Science.gov

Sample records for 17-mer dna substrate

  1. Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments

    NASA Technical Reports Server (NTRS)

    Ojha, R. P.; Dhingra, M. M.; Sarma, M. H.; Myer, Y. P.; Setlik, R. F.; Shibata, M.; Kazim, A. L.; Ornstein, R. L.; Rein, R.; Turner, C. J.; Sarma, R. H.

    1997-01-01

    The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex

  2. Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments.

    PubMed

    Ojha, R P; Dhingra, M M; Sarma, M H; Myer, Y P; Setlik, R F; Shibata, M; Kazim, A L; Ornstein, R L; Rein, R; Turner, C J; Sarma, R H

    1997-10-01

    The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex

  3. A 17-mer Membrane-Active MSI-78 Derivative with Improved Selectivity toward Bacterial Cells.

    PubMed

    Monteiro, Claudia; Pinheiro, Marina; Fernandes, Mariana; Maia, Sílvia; Seabra, Catarina L; Ferreira-da-Silva, Frederico; Reis, Salette; Gomes, Paula; Martins, M Cristina L

    2015-08-01

    Antimicrobial peptides are widely recognized as an excellent alternative to conventional antibiotics. MSI-78, a highly effective and broad spectrum AMP, is one of the most promising AMPs for clinical application. In this study, we have designed shorter derivatives of MSI-78 with the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer derivatives were created by truncating MSI-78 at the N- and/or C-termini, while spanning MSI-78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20) (KFLKKAKKFGKAFVKIL), which was demonstrated to be as effective as MSI-78 against the Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa. This shorter derivative is more selective toward bacterial cells as it was less toxic to erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately to cell death. These features point to a mechanism of action similar to the one described for the lead peptide MSI-78. PMID:26066462

  4. The influence of substrate on DNA transfer and extraction efficiency.

    PubMed

    Verdon, Timothy J; Mitchell, R John; van Oorschot, Roland A H

    2013-01-01

    The circumstances surrounding deposition of DNA profiles are increasingly becoming an issue in court proceedings, especially whether or not the deposit was made by primary transfer. In order to improve the currently problematic evaluation of transfer scenarios in court proceedings, we examined the influence a variety of nine substrate types (six varieties of fabric, plywood, tarpaulin, and plastic sheets) has on DNA transfer involving blood. DNA transfer percentages were significantly higher (p=0.03) when the primary substrate was of non-porous material (such as tarpaulin, plastic or, to a lesser degree, wood) and the secondary substrate porous (such as fabrics). These findings on transfer percentages confirm the results of previous studies. Fabric composition was also shown to have a significant (p=0.03) effect on DNA transfer; when experiments were performed with friction from a variety of fabrics to a specific weave of cotton, transfer percentages ranged from 4% (flannelette) to 94% (acetate). The propensity for the same nine substrates to impact upon the efficiency of DNA extraction procedures was also examined. Significant (p=0.03) differences were found among the extraction efficiencies from different materials. When 15μL of blood was deposited on each of the substrates, the lowest quantity of DNA was extracted from plastic (20ng) and the highest quantities extracted from calico and flannelette (650ng). Significant (p<0.05) differences also exist among the DNA extraction yield from different initial blood volumes from all substrates. Also, significantly greater (p<0.05) loss of DNA was seen during concentration of extracts with higher compared to lower initial quantities of DNA. These findings suggest that the efficiency of extraction and concentration impacts upon the final amount of DNA available for analysis and that consideration of these effects should not be ignored. The application of correction factors to adjust for any variation among extraction and

  5. Recombination between linear double-stranded DNA substrates in vivo

    PubMed Central

    Narayanan, Kumaran; Sim, Edmund Ui-Hang; Ravin, Nikolai V.; Lee, Choon-Weng

    2009-01-01

    Recombineering technology in E. coli enables targeting of linear donor DNA to circular recipient DNA using short shared homology sequences. In this work, we demonstrate that recombineering is also able to support recombination between a pair of linear DNA substrates (linear/linear recombineering) in vivo in E. coli. Linear DNA up to 100 kb is accurately modified and remains intact without undergoing rearrangements after recombination. This system will be valuable for direct in vivo manipulation of large linear DNA including the N15 and PY54 prophages and linear animal viruses, and for assembly of linear constructs as artificial chromosome vectors. PMID:19454252

  6. Modeling of flap endonuclease interactions with DNA substrate.

    PubMed

    Allawi, Hatim T; Kaiser, Michael W; Onufriev, Alexey V; Ma, Wu-Po; Brogaard, Andrew E; Case, David A; Neri, Bruce P; Lyamichev, Victor I

    2003-05-01

    Structure-specific 5' nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5' flap endonuclease from Pyrococcus furiosus in its complex with DNA. The model is based on the known X-ray structure of the enzyme and a variety of biochemical and molecular dynamics (MD) data utilized in the form of distance restraints between the enzyme and the DNA. Contacts between the 5' flap endonuclease and the sugar-phosphate backbone of the overlap flap substrate were identified using enzyme activity assays on substrates with methylphosphonate or 2'-O-methyl substitutions. The enzyme footprint extends two to four base-pairs upstream and eight to nine base-pairs downstream of the cleavage site, thus covering 10-13 base-pairs of duplex DNA. The footprint data are consistent with a model in which the substrate is bound in the DNA-binding groove such that the downstream duplex interacts with the helix-hairpin-helix motif of the enzyme. MD simulations to identify the substrate orientation in this model are consistent with the results of the enzyme activity assays on the methylphosphonate and 2'-O-methyl-modified substrates. To further refine the model, 5' flap endonuclease variants with alanine point substitutions at amino acid residues expected to contact phosphates in the substrate and one deletion mutant were tested in enzyme activity assays on the methylphosphonate-modified substrates. Changes in the enzyme footprint observed for two point mutants, R64A and R94A, and for the deletion mutant in the enzyme's beta(A)/beta(B) region, were interpreted as being the result of specific interactions in the enzyme/DNA complex

  7. Substrate-dependent millisecond domain motions in DNA polymerase β

    PubMed Central

    Berlow, Rebecca B.; Swain, Monalisa; Dalal, Shibani; Sweasy, Joann B.; Loria, J. Patrick

    2012-01-01

    DNA polymerase β (Pol β) is a 39 kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol β have been linked to various cancers and these mutations further correlated with altered Pol β enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol β repair function and several studies have implicated conformational changes in Pol β as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol β have been determined by solution NMR relaxation dispersion for the apo and substrate, binary forms of Pol β. In apo Pol β, molecular motions, primarily isolated to the DNA lyase domain, are observed to occur at 1400 s–1. Additional analysis suggests that these motions allow apo Pol β to sample a conformation similar to the gapped, DNA substrate bound form. Upon binding DNA, these lyase domain motions are significantly quenched whereas evidence for conformational motions in the polymerase domain become apparent. These NMR studies suggest an alteration in the dynamic landscape of Pol β due to substrate binding. Moreover, a number of the flexible residues identified in this work are also the location of residues, which upon mutation, lead to cancer phenotypes in vivo, which may be due to the intimate role of protein motions in Pol β fidelity. PMID:22446382

  8. The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    PubMed Central

    Bauer, Robert J.; Evans, Thomas C.; Lohman, Gregory J. S.

    2016-01-01

    DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site. PMID:26954034

  9. Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

    PubMed Central

    Balasingham, Seetha V.; Zegeye, Ephrem Debebe; Homberset, Håvard; Rossi, Marie L.; Laerdahl, Jon K.; Bohr, Vilhelm A.; Tønjum, Tone

    2012-01-01

    XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB. PMID:22615856

  10. Electrophoresis of DNA on a disordered two-dimensional substrate

    NASA Astrophysics Data System (ADS)

    Reichhardt, C. J. Olson; Reichhardt, C.

    2006-11-01

    We propose a method for electrophoretic separation of DNA in which adsorbed polymers are driven over a disordered two-dimensional substrate which contains attractive sites for the polymers. Using simulations of a model for long polymer chains, we show that the mobility increases with polymer length, in contrast to gel electrophoresis techniques, and that separation can be achieved for a range of length scales. We demonstrate that the separation mechanism relies on steric interactions between polymer segments, which prevent substrate disorder sites from trapping more than one DNA segment each. Since thermal activation does not play a significant role in determining the polymer mobility, band broadening due to diffusion can be avoided in our separation method.

  11. Electrophoresis of DNA on a disordered two-dimensional substrate

    NASA Astrophysics Data System (ADS)

    Olson Reichhardt, Cynthia J.; Reichhardt, Charles

    2007-03-01

    We propose a method for electrophoretic separation of DNA in which adsorbed polymers are driven over a disordered two-dimensional substrate which contains attractive sites for the polymers. Using simulations of a model for long polymer chains, we show that the mobility increases with polymer length, in contrast to gel electrophoresis techniques, and that separation can be achieved for a range of length scales. We demonstrate that the separation relies on steric interactions between polymer segments, which prevent substrate disorder sites from trapping more than one DNA segment each. Since thermal activation does not play a significant role in determining the polymer mobility, band broadening due to diffusion can be avoided in our separation method. [1] Phys. Rev. E 74, 051908 (2006).

  12. Electrophoresis of DNA on a disordered two-dimensional substrate

    NASA Astrophysics Data System (ADS)

    Olson Reichhardt, Cynthia J.; Reichhardt, Charles

    2006-03-01

    We propose a new method for electrophoretic separation of DNA in which adsorbed polymers are driven over a disordreed two-dimensional substrate which contains attractive sites for the polymers. Using simulations of a model for long polymer chains, we show that the mobility increases with polymer length, in contrast to gel electrophoresis techniques, and that separation can be achieved for a range of length scales. We demonstrate that the separation mechanism relies on excluded volume interactions between polymer segments.

  13. Modulating the Substrate Selectivity of DNA Aptamers Using Surfactants.

    PubMed

    Peterson, Amberlyn M; Jahnke, Frank M; Heemstra, Jennifer M

    2015-11-01

    Nucleic acid aptamers have a number of advantages compared to antibodies, including greater ease of production and increased thermal stability. We hypothesized that aptamers may also be capable of functioning in the presence of high concentrations of surfactants, which readily denature antibodies and other protein-based affinity reagents. Here we report the first systematic investigation into the compatibility of DNA aptamers with surfactants. We find that neutral and anionic surfactants have only a minor impact on the ability of aptamers to fold and bind hydrophilic target molecules. Additionally, we demonstrate that surfactants can be utilized to modulate the substrate binding preferences of aptamers, likely due to the sequestration of hydrophobic target molecules within micelles. The compatibility of aptamers with commonly used surfactants is anticipated to expand their scope of potential applications, and the ability to modulate the substrate binding preferences of aptamers using a simple additive provides a novel route to increasing their selectivity in analytical applications. PMID:26465173

  14. Neil DNA glycosylases promote substrate turnover by Tdg during DNA demethylation

    PubMed Central

    Arab, Khelifa; Kienhöfer, Sabine; von Seggern, Annika; Niehrs, Christof

    2016-01-01

    DNA 5-methylcytosine is a dynamic epigenetic mark which plays important roles in development and disease. In the Tet-Tdg demethylation pathway, methylated cytosine is iteratively oxidized by Tet dioxygenases and unmodified cytosine is restored via thymine DNA glycosylase (Tdg). Here we show that human NEIL1 and NEIL2 DNA glycosylases coordinate abasic site processing during TET–TDG DNA demethylation. NEIL1 and NEIL2 cooperate with TDG during base excision: TDG occupies the abasic site and is displaced by NEILs, which further process the baseless sugar, thereby stimulating TDG substrate turnover. In early Xenopus embryos Neil2 cooperates with Tdg to remove oxidized methylcytosines and to specify neural crest development together with Tet3. Thus, Neils function as AP lyases in the coordinated AP site hand-over during oxidative DNA demethylation. PMID:26751644

  15. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    SciTech Connect

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A.; Lees-Miller, S.P.; Lintott, L.G.; Sakaguchi, Kazuyasu; Appella, E.

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  16. Simple method of DNA stretching on glass substrate for fluorescence image and spectroscopy

    NASA Astrophysics Data System (ADS)

    Neupane, Guru P.; Dhakal, Krishna P.; Lee, Hyunsoo; Guthold, Martin; Joseph, Vincent S.; Hong, Jong-Dal; Kim, Jeongyong

    2013-05-01

    Study of biological molecule DNA has contributed to developing many breaking thoughts and wide applications in multidisciplinary fields, such as genomic, medical, sensing and forensic fields. Stretching of DNA molecules is an important supportive tool for AFM or spectroscopic studies of DNA in a single molecular level. In this article, we established a simple method of DNA stretching (to its full length) that occurred on a rotating negatively-charged surface of glass substrate. The isolation of a single DNA molecule was attained by the two competitive forces on DNA molecules, that is, the electrostatic attraction developed between the positively charged YOYO-1 stained DNA and the negatively charged substrate, and the centrifugal force of the rotating substrate, which separates the DNA aggregates into the single molecule. Density of stretched DNA molecules was controlled by selecting the specific parameters such as spinning time and rates, loading volume of DNA-dye complex solution etc. The atomic force microscopy image exhibited a single DNA molecule on the negatively-charged substrate in an isolated state. Further, the photoluminescence spectra of a single DNA molecule stained with YOYO-1 were achieved using the method developed in the present study, which is strongly believed to effectively support the spectroscopic analysis of DNA in a single molecular level.

  17. Photonic translation of DNAs between microscopic beads and a substrate for a photonic DNA memory

    NASA Astrophysics Data System (ADS)

    Ogura, Yusuke; Beppu, Taro; Shogenji, Rui; Tanida, Jun

    2006-08-01

    A DNA memory is a storage system utilizing inherent features of DNA, which is promising as a fundamental technology of nanoscale computing. Realizing a practical DNA memory requires establishment of a method for accessing to and controlling certain DNA strands among a lot of strands in a solution with high accuracy and selectivity. For addressing this issue, we have proposed a DNA memory using photonic techniques: the photonic DNA memory. Manipulation of information by using DNAs on a nanoscale and light on a microscale is effective in achieving a high capacity and flexible memory. This paper reports on experimental results of photonic translation of DNAs containing data between microscopic beads and a substrate. The technique is expected to be useful in writing, transferring, and reading necessary information in a photonic DNA memory effectively. In the experiments, we prepared a glass substrate coated with titanylphthalocyanine for light absorption and gold for DNA attachment. Data container DNA strands, which were labeled by fluorescence-dye for observation, were attached on the substrate by hybridization with their complementary strands immobilized on the substrate; then a solution containing 6-micrometer-diameter beads on which DNA strands including the complementary sequence of the data container DNA was placed on the substrate. After a bead was irradiated with a laser beam and translated on the substrate, the fluorescence intensity of the substrate decreased and that of the bead increased. The result indicates that the data container DNA was moved from the substrate to the bead owing to change of the temperature of the solution at the irradiated area.

  18. Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores.

    PubMed

    Nikiforov, Theo T; Roman, Steven

    2015-05-15

    Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3' ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5' phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5' extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido-pyrimidine-DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions). PMID:25728944

  19. Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

    SciTech Connect

    Lee, Seongmin; Verdine, Gregory L.

    2010-01-14

    Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases have been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.

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

    PubMed

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

    2003-01-01

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

  1. Label free detection of DNA on Au/ZnO/Ag hybrid structure based SERS substrate

    NASA Astrophysics Data System (ADS)

    Pal, Anil Kumar; Mohan, D. Bharathi

    2016-04-01

    Au/ZnO/Ag based SERS substrate was fabricated for the label free detection of DNA of Escherichia Coli bacteria. The SERS substrate was fabricated by growing ZnO nanorod arrays on thermally evaporated ultrathin Ag film of 5 nm thickness using hydrothermal process. Non-spherical like Au nanoparticles were decorated on ZnO nanorod arrays by sputtering technique with sputtering time of 45 sec. The surface of Au/ZnO/Ag was observed to be nearly superhydrophobic exhibiting the contact angle of 144 °. A low volume (5 µl) of aqueous solution of DNA of laboratory strain Escherichia Coli with very low concentration was adsorbed on fabricated SERS substrate by drop casting. The SERS detection of DNA molecules was achieved up to lower concentration of 10-8 M due to strong local electric field enhancement at the nanometer gap among Au nanoparticles and superhydrophobic nature of Au/ZnO/Ag surface.

  2. Influence of Stream Bottom Substrate on Retention and Transport of Vertebrate Environmental DNA.

    PubMed

    Jerde, Christopher L; Olds, Brett P; Shogren, Arial J; Andruszkiewicz, Elizabeth A; Mahon, Andrew R; Bolster, Diogo; Tank, Jennifer L

    2016-08-16

    While environmental DNA (eDNA) is now being regularly used to detect rare and elusive species, detection in lotic environments comes with a caveat: The species being detected is likely some distance upstream from the point of sampling. Here, we conduct a series of seminatural stream experiments to test the sensitivity of new digital droplet PCR (ddPCR) to detect low concentrations of eDNA in a lotic system, measure the residence time of eDNA compared to a conservative tracer, and we model the transport of eDNA in this system. We found that while ddPCR improves our sensitivity of detection, the residence time and transport of eDNA does not follow the same dynamics as the conservative tracer and necessitates a more stochastic framework for modeling eDNA transport. There was no evidence for differences in the transport of eDNA due to substrate type. The relatively large amount of unexplained variability in eDNA transport reveals the need for uncovering mechanisms and processes by which eDNA is transported downstream leading to species detections, particularly when inferences are to be made in natural systems where eDNA is being used for conservation management. PMID:27409250

  3. DNA sequencing using polymerase substrate-binding kinetics

    PubMed Central

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

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  4. DNA sequencing using polymerase substrate-binding kinetics.

    PubMed

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

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  5. Systematic study for DNA recovery and profiling from common IED substrates: From laboratory to casework.

    PubMed

    Phetpeng, Sukanya; Kitpipit, Thitika; Thanakiatkrai, Phuvadol

    2015-07-01

    Improvised explosive devices (IEDs) made from household items are encountered in terrorist attacks worldwide. Assembling an IED leaves trace DNA on its components, but deflagration degrades DNA. To maximize the amount of DNA recovered, a systematic evaluation of DNA collection methods was carried out and the most efficient methods were implemented with IED casework evidence as a validation exercise. Six swab types and six moistening agents were used to collect dried buffy coat stains on four common IED substrates. The most efficient swab/moistening agent combinations were then compared with tape-lifting using three brands of adhesive tape and also with direct DNA extraction from evidence. The most efficient collection methods for different IED substrates (post-study protocol) were then implemented for IED casework and compared with the pre-study protocol using 195 pieces of IED evidence. There was no single best swab type or moistening agent. Swab type had the largest effect on DNA recovery percentages, but moistening agents, substrates, and the interactions between factors all affected DNA recovery. The most efficient swab/moistening agent combinations performed equally well when compared with the best adhesive tape and direct extraction. The post-study protocol significantly improved STR profiles obtained from IED evidence. This paper outlines a comprehensive study of DNA collection methods for trace DNA and the validation of the most efficient collection methods with IED evidence. The findings from both parts of this study emphasize the need to continuously re-evaluate standard operating protocols with empirical studies. PMID:25828367

  6. Influence of disordered packing pattern on elastic modulus of single-stranded DNA film on substrate.

    PubMed

    Meng, W L; Zhang, N H; Tang, H S; Tan, Z Q

    2015-10-01

    Determining mechanical properties of single-stranded DNA film grafted on gold surface is critical for analysis and design of DNA-microcantilever biosensors. However, it remains an open issue to quantify the relations among the disordered packing patterns of DNA chains, the mechanical properties of DNA film and the resultant biodetection signals. In this paper, first, the bending experiment of microcantilever is carried out to provide the basic data for a refined multi-scale model of microcantilever deflection induced by ssDNA immobilization. In the model, the complicated interactions in DNA film (consisting of DNA, water molecules and salt ions) are simplified as effective interactions among coarse-grained soft cylinders, which can reveal the varieties of DNA structure in the circumstances of different lengths and salt concentrations; Ohshima's distribution of net charge density is employed to incorporate compositional variations of salt ions along the thickness direction into the Strey's mesoscopic empirical potential on molecular interactions in DNA solutions, and the related model parameters for ssDNA film on substrate are obtained from the curve fitting with our microcantilever bending experiment. Second, the effect of nanoscopic distribution of DNA chains on elastic modulus of ssDNA film is studied by a thought experiment of uniaxial compression, and the disordered patterns of DNA chains are generated by Monte Carlo method. Simulation results point out that nanoscale ssDNA film shows size effect, gradient and diversity in elastic modulus and can achieve maximum stiffness by preferring a disordered and energetically favorable packing pattern collectively induced by electrostatic force, hydration force and configurational entropy. PMID:25749909

  7. In Vitro Selection of Optimal DNA Substrates for Ligation by a Water-Soluble Carbodiimide

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1994-01-01

    We have used in vitro selection to investigate the sequence requirements for efficient template-directed ligation of oligonucleotides at 0 deg C using a water-soluble carbodiimide as condensing agent. We find that only 2 bp at each side of the ligation junction are needed. We also studied chemical ligation of substrate ensembles that we have previously selected as optimal by RNA ligase or by DNA ligase. As anticipated, we find that substrates selected with DNA ligase ligate efficiently with a chemical ligating agent, and vice versa. Substrates selected using RNA ligase are not ligated by the chemical condensing agent and vice versa. The implications of these results for prebiotic chemistry are discussed.

  8. Detection of specific DNA using a microfluidic device featuring tethered poly(N-isopropylacrylamide) on a silicon substrate

    NASA Astrophysics Data System (ADS)

    Chen, Jem-Kun; Li, Jun-Yan

    2010-08-01

    In this study, we grafted thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) onto a Si substrate as the medium in a microfluidic device to detect specific DNA molecules [human genomic DNA (hgDNA528), 528 bp] at extremely low concentrations (down to 2 ng/μl). After using the polymerase chain reaction to amplify the released human gDNA signal from the tethered PNIPAAm on the substrate, the amplified human gDNA molecules were characterized through agarose gel electrophoresis. The tethered PNIPAAm in the fluid device allowed the precise detection of the human gDNA.

  9. DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

    PubMed Central

    Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

    2016-01-01

    Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

  10. Size-Selective Nanoparticle Assembly on Substrates by DNA Density Patterning.

    PubMed

    Myers, Benjamin D; Lin, Qing-Yuan; Wu, Huanxin; Luijten, Erik; Mirkin, Chad A; Dravid, Vinayak P

    2016-06-28

    The vision of nanoscale self-assembly research is the programmable synthesis of macroscale structures with controlled long and short-range order that exhibit a desired set of properties and functionality. However, strategies to reliably isolate and manipulate the nanoscale building blocks based on their size, shape, or chemistry are still in their infancy. Among the promising candidates, DNA-mediated self-assembly has enabled the programmable assembly of nanoparticles into complex architectures. In particular, two-dimensional assembly on substrates has potential for the development of integrated functional devices and analytical systems. Here, we combine the high-resolution patterning capabilities afforded by electron-beam lithography with the DNA-mediated assembly process to enable direct-write grayscale DNA density patterning. This method allows modulation of the functionally active DNA surface density to control the thermodynamics of interactions between nanoparticles and the substrate. We demonstrate that size-selective directed assembly of nanoparticle films from solutions containing a bimodal distribution of particles can be realized by exploiting the cooperativity of DNA binding in this system. To support this result, we study the temperature-dependence of nanoparticle assembly, analyze the DNA damage by X-ray photoelectron spectroscopy and fluorescence microscopy, and employ molecular dynamics simulations to explore the size-selection behavior. PMID:27192324

  11. Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase

    PubMed Central

    Jarchow-Choy, Sarah K.; Krueger, Andrew T.; Liu, Haibo; Gao, Jianmin; Kool, Eric T.

    2011-01-01

    Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3′-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson–Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)n polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)n, fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA. PMID:20947563

  12. DNA with Damage in Both Strands as Affinity Probes and Nucleotide Excision Repair Substrates.

    PubMed

    Lukyanchikova, N V; Petruseva, I O; Evdokimov, A N; Silnikov, V N; Lavrik, O I

    2016-03-01

    Nucleotide excision repair (NER) is a multistep process of recognition and elimination of a wide spectrum of damages that cause significant distortions in DNA structure, such as UV-induced damage and bulky chemical adducts. A series of model DNAs containing new bulky fluoro-azidobenzoyl photoactive lesion dC(FAB) and well-recognized nonnucleoside lesions nFlu and nAnt have been designed and their interaction with repair proteins investigated. We demonstrate that modified DNA duplexes dC(FAB)/dG (probe I), dC(FAB)/nFlu+4 (probe II), and dC(FAB)/nFlu-3 (probe III) have increased (as compared to unmodified DNA, umDNA) structure-dependent affinity for XPC-HR23B (Kdum > KdI > KdII ≈ KdIII) and differentially crosslink to XPC and proteins of NER-competent extracts. The presence of dC(FAB) results in (i) decreased melting temperature (ΔTm = -3°C) and (ii) 12° DNA bending. The extended dC(FAB)/dG-DNA (137 bp) was demonstrated to be an effective NER substrate. Lack of correlation between the affinity to XPC-HR23B and substrate properties of the model DNA suggests a high impact of the verification stage on the overall NER process. In addition, DNAs containing closely positioned, well-recognized lesions in the complementary strands represent hardly repairable (dC(FAB)/nFlu+4, dC(FAB)/nFlu-3) or irreparable (nFlu/nFlu+4, nFlu/nFlu-3, nAnt/nFlu+4, nAnt/nFlu-3) structures. Our data provide evidence that the NER system of higher eukaryotes recognizes and eliminates damaged DNA fragments on a multi-criterion basis. PMID:27262196

  13. High specific surface gold electrode on polystyrene substrate: Characterization and application as DNA biosensor.

    PubMed

    Yang, Zhiliu; Liu, Yichen; Lu, Wei; Yuan, Qingpan; Wang, Wei; Pu, Qiaosheng; Yao, Bo

    2016-05-15

    In the past decades, many efforts have been made to improve the sensitivity and specificity of electrochemical DNA biosensors. However, it is still strongly required to develop disposable and reliable DNA biosensors for wide and practical application. In this article, we reported superior electrochemical properties of an integrated plastic-gold electrode (PGE) fabricated in-house by chemical plating on polystyrene substrate. PGEs were found having extremely high capacity of DNA immobilization compared with gold electrodes fabricated by standard sputtering based photolithography. Unique nano-structured surface was observed on PGEs through morphology techniques, which would to some extend give an explanation to higher capacity of DNA immobilization on PGEs. A probable mechanism of carboxylic acid produced on polystyrene substrate after exposure to UV irradiation was proposed and discussed for the first time. This biosensor was applied to detection and manipulate of DNA hybridization. Detection limit of 7.2×10(-11)M and 1-500nM of linearity range was obtained. PMID:26992524

  14. Swabs as DNA collection devices for sampling different biological materials from different substrates.

    PubMed

    Verdon, Timothy J; Mitchell, Robert J; van Oorschot, Roland A H

    2014-07-01

    Currently, there is a variety of swabs for collection of biological evidence from crime scenes, but their comparative efficiency is unknown. Here, we report the results of an investigation into the efficiency of different swab types to collect blood, saliva and touch DNA from a range of substrates. The efficiency of extracting blood and saliva from each swab type was also tested. Some swabs were significantly more effective than others for sampling biological materials from different substrates. Swabs with the highest sampling efficiency, however, often did not have the highest extraction efficiency. Observations were recorded regarding practicality of each swab in a variety of situations. Our study demonstrates that selection of sampling device impacts greatly upon successful collection and extraction of DNA. We present guidelines to assist in evaluation of swab choice. PMID:24502761

  15. Design of Novel Relaxase Substrates Based on Rolling Circle Replicases for Bioconjugation to DNA Nanostructures

    PubMed Central

    Sagredo, Sandra; de la Cruz, Fernando; Moncalián, Gabriel

    2016-01-01

    During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence (nic site). Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures. PMID:27027740

  16. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

    NASA Astrophysics Data System (ADS)

    Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G.; Woodbury, Neal W.; Yan, Hao

    2014-07-01

    Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

  17. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm.

    PubMed

    Fu, Jinglin; Yang, Yuhe Renee; Johnson-Buck, Alexander; Liu, Minghui; Liu, Yan; Walter, Nils G; Woodbury, Neal W; Yan, Hao

    2014-07-01

    Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity. PMID:24859813

  18. Simulation of Epitaxial Growth of DNA-nanoparticle Superlattices on Pre-patterned Substrates

    NASA Astrophysics Data System (ADS)

    Pan, Saijie; Li, Ting; Olvera de La Cruz, Monica

    2015-03-01

    DNA self-assembly is a well-developed approach towards the construction of a great variety of nanoarchitectures. E-beam lithography is widely used for high-resolution nanoscale patterning. Recently, a new technique combining the two methods was developed to epitaxially grow DNA-mediated nanoparticle superlattices on a pre-patterned surface. Here we use multi-scale simulations to study and predict the formation and defects of the absorbed superlattice monolayer. We demonstrate that the epitaxial growth is enthalpy driven and show that the anisotropy of the DNA-mediated substrates leads to structure defects. We develop design rules to dramatically reduce defects of the attached layer. Ultimately, with the assist of our simulation, this technique will open the door for the construction of well-ordered, three-dimensional novel metamaterials. This work was supported by the the Air Force Office of Scientific Research (AFOSR) Multidisciplinary University Research Initiative (MURI) FA9550-11-1-0275.

  19. Modelling the transport of environmental DNA through a porous substrate using continuous flow-through column experiments.

    PubMed

    Shogren, Arial J; Tank, Jennifer L; Andruszkiewicz, Elizabeth A; Olds, Brett; Jerde, Christopher; Bolster, Diogo

    2016-06-01

    Detecting environmental DNA (eDNA) in water samples is a powerful tool in determining the presence of rare aquatic species. However, many open questions remain as to how biological and physical conditions in flowing waters influence eDNA. Motivated by what one might find in a stream/river benthos we conducted experiments in continuous flow columns packed with porous substrates to explore eDNA transport and ask whether substrate type and the presence of colonized biofilms plays an important role for eDNA retention. To interpret our data, and for modelling purposes, we began with the assumption that eDNA could be treated as a classical tracer. Comparing our experimental data with traditional transport models, we found that eDNA behaves anomalously, displaying characteristics of a heterogeneous, polydisperse substance with particle-like behaviour that can be filtered by the substrate. Columns were quickly flushed of suspended eDNA particles while a significant amount of particles never made it through and were retained in the column, as calculated from a mass balance. Suspended eDNA was exported through the column, regardless of biofilm colonization. Our results indicate that the variable particle size of eDNA results in stochastic retention, release and transport, which may influence the interpretation eDNA detection in biological systems. PMID:27251680

  20. Highly Specific, Bi-substrate-Competitive Src Inhibitors from DNA-Templated Macrocycles

    PubMed Central

    Georghiou, George; Kleiner, Ralph E.; Pulkoski-Gross, Michael

    2011-01-01

    Protein kinases are attractive therapeutic targets, but their high sequence and structural conservation complicates the development of specific inhibitors. We recently discovered from a DNA-templated macrocycle library inhibitors with unusually high selectivity among Src-family kinases. Starting from these compounds, we developed and characterized in molecular detail potent macrocyclic inhibitors of Src kinase and its cancer-associated gatekeeper mutant. We solved two co-crystal structures of macrocycles bound to Src kinase. These structures reveal the molecular basis of the combined ATP- and substrate peptide-competitive inhibitory mechanism and the remarkable kinase specificity of the compounds. The most potent compounds inhibit Src activity in cultured mammalian cells. Our work establishes that macrocycles can inhibit protein kinases through a bi-substrate competitive mechanism with high potency and exceptional specificity, reveals the precise molecular basis for their desirable properties, and provides new insights into the development of Src-specific inhibitors with potential therapeutic relevance. PMID:22344177

  1. Oligodeoxynucleotides as substrates for O/sup 6/-alkylguanine-DNA alkyltransferase (AGT)

    SciTech Connect

    Scicchitano, D.; Jones, R.A.; Kuzmich, S.; Gaffney, B.; Lasko, D.; Essigmann, J.; Pegg, A.E.

    1986-05-01

    AGT is a DNA repair protein which is known to catalyze the transfer of alkyl groups from the O/sup 6/-position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence. The authors have examined the ability of this protein isolated from both E. coli and mammalian cells to carry out this reaction using oligodeoxynucleotides containing O/sup 6/-methylguanine (m/sup 6/G). Dodecadeoxynucleotides of the sequence 5'-dCGBGAATTCm/sup 6/GCG-3' where B is any one of the normal 4 bases were all repaired very rapidly with 50% repair in less than 15 sec at 0/sup 0/C. The hexadeoxynucleotide 5'-dCGCm/sup 6/GCG-3' was repaired slightly more slowly with 50% removal requiring 7 min at 0/sup 0/C and 1.5 min at 37/sup 0/C. The tetradeoxynucleotide 5'-dTm/sup 6/GCA-3' was also a substrate for the protein but was repaired much more slowly requiring 45 min for 50% repair at 37/sup 0/C. These results indicate that (a) the AGT has a strong but not absolute preference for double stranded DNA substrates; (b) the repair of m/sup 6/G is independent of the base opposite the lesion; and (c) that very short oligodeoxynucleotides are substrates for repair by this protein. The latter property was used to set up a very sensitive assay procedure for the AGT. The 5' end of the 5'-dTm/sup 6/GCA-3' was labeled with /sup 32/P by use of polynucleotide kinase and the formation of (/sup 32/P)-5'-dTGACA-3' monitored by separating the methylated form from the unmethylated form by HPLC.

