealed that Z-DNA was widely existed and may possessed diversity of function in vivo,including gene expression and regulation,chromosomal breaks,recombination,antivirus defense and virus generation and so on.

  16. DNA as Tunable Adaptor for siRNA Polyplex Stabilization and Functionalization.

    Heissig, Philipp; Klein, Philipp M; Hadwiger, Philipp; Wagner, Ernst

    2016-01-01

    siRNA and microRNA are promising therapeutic agents, which are engaged in a natural mechanism called RNA interference that modulates gene expression posttranscriptionally. For intracellular delivery of such nucleic acid triggers, we use sequence-defined cationic polymers manufactured through solid phase chemistry. They consist of an oligoethanamino amide core for siRNA complexation and optional domains for nanoparticle shielding and cell targeting. Due to the small size of siRNA, electrostatic complexes with polycations are less stable, and consequently intracellular delivery is less efficient. Here we use DNA oligomers as adaptors to increase size and charge of cargo siRNA, resulting in increased polyplex stability, which in turn boosts transfection efficiency. Extending a single siRNA with a 181-nucleotide DNA adaptor is sufficient to provide maximum gene silencing aided by cationic polymers. Interestingly, this simple strategy was far more effective than merging defined numbers (4-10) of siRNA units into one DNA scaffolded construct. For DNA attachment, the 3' end of the siRNA passenger strand was beneficial over the 5' end. The impact of the attachment site however was resolved by introducing bioreducible disulfides at the connection point. We also show that DNA adaptors provide the opportunity to readily link additional functional domains to siRNA. Exemplified by the covalent conjugation of the endosomolytic influenza peptide INF-7 to siRNA via a DNA backbone strand and complexing this construct with a targeting polymer, we could form a highly functional polyethylene glycol-shielded polyplex to downregulate a luciferase gene in folate receptor-positive cells. PMID:26928236

  17. The breast cancer susceptibility gene BRCA1: DNA repair and other functions

    BRCA1 is a tumour suppressor gene. Germline mutations in BRCA1 confer susceptibility to breast and ovarian cancer and levels of BRCA1 mRNA and/or protein are reduced in a significant proportion of sporadic breast tumours. The product of this gene is a large multifunctional nuclear phosphoprotein that has been implicated in the regulation of cell cycle progression, apoptosis, transcription and DNA repair. Thus BRCA1 is thought to function as a genomic caretaker, responding to DNA damage by halting cell-cycle progression and activating DNA repair or cell death pathways. Evidence of a role for BRCA1 in DNA repair includes the identification of a novel C-terminal amino acid sequence motif (BRCT) common to a broad range of DNA repair proteins, the observation that the BRCA1 protein interacts with a number of DNA repair proteins, including Rad50, and the demonstration of defective double-strand break repair by homologous recombination and genetic instability in BRCA1-deficient cells. Loss of BRCA1 contributes to breast tumourigenesis by inducing genomic instability. The consistent histological phenotype of BRCA1 tumours, including their high-grade, pushing margins and syncytial appearance, together with the results of differential-expression analyses, indicate that the mutations that accumulate in these tumours are far from random. At present however the pathway between BRCA1 loss and BRCA1-mediated tumour development is poorly understood. In an attempt to address this we have studied the cellular and molecular effects of disrupting BRCA1 function. Results from this analysis and our studies on the regulation of BRCA1 expression will be presented

  18. The role of DNA methylation in directing the functional organization of the cancer epigenome

    Lay, Fides D.; Liu, Yaping; Kelly, Theresa K.; Witt, Heather; Farnham, Peggy J.

    2015-01-01

    The holistic role of DNA methylation in the organization of the cancer epigenome is not well understood. Here we perform a comprehensive, high-resolution analysis of chromatin structure to compare the landscapes of HCT116 colon cancer cells and a DNA methylation-deficient derivative. The NOMe-seq accessibility assay unexpectedly revealed symmetrical and transcription-independent nucleosomal phasing across active, poised, and inactive genomic elements. DNA methylation abolished this phasing primarily at enhancers and CpG island (CGI) promoters, with little effect on insulators and non-CGI promoters. Abolishment of DNA methylation led to the context-specific reestablishment of the poised and active states of normal colon cells, which were marked in methylation-deficient cells by distinct H3K27 modifications and the presence of either well-phased nucleosomes or nucleosome-depleted regions, respectively. At higher-order genomic scales, we found that long, H3K9me3-marked domains had lower accessibility, consistent with a more compact chromatin structure. Taken together, our results demonstrate the nuanced and context-dependent role of DNA methylation in the functional, multiscale organization of cancer epigenomes. PMID:25747664

  19. Lipid nanocapsules functionalized with polyethyleneimine for plasmid DNA and drug co-delivery and cell imaging

    Skandrani, Nadia; Barras, Alexandre; Legrand, Dominique; Gharbi, Tijani; Boulahdour, Hatem; Boukherroub, Rabah

    2014-06-01

    The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene delivery systems. The cationic LNCs were produced by a phase inversion process with a nominal size of 25 nm and subsequently modified with PEI chains using a transacylation reaction. The functionalization process allowed good control over the nanoscale particle size (26.2 +/- 3.9 nm) with monodisperse size characteristics (PI < 0.2) and positive surface charge up to +18.7 mV. The PEI-modified LNCs (LNC25-T) displayed good buffering capacity. Moreover, the cationic LNC25-T were able to condense DNA and form complexes via electrostatic interactions in a typical weight ratio-dependent relationship. It was found that the mean diameter of LNC25-T/pDNA complexes increased to ~40-50 nm with the LNC25-T/pDNA ratio from 1 to 500. Gel electrophoresis and cell viability experiments showed that the LNC25-T/pDNA complexes had high stability with no cytotoxicity due to the anchored PEI polymers on the surface of LNCs. Finally, the transfection efficiency of the LNC25-T/pDNA complexes was studied and evaluated on HEK cell lines in comparison with free PEI/pDNA polyplexes. The combination of cationic LNCs with pDNA exhibited more than a 2.8-fold increase in transfection efficiency compared to the standard free PEI/pDNA polyplexes at the same PEI concentrations. Moreover, we have demonstrated that LNC25-T/pDNA loaded with a hydrophobic drug, paclitaxel, showed high drug efficacy. The high transfection efficiency combined with the potential of simultaneous co-delivery of hydrophobic drugs, relatively small size of LNC25-T/pDNA complexes, and fluorescence imaging can be crucial for gene therapy, as small particle sizes may be more favorable for in vivo studies.The paper reports on the preparation of lipid nanocapsules (LNCs) functionalized with poly(ethyleneimine) (PEI) moieties and their successful use as drug and gene

  20. Structure-function analysis of ribonucleotide bypass by B family DNA replicases

    Clausen, Anders R.; Murray, Michael S.; Passer, Andrew R.; Pedersen, Lars C.; Kunkel, Thomas A. [NIH

    2013-11-01

    Ribonucleotides are frequently incorporated into DNA during replication, they are normally removed, and failure to remove them results in replication stress. This stress correlates with DNA polymerase (Pol) stalling during bypass of ribonucleotides in DNA templates. Here we demonstrate that stalling by yeast replicative Pols δ and ε increases as the number of consecutive template ribonucleotides increases from one to four. The homologous bacteriophage RB69 Pol also stalls during ribonucleotide bypass, with a pattern most similar to that of Pol ε. Crystal structures of an exonuclease-deficient variant of RB69 Pol corresponding to multiple steps in single ribonucleotide bypass reveal that increased stalling is associated with displacement of Tyr391 and an unpreferred C2´-endo conformation for the ribose. Even less efficient bypass of two consecutive ribonucleotides in DNA correlates with similar movements of Tyr391 and displacement of one of the ribonucleotides along with the primer-strand DNA backbone. These structure–function studies have implications for cellular signaling by ribonucleotides, and they may be relevant to replication stress in cells defective in ribonucleotide excision repair, including humans suffering from autoimmune disease associated with RNase H2 defects.

  1. Activation of different split functionalities on re-association of RNA-DNA hybrids

    Afonin, Kirill A.; Viard, Mathias; Martins, Angelica N.; Lockett, Stephen J.; Maciag, Anna E.; Freed, Eric O.; Heldman, Eliahu; Jaeger, Luc; Blumenthal, Robert; Shapiro, Bruce A.

    2013-04-01

    Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of `smart' nucleic acid-based nanoparticles and switches for various biomedical applications.

  2. A closed parameterization of DNA-damage by charged particles as a function of energy

    D, Frank Van den Heuvel Ph

    2013-01-01

    Purpose: To present a closed formalism calculating charged particle radiation damage induced in DNA, based on a simplified molecular model. The formalism is valid for all types of charged particles and due to its closed nature is suited to provide fast conversion of dose to DNA-damage. Methods: The induction of complex DNA--damaged is modelled using the standard scattering theory with a simplified effective potential. This leads to a proposal to use the Breit-Wigner expression to model the probability of the complex damage inelastic scatter as a function of kinetic energy of the scattered particle. A microscopic phenomenological Monte Carlo code is used to predict the damage to a DNA molecule embedded in a cell. The model is fit to the result of the simulation for four particles: electrons, protons, alpha--particles, and Carbon ions. The model is then used to predict the damage in a cell as a function of kinetic energy. Finally, a framework is proposed and implemented to provide data that can be assessed expe...

  3. Across-Platform Imputation of DNA Methylation Levels Incorporating Nonlocal Information Using Penalized Functional Regression

    Zhang, Guosheng; Huang, Kuan-Chieh; Xu, Zheng; Tzeng, Jung-Ying; Conneely, Karen N.; Guan, Weihua; Kang, Jian; Li, Yun

    2016-01-01

    DNA methylation is a key epigenetic mark involved in both normal development and disease progression. Recent advances in high-throughput technologies have enabled genome-wide profiling of DNA methylation. However, DNA methylation profiling often employs different designs and platforms with varying resolution, which hinders joint analysis of methylation data from multiple platforms. In this study, we propose a penalized functional regression model to impute missing methylation data. By incorporating functional predictors, our model utilizes information from nonlocal probes to improve imputation quality. Here, we compared the performance of our functional model to linear regression and the best single probe surrogate in real data and via simulations. Specifically, we applied different imputation approaches to an acute myeloid leukemia dataset consisting of 194 samples and our method showed higher imputation accuracy, manifested, for example, by a 94% relative increase in information content and up to 86% more CpG sites passing post-imputation filtering. Our simulated association study further demonstrated that our method substantially improves the statistical power to identify trait-associated methylation loci. These findings indicate that the penalized functional regression model is a convenient and valuable imputation tool for methylation data, and it can boost statistical power in downstream epigenome-wide association study (EWAS). PMID:27061717

  4. A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation.

    Fujimori, Hiroaki; Sato, Akira; Kikuhara, Sota; Wang, Junhui; Hirai, Takahisa; Sasaki, Yuka; Murakami, Yasufumi; Okayasu, Ryuichi; Masutani, Mitsuko

    2015-01-01

    A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1β-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX accumulation and showed a reduced level of γH2AX induction after γ-irradiation. DNMT3B interacted with HP1β in non-irradiated conditions, whereas irradiation abrogated the DNMT3B/HP1β complex but induced interaction between DNMT3B and H2AX. Consistent with radiosensitization, TP63, BAX, PUMA and NOXA expression was induced after γ-irradiation in DNMT3B knockdown cells. Together with the observation that H2AX overexpression canceled radiosensitization by DNMT3B RNAi, these results suggest that DNMT3B RNAi induced radiosensitization through impairment of damage-dependent HP1β foci formation and efficient γH2AX-induction mechanisms including H2AX accumulation. Enhanced radiosensitivity by DNMT3B RNAi was also observed in a tumor xenograft model. Taken together, the current study implies that comprehensive screening accompanied by a cluster analysis enabled the identification of radiosensitization targets. Downregulation of DNMT3B, one of the targets identified using this method, radiosensitizes cancer cells by disturbing multiple DNA damage responses. PMID:26667181

  5. Functional Expression of a DNA-Topoisomerase IB from Cryptosporidium parvum

    César Ordóñez; Javier Alfonso; Rafael Balaña-Fouce; David Ordóñez

    2009-01-01

    Cryptosporidium parvum, one of the most important causative organisms of human diarrheas during childhood, contains a monomeric DNA-topoisomerase IB (CpTopIB) in chromosome 7. Heterologous expression of CpTopIB gene in a budding yeast strain lacking this activity proves that the cryptosporidial enzyme is functional in vivo. The enzymatic activity is comprised in a single polypeptide, which contains all the structural features defining a fully active TopIB. Relaxation activity of the yeast ext...

  6. Modeling structure-function relationships in synthetic DNA sequences using attribute grammars.

    Yizhi Cai

    2009-10-01

    Full Text Available Recognizing that certain biological functions can be associated with specific DNA sequences has led various fields of biology to adopt the notion of the genetic part. This concept provides a finer level of granularity than the traditional notion of the gene. However, a method of formally relating how a set of parts relates to a function has not yet emerged. Synthetic biology both demands such a formalism and provides an ideal setting for testing hypotheses about relationships between DNA sequences and phenotypes beyond the gene-centric methods used in genetics. Attribute grammars are used in computer science to translate the text of a program source code into the computational operations it represents. By associating attributes with parts, modifying the value of these attributes using rules that describe the structure of DNA sequences, and using a multi-pass compilation process, it is possible to translate DNA sequences into molecular interaction network models. These capabilities are illustrated by simple example grammars expressing how gene expression rates are dependent upon single or multiple parts. The translation process is validated by systematically generating, translating, and simulating the phenotype of all the sequences in the design space generated by a small library of genetic parts. Attribute grammars represent a flexible framework connecting parts with models of biological function. They will be instrumental for building mathematical models of libraries of genetic constructs synthesized to characterize the function of genetic parts. This formalism is also expected to provide a solid foundation for the development of computer assisted design applications for synthetic biology.

  7. Mapping charge to function relationships of the DNA mimic protein Ocr

    Kanwar, Nisha

    2014-01-01

    This thesis investigates the functional consequences of neutralising the negative charges on the bacteriophage T7 antirestriction protein ocr. The ocr molecule is a small highly negatively charged, protein homodimer that mimics a short DNA duplex upon binding to the Type I Restriction Modification (RM) system. Thus, ocr facilitates phage infection by binding to and inactivating the host RM system. The aim of this study was to analyse the effect of reducing the negative charge o...

  8. Rad54 functions as a Heteroduplex DNA Pump Modulated by Its DNA Substrates and Rad51 during D-loop Formation in Homologous Recombination

    Wright, William Douglass; Heyer, Wolf-Dietrich

    2014-01-01

    The displacement loop (D-loop) is the product of homology search and DNA strand invasion, constituting a central intermediate in homologous recombination (HR). In eukaryotes, Rad51 recombinase is assisted in D-loop formation by the Rad54 motor protein. Curiously, Rad54 also disrupts D-loops. How these opposing activities are coordinated toward productive recombination is unknown. Moreover, a seemingly disparate function Rad54 is removal of Rad51 from heteroduplex DNA (hDNA) to allow HR-associ...

  9. Functional interactions and signaling properties of mammalian DNA mismatch repair proteins.

    Bellacosa, A

    2001-11-01

    The mismatch repair (MMR) system promotes genomic fidelity by repairing base-base mismatches, insertion-deletion loops and heterologies generated during DNA replication and recombination. This function is critically dependent on the assembling of multimeric complexes involved in mismatch recognition and signal transduction to downstream repair events. In addition, MMR proteins coordinate a complex network of physical and functional interactions that mediate other DNA transactions, such as transcription-coupled repair, base excision repair and recombination. MMR proteins are also involved in activation of cell cycle checkpoint and induction of apoptosis when DNA damage overwhelms a critical threshold. For this reason, they play a role in cell death by alkylating agents and other chemotherapeutic drugs, including cisplatin. Inactivation of MMR genes in hereditary and sporadic cancer is associated with a mutator phenotype and inhibition of apoptosis. In the future, a deeper understanding of the molecular mechanisms and functional interactions of MMR proteins will lead to the development of more effective cancer prevention and treatment strategies. PMID:11687886

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

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

    2004-01-01

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

  11. Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum

    Vijay Swahari

    2016-01-01

    Full Text Available Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells.

  12. Functionalized tetrapod-like ZnO nanostructures for plasmid DNA purification, polymerase chain reaction and delivery

    Functionalized tetrapodal ZnO nanostructures are tested in plasmid DNA experiments (1) as a solid-phase adsorbent for plasmid DNA purification (2) as improving reagents in a polymerase chain reaction (PCR) and (3) as novel carriers for gene delivery. The amino-modification, the tetrapod-like shape of the nanostructure and its high biocompatibility all contribute to measurements showing promise for applications. A sol-gel method is used for silica coating and amino-modification. Plasmid DNA is purified through reversible conjugations of amino-modified ZnO tetrapods with DNA. Also, as additional reagents, functionalized tetrapods are shown to improve the amount of PCR product. For transfection, ZnO tetrapods provide some protection against deoxyribonuclease cleavage of plasmid DNA and deliver plasmid DNA into cells with little cytotoxicity

  13. Powering the programmed nanostructure and function of gold nanoparticles with catenated DNA machines

    Elbaz, Johann; Cecconello, Alessandro; Fan, Zhiyuan; Govorov, Alexander O.; Willner, Itamar

    2013-01-01

    DNA nanotechnology is a rapidly developing research area in nanoscience. It includes the development of DNA machines, tailoring of DNA nanostructures, application of DNA nanostructures for computing, and more. Different DNA machines were reported in the past and DNA-guided assembly of nanoparticles represents an active research effort in DNA nanotechnology. Several DNA-dictated nanoparticle structures were reported, including a tetrahedron, a triangle or linear nanoengineered nanoparticle str...

  14. Clustering of DNA words and biological function: a proof of principle.

    Hackenberg, Michael; Rueda, Antonio; Carpena, Pedro; Bernaola-Galván, Pedro; Barturen, Guillermo; Oliver, José L

    2012-03-21

    Relevant words in literary texts (key words) are known to be clustered, while common words are randomly distributed. Given the clustered distribution of many functional genome elements, we hypothesize that the biological text per excellence, the DNA sequence, might behave in the same way: k-length words (k-mers) with a clear function may be spatially clustered along the one-dimensional chromosome sequence, while less-important, non-functional words may be randomly distributed. To explore this linguistic analogy, we calculate a clustering coefficient for each k-mer (k=2-9bp) in human and mouse chromosome sequences, then checking if clustered words are enriched in the functional part of the genome. First, we found a positive general trend relating clustering level and word enrichment within exons and Transcription Factor Binding Sites (TFBSs), while a much weaker relation exists for repeats, and no relation at all exists for introns. Second, we found that 38.45% of the 200 top-clustered 8-mers, but only 7.70% of the non-clustered words, are represented in known motif databases. Third, enrichment/depletion experiments show that highly clustered words are significantly enriched in exons and TFBSs, while they are depleted in introns and repetitive DNA. Considering exons and TFBSs together, 1417 (or 72.26%) in human and 1385 (or 72.97%) in mouse of the top-clustered 8-mers showed a statistically significant association to either exons or TFBSs, thus strongly supporting the link between word clustering and biological function. Lastly, we identified a subset of clustered, diagnostic words that are enriched in exons but depleted in introns, and therefore might help to discriminate between these two gene regions. The clustering of DNA words thus appears as a novel principle to detect functionality in genome sequences. As evolutionary conservation is not a prerequisite, the proof of principle described here may open new ways to detect species-specific functional DNA sequences

  15. Metal nanoparticles and DNA co-functionalized single-walled carbon nanotube gas sensors

    Metal/DNA/SWNT hybrid nanostructure-based gas sensor arrays were fabricated by means of ink jet printing of metal ion chelated DNA/SWNTs on microfabricated electrodes, followed by electroless deposition to reduce metal ions to metal. DNA served as a dispersing agent to effectively solubilize pristine SWNTs in water and as metal ion chelating centers for the formation of nanoparticles. Noble metals including palladium, platinum, and gold were used because the high binding affinity toward specific analytes enhances the selectivity and sensitivity. The sensitivity and selectivity of the gas sensors toward various gases such as H2, H2S, NH3, and NO2 were determined at room temperature. Sensing results indicated the enhancement of the sensitivity and selectivity toward certain analytes by functionalizing with different metal nanoparticles (e.g., Pd/DNA/SWNTs for H2 and H2S). The combined responses give a unique pattern or signature for each analyte by which the system can identify and quantify an individual gas. (paper)

  16. Metal nanoparticles and DNA co-functionalized single-walled carbon nanotube gas sensors

    Su, Heng C.; Zhang, Miluo; Bosze, Wayne; Lim, Jae-Hong; Myung, Nosang V.

    2013-12-01

    Metal/DNA/SWNT hybrid nanostructure-based gas sensor arrays were fabricated by means of ink jet printing of metal ion chelated DNA/SWNTs on microfabricated electrodes, followed by electroless deposition to reduce metal ions to metal. DNA served as a dispersing agent to effectively solubilize pristine SWNTs in water and as metal ion chelating centers for the formation of nanoparticles. Noble metals including palladium, platinum, and gold were used because the high binding affinity toward specific analytes enhances the selectivity and sensitivity. The sensitivity and selectivity of the gas sensors toward various gases such as H2, H2S, NH3, and NO2 were determined at room temperature. Sensing results indicated the enhancement of the sensitivity and selectivity toward certain analytes by functionalizing with different metal nanoparticles (e.g., Pd/DNA/SWNTs for H2 and H2S). The combined responses give a unique pattern or signature for each analyte by which the system can identify and quantify an individual gas.

  17. A combined DFT/Green’s function study on electrical conductivity through DNA duplex between Au electrodes

    Tsukamoto, Takayuki; Ishikawa, Yasuyuki; Sengoku, Yasuo; Kurita, Noriyuki

    2009-01-01

    Electrical conducting properties of DNA duplexes sandwiched between Au electrodes have been investigated by use of first-principles molecular simulation based on DFT and Green’s function to elucidate the origin of their base sequence dependence. The theoretically simulated effects of DNA base sequence on the electrical conducting properties are in qualitative agreement with experiment. The HOMOs localized on Guanine bases have the major contribution to the electrical conductivity through DNA ...

  18. A combined DFT/Green’s function study on electrical conductivity through DNA duplex between Au electrodes

    Tsukamoto, Takayuki; Ishikawa, Yasuyuki; Sengoku, Yasuo; Kurita, Noriyuki

    2009-06-01

    Electrical conducting properties of DNA duplexes sandwiched between Au electrodes have been investigated by use of first-principles molecular simulation based on DFT and Green's function to elucidate the origin of their base sequence dependence. The theoretically simulated effects of DNA base sequence on the electrical conducting properties are in qualitative agreement with experiment. The HOMOs localized on Guanine bases have the major contribution to the electrical conductivity through DNA duplexes.

  19. The effects of proliferation and DNA damage on hematopoietic stem cell function determine aging.

    Khurana, Satish

    2016-07-01

    In most of the mammalian tissues, homeostasis as well as injury repair depend upon a small number of resident adult stem cells. The decline in tissue/organ function in aged organisms has been directly linked with poorly functioning stem cells. Altered function of hematopoietic stem cells (HSCs) is at the center of an aging hematopoietic system, a tissue with high cellular turnover. Poorly engrafting, myeloid-biased HSCs with higher levels of DNA damage accumulation are the hallmark features of an aged hematopoietic system. These cells show a higher proliferation rate than their younger counterparts. It was proposed that quiescence of these cells over long period of time leads to accumulation of DNA damage, eventually resulting in poor function/pathological conditions in hematopoietic system. However, various mouse models with premature aging phenotype also show highly proliferative HSCs. This review examines the evidence that links proliferation of HSCs with aging, which leads to functional changes in the hematopoietic system. Developmental Dynamics 245:739-750, 2016. © 2016 Wiley Periodicals, Inc. PMID:26813236

  20. Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen.

    Hu, Xiaoyi V; Rodrigues, Tânia M A; Tao, Haiyan; Baker, Robert K; Miraglia, Loren; Orth, Anthony P; Lyons, Gary E; Schultz, Peter G; Wu, Xu

    2010-08-24

    DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells. PMID:20696907

  1. Towards understanding the evolution and functional diversification of DNA-containing plant organelles.

    Leister, Dario

    2016-01-01

    Plastids and mitochondria derive from prokaryotic symbionts that lost most of their genes after the establishment of endosymbiosis. In consequence, relatively few of the thousands of different proteins in these organelles are actually encoded there. Most are now specified by nuclear genes. The most direct way to reconstruct the evolutionary history of plastids and mitochondria is to sequence and analyze their relatively small genomes. However, understanding the functional diversification of these organelles requires the identification of their complete protein repertoires - which is the ultimate goal of organellar proteomics. In the meantime, judicious combination of proteomics-based data with analyses of nuclear genes that include interspecies comparisons and/or predictions of subcellular location is the method of choice. Such genome-wide approaches can now make use of the entire sequences of plant nuclear genomes that have emerged since 2000. Here I review the results of these attempts to reconstruct the evolution and functions of plant DNA-containing organelles, focusing in particular on data from nuclear genomes. In addition, I discuss proteomic approaches to the direct identification of organellar proteins and briefly refer to ongoing research on non-coding nuclear DNAs of organellar origin (specifically, nuclear mitochondrial DNA and nuclear plastid DNA). PMID:26998248

  2. Production of ROS and its effects on mitochondrial and nuclear DNA, human spermatozoa, and sperm function

    Hardi Darmawan

    2007-06-01

    Full Text Available Over the past few decades many researchers studying the causes of male infertility have recently focused on the role played by reactive oxygen species (ROS – highly reactive oxidizing agents belonging to the class of free radicals. If ROS levels rise, oxidative stress (OS occurs, which results in oxygen and oxygen derived oxidants, and in turn increases the rates of cellular damage. In human, ROS are produced by a variety of semen components, and antioxidants in the seminal fluid keep their level balance. Small amounts of ROS help spermatozoa acquire their necessary fertilizing capabilities. Many researches showed that ROS attack DNA integrity in the sperm nucleus by causing base modification, DNA strand breaks, and chromatin cross linking. The DNA damage induced excessive levels of ROS and might accelerate the process of germ cell apoptosis leading to a decline in sperm counts associated with male infertility. This paper will review the molecular (cellular origins of ROS in human semen, how ROS damage sperm nuclear DNA, and how such DNA damage contributes to male infertility. Increased ROS production by spermatozoa is associated with a decreased mitochondrial membrane potential (MMP, which is an important indicator of functional integrity of the spermatozoa. Germ cell apoptosis is essential for normal spermatogenesis and its dysregulation may lead to male infertility. Thus, understanding the causes and mechanisms of germ cell apoptosis is of major importance in preventing male reproductive problems. Levels of apoptosis in mature spermatozoa that were significantly correlated with levels of seminal ROS determined by chemiluminescence assay indicate the linkage between ROS and male fertility problems. (Med J Indones 2007; 16:127-33 Keywords: Apoptosis, infertility, free radicals

  3. Functionalized Nanostructures: Redox-Active Porphyrin Anchors for Supramolecular DNA Assemblies

    Börjesson, Karl

    2010-09-28

    We have synthesized and studied a supramolecular system comprising a 39-mer DNA with porphyrin-modified thymidine nucleosides anchored to the surface of large unilamellar vesicles (liposomes). Liposome porphyrin binding characteristics, such as orientation, strength, homogeneity, and binding site size, was determined, suggesting that the porphyrin is well suited as a photophysical and redox-active lipid anchor, in comparison to the inert cholesterol anchor commonly used today. Furthermore, the binding characteristics and hybridization capabilities were studied as a function of anchor size and number of anchoring points, properties that are of importance for our future plans to use the addressability of these redox-active nodes in larger DNA-based nanoconstructs. Electron transfer from photoexcited porphyrin to a lipophilic benzoquinone residing in the lipid membrane was characterized by steady-state and time-resolved fluorescence and verified by femtosecond transient absorption. © 2010 American Chemical Society.