  2. Directional Regulation of Enzyme Pathways through the Control of Substrate Channeling on a DNA Origami Scaffold.

    PubMed

    Ke, Guoliang; Liu, Minghui; Jiang, Shuoxing; Qi, Xiaodong; Yang, Yuhe Renee; Wootten, Shaun; Zhang, Fei; Zhu, Zhi; Liu, Yan; Yang, Chaoyong James; Yan, Hao

    2016-06-20

    Artificial multi-enzyme systems with precise and dynamic control over the enzyme pathway activity are of great significance in bionanotechnology and synthetic biology. Herein, we exploit a spatially addressable DNA nanoplatform for the directional regulation of two enzyme pathways (G6pDH-MDH and G6pDH-LDH) through the control of NAD(+) substrate channeling by specifically shifting NAD(+) between the two enzyme pairs. We believe that this concept will be useful for the design of regulatory biological circuits for synthetic biology and biomedicine. PMID:27159899

  3. Two-Dimensional Crystal Structure Formed by Two Components of DNA Nanoparticles on a Substrate

    NASA Astrophysics Data System (ADS)

    Katsuno, Hiroyasu; Maegawa, Yuya; Sato, Masahide

    2016-07-01

    We study the two-dimensional crystal structure of two components of DNA nanoparticles on a substrate by Brownian dynamics simulation. We use the Lennard-Jones potential as the interaction potential between particles and assume that the interaction length between different types of particles, σAB, is smaller than that between the same types of particles, σ. Two types of particles form an alloy structure. With decreasing σAB/σ, the crystal structure changes from a triangular lattice, to a square lattice, a honeycomb lattice, a rectangular lattice, and a triangular lattice.

  4. Oriented Chiral DNA-Silica Film Guided by a Natural Mica Substrate.

    PubMed

    Cao, Yuanyuan; Kao, Kunche; Mou, Chungyuan; Han, Lu; Che, Shunai

    2016-02-01

    The formation of highly ordered chiral organic/inorganic films with high density and long-range orientation is important in constructing chiral devices, such as broadband polarization devices, liquid-crystal displays, or negative-reflection materials. A feasible strategy is presented to fabricate three-dimensional mesostructured chiral DNA-silica assemblies into large-scale oriented arrangements. The highly ordered film was aligned by a mica crystal substrate with the bridging effect of suitable divalent metal ions, followed by the growth of the DNA-silica composite by bottom-up assembly with a "quartet templating" method. This simple and effective route would perform well in the alignment and arrangement of highly charged biomolecules, such as polypeptides, proteins, viruses, and their inorganic assemblies, and furthermore could allow the fabrication of chiral optical materials with long-range ordering. PMID:26836337

  5. Analysis of substrate specificity of Schizosaccharomyces pombe Mag1 alkylpurine DNA glycosylase

    SciTech Connect

    Adhikary, Suraj; Eichman, Brandt F.

    2014-10-02

    DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity, although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N{sup 6}-ethenoadenine ({var_epsilon}A) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their {var_epsilon}A activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.

  6. DNA Substrate-Induced Activation of the Agrobacterium VirB/VirD4 Type IV Secretion System

    PubMed Central

    Cascales, Eric; Atmakuri, Krishnamohan; Sarkar, Mayukh K.

    2013-01-01

    The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface. PMID:23564169

  7. Uracil DNa-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs.

    PubMed

    Krokan, H; Wittwer, C U

    1981-06-11

    Uracil-DNA glycosylase was partially purified from HeLa cells. Various substrates containing [3H]dUMP residues were prepared by nick-translation of calf thymus DNA. The standard substrate was double-stranded DNA with [3H]dUMP located internally in the chain. Compared to the release of uracil from this substrate, a 3-fold increase in the rate was seen with single-stranded DNA, and a 20-fold reduction in the rate was observed when the [3H]dUMP-residue was located at the 3'end. The rate of [3H]uracil release decreased progressively when one, two or three of the dNMP residues were replaced by the corresponding rNMP; in the extreme case when the substrate contained [3H]dUMP in addition to rCMP, rGMP, and rAMP, the rate of [3H]uracil release was less than 3% of that of the control. The enzyme was inhibited to the same extent by uracil and the uracil analogs 6-aminouracil and 5-azauracil, but very weakly, or not at all, by 5 other analogs. Our results suggest strongly that uracil-DNA glycosylase has a high degree of selectivity for uracil in dUMP residues located internally in DNA chains and that the recognition of the correct substrate also depends on the residues flanking dUMP being deoxyribonucleotides. PMID:7279657

  8. Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

    PubMed Central

    Kuznetsov, Nikita A.; Koval, Vladimir V.; Zharkov, Dmitry O.; Nevinsky, Georgy A.; Douglas, Kenneth T.; Fedorova, Olga S.

    2005-01-01

    Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG. PMID:16024742

  9. hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks

    SciTech Connect

    Berglund, Fredrik M.; Clarke, Paul R.

    2009-03-27

    Cellular responses to DNA damage are orchestrated by the large phosphoinositol-3-kinase related kinases ATM, ATR and DNA-PK. We have developed a cell-free system to dissect the biochemical mechanisms of these kinases. Using this system, we identify heterogeneous nuclear ribonucleoprotein U (hnRNP-U), also termed scaffold attachment factor A (SAF-A), as a specific substrate for DNA-PK. We show that hnRNP-U is phosphorylated at Ser59 by DNA-PK in vitro and in cells in response to DNA double-strand breaks. Phosphorylation of hnRNP-U suggests novel functions for DNA-PK in the response to DNA damage.

  10. Interactions of the SAP Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Carra, Claudio; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

    2007-01-01

    NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku70/80 complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The SAP domain of Ku70 (residues 556-609), contains an a helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the SAP/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the SAP domain of Ku70 and a 10 base pairs DNA duplex. Large-scale conformational changes were observed and some putative binding modes were suggested based on energetic analysis. These modes are consistent with previous experimental investigations. In addition, the results indicate that cooperation of SAP with other domains of Ku70/80 is necessary to explain the high affinity of binding as observed in experiments.

  11. Plant DNA: A new substrate for carbon stable isotope analysis and a potential paleoenvironmental indicator

    NASA Astrophysics Data System (ADS)

    Jahren, A. Hope; Petersen, Gitte; Seberg, Ole

    2004-03-01

    The δ13C value of fossil plant materials can be used to gain insight into the dominant photosynthetic pathway, as well as other environmental attributes, of ancient plant ecosystems. Nucleotide sequences from land plant nucleic acids extracted from 400 ka fossil sediments have been recognized as the oldest authenticated fossil deoxyribonucleic acid (DNA), making the inference of plant taxonomy possible in substrates devoid of plant macrofossils and microfossils. If the C isotope relationship between bulk plant tissue and associated plant nucleic acids were known, fossil plant nucleic acids could be analyzed for δ13C value and used as land plant isotopic substrates within mixed organic material. Toward this end, we present δ13C analyses of nucleic acids isolated from 12 higher plant species that span the full phylogenetic diversity of seed plants. Extracted nucleic acids were dominated by double-stranded DNA containing fragments of rbcL gene ˜350 base pairs in length. The C isotope compositions of plant nucleic acids were found to be enriched in 13C relative to bulk plant tissue by a constant value = 1.39‰. This study represents the first comparison of the δ13C value of nucleic acids to the δ13C value of bulk tissue for multicellular organisms; our results contrasted with the minimal fractionations reported for microorganisms. Because the isotopic enrichment is constant across tracheophytes, the δ13C value of fossil plant DNA can be used as a paleoenvironmental indicator, eliminating the need for morphological recognition of fossil plant material in paleoenvironmental studies.

  12. Plant DNA: A new substrate for carbon stable isotope analysis and a potential paleoenvironmental indicator

    NASA Astrophysics Data System (ADS)

    Jahren, H.; Petersen, G.; Seberg, O.

    2003-12-01

    The δ 13C value of fossil plant materials can be used to gain insight into the dominant photosynthetic pathway, as well as other environmental attributes, of ancient plant ecosystems. Nucleotide sequences from land-plant nucleic acids extracted from 400 ka sediments have been recognized as the oldest authenticated fossil DNA, making the inference of plant taxonomy possible in substrates devoid of plant macro- and microfossils. If the C isotope relationship between bulk plant tissue and associated plant nucleic acids were known, fossil plant nucleic acids could be analyzed for δ 13C values and used as land-plant isotopic substrates within mixed organic material. Toward this end, we present δ 13C analyses of nucleic acids isolated from 12 higher-plant species that span the full phylogenetic diversity of seed plants. Extracted nucleic acids were dominated by double-stranded DNA containing fragments of rbcL gene ˜ 350 base pairs in length. The C isotope compositions of plant nucleic acids were found to be enriched in 13C relative to bulk plant tissue by a constant value (1.39 ‰ ). This study represents the first comparison of the δ 13C value of nucleic acids to the δ 13C value of bulk tissue for multicellular organisms; our results contrasted with the minimal fractionations reported for microorganisms. Because the isotopic enrichment δ 13C is constant across tracheophytes, the δ 13C value of fossil plant DNA can be used as a paleoenvironmental indicator, eliminating the need for morphological recognition of fossil plant material in paleoenvironmental studies.

  13. Monitoring conformational heterogeneity of the lid of DnaK substrate-binding domain during its chaperone cycle.

    PubMed

    Banerjee, Rupa; Jayaraj, Gopal Gunanathan; Peter, Joshua Jebakumar; Kumar, Vignesh; Mapa, Koyeli

    2016-08-01

    DnaK or Hsp70 of Escherichia coli is a master regulator of the bacterial proteostasis network. Allosteric communication between the two functional domains of DnaK, the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate- or peptide-binding domain (SBD) regulate its activity. X-ray crystallography and NMR studies have provided snapshots of distinct conformations of Hsp70 proteins in various physiological states; however, the conformational heterogeneity and dynamics of allostery-driven Hsp70 activity remains underexplored. In this work, we employed single-molecule Förster resonance energy transfer (sm-FRET) measurements to capture distinct intradomain conformational states of a region within the DnaK-SBD known as the lid. Our data conclusively demonstrate prominent conformational heterogeneity of the DnaK lid in ADP-bound states; in contrast, the ATP-bound open conformations are homogeneous. Interestingly, a nonhydrolysable ATP analogue, AMP-PNP, imparts heterogeneity to the lid conformations mimicking the ADP-bound state. The cochaperone DnaJ confers ADP-like heterogeneous lid conformations to DnaK, although the presence of the cochaperone accelerates the substrate-binding rate by a hitherto unknown mechanism. Irrespective of the presence of DnaJ, binding of a peptide substrate to the DnaK-SBD leads to prominent lid closure. Lid closure is only partial upon binding to molten globule-like authentic cellular substrates, probably to accommodate non-native substrate proteins of varied structures. PMID:27248857

  14. Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase: SUBSTRATE SPECIFICITY, DNA BRANCH MIGRATION, AND ABILITY TO OVERCOME BLOCKADES TO DNA UNWINDING.

    PubMed

    Khan, Irfan; Crouch, Jack D; Bharti, Sanjay Kumar; Sommers, Joshua A; Carney, Sean M; Yakubovskaya, Elena; Garcia-Diaz, Miguel; Trakselis, Michael A; Brosh, Robert M

    2016-07-01

    Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5' or 3' single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5' to 3' directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding. PMID:27226550

  15. Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

    NASA Technical Reports Server (NTRS)

    Winters, T. A.; Russell, P. S.; Kohli, M.; Dar, M. E.; Neumann, R. D.; Jorgensen, T. J.

    1999-01-01

    Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

  16. Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

    PubMed

    Jiang, Jingwei; Zhou, Zunchun; Dong, Ying; Sun, Hongjuan; Chen, Zhong; Yang, Aifu; Gao, Shan; Wang, Bai; Jiang, Bei; Guan, Xiaoyan

    2014-02-01

    Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses. PMID:24355405

  17. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis.

    PubMed

    Abeldenov, Sailau; Talhaoui, Ibtissam; Zharkov, Dmitry O; Ishchenko, Alexander A; Ramanculov, Erlan; Saparbaev, Murat; Khassenov, Bekbolat

    2015-09-01

    Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3'→5' exonuclease activities. However, it remains unclear whether these enzymes hold 3'-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3'-repair phosphodiesterase and 3'-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37 °C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3'-blocking sugar-phosphate and 3'-phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM=440 and 1280 μM(-1)∙min(-1), respectively), while MtbNfo exhibits much lower 3'-repair activities (kcat/KM=0.26 and 0.65 μM(-1)∙min(-1), respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role

  18. Aphidicolin resistance in herpes simplex virus type 1 appears to alter substrate specificity in the DNA polymerase

    SciTech Connect

    Hall, J.D.; Woodward, S.

    1989-06-01

    The authors describe novel mutants of herpes simplex virus which are resistant to aphidicolin. Their mutant phenotypes suggest that they encode DNA polymerases with altered substrate recognition. This conclusion is based on their abnormal sensitivity to polymerase inhibitors and to the abnormal mutation rates exhibited by two of the mutants.

  19. Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme

    PubMed Central

    Lagunavicius, Arunas; Kiveryte, Zivile; Zimbaite-Ruskuliene, Vilma; Radzvilavicius, Tomas; Janulaitis, Arvydas

    2008-01-01

    Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3′→5′ DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3′ to 5′ polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is ∼10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection. PMID:18230765

  20. Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

    SciTech Connect

    Holland, P.; Hollis, T

    2010-01-01

    In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  1. Photoelectron imaging of viruses and DNA: evaluation of substrates by unidirectional low angle shadowing and photoemission current measurements.

    PubMed Central

    Birrell, G B; Habliston, D L; Griffith, O H

    1994-01-01

    Photoelectron imaging (photoelectron emission microscopy, PEM or PEEM) is a promising high resolution surface-sensitive technique for biophysical studies. At present, image quality is often limited by the underlying substrate. For photoelectron imaging, the substrate must be electrically conductive, low in electron emission, and relatively flat. A number of conductive substrate materials with relatively low electron emission were examined for surface roughness. Low angle, unidirectional shadowing of the specimens followed by photoelectron microscopy was found to be an effective way to test the quality of substrate surfaces. Optimal results were obtained by depositing approximately 0.1 nm of platinum-palladium (80:20) at an angle of 3 degrees. Among potential substrates for photoelectron imaging, silicon and evaporated chromium surfaces were found to be much smoother than evaporated magnesium fluoride, which initially appeared promising because of its very low electron emission. The best images were obtained with a chromium substrate coated with a thin layer of dextran derivatized with spermidine, which facilitated the spreading and adhesion of biomolecules to the surfaces. Making use of this substrate, improved photoelectron images are reported for tobacco mosaic virus particles and DNA-recA complexes. Images FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:7858141

  2. Fabrication of a new substrate for atomic force microscopic observation of DNA molecules from an ultrasmooth sapphire plate.

    PubMed Central

    Yoshida, K; Yoshimoto, M; Sasaki, K; Ohnishi, T; Ushiki, T; Hitomi, J; Yamamoto, S; Sigeno, M

    1998-01-01

    A new stable substrate applicable to the observation of DNA molecules by atomic force microscopy (AFM) was fabricated from a ultrasmooth sapphire (alpha-Al2O3 single crystal) plate. The atomically ultrasmooth sapphire as obtained by high-temperature annealing has hydrophobic surfaces and could not be used for the AFM observation of DNA. However, sapphire treated with Na3PO4 aqueous solution exhibited a hydrophilic character while maintaining a smooth surface structure. The surface of the wet-treated sapphire was found by x-ray photoelectron spectroscopy and AFM to be approximately 0.3 nm. The hydrophilic surface character of the ultrasmooth sapphire plate made it easy for DNA molecules to adhere to the plate. Circular molecules of the plasmid DNA could be imaged by AFM on the hydrophilic ultrasmooth sapphire plate. PMID:9545030

  3. Enzymological and Structural Studies of the Mechanism of Promiscuous Substrate Recognition by the Oxidative DNA Repair Enzyme AlkB

    SciTech Connect

    Yu, B.; Hunt, J

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this 'kcat/Km compensation,' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding.

  4. Kinetics of Oligonucleotide Hybridization to DNA Probe Arrays on High-Capacity Porous Silica Substrates

    PubMed Central

    Glazer, Marc I.; Fidanza, Jacqueline A.; McGall, Glenn H.; Trulson, Mark O.; Forman, Jonathan E.; Frank, Curtis W.

    2007-01-01

    We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of ∼23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-μm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-μm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22°C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (ka, kd, and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22°C is described well by the predictive equations for

  5. Evaluation of circular DNA substrates for whole genome amplification prior to forensic analysis.

    PubMed

    Tate, Courtney M; Nuñez, Ada N; Goldstein, Cori A; Gomes, Iva; Robertson, James M; Kavlick, Mark F; Budowle, Bruce

    2012-03-01

    Forensic biological evidence often contains low quantities of DNA or substantially degraded DNA which makes samples refractory to genotype analysis. One approach that shows promise to overcome the limited quantity of DNA is whole genome amplification (WGA). One WGA technique, termed rolling circle amplification (RCA), involves the amplification of circular DNA fragments and this study evaluates a single-stranded (ss) DNA ligase enzyme for generating circular DNA templates for RCA WGA. Fast, efficient ligation of several sizes of ssDNA templates was achieved. The enzyme also ligated double-stranded (ds) DNA templates, a novel activity not previously reported. Adapter sequences containing optimal terminal nucleotide ends for increased ligation efficiency were designed and ligation of adapters to template DNA was optimized. Increased amplification of DNA templates was observed following WGA; however, no amplification advantage for ssDNA ligase treatment of templates was evident compared to linear templates. A multi-step process to utilize ssDNA ligase prior to WGA was developed and short tandem repeat (STR) analysis of simulated low template (LT) and fragmented DNA was evaluated. The process resulted in the loss of template DNA and failed STR analysis whereas input of linear genomic DNA template directly into WGA prior to STR analysis improved STR genotyping results compared to non-WGA treated samples. Inclusion of an extreme thermostable single-stranded DNA binding protein (SSB) during WGA also increased DNA yields. While STR artifacts such as peak imbalance, drop-in, and dropout persisted, WGA shows potential for successful genetic profiling of LT and fragmented DNA samples. Further research and development is warranted prior to use of WGA in forensic casework. PMID:21570374

  6. Structure of a DNA Repair Substrate Containing an Alkyl Interstrand Crosslink at 1.65 Å Resolution†‡

    PubMed Central

    Swenson, Matthew C.; Paranawithana, Shanthi R.; Miller, Paul S.; Kielkopf, Clara L.

    2008-01-01

    Chemotherapeutic alkylating agents, such as bifunctional nitrogen mustards and cisplatins, generate interstrand DNA crosslinks that inhibit cell proliferation by arresting DNA transcription and replication. A synthetic N4C-ethyl-N4C interstrand crosslink between opposing cytidines mimics the DNA damage produced by this class of clinically important compounds, and can be synthesized in large quantities to study the repair, physical properties, and structures of these DNA adducts. The X-ray structure of a DNA duplex d(CCAAC*GTTGG)2 containing a synthetic N4C-ethyl-N4C interstrand crosslink between the cytosines of the central CpG step (*) has been determined at 1.65 Å resolution. This structure reveals that the ethyl crosslink in the CpG major groove does not significantly disrupt the B-form DNA helix. Comparison of the N4C-ethyl-N 4C crosslinked structure with the structure of an uncrosslinked oligonucleotide of the same sequence reveals that the crosslink selectively stabilizes a pre-existing alternative conformation. The conformation preferred by the crosslinked DNA is constrained by the geometry of the ethyl group bridging the cytosine amines. Characteristics of the crosslinked CpG step include subtle differences in the roll of the base pairs, optimized Watson-Crick hydrogen bonds, and loss of a divalent cation binding site. Given that the N4C-ethyl-N4C crosslink stabilizes a pre-existing conformation of the CpG step, this synthetically accessible substrate presents an ideal model system for studying the genomic effects of covalently coupling the DNA strands, independent of gross alterations in DNA structure. PMID:17375936

  7. Dynamics of Bleomycin Interaction with a Strongly Bound Hairpin DNA Substrate, and Implications for Cleavage of the Bound DNA

    PubMed Central

    Bozeman, Trevor C.; Nanjunda, Rupesh; Tang, Chenhong; Liu, Yang; Segerman, Zachary J.; Zaleski, Paul A.; Wilson, W. David; Hecht, Sidney M.

    2013-01-01

    Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe•BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B2 binding to a strongly bound hairpin DNA, to define the effects of Fe3+, salt and temperature on BLM–DNA interaction. One strong primary DNA binding site, and at least one much weaker site was documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus enhanced binding to a given site does not necessarily result in increased DNA degradation at that site, i.e. for strongly bound DNAs, the facility of DNA cleavage must involve parameters in addition to the intrinsic rate of C-4′ H atom abstraction from DNA sugars. PMID:23072568

  8. A source of the single-stranded DNA substrate for activation-induced deaminase during somatic hypermutation.

    PubMed

    Wang, Xiaohua; Fan, Manxia; Kalis, Susan; Wei, Lirong; Scharff, Matthew D

    2014-01-01

    During somatic hypermutation (SHM), activation-induced deaminase (AID) mutates deoxycytidine on single-stranded DNA (ssDNA) generated by the transcription machinery, but the detailed mechanism remains unclear. Here we report a higher abundance of RNA polymerase II (Pol II) at the immunoglobulin heavy-chain variable (Igh-V) region compared with the constant region and partially transcribed Igh RNAs, suggesting a slower Pol II progression at Igh-V that could result in some early/premature transcription termination after prolonged pausing/stalling of Pol II. Knocking down RNA-exosome complexes, which could decrease premature transcription termination, leads to decreased SHM. Knocking down Spt5, which can augment premature transcription termination, leads to increase in both, SHM and the abundance of ssDNA substrates. Collectively, our data support the model that, following the reduction of Pol II progression (pausing or stalling) at the Igh-V, additional steps such as premature transcription termination are involved in providing ssDNA substrates for AID during SHM. PMID:24923561

  9. Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs.

    PubMed

    Alian, Akram; Griner, Sarah L; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D; Stroud, Robert M

    2009-05-19

    HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN-DNA complexes that form disulfide linkages between 5'-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors. PMID:19416821

  10. Detection of DNA damage: effect of thymidine glycol residues on the thermodynamic, substrate and interfacial acoustic properties of oligonucleotide duplexes.

    PubMed

    Yang, F; Romanova, E; Kubareva, E; Dolinnaya, N; Gajdos, V; Burenina, O; Fedotova, E; Ellis, J S; Oretskaya, T; Hianik, T; Thompson, M

    2009-01-01

    Thymidine glycol residues in DNA are biologically active oxidative molecular damage sites caused by ionizing radiation and other factors. One or two thymidine glycol residues were incorporated in 19- to 31-mer DNA fragments during automatic oligonucleotide synthesis. These oligonucleotide models were used to estimate the effect of oxidized thymidines on the thermodynamic, substrate and interfacial acoustic properties of DNA. UV-monitoring melting data revealed that modified residues in place of thymidines destabilize the DNA double helix by 8-22 degrees C, depending on the number of lesions, the length of oligonucleotide duplexes and their GC-content. The diminished hybridizing capacity of modified oligonucleotides is presumably due to the loss of aromaticity and elevated hydrophilicity of thymine glycol in comparison to the thymine base. According to circular dichroism (CD) data, the modified DNA duplexes retain B-form geometry, and the thymidine glycol residue introduces only local perturbations limited to the lesion site. The rate of DNA hydrolysis by restriction endonucleases R.MvaI, R.Bst2UI, R.MspR9I and R.Bme1390I is significantly decreased as the thymidine glycol is located in the central position of the double-stranded recognition sequences 5'-CC / WGG-3' (W = A, T) or 5'-CC / NGG-3' (N = A, T, G, C) adjacent to the cleavage site. On the other hand, the catalytic properties of enzymes R.Psp6I and R.BstSCI recognizing the similar sequence are not changed dramatically, since their cleavage site is separated from the point of modification by several base-pairs. Data obtained by gel-electrophoretic analysis of radioactive DNA substrates were confirmed by direct spectrophotometric assay developed by the authors. The effect of thymidine glycol was also observed on DNA hybridization at the surface of a thickness-shear mode acoustic wave device. A 1.9-fold decrease in the rate of duplex formation was noted for oligonucleotides carrying one or two thymidine glycol

  11. Detection of target DNA using photo-reactive protoporphyrin moeity on a nanocomposite substrate

    NASA Astrophysics Data System (ADS)

    Das, Sumana; Mishra, Madhusmita; Vasireddi, Ramakrishna; Roy Mahapatra, D.

    2014-03-01

    Detection of pathogens from infected biological samples through conventional process involves cell lysis and purification. The main objective of this work is to minimize the time and sample loss, as well as to increase the efficiency of detection of biomolecules. Electrical lysis of medical sample is performed in a closed microfluidic channel in a single integrated platform where the downstream analysis of the sample is possible. The device functions involve, in a sequence, flow of lysate from lysis chamber passed through a thermal denaturation counter where dsDNA is denatured to ssDNA, which is controlled by heater unit. A functionalized binding chamber of ssDNA is prepared by using ZnO nanorods as the matrix and functionalized with bifunctional carboxylic acid, 16-(2-pyridyldithiol) hexadecanoic acid (PDHA) which is further attached to a linker molecule 1-ethyl-3-(3-dimethylaminopropyl) (EDC). Linker moeity is then covalently bound to photoreactive protoporphyrin (PPP) molecule. The photolabile molecule protoporphyrin interacts with -NH2 labeled single stranded DNA (ssDNA) which thus acts as a probe to detect complimentary ssDNA from target organisms. Thereafter the bound DNA with protoporphyrin is exposed to an LED of particular wavelength for a definite period of time and DNA was eluted and analyzed. UV/Vis spectroscopic analysis at 260/280 nm wavelength confirms the purity and peak at 260 nm is reconfirmed for the elution of target DNA. Quantitative and qualitative data obtained from the current experiments show highly selective detection of biomolecule such as DNA which have large number of future applications in Point-of-Care devices.

  12. Tyrosyl-DNA phosphodiesterase I catalytic mutants reveal an alternative nucleophile that can catalyze substrate cleavage.

    PubMed

    Comeaux, Evan Q; Cuya, Selma M; Kojima, Kyoko; Jafari, Nauzanene; Wanzeck, Keith C; Mobley, James A; Bjornsti, Mary-Ann; van Waardenburg, Robert C A M

    2015-03-01

    Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3'-DNA adducts, such as the 3'-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His(nuc)) that attacks DNA adducts to form a covalent 3'-phosphohistidyl intermediate and a general acid/base His (His(gab)), which resolves the Tdp1-DNA linkage. A His(nuc) to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His(gab) to Arg. However, here we report that expression of the yeast His(nuc)Ala (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His(gab) mutants, including H432N and the SCAN1-related H432R. Moreover, the His(nuc)Ala mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His(nuc)Phe mutant was catalytically inactive and suppressed His(gab) mutant-induced toxicity. These data suggest that the activity of another nucleophile when His(nuc) is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His(nuc), can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate. PMID:25609251

  13. Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

    PubMed

    Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

    2015-11-17

    The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools. PMID:26494264

  14. Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.

  15. Eggshell membrane: A natural substrate for immobilization and detection of DNA.

    PubMed

    Ray, Preetam Guha; Roy, Somenath

    2016-02-01

    Chemically modified eggshell membranes (ESM) have been explored as potentially novel platforms for immobilization of oligonucleotides and subsequent detection of target DNA. The fibrous network of the native ESM as well those functionalized with acetic acid or n-butyl acetate has been examined by field-emission scanning electron microscopy (FESEM). The formation of surface functional moieties has been confirmed by Fourier-transform infrared spectroscopy (FTIR). DNA molecules, with an end terminal -NH2 group (at 5' end) have been immobilized on the chemically modified ESM surface. The effect of surface modification on the DNA immobilization efficiency has been investigated using fluorescence microscopy and atomic force microscopy (AFM). The above studies concurrently suggest that functionalization of ESM with n-butyl acetate causes a better homogeneity of the DNA probes on the membrane surface. On-chip hybridization of the target DNA with the surface bound capture probes has been performed on the functionalized membranes. It is observed that n-butyl acetate modification of ESM pushes the limit of detection (LOD) of the DNA sensors by at least an order of magnitude compared to the other modification method. PMID:26652390

  16. Interactions of the C-terminal Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

    2007-01-01

    NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku(sub 70/80) complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The C-terminal domain of Ku70 (Ku70c, residues 559-609), contains an helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the Ku70c/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the subunit Ku70c and a 14 base pairs DNA duplex, whose starting structures are designed to be variable so as to mimic their different binding modes. By analyzing conformational changes and energetic properties of the complex during MD simulations, we found that interactions are preferred at DNA ends, and within the major groove, which is consistent with previous experimental investigations. In addition, the results indicate that cooperation of Ku70c with other subunits of Ku(sub 70/80) is necessary to explain the high affinity of binding as observed in experiments.

  17. Products and substrate/template usage of vaccinia virus DNA primase

    SciTech Connect

    De Silva, Frank S.; Paran, Nir; Moss, Bernard

    2009-01-05

    Vaccinia virus encodes a 90-kDa protein conserved in all poxviruses, with DNA primase and nucleoside triphosphatase activities. DNA primase products, synthesized with a single stranded {phi}X174 DNA template, were resolved as dinucleotides and long RNAs on denaturing polyacrylamide and agarose gels. Following phosphatase treatment, the dinucleotides GpC and ApC in a 4:1 ratio were identified by nearest neighbor analysis in which {sup 32}P was transferred from [{alpha}-{sup 32}P]CTP to initiating purine nucleotides. Differences in the nucleotide binding sites for initiation and elongation were suggested by the absence of CpC and UpC dinucleotides as well as the inability of deoxynucleotides to mediate primer synthesis despite their incorporation into mixed RNA/DNA primers. Strong primase activity was detected with an oligo(dC) template. However, there was only weak activity with an oligo(dT) template and none with oligo(dA) or oligo(dG). The absence of stringent template specificity is consistent with a role for the enzyme in priming DNA synthesis at the replication fork.

  18. Saccharomyces cerevisiae Hrq1 requires a long 3 Prime -tailed DNA substrate for helicase activity

    SciTech Connect

    Kwon, Sung-Hun; Choi, Do-Hee; Lee, Rina; Bae, Sung-Ho

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Hrq1 has intrinsic 3 Prime -5 Prime helicase and DNA strand annealing activities. Black-Right-Pointing-Pointer Hrq1 requires a long 3 Prime -tail for efficient DNA unwinding. Black-Right-Pointing-Pointer Helicase activity of Hrq1 is stimulated by a fork structure. Black-Right-Pointing-Pointer Hrq1 is a moderately processive helicase. -- Abstract: RecQ helicases are well conserved proteins from bacteria to human and function in various DNA metabolism for maintenance of genome stability. Five RecQ helicases are found in humans, whereas only one RecQ helicase has been described in lower eukaryotes. However, recent studies predicted the presence of a second RecQ helicase, Hrq1, in fungal genomes and verified it as a functional gene in fission yeast. Here we show that 3 Prime -5 Prime helicase activity is intrinsically associated with Hrq1 of Saccharomyces cerevisiae. We also determined several biochemical properties of Hrq1 helicase distinguishable from those of other RecQ helicase members. Hrq1 is able to unwind relatively long duplex DNA up to 120-bp and is significantly stimulated by a preexisting fork structure. Further, the most striking feature of Hrq1 is its absolute requirement for a long 3 Prime -tail ( Greater-Than-Or-Slanted-Equal-To 70-nt) for efficient unwinding of duplex DNA. We also found that Hrq1 has potent DNA strand annealing activity. Our results indicate that Hrq1 has vigorous helicase activity that deserves further characterization to expand our understanding of RecQ helicases.

  19. Direct visualization of triplex DNA molecular dynamics by fluorescence resonance energy transfer and atomic force microscopy measurements

    NASA Astrophysics Data System (ADS)

    Chang, Chia-Ching; Lin, Po-Yen; Chen, Yen-Fu; Chang, Chia-Seng; Kan, Lou-Sing

    2007-11-01

    We have detected the dynamics of 17-mer DNA triplex dissociation mechanism at the molecular level. Fluorescence resonance energy transfer (FRET) was used as an indicator of intermolecular interaction in nanometer range, whereas atomic force microscopy (AFM) was employed to address single molecule with sub-angstrom precision. The maximum rupture force of DNA triplex was found at pH 4.65, consistent with macroscopic observations. These results indicated that the FRET together with an AFM detection system could be used to reveal the DNA triplex interaction in nanometer scale unambiguously.

  20. In vitro selection of optimal DNA substrates for T4 RNA ligase

    NASA Technical Reports Server (NTRS)

    Harada, Kazuo; Orgel, Leslie E.

    1993-01-01

    We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase. We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

  1. Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

    PubMed Central

    Alian, Akram; Griner, Sarah L.; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D.; Stroud, Robert M.

    2009-01-01

    HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN–DNA complexes that form disulfide linkages between 5′-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors. PMID:19416821

  2. Silver colloids as plasmonic substrates for direct label-free surface-enhanced Raman scattering analysis of DNA.

    PubMed

    Torres-Nuñez, A; Faulds, K; Graham, D; Alvarez-Puebla, R A; Guerrini, L

    2016-08-15

    Ultrasensitive direct SERS analysis offers a powerful analytical tool for the structural characterization and classification of nucleic acids. However, acquisition of reliable spectral fingerprints of such complex biomolecules poses important challenges. In recent years, many efforts have been devoted to overcome these limitations, mainly implementing silver colloids as plasmonic substrates. However, a reliable cross-comparison of results reported in the recent literature is extremely hard to achieve, mostly due to the broad set of different surface properties of the plasmonic nanoparticles. Herein, we perform a thorough investigation of the role played by the metal/liquid interface composition of silver colloids in the direct label-free SERS analysis of DNA. Target molecules of increasing complexity, from short homopolymeric strands to long genomic duplexes, were used as probes. We demonstrate how apparently subtle changes in the colloidal surface chemistry can dramatically modify the affinity and the final SERS spectral profile of DNA. This has significant implications for the future design of new analytical strategies for the detection of DNA using SERS without labels. PMID:27213770

  3. Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate.

    PubMed Central

    Focher, F; Maga, G; Bendiscioli, A; Capobianco, M; Colonna, F; Garbesi, A; Spadari, S

    1995-01-01

    L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides. Images PMID:7544886

  4. Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate.