  4. O6-methylguanine-DNA methyltransferase (MGMT): can function explain a suicidal mechanism?

    Gouws, Chrisna; Pretorius, Pieter J

    2011-11-01

    Why does O(6)-methylguanine-DNA methyltransferase (MGMT), an indispensable DNA repair enzyme, have a mechanism which seems to run counter to its importance? This enzyme is key to the removal of detrimental alkyl adducts from guanine bases. Although the mechanism is well known, an unusual feature surrounds its mode of action, which is its so-called suicidal endpoint. In addition, induction of MGMT is highly variable and its kinetics is atypical. These features raise some questions on the seemingly paradoxical mechanism. In this manuscript we point out that, although there is ample literature regarding the "how" of the MGMT enzyme, we found a lack of information on "why" this specific mechanism is in place. We then ask whether we know all there is to know about MGMT, or if perhaps there is a further as yet unknown function for MGMT, or if the suicidal mechanism may play some kind of protective role in the cell. PMID:21864987

  5. Antiviral immunity in fish – functional analysis using DNA vaccination as a tool

    Lorenzen, Niels; Lorenzen, Ellen; Einer-Jensen, Katja;

    2013-01-01

    cellular components both play a role in the long lasting protection. The similarity of the functional immune response profile to that induced by a natural virus infection is striking and is most likely one of the major reasons for the efficacy of the rhabdovirus DNA vaccines. Although other elements like...... fingerlings. Vaccination of fish at an early stage appears advantageous, since larger fish require higher doses of vaccine to be protected. Even in fish with an average size of 0.5 g at the time of vaccination, good protection can be obtained. Interestingly, immunity is established already a few days after...... CpG motifs in the plasmid backbone sequence might play a role, the viral G protein appears to have an inherent ability to stimulate innate immune mechanisms by receptors and pathways that still remain to be characterized in detail. Immunity to VHS in rainbow trout can be induced by DNA vaccination...

  6. In vitro transcription and translation inhibition via DNA functionalized gold nanoparticles

    Conde, J; Baptista, P V [Centro de Investigacao em Genetica Molecular Humana (CIGMH), Departamento de Ciencias da Vida, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); De la Fuente, J M, E-mail: pmvb@fct.unl.pt [Instituto de Nanociencia de Aragon, Universidad de Zaragoza, Pedro Cerbuna 12, 50009 Zaragoza (Spain)

    2010-12-17

    The use of gold nanoparticles (AuNPs) has been gaining momentum as vectors for gene silencing strategies, combining the AuNPs' ease of functionalization with DNA and/or siRNA, high loading capacity and fast uptake by target cells. Here, we used AuNP functionalized with thiolated oligonucleotides to specifically inhibit transcription in vitro, demonstrating the synergetic effect between AuNPs and a specific antisense sequence that blocks the T7 promoter region. Also, AuNPs efficiently protect the antisense oligonucleotide against nuclease degradation, which can thus retain its inhibitory potential. In addition, we demonstrate that AuNPs functionalized with a thiolated oligonucleotide complementary to the ribosome binding site and the start codon, effectively shut down in vitro translation. Together, these two approaches can provide for a simple yet robust experimental set up to test for efficient gene silencing of AuNP-DNA conjugates. What is more, these results show that appropriate functionalization of AuNPs can be used as a dual targeting approach to an enhanced control of gene expression-inhibition of both transcription and translation.

  7. Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

    Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin;

    2015-01-01

    infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique......-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from...... clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones...

  8. Functional homology between the sequence-specific DNA-binding proteins nuclear factor I from HeLa cells and the TGGCA protein from chicken liver.

    Leegwater, P.A.; van der Vliet, P C; Rupp, R A; Nowock, J; Sippel, A E

    1986-01-01

    Nuclear factor I from HeLa cells, a protein with enhancing function in adenovirus DNA replication, and the chicken TGGCA protein are specific DNA-binding proteins that were first detected by independent methods and that appeared to have similar DNA sequence specificity. To test whether they are homologous proteins from different species we have compared (i) their DNA binding properties and (ii) their function in reconstituted adenovirus DNA replication systems. Using deletion and substitution...

  9. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

    Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

    2009-05-11

    The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

  10. First-principles photoemission spectroscopy of DNA and RNA nucleobases from Koopmans-compliant functionals

    Nguyen, Ngoc Linh; Ferretti, Andrea; Marzari, Nicola

    2016-01-01

    The need to interpret ultraviolet photoemission data strongly motivates the refinement of first-principles techniques able to accurately predict spectral properties. In this work we employ Koopmans-compliant functionals, constructed to enforce piecewise linearity in approximate density functionals, to calculate the structural and electronic properties of DNA and RNA nucleobases. Our results show that not only ionization potentials and electron affinities are accurately predicted with mean absolute errors < 0.1 eV, but also that calculated photoemission spectra are in excellent agreement with experimental ultraviolet photoemission spectra. In particular, the role and contribution of different tautomers to the photoemission spectra are highlighted and discussed in detail. The structural properties of nucleobases are also investigated, showing an improved description with respect to local and semilocal density-functional theory. Methodologically, our results further consolidate the role of Koopmans-compliant ...

  11. Auditory analysis of xeroderma pigmentosum 1971–2012: hearing function, sun sensitivity and DNA repair predict neurological degeneration

    Totonchy, Mariam B.; Tamura, Deborah; Pantell, Matthew S.; Zalewski, Christopher; Bradford, Porcia T.; Merchant, Saumil N.; Nadol, Joseph; Khan, Sikandar G.; Schiffmann, Raphael; Pierson, Tyler Mark; Wiggs, Edythe; Griffith, Andrew J.; DiGiovanna, John J.; Kraemer, Kenneth H.; Brewer, Carmen C.

    2013-01-01

    To assess the role of DNA repair in maintenance of hearing function and neurological integrity, we examined hearing status, neurological function, DNA repair complementation group and history of acute burning on minimal sun exposure in all patients with xeroderma pigmentosum, who had at least one complete audiogram, examined at the National Institutes of Health from 1971 to 2012. Seventy-nine patients, aged 1–61 years, were diagnosed with xeroderma pigmentosum (n = 77) or xeroderma pigmentosu...

  12. SETD2 loss-of-function promotes renal cancer branched evolution through replication stress and impaired DNA repair

    Kanu, N.; Grönroos, E.; Martinez, P.;

    2015-01-01

    proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal......, suppression of replication stress and the coordination of DNA repair.......-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication...

  13. Structure-function analysis indicates that sumoylation modulates DNA-binding activity of STAT1

    Grönholm Juha

    2012-10-01

    Full Text Available Abstract Background STAT1 is an essential transcription factor for interferon-γ-mediated gene responses. A distinct sumoylation consensus site (ψKxE 702IKTE705 is localized in the C-terminal region of STAT1, where Lys703 is a target for PIAS-induced SUMO modification. Several studies indicate that sumoylation has an inhibitory role on STAT1-mediated gene expression but the molecular mechanisms are not fully understood. Results Here, we have performed a structural and functional analysis of sumoylation in STAT1. We show that deconjugation of SUMO by SENP1 enhances the transcriptional activity of STAT1, confirming a negative regulatory effect of sumoylation on STAT1 activity. Inspection of molecular model indicated that consensus site is well exposed to SUMO-conjugation in STAT1 homodimer and that the conjugated SUMO moiety is directed towards DNA, thus able to form a sterical hindrance affecting promoter binding of dimeric STAT1. In addition, oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant has higher DNA-binding activity on STAT1 responsive gene promoters than wild-type STAT1. Furthermore, sumoylation deficient STAT1 E705Q mutant displayed enhanced histone H4 acetylation on interferon-γ-responsive promoter compared to wild-type STAT1. Conclusions Our results suggest that sumoylation participates in regulation of STAT1 responses by modulating DNA-binding properties of STAT1.

  14. Bidirectional gene sequences with similar homology to functional proteins of alkane degrading bacterium pseudomonas fredriksbergensis DNA

    The potential for two overlapping fragments of DNA from a clone of newly isolated alkanes degrading bacterium Pseudomonas frederiksbergensis encoding sequences with similar homology to two parts of functional proteins is described. One strand contains a sequence with high homology to alkanes monooxygenase (alkB), a member of the alkanes hydroxylase family, and the other strand contains a sequence with some homology to alcohol dehydrogenase gene (alkJ). Overlapping of the genes on opposite strands has been reported in eukaryotic species, and is now reported in a bacterial species. The sequence comparisons and ORFS results revealed that the regulation and the genes organization involved in alkane oxidation represented in Pseudomonas frederiksberghensis varies among the different known alkane degrading bacteria. The alk gene cluster containing homologues to the known alkane monooxygenase (alkB), and rubredoxin (alkG) are oriented in the same direction, whereas alcohol dehydrogenase (alkJ) is oriented in the opposite direction. Such genomes encode messages on both strands of the DNA, or in an overlapping but different reading frames, of the same strand of DNA. The possibility of creating novel genes from pre-existing sequences, known as overprinting, which is a widespread phenomenon in small viruses. Here, the origin and evolution of the gene overlap to bacteriophages belonging to the family Microviridae have been investigated. Such a phenomenon is most widely described in extremely small genomes such as those of viruses or small plasmids, yet here is a unique phenomenon. (author)

  15. Ionization and fragmentation of DNA-RNA bases: a density functional theory study

    Ionizing radiation (IR) cross human tissue, deposit energy and dissipate fragmenting molecules. The resulting fragments may be highlighted by mass spectrometry. Despite the amount of information obtained experimentally by the interpretation of the mass spectrum, experience alone cannot answer all the questions of the mechanism of fragmentation of DNA/RNA bases and a theoretical study is a complement to this information. A theoretical study allows us to know the weakest bonds in the molecule during ionization and thus may help to provide mechanisms of dissociation and produced fragments. The purpose of this work, using the DFT with the PBE functional, is to study the ionization and fragmentation mechanisms of DNA/RNA bases (Uracil, Cytosine, Adenine and Guanine) and to identify the cations corresponding to each peak in mass spectra. For all RNA bases, the retro Diels-Alder reaction (elimination of HNCO or NCO*) is a major route for dissociating, with the exception of adenine for which there is no atom oxygen in its structure. Loss of NH3 (NH2*) molecule is another common way to all bases that contain amine group. The possibility of the loss of hydrogen from the cations is also investigated, as well as the dissociation of dehydrogenated cations and protonated uracil. This work shows the interest of providing DFT calculation in the interpretation of mass spectra of DNA bases. (author)

  16. Preparation of functional spherical polysilsesquioxane/gold nanoparticle composites and their applications in DNA assay

    Functional spherical solid and hollow particles of polysilsesquioxanes (PSQs) containing amine, thiol, and vinyl groups were prepared by polymerizing organotrialkoxysilanes (OTASs) containing corresponding chemical groups. Fluorescent PSQ particles were prepared by physically entrapping Rhodamine 6G, Coumarin 7, and Fluoresceine sodium salts. The intensity of fluorescent light increased initially with increasing amount of entrapped fluorophores and then leveled off or decreased slightly after reaching a maximum value. PSQ particles containing gold nanoparticles (GNPs), both inside and on the surface, were prepared by the in situ reduction of gold ions by the PSQ particles. When the reduction reaction was carried out for extended periods of time, the GNP that had formed inside the poly(3-mercaptopropyl)silsesquioxane (PMPSQ) and polyvinylsilsesequioxane (PVSQ) particles underwent interesting morphological changes. PSQ particles containing amine and thiol groups fixed the GNPs on the surface, which could be utilized further in binding amine-modified oligo-DNA strands. The aggregation of PSQ/GNP particles combined with complementary oligo-DNA strands was examined to demonstrate that these particles could be applied to DNA assays and isolation. The particles were characterized by scanning electron microscopy, transmission electron microscopy, solid state nuclear magnetic resonance spectroscopy, ultraviolet/visible spectroscopy, and fluorescence microscopy.

  17. Repetitive Sequences in Plant Nuclear DNA:Types, Distribution, Evolution and Function

    Shweta Mehrotra; Vinod Goyal

    2014-01-01

    Repetitive DNA sequences are a major component of eukaryotic genomes and may account for up to 90% of the genome size. They can be divided into minisatellite, microsatellite and satellite sequences. Satellite DNA sequences are considered to be a fast-evolving component of eukaryotic genomes, comprising tandemly-arrayed, highly-repetitive and highly-conserved monomer sequences. The monomer unit of satellite DNA is 150-400 base pairs (bp) in length. Repetitive sequences may be species- or genus-specific, and may be centromeric or subtelomeric in nature. They exhibit cohesive and concerted evolution caused by molecular drive, leading to high sequence homogeneity. Repetitive sequences accumulate variations in sequence and copy number during evolution, hence they are important tools for taxonomic and phylogenetic studies, and are known as‘‘tuning knobs’’ in the evolution. Therefore, knowledge of repetitive sequences assists our understanding of the organization, evolution and behavior of eukaryotic genomes. Repetitive sequences have cytoplasmic, cellular and developmental effects and play a role in chromosomal recombination. In the post-genomics era, with the introduction of next-generation sequencing tech-nology, it is possible to evaluate complex genomes for analyzing repetitive sequences and decipher-ing the yet unknown functional potential of repetitive sequences.

  18. Functional importance of the DNA binding activity of Candida albicans Czf1p.

    Ivana Petrovska

    Full Text Available The human opportunistic pathogen Candida albicans undergoes a reversible morphological transition between the yeast and hyphal states in response to a variety of signals. One such environmental trigger is growth within a semisolid matrix such as agar medium. This growth condition is of interest because it may mimic the growth of C. albicans in contact with host tissue during infection. During growth within a semisolid matrix, hyphal growth is positively regulated by the transcriptional regulator Czf1p and negatively by a second key transcriptional regulator, Efg1p. Genetic studies indicate that Czf1p, a member of the zinc-cluster family of transcriptional regulators, exerts its function by opposing the inhibitory influence of Efg1p on matrix-induced filamentous growth. We examined the importance of the two known activities of Czf1p, DNA-binding and interaction with Efg1p. We found that the two activities were separable by mutation allowing us to demonstrate that the DNA-binding activity of Czf1p was essential for its role as a positive regulator of morphogenesis. Surprisingly, however, interactions with Efg1p appeared to be largely dispensable. Our studies provide the first evidence of a key role for the DNA-binding activity of Czf1p in the morphological yeast-to-hyphal transition triggered by matrix-embedded growth.

  19. Functional interfaces for biomimetic energy harvesting: CNTs-DNA matrix for enzyme assembly.

    Hjelm, Rachel M E; Garcia, Kristen E; Babanova, Sofia; Artyushkova, Kateryna; Matanovic, Ivana; Banta, Scott; Atanassov, Plamen

    2016-05-01

    The development of 3D structures exploring the properties of nano-materials and biological molecules has been shown through the years as an effective path forward for the design of advanced bio-nano architectures for enzymatic fuel cells, photo-bio energy harvesting devices, nano-biosensors and bio-actuators and other bio-nano-interfacial architectures. In this study we demonstrate a scaffold design utilizing carbon nanotubes, deoxyribose nucleic acid (DNA) and a specific DNA binding transcription factor that allows for directed immobilization of a single enzyme. Functionalized carbon nanotubes were covalently bonded to a diazonium salt modified gold surface through carbodiimide chemistry creating a brush-type nanotube alignment. The aligned nanotubes created a highly ordered structure with high surface area that allowed for the attachment of a protein assembly through a designed DNA scaffold. The enzyme immobilization was controlled by a zinc finger (ZNF) protein domain that binds to a specific dsDNA sequence. ZNF 268 was genetically fused to the small laccase (SLAC) from Streptomyces coelicolor, an enzyme belonging to the family of multi-copper oxidases, and used to demonstrate the applicability of the developed approach. Analytical techniques such as X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and enzymatic activity analysis, allowed characterization at each stage of development of the bio-nano architecture. This article is part of a Special Issue entitled Biodesign for Bioenergetics - the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. PMID:26751397

  20. Activation of different split functionalities on re-association of RNA-DNA hybrids.

    Afonin, Kirill A; Viard, Mathias; Martins, Angelica N; Lockett, Stephen J; Maciag, Anna E; Freed, Eric O; Heldman, Eliahu; Jaeger, Luc; Blumenthal, Robert; Shapiro, Bruce A

    2013-04-01

    Split-protein systems, an approach that relies on fragmentation of proteins with their further conditional re-association to form functional complexes, are increasingly used for various biomedical applications. This approach offers tight control of protein functions and improved detection sensitivity. Here we report a similar technique based on a pair of RNA-DNA hybrids that can be used generally for triggering different split functionalities. Individually, each hybrid is inactive but when two cognate hybrids re-associate, different functionalities are triggered inside mammalian cells. As a proof of concept, this work mainly focuses on the activation of RNA interference. However, the release of other functionalities (such as resonance energy transfer and RNA aptamer) is also shown. Furthermore, in vivo studies demonstrate a significant uptake of the hybrids by tumours together with specific gene silencing. This split-functionality approach presents a new route in the development of 'smart' nucleic acid-based nanoparticles and switches for various biomedical applications. PMID:23542902

  1. Search for DNA conformational features for functional sites. Investigation of the TATA box

    Ponomarenko, M.P.; Ponomarenko, J.V.; Kel, A.E.; Kolchanov, N.A. [Institute of Cytology and Genetics, Novosibirsk (Russian Federation)

    1996-12-31

    A method for searching for DNA conformational features significant for functional sites is developed. The method uses helical angles averaged for known X-ray structures. Nucleotide sequences are assigned mean angles in a given region. Choice of the significant angles is based on their capabilities to discriminate functional sites from random sequences. The yeast, invertebrate, and vertebrate TATA boxes are analyzed using this method. Regions neighboring the TATA boxes are found to have smaller helical twist and roll angles. The results agree with the experimental data on Dickerson-Drew dodecamers. There is a significant decrease in the length of a small roll angle region with increasing complexity of taxon organization. 28 refs., 3 figs., 3 tabs.

  2. Amino-functionalized DNA: the properties of C5-amino-alkyl substituted 2′-deoxyuridines and their application in DNA triplex formation

    Brazier, John A.; Shibata, Takayuki; Townsley, John; Taylor, Brian F.; Frary, Elaine; Williams, Nicholas H.; Williams, David M.

    2005-01-01

    The incorporation of C5-amino-modified 2′-deoxyuridine analogues into DNA have found application in nucleic acid labelling, the stabilization of nucleic acid structures, functionalization of nucleic acid aptamers and catalysts, and the investigation of sequence-specific DNA bending. In this study, we describe the physicochemical properties of four different C5-amino-modified 2′-deoxyuridines in which the amino group is tethered to the base via a 3-carbon alkyl, Z- or E-alkenyl or alkynyl link...

  3. Methamidophos alters sperm function and DNA at different stages of spermatogenesis in mice

    Urióstegui-Acosta, Mayrut; Hernández-Ochoa, Isabel [Departamento de Toxicología, CINVESTAV-IPN, D.F. (Mexico); Sánchez-Gutiérrez, Manuel [Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Hidalgo (Mexico); Piña-Guzmán, Belem [Instituto Politécnico Nacional-UPIBI, D.F. (Mexico); Rafael-Vázquez, Leticia; Solís-Heredia, M.J.; Martínez-Aguilar, Gerardo [Departamento de Toxicología, CINVESTAV-IPN, D.F. (Mexico); Quintanilla-Vega, Betzabet, E-mail: mquintan@cinvestav.mx [Departamento de Toxicología, CINVESTAV-IPN, D.F. (Mexico)

    2014-09-15

    Methamidophos (MET) is a highly toxic organophosphate (OP) pesticide that is widely used in developing countries. MET has male reproductive effects, including decreased fertility. We evaluated MET effects on sperm quality, fertilization and DNA integrity, exploring the sensitivity of different stages of spermatogenesis. Adult male mice received MET (3.75 or 5 mg/kg-bw/ip/day/4 days) and were euthanized 1, 28 or 45 days post-treatment (dpt) to evaluate MET's effects on epididymal maturation, meiosis or mitosis, respectively. Spermatozoa were obtained from the cauda epididymis–vas deferens and were evaluated for sperm quality, acrosome reaction (AR; Coomassie staining), mitochondrial membrane potential (by JC-1), DNA damage (comet assay), oxidative damage (malondialdehyde (MDA) production), in vitro fertilization and protein phosphorylation (immunodetection), and erythrocyte acetylcholinesterase (AChE) activity. At 1-dpt, MET inhibited AChE (43–57%) and increased abnormal cells (6%). While at 28- and 45-dpt, sperm motility and viability were significantly reduced with an increasing MET dose, and abnormal morphology increased at 5 mg/kg/day/4 days. MDA and mitochondrial activity were not affected at any dose or time. DNA damage (OTM and %DNA) was observed at 5 mg/kg/day/4 days in a time-dependent manner, whereas both parameters were altered in cells from mice exposed to 3.75 mg/kg/day/4 days only at 28-dpt. Depending on the time of collection, initial-, spontaneous- and induced-AR were altered at 5 mg/kg/day/4 days, and the fertilization capacity also decreased. Sperm phosphorylation (at serine and tyrosine residues) was observed at all time points. Data suggest that meiosis and mitosis are the more sensitive stages of spermatogenesis for MET reproductive toxicity compared to epididymal maturation. - Highlights: • Methamidophos alters sperm cell function at different stages of spermatogenesis. • Testicular stages of spermatogenesis are more sensitive to

  4. Methamidophos alters sperm function and DNA at different stages of spermatogenesis in mice

    Methamidophos (MET) is a highly toxic organophosphate (OP) pesticide that is widely used in developing countries. MET has male reproductive effects, including decreased fertility. We evaluated MET effects on sperm quality, fertilization and DNA integrity, exploring the sensitivity of different stages of spermatogenesis. Adult male mice received MET (3.75 or 5 mg/kg-bw/ip/day/4 days) and were euthanized 1, 28 or 45 days post-treatment (dpt) to evaluate MET's effects on epididymal maturation, meiosis or mitosis, respectively. Spermatozoa were obtained from the cauda epididymis–vas deferens and were evaluated for sperm quality, acrosome reaction (AR; Coomassie staining), mitochondrial membrane potential (by JC-1), DNA damage (comet assay), oxidative damage (malondialdehyde (MDA) production), in vitro fertilization and protein phosphorylation (immunodetection), and erythrocyte acetylcholinesterase (AChE) activity. At 1-dpt, MET inhibited AChE (43–57%) and increased abnormal cells (6%). While at 28- and 45-dpt, sperm motility and viability were significantly reduced with an increasing MET dose, and abnormal morphology increased at 5 mg/kg/day/4 days. MDA and mitochondrial activity were not affected at any dose or time. DNA damage (OTM and %DNA) was observed at 5 mg/kg/day/4 days in a time-dependent manner, whereas both parameters were altered in cells from mice exposed to 3.75 mg/kg/day/4 days only at 28-dpt. Depending on the time of collection, initial-, spontaneous- and induced-AR were altered at 5 mg/kg/day/4 days, and the fertilization capacity also decreased. Sperm phosphorylation (at serine and tyrosine residues) was observed at all time points. Data suggest that meiosis and mitosis are the more sensitive stages of spermatogenesis for MET reproductive toxicity compared to epididymal maturation. - Highlights: • Methamidophos alters sperm cell function at different stages of spermatogenesis. • Testicular stages of spermatogenesis are more sensitive to

  5. DNA supercoiling inhibits DNA knotting.

    Burnier Y.; Dorier J.; Stasiak A.

    2008-01-01

    Despite the fact that in living cells DNA molecules are long and highly crowded, they are rarely knotted. DNA knotting interferes with the normal functioning of the DNA and, therefore, molecular mechanisms evolved that maintain the knotting and catenation level below that which would be achieved if the DNA segments could pass randomly through each other. Biochemical experiments with torsionally relaxed DNA demonstrated earlier that type II DNA topoisomerases that permit inter- and intramolecu...

  6. Structural and functional studies of a large winged Z-DNA-binding domain of Danio rerio protein kinase PKZ.

    Subramani, Vinod Kumar; Kim, Doyoun; Yun, Kyunghee; Kim, Kyeong Kyu

    2016-07-01

    The Z-DNA-binding domain of PKZ from zebrafish (Danio rerio; drZαPKZ ) contains the largest β-wing among known Z-DNA-binding domains. To elucidate the functional implication of the β-wing, we solved the crystal structure of apo-drZαPKZ . Structural comparison with its Z-DNA-bound form revealed a large conformational change within the β-wing during Z-DNA binding. Biochemical studies of protein mutants revealed that two basic residues in the β-wing are responsible for Z-DNA recognition as well as fast B-Z transition. Therefore, the extra basic residues in the β-wing of drZαPKZ are necessary for the fast B-Z transition activity. PMID:27265117

  7. DNA barcoding of life: a classification of uses according to function and scale after ten years of development

    Nancai Pei

    2013-09-01

    Full Text Available DNA barcoding technology provides molecular information, standard dataset platforms, and universal technical regulations for modern biological research. We briefly review the history of DNA barcoding between 2003 and 2012, and classify DNA barcoding into three types of biological function: basic function (e.g., storing data, and identifying species, extending function (e.g., building phylogenies, serving specific subjects, and compiling biological atlas and potential function (e.g., revealing cryptic species. We sort DNA barcoding studies at three levels: clade scale (e.g., familial and/or generic taxa, community scale (e.g., biotic communities in nature reserves and permanent forest dynamics plots, and regional scale (e.g., biodiversity hotpots. We further list ten major research programs proposed by the International Barcode of Life, which are related to DNA barcoding approaches from the prospective of systematics and taxonomy, biodiversity conservation, evolutionary ecology and phylogenetics, and the construction of digital platforms. We appreciate the huge capability of barcoding technology in the field of biological sciences, and also realize the challenges of DNA barcoding utilizations in multidisciplinary studies and the essential to add more tests before the large-scale applications.

  8. Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

    Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan; Dorsett, Yair; Bredemeyer, Andrea; George, Rosmy; Callen, Elsa; Daniel, Jeremy A; Osipovich, Oleg; Oltz, Eugene M; Bassing, Craig H; Nussenzweig, Andre; Lees-Miller, Susan; Hammel, Michal; Chen, Benjamin P C; Sleckman, Barry P

    2013-01-01

    V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are...... serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA...... when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA...

  9. DNA methylation status of nuclear-encoded mitochondrial genes underlies the tissue-dependent mitochondrial functions

    Takasugi Masaki; Yagi Shintaro; Hirabayashi Keiji; Shiota Kunio

    2010-01-01

    Abstract Background Mitochondria are semi-autonomous, semi-self-replicating organelles harboring their own DNA (mitochondrial DNA, mtDNA), and their dysregulation is involved in the development of various diseases. While mtDNA does not generally undergo epigenetic modifications, almost all mitochondrial proteins are encoded by nuclear DNA. However, the epigenetic regulation of nuclear-encoded mitochondrial genes (nuclear mt genes) has not been comprehensively analyzed. Results We analyzed the...

  10. Electrochemical functionalization of polypyrrole through amine oxidation of poly(amidoamine) dendrimers: Application to DNA biosensor.