    PubMed

    Focher, F; Maga, G; Bendiscioli, A; Capobianco, M; Colonna, F; Garbesi, A; Spadari, S

    1995-08-11

    L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides. PMID:7544886

  5. Enzymatic Reaction with Unnatural Substrates: DNA Photolyase (Escherichia coli) Recognizes and Reverses Thymine [2+2] Dimers in the DNA Strand of a DNA/PNA Hybrid Duplex

    NASA Astrophysics Data System (ADS)

    Ramaiah, Danaboyina; Kan, Yongzhi; Koch, Troels; Orum, Henrik; Schuster, Gary B.

    1998-10-01

    Peptide nucleic acids (PNA) are mimics with normal bases connected to a pseudopeptide chain that obey Watson--Crick rules to form stable duplexes with itself and natural nucleic acids. This has focused attention on PNA as therapeutic or diagnostic reagents. Duplexes formed with PNA mirror some but not all properties of DNA. One fascinating aspect of PNA biochemistry is their reaction with enzymes. Here we show an enzyme reaction that operates effectively on a PNA/DNA hybrid duplex. A DNA oligonucleotide containing a cis, syn-thymine [2+2] dimer forms a stable duplex with PNA. The hybrid duplex is recognized by photolyase, and irradiation of the complex leads to the repair of the thymine dimer. This finding provides insight into the enzyme mechanism and provides a means for the selective repair of thymine photodimers.

  6. Fluorescence of quantum dots on e-beam patterned and DNA origami substrates

    NASA Astrophysics Data System (ADS)

    Corrigan, Timothy D.; Kessinger, Matthew; Kidd, Jesse; Neff, David; Rahman, Masudur; Norton, Michael L.

    2015-05-01

    Attachment of quantum dots or fluorescent molecules to gold nanoparticles has a variety of optical labeling and sensory applications. In this study, we use both e-beam lithography and DNA origami to examine the fluorescence enhancement of fluorescent molecules and quantum dots with a systematic approach to understanding the contribution of gold nanoparticle size and interparticle spacing. The unique design of our patterns allows us to study the effects of size and spacing of the gold nanoparticles on the enhancement of fluorescence in one quick study with constant conditions - removing undesirable effects such as differences in concentration of quantum dots or other chemistry differences that plague multiple experiments. We also discuss the fluorescence and bonding of CdSe/ZnS quantum dots to both gold as well as DNA for use in self assembled DNA constructs. Specifically, bioconjugated CdSe/ZnS core/shell quantum dots were synthesized and functionalized with MPA using both traditional ligand exchange as well as newly developed in situ functionalization techniques used to increase the quantum yield of the quantum dots. We will present fluorescent images showing results of optimal size and spacing for fluorescence as well as demonstrating attachment chemistry of the quantum dots.

  7. Fabrication of large-scale gold nanoplate films as highly active SERS substrates for label-free DNA detection.

    PubMed

    Bi, Liyan; Rao, Yanying; Tao, Qin; Dong, Jian; Su, Ting; Liu, Fangjing; Qian, Weiping

    2013-05-15

    We introduce a simple but robust label-free method to detect DNA based on large-scale gold nanoplate (GNP) films with tunable localized surface plasmon resonance (LSPR) and highly surface-enhanced Raman scattering (SERS) activity. The common probe molecule, Neil Blue A sulfate (NBA) is used for testing the SERS activity of the GNP films at very low concentrations. It is found that the SERS properties are highly dependent on the edge lengths of gold nanoplate and gold nanoplate density in the films. Multiple-layer GNP films which are constructed by gold nanoplate with an edge length of 134±6nm have the density of 916±40GNPsGNPs/spot. It shows the highest signal intensity with SERS enhancement factor (EF) as high as 5.4×10(7) and also has excellent stability, reproducibility and repeatability. The optimized SERS-active substrate with the largest enhancement ability could be used to detect double-strand DNA without a dye label, and the detection limit is down to 10(-6)mg/mL. PMID:23306075

  8. Polyphenol oxidase and herbivore defense in trembling aspen (Populus tremuloides): cDNA cloning, expression, and potential substrates.

    PubMed

    Haruta, Miyoshi; Pedersen, Jens A.; Constabel, C. Peter

    2001-08-01

    The biochemical anti-herbivore defense of trembling aspen (Populus tremuloides Michx.) was investigated in a molecular analysis of polyphenol oxidase (PPO; EC 1.10.3.2). A PPO cDNA was isolated from a trembling aspen wounded leaf cDNA library and its nucleotide sequence determined. Southern analysis indicated the presence of two PPO genes in the trembling aspen genome. Expression of PPO was found to be induced after herbivory by forest tent caterpillar, by wounding, and by methyl jasmonate treatment. Wound induction was systemic, and occurred in unwounded leaves on wounded plants. This pattern of expression is consistent with a role of this enzyme in insect defense. A search for potential PPO substrates in ethanolic aspen leaf extracts using electron spin resonance (ESR) found no pre-existing diphenolic compounds. However, following a brief delay and several additions of oxygen, an ESR signal specific for catechol was detected. The source of this catechol was most likely the aspen phenolic glycosides tremulacin or salicortin which decomposed during ESR experiments. This was subsequently confirmed in experiments using pure salicortin. PMID:11473716

  9. FANCJ helicase uniquely senses oxidative base damage in either strand of duplex DNA and is stimulated by replication protein A to unwind the damaged DNA substrate in a strand-specific manner.

    PubMed

    Suhasini, Avvaru N; Sommers, Joshua A; Mason, Aaron C; Voloshin, Oleg N; Camerini-Otero, R Daniel; Wold, Marc S; Brosh, Robert M

    2009-07-01

    FANCJ mutations are genetically linked to the Fanconi anemia complementation group J and predispose individuals to breast cancer. Understanding the role of FANCJ in DNA metabolism and how FANCJ dysfunction leads to tumorigenesis requires mechanistic studies of FANCJ helicase and its protein partners. In this work, we have examined the ability of FANCJ to unwind DNA molecules with specific base damage that can be mutagenic or lethal. FANCJ was inhibited by a single thymine glycol, but not 8-oxoguanine, in either the translocating or nontranslocating strands of the helicase substrate. In contrast, the human RecQ helicases (BLM, RECQ1, and WRN) display strand-specific inhibition of unwinding by the thymine glycol damage, whereas other DNA helicases (DinG, DnaB, and UvrD) are not significantly inhibited by thymine glycol in either strand. In the presence of replication protein A (RPA), but not Escherichia coli single-stranded DNA-binding protein, FANCJ efficiently unwound the DNA substrate harboring the thymine glycol damage in the nontranslocating strand; however, inhibition of FANCJ helicase activity by the translocating strand thymine glycol was not relieved. Strand-specific stimulation of human RECQ1 helicase activity was also observed, and RPA bound with high affinity to single-stranded DNA containing a single thymine glycol. Based on the biochemical studies, we propose a model for the specific functional interaction between RPA and FANCJ on the thymine glycol substrates. These studies are relevant to the roles of RPA, FANCJ, and other DNA helicases in the metabolism of damaged DNA that can interfere with basic cellular processes of DNA metabolism. PMID:19419957

  10. TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site.

    PubMed

    Azhibek, Dulat; Skvortsov, Dmitry; Andreeva, Anna; Zatsepin, Timofei; Arutyunyan, Alexandr; Zvereva, Maria; Dontsova, Olga

    2016-06-01

    Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G-rich sequence, and with noncoding RNA, Telomeric repeat-containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2'-OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G-quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate - the 3'-end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3'-end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26669798

  11. Long-lived crowded-litter mice have an age-dependent increase in protein synthesis to DNA synthesis ratio and mTORC1 substrate phosphorylation

    PubMed Central

    Bruns, Danielle R.; Peelor, Frederick F.; Biela, Laurie M.; Miller, Richard A.; Hamilton, Karyn L.; Miller, Benjamin F.

    2014-01-01

    Increasing mouse litter size [crowded litter (CL)] presumably imposes a transient nutrient stress during suckling and extends lifespan through unknown mechanisms. Chronic calorically restricted and rapamycin-treated mice have decreased DNA synthesis and mTOR complex 1 (mTORC1) signaling but maintained protein synthesis, suggesting maintenance of existing cellular structures. We hypothesized that CL would exhibit similar synthetic and signaling responses to other long-lived models and, by comparing synthesis of new protein to new DNA, that insight may be gained into the potential preservation of existing cellular structures in the CL model. Protein and DNA synthesis was assessed in gastroc complex, heart, and liver of 4- and 7-mo CL mice. We also examined mTORC1 signaling in 3- and 7-mo aged animals. Compared with controls, 4-mo CL had greater DNA synthesis in gastroc complex with no differences in protein synthesis or mTORC1 substrate phosphorylation across tissues. Seven-month CL had less DNA synthesis than controls in heart and greater protein synthesis and mTORC1 substrate phosphorylation across tissues. The increased new protein-to-new DNA synthesis ratio suggests that new proteins are synthesized more so in existing cells at 7 mo, differing from 4 mo, in CL vs. controls. We propose that, in CL, protein synthesis shifts from being directed toward new cells (4 mo) to maintenance of existing cellular structures (7 mo), independently of decreased mTORC1. PMID:25205819

  12. Structural basis for the substrate selectivity of PvuRts1I, a 5-hydroxymethylcytosine DNA restriction endonuclease

    PubMed Central

    Shao, Chen; Wang, Chengliang; Zang, Jianye

    2014-01-01

    5-Hydroxymethylation is a curious modification of cytosine that was discovered some decades ago, but its functional role in eukaryotes still awaits elucidation. 5-Hydroxymethyl­cytosine is an epigenetic marker that is crucial for multiple biological processes. The profile is altered under certain disease conditions such as cancer, Huntington’s disease and Alzheimer’s disease. Using the DNA-modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq), the hydroxymethylome can be deciphered at the resolution of individual bases. The method is based on the enzymatic properties of AbaSI, a member of the PvuRts1I family of endonucleases. PvuRts1I is a modification-dependent endonuclease with high selectivity for 5-hydroxymethyl­cytosine over 5-methylcytosine and cytosine. In this study, the crystal structure of PvuRts1I was determined in order to understand and improve the substrate selectivity. A nuclease domain and an SRA-like domain are located at the N- and C-termini, respectively. Through comparison with other SRA-domain structures, the SRA-like domain was proposed to be the 5-hmC recognition module. Several mutants of PvuRts1I with enzymatic activity restricted to 5-hydroxymethylcytosine only were generated based on the structural analysis, and these enzyme variants are appropriate for separating the hydroxymethylome from the wider methylome. PMID:25195760

  13. Differential Incorporation of Carbon Substrates among Microbial Populations Identified by Field-Based, DNA Stable-Isotope Probing in South China Sea

    PubMed Central

    Xie, Xiabing; Jiao, Nianzhi

    2016-01-01

    To determine the adapted microbial populations to variant dissolved organic carbon (DOC) sources in the marine environment and improve the understanding of the interaction between microorganisms and marine DOC pool, field-based incubation experiments were carried out using supplemental 13C-labeled typical substrates D-glucose and D-glucosamine (D-Glc and D-GlcN, respectively), which are two important components in marine DOC pool in the South China Sea. 13C- and 12C-DNA were then fractionated by ultracentrifugation and the microbial community was analyzed by terminal-restriction fragment length polymorphism and 454 pyrosequencing of 16S rRNA gene. 12C-DNA-based communities showed relatively high similarities with their corresponding in situ communities, and their bacterial diversities were generally higher than 13C-DNA-based counterparts. Distinct differences in community composition were found between 13C- and 12C-DNA-based communities and between two substrate-supplemented 13C-DNA-based communities; these differences distinctly varied with depth and site. In most cases, there were more genera with relative abundances of >0.1% in D-Glc-incorporating communities than in D-GlcN-incorporating communities. The Roseobacter clade was one of the prominent actively substrate-incorporating bacterial populations in all 13C-DNA-based communities. Vibrio was another prominent actively D-GlcN-incorporating bacterial population in most incubations. However notably, different OTUs dominated this clade or genus in different treatments at different depths. Altogether, these results suggested that there were taxa-specific differences in DOC assimilations and, moreover, their differences varied among the typical water masses, which could have been caused by the variant compositions of original bacterial communities from different hydrological environments. This implies that ecologically, the levels of labile or recalcitrance of DOC can be maintained only in a specific environmental

  14. Highly selective and sensitive detection of miRNA based on toehold-mediated strand displacement reaction and DNA tetrahedron substrate.

    PubMed

    Li, Wei; Jiang, Wei; Ding, Yongshun; Wang, Lei

    2015-09-15

    MicroRNAs (miRNAs) play important roles in a variety of biological processes and have been regarded as tumor biomarkers in cancer diagnosis and prognosis. In this work, a single-molecule counting method for miRNA analysis was proposed based on toehold-mediated strand displacement reaction (SDR) and DNA tetrahedron substrate. Firstly, a specially designed DNA tetrahedron was assembled with a hairpin at one of the vertex, which has an overhanging toehold domain. Then, the DNA tetrahedron was immobilized on the epoxy-functional glass slide by epoxy-amine reaction, forming a DNA tetrahedron substrate. Next, the target miRNA perhybridized with the toehold domain and initiated a strand displacement reaction along with the unfolding of the hairpin, realizing the selective recognization of miRNA. Finally, a biotin labeled detection DNA was hybridized with the new emerging single strand and the streptavidin coated QDs were used as fluorescent probes. Fluorescent images were acquired via epi-fluorescence microscopy, the numbers of fluorescence dots were counted one by one for quantification. The detection limit is 5 fM, which displayed an excellent sensitivity. Moreover, the proposed method which can accurately be identified the target miRNA among its family members, demonstrated an admirable selectivity. Furthermore, miRNA analysis in total RNA samples from human lung tissues was performed, suggesting the feasibility of this method for quantitative detection of miRNA in biomedical research and early clinical diagnostics. PMID:25950935

  15. DNA repair by photolyase: a novel substrate with low background absorption around 265 nm for transient absorption studies in the UV.

    PubMed

    Thiagarajan, Viruthachalam; Villette, Sandrine; Espagne, Agathe; Eker, Andre P M; Brettel, Klaus; Byrdin, Martin

    2010-01-19

    CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH(-)) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH(-)). Flash-induced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase. PMID:20000331

  16. Structure and Specificity of the Bacterial Cysteine Methyltransferase Effector NleE Suggests a Novel Substrate in Human DNA Repair Pathway

    PubMed Central

    Wan, Xiaobo; Chen, Jing; Hu, Liyan; Ding, Xiaojun; Li, Lin; Karar, Jayashree; Peng, Hongzhuang; Chen, She; Huang, Niu; Rauscher, Frank J.; Shao, Feng

    2014-01-01

    Enteropathogenic E. coli (EPEC) and related enterobacteria rely on a type III secretion system (T3SS) effector NleE to block host NF-κB signaling. NleE is a first in class, novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates a zinc-coordinating cysteine in the Npl4-like Zinc Finger (NZF) domains in TAB2/3 adaptors in the NF-κB pathway, but its mechanism of action and other human substrates are unknown. Here we solve crystal structure of NleE-SAM complex, which reveals a methyltransferase fold different from those of known ones. The SAM, cradled snugly at the bottom of a deep and narrow cavity, adopts a unique conformation ready for nucleophilic attack by the methyl acceptor. The substrate NZF domain can be well docked into the cavity, and molecular dynamic simulation indicates that Cys673 in TAB2-NZF is spatially and energetically favorable for attacking the SAM. We further identify a new NleE substrate, ZRANB3, that functions in PCNA binding and remodeling of stalled replication forks at the DNA damage sites. Specific inactivation of the NZF domain in ZRANB3 by NleE offers a unique opportunity to suggest that ZRANB3-NZF domain functions in DNA repair processes other than ZRANB3 recruitment to DNA damage sites. Our analyses suggest a novel and unexpected link between EPEC infection, virulence proteins and genome integrity. PMID:25412445

  17. DNA Damage: Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

    PubMed Central

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-01

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N1-methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ-and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained. PMID:24339041

  18. The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1

    PubMed Central

    Herbert, Alan; Rich, Alexander

    2001-01-01

    RNA editing changes the read-out of genetic information, increasing the number of different protein products that can be made from a single gene. One form involves the deamination of adenosine to form inosine, which is subsequently translated as guanosine. The reaction requires a double-stranded RNA (dsRNA) substrate and is catalyzed by the adenosine deaminase that act on dsRNA (ADAR) family of enzymes. These enzymes possess dsRNA-binding domains (DRBM) and a catalytic domain. ADAR1 so far has been found only in vertebrates and is characterized by two Z-DNA-binding motifs, the biological function of which remains unknown. Here the role of the various functional domains of ADAR1 in determining the editing efficiency and specificity of ADAR1 is examined in cell-based assays. A variety of dsRNA substrates was tested. It was found that a 15-bp dsRNA stem with a single base mismatch was sufficient for editing. The particular adenosine modified could be varied by changing the position of the mismatch. Editing efficiency could be increased by placing multiple pyrimidines 5′ to the edited adenosine. With longer substrates, editing efficiency also increased and was partly due to the use of DRBMs. Additional editing sites were also observed that clustered on the complementary strand 11–15 bp from the first. An unexpected finding was that the DRBMs are not necessary for the editing of the shorter 15-bp substrates. However, mutation of the Z-DNA-binding domains of ADAR1 decreased the efficiency with which such a substrate was edited. PMID:11593027

  19. Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

    PubMed Central

    Knutzon, D S; Lardizabal, K D; Nelsen, J S; Bleibaum, J L; Davies, H M; Metz, J G

    1995-01-01

    Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme. PMID:8552723

  20. A New Family of HEAT-Like Repeat Proteins Lacking a Critical Substrate Recognition Motif Present in Related DNA Glycosylases.

    PubMed

    Mullins, Elwood A; Shi, Rongxin; Kotsch, Lyle A; Eichman, Brandt F

    2015-01-01

    DNA glycosylases are important repair enzymes that eliminate a diverse array of aberrant nucleobases from the genomes of all organisms. Individual bacterial species often contain multiple paralogs of a particular glycosylase, yet the molecular and functional distinctions between these paralogs are not well understood. The recently discovered HEAT-like repeat (HLR) DNA glycosylases are distributed across all domains of life and are distinct in their specificity for cationic alkylpurines and mechanism of damage recognition. Here, we describe a number of phylogenetically diverse bacterial species with two orthologs of the HLR DNA glycosylase AlkD. One ortholog, which we designate AlkD2, is substantially less conserved. The crystal structure of Streptococcus mutans AlkD2 is remarkably similar to AlkD but lacks the only helix present in AlkD that penetrates the DNA minor groove. We show that AlkD2 possesses only weak DNA binding affinity and lacks alkylpurine excision activity. Mutational analysis of residues along this DNA binding helix in AlkD substantially reduced binding affinity for damaged DNA, for the first time revealing the importance of this structural motif for damage recognition by HLR glycosylases. PMID:25978435

  1. Reactive Microcontact Printing of DNA Probes on (DMA-NAS-MAPS) Copolymer-Coated Substrates for Efficient Hybridization Platforms.

    PubMed

    Castagna, Rossella; Bertucci, Alessandro; Prasetyanto, Eko Adi; Monticelli, Marco; Conca, Dario Valter; Massetti, Matteo; Sharma, Parikshit Pratim; Damin, Francesco; Chiari, Marcella; De Cola, Luisa; Bertacco, Riccardo

    2016-04-01

    High-performing hybridization platforms fabricated by reactive microcontact printing of DNA probes are presented. Multishaped PDMS molds are used to covalently bind oligonucleotides over a functional copolymer (DMA-NAS-MAPS) surface. Printed structures with minimum width of about 1.5 μm, spaced by 10 μm, are demonstrated, with edge corrugation lower than 300 nm. The quantification of the immobilized surface probes via fluorescence imaging gives a remarkable concentration of 3.3 × 10(3) oligonucleotides/μm(2), almost totally active when used as probes in DNA-DNA hybridization assays. Indeed, fluorescence and atomic force microscopy show a 95% efficiency in target binding and uniform DNA hybridization over printed areas. PMID:26972953

  2. PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage

    PubMed Central

    Tsanov, Nikolay; Kermi, Chames; Coulombe, Philippe; Van der Laan, Siem; Hodroj, Dana; Maiorano, Domenico

    2014-01-01

    Proliferating cell nuclear antigen (PCNA) is a well-known scaffold for many DNA replication and repair proteins, but how the switch between partners is regulated is currently unclear. Interaction with PCNA occurs via a domain known as a PCNA-Interacting Protein motif (PIP box). More recently, an additional specialized PIP box has been described, the « PIP degron », that targets PCNA-interacting proteins for proteasomal degradation via the E3 ubiquitin ligase CRL4Cdt2. Here we provide evidence that CRL4Cdt2-dependent degradation of PIP degron proteins plays a role in the switch of PCNA partners during the DNA damage response by facilitating accumulation of translesion synthesis DNA polymerases into nuclear foci. We show that expression of a nondegradable PIP degron (Cdt1) impairs both Pol η and Pol κ focus formation on ultraviolet irradiation and reduces cell viability, while canonical PIP box-containing proteins have no effect. Furthermore, we identify PIP degron-containing peptides from several substrates of CRL4Cdt2 as efficient inhibitors of Pol η foci formation. By site-directed mutagenesis we show that inhibition depends on a conserved threonine residue that confers high affinity for PCNA-binding. Altogether these findings reveal an important regulative role for the CRL4Cdt2 pathway in the switch of PCNA partners on DNA damage. PMID:24423875

  3. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  4. Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques.

    PubMed

    Tassaneeyakul, W; Veronese, M E; Birkett, D J; Gonzalez, F J; Miners, J O

    1993-12-01

    The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylation of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigated using cDNA expression and liver microsomal kinetic and inhibitor techniques. 4NP hydroxylation by human liver microsomes and cDNA-expressed human CYP2E1 exhibited Michaelis-Menten kinetics; the respective apparent Km values were 30 +/- 7 and 21 microM. Mutual competitive inhibition was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent Km and Ki values for each individual compound. 4NP and CZ hydroxylase activities in microsomes from 18 liver donors varied to a similar extent (3.3- and 3.0-fold, respectively) and 4NP hydroxylase activity correlated significantly (rs > or = 0.75, P < 0.005) with both CZ hydroxylation and immunoreactive CYP2E1 content. The prototypic CYP2E1 inhibitor, diethyldithiocarbamate, was a potent inhibitor of 4NC formation and decreased 4NP hydroxylation by cDNA-expressed CYP2E1 and human liver microsomes in parallel. Probes for other human CYP isoforms namely (alpha-naphthoflavone, coumarin, sulphaphenazole, quinidine, troleandomycin and mephenytoin) caused < 15% inhibition of liver microsomal 4NP hydroxylation. These data confirm that, as in animal species, 4NP hydroxylation is catalysed largely by CYP2E1 in human liver and 4NP may therefore be used as an in vitro substrate probe for the human enzyme. PMID:8267647

  5. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  6. Activation-induced cytidine deaminase-mediated sequence diversification is transiently targeted to newly integrated DNA substrates.

    PubMed

    Yang, Shu Yuan; Fugmann, Sebastian D; Gramlich, Hillary S; Schatz, David G

    2007-08-31

    The molecular features that allow activation-induced cytidine deaminase (AID) to target Ig and certain non-Ig genes are not understood, although transcription has been implicated as one important parameter. We explored this issue by testing the mutability of a non-Ig transcription cassette in Ig and non-Ig loci of the chicken B cell line DT40. The cassette did not act as a stable long term mutation target but was able to be mutated in an AID-dependent manner for a limited time post-integration. This indicates that newly integrated DNA has molecular characteristics that render it susceptible to modification by AID, with implications for how targeting and mis-targeting of AID occurs. PMID:17613522

  7. The expanded mtDNA phylogeny of the Franco-Cantabrian region upholds the pre-neolithic genetic substrate of Basques.

    PubMed

    Cardoso, Sergio; Valverde, Laura; Alfonso-Sánchez, Miguel A; Palencia-Madrid, Leire; Elcoroaristizabal, Xabier; Algorta, Jaime; Catarino, Susana; Arteta, David; Herrera, Rene J; Zarrabeitia, María Teresa; Peña, José A; de Pancorbo, Marian M

    2013-01-01

    The European genetic landscape has been shaped by several human migrations occurred since Paleolithic times. The accumulation of archaeological records and the concordance of different lines of genetic evidence during the last two decades have triggered an interesting debate concerning the role of ancient settlers from the Franco-Cantabrian region in the postglacial resettlement of Europe. Among the Franco-Cantabrian populations, Basques are regarded as one of the oldest and more intriguing human groups of Europe. Recent data on complete mitochondrial DNA genomes focused on macrohaplogroup R0 revealed that Basques harbor some autochthonous lineages, suggesting a genetic continuity since pre-Neolithic times. However, excluding haplogroup H, the most representative lineage of macrohaplogroup R0, the majority of maternal lineages of this area remains virtually unexplored, so that further refinement of the mtDNA phylogeny based on analyses at the highest level of resolution is crucial for a better understanding of the European prehistory. We thus explored the maternal ancestry of 548 autochthonous individuals from various Franco-Cantabrian populations and sequenced 76 mitogenomes of the most representative lineages. Interestingly, we identified three mtDNA haplogroups, U5b1f, J1c5c1 and V22, that proved to be representative of Franco-Cantabria, notably of the Basque population. The seclusion and diversity of these female genetic lineages support a local origin in the Franco-Cantabrian area during the Mesolithic of southwestern Europe, ~10,000 years before present (YBP), with signals of expansions at ~3,500 YBP. These findings provide robust evidence of a partial genetic continuity between contemporary autochthonous populations from the Franco-Cantabrian region, specifically the Basques, and Paleolithic/Mesolithic hunter-gatherer groups. Furthermore, our results raise the current proportion (≈ 15%) of the Franco-Cantabrian maternal gene pool with a putative pre

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

    PubMed Central

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

    2015-01-01

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

  9. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

    PubMed Central

    Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

    2015-01-01

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  10. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

  11. The Expanded mtDNA Phylogeny of the Franco-Cantabrian Region Upholds the Pre-Neolithic Genetic Substrate of Basques

    PubMed Central

    Cardoso, Sergio; Valverde, Laura; Alfonso-Sánchez, Miguel A.; Palencia-Madrid, Leire; Elcoroaristizabal, Xabier; Algorta, Jaime; Catarino, Susana; Arteta, David; Herrera, Rene J.; Zarrabeitia, María Teresa; Peña, José A.; de Pancorbo, Marian M.

    2013-01-01

    The European genetic landscape has been shaped by several human migrations occurred since Paleolithic times. The accumulation of archaeological records and the concordance of different lines of genetic evidence during the last two decades have triggered an interesting debate concerning the role of ancient settlers from the Franco-Cantabrian region in the postglacial resettlement of Europe. Among the Franco-Cantabrian populations, Basques are regarded as one of the oldest and more intriguing human groups of Europe. Recent data on complete mitochondrial DNA genomes focused on macrohaplogroup R0 revealed that Basques harbor some autochthonous lineages, suggesting a genetic continuity since pre-Neolithic times. However, excluding haplogroup H, the most representative lineage of macrohaplogroup R0, the majority of maternal lineages of this area remains virtually unexplored, so that further refinement of the mtDNA phylogeny based on analyses at the highest level of resolution is crucial for a better understanding of the European prehistory. We thus explored the maternal ancestry of 548 autochthonous individuals from various Franco-Cantabrian populations and sequenced 76 mitogenomes of the most representative lineages. Interestingly, we identified three mtDNA haplogroups, U5b1f, J1c5c1 and V22, that proved to be representative of Franco-Cantabria, notably of the Basque population. The seclusion and diversity of these female genetic lineages support a local origin in the Franco-Cantabrian area during the Mesolithic of southwestern Europe, ∼10,000 years before present (YBP), with signals of expansions at ∼3,500 YBP. These findings provide robust evidence of a partial genetic continuity between contemporary autochthonous populations from the Franco-Cantabrian region, specifically the Basques, and Paleolithic/Mesolithic hunter-gatherer groups. Furthermore, our results raise the current proportion (≈15%) of the Franco-Cantabrian maternal gene pool with a putative pre

  12. Cleaving DNA with DNA

    NASA Astrophysics Data System (ADS)

    Carmi, Nir; Balkhi, Shameelah R.; Breaker, Ronald R.

    1998-03-01

    A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This ``deoxyribozyme'' can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple ``restriction enzymes'' for the site-specific cleavage of single-stranded DNA.

  13. Electrochemical properties of interstrand cross-linked DNA duplexes labeled with Nile blue.

    PubMed

    Mie, Yasuhiro; Kowata, Keiko; Kojima, Naoshi; Komatsu, Yasuo

    2012-12-11

    DNA molecules have attracted considerable attention as functional materials in various fields such as electrochemical sensors with redox-labeled DNA. However, the recently developed interstrand cross-link (ICL) technique for double-stranded DNA can adequately modify the electronic properties inside the duplex. Hence, the electrochemical investigation of ICL-DNA helps us to understand the electron transfer of redox-labeled DNA at an electrode surface, which would develop useful sensors. In this study, the first insight into this matter is presented. We prepared 17-mer DNA duplexes incorporating Nile blue (NB-DNA) at one end as a redox marker and a disulfide tether at the other end for immobilization onto an electrode. The duplexes were covalently cross-linked by bifunctional cross-linkers that utilize either a propyl or naphthalene residue to replace a base pair. Their electrochemical responses at the electrode surface were compared to evaluate the effect of the ICL on the electron-transfer reactions of the redox-labeled DNA duplexes. A direct transfer of electrons between NB and the electrode was observed for a standard DNA, as previously reported, whereas interstrand cross-linked DNA (CL-DNA) strands showed a decrease in the direct electron-transfer pathway. This is expected to result from constraining the elastic bending/flexibility of the duplex caused by the covalent cross-links. Interestingly, the CL-DNA incorporating naphthalene residues exhibited additional voltammetric peaks derived from DNA-mediated electron transfer (through base π stacking), which was not observed in the mismatched CL-DNA. The present results indicate that the ICL significantly affects electron transfer in the redox-labeled DNA at the electrode and can be an important determinant for electrochemical signaling in addition to its role in stabilizing the duplex structure. PMID:23153070

  14. Interaction of the type I methyltransferase M.EcoR124I with modified DNA substrates: sequence discrimination and base flipping.

    PubMed Central

    Mernagh, D R; Taylor, I A; Kneale, G G

    1998-01-01

    We have analysed the DNA-protein contacts made between the type I DNA methyltransferase M.EcoR124I and its recognition sequence. The effects of base modifications have been probed by measuring the affinity of M.EcoR124I for the modified sequences relative to that for the wild-type sequence by using gel-retardation competition assays. These results, along with those from methylation interference footprinting and photo-affinity cross-linking have identified the location of potential DNA contacts within the DNA recognition site. Substitution of 6-thioguanosine for each of the three specific guanines in the recognition sequence leads to a large (10-20-fold) decrease in the strength of DNA binding, indicating the importance of hydrogen-bonding interactions in the major groove of DNA. In contrast, replacement of either (or both) of the adenines at the target site for methylation by the enzyme, to produce either a base pair mismatch or loss of the base, leads to a marked increase in DNA-binding affinity. The results strongly support the proposal that type I methyltransferases employ a base-flipping mechanism to methylate their target base. PMID:9841886

  15. The ATM substrate KAP1 controls DNA repair in heterochromatin: Regulation by HP1 proteins and Serine 473/824 phosphorylation

    PubMed Central

    White, D. E.; Rafalska-Metcalf, I. U.; Ivanov, A. V.; Corsinotti, A.; Peng, H.; Lee, S. C.; Trono, D.; Janicki, S. M.; Rauscher, F. J.

    2016-01-01

    The repair of DNA damage in highly compact, transcriptionally silent heterochromatin requires that repair and chromatin packaging machineries be tightly coupled and regulated. KAP1 is a heterochromatin protein and co-repressor which binds to HP1 during gene silencing, but is also robustly phosphorylated by ATM at serine 824 in response to DNA damage. The interplay between HP1-KAP1 binding/ATM phosphorylation during DNA repair is not known. We show that HP1α and unmodified KAP1 are enriched in endogenous heterochromatic loci and at a silent transgene prior to damage. Following damage, γH2AX and pKAP1-s824 rapidly increase and persist at these loci. Cells which lack HP1 fail to form discreet pKAP1-s824 foci after damage but levels are higher and more persistent. KAP1 is phosphorylated at Serine 473 in response to DNA damage and its levels are also modulated by HP1. Unlike pKAP1-s824, pKAP1-s473 does not accumulate at damage foci but is diffusely localized in the nucleus. While HP1 association tempers KAP1 phosphorylation, this interaction also slows the resolution of γH2AX foci. Thus, HP1-dependent regulation of KAP1 influences DNA repair in heterochromatin. PMID:22205726

  16. Protease Activity of PprI Facilitates DNA Damage Response: Mn(2+)-Dependence and Substrate Sequence-Specificity of the Proteolytic Reaction

    PubMed Central

    Lu, Huiming; Lin, Lin; Wang, Liangyan; Xu, Hong; Cui, Xianyan; Zhang, Hui; Li, Tingting; Hua, Yuejin

    2015-01-01

    The extremophilic bacterium Deinococcus radiodurans exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the Deinococcus-Thermus family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR) proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+)-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes’ expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair in vivo. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium Escherichia coli. This PprI-mediated pathway in D. radiodurans is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism. PMID:25811789

  17. Protease activity of PprI facilitates DNA damage response: Mn2+-dependence and substrate sequence-specificity of the proteolytic reaction.

    PubMed

    Wang, Yunguang; Xu, Qiang; Lu, Huiming; Lin, Lin; Wang, Liangyan; Xu, Hong; Cui, Xianyan; Zhang, Hui; Li, Tingting; Hua, Yuejin

    2015-01-01

    The extremophilic bacterium Deinococcus radiodurans exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the Deinococcus-Thermus family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR) proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+)-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes' expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair in vivo. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium Escherichia coli. This PprI-mediated pathway in D. radiodurans is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism. PMID:25811789

  18. [cDNA cloning, expression and determination of substrate specificity of mice selenocysteine-containing protein SelV (Selenoprotein V)].

    PubMed

    Varlamova, E G; Novoselov, S V; Novoselov, V I

    2015-01-01

    To date various bioinformatics tools allowed to identify 25 selenocysteine-containing mammalian proteins. The name of these proteins assumes that they contain the amino acid selenocysteine (Sec). Functionally characterized selenocysteine-containing proteins are oxidoreductases with various functions, including glutathione peroxidases, thioredoxin reductases, deiodinases etc. However, the functions of more than half of identified proteins are still unclear, and mammalian selenoprotein SeIV is among them. We studied the selV in all stages of postnatal development with the maximum level of mRNA expression during puberty, whereas in adult mice (8-18 months) we observed a gradual decrease of expression. In order to get closer to the functional role of Selenoprotein V, we have carried out experiments on the substrate specificity and enzymatic activity measurement of this selenocysteine-containing protein. It was shown that SelV posseses glutathionperoxidase and thioredoxinreductase activities. PMID:26510596

  19. Interaction between the N-terminal domain of human DNA topoisomerase I and the arginine-serine domain of its substrate determines phosphorylation of SF2/ASF splicing factor.

    PubMed Central

    Labourier, E; Rossi, F; Gallouzi, I E; Allemand, E; Divita, G; Tazi, J

    1998-01-01

    Human DNA topoisomerase I, known for its DNA-relaxing activity, is possibly one of the kinases phosphorylating members of the SR protein family of splicing factors, in vivo. Little is known about the mechanism of action of this novel kinase. Using the prototypical SR protein SF2/ASF (SRp30a) as model substrate, we demonstrate that serine residues phosphorylated by topo I/kinase exclusively located within the most extended arginine-serine repeats of the SF2/ASF RS domain. Unlike other kinases such as cdc2 and SRPK1, which also phosphorylated serines at the RS domain, topo I/kinase required several SR dipeptide repeats. These repeats possibly contribute to a versatile structure in the RS domain thereby facilitating phosphorylation. Furthermore, far-western, fluorescence spectroscopy and kinase assays using the SF2/ASF mutants, demonstrated that kinase activity and binding were tightly coupled. Since the deletion of N-terminal 174 amino acids of Topo I destroys SF2/ASF binding and kinase activity but not ATP binding, we conclude that at least two distinct domains of Topo I are necessary for kinase activity: one in the C-terminal region contributing to the ATP binding site and the other one in the N-terminal region that allows binding of SF2/ASF. PMID:9611241

  20. EcoRV catalysis with a pre-bent substrate

    NASA Astrophysics Data System (ADS)

    Sanchez, Daniel S.; Zocchi, Giovanni

    2015-05-01

    Enzymes are deformable molecules which often adapt their conformation to the substrate's geometry. In the case of restriction enzymes acting on DNA, the substrate (DNA) is deformable also. While it is well established that external mechanical stress exerted on an enzyme modulates the enzymatic activity, the effect of mechanically stressing the substrate is less explored. Here we present the restriction enzyme EcoRV with a series of pre-bent DNA substrates and observe modulation of the overall speed of the enzymatic reaction by up to a factor 50. While in all cases the reaction is slowed down compared to the unstressed substrate, we observe peaks in the reaction rate as we vary the state of stress of the DNA substrate. Both bending moments and torsional moments on the DNA substrate affect the reaction rate.

  1. DNAzymes in DNA Nanomachines and DNA Analysis

    NASA Astrophysics Data System (ADS)

    He, Yu; Tian, Ye; Chen, Yi; Mao, Chengde

    This chapter discusses our efforts in using DNAzymes in DNA nano-machines and DNA analysis systems. 10-23 DNAzymes can cleave specific phos-phodiester bonds in RNA. We use them to construct an autonomous DNA-RNA chimera nanomotor, which constantly extracts chemical energy from RNA substrates and transduces the energy into a mechanical motion: cycles of contraction and extension. The motor's motion can be reversibly turned on and off by a DNA analogue (brake) of the RNA substrate. Addition and removal of the brake stops and restarts, respectively, the motor's motion. Furthermore, when the RNA substrates are preorganized into a one-dimensional track, a DNAzyme can continuously move along the track so long as there are substrates available ahead. Based on a similar mechanism, a novel DNA detection system has been developed. A target DNA activates a DNAzyme to cleave RNA-containing molecular beacons (MB), which generates an enhanced fluorescence signal. A following work integrates two steps of signal amplifications: a rolling-circle amplification (RCA) to synthesize multiple copies of DNAzymes, and the DNAzymes catalyze a chemical reaction to generate a colorimetric signal. This method allows detection of DNA analytes whose concentration is as low as 1 pM.

  2. Respiration of 13C-Labeled Substrates Added to Soil in the Field and Subsequent 16S rRNA Gene Analysis of 13C-Labeled Soil DNA

    PubMed Central

    Padmanabhan, P.; Padmanabhan, S.; DeRito, C.; Gray, A.; Gannon, D.; Snape, J. R.; Tsai, C. S.; Park, W.; Jeon, C.; Madsen, E. L.

    2003-01-01

    Our goal was to develop a field soil biodegradation assay using 13C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived 13C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of 13C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for 13CO2 respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of 13CO2 emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF6, that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired 13CO2. Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of 13CO2 released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of 13C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with 13C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter

  3. DNA-cell conjugates

    DOEpatents

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki

    2016-05-03

    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  4. Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

    PubMed

    Praseeda, Mullasseril; Pradeep, Kurup K; Krupa, Ananth; Krishna, S Sri; Leena, Suseela; Kumar, R Rajeev; Cheriyan, John; Mayadevi, Madhavan; Srinivasan, Narayanaswamy; Omkumar, Ramakrishnapillai V

    2004-03-01

    CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation. PMID:14558884

  5. DNA ELECTROPHORESIS AT SURFACES

    SciTech Connect

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  6. Power electronics substrate for direct substrate cooling

    DOEpatents

    Le, Khiet; Ward, Terence G.; Mann, Brooks S.; Yankoski, Edward P.; Smith, Gregory S.

    2012-05-01

    Systems and apparatus are provided for power electronics substrates adapted for direct substrate cooling. A power electronics substrate comprises a first surface configured to have electrical circuitry disposed thereon, a second surface, and a plurality of physical features on the second surface. The physical features are configured to promote a turbulent boundary layer in a coolant impinged upon the second surface.

  7. DNA Oligonucleotide 3'-Phosphorylation by a DNA Enzyme.

    PubMed

    Camden, Alison J; Walsh, Shannon M; Suk, Sarah H; Silverman, Scott K

    2016-05-10

    T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3'-phosphorylation, using a 5'-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3'-phosphate (forming 3'-MeImp) and subsequent splint ligation with a 5'-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3'-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3'-kinase deoxyribozyme, obtained from an N40 random pool and named 3'Kin1, can 3'-phosphorylate nearly any DNA oligonucleotide substrate for which the 3'-terminus has the sequence motif 5'-NKR-3', where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3'-phosphorylate DNA oligonucleotides. PMID:27063020

  8. Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

    PubMed Central

    Hirano, Nobutaka; Ohshima, Hiroyuki; Takahashi, Hideo

    2006-01-01

    Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5′ phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5′-dTdCdA-3′ but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5′-dTdCdA-3′, which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA. PMID:16971463

  9. Human DNA polymerase α in binary complex with a DNA:DNA template-primer

    PubMed Central

    Coloma, Javier; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2016-01-01

    The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer – with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes. PMID:27032819

  10. Butanol formation from gaseous substrates.

    PubMed

    Dürre, Peter

    2016-03-01

    Mostly, butanol is formed as a product by saccharolytic anaerobes, employing the so-called ABE fermentation (for acetone-butanol-ethanol). However, this alcohol can also be produced from gaseous substrates such as syn(thesis) gas (major components are carbon monoxide and hydrogen) by autotrophic acetogens. In view of economic considerations, a biotechnological process based on cheap and abundant gases such as CO and CO2 as a carbon source is preferable to more expensive sugar or starch fermentation. In addition, any conflict for use of substrates that can also serve as human nutrition is avoided. Natural formation of butanol has been found with, e.g. Clostridium carboxidivorans, while metabolic engineering for butanol production was successful using, e.g. C. ljungdahlii. Production of butanol from CO2 under photoautotrophic conditions was also possible by recombinant DNA construction of a respective cyanobacterial Synechococcus sp. PCC 7942 strain. PMID:26903012

  11. Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 domain bound to a palindromic SBE DNA element.

    PubMed

    Baburajendran, Nithya; Palasingam, Paaventhan; Ng, Calista Keow Leng; Jauch, Ralf; Kolatkar, Prasanna R

    2009-11-01

    The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 angstrom resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 angstrom, alpha = beta = gamma = 90 degrees. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit. PMID:19923727

  12. Electrochemical DNA sensor-based strategy for sensitive detection of DNA demethylation and DNA demethylase activity.

    PubMed

    Shen, Qingming; Fan, Mengxing; Yang, Yin; Zhang, Hui

    2016-08-31

    DNA demethylation and demethylase activity play important roles in DNA self-repair, and their detection is key to early diagnosis of fatal diseases. Herein, a facile electrochemical DNA (E-DNA) sensor was developed for the sensitive detection of DNA demethylation and demethylase activity based on an enzyme cleavage strategy. The thiol modified hemi-methylated hairpin probe DNA (pDNA) was self-assembled on a Au electrode surface through the formation of AuS bonds. The hemi-methylated pDNA served as the substrate of DNA demethylase (using methyl-CpG-binding domain protein 2 (MBD2) as an example). Following demethylation, the hairpin stem was then recognized and cleaved by BstUI endonuclease. The ferrocene carboxylic acid (FcA)-tagged pDNA strands were released into the buffer solution from the electrode surface, resulting in a significant decrease of electrochemical signal and providing a means to observe DNA demethylation. The activity of DNA demethylase was analyzed in the concentration ranging from 0.5 to 500 ng mL(-1) with a limit of detection as low as 0.17 ng mL(-1). With high specificity and sensitivity, rapid response, and low cost, this simple E-DNA sensor provides a unique platform for the sensitive detection of DNA demethylation, DNA demethylase activity, and related molecular diagnostics and drug screening. PMID:27506345

  13. Active DNA demethylation by DNA repair: Facts and uncertainties.

    PubMed

    Schuermann, David; Weber, Alain R; Schär, Primo

    2016-08-01

    Pathways that control and modulate DNA methylation patterning in mammalian cells were poorly understood for a long time, although their importance in establishing and maintaining cell type-specific gene expression was well recognized. The discovery of proteins capable of converting 5-methylcytosine (5mC) to putative substrates for DNA repair introduced a novel and exciting conceptual framework for the investigation and ultimate discovery of molecular mechanisms of DNA demethylation. Against the prevailing notion that DNA methylation is a static epigenetic mark, it turned out to be dynamic and distinct mechanisms appear to have evolved to effect global and locus-specific DNA demethylation. There is compelling evidence that DNA repair, in particular base excision repair, contributes significantly to the turnover of 5mC in cells. By actively demethylating DNA, DNA repair supports the developmental establishment as well as the maintenance of DNA methylation landscapes and gene expression patterns. Yet, while the biochemical pathways are relatively well-established and reviewed, the biological context, function and regulation of DNA repair-mediated active DNA demethylation remains uncertain. In this review, we will thus summarize and critically discuss the evidence that associates active DNA demethylation by DNA repair with specific functional contexts including the DNA methylation erasure in the early embryo, the control of pluripotency and cellular differentiation, the maintenance of cell identity, and the nuclear reprogramming. PMID:27247237

  14. Fabricating nanoscale DNA patterns with gold nanowires.

    PubMed

    Chen, Yulin; Kung, Sheng-Chin; Taggart, David K; Halpern, Aaron R; Penner, Reginald M; Corn, Robert M

    2010-04-15

    Surface patterns of single-stranded DNA (ssDNA) consisting of nanoscale lines as thin as 40 nm were fabricated on polymer substrates for nanotechnology and bioaffinity sensing applications. Large scale arrays (with areas up to 4 cm(2)) of ssDNA "nanolines" were created on streptavidin-coated polymer (PDMS) surfaces by transferring biotinylated ssDNA from a master pattern of gold nanowires attached to a glass substrate. The gold nano-wires were first formed on the glass substrate by the process of lithographically patterned nanowire electrodeposition (LPNE), and then "inked" with biotinylated ssDNA by hybridization adsorption to a thiol-modified ssDNA monolayer attached to the gold nanowires. The transferred ssDNA nanolines were capable of hybridizing with ssDNA from solution to form double-stranded DNA (dsDNA) patterns; a combination of fluorescence and atomic force microscopy (AFM) measurements were used to characterize the dsDNA nanoline arrays. To demonstrate the utility of these surfaces for biosensing, optical diffraction measurements of the hybridization adsorption of DNA-coated gold nanoparticles onto the ssDNA nanoline arrays were used to detect a specific target sequence of unlabeled ssDNA in solution. PMID:20337428

  15. Coated substrates and process

    DOEpatents

    Chu, Wei-kan; Childs, Charles B.

    1991-01-01

    Disclosed herein is a coated substrate and a process for forming films on substrates and for providing a particularly smooth film on a substrate. The method of this invention involves subjecting a surface of a substrate to contact with a stream of ions of an inert gas having sufficient force and energy to substantially change the surface characteristics of said substrate, and then exposing a film-forming material to a stream of ions of an inert gas having sufficient energy to vaporize the atoms of said film-forming material and to transmit the vaporized atoms to the substrate surface with sufficient force to form a film bonded to the substrate. This process is particularly useful commercially because it forms strong bonds at room temperature. This invention is particularly useful for adhering a gold film to diamond and forming ohmic electrodes on diamond, but also can be used to bond other films to substrates.

  16. Ribbed electrode substrates

    DOEpatents

    Breault, Richard D.; Goller, Glen J.

    1983-01-01

    A ribbed substrate for an electrochemical cell electrode is made from a mixture of carbon fibers and carbonizable resin and has a mean pore size in the ribs which is 60-75% of the mean pore size of the web portions of the substrate which interconnect the ribs. Preferably the mean pore size of the web portion is 25-45 microns; and, if the substrate includes edge seals parallel to the ribs, the edge seals preferably have a mean pore size no greater than about ten microns. Most preferably the substrate has the same ratio of carbon fibers to polymeric carbon in all areas, including the ribs, webs, and edge seals. A substrate according to the present invention will have better overall performance than prior art substrates and minimizes the substrate thickness required for the substrate to perform all its functions well.

  17. Polished polymide substrate

    DOEpatents

    Farah, John; Sudarshanam, Venkatapuram S.

    2003-05-13

    Polymer substrates, in particular polyimide substrates, and polymer laminates for optical applications are described. Polyimide substrates are polished on one or both sides depending on their thickness, and single-layer or multi-layer waveguide structures are deposited on the polished polyimide substrates. Optical waveguide devices are machined by laser ablation using a combination of IR and UV lasers. A waveguide-fiber coupler with a laser-machined groove for retaining the fiber is also disclosed.

  18. Recovery of EUVL substrates

    SciTech Connect

    Vernon, S.P.; Baker, S.L.

    1995-01-19

    Mo/Si multilayers, were removed from superpolished zerodur and fused silica substrates with a dry etching process that, under suitable processing conditions, produces negligible change in either the substrate surface figure or surface roughness. Full recovery of the initial normal incidence extreme ultra-violet (EUV) reflectance response has been demonstrated on reprocessed substrates.

  19. Chemiluminescent and chemiluminescence resonance energy transfer (CRET) detection of DNA, metal ions, and aptamer-substrate complexes using hemin/G-quadruplexes and CdSe/ZnS quantum dots.

    PubMed

    Freeman, Ronit; Liu, Xiaoqing; Willner, Itamar

    2011-08-01

    Nucleic acid subunits consisting of fragments of the horseradish peroxidase (HRP)-mimicking DNAzyme and aptamer domains against ATP or sequences recognizing Hg(2+) ions self-assemble, in the presence of ATP or Hg(2+), into the active hemin-G-quadruplex DNAzyme structure. The DNAzyme-generated chemiluminescence provides the optical readout for the sensing events. In addition, the DNAzyme-stimulated chemiluminescence resonance energy transfer (CRET) to CdSe/ZnS quantum dots (QDs) is implemented to develop aptamer or DNA sensing platforms. The self-assembly of the ATP-aptamer subunits/hemin-G-quadruplex DNAzyme, where one of the aptamer subunits is functionalized with CdSe/ZnS QDs, leads to the CRET signal. Also, the functionalization of QDs with a hairpin nucleic acid that includes the G-quadruplex sequence in a ''caged'' configuration is used to analyze DNA. The opening of the hairpin structure by the target DNA assembles the hemin-G-quadruplex DNAzyme that stimulates the CRET signal. By the application of three different sized QDs functionalized with different hairpins, the multiplexed analysis of three different DNA targets is demonstrated by the generation of three different CRET luminescence signals. PMID:21678959

  20. DNA Templating of Au Nanowires

    NASA Astrophysics Data System (ADS)

    Wood, David; Braun, Gary; Inagaki, Katsuhiko

    2005-03-01

    We have developed a process for fabricating nanoscale wires using DNA templates. The templates were subsequently decorated with gold nanoparticles to make metallic wires. We have successfully deposited linear, straight sections of random (λ-phage) and regular-repeat sequences of DNA, of various lengths, on oxidized silicon substrates. We have also successfully deposited thiolated DNA on gold electrodes, allowing the DNA to electrically bridge gaps between electrode pairs. Electrode gaps ranged from 50 nm to 300 nm, fabricated using electron beam lithography. We decorated the DNA with gold nanoparticles with diameters in the range of 1-13 nm, and have used the nanoparticles as nucleation sites for the growth of continuous gold wires. We have performed AFM characterization of all surfaces and structures. In addition, we have performed current-voltage measurements on the undecorated DNA, the nanoparticle-decorated DNA, and the gold nanowires.

  1. DNA repair

    SciTech Connect

    Friedberg, E.C.; Hanawalt, P.C. )

    1988-01-01

    Topics covered in this book included: Eukaryote model systems for DNA repair study; Sensitive detection of DNA lesions and their repair; and Defined DNA sequence probes for analysis of mutagenesis and repair.

  2. Quantitive DNA Fiber Mapping

    SciTech Connect

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  3. Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

    PubMed Central

    Sharma, Nynne; Cai, Yujia; Bak, Rasmus O; Jakobsen, Martin R; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

    2013-01-01

    DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system. PMID:23443502

  4. DNA materials: bridging nanotechnology and biotechnology.

    PubMed

    Yang, Dayong; Hartman, Mark R; Derrien, Thomas L; Hamada, Shogo; An, Duo; Yancey, Kenneth G; Cheng, Ru; Ma, Minglin; Luo, Dan

    2014-06-17

    CONSPECTUS: In recent decades, DNA has taken on an assortment of diverse roles, not only as the central genetic molecule in biological systems but also as a generic material for nanoscale engineering. DNA possesses many exceptional properties, including its biological function, biocompatibility, molecular recognition ability, and nanoscale controllability. Taking advantage of these unique attributes, a variety of DNA materials have been created with properties derived both from the biological functions and from the structural characteristics of DNA molecules. These novel DNA materials provide a natural bridge between nanotechnology and biotechnology, leading to far-ranging real-world applications. In this Account, we describe our work on the design and construction of DNA materials. Based on the role of DNA in the construction, we categorize DNA materials into two classes: substrate and linker. As a substrate, DNA interfaces with enzymes in biochemical reactions, making use of molecular biology's "enzymatic toolkit". For example, employing DNA as a substrate, we utilized enzymatic ligation to prepare the first bulk hydrogel made entirely of DNA. Using this DNA hydrogel as a structural scaffold, we created a protein-producing DNA hydrogel via linking plasmid DNA onto the hydrogel matrix through enzymatic ligation. Furthermore, to fully make use of the advantages of both DNA materials and polymerase chain reaction (PCR), we prepared thermostable branched DNA that could remain intact even under denaturing conditions, allowing for their use as modular primers for PCR. Moreover, via enzymatic polymerization, we have recently constructed a physical DNA hydrogel with unique internal structure and mechanical properties. As a linker, we have used DNA to interface with other functional moieties, including gold nanoparticles, clay minerals, proteins, and lipids, allowing for hybrid materials with unique properties for desired applications. For example, we recently designed a

  5. DNA-PK assay

    DOEpatents

    Anderson, Carl W.; Connelly, Margery A.

    2004-10-12

    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  6. Electrochemical detection of DNA 3'-phosphatases based on surface-extended DNA nanotail strategy.

    PubMed

    Wu, Dan; Li, Chao; Hu, Xiaolu; Mao, Xiaoxia; Li, Genxi

    2016-06-14

    Determination of DNA dephosphorylation is of great value due to its vital role in many cellular processes. Here we report a surface-extended DNA nanotail strategy for simple and ultrasensitive detection of DNA 3'-phosphatases by terminal deoxynucleotidyl transferase (TdT) mediated signal amplification. In this work, DNA probes labeled with thiols at their 5' terminals and phosphoryls at 3' terminals are immobilized on gold electrode and are used as substrates for DNA 3'-phosphatases, taking T4 polynucleotide kinase phosphatase (T4PNKP) as an example. T4PNKP can catalyze the dephosphorylation reaction of the substrate DNA, followed by the formation of a long DNA strand by TdT on its 3' terminal hydroxyl, leading to an evident chronocoulometry signal enhancement. The proposal presents a considerable analytical performance with low detection limit and wide linear range, making it promise to be applied in the fields of DNA dephosphorylation related processes, drug discovery, and clinical diagnostics. PMID:27181641

  7. Fabrication of patterned DNA surfaces.

    PubMed Central

    Chrisey, L A; O'Ferrall, C E; Spargo, B J; Dulcey, C S; Calvert, J M

    1996-01-01

    Two photolithographic methods are described for the formation of patterned single or multiple DNA species on SiO2 substrates. In the first approach, substrates are treated with a photochemically labile organosilane monolayer film. Irradiation of these surfaces with patterned deep UV (193 nm) light results in patterned chemically reactive groups which are then reacted with heterobifunctional crosslinking molecules. Covalent attachment of modified synthetic DNA oligomers to the crosslinker results in stable DNA patterns. Alternatively, a photoresist is spin-coated over a silane film which had been previously modified with the heterobifunctional crosslinker. Upon patterned irradiation and subsequent development, the underlying crosslinker-modified layer is revealed, and is then reacted with a chemically modified DNA. Feature dimensions to 1 micron are observed when a single fluorescent DNA is attached to the surface. By performing sequential exposures, we have successfully immobilized two distinguishable DNA oligomers on a single surface. Synthetic DNA immobilized in this manner retains the ability to hybridize to its complementary strand, suggesting that these approaches may find utility in the development of miniaturized DNA-based biosensors. PMID:8760891

  8. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    PubMed Central

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K.

    2014-01-01

    Loss of WRN function causes Werner Syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRN’s DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor hydroxyurea. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency. PMID:24965941

  9. Tunnelling microscopy of DNA

    NASA Astrophysics Data System (ADS)

    Selci, Stefano; Cricenti, Antonio

    1991-01-01

    Uncoated DNA molecules marked with an activated tris (1-aziridinyl) phosphine oxide (TAPO) solution were deposited on gold substrates and imaged in air with a high resolution Scanning Tunnelling Microscope (STM). The STM operated simultaneously in the constant-current and gap-modulated mode. Highly reproducible STM images have been obtained and interpreted in terms of expected DNA structure. The main periodicity, regularly presented in molecules several hundred Ångstrom long, ranges from 25 Å to 35 Å with an average diameter of 22 Å. Higher resolution images of the minor groove have revealed the phosphate groups along the DNA backbones. Constant-current images of TAPO deposited on gold show a crystalline structure of rows of molecules with a side-by-side spacing of 3 Å.

  10. Targeting DNA with "light-up" pyrimidine triple-helical forming oligonucleotides conjugated to stabilizing fluorophores (LU-TFOs).

    PubMed

    Renard, Brice-Loïc; Lartia, Rémy; Asseline, Ulysse

    2008-12-01

    The synthesis of triple-helix-forming oligonucleotides (TFOs) linked to a series of cyanine monomethines has been performed. Eight cyanines including one thiocyanine, four thiazole orange analogues, and three quinocyanines were attached to the 5'-end of 10-mer pyrimidine TFOs. The binding properties of these modified TFOs with their double-stranded DNA target were studied by absorption and steady-state fluorescence spectroscopy. The stability of the triplex structures depended on the cyanine structure and the linker size used to connect both entities. The most efficient cyanines able to stabilize the triplex structures, when attached at the 5'-end of the TFO, have been incorporated at both ends and provided triplex structures with increased stability. Fluorescence studies have shown that for the TFOs involving one cyanine, an important intensity increase (up to 37-fold) in the fluorescent signal was observed upon their hybridization with the double-stranded target, proving hybridization. The conjugates involving thiazole orange attached by the benzothiazole ring provided the most balanced properties in terms of triplex stabilization, fluorescence intensity and fluorescence enhancement upon hybridization with the double-stranded target. In order to test the influence of different parameters such as the TFO sequence and length, thiazole orange was used to label 17-mer TFOs. Hybridizations of these TFOs with different duplexes, designed to study the influence of mismatches at both internal and terminal positions on the triplex structures, confirmed the possibility of triplex formation without loss of specificity together with a strong fluorescence enhancement (up to 13-fold). PMID:19005602

  11. Lightweight Substrates For Mirrors

    NASA Technical Reports Server (NTRS)

    Brown, D. Kyle

    1991-01-01

    New substrate uses conventional quasi-isotropic fabric laminate with surfacing layer of carbon-fiber paper consisting of randomly oriented chopped carbon fibers. Layered structure of fabric and paper relatively easy to manufacture. When impregnated with carbon, structure rigid and stable. Substrates of this type made quite thin, thus keeping areal weights to minimum. Mirrors of this type made faster, and cost less, than predecessors.

  12. Fossil avian eggshell preserves ancient DNA

    PubMed Central

    Oskam, Charlotte L.; Haile, James; McLay, Emma; Rigby, Paul; Allentoft, Morten E.; Olsen, Maia E.; Bengtsson, Camilla; Miller, Gifford H.; Schwenninger, Jean-Luc; Jacomb, Chris; Walter, Richard; Baynes, Alexander; Dortch, Joe; Parker-Pearson, Michael; Gilbert, M. Thomas P.; Holdaway, Richard N.; Willerslev, Eske; Bunce, Michael

    2010-01-01

    Owing to exceptional biomolecule preservation, fossil avian eggshell has been used extensively in geochronology and palaeodietary studies. Here, we show, to our knowledge, for the first time that fossil eggshell is a previously unrecognized source of ancient DNA (aDNA). We describe the successful isolation and amplification of DNA from fossil eggshell up to 19 ka old. aDNA was successfully characterized from eggshell obtained from New Zealand (extinct moa and ducks), Madagascar (extinct elephant birds) and Australia (emu and owl). Our data demonstrate excellent preservation of the nucleic acids, evidenced by retrieval of both mitochondrial and nuclear DNA from many of the samples. Using confocal microscopy and quantitative PCR, this study critically evaluates approaches to maximize DNA recovery from powdered eggshell. Our quantitative PCR experiments also demonstrate that moa eggshell has approximately 125 times lower bacterial load than bone, making it a highly suitable substrate for high-throughput sequencing approaches. Importantly, the preservation of DNA in Pleistocene eggshell from Australia and Holocene deposits from Madagascar indicates that eggshell is an excellent substrate for the long-term preservation of DNA in warmer climates. The successful recovery of DNA from this substrate has implications in a number of scientific disciplines; most notably archaeology and palaeontology, where genotypes and/or DNA-based species identifications can add significantly to our understanding of diets, environments, past biodiversity and evolutionary processes. PMID:20219731

  13. Transcription rates in DNA brushes.

    PubMed

    Yamamoto, Tetsuya; Safran, S A

    2015-04-21

    We theoretically predict the rate of transcription (TX) in DNA brushes by introducing the concept of TX dipoles that takes into account the unidirectional motion of enzymes (RNAP) along DNA during transcription as correlated pairs of sources and sinks in the relevant diffusion equation. Our theory predicts that the TX rates dramatically change upon the inversion of the orientation of the TX dipoles relative to the substrate because TX dipoles modulate the concentrations of RNAP in the solution. Comparing our theory with experiments suggests that, in some cases, DNA chain segments are relatively uniformly distributed in the brush, in contrast to the parabolic profile expected for flexible polymer brushes. PMID:25736601

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

    PubMed Central

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

    2015-01-01

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

  15. Mitochondrial DNA.

    ERIC Educational Resources Information Center

    Wright, Russell G.; Bottino, Paul J.

    1986-01-01

    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  16. DNA Banking

    SciTech Connect

    Reilly, P.R. )

    1992-11-01

    The author is involved in the ethical, legal, and social issues of banking of DNA and data from DNA analysis. In his attempt to determine the extent of DNA banking in the U.S., the author surveyed some commercial companies performing DNA banking services. This article summarizes the results of that survey, with special emphasis on the procedures the companies use to protect the privacy of individuals. 4 refs.

  17. Dna Sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1995-04-25

    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  18. Bonded semiconductor substrate

    DOEpatents

    Atwater, Jr.; Harry A. , Zahler; James M.

    2010-07-13

    Ge/Si and other nonsilicon film heterostructures are formed by hydrogen-induced exfoliation of the Ge film which is wafer bonded to a cheaper substrate, such as Si. A thin, single-crystal layer of Ge is transferred to Si substrate. The bond at the interface of the Ge/Si heterostructures is covalent to ensure good thermal contact, mechanical strength, and to enable the formation of an ohmic contact between the Si substrate and Ge layers. To accomplish this type of bond, hydrophobic wafer bonding is used, because as the invention demonstrates the hydrogen-surface-terminating species that facilitate van der Waals bonding evolves at temperatures above 600.degree. C. into covalent bonding in hydrophobically bound Ge/Si layer transferred systems.

  19. Decontamination of metal substrates

    SciTech Connect

    Vincent, L.D.

    1998-12-31

    A brief look at the history of surface corrosion and contamination of steel is important for understanding the best approach to proper cleaning of substrates prior to surface preparation and application of coatings and linings, particularly in immersion conditions such as encountered in railroad hopper and tank cars. All contaminants contribute to reduction of the coating or lining`s capacity to either protect the substrate or prevent contamination of the liquid cargo. This paper will explore the types of tests available to determine the levels of contamination, particularly sulfides, sulfates and chlorides, along with suggested methods to reduce theses contaminants to acceptable levels.

  20. Biaxially textured composite substrates

    DOEpatents

    Groves, James R.; Foltyn, Stephen R.; Arendt, Paul N.

    2005-04-26

    An article including a substrate, a layer of a metal phosphate material such as an aluminum phosphate material upon the surface of the substrate, and a layer of an oriented cubic oxide material having a rock-salt-like structure upon the metal phosphate material layer is provided together with additional layers such as a HTS top-layer of YBCO directly upon a layer of a buffer material such as a SrTi.sub.x Ru.sub.1-x O.sub.3 layer.

  1. Multiple alternative substrate kinetics.

    PubMed

    Anderson, Vernon E

    2015-11-01

    The specificity of enzymes for their respective substrates has been a focal point of enzyme kinetics since the initial characterization of metabolic chemistry. Various processes to quantify an enzyme's specificity using kinetics have been utilized over the decades. Fersht's definition of the ratio kcat/Km for two different substrates as the "specificity constant" (ref [7]), based on the premise that the important specificity existed when the substrates were competing in the same reaction, has become a consensus standard for enzymes obeying Michaelis-Menten kinetics. The expansion of the theory for the determination of the relative specificity constants for a very large number of competing substrates, e.g. those present in a combinatorial library, in a single reaction mixture has been developed in this contribution. The ratio of kcat/Km for isotopologs has also become a standard in mechanistic enzymology where kinetic isotope effects have been measured by the development of internal competition experiments with extreme precision. This contribution extends the theory of kinetic isotope effects to internal competition between three isotopologs present at non-tracer concentrations in the same reaction mix. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment. PMID:26051088

  2. Substrate system for spray forming

    DOEpatents

    Chu, Men G.; Chernicoff, William P.

    2002-01-01

    A substrate system for receiving a deposit of sprayed metal droplets including a movable outer substrate on which the sprayed metal droplets are deposited. The substrate system also includes an inner substrate disposed adjacent the outer substrate where the sprayed metal droplets are deposited on the outer substrate. The inner substrate includes zones of differing thermal conductivity to resist substrate layer porosity and to resist formation of large grains and coarse constituent particles in a bulk layer of the metal droplets which have accumulated on the outer substrate. A spray forming apparatus and associated method of spray forming a molten metal to form a metal product using the substrate system of the invention is also provided.

  3. Substrate system for spray forming

    DOEpatents

    Chu, Men G.; Chernicoff, William P.

    2000-01-01

    A substrate system for receiving a deposit of sprayed metal droplets including a movable outer substrate on which the sprayed metal droplets are deposited. The substrate system also includes an inner substrate disposed adjacent the outer substrate where the sprayed metal droplets are deposited on the outer substrate. The inner substrate includes zones of differing thermal conductivity to resist substrate layer porosity and to resist formation of large grains and coarse constituent particles in a bulk layer of the metal droplets which have accumulated on the outer substrate. A spray forming apparatus and associated method of spray forming a molten metal to form a metal product using the substrate system of the invention is also provided.