    Miodek, Anna; Mejri-Omrani, Nawel; Khoder, Rabih; Korri-Youssoufi, Hafsa

    2016-07-01

    Electrochemical patterning method has been developed to fabricate composite based on polypyrrole (PPy) film and poly(amidoamine) dendrimers of fourth generation (PAMAM G4). PPy layer was generated using electrochemical polymerization of pyrrole on a gold electrode. PPy film was then modified with PAMAM G4 using amines electro-oxidation method. Covalent bonding of PAMAM G4 and the formation of PPy-PAMAM composite was characterized using Fourier Transform Infrared Spectroscopy (FT-IR) and X-ray Photoelectron Spectroscopy (XPS). Ferrocenyl groups were then attached to such surface as a redox marker. Electrochemical properties of the modified nanomaterial (PPy-PAMAM-Fc) were studied using both amperometric and impedimetric methods to demonstrate the efficiency of electron transfer through the modified PPy layer. The obtained electrical and electrochemical properties were compared to a composite where PPy bearing carboxylic acid functions was chemically modified with PAMAM G4 by covalent attachment through formation of amid bond (PPy-CONH-PAMAM). The above mentioned studies showed that electrochemical patterning does not disturb the electronic properties of PPy. The effect of the number of functional groups introduced by the electrochemical patterning was demonstrated through the association of various compounds (ethylenediamine, PAMAM G2 and PAMAM G6). We demonstrated that such compounds could be applied in the biosensors technology. The modified PPy-PAMAM-Fc was evaluated as a platform for DNA sensing. High performance in the DNA detection by variation of the electrochemical signal of ferrocene was obtained with detection limit of 0.4 fM. Furthermore, such approach of electrochemical patterning by oxidation of amines could be applied for chemical modification of PPy and open a new way in various biosensing application involving functionalized PPy. PMID:27154698

  11. A Novel Aspect of Tumorigenesis-BMI1 Functions in Regulating DNA Damage Response.

    Lin, Xiaozeng; Ojo, Diane; Wei, Fengxiang; Wong, Nicholas; Gu, Yan; Tang, Damu

    2015-01-01

    BMI1 plays critical roles in maintaining the self-renewal of hematopoietic, neural, intestinal stem cells, and cancer stem cells (CSCs) for a variety of cancer types. BMI1 promotes cell proliferative life span and epithelial to mesenchymal transition (EMT). Upregulation of BMI1 occurs in multiple cancer types and is associated with poor prognosis. Mechanistically, BMI1 is a subunit of the Polycomb repressive complex 1 (PRC1), and binds the catalytic RING2/RING1b subunit to form a functional E3 ubiquitin ligase. Through mono-ubiquitination of histone H2A at lysine 119 (H2A-K119Ub), BMI1 represses multiple gene loci; among these, the INK4A/ARF locus has been most thoroughly investigated. The locus encodes the p16INK4A and p14/p19ARF tumor suppressors that function in the pRb and p53 pathways, respectively. Its repression contributes to BMI1-derived tumorigenesis. BMI1 also possesses other oncogenic functions, specifically its regulative role in DNA damage response (DDR). In this process, BMI1 ubiquitinates histone H2A and γH2AX, thereby facilitating the repair of double-stranded DNA breaks (DSBs) through stimulating homologous recombination and non-homologous end joining. Additionally, BMI1 compromises DSB-induced checkpoint activation independent of its-associated E3 ubiquitin ligase activity. We review the emerging role of BMI1 in DDR regulation and discuss its impact on BMI1-derived tumorigenesis. PMID:26633535

  12. A Novel Aspect of Tumorigenesis—BMI1 Functions in Regulating DNA Damage Response

    Xiaozeng Lin

    2015-12-01

    Full Text Available BMI1 plays critical roles in maintaining the self-renewal of hematopoietic, neural, intestinal stem cells, and cancer stem cells (CSCs for a variety of cancer types. BMI1 promotes cell proliferative life span and epithelial to mesenchymal transition (EMT. Upregulation of BMI1 occurs in multiple cancer types and is associated with poor prognosis. Mechanistically, BMI1 is a subunit of the Polycomb repressive complex 1 (PRC1, and binds the catalytic RING2/RING1b subunit to form a functional E3 ubiquitin ligase. Through mono-ubiquitination of histone H2A at lysine 119 (H2A-K119Ub, BMI1 represses multiple gene loci; among these, the INK4A/ARF locus has been most thoroughly investigated. The locus encodes the p16INK4A and p14/p19ARF tumor suppressors that function in the pRb and p53 pathways, respectively. Its repression contributes to BMI1-derived tumorigenesis. BMI1 also possesses other oncogenic functions, specifically its regulative role in DNA damage response (DDR. In this process, BMI1 ubiquitinates histone H2A and γH2AX, thereby facilitating the repair of double-stranded DNA breaks (DSBs through stimulating homologous recombination and non-homologous end joining. Additionally, BMI1 compromises DSB-induced checkpoint activation independent of its-associated E3 ubiquitin ligase activity. We review the emerging role of BMI1 in DDR regulation and discuss its impact on BMI1-derived tumorigenesis.

  13. First-principles photoemission spectroscopy in DNA and RNA nucleobases from Koopmans-compliant functionals

    Nguyen, Ngoc Linh; Borghi, Giovanni; Ferretti, Andrea; Marzari, Nicola

    The determination of spectral properties of the DNA and RNA nucleobases from first principles can provide theoretical interpretation for experimental data, but requires complex electronic-structure formulations that fall outside the domain of applicability of common approaches such as density-functional theory. In this work, we show that Koopmans-compliant functionals, constructed to enforce piecewise linearity in energy functionals with respect to fractional occupation-i.e., with respect to charged excitations-can predict not only frontier ionization potentials and electron affinities of the nucleobases with accuracy comparable or superior with that of many-body perturbation theory and high-accuracy quantum chemistry methods, but also the molecular photoemission spectra are shown to be in excellent agreement with experimental ultraviolet photoemsision spectroscopy data. The results highlight the role of Koopmans-compliant functionals as accurate and inexpensive quasiparticle approximations to the spectral potential, which transform DFT into a novel dynamical formalism where electronic properties, and not only total energies, can be correctly accounted for.

  14. The Ku70 DNA-repair protein is involved in centromere function in a grasshopper species.

    Cabrero, Josefa; Bakkali, Mohammed; Navarro-Domínguez, Beatriz; Ruíz-Ruano, Francisco J; Martín-Blázquez, Rubén; López-León, María Dolores; Camacho, Juan Pedro M

    2013-06-25

    The Ku70 protein is involved in numerous cell functions, the nonhomologous end joining (NHEJ) DNA repair pathway being the best known. Here, we report a novel function for this protein in the grasshopper Eyprepocnemis plorans. We observed the presence of large Ku70 foci on the centromeres of meiotic and mitotic chromosomes during the cell cycle stages showing the highest centromeric activity (i.e., metaphase and anaphase). The fact that colchicine treatment prevented centromeric location of Ku70, suggests a microtubule-dependent centromeric function for Ku70. Likewise, the absence of Ku70 at metaphase-anaphase centromeres from three males whose Ku70 gene had been knocked down using interference RNA, and the dramatic increase in the frequency of polyploid spermatids observed in these males, suggest that the centromeric presence of Ku70 is required for normal cytokinesis in this species. The centromeric function of Ku70 was not observed in 14 other grasshopper and locust species, or in the mouse, thus suggesting that it is an autapomorphy in E. plorans. PMID:23797468

  15. Structure and Function of the PriC DNA Replication Restart Protein.

    Wessel, Sarah R; Cornilescu, Claudia C; Cornilescu, Gabriel; Metz, Alice; Leroux, Maxime; Hu, Kaifeng; Sandler, Steven J; Markley, John L; Keck, James L

    2016-08-26

    Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the single-stranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii PriC forms a compact bundle of α-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB·DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB·DnaC to SSB/ssDNA sites present at stalled replication forks. PMID:27382050

  16. Integral parametrization of the Kinetics of Crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

    In this paper we present results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8-MOP) concentration. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. the experimental results showed a three-stage profile when represented as a function of the natural logarithms of 8-MOP concentration. an integral parametrization which allows a simultaneous fit of the three observed stages is presented here. the theoretical values of crosslink production determined from the fit are useful to asses the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (Author) 24 refs

  17. Functional relevance of the histone γH2Ax in the response to DNA damaging agents

    Revet, Ingrid; Feeney, Luzviminda; Bruguera, Stephanie; Wilson, Wade; Dong, Tiffany K.; Oh, Dennis H.; Dankort, David; Cleaver, James E.

    2011-01-01

    The phosphorylation of H2Ax on its S139 site, γH2Ax, is important during DNA double-strand repair and is considered necessary for assembly of repair complexes, but its functional role after other kinds of DNA damage is less clear. We have measured the survival of isogenic mouse cell lines with the H2Ax gene knocked out, and replaced with wild-type or mutant (S139A) H2Ax genes, exposed to a range of agents with varied mechanisms of DNA damage. Knockout and mutant cells were sensitive to γ-rays...

  18. Integral parametrization of the Kinetics of Crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

    Vidania, R. de; Paramio, J. M.; Bauluz, C.

    1986-07-01

    In this paper we present results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8-MOP) concentration. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. the experimental results showed a three-stage profile when represented as a function of the natural logarithms of 8-MOP concentration. an integral parametrization which allows a simultaneous fit of the three observed stages is presented here. the theoretical values of crosslink production determined from the fit are useful to asses the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (Author) 24 refs.

  19. Integral parametrization of the kinetics of crosslink production in plasmid DNA as a function of 8-methoxypsoralen concentration

    The results of crosslink production in pBR322 DNA along a wide range of 8-methoxypsoralen (8 MOP) concentration are presented. Experimental data were obtained as DNA renaturation percentages, from the shift in hyperchromicity after a temperature-dependent denaturation-renaturation process. The experimental results showed a three-stage profile when represented as a function of the natural logarithm of 8-MOP concentration. An integral parametrization which allows a simultaneous fit of the three observed stages is presented here. The theoretical values of crosslink production determined from the fit are useful to assess the genotoxicity of psoralen-induced crosslinks in plasmid DNA. (author)

  20. The DnaJ domain of polyomavirus large T antigen is required to regulate Rb family tumor suppressor function.

    Sheng, Q.; Denis, D; Ratnofsky, M; Roberts, T.M.; DeCaprio, J A; Schaffhausen, B

    1997-01-01

    Tumor suppressors of the retinoblastoma susceptibility gene family regulate cell growth and differentiation. Polyomavirus large T antigens (large T) bind Rb family members and block their function. Mutations of large T sequences conserved with the DnaJ family affect large T binding to a cellular DnaK, heat shock protein 70. The same mutations abolish large T activation of E2F-containing promoters and Rb binding-dependent large T activation of cell cycle progression. Cotransfection of a cellul...

  1. Development of DNA affinity techniques for the functional characterization of purified RNA polymerase II transcription factors

    Affinity adsorption, precipitation, and partitioning techniques have been developed to purify and characterize RNA Pol II transcription components from whole cell extracts (WCE) (HeLa) and nuclear extracts (K562). The titration of these extracts with multicopy constructs of the Ad2 MLP but not pUC8, inhibits transcriptional activity. DNA-binding factors precipitated by this technique are greatly enriched by centrifugation. Using this approach, factors binding to the upstream promoter sequence (UPS) of the Ad2 MLP have been rapidly isolated by Mono Q, Mono S, and DNA affinity chromatography. By U.V. crosslinking to nucleotides containing specific 32P-phosphodiester bonds within the recognition sequence, this factor is identified as a M/sub r/ = 45,000 polypeptide. To generate an assay system for the functional evaluation of single transcription components, a similar approach using synthetic oligonucleotide sequences spanning single promoter binding sites has been developed. The addition of a synthetic 63-mer containing the UPS element of the Ad2 MLP to HeLa WCE inhibited transcription by 60%. The addition of partially purified UPS binding protein, but not RNA Pol II, restored transcriptional activity. The addition of synthetic oligonucleotides containing other regulatory sequences not present in the Ad2 MLP was without effect

  2. Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints.

    Chen, Z Q; Ritzel, R G; Lin, C C; Hodgetts, R B

    1991-07-01

    CR1 is a short interspersed repetitive DNA element originally identified in the domestic chicken (Gallus gallus). However, unlike virtually all other such sequences described to date, CR1 is not confined to one or a few closely related species. It is probably a ubiquitous component of the avian genome, having been detected in representatives of nine orders encompassing a wide spectrum of the class Aves. This identification was made possible by using the polymerase chain reaction (PCR), which revealed interspecific similarities not detected by conventional Southern analysis. DNA sequence comparisons between a CR1 element isolated from a sarus crane (Grus antigone) and those isolated from an emu (Dromaius novaehollandiae) showed that two short highly conserved regions are present. These are included within two regions previously characterized in the CR1 units of domestic fowl. One of these behaves as a transcriptional silencer and the other is a binding site for a nuclear protein. Our observations suggest that CR1 has evolved under functional constraints and that interspersed repetitive sequences as a class may constitute a more significant component of the eukaryotic genome than is generally acknowledged. PMID:1829530

  3. Polyglycerol-functionalized nanodiamond as a platform for gene delivery: Derivatization, characterization, and hybridization with DNA

    Li Zhao

    2014-03-01

    Full Text Available A gene vector consisting of nanodiamond, polyglycerol, and basic polypeptide (ND-PG-BPP has been designed, synthesized, and characterized. The ND-PG-BPP was synthesized by PG functionalization of ND through ring-opening polymerization of glycidol on the ND surface, multistep organic transformations (–OH → –OTs (tosylate → –N3 in the PG layer, and click conjugation of the basic polypeptides (Arg8, Lys8 or His8 terminated with propargyl glycine. The ND-PG-BPP exhibited good dispersibility in water (>1.0 mg/mL and positive zeta potential ranging from +14.2 mV to +44.1 mV at neutral pH in Milli-Q water. It was confirmed by gel retardation assay that ND-PG-Arg8 and ND-PG-Lys8 with higher zeta potential hybridized with plasmid DNA (pDNA through electrostatic attraction, making them promising as nonviral vectors for gene delivery.