  4. DNA Immunization

    PubMed Central

    Wang, Shixia; Lu, Shan

    2013-01-01

    DNA immunization was discovered in early 1990s and its use has been expanded from vaccine studies to a broader range of biomedical research, such as the generation of high quality polyclonal and monoclonal antibodies as research reagents. In this unit, three common DNA immunization methods are described: needle injection, electroporation and gene gun. In addition, several common considerations related to DNA immunization are discussed. PMID:24510291

  5. DNA helicases: 'inching forward'.

    PubMed

    Soultanas, P; Wigley, D B

    2000-02-01

    Recently determined crystal structures of PcrA helicase complexed with a DNA substrate have revealed details of the helicase mechanism. PcrA and UvrD helicases have been shown to be functional as monomers, challenging previous suggestions that all helicases are required to be oligomeric. Crystal structures of the hexameric helicases RepA and T7 gene 4 explain the formation of hexameric assemblies from identical monomers with RecA-like folds, but their molecular mechanism remains elusive. PMID:10679457

  6. Terminal Deoxynucleotidyl Transferase: The Story of a Misguided DNA Polymerase

    PubMed Central

    Motea, Edward A.; Berdis, Anthony J.

    2009-01-01

    Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis. PMID:19596089

  7. DNA ALTERATIONS

    EPA Science Inventory

    The exposure of an organism to genotoxic chemicals may induce a cascade of genetic events. nitially, structural alterations to DNA are formed. ext, the DNA damage is processed and subsequently expressed in mutant gene products. inally, diseases result from the genetic damage. he ...

  8. DNA: Polymer and molecular code

    NASA Astrophysics Data System (ADS)

    Shivashankar, G. V.

    1999-10-01

    gene expression a prime example of a biological code. We developed a novel method of making DNA micro- arrays, the so-called DNA chip. Using the optical tweezer concept, we were able to pattern biomolecules on a solid substrate, developing a new type of sub-micron laser lithography. A laser beam is focused onto a thin gold film on a glass substrate. Laser ablation of gold results in local aggregation of nanometer scale beads conjugated with small DNA oligonucleotides, with sub-micron resolution. This leads to specific detection of cDNA and RNA molecules. We built a simple micro-array fabrication and detection in the laboratory, based on this method, to probe addressable pools (genes, proteins or antibodies). We have lately used molecular beacons (single stranded DNA with a stem-loop structure containing a fluorophore and quencher), for the direct detection of unlabelled mRNA. As a first step towards a study of the dynamics of the biological code, we have begun to examine the patterns of gene expression during virus (T7 phage) infection of E-coli bacteria.

  9. Enzyme analysis for Pompe disease in leukocytes; superior results with natural substrate compared with artificial substrates.

    PubMed

    van Diggelen, O P; Oemardien, L F; van der Beek, N A M E; Kroos, M A; Wind, H K; Voznyi, Y V; Burke, D; Jackson, M; Winchester, B G; Reuser, A J J

    2009-06-01

    Enzyme analysis for Pompe disease in leukocytes has been greatly improved by the introduction of acarbose, a powerful inhibitor of interfering alpha-glucosidases, which are present in granulocytes but not in lymphocytes. Here we show that the application of acarbose in the enzymatic assay employing the artificial substrate 4-methylumbelliferyl-alpha-D: -glucoside (MU-alphaGlc) is insufficient to clearly distinguish patients from healthy individuals in all cases. Also, the ratios of the activities without/with acarbose only marginally discriminated Pompe patients and healthy individuals. By contrast, when the natural substrate glycogen is used, the activity in leukocytes from patients (n = 82) with Pompe disease is at most 17% of the lowest control value. The use of artificial substrate in an assay with isolated lymphocytes instead of total leukocytes is a poor alternative as blood samples older than one day invariably yield lymphocyte preparations that are contaminated with granulocytes. To diagnose Pompe disease in leukocytes we recommend the use of glycogen as substrate in the presence of acarbose. This assay unequivocally excludes Pompe disease. To also exclude pseudo-deficiency of acid alpha-glucosidase caused by the sequence change c.271G>A (p.D91N or GAA2; homozygosity in approximately 1:1000 caucasians), a second assay employing MU-alphaGlc substrate plus acarbose or DNA analysis is required. PMID:19387865

  10. DNA affinity cleaving analysis of homeodomain-DNA interaction: identification of homeodomain consensus sites in genomic DNA.

    PubMed Central

    Shang, Z; Ebright, Y W; Iler, N; Pendergrast, P S; Echelard, Y; McMahon, A P; Ebright, R H; Abate, C

    1994-01-01

    We have incorporated the DNA-cleaving moiety o-phenanthroline-copper at amino acid 10 of the Msx-1 homeodomain, and we have analyzed site-specific DNA cleavage by the resulting Msx-1 derivative. We show that amino acid 10 of the Msx-1 homeodomain is close to the 5' end of the consensus DNA site 5'-(C/G)TAATTG-3' in the Msx-1-DNA complex. Our results indicate that the orientation of the Msx-1 homeodomain relative to DNA is analogous to the orientation of the engrailed and Antennapedia homeodomains. We show further that DNA affinity cleaving permits identification of consensus DNA sites for Msx-1 in kilobase DNA substrates. The specificity of the approach enabled us to identify an Msx-1 consensus DNA site within the transcriptional control region of the developmental regulatory gene Wnt-1. We propose that incorporation of o-phenanthroline-copper at amino acid 10 of a homeodomain may provide a generalizable strategy to determine the orientation of a homeodomain relative to DNA and to identify homeodomain consensus DNA sites in genomic DNA. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7904065

  11. Mechanisms of DNA Motor Proteins (Helicases)

    NASA Astrophysics Data System (ADS)

    Lohman, Timothy M.

    1996-03-01

    DNA helicases are ubiquitous motor proteins that couple the binding and hydrolysis of NTP to the unwinding of duplex (ds) DNA to form the single stranded (ss) DNA intermediates that are required for replication, recombination and repair. We are studying the DNA unwinding mechanisms catalyzed by two helicases from E. coli: Rep and Helicase II (UvrD) by examining the linkage of DNA binding, protein dimerization and nucleotide binding using both thermodynamic and kinetic approaches. A dimer of the Rep protein is the active form of the helicase; however, the dimer forms only upon binding either ss or ds DNA. There are significant cooperative interactions between the two DNA binding sites on the dimer and nucleotides (ATP, ADP) allosterically control the stabilities of the DNA ligation states of the Rep dimer. Based on these studies we have proposed an "active, rolling" mechanism for the Rep dimer unwinding of duplex DNA. An essential intermediate is a complex, in which ss- and ds-DNA bind simultaneously to each subunit of a Rep dimer. This model predicts that Rep helicase translocation along DNA is coupled to ATP binding, whereas ATP hydrolysis drives unwinding of multiple DNA base pairs for each catalytic event. Rapid chemical quench-flow and stopped-flow fluorescence studies of Rep and UvrD- catalyzed DNA unwinding of a series of non-natural DNA substrates support the "active, rolling" mechanism and rule out a strictly "passive" mechanism of unwinding. Kinetic studies of DNA and nucleotide binding and ATP hydrolysis by wild type and mutant Rep proteins will be discussed that bear on the coupling of ATP binding and hydrolysis to translocation along DNA and DNA unwinding.

  12. Synthesis of DNA oligonucleotides containing C5-ethynylbenzenesulfonamide-modified nucleotides (EBNA) by polymerases towards the construction of base functionalized nucleic acids.

    PubMed

    Goubet, Astrid; Chardon, Antoine; Kumar, Pawan; Sharma, Pawan K; Veedu, Rakesh N

    2013-02-01

    C5-Ethynylbenzenesulfonamide-modified nucleotide (EBNA) was investigated as substrate of various DNA polymerases. The experiments revealed that KOD, Phusion and Klenow DNA polymerases successfully accepted EBNA-T nucleotide as a substrate and yielded the fully extended DNA. KOD DNA polymerase was found to be the most efficient enzyme to furnish EBNA-T containing DNA in good yields. Phusion DNA polymerase efficiently amplified the template containing EBNA-T nucleotides by PCR. PMID:23265899

  13. [DNA computing].

    PubMed

    Błasiak, Janusz; Krasiński, Tadeusz; Popławski, Tomasz; Sakowski, Sebastian

    2011-01-01

    Biocomputers can be an alternative for traditional "silicon-based" computers, which continuous development may be limited due to further miniaturization (imposed by the Heisenberg Uncertainty Principle) and increasing the amount of information between the central processing unit and the main memory (von Neuman bottleneck). The idea of DNA computing came true for the first time in 1994, when Adleman solved the Hamiltonian Path Problem using short DNA oligomers and DNA ligase. In the early 2000s a series of biocomputer models was presented with a seminal work of Shapiro and his colleguas who presented molecular 2 state finite automaton, in which the restriction enzyme, FokI, constituted hardware and short DNA oligomers were software as well as input/output signals. DNA molecules provided also energy for this machine. DNA computing can be exploited in many applications, from study on the gene expression pattern to diagnosis and therapy of cancer. The idea of DNA computing is still in progress in research both in vitro and in vivo and at least promising results of these research allow to have a hope for a breakthrough in the computer science. PMID:21735816

  14. DNA Polymerase β Ribonucleotide Discrimination

    PubMed Central

    Cavanaugh, Nisha A.; Beard, William A.; Wilson, Samuel H.

    2010-01-01

    DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxy-counterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase β to utilize nucleotides with modified sugars. DNA polymerase β readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3′-endo sugar pucker facilitates nucleotide insertion, whereas a 2′-endo conformation inhibits insertion. PMID:20519499

  15. Nitrification in a zeoponic substrate

    NASA Technical Reports Server (NTRS)

    McGilloway, R. L.; Weaver, R. W.; Ming, D. W.; Gruener, J. E.

    2003-01-01

    Clinoptilolite is a zeolite mineral with high cation exchange capacity used in zeoponic substrates that have been proposed as a solid medium for growing plants or as a fertilizer material. The kinetics of nitrification has not been measured for NH4+ saturated zeoponic substrate. Experiments were conducted to evaluate the production of NO2- and NO3-, and nitrifier populations in zeoponic substrates. Small columns were filled with zeoponic substrate inoculated with a commercial inoculum or soil enrichment culture of nitrifying bacteria. In addition to column studies, a growth chamber study was conducted to evaluate the kinetics of nitrification in zeoponic substrates used to grow radishes (Raphanus sativus L.). The zeoponic substrate provided a readily available source of NH4+, and nitrifying bacteria were active in the substrate. Ammonium oxidation rates in column studies ranged from 5 to 10 micrograms N g-1 substrate h-1, and NO2- oxidation rates were 2 to 9.5 micrograms N g-1 substrate h-1. Rates determined from the growth chamber study were approximately 1.2 micrograms N g-1 substrate h-1. Quantities of NH4+ oxidized to NO2- and NO3- in inoculated zeoponic substrate were in excess of plant up-take. Acidification as a result of NH4+ oxidation resulted in a pH decline, and the zeoponic substrate showed limited buffering capacity.

  16. Dancing DNA.

    ERIC Educational Resources Information Center

    Pennisi, Elizabeth

    1991-01-01

    An imaging technique that uses fluorescent dyes and allows scientists to track DNA as it moves through gels or in solution is described. The importance, opportunities, and implications of this technique are discussed. (KR)

  17. Maintainable substrate carrier for electroplating

    SciTech Connect

    Chen, Chen-An; Abas, Emmanuel Chua; Divino, Edmundo Anida; Ermita, Jake Randal G.; Capulong, Jose Francisco S.; Castillo, Arnold Villamor; Ma; Diana Xiaobing

    2012-07-17

    One embodiment relates to a substrate carrier for use in electroplating a plurality of substrates. The carrier includes a non-conductive carrier body on which the substrates are placed and conductive lines embedded within the carrier body. A plurality of conductive clip attachment parts are attached in a permanent manner to the conductive lines embedded within the carrier body. A plurality of contact clips are attached in a removable manner to the clip attachment parts. The contact clips hold the substrates in place and conductively connecting the substrates with the conductive lines. Other embodiments, aspects and features are also disclosed.

  18. Maintainable substrate carrier for electroplating

    DOEpatents

    Chen, Chen-An; Abas, Emmanuel Chua; Divino, Edmundo Anida; Ermita, Jake Randal G.; Capulong, Jose Francisco S.; Castillo, Arnold Villamor; Ma, Diana Xiaobing

    2016-08-02

    One embodiment relates to a substrate carrier for use in electroplating a plurality of substrates. The carrier includes a non-conductive carrier body on which the substrates are placed and conductive lines embedded within the carrier body. A plurality of conductive clip attachment parts are attached in a permanent manner to the conductive lines embedded within the carrier body. A plurality of contact clips are attached in a removable manner to the clip attachment parts. The contact clips hold the substrates in place and conductively connecting the substrates with the conductive lines. Other embodiments, aspects and features are also disclosed.

  19. Unravelling DNA

    NASA Astrophysics Data System (ADS)

    Conroy, Rs; Danilowicz, C.

    2004-04-01

    The forces involved in the biology of life are carefully balanced between stopping thermal fluctuations ripping our DNA apart and having bonds weak enough to allow enzymes to function. The application of recently developed techniques for measuring piconewton forces and imaging at the nanometre scale on a molecule-by-molecule basis has dramatically increased the impact of single-molecule biophysics. This article describes the most commonly used techniques for imaging and manipulating single biomolecules. Using these techniques, the mechanical properties of DNA can be investigated, for example through measurements of the forces required to stretch and unzip the DNA double helix. These properties determine the ease with which DNA can be folded into the cell nucleus and the size and complexity of the accompanying cellular machinery. Part of this cellular machinery is enzymes, which manipulate, repair and transcribe the DNA helix. Enzymatic function is increasingly being investigated at the single molecule level to give better understanding of the forces and processes involved in the genetic cycle. One of the challenges is to transfer this understanding of single molecules into living systems. Already there have been some notable successes, such as the development of techniques for gene expression through the application of mechanical forces to cells, and the imaging and control of viral infection of a cell. This understanding and control of DNA has also been used to design molecules, which can self-assemble into a range of structures.

  20. DNA detection using origami paper analytical devices

    PubMed Central

    Ellington, Andrew D.; Crooks, Richard M.

    2013-01-01

    We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs. analyte concentration was observed with an extrapolated limit of detection < 5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems. PMID:24070108

  1. DNA detection using origami paper analytical devices.

    PubMed

    Scida, Karen; Li, Bingling; Ellington, Andrew D; Crooks, Richard M

    2013-10-15

    We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs analyte concentration was observed with an extrapolated limit of detection <5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems. PMID:24070108

  2. What Is Mitochondrial DNA?

    MedlinePlus

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  3. Substrate inhibition of transketolase.

    PubMed

    Solovjeva, Olga N; Kovina, Marina V; Kochetov, German A

    2016-03-01

    We studied the influence of the acceptor substrate of transketolase on the activity of the enzyme in the presence of reductants. Ribose-5-phosphate in the presence of cyanoborohydride decreased the transketolase catalytic activity. The inhibition is caused by the loss of catalytic function of the coenzyme-thiamine diphosphate. Similar inhibitory effect was observed in the presence of NADPH. This could indicate its possible regulatory role not only towards transketolase, but also towards the pentose phosphate pathway of carbohydrate metabolism overall, taking into account the fact that it inhibits not only transketolase but also another enzyme of the pentose phosphate pathway--glucose 6-phosphate dehydrogenase [Eggleston L.V., Krebs H.A. Regulation of the pentose phosphate cycle, Biochem. J. 138 (1974) 425-435]. PMID:26708478

  4. Accelerating peroxidase mimicking nanozymes using DNA

    NASA Astrophysics Data System (ADS)

    Liu, Biwu; Liu, Juewen

    2015-08-01

    DNA-capped iron oxide nanoparticles are nearly 10-fold more active as a peroxidase mimic for TMB oxidation than naked nanoparticles. To understand the mechanism, the effect of DNA length and sequence is systematically studied, and other types of polymers are also compared. This rate enhancement is more obvious with longer DNA and, in particular, poly-cytosine. Among the various polymer coatings tested, DNA offers the highest rate enhancement. A similar acceleration is also observed for nanoceria. On the other hand, when the positively charged TMB substrate is replaced by the negatively charged ABTS, DNA inhibits oxidation. Therefore, the negatively charged phosphate backbone and bases of DNA can increase TMB binding by the iron oxide nanoparticles, thus facilitating the oxidation reaction in the presence of hydrogen peroxide.DNA-capped iron oxide nanoparticles are nearly 10-fold more active as a peroxidase mimic for TMB oxidation than naked nanoparticles. To understand the mechanism, the effect of DNA length and sequence is systematically studied, and other types of polymers are also compared. This rate enhancement is more obvious with longer DNA and, in particular, poly-cytosine. Among the various polymer coatings tested, DNA offers the highest rate enhancement. A similar acceleration is also observed for nanoceria. On the other hand, when the positively charged TMB substrate is replaced by the negatively charged ABTS, DNA inhibits oxidation. Therefore, the negatively charged phosphate backbone and bases of DNA can increase TMB binding by the iron oxide nanoparticles, thus facilitating the oxidation reaction in the presence of hydrogen peroxide. Electronic supplementary information (ESI) available: Methods, TEM, UV-vis and DLS data. See DOI: 10.1039/c5nr04176g

  5. Substrate effect on the enhanced biodegradation of carbon tetrachloride

    NASA Astrophysics Data System (ADS)

    Wu, Shian C.; Doong, Rueyan

    1993-03-01

    The effects of different substrates on the biotransformation of heavily chlorinated hydrocarbons under anaerobic conditions were investigated to evaluate the feasibility of in-situ bioremediation of the contaminated groundwaters by amending different substrates. The substrate-fed batches were anaerobically incubated with the addition of either acetate, glucose, methanol, or dissolved organic matter (DOM) with concentrations ranging from 10 to 30 mg/L. Experimental results demonstrated that the effect of the substrates on the dechlorination of the compounds varied. The removal efficiency was observed greatest for glucose- then methanol- and acetate-fed batches and least for DOM-fed batches. The sequence of the enhancement efficiency is consistent with the sequence of the reducing potentials of these substrates. Changing the substrate concentration could vary the dechlorination capability of the system. The viable counts of microorganisms determined by the direct epifluorescence counting technique showed that the batches with higher concentration of the supplemental substrate produced higher bacterial cell numbers. Moreover, from microscopic observations, different compositions of bacterial population were found. Small-sized bacteria with spheric shape were observed when culture bottles were amended with either acetate or DOM, whereas large-sized bacteria with rod-shape were predominant for bottles amended with glucose. Also, higher DNA contents were demonstrated for glucose-fed batches.

  6. Environmental DNA mapping of Zebra Mussel populations

    USGS Publications Warehouse

    Amberg, Jon; Merkes, Christopher

    2016-01-01

    Environmental DNA (eDNA) has become a popular tool for detecting aquatic invasive species, but advancements have made it possible to potentially answer other questions like reproduction, movement, and abundance of the targeted organism. In this study we developed a Zebra Mussel (Dreissena polymorpha) eDNA protocol. We then determined if this assay could be used to help determine Zebra Mussel biomass in a lake with a well-established population of Zebra Mussels and a lake with an emerging population of mussels. Our eDNA assay detected DNA of Zebra Mussels but not DNA from more than 20 other species of fish and mussels, many commonly found in Minnesota waters. Our assay did not predict biomass. We did find that DNA from Zebra Mussels accumulated in softer substrates in both lakes, even though the mussels were predominately on the harder substrates. Therefore, we concluded that eDNA may be useful to detect the presence of Zebra Mussels in these lakes but our assay/approach could not predict biomass.

  7. Ancient DNA

    PubMed Central

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    In the past two decades, ancient DNA research has progressed from the retrieval of small fragments of mitochondrial DNA from a few late Holocene specimens, to large-scale studies of ancient populations, phenotypically important nuclear loci, and even whole mitochondrial genome sequences of extinct species. However, the field is still regularly marred by erroneous reports, which underestimate the extent of contamination within laboratories and samples themselves. An improved understanding of these processes and the effects of damage on ancient DNA templates has started to provide a more robust basis for research. Recent methodological advances have included the characterization of Pleistocene mammal populations and discoveries of DNA preserved in ancient sediments. Increasingly, ancient genetic information is providing a unique means to test assumptions used in evolutionary and population genetics studies to reconstruct the past. Initial results have revealed surprisingly complex population histories, and indicate that modern phylogeographic studies may give misleading impressions about even the recent evolutionary past. With the advent and uptake of appropriate methodologies, ancient DNA is now positioned to become a powerful tool in biological research and is also evolving new and unexpected uses, such as in the search for extinct or extant life in the deep biosphere and on other planets. PMID:15875564

  8. DNA vaccines

    NASA Astrophysics Data System (ADS)

    Gregersen, Jens-Peter

    2001-12-01

    Immunization by genes encoding immunogens, rather than with the immunogen itself, has opened up new possibilities for vaccine research and development and offers chances for new applications and indications for future vaccines. The underlying mechanisms of antigen processing, immune presentation and regulation of immune responses raise high expectations for new and more effective prophylactic or therapeutic vaccines, particularly for vaccines against chronic or persistent infectious diseases and tumors. Our current knowledge and experience of DNA vaccination is summarized and critically reviewed with particular attention to basic immunological mechanisms, the construction of plasmids, screening for protective immunogens to be encoded by these plasmids, modes of application, pharmacokinetics, safety and immunotoxicological aspects. DNA vaccines have the potential to accelerate the research phase of new vaccines and to improve the chances of success, since finding new immunogens with the desired properties is at least technically less demanding than for conventional vaccines. However, on the way to innovative vaccine products, several hurdles have to be overcome. The efficacy of DNA vaccines in humans appears to be much less than indicated by early studies in mice. Open questions remain concerning the persistence and distribution of inoculated plasmid DNA in vivo, its potential to express antigens inappropriately, or the potentially deleterious ability to insert genes into the host cell's genome. Furthermore, the possibility of inducing immunotolerance or autoimmune diseases also needs to be investigated more thoroughly, in order to arrive at a well-founded consensus, which justifies the widespread application of DNA vaccines in a healthy population.

  9. Reprint of "Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair".

    PubMed

    Çağlayan, Melike; Wilson, Samuel H

    2015-12-01

    DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER. PMID:26596511

  10. PLZT capacitor on glass substrate

    DOEpatents

    Fairchild, M. Ray; Taylor, Ralph S.; Berlin, Carl W.; Wong, Celine W. K.; Ma, Beihai; Balachandran, Uthamalingam

    2016-01-05

    A lead-lanthanum-zirconium-titanate (PLZT) capacitor on a substrate formed of glass. The first metallization layer is deposited on a top side of the substrate to form a first electrode. The dielectric layer of PLZT is deposited over the first metallization layer. The second metallization layer deposited over the dielectric layer to form a second electrode. The glass substrate is advantageous as glass is compatible with an annealing process used to form the capacitor.

  11. PLZT capacitor on glass substrate

    DOEpatents

    Fairchild, Manuel Ray; Taylor, Ralph S.; Berlin, Carl W.; Wong, Celine Wk; Ma, Beihai; Balachandran, Uthamalingam

    2016-03-29

    A lead-lanthanum-zirconium-titanate (PLZT) capacitor on a substrate formed of glass. The first metallization layer is deposited on a top side of the substrate to form a first electrode. The dielectric layer of PLZT is deposited over the first metallization layer. The second metallization layer deposited over the dielectric layer to form a second electrode. The glass substrate is advantageous as glass is compatible with an annealing process used to form the capacitor.

  12. The bacterial DnaC helicase loader is a DnaB ring breaker

    PubMed Central

    Arias-Palomo, Ernesto; O’Shea, Valerie L.; Hood, Iris V.; Berger, James M.

    2013-01-01

    Summary Dedicated AAA+ ATPases help deposit hexameric ring-shaped helicases onto DNA to promote replication in cellular organisms. To understand how loading occurs, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the ATP-bound structure of the intact E. coli DnaB•DnaC helicase/loader complex. The 480 kDa dodecamer forms a three-tiered assembly, in which DnaC adopts a spiral configuration that remodels N-terminal scaffolding and C-terminal motor regions of DnaB to produce a clear break in the helicase ring. Surprisingly, DnaC’s AAA+ fold is dispensable for ring remodeling, as the isolated helicase-binding domain of DnaC can both load DnaB onto DNA and increase the efficiency by which the helicase acts on substrates in vitro. Our data demonstrate that DnaC opens DnaB by a mechanism akin to that of polymerase clamp loaders, and indicate that bacterial replicative helicases, like their eukaryotic counterparts, possess auto-regulatory elements that influence how the hexameric motor domains are loaded onto and unwind DNA. PMID:23562643

  13. Pedestal substrate for coated optics

    DOEpatents

    Hale, Layton C.; Malsbury, Terry N.; Patterson, Steven R.

    2001-01-01

    A pedestal optical substrate that simultaneously provides high substrate dynamic stiffness, provides low surface figure sensitivity to mechanical mounting hardware inputs, and constrains surface figure changes caused by optical coatings to be primarily spherical in nature. The pedestal optical substrate includes a disk-like optic or substrate section having a top surface that is coated, a disk-like base section that provides location at which the substrate can be mounted, and a connecting cylindrical section between the base and optics or substrate sections. The connecting cylindrical section may be attached via three spaced legs or members. However, the pedestal optical substrate can be manufactured from a solid piece of material to form a monolith, thus avoiding joints between the sections, or the disk-like base can be formed separately and connected to the connecting section. By way of example, the pedestal optical substrate may be utilized in the fabrication of optics for an extreme ultraviolet (EUV) lithography imaging system, or in any optical system requiring coated optics and substrates with reduced sensitivity to mechanical mounts.

  14. Sealed substrate carrier for electroplating

    DOEpatents

    Ganti, Kalyana Bhargava

    2012-07-17

    One embodiment relates to a substrate carrier for use in electroplating a plurality of substrates. The substrate carrier includes a non-conductive carrier body on which the substrates are held, and conductive lines are embedded within the carrier body. A conductive bus bar is embedded into a top side of the carrier body and is conductively coupled to the conductive lines. A thermoplastic overmold covers a portion of the bus bar, and there is a plastic-to-plastic bond between the thermoplastic overmold and the non-conductive carrier body. Other embodiments, aspects and features are also disclosed.

  15. DNA codes

    SciTech Connect

    Torney, D. C.

    2001-01-01

    We have begun to characterize a variety of codes, motivated by potential implementation as (quaternary) DNA n-sequences, with letters denoted A, C The first codes we studied are the most reminiscent of conventional group codes. For these codes, Hamming similarity was generalized so that the score for matched letters takes more than one value, depending upon which letters are matched [2]. These codes consist of n-sequences satisfying an upper bound on the similarities, summed over the letter positions, of distinct codewords. We chose similarity 2 for matches of letters A and T and 3 for matches of the letters C and G, providing a rough approximation to double-strand bond energies in DNA. An inherent novelty of DNA codes is 'reverse complementation'. The latter may be defined, as follows, not only for alphabets of size four, but, more generally, for any even-size alphabet. All that is required is a matching of the letters of the alphabet: a partition into pairs. Then, the reverse complement of a codeword is obtained by reversing the order of its letters and replacing each letter by its match. For DNA, the matching is AT/CG because these are the Watson-Crick bonding pairs. Reversal arises because two DNA sequences form a double strand with opposite relative orientations. Thus, as will be described in detail, because in vitro decoding involves the formation of double-stranded DNA from two codewords, it is reasonable to assume - for universal applicability - that the reverse complement of any codeword is also a codeword. In particular, self-reverse complementary codewords are expressly forbidden in reverse-complement codes. Thus, an appropriate distance between all pairs of codewords must, when large, effectively prohibit binding between the respective codewords: to form a double strand. Only reverse-complement pairs of codewords should be able to bind. For most applications, a DNA code is to be bi-partitioned, such that the reverse-complementary pairs are separated

  16. Methods for immobilizing nucleic acids on a gel substrate

    DOEpatents

    Mirzabekov, Andrei Darievich; Proudnikov, Dimitri Y.; Timofeev, Edward N.; Kochetkova, Svetlana V.; Florentiev, Vladimir L.; Shick, Valentine V.

    1999-01-01

    A method for labeling oligonucleotide molecules, and for immobilizing oligonucleotide and DNA molecules is provided comprising modifying the molecules to create a chemically active group, and contacting activated fluorescent dyes to the region. A method for preparing an immobilization substrate is also provided comprising modifying a gel to contain desired functional groups which covalently interact with certain moieties of the oligonucleotide molecules. A method for immobilizing biomolecules and other molecules within a gel by copolymerization of allyl-substituted oligonucleotides, DNA and proteins with acrylamide is also provided.

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

    SciTech Connect

    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.

  18. HIV-1 Integrase-DNA Recognition Mechanisms

    PubMed Central

    Kessl, Jacques J.; McKee, Christopher J.; Eidahl, Jocelyn O.; Shkriabai, Nikolozi; Katz, Ari; Kvaratskhelia, Mamuka

    2009-01-01

    Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review. PMID:21994566

  19. DNA computing.

    PubMed

    Gibbons, A; Amos, M; Hodgson, D

    1997-02-01

    DNA computation is a novel and exciting recent development at the interface of computer science and molecular biology. We describe the current activity in this field following the seminal work of Adleman, who recently showed how techniques of molecular biology may be applied to the solution of a computationally intractable problem. PMID:9013647

  20. DNA Music.

    ERIC Educational Resources Information Center

    Miner, Carol; della Villa, Paula

    1997-01-01

    Describes an activity in which students reverse-translate proteins from their amino acid sequences back to their DNA sequences then assign musical notes to represent the adenine, guanine, cytosine, and thymine bases. Data is obtained from the National Institutes of Health (NIH) on the Internet. (DDR)

  1. DNA Investigations.

    ERIC Educational Resources Information Center

    Mayo, Ellen S.; Bertino, Anthony J.

    1991-01-01

    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  2. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  3. DNA molecules sticking on a vicinal Si(111) surface observed by noncontact atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Arai, Toyoko; Tomitori, Masahiko; Saito, Masato; Tamiya, Eiichi

    2002-03-01

    The DNA molecules on a vicinal Si(111) substrate with steps of single and double bi-atomic layers are imaged by noncontact atomic force microscopy (nc-AFM) in ultrahigh vacuum. The water solution containing pBR322 plasmid DNA molecules digested by Cla I is dropped on the substrate in a pure nitrogen atmosphere in a glove box, which is connected to the introduction chamber of the AFM. The ends of DNA molecules are frequently folded and pinned at the steps on the substrate, and the DNA strings often lie along the step. The chemical and dipole interactions between the DNA and the semiconductor substrate seem to play an important role in folding, pinning and sticking on the Si(111) substrate.

  4. Carbon nanotubes on a substrate

    DOEpatents

    Gao, Yufei [Kennewick, WA; Liu, Jun [West Richland, WA

    2002-03-26

    The present invention includes carbon nanotubes whose hollow cores are 100% filled with conductive filler. The carbon nanotubes are in uniform arrays on a conductive substrate and are well-aligned and can be densely packed. The uniformity of the carbon nanotube arrays is indicated by the uniform length and diameter of the carbon nanotubes, both which vary from nanotube to nanotube on a given array by no more than about 5%. The alignment of the carbon nanotubes is indicated by the perpendicular growth of the nanotubes from the substrates which is achieved in part by the simultaneous growth of the conductive filler within the hollow core of the nanotube and the densely packed growth of the nanotubes. The present invention provides a densely packed carbon nanotube growth where each nanotube is in contact with at least one nearest-neighbor nanotube. The substrate is a conductive substrate coated with a growth catalyst, and the conductive filler can be single crystals of carbide formed by a solid state reaction between the substrate material and the growth catalyst. The present invention further provides a method for making the filled carbon nanotubes on the conductive substrates. The method includes the steps of depositing a growth catalyst onto the conductive substrate as a prepared substrate, creating a vacuum within a vessel which contains the prepared substrate, flowing H2/inert (e.g. Ar) gas within the vessel to increase and maintain the pressure within the vessel, increasing the temperature of the prepared substrate, and changing the H2/Ar gas to ethylene gas such that the ethylene gas flows within the vessel. Additionally, varying the density and separation of the catalyst particles on the conductive substrate can be used to control the diameter of the nanotubes.

  5. Multichannel arrays on polymer substrates: toward a disposable proteomics chip

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Ehrfeld, Wolfgang; Pommersheim, Rainer

    1999-03-01

    Miniaturization is dramatically changing the shape of biotechnology. After the first wave of discoveries inventions in the field of analytical methods and DNA-probes on silicon chips, the trend in recent years has been to more complex and integrated systems in terms of microfabrication for production purposes mainly focused on polymer substrates. Additionally, an increased complexity in the biochemical functionality for tasks like cell handling, cell lysis, polymerase chain reaction, DNA-sequencing and recently in the field of proteomics research can be observed. In this paper we describe the practical approach to a polymer substrate based, microfabricated chip-based multichannel array for 2D capillary electrophoresis. This chip can be fabricated by classical mass production techniques like hot embossing or injection modeling, and has the potential for on-chip-integration of electrodes and detection system.

  6. Mechanism of DNA loading by the DNA repair helicase XPD.

    PubMed

    Constantinescu-Aruxandei, Diana; Petrovic-Stojanovska, Biljana; Penedo, J Carlos; White, Malcolm F; Naismith, James H

    2016-04-01

    The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5' to 3' helicase with an essential iron-sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase fromThermoplasma acidophilumhas been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD fromSulfolobus acidocaldiariusthat lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD. PMID:26896802

  7. Palladium on Plastic Substrates for Plasmonic Devices

    PubMed Central

    Zuppella, Paola; Pasqualotto, Elisabetta; Zuccon, Sara; Gerlin, Francesca; Corso, Alain Jody; Scaramuzza, Matteo; De Toni, Alessandro; Paccagnella, Alessandro; Pelizzo, Maria Guglielmina

    2015-01-01

    Innovative chips based on palladium thin films deposited on plastic substrates have been tested in the Kretschmann surface plasmon resonance (SPR) configuration. The new chips combine the advantages of a plastic support that is interesting and commercially appealing and the physical properties of palladium, showing inverted surface plasmon resonance (ISPR). The detection of DNA chains has been selected as the target of the experiment, since it can be applied to several medical early diagnostic tools, such as different biomarkers of cancers or cystic fibrosis. The results are encouraging for the use of palladium in SPR-based sensors of interest for both the advancement of biodevices and the development of hydrogen sensors. PMID:25585102

  8. Electrical switching of DNA monolayers investigated by surface plasmon resonance.

    PubMed

    Yang, Xiaohai; Wang, Qing; Wang, Kemin; Tan, Weihong; Yao, Jing; Li, Huimin

    2006-06-20

    The switching of DNA monolayers between a "lying" and a "standing" state initiated by applying electric field, and the subsequent DNA hybridization at different states were investigated in a contactless, label-free mode by surface plasmon resonance (SPR) technique. The results showed that the strength of the electric field and surface coverage could influence the switching of DNA monolayers. In addition, it was found that DNA hybridization efficiency could be enhanced or decreased when DNA probes stood straight up or lay flat on the gold surface, depending on the potential of the gold substrate. The enhancement of DNA hybridization efficiency reached the maximum when surface coverage reached 5.87 x 10(12) molecules/cm(2) and the potential of gold substrate was more negative than -0.7 V (versus ITO-coated glass). The research may be helpful for the construction of sensitive biosensors, biochips, and nanoscale electronic devices. PMID:16768490

  9. Substrate With Low Secondary Emissions

    NASA Technical Reports Server (NTRS)

    Jensen, Kenneth A. (Inventor); Curren, Arthur N. (Inventor); Roman, Robert F. (Inventor)

    2000-01-01

    The present invention is directed to a method and apparatus for producing a highly -textured surface on a copper substrate -with only extremely small amounts of texture-inducing seeding or masking material. The texture-inducing seeding material is delivered to the copper substrate electrically switching the seeding material in and out of a circuit loop.

  10. Photoresist substrate having robust adhesion

    DOEpatents

    Dentinger, Paul M.

    2005-07-26

    A substrate material for LIGA applications w hose general composition is Ti/Cu/Ti/SiO.sub.2. The SiO.sub.2 is preferably applied to the Ti/Cu/Ti wafer as a sputtered coating, typically about 100 nm thick. This substrate composition provides improved adhesion for epoxy-based photoresist materials, and particularly the photoresist material SU-8.

  11. MTH1 Substrate Recognition--An Example of Specific Promiscuity.

    PubMed

    Nissink, J Willem M; Bista, Michal; Breed, Jason; Carter, Nikki; Embrey, Kevin; Read, Jonathan; Winter-Holt, Jon J

    2016-01-01

    MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1. PMID:26999531

  12. MTH1 Substrate Recognition—An Example of Specific Promiscuity

    PubMed Central

    Nissink, J. Willem M.; Bista, Michal; Breed, Jason; Carter, Nikki; Embrey, Kevin; Read, Jonathan; Winter-Holt, Jon J.

    2016-01-01

    MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1. PMID:26999531

  13. Formation of DNA triple helices inhibits DNA unwinding by the SV40 large T-antigen helicase.

    PubMed Central

    Peleg, M; Kopel, V; Borowiec, J A; Manor, H

    1995-01-01

    Previous studies have indicated that d(TC)n.d(GA)n microsatellites may serve as arrest signals for mammalian DNA replication through the ability of such sequences to form DNA triple helices and thereby inhibit replication enzymes. To further test this hypothesis, we examined the ability of d(TC)i.d(GA)i.d(TC)i triplexes to inhibit DNA unwinding in vitro by a model eukaryotic DNA helicase, the SV40 large T-antigen. DNA substrates that were able to form triplexes, and non-triplex-forming control substrates, were tested. We found that the presence of DNA triplexes, as assayed by endonuclease S1 and osmium tetroxide footprinting, significantly inhibited DNA unwinding by T-antigen. Strong inhibition was observed not only at acidic pH values, in which the triplexes were most stable, but also at physiological pH values in the range 6.9-7.2. Little or no inhibition was detected at pH 8.7. Based on these results, and on previous studies of DNA polymerases, we suggest that DNA triplexes may form in vivo and cause replication arrest through a dual inhibition of duplex unwinding by DNA helicases and of nascent strand synthesis by DNA polymerases. DNA triplexes also have the potential to inhibit recombination and repair processes in which helicases and polymerases are involved. Images PMID:7753619

  14. Method for imaging informational biological molecules on a semiconductor substrate

    NASA Technical Reports Server (NTRS)

    Coles, L. Stephen (Inventor)

    1994-01-01

    Imaging biological molecules such as DNA at rates several times faster than conventional imaging techniques is carried out using a patterned silicon wafer having nano-machined grooves which hold individual molecular strands and periodically spaced unique bar codes permitting repeatably locating all images. The strands are coaxed into the grooves preferably using gravity and pulsed electric fields which induce electric charge attraction to the molecular strands in the bottom surfaces of the grooves. Differential imaging removes substrate artifacts.

  15. Composite substrate for bipolar electrodes

    DOEpatents

    Tekkanat, B.; Bolstad, J.J.

    1992-12-22

    Substrates for electrode systems, particularly those to be used for bipolar electrodes in zinc-bromine batteries, are disclosed. The substrates preferably include carbon-black as a conductive filler in a polymeric matrix, with reinforcing materials such as glass fibers. Warpage of the zinc-bromine electrodes which was experienced in the prior art and which was believed to be caused by physical expansion of the electrodes due to bromine absorption by the carbon-black, is substantially eliminated when new substrate fabrication techniques are employed. In the present invention, substrates are prepared using a lamination process known as glass mat reinforced thermoplastics technology or, in an alternate embodiment, the substrate is made using a slurry process. 4 figs.

  16. Composite substrate for bipolar electrodes

    DOEpatents

    Tekkanat, Bora; Bolstad, James J.

    1992-12-22

    Substrates for electrode systems, particularly those to be used for bipolar electrodes in zinc-bromine batteries, are disclosed. The substrates preferably include carbon-black as a conductive filler in a polymeric matrix, with reinforcing materials such as glass fibers. Warpage of the zinc-bromine electrodes which was experienced in the prior art and which was believed to be caused by physical expansion of the electrodes due to bromine absorption by the carbon-black, is substantially eliminated when new substrate fabrication techniques are employed. In the pesent invention, substrates are prepared using a lamination process known as glass mat reinforced thermoplastics technology or, in an alternate embodiment, the substrate is made using a slurry process.

  17. Electrospray Ionization on Solid Substrates

    PubMed Central

    So, Pui-Kin; Hu, Bin; Yao, Zhong-Ping

    2014-01-01

    Development of electrospray ionization on solid substrates (solid-substrate ESI) avoids the clogging problem encountered in conventional capillary-based ESI, allows more convenient sampling and permits new applications. So far, solid-substrate ESI with various materials, e.g., metals, paper, wood, fibers and biological tissue, has been developed, and applications ranging from analysis of pure compounds to complex mixtures as well as in vivo study were demonstrated. Particularly, the capability of solid-substrate ESI in direct analysis of complex samples, e.g., biological fluids and foods, has significantly facilitated mass spectrometric analysis in real-life applications and led to increasingly important roles of these techniques nowadays. In this review, various solid-substrate ESI techniques and their applications are summarized and the prospects in this field are discussed. PMID:26819900

  18. A new structural framework for integrating replication protein A into DNA processing machinery

    SciTech Connect

    Brosey, Chris; Yan, Chunli; Tsutakawa, Susan; Heller, William; Rambo, Robert; Tainer, John; Ivanov, Ivaylo; Chazin, Walter

    2013-01-17

    By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.

  19. Development of an optical biosensor based on surface-enhanced Raman scattering for DNA analysis

    NASA Astrophysics Data System (ADS)

    Yigit, Tugce; Akdogan, Ebru; Karagoz, Isık. Didem; Kahraman, Mehmet

    2016-03-01

    Rapid, accurate and sensitive DNA analysis is critically important for the diagnostic of genetic diseases. The most common method preferred in practice is fluorescence based microarrays to analyze the DNA. However, there exist some disadvantages related to the above-mentioned method such as the overlapping of the fluorescence emission wavelengths that can diminish in the performance of multiplexing, needed to obtain fluorescence spectra from each dye and photo degradation. In this study, a novel SERS based DNA analysis approach, which is Raman active dye-free and independent of SERS substrate properties, is developed. First, the single strand DNA probe is attached to the SERS substrate and half of the complimentary DNA is attached to gold nanoparticles, as well. We hypothesize that in the presence of target DNA, the complimentary DNA coupled colloids will bind to the SERS substrate surface via hybridization of single strand target DNA. To test this hypothesis, we used UV/Vis spectroscopy, atomic for microscopy (AFM) and dynamic light scattering (DLS). DNA analysis is demonstrated by a peak shift of the certain peak of the small molecules attached to the SERS substrate surface instead of SERS spectrum obtained in the presence of target DNA from the Raman reporter molecules. The degree of peak shifting will be used for the quantification of the target DNA in the sample. Plasmonic properties of SERS substrates and reproducibility issues will not be considerable due to the use of peak shifting instead of peak intensity for the qualitative analysis.

  20. Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity

    PubMed Central

    Levikova, Maryna; Klaue, Daniel; Seidel, Ralf; Cejka, Petr

    2013-01-01

    Dna2 is a nuclease-helicase involved in several key pathways of eukaryotic DNA metabolism. The potent nuclease activity of Saccharomyces cerevisiae Dna2 was reported to be required for all its in vivo functions tested to date. In contrast, its helicase activity was shown to be weak, and its inactivation affected only a subset of Dna2 functions. We describe here a complex interplay of the two enzymatic activities. We show that the nuclease of Dna2 inhibits its helicase by cleaving 5′ flaps that are required by the helicase domain for loading onto its substrate. Mutational inactivation of Dna2 nuclease unleashes unexpectedly vigorous DNA unwinding activity, comparable with that of the most potent eukaryotic helicases. Thus, the ssDNA-specific nuclease activity of Dna2 limits and controls the enzyme's capacity to unwind dsDNA. We postulate that regulation of this interplay could modulate the biochemical properties of Dna2 and thus license it to carry out its distinct cellular functions. PMID:23671118

  1. Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE

    PubMed Central

    Sen, Doyel; Patel, Gayatri; Patel, Smita S.

    2016-01-01

    A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE—a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA. PMID:26887820

  2. Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE.

    PubMed

    Sen, Doyel; Patel, Gayatri; Patel, Smita S

    2016-05-19

    A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE-a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA. PMID:26887820

  3. Isolation of Discrete Nanoparticle-DNA Conjugates for Plasmonic Applications

    SciTech Connect

    Alivisatos, Paul; Claridge, Shelley A.; Liang, Huiyang W.; Basu, Sourav Roger; Frechet, Jean M.J.; Alivisatos, A. Paul

    2008-04-11

    Discrete DNA-gold nanoparticle conjugates with DNA lengths as short as 15 bases for both 5 nm and 20 nm gold particles have been purified by anion-exchange HPLC. Conjugates comprising short DNA (<40 bases) and large gold particles (>_ 20 nm) are difficult to purify by other means, and are potential substrates for plasmon coupling experiments. Conjugate purity is demonstrated by hybridizing complementary conjugates to form discrete structures, which are visualized by TEM.

  4. Predicting the substrates of cloned plant O-methyltransferases.

    PubMed

    Schröder, Gudrun; Wehinger, Elke; Schröder, Joachim

    2002-01-01

    Plant O-methyltransferases (OMTs) have important roles in secondary metabolite biosynthesis. Sequencing projects and homology-based cloning strategies yield sequences for proteins with similarities to known OMTs, but the identification of the physiological substrates is not trivial. We investigated with a cDNA cloned from Catharanthus roseus the possibilities for predicting the substrates of OMTs, using the information from previous work and two newly identified motifs that were based on information from the crystal structures of two plant OMTs. The results, confirmed by functional analysis of the recombinant protein, indicated that a careful analysis of the deduced protein sequence can provide clues for predicting the substrates of cloned OMTs. PMID:11754938

  5. Coordinated DNA dynamics during the human telomerase catalytic cycle

    NASA Astrophysics Data System (ADS)

    Parks, Joseph W.; Stone, Michael D.

    2014-06-01

    The human telomerase reverse transcriptase (hTERT) utilizes a template within the integral RNA subunit (hTR) to direct extension of telomeres. Telomerase exhibits repeat addition processivity (RAP) and must therefore translocate the nascent DNA product into a new RNA:DNA hybrid register to prime each round of telomere repeat synthesis. Here, we use single-molecule FRET and nuclease protection assays to monitor telomere DNA structure and dynamics during the telomerase catalytic cycle. DNA translocation during RAP proceeds through a previously uncharacterized kinetic substep during which the 3‧-end of the DNA substrate base pairs downstream within the hTR template. The rate constant for DNA primer realignment reveals this step is not rate limiting for RAP, suggesting a second slow conformational change repositions the RNA:DNA hybrid into the telomerase active site and drives the extrusion of the 5‧-end of the DNA primer out of the enzyme complex.

  6. Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication.

    PubMed

    Yeo, Jung Eun; Wickramaratne, Susith; Khatwani, Santoshkumar; Wang, Yen-Chih; Vervacke, Jeffrey; Distefano, Mark D; Tretyakova, Natalia Y

    2014-08-15

    DNA-protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA-protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts. PMID:24918113

  7. Preparation of DNA-crosslinked polyacrylamide hydrogels.

    PubMed

    Previtera, Michelle L; Langrana, Noshir A

    2014-01-01

    Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels. PMID:25226067

  8. Substrate-Sensitive Graphene Oxidation.

    PubMed

    Zhang, Zhuhua; Yin, Jun; Liu, Xiaofei; Li, Jidong; Zhang, Jiahuan; Guo, Wanlin

    2016-03-01

    The inertness of graphene toward reaction with ambient molecules is essential for realizing durable devices with stable performance. Many device applications require graphene to contact with substrates, but whose impact on the chemical property of graphene has been largely overlooked. Here, we combine comprehensive first-principles analyses with experiments to show that graphene oxidation is highly sensitive to substrates. Graphene remains inert on SiO2 and hexagonal boron nitride but becomes increasingly weak against oxidation on metal substrates because of enhanced charge transfer and chemical interaction between them. In particular, Ni and Co substrates lead to spontaneous oxidation of graphene, while a Cu substrate maximally promotes the oxygen diffusion on graphene, with an estimated diffusivity 13 orders of magnitude higher than that on freestanding graphene. Bilayer graphene is revealed to have high oxidation resistance independent of substrate and thus is a better choice for high-performance nanoelectronics. Our findings should be extendable to a wide spectrum of chemical functionalizations of two-dimensional materials mediated by substrates. PMID:26884318

  9. Peptide nucleic acid (PNA) is capable of enhancing hammerhead ribozyme activity with long but not with short RNA substrates.

    PubMed Central

    Jankowsky, E; Strunk, G; Schwenzer, B

    1997-01-01

    Long RNA substrates are inefficiently cleaved by hammerhead ribozymes in trans. Oligonucleotide facilitators capable of affecting the ribozyme activity by interacting with the substrates at the termini of the ribozyme provide a possibility to improve ribozyme mediated cleavage of long RNA substrates. We have examined the effect of PNA as facilitator in vitro in order to test if even artificial compounds have facilitating potential. Effects of 12mer PNA- (peptide nucleic acid), RNA- and DNA-facilitators of identical sequence were measured with three substrates containing either 942, 452 or 39 nucleotides. The PNA facilitator enhances the ribozyme activity with both, the 942mer and the 452mer substrate to a slightly smaller extent than RNA and DNA facilitators. This effect was observed up to PNA facilitator:substrate ratios of 200:1. The enhancement becomes smaller as the PNA facilitator:substrate ratio exceeds 200:1. With the 39mer substrate, the PNA facilitator decreases the ribozyme activity by more than 100-fold, even at PNA facilitator:substrate ratios of 1:1. Although with long substrates the effect of the PNA facilitator is slightly smaller than the effect of identical RNA or DNA facilitators, PNA may be a more practical choice for potential applications in vivo because PNA is much more resistant to degradation by cellular enzymes. PMID:9207013

  10. Method of processing a substrate

    DOEpatents

    Babayan, Steven E.; Hicks, Robert F.

    2008-02-12

    The invention is embodied in a plasma flow device or reactor having a housing that contains conductive electrodes with openings to allow gas to flow through or around them, where one or more of the electrodes are powered by an RF source and one or more are grounded, and a substrate or work piece is placed in the gas flow downstream of the electrodes, such that said substrate or work piece is substantially uniformly contacted across a large surface area with the reactive gases emanating therefrom. The invention is also embodied in a plasma flow device or reactor having a housing that contains conductive electrodes with openings to allow gas to flow through or around them, where one or more of the electrodes are powered by an RF source and one or more are grounded, and one of the grounded electrodes contains a means of mixing in other chemical precursors to combine with the plasma stream, and a substrate or work piece placed in the gas flow downstream of the electrodes, such that said substrate or work piece is contacted by the reactive gases emanating therefrom. In one embodiment, the plasma flow device removes organic materials from a substrate or work piece, and is a stripping or cleaning device. In another embodiment, the plasma flow device kills biological microorganisms on a substrate or work piece, and is a sterilization device. In another embodiment, the plasma flow device activates the surface of a substrate or work piece, and is a surface activation device. In another embodiment, the plasma flow device etches materials from a substrate or work piece, and is a plasma etcher. In another embodiment, the plasma flow device deposits thin films onto a substrate or work piece, and is a plasma-enhanced chemical vapor deposition device or reactor.

  11. Escherichia coli MutL loads DNA helicase II onto DNA.

    PubMed

    Mechanic, L E; Frankel, B A; Matson, S W

    2000-12-01

    Previous studies have shown that MutL physically interacts with UvrD (DNA helicase II) (Hall, M. C., Jordan, J. R., and Matson, S. W. (1998) EMBO J. 17, 1535-1541) and dramatically stimulates the unwinding reaction catalyzed by UvrD in the presence and absence of the other protein components of the methyl-directed mismatch repair pathway (Yamaguchi, M., Dao, V., and Modrich, P. (1998) J. Biol. Chem. 273, 9197-9201). The mechanism of this stimulation was investigated using DNA binding assays, single-turnover helicase assays, and unwinding assays involving long duplex DNA substrates. The results indicate that MutL binds DNA and loads UvrD onto the DNA substrate. The interaction between MutL and DNA and that between MutL and UvrD are both important for stimulation of UvrD-catalyzed unwinding. MutL does not clamp UvrD onto the substrate; and therefore, the processivity of unwinding is not increased in the presence of MutL. The implications of these results are discussed, and models are presented for the mechanism of MutL stimulation as well as for the role of MutL as a master coordinator in the methyl-directed mismatch repair pathway. PMID:10984488

  12. Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases.

    PubMed

    Welter, Moritz; Verga, Daniela; Marx, Andreas

    2016-08-16

    DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High-fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high-fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template-dependent DNA synthesis, despite this "cargo" being more than 100-fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked-eye detection of DNA and RNA at single nucleotide resolution. PMID:27392211

  13. Study of DNA adsorption on mica surfaces using a surface force apparatus

    NASA Astrophysics Data System (ADS)

    Kan, Yajing; Tan, Qiyan; Wu, Gensheng; Si, Wei; Chen, Yunfei

    2015-02-01

    We report our studies on the adsorption properties of double-stranded DNA molecules on mica surfaces in a confined environment using a surface force apparatus. Specifically, we studied the influence of cation species and concentrations on DNA adsorption properties. Our results indicated that divalent cations (Mg2+ and Co2+) preferred to form uniform and moderately dense DNA layers on a mica substrate. By measuring the interactions between DNA-coated mica and bare mica in an aqueous solution, obvious adhesion was observed in a cobalt chloride solution, possibly due to the ion-correlation attraction between negatively charged DNA and the mica surface. Furthermore, the interaction differences that were observed with MgCl2 and CoCl2 solutions reveal that the specific adsorption behaviors of DNA molecules on a mica substrate were mediated by these two salts. Our results are helpful to elucidate the dynamics of DNA binding on a solid substrate.

  14. Structure-Specific nuclease activities of Artemis and the Artemis: DNA-PKcs complex

    PubMed Central

    Chang, Howard H.Y.; Lieber, Michael R.

    2016-01-01

    Artemis is a vertebrate nuclease with both endo- and exonuclease activities that acts on a wide range of nucleic acid substrates. It is the main nuclease in the non-homologous DNA end-joining pathway (NHEJ). Not only is Artemis important for the repair of DNA double-strand breaks (DSBs) in NHEJ, it is essential in opening the DNA hairpin intermediates that are formed during V(D)J recombination. Thus, humans with Artemis deficiencies do not have T- or B-lymphocytes and are diagnosed with severe combined immunodeficiency (SCID). While Artemis is the only vertebrate nuclease capable of opening DNA hairpins, it has also been found to act on other DNA substrates that share common structural features. Here, we discuss the key structural features that all Artemis DNA substrates have in common, thus providing a basis for understanding how this structure-specific nuclease recognizes its DNA targets. PMID:27198222

  15. Boron hydride polymer coated substrates

    DOEpatents

    Pearson, Richard K.; Bystroff, Roman I.; Miller, Dale E.

    1987-01-01

    A method is disclosed for coating a substrate with a uniformly smooth layer of a boron hydride polymer. The method comprises providing a reaction chamber which contains the substrate and the boron hydride plasma. A boron hydride feed stock is introduced into the chamber simultaneously with the generation of a plasma discharge within the chamber. A boron hydride plasma of ions, electrons and free radicals which is generated by the plasma discharge interacts to form a uniformly smooth boron hydride polymer which is deposited on the substrate.

  16. Boron hydride polymer coated substrates

    DOEpatents

    Pearson, R.K.; Bystroff, R.I.; Miller, D.E.

    1986-08-27

    A method is disclosed for coating a substrate with a uniformly smooth layer of a boron hydride polymer. The method comprises providing a reaction chamber which contains the substrate and the boron hydride plasma. A boron hydride feed stock is introduced into the chamber simultaneously with the generation of a plasma discharge within the chamber. A boron hydride plasma of ions, electrons and free radicals which is generated by the plasma discharge interacts to form a uniformly smooth boron hydride polymer which is deposited on the substrate.

  17. Direct cooled power electronics substrate

    DOEpatents

    Wiles, Randy H [Powell, TN; Wereszczak, Andrew A [Oak Ridge, TN; Ayers, Curtis W [Kingston, TN; Lowe, Kirk T [Knoxville, TN

    2010-09-14

    The disclosure describes directly cooling a three-dimensional, direct metallization (DM) layer in a power electronics device. To enable sufficient cooling, coolant flow channels are formed within the ceramic substrate. The direct metallization layer (typically copper) may be bonded to the ceramic substrate, and semiconductor chips (such as IGBT and diodes) may be soldered or sintered onto the direct metallization layer to form a power electronics module. Multiple modules may be attached to cooling headers that provide in-flow and out-flow of coolant through the channels in the ceramic substrate. The modules and cooling header assembly are preferably sized to fit inside the core of a toroidal shaped capacitor.

  18. Purcell factor based understanding of enhancements in surface plasmon-coupled emission with DNA architectures.

    PubMed

    Venkatesh, S; Badiya, Pradeep Kumar; Ramamurthy, Sai Sathish

    2016-01-14

    We demonstrate the successful application of DNA thin films as dynamic bio-spacers in a surface plasmon-coupled emission platform. Site-directed DNA modification using silver and carbon nanomaterials resulted in an amplified Purcell factor (PF) and >130-fold fluorescence enhancements. We present unique architectures of DNA as a plasmonic spacer in metal-dielectric-metal substrates. PMID:26651026

  19. Raman-based system for DNA sequencing-mapping and other separations

    DOEpatents

    Vo-Dinh, Tuan

    1994-01-01

    DNA sequencing and mapping are performed by using a Raman spectrometer with a surface enhanced Raman scattering (SERS) substrate to enhance the Raman signal. A SERS label is attached to a DNA fragment and then analyzed with the Raman spectrometer to identify the DNA fragment according to characteristics of the Raman spectrum generated.

  20. Raman-based system for DNA sequencing-mapping and other separations

    DOEpatents

    Vo-Dinh, T.

    1994-04-26

    DNA sequencing and mapping are performed by using a Raman spectrometer with a surface enhanced Raman scattering (SERS) substrate to enhance the Raman signal. A SERS label is attached to a DNA fragment and then analyzed with the Raman spectrometer to identify the DNA fragment according to characteristics of the Raman spectrum generated. 11 figures.

  1. Film Growth on Nanoporous Substrate

    NASA Astrophysics Data System (ADS)

    Zhang, Xue; Joy, James; Zhao, Chenwei; Xu, J. M.; Valles, James

    Self-ordered nanoporous anodic aluminum oxide (AAO) provides an easy way to fabricate nano structured material, such as nano wires and nano particles. We employ AAO as substrates and focus on the thermally evaporated film growth on the surface of the substrate. With various materials deposited onto the substrate, we find the films show different structures, e,g. ordered array of nano particles for Lead and nanohoneycomb structure for Silver. We relate the differing behaviors to the difference of surface energy and diffusion constant. To verify this, the effect of substrate temperature on the film growth has been explored and the structure of the film has been successfully changed through the process. We are grateful for the support of NSF Grants No. DMR-1307290.

  2. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  3. The structural basis for substrate recognition by mammalian polynucleotide kinase 3’ phosphatase

    PubMed Central

    Garces, Fernando; Pearl, Laurence H.; Oliver, Antony W.

    2016-01-01

    Mammalian polynucleotide kinase 3’ phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the non-homologous end-joining (NHEJ) and base-excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3’-phosphates from, or by phosphorylating 5’-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3’-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end-seeking. The structure of dsDNA bound to the 5’-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA-ends in the context of single-strand and double-strand breaks, and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites. PMID:22055185

  4. Sequencing mitochondrial DNA polymorphisms by hybridization

    SciTech Connect

    Chee, M.S.; Lockhart, D.J.; Hubbell, E.

    1994-09-01

    We have investigated the use of DNA chips for genetic analysis, using human mitochondrial DNA (mtDNA) as a model. The DNA chips are made up of ordered arrays of DNA oligonucleotide probes, synthesized on a glass substrate using photolithographic techniques. The synthesis site for each different probe is specifically addressed by illumination of the substrate through a photolithographic mask, achieving selective deprotection Nucleoside phosphoramidites bearing photolabile protecting groups are coupled only to exposed sites. Repeated cycles of deprotection and coupling generate all the probes in parallel. The set of 4{sup N} N-mer probes can be synthesized in only 4N steps. Any subset can be synthesized in 4N steps. Any subset can be synthesized in 4N or fewer steps. Sequences amplified from the D-loop region of human mitochondrial DNA (mtDNA) were fluorescently labelled and hybridized to DNA chips containing probes specific for mtDNA. Each nucleotide of a 1.3 kb region spanning the D loop is represented by four probes on the chip. Each probe has a different base at the position of interest: together they comprise a set of A, C, G and T probes which are otherwise identical. In principle, only one probe-target hybrid will be a perfect match. The other three will be single base mismatches. Fluorescence imaging of the hybridized chip allows quantification of hybridization signals. Heterozygous mixtures of sequences can also be characterized. We have developed software to quantitate and interpret the hybridization signals, and to call the sequence automatically. Results of sequence analysis of human mtDNAs will be presented.

  5. Substrate channeling in proline metabolism

    PubMed Central

    Arentson, Benjamin W.; Sanyal, Nikhilesh; Becker, Donald F.

    2012-01-01

    Proline metabolism is an important pathway that has relevance in several cellular functions such as redox balance, apoptosis, and cell survival. Results from different groups have indicated that substrate channeling of proline metabolic intermediates may be a critical mechanism. One intermediate is pyrroline-5-carboxylate (P5C), which upon hydrolysis opens to glutamic semialdehyde (GSA). Recent structural and kinetic evidence indicate substrate channeling of P5C/GSA occurs in the proline catabolic pathway between the proline dehydrogenase and P5C dehydrogenase active sites of bifunctional proline utilization A (PutA). Substrate channeling in PutA is proposed to facilitate the hydrolysis of P5C to GSA which is unfavorable at physiological pH. The second intermediate, gamma-glutamyl phosphate, is part of the proline biosynthetic pathway and is extremely labile. Substrate channeling of gamma-glutamyl phosphate is thought to be necessary to protect it from bulk solvent. Because of the unfavorable equilibrium of P5C/GSA and the reactivity of gamma-glutamyl phosphate, substrate channeling likely improves the efficiency of proline metabolism. Here, we outline general strategies for testing substrate channeling and review the evidence for channeling in proline metabolism. PMID:22201749

  6. Wrinkled DNA.

    PubMed Central

    Arnott, S; Chandrasekaran, R; Puigjaner, L C; Walker, J K; Hall, I H; Birdsall, D L; Ratliff, R L

    1983-01-01

    The B form of poly d(GC):poly d(GC) in orthorhombic microcrystallites in oriented fibers has a secondary structure in which a dinucleotide is the repeated motif rather than a mononucleotide as in standard, smooth B DNA. One set of nucleotides (probably GpC) has the same conformations as the smooth form but the alternate (CpG) nucleotides have a different conformation at C3'-O3'. This leads to a distinctive change in the orientation of the phosphate groups. Similar perturbations can be detected in other poly d(PuPy):poly d(PuPy) DNAs such as poly d(IC):poly d(IC) and poly d(AT):poly d(AT) in their D forms which have tetragonal crystal environments. This suggests that such perturbations are intrinsic to all stretches of duplex DNA where purines and pyrimidines alternate and may play a role in the detection and exploitation of such sequences by regulatory proteins. Images PMID:6572358

  7. Optical DNA

    NASA Astrophysics Data System (ADS)

    Vijaywargi, Deepak; Lewis, Dave; Kirovski, Darko

    A certificate of authenticity (COA) is an inexpensive physical object with a random and unique structure S which is hard to near-exactly replicate. An inexpensive device should be able to scan object’s physical “fingerprint,” a set of features that represents S. In this paper, we explore one set of requirements that optical media such as DVDs should satisfy, to be considered as COAs. As manufacturing of such media produces inevitable errors, we use the locations and count of these errors as a “fingerprint” for each optical disc: its optical DNA. The “fingerprint” is signed using publisher’s private-key and the resulting signature is stored onto the optical medium using a post-production process. Standard DVD players with altered firmware that includes publisher’s public-key, should be able to verify the authenticity of DVDs protected with optical DNA. Our key finding is that for the proposed protocol, only DVDs with exceptional wear-and-tear characteristics would result in an inexpensive and viable anti-counterfeiting technology.

  8. Switching from single-stranded to double-stranded DNA limits the unwinding processivity of ring-shaped T7 DNA helicase

    PubMed Central

    Jeong, Yong-Joo; Rajagopal, Vaishnavi; Patel, Smita S.

    2013-01-01

    Phage T7 helicase unwinds double-stranded DNA (dsDNA) by encircling one strand while excluding the complementary strand from its central channel. When T7 helicase translocates on single-stranded DNA (ssDNA), it has kilobase processivity; yet, it is unable to processively unwind linear dsDNA, even 60 base-pairs long. Particularly, the GC-rich dsDNAs are unwound with lower amplitudes under single-turnover conditions. Here, we provide evidence that T7 helicase switches from ssDNA to dsDNA during DNA unwinding. The switching propensity is higher when dsDNA is GC-rich or when the 3′-overhang of forked DNA is <15 bases. Once helicase encircles dsDNA, it travels along dsDNA and dissociates from the end of linear DNA without strand separation, which explains the low unwinding amplitude of these substrates. Trapping the displaced strand with ssDNA binding protein or changing its composition to morpholino oligomer that does not interact with helicase increases the unwinding amplitude. We conclude that the displaced strand must be continuously excluded and kept away from the central channel for processive DNA unwinding. The finding that T7 helicase can switch from ssDNA to dsDNA binding mode during unwinding provides new insights into ways of limiting DNA unwinding and triggering fork regression when stalled forks need to be restarted. PMID:23446275

  9. Protein-protein interactions in DNA mismatch repair.

    PubMed

    Friedhoff, Peter; Li, Pingping; Gotthardt, Julia

    2016-02-01

    The principal DNA mismatch repair proteins MutS and MutL are versatile enzymes that couple DNA mismatch or damage recognition to other cellular processes. Besides interaction with their DNA substrates this involves transient interactions with other proteins which is triggered by the DNA mismatch or damage and controlled by conformational changes. Both MutS and MutL proteins have ATPase activity, which adds another level to control their activity and interactions with DNA substrates and other proteins. Here we focus on the protein-protein interactions, protein interaction sites and the different levels of structural knowledge about the protein complexes formed with MutS and MutL during the mismatch repair reaction. PMID:26725162

  10. Single DNA Molecule Patterning for High-Throughput Epigenetic Mapping

    PubMed Central

    Cerf, Aline; Cipriany, Benjamin R.; Benítez, Jaime J.; Craighead, Harold G.

    2013-01-01

    We present a method for profiling the 5-methyl cytosine distribution on single DNA molecules. Our method combines soft-lithography and molecular elongation to form ordered arrays of more than 250,000 individual DNA molecules immobilized on a solid substrate. The methylation state of the DNA is detected and mapped by binding of fluorescently labeled methyl-CpG binding domain peptides to the elongated dsDNA molecules and imaging of their distribution. The stretched molecules are fixed in their extended configuration by adsorption onto the substrate so analysis can be performed with high spatial resolution and signal averaging. We further prove this technique allows imaging of DNA molecules with different methylation states. PMID:21981444

  11. Mapping protease substrates using a biotinylated phage substrate library.

    SciTech Connect

    Scholle, M. D.; Kriplani, U.; Pabon, A.; Sishtla, K.; Glucksman, M. J.; Kay, B. K.; Biosciences Division; Chicago Medical School

    2005-05-05

    We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

  12. Fungal Diversity Is Not Determined by Mineral and Chemical Differences in Serpentine Substrates

    PubMed Central

    Daghino, Stefania; Murat, Claude; Sizzano, Elisa; Girlanda, Mariangela; Perotto, Silvia

    2012-01-01

    The physico-chemical properties of serpentine soils lead to strong selection of plant species. Whereas many studies have described the serpentine flora, little information is available on the fungal communities dwelling in these sites. Asbestos minerals, often associated with serpentine rocks, can be weathered by serpentine-isolated fungi, suggesting an adaptation to this substrate. In this study, we have investigated whether serpentine substrates characterized by the presence of rocks with distinct mineral composition could select for different fungal communities. Both fungal isolation and 454 pyrosequencing of amplicons obtained from serpentine samples following direct DNA extraction revealed some fungal taxa shared by the four ophiolitic substrates, but also highlighted several substrate-specific taxa. Bootstrap analysis of 454 OTU abundances indicated weak clustering of fungal assemblages from the different substrates, which did not match substrate classification based on exchangeable macronutrients and metals. Intra-substrate variability, as assessed by DGGE profiles, was similar across the four serpentine substrates, and comparable to inter-substrate variability. These findings indicate the absence of a correlation between the substrate (mineral composition and available cations) and the diversity of the fungal community. Comparison of culture-based and culture-independent methods supports the higher taxonomic precision of the former, as complementation of the better performance of the latter. PMID:23028507

  13. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  14. Site-directed, on-surface assembly of DNA nanostructures.