  4. Impact of radiotherapy on PBMCs DNA repair capacity - Use of a multiplexed functional repair assay

    Radiation therapy is an essential part of cancer treatment as about 50% of patients will receive radiations at least once. Significant broad variation in radiosensitivity has been demonstrated in patients. About 5-10% of patients develop acute toxicity after radiotherapy. Therefore there is a need for the identification of markers able to predict the occurrence of adverse effects and thus adapt the radiotherapy regimen for radiosensitive patients. As a first step toward this goal, and considering the DNA repair defects associated with hypersensitivity radiation syndromes, we investigated the DNA repair phenotype of patients receiving radiotherapy. More precisely, we used a functional repair assay on support to follow the evolution of the glycosylases/AP endonuclease activities of PBMCs extracts of a series of patients during the time course of radiotherapy. For each patient, we collected one PBMCs sample before the first radiotherapy application (S1) and three samples after (S2 to S4) (one day and one week after application 1, and one at the end of the radiotherapy protocol). These four samples have been analysed for 11 donors. Clustering analyses of the results demonstrated a great heterogeneity of responses among the patients. Interestingly, this heterogeneity decreased between S1 and S4 where only 2 classes of patients remained if we except one patient that exhibited an atypical DNA repair phenotype. Furthermore, we showed that repair of several oxidized bases significantly increased between S1 and S3 or S4 (8oxoG, thymine glycol, A paired with 8oxoG), suggesting an adaptation of patients repair systems to the oxidative stress generated by the ionising radiations. Our preliminary results provided evidence that the DNA repair phenotype was impacted by the radiotherapy regimen. Further characterization of patients with known repair defects are needed to determine if atypical repair phenotypes could be associated with radiotherapy complications. Finally

  5. Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

    Carmen Fernandez-Fernandez

    Full Text Available DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA. We found that the expression of the DnaA(R357A mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

  6. Functional Expression of a DNA-Topoisomerase IB from Cryptosporidium parvum

    César Ordóñez

    2009-01-01

    Full Text Available Cryptosporidium parvum, one of the most important causative organisms of human diarrheas during childhood, contains a monomeric DNA-topoisomerase IB (CpTopIB in chromosome 7. Heterologous expression of CpTopIB gene in a budding yeast strain lacking this activity proves that the cryptosporidial enzyme is functional in vivo. The enzymatic activity is comprised in a single polypeptide, which contains all the structural features defining a fully active TopIB. Relaxation activity of the yeast extracts was detected only when CpTopIB ORF was expressed in a yeast expression system showing time and protein dependence under steady state kinetic conditions. The susceptibility of CpTopIB-transformed yeast to the irreversible inhibitor camptothecin and its water-soluble derivatives (topotecan and SN-38 was assessed.

  7. Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA.

    Philippe A Parone

    Full Text Available Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS. At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.

  8. The murine biglycan: Complete cDNA cloning, genomic organization, promoter function, and expression

    Wegrowski, Y.; Pillarisetti, J.; Danielson, K.G.; Iozzo, R.V. [Thomas Jefferson Univ., Philadelphia, PA (United States); Suzuki, S. [Univ. of Southern California, Los Angeles, CA (United States)

    1995-11-01

    Biglycan is a ubiquitous chondroitin/dermatan sulfate proteoglycan that belongs to a growing family of proteins harboring leucine-rich repeats. We have cloned and sequenced the cDNA containing the complete murine biglycan, elucidated its genomic organization, and demonstrated functional promoter activity of its 5{prime} flanking region. The deduced biglycan protein core was highly conserved across species. However, the mouse biglycan (Bgn) gene was significantly larger than the human counterpart, primarily because of a large > 4.5-kb intron between exons 1 and 2. The mouse Bgn gene spanned over 9.5 kb of continuous DNA and comprised 8 exons, with a perfectly conserved intron/exon organization vis-a-vis the human counterpart. The promoter region was enriched in GC dinucleotide and contained numerous cis-acting elements including binding sites for SP-1, AP-1, and AP-2 factors. It lacked TATA and CAAT boxes typical of housekeeping genes. In support of this, primer extension analysis showed the existence of multiple transcription start sites. Transient cell transfection assays with a construct comprising the 548 hp upstream of the major transcription start site fused to the chloramphenicol acetyl transferase reporter gene showed functional promoter activity. Internal and 5{prime} deletion constructs showed that the distal promoter of the Bgn gene was required for full transcriptional activity. In contrast to the homologous proteoglycan decorin, the highest expression of biglycan mRNA was observed in lung, liver, and spleen of adult mouse and the lowest in skin, heart, and kidney. These results will be useful for the study of biglycan gene regulation and for the generation of mice with targeted null mutation of the Bgn gene. 56 refs., 7 figs., 1 tab.

  9. Inhibition of immunological function mediated DNA damage of alveolar macrophages caused by cigarette smoke in mice.

    Ishida, Takahiro; Hirono, Yuriko; Yoshikawa, Kenichi; Hutei, Yoshimi; Miyagawa, Mayuko; Sakaguchi, Ikuyo; Pinkerton, Kent E; Takeuchi, Minoru

    2009-12-01

    Exposure to cigarette smoke impairs the pulmonary immune system, including alveolar macrophage function, although the mechanisms by which this occurs are not fully elucidated. This study investigates the effect of cigarette smoke exposure on the antigen-presenting activity of alveolar macrophages, which is required for antigen-specific response to T cells. C57BL/6 mice were exposed to cigarette smoke for 10 days using a Hamburg II smoking machine, and alveolar macrophages were obtained by bronchoalveolar lavage. The antigen-presenting activity of alveolar macrophages was significantly inhibited in mice exposed to cigarette smoke compared with mice not exposed to cigarette smoke. Major histocompatibility complex class II cell surface molecule-positive cells, B7-1 molecule-positive cells, and interleukin-1beta messenger RNA gene expression in alveolar macrophages were significantly decreased in mice exposed to cigarette smoke compared with mice not exposed to cigarette smoke. In contrast, DNA damage and generation of superoxide and hydrogen peroxide in alveolar macrophages were significantly increased by cigarette smoke exposure. These results suggest that inhibition of the antigen-presenting activity of alveolar macrophages may result from decreased expression of major histocompatibility complex class II and B7-1 molecules and interleukin-1beta messenger RNA gene expression following cigarette smoke exposure. Furthermore, inhibition of antigen presentation in alveolar macrophage may result from DNA damage induced by excessive amounts of reactive oxygen species being generated by alveolar macrophages following cigarette smoke exposure. These findings suggest that cigarette smoke impairs the immunological function of alveolar macrophages and, as a result, increases the risk for pulmonary infection. PMID:19922407

  10. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

    Angela Brieger

    Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

  11. Global functional analysis of nucleophosmin in Taxol response, cancer, chromatin regulation, and ribosomal DNA transcription

    Analysis of lung cancer response to chemotherapeutic agents showed the accumulation of a Taxol-induced protein that reacted with an anti-phospho-MEK1/2 antibody. Mass spectroscopy identified the protein as nucleophosmin/B23 (NPM), a multifunctional protein with diverse roles: ribosome biosynthesis, p53 regulation, nuclear-cytoplasmic shuttling, and centrosome duplication. Our work demonstrates that following cellular exposure to mitosis-arresting agents, NPM is phosphorylated and its chromatographic property is altered, suggesting changes in function during mitosis. To determine the functional relevance of NPM, its expression in tumor cells was reduced by siRNA. Cells with reduced NPM were treated with Taxol followed by microarray profiling accompanied by gene/protein pathway analyses. These studies demonstrate several expected and unexpected consequences of NPM depletion. The predominant downstream effectors of NPM are genes involved in cell proliferation, cancer, and the cell cycle. In congruence with its role in cancer, NPM is over-expressed in primary malignant lung cancer tissues. We also demonstrate a role for NPM in the expression of genes encoding SET (TAF1β) and the histone methylase SET8. Additionally, we show that NPM is required for a previously unobserved G2/M upregulation of TAF1A, which encodes the rDNA transcription factor TAFI48. These results demonstrate multi-faceted functions of NPM that can affect cancer cells

  12. Electrical DNA sequencing by graphene edges functionalized with H or N atoms

    Amorim, Rodrigo G.; Scheicheir, Ralph H.

    2014-03-01

    The current technology of DNA sequencing needs to be revolutionized in order to be sufficiently cost-efficient for widespread application in healthcare and genomic research. One of the most promising proposals is to use a solid-state nanodevice based on graphene due to its atomically thin edges which would readily enable single-nucleobase resolution in transverse conductance measurements. We used ab initio calculations based on Density Functional Theory combined with the non-equilibrium Green's function method to study how the capability of a graphene nanogap to electrically sense the four nucleobases (Adenine, Cytosine, Guanine and Thymine) is affected by different passivation (H or N) of the graphene edges. We will show how, for the same nucleobase, the zero bias conductance can be increased by five orders of magnitude when N atoms are chosen for functionalization over H atoms. Other aspects investigated by us concern the translational process of nucleobases through the nanogap and the corresponding spatial resolution due to diminishing transmittance as the nucleobase moves out of the gap.

  13. DNA Structures Generated during Recombination Initiated by Mismatch Repair of Uv-Irradiated Nonreplicating Phage DNA in Escherichia Coli: Requirements for Helicase, Exonucleases, and Recf and Recbcd Functions

    Feng, W. Y.; Hays, J B

    1995-01-01

    During infection of homoimmune Escherichia coli lysogens (``repressed infections''), undamaged non-replicating λ phage DNA circles undergo very little recombination. Prior UV irradiation of phages dramatically elevates recombinant frequencies, even in bacteria deficient in UvrABC-mediated excision repair. We previously reported that 80-90% of this UvrABC-independent recombination required MutHLS function and unmethylated d(GATC) sites, two hallmarks of methyl-directed mismatch repair. We now ...

  14. Coupling between Histone Conformations and DNA Geometry in Nucleosomes on a Microsecond Timescale: Atomistic Insights into Nucleosome Functions.

    Shaytan, Alexey K; Armeev, Grigoriy A; Goncearenco, Alexander; Zhurkin, Victor B; Landsman, David; Panchenko, Anna R

    2016-01-16

    An octamer of histone proteins wraps about 200bp of DNA into two superhelical turns to form nucleosomes found in chromatin. Although the static structure of the nucleosomal core particle has been solved, details of the dynamic interactions between histones and DNA remain elusive. We performed extensively long unconstrained, all-atom microsecond molecular dynamics simulations of nucleosomes including linker DNA segments and full-length histones in explicit solvent. For the first time, we were able to identify and characterize the rearrangements in nucleosomes on a microsecond timescale including the coupling between the conformation of the histone tails and the DNA geometry. We found that certain histone tail conformations promoted DNA bulging near its entry/exit sites, resulting in the formation of twist defects within the DNA. This led to a reorganization of histone-DNA interactions, suggestive of the formation of initial nucleosome sliding intermediates. We characterized the dynamics of the histone tails upon their condensation on the core and linker DNA and showed that tails may adopt conformationally constrained positions due to the insertion of "anchoring" lysines and arginines into the DNA minor grooves. Potentially, these phenomena affect the accessibility of post-translationally modified histone residues that serve as important sites for epigenetic marks (e.g., at H3K9, H3K27, H4K16), suggesting that interactions of the histone tails with the core and linker DNA modulate the processes of histone tail modifications and binding of the effector proteins. We discuss the implications of the observed results on the nucleosome function and compare our results to different experimental studies. PMID:26699921

  15. A DNA nanocapsule with aptamer-controlled open-closure function for targeted delivery

    Bentin, Thomas

    2012-01-01

    A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces.......A DNA capsule fitted with aptamer controlled target sensing has been "woven" using a 7308-base single-stranded DNA "thread" and 196 staple oligonucleotides. The capsule enables logic-gated molecular cargo delivery to targeted cell surfaces....

  16. Survival and mitochondrial function in septic patients according to mitochondrial DNA haplogroup

    Lorente, Leonardo; Iceta, Ruth; Martín, María M.; López-Gallardo, Esther; Solé-Violán, Jordi; Blanquer, José; Labarta, Lorenzo; Díaz, César; Jiménez, Alejandro; Montoya, Julio; Ruiz-Pesini, Eduardo

    2012-01-01

    Introduction We recently found that platelet cytochrome c oxidase (COX) activities and quantities in 6-month-survival septic patients are significantly higher than those of patients who died before 6 months. Other studies suggested that the mitochondrial DNA (mtDNA) genotype could play a major role in sepsis survival. Given that COX catalytic subunits are encoded by mtDNA, the objective of the present study was to explore whether mtDNA population genetic variation could affect COX activity an...

  17. Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

    Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair

  18. Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

    Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Zhang, Minjie [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua; Yang, Caiyun [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Meng, Hao; Wang, Dong; Chang, Shuang [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Xu, Ye [Department of Radiation Oncology, Division of Genomic Stability, Dana Farber Cancer Institute, Harvard Medical School, MA 02134 (United States); Price, Brendan, E-mail: Brendan_Price@dfci.harvard.edu [Department of Radiation Oncology, Division of Genomic Stability, Dana Farber Cancer Institute, Harvard Medical School, MA 02134 (United States); Sun, Yingli, E-mail: sunyl@big.ac.cn [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-10-03

    Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage. Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.

  19. Electrochemical Characterization of O2 Plasma Functionalized Multi-Walled Carbon Nanotube Electrode for Legionella pneumophila DNA Sensor

    Park, Eun Jin; Lee, Jun-Yong; Hyup Kim, Jun; Kug Kim, Sun; Lee, Cheol Jin; Min, Nam Ki

    2010-08-01

    An electrochemical DNA sensor for Legionella pneumophila detection was constructed using O2 plasma functionalized multi-walled carbon nanotube (MWCNT) film as a working electrode (WE). The cyclic voltammetry (CV) results revealed that the electrocatalytic activity of plasma functionalized MWCNT (pf-MWCNT) significantly changed depending on O2 plasma treatment time due to some oxygen containing functional groups on the pf-MWCNT surface. Scanning electron microscope (SEM) images and X-ray photoelectron spectroscopy (XPS) spectra were also presented the changes of their surface morphologies and oxygen composition before and after plasma treatment. From a comparison study, it was found that the pf-MWCNT WEs had higher electrocatalytic activity and more capability of probe DNA immobilization: therefore, electrochemical signal changes by probe DNA immobilization and hybridization on pf-MWCNT WEs were larger than on Au WEs. The pf-MWCNT based DNA sensor was able to detect a concentration range of 10 pM-100 nM of target DNA to detect L. pneumophila.