    PubMed

    Meyer, Rebecca; Saccà, Barbara; Niemeyer, Christof M

    2015-10-01

    Two-dimensional DNA lattices have been assembled from DNA double-crossover (DX) motifs on DNA-encoded surfaces in a site-specific manner. The lattices contained two types of single-stranded protruding arms pointing into opposite directions of the plane. One type of these protruding arms served to anchor the DNA lattice on the solid support through specific hybridization with surface-bound, complementary capture oligomers. The other type of arms allowed for further attachment of DNA-tethered probe molecules on the opposite side of the lattices exposed to the solution. Site-specific lattice assembly and attachment of fluorophore-labeled oligonucleotides and DNA-protein conjugates was demonstrated using DNA microarrays on flat, transparent mica substrates. Owing to their programmable orientation and addressability over a broad dynamic range from the nanometer to the millimeter length scale, such supramolecular architecture might be used for presenting biomolecules on surfaces, for instance, in biosensor applications. PMID:26306556

  15. Method and apparatus for synthesis of arrays of DNA probes

    DOEpatents

    Cerrina, Francesco; Sussman, Michael R.; Blattner, Frederick R.; Singh-Gasson, Sangeet; Green, Roland

    2002-04-23

    The synthesis of arrays of DNA probes sequences, polypeptides, and the like is carried out using a patterning process on an active surface of a substrate. An image is projected onto the active surface of the substrate utilizing an image former that includes a light source that provides light to a micromirror device comprising an array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two positions. Projection optics receives the light reflected from the micromirrors along an optical axis and precisely images the micromirrors onto the active surface of the substrate, which may be used to activate the surface of the substrate. The first level of bases may then be applied to the substrate, followed by development steps, and subsequent exposure of the substrate utilizing a different pattern of micromirrors, with further repeats until the elements of a two dimensional array on the substrate surface have an appropriate base bound thereto. The micromirror array can be controlled in conjunction with a DNA synthesizer supplying appropriate reagents to a flow cell containing the active substrate to control the sequencing of images presented by the micromirror array in coordination of the reagents provided to the substrate.

  16. Programmed DNA Self-Assembly and Logic Circuits

    NASA Astrophysics Data System (ADS)

    Li, Wei

    DNA is a unique, highly programmable and addressable biomolecule. Due to its reliable and predictable base recognition behavior, uniform structural properties, and extraordinary stability, DNA molecules are desirable substrates for biological computation and nanotechnology. The field of DNA computation has gained considerable attention due to the possibility of exploiting the massive parallelism that is inherent in natural systems to solve computational problems. This dissertation focuses on building novel types of computational DNA systems based on both DNA reaction networks and DNA nanotechnology. A series of related research projects are presented here. First, a novel, three-input majority logic gate based on DNA strand displacement reactions was constructed. Here, the three inputs in the majority gate have equal priority, and the output will be true if any two of the inputs are true. We subsequently designed and realized a complex, 5-input majority logic gate. By controlling two of the five inputs, the complex gate is capable of realizing every combination of OR and AND gates of the other 3 inputs. Next, we constructed a half adder, which is a basic arithmetic unit, from DNA strand operated XOR and AND gates. The aim of these two projects was to develop novel types of DNA logic gates to enrich the DNA computation toolbox, and to examine plausible ways to implement large scale DNA logic circuits. The third project utilized a two dimensional DNA origami frame shaped structure with a hollow interior where DNA hybridization seeds were selectively positioned to control the assembly of small DNA tile building blocks. The small DNA tiles were directed to fill the hollow interior of the DNA origami frame, guided through sticky end interactions at prescribed positions. This research shed light on the fundamental behavior of DNA based self-assembling systems, and provided the information necessary to build programmed nanodisplays based on the self-assembly of DNA.

  17. DNA computation: theory, practice, and prospects.

    PubMed

    Maley, C C

    1998-01-01

    L. M. Adleman launched the field of DNA computing with a demonstration in 1994 that strands of DNA could be used to solve the Hamiltonian path problem for a simple graph. He also identified three broad categories of open questions for the field. First, is DNA capable of universal computation? Second, what kinds of algorithms can DNA implement? Third, can the error rates in the manipulations of the DNA be controlled enough to allow for useful computation? In the two years that have followed, theoretical work has shown that DNA is in fact capable of universal computation. Furthermore, algorithms for solving interesting questions, like breaking the Data Encryption Standard, have been described using currently available technology and methods. Finally, a few algorithms have been proposed to handle some of the apparently crippling error rates in a few of the common processes used to manipulate DNA. It is thus unlikely that DNA computation is doomed to be only a passing curiosity. However, much work remains to be done on the containment and correction of errors. It is far from clear if the problems in the error rates can be solved sufficiently to ever allow for general-purpose computation that will challenge the more popular substrates for computation. Unfortunately, biological demonstrations of the theoretical results have been sadly lacking. To date, only the simplest of computations have been carried out in DNA. To make significant progress, the field will require both the assessment of the practicality of the different manipulations of DNA and the implementation of algorithms for realistic problems. Theoreticians, in collaboration with experimentalists, can contribute to this research program by settling on a small set of practical and efficient models for DNA computation. PMID:10021747

  18. DNA strand displacement, strand annealing and strand swapping by the Drosophila Bloom's syndrome helicase.

    PubMed

    Weinert, Brian T; Rio, Donald C

    2007-01-01

    Genetic analysis of the Drosophila Bloom's syndrome helicase homolog (mus309/DmBLM) indicates that DmBLM is required for the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination. Here we report the first biochemical study of DmBLM. Recombinant, epitope-tagged DmBLM was expressed in Drosophila cell culture and highly purified protein was prepared from nuclear extracts. Purified DmBLM exists exclusively as a high molecular weight ( approximately 1.17 MDa) species, is a DNA-dependent ATPase, has 3'-->5' DNA helicase activity, prefers forked substrate DNAs and anneals complementary DNAs. High-affinity DNA binding is ATP-dependent and low-affinity ATP-independent interactions contribute to forked substrate DNA binding and drive strand annealing. DmBLM combines DNA strand displacement with DNA strand annealing to catalyze the displacement of one DNA strand while annealing a second complementary DNA strand. PMID:17272294

  19. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action. PMID:26045555

  20. Porous substrates filled with nanomaterials

    DOEpatents

    Worsley, Marcus A.; Baumann, Theodore F.; Satcher, Jr., Joe H.; Stadermann, Michael

    2014-08-19

    A composition comprising: at least one porous carbon monolith, such as a carbon aerogel, comprising internal pores, and at least one nanomaterial, such as carbon nanotubes, disposed uniformly throughout the internal pores. The nanomaterial can be disposed in the middle of the monolith. In addition, a method for making a monolithic solid with both high surface area and good bulk electrical conductivity is provided. A porous substrate having a thickness of 100 microns or more and comprising macropores throughout its thickness is prepared. At least one catalyst is deposited inside the porous substrate. Subsequently, chemical vapor deposition is used to uniformly deposit a nanomaterial in the macropores throughout the thickness of the porous substrate. Applications include electrical energy storage, such as batteries and capacitors, and hydrogen storage.

  1. Coatings on reflective mask substrates

    DOEpatents

    Tong, William Man-Wai; Taylor, John S.; Hector, Scott D.; Mangat, Pawitter J. S.; Stivers, Alan R.; Kofron, Patrick G.; Thompson, Matthew A.

    2002-01-01

    A process for creating a mask substrate involving depositing: 1) a coating on one or both sides of a low thermal expansion material EUVL mask substrate to improve defect inspection, surface finishing, and defect levels; and 2) a high dielectric coating, on the backside to facilitate electrostatic chucking and to correct for any bowing caused by the stress imbalance imparted by either other deposited coatings or the multilayer coating of the mask substrate. An film, such as TaSi, may be deposited on the front side and/or back of the low thermal expansion material before the material coating to balance the stress. The low thermal expansion material with a silicon overlayer and a silicon and/or other conductive underlayer enables improved defect inspection and stress balancing.

  2. Comparative Study of Atomic Force Imaging of DNA on Graphite and Mica Surfaces

    NASA Astrophysics Data System (ADS)

    Klinov, Dmitry; Dwir, Benjamin; Kapon, Eli; Borovok, Natalia; Molotsky, Tatiana; Kotlyar, Alexander

    2006-09-01

    Various DNA-based structures (single-, double-, triple-stranded and quadruplex-DNA) were characterized using non-contact atomic-force microscopy on two substrates: modified highly-oriented pyrolitic graphite (HOPG) and mica. Deposition on mica, a conventional substrate used in studies of bio-molecules, results in strong deformation of all above types of molecules while deposition on modified HOPG affects the morphology of DNA much less compared to mica. This is demonstrated by a larger measured height of the DNA molecules deposited on HOPG, as compared to mica, and an increased flexibility of the molecules, evidenced by a shorter molecular end-to-end distance on HOPG. The estimated heights of the triplex and the quadruplex DNA measured on HOPG are similar to the diameter of these molecules in liquid. We thus conclude that modified HOPG is a substrate more suitable than mica for AFM characterization of DNA morphology.

  3. Quantitative analysis of molecular-level DNA crystal growth on a 2D surface

    PubMed Central

    Lee, Junwye; Hamada, Shogo; Hwang, Si Un; Amin, Rashid; Son, Junyoung; Dugasani, Sreekantha Reddy; Murata, Satoshi; Park, Sung Ha

    2013-01-01

    Crystallization is an essential process for understanding a molecule's aggregation behavior. It provides basic information on crystals, including their nucleation and growth processes. Deoxyribonucleic acid (DNA) has become an interesting building material because of its remarkable properties for constructing various shapes of submicron-scale DNA crystals by self-assembly. The recently developed substrate-assisted growth (SAG) method produces fully covered DNA crystals on various substrates using electrostatic interactions and provides an opportunity to observe the overall crystallization process. In this study, we investigated quantitative analysis of molecular-level DNA crystallization using the SAG method. Coverage and crystal size distribution were studied by controlling the external parameters such as monomer concentration, annealing temperature, and annealing time. Rearrangement during crystallization was also discussed. We expect that our study will provide overall picture of the fabrication process of DNA crystals on the charged substrate and promote practical applications of DNA crystals in science and technology. PMID:23817625

  4. Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases.

    PubMed

    Su, Yan; Peter Guengerich, F

    2016-01-01

    Pre-steady-state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step-by-step procedure for pre-steady-state kinetic analysis of single-nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF-3 rapid quench-flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre-steady-state assays on an RQF-3 rapid quench-flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. © 2016 by John Wiley & Sons, Inc. PMID:27248785

  5. Nanostructured Substrates for Optical Sensing

    PubMed Central

    Kemling, Jonathan W.; Qavi, Abraham J.; Bailey, Ryan C.

    2011-01-01

    Sensors that change color have the advantages of versatility, ease of use, high sensitivity, and low cost. The recent development of optically based chemical sensing platforms has increasingly employed substrates manufactured with advanced processing or fabrication techniques to provide precise control over shape and morphology of the sensor micro- and nano-structure. New sensors have resulted with improved capabilities for a number of sensing applications, including the detection of biomolecules and environmental monitoring. This perspective focuses on recent optical sensor devices that utilize nanostructured substrates. PMID:22174955

  6. Silicon cladding for mirror substrates

    NASA Astrophysics Data System (ADS)

    Duston, Christopher J.; Gunda, Nilesh; Schwartz, Jay R.; Robichaud, Joseph L.

    2009-08-01

    To reduce the finishing costs of silicon carbide mirror substrates, silicon claddings are applied allowing the surfaces to be more easily diamond turned and polished than the bare chemical vapor deposited (CVD) silicon carbide or bimodal reaction bonded SiC (RB-SiC). The benefits of using silicon as the optical face will be reviewed as will the process for applying plasma enhanced chemical vapor (PE-CVD) deposited amorphous silicon cladding on substrates. Using one mirror as an example, the successful finishing results will be shared.

  7. Adsorption on a stepped substrate

    NASA Astrophysics Data System (ADS)

    Merikoski, J.; Timonen, J.; Kaski, K.

    1994-09-01

    The effect of substrate steps on the adsorption of particles is considered. The problem is formulated as a lattice-gas model with nearest neighbor interactions and it is studied by a numerical transfer-matrix method. In particular, the influence of the substrate-induced row potential on adsorbed monolayers is discussed. It is found that strong row-transition-like features appear in the presence of a row potential and it is suggested that these may be seen in adsorption on vicinal faces.

  8. Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates.

    PubMed

    Kusevic, Denis; Kudithipudi, Srikanth; Jeltsch, Albert

    2016-03-18

    Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1. PMID:26797129

  9. Mitochondrial DNA replacement versus nuclear DNA persistence

    NASA Astrophysics Data System (ADS)

    Serva, Maurizio

    2006-10-01

    In this paper we consider two populations whose generations are not overlapping and whose size is large. The number of males and females in both populations is constant. Any generation is replaced by a new one and any individual has two parents concerning nuclear DNA and a single one (the mother) concerning mtDNA. Moreover, at any generation some individuals migrate from the first population to the second. In a finite random time T, the mtDNA of the second population is completely replaced by the mtDNA of the first. In the same time, the nuclear DNA is not completely replaced and a fraction F of the ancient nuclear DNA persists. We compute both T and F. Since this study shows that complete replacement of mtDNA in a population is compatible with the persistence of a large fraction of nuclear DNA, it may have some relevance for the 'out of Africa'/multiregional debate in palaeoanthropology.

  10. DNA modifications: Another stable base in DNA

    NASA Astrophysics Data System (ADS)

    Brazauskas, Pijus; Kriaucionis, Skirmantas

    2014-12-01

    Oxidation of 5-methylcytosine has been proposed to mediate active and passive DNA demethylation. Tracking the history of DNA modifications has now provided the first solid evidence that 5-hydroxymethylcytosine is a stable epigenetic modification.

  11. Thermodynamics of the DNA Damage Repair Steps of Human 8-Oxoguanine DNA Glycosylase

    PubMed Central

    Kuznetsov, Nikita A.; Kuznetsova, Alexandra A.; Vorobjev, Yuri N.; Krasnoperov, Lev N.; Fedorova, Olga S.

    2014-01-01

    Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van’t Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves. PMID:24911585

  12. Simulations of DNA topoisomerase 1B bound to supercoiled DNA reveal changes in the flexibility pattern of the enzyme and a secondary protein–DNA binding site

    PubMed Central

    D'Annessa, Ilda; Coletta, Andrea; Sutthibutpong, Thana; Mitchell, Jonathan; Chillemi, Giovanni; Harris, Sarah; Desideri, Alessandro

    2014-01-01

    Human topoisomerase 1B has been simulated covalently bound to a negatively supercoiled DNA minicircle, and its behavior compared to the enzyme bound to a simple linear DNA duplex. The presence of the more realistic supercoiled substrate facilitates the formation of larger number of protein–DNA interactions when compared to a simple linear duplex fragment. The number of protein–DNA hydrogen bonds doubles in proximity to the active site, affecting all of the residues in the catalytic pentad. The clamp over the DNA, characterized by the salt bridge between Lys369 and Glu497, undergoes reduced fluctuations when bound to the supercoiled minicircle. The linker domain of the enzyme, which is implicated in the controlled relaxation of superhelical stress, also displays an increased number of contacts with the minicircle compared to linear DNA. Finally, the more complex topology of the supercoiled DNA minicircle gives rise to a secondary DNA binding site involving four residues located on subdomain III. The simulation trajectories reveal significant changes in the interactions between the enzyme and the DNA for the more complex DNA topology, which are consistent with the experimental observation that the protein has a preference for binding to supercoiled DNA. PMID:25056319

  13. Synthesis of DNA

    DOEpatents

    Mariella, Jr., Raymond P.

    2008-11-18

    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  14. Sperm DNA oxidative damage and DNA adducts.

    PubMed

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm

  15. Engineered DNA ligases with improved activities in vitro.

    PubMed

    Wilson, Robert H; Morton, Susan K; Deiderick, Heather; Gerth, Monica L; Paul, Hayden A; Gerber, Ilana; Patel, Ankita; Ellington, Andrew D; Hunicke-Smith, Scott P; Patrick, Wayne M

    2013-07-01

    The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by ∼160% in blunt end 'vector + insert' cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases. PMID:23754529

  16. Scanning Electrochemical Microscopy of DNA Monolayers Modified with Nile Blue

    PubMed Central

    Gorodetsky, Alon A.; Hammond, William J.; Hill, Michael G.; Slowinski, Krzysztof; Barton, Jacqueline K.

    2009-01-01

    Scanning electrochemical microscopy (SECM) is used to probe long-range charge transport (CT) through DNA monolayers containing the redox-active Nile Blue (NB) intercalator covalently affixed at a specific location in the DNA film. At substrate potentials negative of the formal potential of covalently attached NB, the electrocatalytic reduction of Fe(CN)63− generated at the SECM tip is observed only when NB is located at the DNA/solution interface; for DNA films containing NB in close proximity to the DNA/electrode interface, the electrocatalytic effect is absent. This behavior is consistent with both rapid DNA-mediated CT between the NB intercalator and the gold electrode as well as a rate-limiting electron transfer between NB and the solution phase Fe(CN)63−. The DNA-mediated nature of the catalytic cycle is confirmed through sequence-specific and localized detection of attomoles of TATA-binding protein, a transcription factor that severely distorts DNA upon binding. Importantly, the strategy outlined here is general and allows for the local investigation of the surface characteristics of DNA monolayers both in the absence and in the presence of DNA binding proteins. These experiments highlight the utility of DNA-modified electrodes as versatile platforms for SECM detection schemes that take advantage of CT mediated by the DNA base pair stack. PMID:19053641

  17. Substrate for thin silicon solar cells

    DOEpatents

    Ciszek, Theodore F.

    1998-01-01

    A substrate for a photovoltaic device wherein the substrate is the base upon which photosensitive material is to be grown and the substrate comprises an alloy having boron in a range from 0.1 atomic % of the alloy to 1.3 atomic % of the alloy and the substrate has a resistivity less than 3.times.10.sup.-3 ohm-cm.

  18. Semiconductor films on flexible iridium substrates

    DOEpatents

    Goyal, Amit

    2005-03-29

    A laminate semiconductor article includes a flexible substrate, an optional biaxially textured oxide buffer system on the flexible substrate, a biaxially textured Ir-based buffer layer on the substrate or the buffer system, and an epitaxial layer of a semiconductor. Ir can serve as a substrate with an epitaxial layer of a semiconductor thereon.

  19. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  20. DNA polymerases and cancer

    PubMed Central

    Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.

    2013-01-01

    There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies. PMID:21258395

  1. Metallization of DNA and DNA Origami Using a Palladium Seeding Method

    NASA Astrophysics Data System (ADS)

    Geng, Yanli

    In this dissertation, I developed a Pd seeding method in association with electroless plating, to successfully metallize both lambda DNA and DNA origami templates on different surfaces. On mica surfaces, this method offered a fast, simple process, and the ability to obtain a relatively high yield of metallized DNA nanostructures. When using lambda DNA as the templates, I studied the effect of Pd(II) activation time on the seed height and density, and an optimal activation time between 10 and 30 min was obtained. Based on the Pd seeds formed on DNA, as well as a Pd electroless plating solution, continuous Pd nanowires that had an average diameter of ˜28 nm were formed with good selectivity on lambda DNA. The selected Pd activation time was also applied to metallize "T"-shape DNA origami, and Au coated branched nanostructures with a length between 200-250 nm, and wire diameters of ˜40 nm were also fabricated. In addition, I found that the addition of Mg2+ ion into the reducing agent and electroless plating solution could benefit the surface retention of Pd seeded DNA and Au plated DNA structures. This work indicated that DNA molecules were promising templates to fabricate metal nanostructures; moreover, the formation of Au metallized branched nanostructures showed progress towards nanodevice fabrication using DNA origami. Silicon surfaces were also used as the substrates for DNA metallization. More complex circular circuit DNA origami templates were used. To obtain high enough seed density, multiple Pd seeding steps were applied which showed good selectivity and the seeded DNA origami remained on the surface after seeding steps. I used distribution analysis of seed height to study the effect of seeding steps on both average height and the uniformity of the Pd seeds. Four-repeated palladium seedings were confirmed to be optimal by the AFM images, seed height distribution analysis, and Au electroless plating results. Both Au and Cu metallized circular circuit design

  2. Flexible substrate for printed wiring

    NASA Technical Reports Server (NTRS)

    Asakura, M.; Yabe, K.; Tanaka, H.; Soda, A.

    1982-01-01

    A very flexible substrate for printed wiring is disclosed which is composed of a blend of phenoxy resin-polyisocyanate-brominated epoxy resin in which the equivalent ration of the functional groups is hydroxyl grouped: isocyanate group: epoxy group = 1:0.2 to 2:0.5 to 3. The product has outstanding solder resistance and is applied to metal without using adhesives.

  3. Synthetic substrates for enzyme analysis

    DOEpatents

    Bissell, E.R.; Mitchell, A.R.; Pearson, K.W.; Smith, R.E.

    1983-06-14

    Synthetic substrates are provided which may be represented as A-D. The A moiety includes an amino acid, polypeptide, or derivative. The D moiety includes 7-amino coumarin derivatives having an electron withdrawing substituent group at the 3 position carbon or fused between the 3 and 4 position carbons. No Drawings

  4. Synthetic substrates for enzyme analysis

    DOEpatents

    Bissell, Eugene R.; Mitchell, Alexander R.; Pearson, Karen W.; Smith, Robert E.

    1983-01-01

    Synthetic substrates are provided which may be represented as A-D. The A moiety thereof includes an amino acid, polypeptide, or derivative thereof. The D moiety thereof includes 7-amino coumarin derivatives having an electron withdrawing substituent group at the 3 position carbon or fused between the 3 and 4 position carbons.

  5. Alternate Substrates for Bedding Plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this work was to evaluate fresh Clean Chip Residual (CCR) as a substrate component for production of container-grown annuals. Loblolly pine trees (Pinus taeda) (12 year old planted pine plantation) were thinned and processed for clean chips using a total tree harvester. The residual...

  6. Cellulosic Substrates and Challenges Ahead

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cost of production of butanol (acetone-butanol-ethanol; or ABE) is determined by feedstock prices, fermentation, recovery, by-product credits and the waste water treatment. Along these lines, we have an intensive research program on the use of various agricultural substrates, fermentation strate...

  7. Dynamics of the T4 Bacteriophage DNA Packasome Motor

    PubMed Central

    Dixit, Aparna; Ray, Krishanu; Lakowicz, Joseph R.; Black, Lindsay W.

    2011-01-01

    Conserved bacteriophage ATP-based DNA translocation motors consist of a multimeric packaging terminase docked onto a unique procapsid vertex containing a portal ring. DNA is translocated into the empty procapsid through the portal ring channel to high density. In vivo the T4 phage packaging motor deals with Y- or X-structures in the replicative concatemer substrate by employing a portal-bound Holliday junction resolvase that trims and releases these DNA roadblocks to packaging. Here using dye-labeled packaging anchored 3.7-kb Y-DNAs or linear DNAs, we demonstrate FRET between the dye-labeled substrates and GFP portal-containing procapsids and between GFP portal and single dye-labeled terminases. We show using FRET-fluorescence correlation spectroscopy that purified T4 gp49 endonuclease VII resolvase can release DNA compression in vitro in prohead portal packaging motor anchored and arrested Y-DNA substrates. In addition, using active terminases labeled at the N- and C-terminal ends with a single dye molecule, we show by FRET distance of the N-terminal GFP-labeled portal protein containing prohead at 6.9 nm from the N terminus and at 5.7 nm from the C terminus of the terminase. Packaging with a C-terminal fluorescent terminase on a GFP portal prohead, FRET shows a reduction in distance to the GFP portal of 0.6 nm in the arrested Y-DNA as compared with linear DNA; the reduction is reversed by resolvase treatment. Conformational changes in both the motor proteins and the DNA substrate itself that are associated with the power stroke of the motor are consistent with a proposed linear motor employing a terminal-to-portal DNA grip-and-release mechanism. PMID:21454482

  8. Crystal structure of a DNA catalyst.

    PubMed

    Ponce-Salvatierra, Almudena; Wawrzyniak-Turek, Katarzyna; Steuerwald, Ulrich; Höbartner, Claudia; Pena, Vladimir

    2016-01-14

    Catalysis in biology is restricted to RNA (ribozymes) and protein enzymes, but synthetic biomolecular catalysts can also be made of DNA (deoxyribozymes) or synthetic genetic polymers. In vitro selection from synthetic random DNA libraries identified DNA catalysts for various chemical reactions beyond RNA backbone cleavage. DNA-catalysed reactions include RNA and DNA ligation in various topologies, hydrolytic cleavage and photorepair of DNA, as well as reactions of peptides and small molecules. In spite of comprehensive biochemical studies of DNA catalysts for two decades, fundamental mechanistic understanding of their function is lacking in the absence of three-dimensional models at atomic resolution. Early attempts to solve the crystal structure of an RNA-cleaving deoxyribozyme resulted in a catalytically irrelevant nucleic acid fold. Here we report the crystal structure of the RNA-ligating deoxyribozyme 9DB1 (ref. 14) at 2.8 Å resolution. The structure captures the ligation reaction in the post-catalytic state, revealing a compact folding unit stabilized by numerous tertiary interactions, and an unanticipated organization of the catalytic centre. Structure-guided mutagenesis provided insights into the basis for regioselectivity of the ligation reaction and allowed remarkable manipulation of substrate recognition and reaction rate. Moreover, the structure highlights how the specific properties of deoxyribose are reflected in the backbone conformation of the DNA catalyst, in support of its intricate three-dimensional organization. The structural principles underlying the catalytic ability of DNA elucidate differences and similarities in DNA versus RNA catalysts, which is relevant for comprehending the privileged position of folded RNA in the prebiotic world and in current organisms. PMID:26735012

  9. SUMO-mediated regulation of DNA damage repair and responses

    PubMed Central

    Sarangi, Prabha; Zhao, Xiaolan

    2015-01-01

    Sumoylation plays important roles during DNA damage repair and responses. Recent broad-scope and substrate-based studies have shed light on the regulation and significance of sumoylation during these processes. An emerging paradigm is that sumoylation of many DNA metabolism proteins is controlled by DNA engagement. Such “on-site modification” can explain low substrate modification levels and has important implications in sumoylation mechanisms and effects. New studies also suggest that sumoylation can regulate a process through an ensemble effect or via major substrates. Additionally, we describe new trends in the functional effects of sumoylation, such as bi-directional changes in biomolecule binding and multi-level coordination with other modifications. These emerging themes and models will stimulate our thinking and research in sumoylation and genome maintenance. PMID:25778614

  10. Transverse charge transport through DNA oligomers in large-area molecular junctions

    NASA Astrophysics Data System (ADS)

    Katsouras, Ilias; Piliego, Claudia; Blom, Paul W. M.; de Leeuw, Dago M.

    2013-09-01

    We investigate the nature of charge transport in deoxyribonucleic acid (DNA) using self-assembled layers of DNA in large-area molecular junctions. A protocol was developed that yields dense monolayers where the DNA molecules are not standing upright, but are lying flat on the substrate. As a result the charge transport is measured not along the DNA molecules but in the transverse direction, across their diameter. The electrical transport data are consistent with the derived morphology. We demonstrate that the charge transport mechanism through DNA is identical to non-resonant tunneling through alkanethiols with identical length, classifying DNA as a dielectric.

  11. Case study: ancient sloth DNA recovered from hairs preserved in paleofeces.

    PubMed

    Clack, Andrew A; Macphee, Ross D E; Poinar, Hendrik N

    2012-01-01

    Ancient hair, which has proved to be an excellent source of well-preserved ancient DNA, is often preserved in paleofeces. Here, we separate and wash hair shafts preserved in a paleofecal specimen believed to be from a Darwin's ground sloth, Mylodon darwinii. After extracting DNA from the recovered and cleaned hair using a protocol optimized for DNA extraction from keratinous substrates, we amplify 12S and 16S rDNA sequences from the DNA extract. As expected, the recovered sequences most closely match previously published sequences of M. darwinii. Our results demonstrate that hair preserved in paleofeces, even from temperate cave environments, is an effective source of ancient DNA. PMID:22237521

  12. MGME1 processes flaps into ligatable nicks in concert with DNA polymerase γ during mtDNA replication.

    PubMed

    Uhler, Jay P; Thörn, Christian; Nicholls, Thomas J; Matic, Stanka; Milenkovic, Dusanka; Gustafsson, Claes M; Falkenberg, Maria

    2016-07-01

    Recently, MGME1 was identified as a mitochondrial DNA nuclease with preference for single-stranded DNA (ssDNA) substrates. Loss-of-function mutations in patients lead to mitochondrial disease with DNA depletion, deletions, duplications and rearrangements. Here, we assess the biochemical role of MGME1 in the processing of flap intermediates during mitochondrial DNA replication using reconstituted systems. We show that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POLγ. MGME1 generates a pool of imprecisely cut products (short flaps, nicks and gaps) that are converted to ligatable nicks by POLγ through extension or excision of the 3'-end strand. This is dependent on the 3'-5' exonuclease activity of POLγ which limits strand displacement activity and enables POLγ to back up to the nick by 3'-5' degradation. We also demonstrate that POLγ-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME1 cleavage and ligation during replication. Our findings have implications for RNA primer removal models, the 5'-end processing of nascent DNA at OriH, and DNA repair. PMID:27220468

  13. MGME1 processes flaps into ligatable nicks in concert with DNA polymerase γ during mtDNA replication

    PubMed Central

    Uhler, Jay P.; Thörn, Christian; Nicholls, Thomas J.; Matic, Stanka; Milenkovic, Dusanka; Gustafsson, Claes M.; Falkenberg, Maria

    2016-01-01

    Recently, MGME1 was identified as a mitochondrial DNA nuclease with preference for single-stranded DNA (ssDNA) substrates. Loss-of-function mutations in patients lead to mitochondrial disease with DNA depletion, deletions, duplications and rearrangements. Here, we assess the biochemical role of MGME1 in the processing of flap intermediates during mitochondrial DNA replication using reconstituted systems. We show that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POLγ. MGME1 generates a pool of imprecisely cut products (short flaps, nicks and gaps) that are converted to ligatable nicks by POLγ through extension or excision of the 3′-end strand. This is dependent on the 3′-5′ exonuclease activity of POLγ which limits strand displacement activity and enables POLγ to back up to the nick by 3′-5′ degradation. We also demonstrate that POLγ-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME1 cleavage and ligation during replication. Our findings have implications for RNA primer removal models, the 5′-end processing of nascent DNA at OriH, and DNA repair. PMID:27220468

  14. Modulation of the chaperone DnaK allosterism by the nucleotide exchange factor GrpE.

    PubMed

    Melero, Roberto; Moro, Fernando; Pérez-Calvo, María Ángeles; Perales-Calvo, Judit; Quintana-Gallardo, Lucía; Llorca, Oscar; Muga, Arturo; Valpuesta, José María

    2015-04-17

    Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaKNBD, with ATP inducing the open, substrate-receptive DnaKSBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaKNBD. DnaKSBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaKNBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaKSBD near DnaKNBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaKSBD, whereas the DnaKNBD-DnaKSBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function. PMID:25739641

  15. PREFACE: Cell-substrate interactions Cell-substrate interactions

    NASA Astrophysics Data System (ADS)

    Gardel, Margaret; Schwarz, Ulrich

    2010-05-01

    One of the most striking achievements of evolution is the ability to build cellular systems that are both robust and dynamic. Taken by themselves, both properties are obvious requirements: robustness reflects the fact that cells are there to survive, and dynamics is required to adapt to changing environments. However, it is by no means trivial to understand how these two requirements can be implemented simultaneously in a physical system. The long and difficult quest to build adaptive materials is testimony to the inherent difficulty of this goal. Here materials science can learn a lot from nature, because cellular systems show that robustness and dynamics can be achieved in a synergetic fashion. For example, the capabilities of tissues to repair and regenerate are still unsurpassed in the world of synthetic materials. One of the most important aspects of the way biological cells adapt to their environment is their adhesive interaction with the substrate. Numerous aspects of the physiology of metazoan cells, including survival, proliferation, differentiation and migration, require the formation of adhesions to the cell substrate, typically an extracellular matrix protein. Adhesions guide these diverse processes both by mediating force transmission from the cell to the substrate and by controlling biochemical signaling pathways. While the study of cell-substrate adhesions is a mature field in cell biology, a quantitative biophysical understanding of how the interactions of the individual molecular components give rise to the rich dynamics and mechanical behaviors observed for cell-substrate adhesions has started to emerge only over the last decade or so. The recent growth of research activities on cell-substrate interactions was strongly driven by the introduction of new physical techniques for surface engineering into traditional cell biological work with cell culture. For example, microcontact printing of adhesive patterns was used to show that cell fate depends

  16. Recent Insight into the Kinetic Mechanisms and Conformational Dynamics of Y-Family DNA Polymerases

    PubMed Central

    2015-01-01

    The kinetic mechanisms by which DNA polymerases catalyze DNA replication and repair have long been areas of active research. Recently discovered Y-family DNA polymerases catalyze the bypass of damaged DNA bases that would otherwise block replicative DNA polymerases and stall replication forks. Unlike DNA polymerases from the five other families, the Y-family DNA polymerases have flexible, solvent-accessible active sites that are able to tolerate various types of damaged template bases and allow for efficient lesion bypass. Their promiscuous active sites, however, also lead to fidelities that are much lower than those observed for other DNA polymerases and give rise to interesting mechanistic properties. Additionally, the Y-family DNA polymerases have several other unique structural features and undergo a set of conformational changes during substrate binding and catalysis different from those observed for replicative DNA polymerases. In recent years, pre-steady-state kinetic methods have been extensively employed to reveal a wealth of information about the catalytic properties of these fascinating noncanonical DNA polymerases. Here, we review many of the recent findings on the kinetic mechanisms of DNA polymerization with undamaged and damaged DNA substrates by the Y-family DNA polymerases, and the conformational dynamics employed by these error-prone enzymes during catalysis. PMID:24716482

  17. DNA Nanotechnology-- Architectures Designed with DNA

    NASA Astrophysics Data System (ADS)

    Han, Dongran

    As the genetic information storage vehicle, deoxyribonucleic acid (DNA) molecules are essential to all known living organisms and many viruses. It is amazing that such a large amount of information about how life develops can be stored in these tiny molecules. Countless scientists, especially some biologists, are trying to decipher the genetic information stored in these captivating molecules. Meanwhile, another group of researchers, nanotechnologists in particular, have discovered that the unique and concise structural features of DNA together with its information coding ability can be utilized for nano-construction efforts. This idea culminated in the birth of the field of DNA nanotechnology which is the main topic of this dissertation. The ability of rationally designed DNA strands to self-assemble into arbitrary nanostructures without external direction is the basis of this field. A series of novel design principles for DNA nanotechnology are presented here, from topological DNA nanostructures to complex and curved DNA nanostructures, from pure DNA nanostructures to hybrid RNA/DNA nanostructures. As one of the most important and pioneering fields in controlling the assembly of materials (both DNA and other materials) at the nanoscale, DNA nanotechnology is developing at a dramatic speed and as more and more construction approaches are invented, exciting advances will emerge in ways that we may or may not predict.