  20. Novel and functional DNA sequence variants within the GATA5 gene promoter in ventricular septal defects

    Ji-Ping Shan; Xiao-Li Wang; Yuan-Gang Qiao; Hong-Xin Wan Yan; Wen-Hui Huang; Shu-Chao Pang; Bo Yan

    2014-01-01

    Background: Congenital heart disease (CHD) is the most common human birth defect. Genetic causes for CHD remain largely unknown. GATA transcription factor 5 (GATA 5) is an essential regulator for the heart development. Mutations in the GATA5 gene have been reported in patients with a variety of CHD. Since misregulation of gene expression have been associated with human diseases, we speculated that changed levels of cardiac transcription factors, GATA5, may mediate the development of CHD. Methods: In this study, GATA5 gene promoter was genetically and functionally analyzed in large cohorts of patients with ventricular septal defect (VSD) (n=343) and ethnic-matched healthy controls (n=348). Results: Two novel and heterozygous DNA sequence variants (DSVs), g.61051165A>G and g.61051463delC, were identified in three VSD patients, but not in the controls. In cultured cardiomyocytes, GATA5 gene promoter activities were significantly decreased by DSV g.61051165A>G and increased by DSV g.61051463delC. Moreover, fathers of the VSD patients carrying the same DSVs had reduced diastolic function of left ventricles. Three SNPs, g.61051279C>T (rs77067995), g.61051327A>C (rs145936691) and g.61051373G>A (rs80197101), and one novel heterozygous DSV, g.61051227C>T, were found in both VSD patients and controls with similar frequencies. Conclusion: Our data suggested that the DSVs in the GATA5 gene promoter may increase the susceptibility to the development of VSD as a risk factor.

  1. Novel and Functional DNA Sequence Variants within the GATA6 Gene Promoter in Ventricular Septal Defects

    Chunyu Li

    2014-07-01

    Full Text Available Congenital heart disease (CHD is the most common birth defect in humans. Genetic causes and underlying molecular mechanisms for isolated CHD remain largely unknown. Studies have demonstrated that GATA transcription factor 6 (GATA6 plays an essential role in the heart development. Mutations in GATA6 gene have been associated with diverse types of CHD. As GATA6 functions in a dosage-dependent manner, we speculated that changed GATA6 levels, resulting from DNA sequence variants (DSVs within the gene regulatory regions, may mediate the CHD development. In the present study, GATA6 gene promoter was genetically and functionally analyzed in large groups of patients with ventricular septal defect (VSD (n = 359 and ethnic-matched healthy controls (n = 365. In total, 11 DSVs, including four SNPs, were identified in VSD patients and controls. Two novel and heterozygous DSVs, g.22169190A>T and g.22169311C>G, were identified in two VSD patients, but in none of controls. In cultured cardiomyocytes, the activities of the GATA6 gene promoter were significantly reduced by the DSVs g.22169190A>T and g.22169311C>G. Therefore, our findings suggested that the DSVs within the GATA6 gene promoter identified in VSD patients may change GATA6 levels, contributing to the VSD development as a risk factor.

  2. Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase

    Yousif, Ashraf S.; Stanlie, Andre; Mondal, Samiran; Honjo, Tasuku; Begum, Nasim A.

    2014-01-01

    Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR. PMID:24591630

  3. Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase.

    Yousif, Ashraf S; Stanlie, Andre; Mondal, Samiran; Honjo, Tasuku; Begum, Nasim A

    2014-03-18

    Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR. PMID:24591630

  4. A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein, Mgm101.

    Nardozzi, Jonathan D; Wang, Xiaowen; Mbantenkhu, MacMillan; Wilkens, Stephan; Chen, Xin Jie

    2012-10-26

    Mgm101 is a Rad52-type recombination protein of bacteriophage origin required for the repair and maintenance of mitochondrial DNA (mtDNA). It forms large oligomeric rings of ∼14-fold symmetry that catalyze the annealing of single-stranded DNAs in vitro. In this study, we investigated the structural elements that contribute to this distinctive higher order structural organization and examined its functional implications. A pair of vicinal cysteines, Cys-216 and Cys-217, was found to be essential for mtDNA maintenance. Mutations to the polar serine, the negatively charged aspartic and glutamic acids, and the hydrophobic amino acid alanine all destabilize mtDNA in vivo. The alanine mutants have an increased propensity of forming macroscopic filaments. In contrast, mutations to aspartic acid drastically destabilize the protein and result in unstructured aggregates with severely reduced DNA binding activity. Interestingly, the serine mutants partially disassemble the Mgm101 rings into smaller oligomers. In the case of the C216S mutant, a moderate increase in DNA binding activity was observed. By using small angle x-ray scattering analysis, we found that Mgm101 forms rings of ∼200 Å diameter in solution, consistent with the structure previously established by transmission electron microscopy. We also found that the C216A/C217A double mutant tends to form broken rings, which likely provide free ends for seeding the growth of the super-stable but functionally defective filaments. Taken together, our data underscore the importance of a delicately maintained ring structure critical for Mgm101 activity. We discuss a potential role of Cys-216 and Cys-217 in regulating Mgm101 function and the repair of damaged mtDNA under stress conditions. PMID:22948312

  5. Structure-function studies of DNA binding domain of response regulator KdpE reveals equal affinity interactions at DNA half-sites.

    Anoop Narayanan

    Full Text Available Expression of KdpFABC, a K(+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABC(BS via the winged helix-turn-helix type DNA binding domain (KdpE(DBD. Exploration of E. coli KdpE(DBD and kdpFABC(BS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpE(DBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpE(DBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpE(DBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins.

  6. Gold Functionalized Mesoporous Silica Nanoparticle Mediated Protein and DNA Codelivery to Plant Cells Via the Biolistic Method

    Martin-Ortigosa, Susana; Valenstein, Justin S.; Lin, Victor S.-Y.; Trewyn, Brian G.; Wang, Kan

    2012-09-11

    The synthesis and characterization of a gold nanoparticle functionalized mesoporous silica nanoparticle (Au-MSN) platform for codelivery of proteins and plasmid DNA to plant tissues using a biolistic particle delivery system is reported. The in vitro uptake and release profiles of fluorescently labeled bovine serum albumin (BSA) and enhanced green fluorescent protein (eGFP) are investigated. As a proof-of-concept demonstration, Au-MSN with large average pore diameters (10 nm) are shown to deliver and subsequently release proteins and plasmid DNA to the same cell after passing through the plant cell wall upon bombardment. Release of fluorescent eGFP indicates the delivery of active, non-denatured proteins to plant cells. This advance represents the first example of biolistic-mediated codelivery of proteins and plasmid DNA to plant cells via gold-functionalized MSN and provides a powerful tool for both fundamental and applied research of plant sciences.

  7. Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions.

    Sakai, H; Yasugi, T; Benson, J D; Dowhanick, J J; Howley, P M

    1996-03-01

    The E2 gene products of papillomavirus play key roles in viral replication, both as regulators of viral transcription and as auxiliary factors that act with E1 in viral DNA replication. We have carried out a detailed structure-function analysis of conserved amino acids within the N-terminal domain of the human papillomavirus type 16 (HPV16) E2 protein. These mutants were tested for their transcriptional activation activities as well as transient DNA replication and E1 binding activities. Analysis of the stably expressed mutants revealed that the transcriptional activation and replication activities of HPV16 E2 could be dissociated. The 173A mutant was defective for the transcriptional activation function but retained wild-type DNA replication activity, whereas the E39A mutant wild-type transcriptional activation function but was defective in transient DNA replication assays. The E39A mutant was also defective for HPV16 E1 binding in vitro, suggesting that the ability of E2 protein to form a complex with E1 appears to be essential for its function as an auxiliary replication factor. PMID:8627680

  8. Helicobacter pylori infection affects mitochondrial function and DNA repair, thus, mediating genetic instability in gastric cells

    Machado, Ana Manuel Dantas; Madsen, Claus Desler; Bøggild, Cecilie Sisse Line;

    2013-01-01

    causes mtDNA mutations and a decrease of mtDNA content. Consequently, we show a decrease of respiration coupled ATP turnover and respiratory capacity and accordingly a lower level and activity of complex I of the electron transport chain. We wanted to investigate if the increased mutational load in the...... mitochondrial genome was caused by down-regulation of mitochondrial DNA repair pathways. We lowered the expression of APE-1 and YB-1, which are believed to be involved in mitochondrial base excision repair and mismatch repair. Our results suggest that both APE-1 and YB-1 are involved in mtDNA repair during H....... pylori infection, furthermore, the results demonstrate that multiple DNA repair activities are involved in protecting mtDNA during infection....

  9. Functional characterization and inhibition of the type II DNA topoisomerase coded by African swine fever virus.

    Coelho, João; Ferreira, Fernando; Martins, Carlos; Leitão, Alexandre

    2016-06-01

    DNA topoisomerases are essential for DNA metabolism and while their role is well studied in prokaryotes and eukaryotes, it is less known for virally-encoded topoisomerases. African swine fever virus (ASFV) is a nucleo-cytoplasmic large DNA virus that infects Ornithodoros ticks and all members of the family Suidae, representing a global threat for pig husbandry with no effective vaccine nor treatment. It was recently demonstrated that ASFV codes for a type II topoisomerase, highlighting a possible target for control of the virus. In this work, the ASFV DNA topoisomerase II was expressed in Saccharomyces cerevisiae and found to efficiently decatenate kDNA and to processively relax supercoiled DNA. Optimal conditions for its activity were determined and its sensitivity to a panel of topoisomerase poisons and inhibitors was evaluated. Overall, our results provide new knowledge on viral topoisomerases and on ASFV, as well as a possible target for the control of this virus. PMID:27060564

  10. Functional evaluation of DNA repair in human biopsies and their relation to other cellular biomarkers

    Slyskova, Jana; Langie, Sabine A. S.; Collins, Andrew R.; Vodicka, Pavel

    2014-01-01

    Thousands of DNA lesions are estimated to occur in each cell every day and almost all are recognized and repaired. DNA repair is an essential system that prevents accumulation of mutations which can lead to serious cellular malfunctions. Phenotypic evaluation of DNA repair activity of individuals is a relatively new approach. Methods to assess base and nucleotide excision repair pathways (BER and NER) in peripheral blood cells based on modified comet assay protocols have been widely applied i...

  11. Relative ultraviolet radiation sensitivity of certain functions of polyoma virus. Stimulation of cell DNA synthesis

    Peritoneal Mouse macrophages were used to study the stimulation of cell DNA synthesis by polyoma virus. Using ultraviolet-irradiated polyoma virus, it was possible to show a difference between the inactivation of infectivity and of induction of DNA synthesis. By statistical analysis of these two phenomena it was found that 39% of the viral genome is necessary for the induction of cell DNA synthesis

  12. A DNA binding winged helix domain in CAF-1 functions with PCNA to stabilize CAF-1 at replication forks

    Zhang, Kuo; Gao, Yuan; Li, Jingjing; Burgess, Rebecca; Han, Junhong; Liang, Huanhuan; Zhang, Zhiguo; Liu, Yingfang

    2016-01-01

    Chromatin assembly factor 1 (CAF-1) is a histone H3–H4 chaperone that deposits newly synthesized histone (H3–H4)2 tetramers during replication-coupled nucleosome assembly. However, how CAF-1 functions in this process is not yet well understood. Here, we report the crystal structure of C terminus of Cac1 (Cac1C), a subunit of yeast CAF-1, and the function of this domain in stabilizing CAF-1 at replication forks. We show that Cac1C forms a winged helix domain (WHD) and binds DNA in a sequence-independent manner. Mutations in Cac1C that abolish DNA binding result in defects in transcriptional silencing and increased sensitivity to DNA damaging agents, and these defects are exacerbated when combined with Cac1 mutations deficient in PCNA binding. Similar phenotypes are observed for corresponding mutations in mouse CAF-1. These results reveal a mechanism conserved in eukaryotic cells whereby the ability of CAF-1 to bind DNA is important for its association with the DNA replication forks and subsequent nucleosome assembly. PMID:26908650

  13. Mitochondria DNA mutations cause sex-dependent development of hypertension and alterations in cardiovascular function

    Golob, Mark J.; Tian, Lian; Wang, Zhijie; Zimmerman, Todd A.; Caneba, Christine A.; Hacker, Timothy A.; Song, Guoqing; Chesler, Naomi C.

    2014-01-01

    Aging is associated with conduit artery stiffening that is a risk factor for and can precede hypertension and ventricular dysfunction. Increases in mitochondria DNA (mtDNA) frequency have been correlated with aging. Mice with a mutation in the encoding domain (D257A) of a proof-reading deficient version of mtDNA polymerase-γ (POLG) have musculoskeletal features of premature aging and a shortened lifespan. However, few studies using these mice have investigated the effects of mtDNA mutations o...