  18. A microchannel electrophoresis DNA sequencing system

    SciTech Connect

    Madabhushi, R S; Warth, T; Balch, J W; Bass, M; Brewer, L R; Copeland, A C; Davidson, J C; Fitch, J P; Kegelmeyer, L M; Kimbrough, J R; McCready, P; Nelson, D; Pastrone, R L; Richardson, P M; Swierkowski, S P; Tarte, L A; Vainer, M

    1999-01-01

    In order to increase the DNA sequencing throughput of the Joint Genome Institute, we have developed a microchannel electrophoresis system. The critical new and unique elements of this system include 1) a process for the production of arrays of 96 and 384 microchannels on bonded glass substrates up to 14 x 58 cm and 2) new sieving media for high resolution and high speed separations. With custom fabrication apparatus, microchannels are etched in a borosilicate substrate, and then fusion bonded to a top substrate 1.1 mm thick that has access holes formed in it. SEM examination shows a typical microchannel to be 40 micrometers deep x 180 micrometers wide by 46 cm long. This technology offers significant advantages over discrete capillaries or conventional slab-gel approaches. High throughput DNA sequencing with over 550 base pairs resolution has been achieved in roughly half the time of conventional sequencers. In February 1999, we begin a pre-production evaluation protocol for the microchannel and for three glass capillary electrophoresis systems (two from industry and one developed by Lawrence Berkeley National Laboratory for the Joint Genome Institute). In order to utilize these instruments for DNA production sequencing, we have been evaluating and implementing software to convert raw electropherograms into called DNA bases with an associated probability of error. Our original intent was to utilize the DNA base calling software known as Plan and Phred developed by the University of Washington. This software has been outstanding for our slab gel electrophoresis systems currently in the production facility. In our tests and evaluations of this software applied to microchannel data, we observed that the electropherograms are of a different statistical and underlying signal structure compared to slab gels. Even with substantial modifications to the software, base calling performance was not satisfactory for the microchannel data. In this paper, we will present o The

  19. DNA Scrunching in the Packaging of Viral Genomes.

    PubMed

    Waters, James T; Kim, Harold D; Gumbart, James C; Lu, Xiang-Jun; Harvey, Stephen C

    2016-07-01

    The motors that drive double-stranded DNA (dsDNA) genomes into viral capsids are among the strongest of all biological motors for which forces have been measured, but it is not known how they generate force. We previously proposed that the DNA is not a passive substrate but that it plays an active role in force generation. This "scrunchworm hypothesis" holds that the motor proteins repeatedly dehydrate and rehydrate the DNA, which then undergoes cyclic shortening and lengthening motions. These are captured by a coupled protein-DNA grip-and-release cycle to rectify the motion and translocate the DNA into the capsid. In this study, we examined the interactions of dsDNA with the dodecameric connector protein of bacteriophage ϕ29, using molecular dynamics simulations on four different DNA sequences, starting from two different conformations (A-DNA and B-DNA). In all four simulations starting with the protein equilibrated with A-DNA in the channel, we observed transitions to a common, metastable, highly scrunched conformation, designated A*. This conformation is very similar to one recently reported by Kumar and Grubmüller in much longer MD simulations on B-DNA docked into the ϕ29 connector. These results are significant for four reasons. First, the scrunched conformations occur spontaneously, without requiring lever-like protein motions often believed to be necessary for DNA translocation. Second, the transition takes place within the connector, providing the location of the putative "dehydrator". Third, the protein has more contacts with one strand of the DNA than with the other; the former was identified in single-molecule laser tweezer experiments as the "load-bearing strand". Finally, the spontaneity of the DNA-protein interaction suggests that it may play a role in the initial docking of DNA in motors like that of T4 that can load and package any sequence. PMID:27214211

  20. Human DNA polymerase. alpha. : Predicted functional domains and relationships with viral DNA polymerases

    SciTech Connect

    Wang, T.S.F.; Wong, S.W.; Korn, D. )

    1989-01-01

    The primary sequence of human DNA polymerase {alpha} deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage {phi}19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DBA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.

  1. Massively parallel determination and modeling of endonuclease substrate specificity

    PubMed Central

    Thyme, Summer B.; Song, Yifan; Brunette, T. J.; Szeto, Mindy D.; Kusak, Lara; Bradley, Philip; Baker, David

    2014-01-01

    We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution. PMID:25389263

  2. Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate

    PubMed Central

    Abid Ali, Ferdos; Renault, Ludovic; Gannon, Julian; Gahlon, Hailey L.; Kotecha, Abhay; Zhou, Jin Chuan; Rueda, David; Costa, Alessandro

    2016-01-01

    The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA. PMID:26888060

  3. Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate

    NASA Astrophysics Data System (ADS)

    Abid Ali, Ferdos; Renault, Ludovic; Gannon, Julian; Gahlon, Hailey L.; Kotecha, Abhay; Zhou, Jin Chuan; Rueda, David; Costa, Alessandro

    2016-02-01

    The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.

  4. Recent Advances in the Structural Mechanisms of DNA Glycosylases

    PubMed Central

    Brooks, Sonja C.; Adhikary, Suraj; Rubinson, Emily H.; Eichman, Brandt F.

    2012-01-01

    DNA glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of DNA. Since the discovery 28 years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. Work over the past several years has unraveled details for how the various DNA glycosylases survey DNA, detect damage within the duplex, select for the correct modification, and catalyze base excision. Here, we provide a broad overview of these latest advances in glycosylase mechanisms gleaned from structural enzymology, highlighting features common to all glycosylases as well as key differences that define their particular substrate specificities. PMID:23076011

  5. Quantitative DNA fiber mapping

    DOEpatents

    Gray, Joe W.; Weier, Heinz-Ulrich G.

    1998-01-01

    The present invention relates generally to the DNA mapping and sequencing technologies. In particular, the present invention provides enhanced methods and compositions for the physical mapping and positional cloning of genomic DNA. The present invention also provides a useful analytical technique to directly map cloned DNA sequences onto individual stretched DNA molecules.

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

    PubMed Central

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

    2014-01-01

    Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role. PMID:25159615

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

    PubMed Central

    Thalhammer, Verena; Längst, Gernot

    2015-01-01

    The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin. PMID:26496704

  8. Recent Development of Nano-Materials Used in DNA Biosensors

    PubMed Central

    Xu, Kai; Huang, Junran; Ye, Zunzhong; Ying, Yibin; Li, Yanbin

    2009-01-01

    As knowledge of the structure and function of nucleic acid molecules has increased, sequence-specific DNA detection has gained increased importance. DNA biosensors based on nucleic acid hybridization have been actively developed because of their specificity, speed, portability, and low cost. Recently, there has been considerable interest in using nano-materials for DNA biosensors. Because of their high surface-to-volume ratios and excellent biological compatibilities, nano-materials could be used to increase the amount of DNA immobilization; moreover, DNA bound to nano-materials can maintain its biological activity. Alternatively, signal amplification by labeling a targeted analyte with nano-materials has also been reported for DNA biosensors in many papers. This review summarizes the applications of various nano-materials for DNA biosensors during past five years. We found that nano-materials of small sizes were advantageous as substrates for DNA attachment or as labels for signal amplification; and use of two or more types of nano-materials in the biosensors could improve their overall quality and to overcome the deficiencies of the individual nano-components. Most current DNA biosensors require the use of polymerase chain reaction (PCR) in their protocols. However, further development of nano-materials with smaller size and/or with improved biological and chemical properties would substantially enhance the accuracy, selectivity and sensitivity of DNA biosensors. Thus, DNA biosensors without PCR amplification may become a reality in the foreseeable future. PMID:22346713

  9. Catalytic decarbonylation of biosourced substrates.

    PubMed

    Ternel, Jérémy; Lebarbé, Thomas; Monflier, Eric; Hapiot, Frédéric

    2015-05-11

    Linear α-olefins (LAO) are one of the main targets in the field of surfactants, lubricants, and polymers. With the depletion of petroleum resources, the production of LAO from renewable feedstocks has gained increasing interest in recent years. In the present study, we demonstrated that Ir catalysts were suitable to decarbonylate a wide range of biosourced substrates under rather mild conditions (160 °C, 5 h reaction time) in the presence of potassium iodide and acetic anhydride. The resulting LAO were obtained with good conversion and selectivity provided that the purity of the substrate, the nature of the ligand, and the amounts of the additives were controlled accurately. The catalytic system could be recovered efficiently by using a Kugelrohr distillation apparatus and recycled. PMID:25855489

  10. Graphene folding on flat substrates

    SciTech Connect

    Chen, Xiaoming; Zhao, Yadong; Ke, Changhong; Zhang, Liuyang; Wang, Xianqiao

    2014-10-28

    We present a combined experimental-theoretical study of graphene folding on flat substrates. The structure and deformation of the folded graphene sheet are experimentally characterized by atomic force microscopy. The local graphene folding behaviors are interpreted based on nonlinear continuum mechanics modeling and molecular dynamics simulations. Our study on self-folding of a trilayer graphene sheet reports a bending stiffness of about 6.57 eV, which is about four times the reported values for monolayer graphene. Our results reveal that an intriguing free sliding phenomenon occurs at the interlayer van der Waals interfaces during the graphene folding process. This work demonstrates that it is a plausible venue to quantify the bending stiffness of graphene based on its self-folding conformation on flat substrates. The findings reported in this work are useful to a better understanding of the mechanical properties of graphene and in the pursuit of its applications.

  11. Wetting phenomena on rough substrates

    NASA Astrophysics Data System (ADS)

    Li, Hao; Kardar, Mehran

    1990-10-01

    We consider wetting phenomena in the vicinity of rough substrates. The quenched random geometry of the substrate is assumed to be a self-affine fractal with a roughness exponent of ζS. Asymptotic critical properties on approaching complete and critical wetting transitions are studied by combining the replica method with scaling and renormalization-group arguments. We find new critical behavior, controlled by a zero-temperature fixed point, when ζS exceeds the thermal roughness exponent of the emerging wetting layer. The possibility of an effective dimensional reduction due to randomness is considered. In two dimensions a number of exact results are obtained by using a many-body transfer-matrix technique.

  12. Methods of repairing a substrate

    NASA Technical Reports Server (NTRS)

    Riedell, James A. (Inventor); Easler, Timothy E. (Inventor)

    2011-01-01

    A precursor of a ceramic adhesive suitable for use in a vacuum, thermal, and microgravity environment. The precursor of the ceramic adhesive includes a silicon-based, preceramic polymer and at least one ceramic powder selected from the group consisting of aluminum oxide, aluminum nitride, boron carbide, boron oxide, boron nitride, hafnium boride, hafnium carbide, hafnium oxide, lithium aluminate, molybdenum silicide, niobium carbide, niobium nitride, silicon boride, silicon carbide, silicon oxide, silicon nitride, tin oxide, tantalum boride, tantalum carbide, tantalum oxide, tantalum nitride, titanium boride, titanium carbide, titanium oxide, titanium nitride, yttrium oxide, zirconium boride, zirconium carbide, zirconium oxide, and zirconium silicate. Methods of forming the ceramic adhesive and of repairing a substrate in a vacuum and microgravity environment are also disclosed, as is a substrate repaired with the ceramic adhesive.

  13. Substrates dissociate dopamine transporter oligomers

    PubMed Central

    Chen, Nianhang; Reith, Maarten E. A.

    2016-01-01

    Substrate-induced endocytic trafficking of DAT has been observed, but little is known about the regulation of DAT oligomerization by substrate. The present study investigates the effect on substrates on DAT oligomerization and explores a potential link with the presence of DAT at the cell surface in HEK-293 cells transiently or stably expressing N-terminal tagged DAT constructs. DA (100 μM) or amphetamine (2–10 μM) reduced Myc-DAT coimmunoprecipitated along with Flag-DAT (oligomeric DAT) in tandem with a reduction in surface DAT determined by biotinylation. DA (10–1000 μM) and amphetamine (0.2–200 μM) reduced DAT oligomerization as assessed by cross-linking with copper sulfate phenanthroline (CuP) or Cu2+. Inhibition of endocytosis by 10 μM phenylarsine oxide (PAO) or 450 mM sucrose counteracted the effect of 10 μM DA or 2 μM amphetamine in reducing DAT cross-linking. In addition to overall similarities between the results with the two cross-linking agents and between the results with the two different endocytosis inhibitors, some differences were noted as well, likely related to the efficiency of the cross-linking process and the sulfhydryl-reactive properties of PAO, respectively. The present results are the first to indicate regulation of oligomerization of an SLC6 transporter, the DAT, by substrates that act at DAT. In addition, the present study opens up the possibility of an important linkage between between oligomerization of DAT and endocytic or other modulatory mechanisms impacting surface DAT. PMID:18088380

  14. Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context*

    PubMed Central

    Zhou, Jia; Liu, Minmin; Fleming, Aaron M.; Burrows, Cynthia J.; Wallace, Susan S.

    2013-01-01

    The telomeric DNA of vertebrates consists of d(TTAGGG)n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA. PMID:23926102

  15. Active matter on asymmetric substrates

    NASA Astrophysics Data System (ADS)

    Olson Reichhardt, C. J.; Drocco, J.; Mai, T.; Wan, M. B.; Reichhardt, C.

    2011-10-01

    For collections of particles in a thermal bath interacting with an asymmetric substrate, it is possible for a ratchet effect to occur where the particles undergo a net dc motion in response to an ac forcing. Ratchet effects have been demonstrated in a variety of systems including colloids as well as magnetic vortices in type-II superconductors. Here we examine the case of active matter or self-driven particles interacting with asymmetric substrates. Active matter systems include self-motile colloidal particles undergoing catalysis, swimming bacteria, artificial swimmers, crawling cells, and motor proteins. We show that a ratchet effect can arise in this type of system even in the absence of ac forcing. The directed motion occurs for certain particle-substrate interaction rules and its magnitude depends on the amount of time the particles spend swimming in one direction before turning and swimming in a new direction. For strictly Brownian particles there is no ratchet effect. If the particles reflect off the barriers or scatter from the barriers according to Snell's law there is no ratchet effect; however, if the particles can align with the barriers or move along the barriers, directed motion arises. We also find that under certain motion rules, particles accumulate along the walls of the container in agreement with experiment. We also examine pattern formation for synchronized particle motion. We discuss possible applications of this system for self-assembly, extracting work, and sorting as well as future directions such as considering collective interactions and flocking models.

  16. Substrate analogues for isoprenoid enzymes

    SciTech Connect

    Stremler, K.E.

    1987-01-01

    Diphosphonate analogues of geranyl diphosphate, resistant to degradation by phosphatases, were found to be alternate substrates for the reaction with farnesyl diphosphate synthetase isolated from avian liver. The difluoromethane analogue was shown to be the better alternate substrate, in agreement with solvolysis results which indicate that the electronegativity of the difluoromethylene unit more closely approximates that of the normal bridging oxygen. The usefulness of the C/sub 10/ difluoro analogue, for detecting low levels of isoprenoid enzymes in the presence of high levels of phosphatase activity, was demonstrated with a cell-free preparation from lemon peel. A series of C/sub 5/ through C/sub 15/ homoallylic and allylic diphosphonates, as well as two 5'-nucleotide diphosphonates, was prepared in high overall yield using the activation-displacement sequence. Radiolabeled samples of several of the allylic diphosphonates were prepared with tritium located at C1. A series of geraniols, stereospecifically deuterated at C1, was prepared. The enantiomeric purities and absolute configurations were determined by derivatization as the mandelate esters for analysis by /sup 1/H NMR. The stereochemistry of the activation-displacement sequence was examined using C1-deuterated substrates.

  17. Phosphonate analogue substrates for enolase.

    PubMed

    Anderson, V E; Cleland, W W

    1990-11-20

    Phosphonate analogues in which the bridge between C-2 and phosphorus is a CH2 group are slow substrates for yeast enolase. The pH variation of the kinetic parameters for the methylene analogue of 2-phosphoglycerate suggests that the substrate binds as a dianion and that Mg2+ can bind subsequently only if a metal ligand and the catalytic base are unprotonated. Primary deuterium isotope effects of 4-8 on V/KMg, but ones of only 1.15-1.32 on V for dehydration, show that proton removal to give the carbanion intermediate largely limits V/KMg and that a slow step follows which largely limits V (presumably carbanion breakdown). Since there is a D2O solvent isotope effect on V for the reverse reaction of 5, but not an appreciable one on the forward reaction, it appears that the slow rates with phosphonate analogues result from the fact that the carbanion intermediate is more stable than that formed from the normal substrates, and its reaction in both directions limits V. Increased stability as a result of replacement of oxygen by carbon at C-2 of the carbanion is the expected chemical behavior. PMID:2271661

  18. High-speed detection of DNA translocation in nanopipettes

    NASA Astrophysics Data System (ADS)

    Fraccari, Raquel L.; Ciccarella, Pietro; Bahrami, Azadeh; Carminati, Marco; Ferrari, Giorgio; Albrecht, Tim

    2016-03-01

    We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface.We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface. Electronic supplementary information (ESI) available: Gel electrophoresis confirming lengths and purity of DNA samples, comparison between Axopatch 200B and custom-built setup, comprehensive low-noise amplifier characterization, representative I-V curves of nanopipettes used, typical scatter plots of τ vs. peak amplitude for the four LDNA's used, table of most probable τ values, a comparison between different fitting models for the DNA translocation time distribution, further details on the stochastic numerical simulation of the scaling statistics and the derivation of the extended

  19. In vitro rejoining of double strand breaks in genomic DNA.

    PubMed

    Iliakis, George; Mladenov, Emil; Cheong, Nge

    2012-01-01

    Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end joining (NHEJ) pathways in higher eukaryotes, and have facilitated the functional characterization of several of its components including DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos, and Artemis. Nevertheless, there is evidence that as of yet uncharacterized repair factors may contribute to the efficiency of NHEJ, for example by modulating the activity of known factors. Also, the discovery of alternative pathways of NHEJ that function as backup to the classical DNA-PK-dependent pathway of NHEJ has added yet another dimension in the set of activities involved. The biochemical characterization of NHEJ in higher eukaryotes has benefited significantly from in vitro plasmid-based end joining assays. However, because of differences in the organization and sequence of genomic and plasmid DNA, and because multiple pathways of NHEJ are operational, it is possible that different factors are preferred for the rejoining of DSBs induced in plasmid versus genomic DNA organized in chromatin. Here, we describe an in vitro assay that allows the study of DSB rejoining in genomic DNA. The assay utilizes as a substrate DSBs induced by various means in genomic DNA prepared from agarose-embedded cells after appropriate lysis. Two extremes in terms of state of DNA organization are described: "naked" DNA and DNA organized in chromatin. We describe the protocols developed to carry out and analyze these in vitro reactions, including procedures for the preparation of cell extract and the preparation of the substrate DNA ("naked" DNA or nuclei). PMID:22941623

  20. Molecular structure of DNA by scanning tunneling microscopy.

    PubMed

    Cricenti, A; Selci, S; Felici, A C; Generosi, R; Gori, E; Djaczenko, W; Chiarotti, G

    1989-09-15

    Uncoated DNA molecules marked with an activated tris(l-aziridinyl) phosphine oxide (TAPO) solution were deposited on gold substrates and imaged in air with the use of a high-resolution scanning tunneling microscope (STM). Constant-current and gap-modulated STM images show clear evidence of the helicity of the DNA structure: pitch periodicity ranges from 25 to 35 angstroms, whereas the average diameter is 20 angstroms. Molecular structure within a single helix turn was also observed. PMID:2781279

  1. Molecular Structure of DNA by Scanning Tunneling Microscopy

    NASA Astrophysics Data System (ADS)

    Cricenti, A.; Selci, S.; Felici, A. C.; Generosi, R.; Gori, E.; Djaczenko, W.; Chiarotti, G.

    1989-09-01

    Uncoated DNA molecules marked with an activated tris(1-aziridinyl) phosphine oxide (TAPO) solution were deposited on gold substrates and imaged in air with the use of a high-resolution scanning tunneling microscope (STM). Constant-current and gap-modulated STM images show clear evidence of the helicity of the DNA structure: pitch periodicity ranges from 25 and 35 angstroms, whereas the average diameter is 20 angstroms. Molecular structure within a single helix turn was also observed.

  2. Fungal Taxa Target Different Carbon Substrates in Harvard Forest Soils

    NASA Astrophysics Data System (ADS)

    Hanson, C. A.; Allison, S. D.; Wallenstein, M. D.; Mellilo, J. M.; Treseder, K. K.

    2006-12-01

    The mineralization of soil organic carbon is a major component of the global carbon cycle and is largely controlled by soil microbial communities. However, little is known about the functional roles of soil microbes or whether different microbial taxa target different carbon substrates under natural conditions. To examine this possibility, we assessed the community composition of active fungi by using a novel nucleotide analog technique in soils from the Harvard Forest. We hypothesized that fungal community composition would shift in response to the addition of different substrates and that specific fungal taxa would respond differentially to particular carbon sources. To test this hypothesis, we added a nucleotide analog probe directly to soils in conjunction with one of five carbon compounds of increasing recalcitrance: glycine, sucrose, cellulose, tannin-protein complex, and lignin. During 48 hour incubations, the nucleotide analog was incorporated into newly replicated DNA of soil organisms that proliferated following the addition of the substrates. In this way, we labeled the DNA of microbes that respond to a particular carbon source. Labeled DNA was isolated and fungal Internal Transcribed Spacer (ITS) regions of ribosomal DNA (rDNA) were sequenced and analyzed to identify active fungi to near-species resolution. Diversity analyses at the ≥97% sequence similarity level indicated that taxonomic richness was greater under cellulose (Shannon Index: 3.23 ± 0.11 with ± 95% CI) and lignin (2.87 ± 0.15) additions than the other treatments (2.34 ± 0.16 to 2.64 ± 0.13). In addition, community composition of active fungi shifted under glycine, sucrose, and cellulose additions. Specifically, the community under glycine was significantly different from communities under control, cellulose, and tannin-protein (P<0.05). Additionally, the sucrose and cellulose communities were marginally different from the control community (P = 0.059 and 0.054, respectively) and

  3. Microstrip dipole antennas on electrically thick substrates

    NASA Astrophysics Data System (ADS)

    Jackson, D. R.; Alexopoulos, N. G.

    1985-10-01

    Printed circuit antennas are attractive radiation sources both at microwave and millimeter wave frequencies. However, for millimeter wave applications where the substrate is likely to be electrically thick, it is important to understand the basic effects of a thick substrate on radiation characteristics. In particular, it is concluded here that dipole radiation properties become sensitive to loss as the substrate becomes thick. Furthermore, the efficiency of dipoles on thick substrates tends to be low, especially as the dielectric constant of the substrate increases. A method of improving both the efficiency and gain can be used for thick substrates, however, which uses a superstrate layer on top of the antenna.

  4. FEN1 participates in repair of the 5'-phosphotyrosyl terminus of DNA single-strand breaks.

    PubMed

    Kametani, Yukiko; Takahata, Chiaki; Narita, Takashi; Tanaka, Kiyoji; Iwai, Shigenori; Kuraoka, Isao

    2016-01-01

    Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells. PMID:26581212

  5. Mechanism and regulation of DNA end resection in eukaryotes.

    PubMed

    Symington, Lorraine S

    2016-01-01

    The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic degradation of the 5'-terminated strands in a process termed end resection. End resection generates 3'-single-stranded DNA tails, substrates for Rad51 to catalyze homologous pairing and DNA strand exchange, and for activation of the DNA damage checkpoint. The commonly accepted view is that end resection occurs by a two-step mechanism. In the first step, Sae2/CtIP activates the Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex to endonucleolytically cleave the 5'-terminated DNA strands close to break ends, and in the second step Exo1 and/or Dna2 nucleases extend the resected tracts to produce long 3'-ssDNA-tailed intermediates. Initiation of resection commits a cell to repair a DSB by HR because long ssDNA overhangs are poor substrates for non-homologous end joining (NHEJ). Thus, the initiation of end resection has emerged as a critical control point for repair pathway choice. Here, I review recent studies on the mechanism of end resection and how this process is regulated to ensure the most appropriate repair outcome. PMID:27098756

  6. Mechanism of repair of 5'-topoisomerase II-DNA adducts by mammalian tyrosyl-DNA phosphodiesterase 2

    SciTech Connect

    Schellenberg, Matthew J; Appel, C Denise; Adhikari, Sanjay; Robertson, Patrick D; Ramsden, Dale A; Williams, R Scott

    2012-10-28

    The topoisomerase II (topo II) DNA incision-and-ligation cycle can be poisoned (for example following treatment with cancer chemotherapeutics) to generate cytotoxic DNA double-strand breaks (DSBs) with topo II covalently conjugated to DNA. Tyrosyl-DNA phosphodiesterase 2 (Tdp2) protects genomic integrity by reversing 5'-phosphotyrosyl–linked topo II–DNA adducts. Here, X-ray structures of mouse Tdp2–DNA complexes reveal that Tdp2 β–2-helix–β DNA damage–binding 'grasp', helical 'cap' and DNA lesion–binding elements fuse to form an elongated protein-DNA conjugate substrate-interaction groove. The Tdp2 DNA-binding surface is highly tailored for engagement of 5'-adducted single-stranded DNA ends and restricts nonspecific endonucleolytic or exonucleolytic processing. Structural, mutational and functional analyses support a single–metal ion catalytic mechanism for the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily and establish a molecular framework for targeted small-molecule blockade of Tdp2-mediated resistance to anticancer topoisomerase drugs.

  7. Regulation of DNA repair by parkin

    SciTech Connect

    Kao, Shyan-Yuan

    2009-05-01

    Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant. Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.

  8. Templating gold nanorods with liquid crystalline DNA

    NASA Astrophysics Data System (ADS)

    De Sio, Luciano; Annesi, Ferdinanda; Placido, Tiziana; Comparelli, Roberto; Bruno, Vincenzo; Pane, Alfredo; Palermo, Giovanna; Curri, Maria Lucia; Umeton, Cesare; Bartolino, Roberto

    2015-02-01

    A liquid crystalline, negatively charged, whole-genome DNA is exploited to organize positively charged gold nanorods (GNRs) by means of electrostatic interaction. A mesoscopic alignment of the composite system along a preferred direction is obtained by casting a droplet of the DNA-nanorods solution onto an untreated glass substrate. Gel electrophoresis analysis enables evaluating the effective electric charge of the system, thus minimizing the DNA fragmentation. Polarized optical microscopy, combined with transmission and scanning electron microscopy, shows that, up to 20% in weight of GNR solution, the system exhibits both a long range order, induced by the liquid crystalline phase of the DNA, and a nanoscale organization, due to the DNA self-assembly. These evidences are confirmed by a polarized spectral analysis, which also points out that the optical properties of GNRs strongly depend on the polarization of the impinging probe light. The capability to organize plasmonic nanoparticles by means of DNA material represents a significant advance towards the realization of life science inspired optical materials.

  9. E. coli Rep oligomers are required to initiate DNA unwinding in vitro.

    PubMed

    Cheng, W; Hsieh, J; Brendza, K M; Lohman, T M

    2001-07-01

    E. coli Rep protein is a 3' to 5' SF1 superfamily DNA helicase which is monomeric in the absence of DNA, but can dimerize upon binding either single-stranded or duplex DNA. A variety of biochemical studies have led to proposals that Rep dimerization is important for its helicase activity; however, recent structural studies of Bacillus stearothermophilus PcrA have led to suggestions that SF1 helicases, such as E. coli Rep and E. coli UvrD, function as monomeric helicases. We have examined the question of whether Rep oligomerization is important for its DNA helicase activity using pre-steady state stopped-flow and chemical quenched-flow kinetic studies of Rep-catalyzed DNA unwinding. The results from four independent experiments demonstrate that Rep oligomerization is required for initiation of DNA helicase activity in vitro. No DNA unwinding is observed when only a Rep monomer is bound to the DNA substrate, even when fluorescent DNA substrates are used that can detect partial unwinding of the first few base-pairs at the ss-ds-DNA junction. In fact, under these conditions, ATP hydrolysis causes dissociation of the Rep monomer from the DNA, rather than DNA unwinding. These studies demonstrate that wild-type Rep monomers are unable to initiate DNA unwinding in vitro, and that oligomerization is required. PMID:11428893

  10. Multipartite control of the DNA translocase, Mfd

    PubMed Central

    Smith, Abigail J.; Pernstich, Christian; Savery, Nigel J.

    2012-01-01

    ATP-dependent nucleic acid helicases and translocases play essential roles in many aspects of DNA and RNA biology. In order to ensure that these proteins act only in specific contexts, their activity is often regulated by intramolecular contacts and interaction with partner proteins. We have studied the bacterial Mfd protein, which is an ATP-dependent DNA translocase that relocates or displaces transcription ECs in a variety of cellular contexts. When bound to RNAP, Mfd exhibits robust ATPase and DNA translocase activities, but when released from its substrate these activities are repressed by autoinhibitory interdomain contacts. In this work, we have identified an interface within the Mfd protein that is important for regulating the activity of the protein, and whose disruption permits Mfd to act indiscriminately at transcription complexes that lack the usual determinants of Mfd specificity. Our results indicate that regulation of Mfd occurs through multiple nodes, and that activation of Mfd may be a multi-stage process. PMID:22904071

  11. Alternative nucleophilic substrates for the endonuclease activities of human immunodeficiency virus type 1 integrase

    SciTech Connect

    Ealy, Julie B.; Sudol, Malgorzata; Krzeminski, Jacek; Amin, Shantu; Katzman, Michael

    2012-11-10

    Retroviral integrase can use water or some small alcohols as the attacking nucleophile to nick DNA. To characterize the range of compounds that human immunodeficiency virus type 1 integrase can accommodate for its endonuclease activities, we tested 45 potential electron donors (having varied size and number or spacing of nucleophilic groups) as substrates during site-specific nicking at viral DNA ends and during nonspecific nicking reactions. We found that integrase used 22 of the 45 compounds to nick DNA, but not all active compounds were used for both activities. In particular, 13 compounds were used for site-specific and nonspecific nicking, 5 only for site-specific nicking, and 4 only for nonspecific nicking; 23 other compounds were not used for either activity. Thus, integrase can accommodate a large number of nucleophilic substrates but has selective requirements for its different activities, underscoring its dynamic properties and providing new information for modeling and understanding integrase.

  12. Cascade DNA logic device programmed ratiometric DNA analysis and logic devices based on a fluorescent dual-signal probe of a G-quadruplex DNAzyme.

    PubMed

    Fan, Daoqing; Zhu, Jinbo; Zhai, Qingfeng; Wang, Erkang; Dong, Shaojun

    2016-03-01

    Herein, two fluorescence sensitive substrates of G-quadruplex/hemin DNAzyme with inverse responses (Scopoletin and Amplex Red) were simultaneously used in one homogeneous system to construct a cascade advanced DNA logic device for the first time (a functional logic device (a three input based DNA calliper) cascade with an advanced non-arithmetic logic gate (1 to 2 decoder)). This cascade logic device was applied to label-free ratiometric target DNA detection and length measurement. PMID:26882417

  13. Molecular basis for the substrate specificity and catalytic mechanism of thymine-7-hydroxylase in fungi.

    PubMed

    Li, Wenjing; Zhang, Tianlong; Ding, Jianping

    2015-11-16

    TET proteins play a vital role in active DNA demethylation in mammals and thus have important functions in many essential cellular processes. The chemistry for the conversion of 5mC to 5hmC, 5fC and 5caC catalysed by TET proteins is similar to that of T to 5hmU, 5fU and 5caU catalysed by thymine-7-hydroxylase (T7H) in the nucleotide anabolism in fungi. Here, we report the crystal structures and biochemical properties of Neurospora crassa T7H. T7H can bind the substrates only in the presence of cosubstrate, and binding of different substrates does not induce notable conformational changes. T7H exhibits comparable binding affinity for T and 5hmU, but 3-