  14. Transcriptional profiling in C. elegans suggests DNA damage dependent apoptosis as an ancient function of the p53 family

    Rothblatt Jonathan

    2008-07-01

    Full Text Available Abstract Background In contrast to the three mammalian p53 family members, p53, which is generally involved in DNA damage responses, and p63 and p73 which are primarily needed for developmental regulation, cep-1 encodes for the single C. elegans p53-like gene. cep-1 acts as a transcription activator in a primordial p53 pathway that involves CEP-1 activation and the CEP-1 dependent transcriptional induction of the worm BH3 only domain encoding genes egl-1 and ced-13 to induce germ cell apoptosis. EGL-1 and CED-13 proteins inactivate Bcl-2 like CED-9 to trigger CED-4 and CED-3 caspase dependent germ cell apoptosis. To address the function of p53 in global transcriptional regulation we investigate genome-wide transcriptional responses upon DNA damage and cep-1 deficiency. Results Examining C. elegans expression profiles using whole genome Affymetrix GeneChip arrays, we found that 83 genes were induced more than two fold upon ionizing radiation (IR. None of these genes, with exception of an ATP ribosylase homolog, encode for known DNA repair genes. Using two independent cep-1 loss of function alleles we did not find genes regulated by cep-1 in the absence of IR. Among the IR-induced genes only three are dependent on cep-1, namely egl-1, ced-13 and a novel C. elegans specific gene. The majority of IR-induced genes appear to be involved in general stress responses, and qRT-PCR experiments indicate that they are mainly expressed in somatic tissues. Interestingly, we reveal an extensive overlap of gene expression changes occurring in response to DNA damage and in response to bacterial infection. Furthermore, many genes induced by IR are also transcriptionally regulated in longevity mutants suggesting that DNA damage and aging induce an overlapping stress response. Conclusion We performed genome-wide gene expression analyses which indicate that only a surprisingly small number of genes are regulated by CEP-1 and that DNA damage induced apoptosis via the

  15. Selective inhibition of influenza virus protein synthesis by inhibitors of DNA function

    Various known inhibitors of cellular DNA function were shown to inhibit cellular RNA synthesis and influenza (fowl plague) virus multiplication. The drugs were investigated for their effect upon the synthesis of influenza virus proteins. According to this effect they could be classified with previously studied compounds as follows: Group I (ethidium bromide, proflavine, and N-nitroquinoline-N-oxide) inhibited both viral and cellular protein synthesis; Group II (nogalomycin, daunomycin and α-amanitin) inhibited viral but not cellular protein synthesis, and all viral proteins were inhibited coordinately; Group III (mithramycin, echinomycin, and actinomycin D) inhibited all viral but not cellular protein synthesis at high concentrations, but at a lower critical concentration inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein preferentially; Group IV(uv irradiation and camptothecin) inhibited the synthesis of viral haemagglutinin, neuraminidase, and M protein, but not other viral proteins, even at high doses. The mode of action of these inhibitors is discussed in relation to the mechanism of the nuclear events upon which influenza virus multiplication is dependent

  16. Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein

    Bendtsen, Jannick Dyrløv; Nilsson, A.S.; Lehnherr, H.

    2002-01-01

    Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium Escherichia coli. A phylogenetic analysis indicated that the P1 ssb gene coexists with its E. coli counterpart as an independent unit and...... does not represent a recent acquirement of the phage. The P1 and E. coli SSB proteins are fully functionally interchangeable. SSB-P1 is nonessential for phage growth in an exponentially growing E. coli host, and it is sufficient to promote bacterial growth in the absence of the E. coli SSB protein....... Expression studies showed that the P1 ssb gene is transcribed only, in an rpoS-independent fashion, during stationary-phase growth in E. coli. Mixed infection experiments demonstrated that a wild-type phage has a selective advantage over an ssb-null mutant when exposed to a bacterial host in the stationary...

  17. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    Yuxin Feng

    Full Text Available Estrogen receptor alpha (ERα, a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

  18. Functional zinc-binding motifs in enzymes and DNA-binding proteins.

    Vallee, B L; Auld, D S

    1992-01-01

    Zinc is now known to be an integral component of a large number and variety of enzymes and proteins involved in virtually all aspects of metabolism, thus accounting for the fact that this element is essential for growth and development. The chemistry of zinc, superficially bland, in reality has turned out to be ideally appropriate and versatile for the unexpected development of multiple and unique chemical structures which biology has used for specific life processes. The present discussion will centre on those distinctive zinc-binding motifs that are critical both to enzyme function and the expression of the genetic message. X-Ray diffraction structure determination of 15 zinc enzymes belonging to IUB classes I-IV provide absolute standards of reference for the identity and nature of zinc ligands in their families. Three types of zinc enzyme binding motifs emerge through analysis of these: catalytic, coactive or cocatalytic, and structural. In contrast to zinc enzymes virtually all DNA-binding proteins contain multiple zinc atoms. With the availability of NMR and X-ray structure analyses three distinct motifs now emerge for those: zinc fingers, twists and clusters. PMID:1290939

  19. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    Feng, Yuxin; Singleton, David; Guo, Chun; Gardner, Amanda; Pakala, Suresh; Kumar, Rakesh; Jensen, Elwood; Zhang, Jinsong; Khan, Sohaib

    2013-01-01

    Estrogen receptor alpha (ERα), a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy. PMID:23874500

  20. Electrical and optical characterization of DNA molecules as a function of concentration in aqueous solution

    Polcari, A.; Romano, P.; Sabatino, L.; Vecchio, E. del; Consales, M.; Cusano, A.; Cutolo, A.; Colantuoni, V.

    2011-04-01

    We present experimental data on the electrical and optical behavior of circular and linear plasmid DNA molecules embedded in an aqueous matrix. From the electrical point of view, the results indicate that the presence of water is essential when the DNA concentration is not sufficient to ensure conduction patterns, consistent with the conduction mechanisms proposed for DNA molecules. In fact, as droplets are layered on the substrate, a nonlinear behavior of the current-voltage curves is observed; when water evaporates, the conductivity decreases to a very low value that resembles the high resistivity of the substrate. As more droplets are added, the current increases giving higher conductivity, as expected, since more molecules participate into the conduction process. The conduction behavior of DNA could be due to a tunneling transport of charges inside the molecule, the tunneling barrier being the adenine-thymine bridge between consecutive guanine-cytosine base pairs. A hopping channel, activated by the water surrounding the molecules, could act as an additional mechanism between different molecules. Optical reflectance measurements, performed in the wavelength range 230-450 nm on dried samples, obtained by complete evaporation of the aqueous matrix containing plasmid DNA under UV-VIS light, reveal absorption in the wavelength range 230-300 nm, typical of DNA-based materials. Moreover, in correspondence of the absorption region, the higher the DNA concentration, the higher the reflectance reduction. This suggests that the DNA concentration strongly influences the spectral behavior of biological layers. Finally, we present preliminary results exploiting the possibility to control the morphological and optical features of DNA layers by means of proper lighting procedures. Both electrical and optical measurements indicate that the DNA concentration is a crucial parameter for technical applications. The results reported here provide a contribution for the possible use

  1. Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase

    Yousif, Ashraf S.; Stanlie, Andre; Mondal, Samiran; Honjo, Tasuku; Begum, Nasim A.

    2014-01-01

    Uracil DNA glycosylase (UNG) has been known as a critical base excision repair protein required for class switch recombination (CSR) and somatic hypermutation (SHM). On the other hand, its precise function in both CSR and SHM is extremely debatable and elusive. Here, we showed that UNG suppresses S region SHM (s-SHM) by recruiting the faithful DNA repair complex and, in the absence of UNG, the error-prone repair complex that induces s-SHM overrides. Moreover, UNG promotes activation-induced c...

  2. Adsorption and desorption characteristics of DNA onto the surface of amino functional mesoporous silica with various particle morphologies.

    Hikosaka, Ryouichi; Nagata, Fukue; Tomita, Masahiro; Kato, Katsuya

    2016-04-01

    Recently, deoxyribonucleic acid (DNA) adsorption on solid materials has been reported for applications such as genetic diagnosis of diseases, gene delivery, and biosensors. Mesoporous silica (MPS) is an excellent carrier because of its high surface area and large pore volume. Functionalization of the MPS surface can be controlled by silane coupling reagents, and the MPS particle morphology can be easily changed by the synthetic conditions. In this study, to evaluate the ability of DNA adsorption on MPS, the MPS surface was functionalized using four reagents, 3-aminopropyltriethoxysilane (-NH2), N-(2-aminoethyl)-3-aminopropyltriethoxysilane (-2ENH2), N-(6-aminohexyl)aminopropyltrimethoxysilane (-2HNH2), and (3-trimethoxysilylpropyl)diethylenetriamine (-3NH2), each having a different number of amino groups and alkyl chain lengths. Moreover, we prepared three types of MPSs with different particle morphologies: sheet-type structure (MPS sheet), spherical MPS (MCM-41s), and nonporous spherical silica. A high adsorption capacity was observed in MPS sheets with -2HNH2 (sheet-2HNH2) and -3NH2 (sheet-3NH2), as well as MCM-41s with -3NH2 (41s-3NH2). The adsorption and desorption rates of DNA on these three MPSs were then examined and the best results were obtained with 41s-3NH2. These results demonstrate that the amino functionalized MPS materials are useful DNA adsorbents. PMID:26764114

  3. Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.

    Morales, Julio C; Richard, Patricia; Rommel, Amy; Fattah, Farjana J; Motea, Edward A; Patidar, Praveen L; Xiao, Ling; Leskov, Konstantin; Wu, Shwu-Yuan; Hittelman, Walter N; Chiang, Cheng-Ming; Manley, James L; Boothman, David A

    2014-04-01

    Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair. PMID:24589584

  4. Magnesium coordination controls the molecular switch function of DNA mismatch repair protein MutS

    J.H.G. Lebbink (Joyce); A. Fish (Alexander); A. Reumer (Annet); G. Natrajan; H.H.K. Winterwerp (Herrie); T.K. Sixma (Titia)

    2010-01-01

    textabstractThe DNA mismatch repair protein MutS acts as a molecular switch. It toggles between ADP and ATP states and is regulated by mismatched DNA. This is analogous to G-protein switches and the regulation of their "on" and "off" states by guanine exchange factors. Although GDP release in monome

  5. Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

    Trujillo, L E; Pupo, E; Miranda, F;

    1996-01-01

    We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assay...

  6. Interactions of Ku70/80 with Double-Strand DNA: Energetic, Dynamics, and Functional Implications

    Hu, Shaowen; Cucinotta, Francis A.

    2010-01-01

    Space radiation is a proficient inducer of DNA damage leading to mutation, aberrant cell signaling, and cancer formation. Ku is among the first responding proteins in nucleus to recognize and bind the DNA double strand breaks (DSBs) whenever they are introduced. Once loaded Ku works as a scaffold to recruit other repair factors of non-homologous end joining and facilitates the following repair processes. The crystallographic study of the Ku70/80 heterodimer indicate the core structure of this protein shows virtually no conformational change after binding with DNA. To investigate the dynamical features as well as the energetic characteristics of Ku-DNA binding, we conduct multi-nanosecond molecular dynamics simulations of a modeled Ku70/80 structure and several complexes with two 24-bp DNA duplexes. Free energy calculations show significant energy differences between the complexes with Ku bound at DSBs and those with Ku associated at an internal site of a chromosome. The results also reveal detailed interactions between different nucleotides and the amino acids along the DNA-binding cradle of Ku, indicating subtle binding preference of Ku at specific DNA sequences. The covariance matrix analyses along the trajectories demonstrate the protein is stimulated to undergo correlated motions of different domains once bound to DNA ends. Additionally, principle component analyses identify these low frequency collective motions suitable for binding with and translocation along duplex DNA. It is proposed that the modification of dynamical properties of Ku upon binding with DSBs may provide a signal for the further recruitment of other repair factors such as DNA-PKcs, XLF, and XRCC4.

  7. Regions within the N-terminal domain of human topoisomerase I exert important functions during strand rotation and DNA binding

    Hougaard, Rikke Frølich; Andersen, Félicie Faucon; Westergaard, Ole;

    2004-01-01

    The human topoisomerase I N-terminal domain is the only part of the enzyme still not crystallized and the function of this domain remains enigmatical. In the present study, we have addressed the specific functions of individual N-terminal regions of topoisomerase I by characterizing mutants lacking......, insensitivity towards the anti-cancer drug camptothecin in relaxation and the inability to ligate blunt end DNA fragments. The mutant lacking amino acid residues 1–202 was impaired in blunt end DNA ligation and showed wild-type sensitivity towards camptothecin in relaxation. Taken together, the presented data...... support a model according to which tryptophane-205 and possibly other residues located between position 191–206 coordinates the restriction of free strand rotation during the topoisomerization step of catalysis. Moreover, tryptophane-205 appears important for the function of the bulk part of the N-terminal...

  8. Theoretical study of excited states of DNA base dimers and tetramers using optimally tuned range-separated density functional theory.

    Sun, Haitao; Zhang, Shian; Zhong, Cheng; Sun, Zhenrong

    2016-03-15

    Excited states of various DNA base dimers and tetramers including Watson-Crick H-bonding and stacking interactions have been investigated by time-dependent density functional theory using nonempirically tuned range-separated exchange (RSE) functionals. Significant improvements are found in the prediction of excitation energies and oscillator strengths, with results comparable to those of high-level coupled-cluster (CC) models (RI-CC2 and EOM-CCSD(T)). The optimally-tuned RSE functional significantly outperforms its non-tuned (default) version and widely-used B3LYP functional. Compared to those high-level CC benchmarks, the large mean absolute deviations of conventional functionals can be attributed to their inappropriate amount of exact exchange and large delocalization errors which can be greatly eliminated by tuning approach. Furthermore, the impacts of H-bonding and π-stacking interactions in various DNA dimers and tetramers are analyzed through peak shift of simulated absorption spectra as well as corresponding change of absorption intensity. The result indicates the stacking interaction in DNA tetramers mainly contributes to the hypochromicity effect. The present work provides an efficient theoretical tool for accurate prediction of optical properties and excited states of nucleobase and other biological systems. © 2015 Wiley Periodicals, Inc. PMID:26666212

  9. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  10. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    Pang, Jinsong, E-mail: pangjs542@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Dong, Mingyue; Li, Ning; Zhao, Yanli [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China); Liu, Bao, E-mail: baoliu@nenu.edu.cn [Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, Changchun, Jilin 130024 (China)

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA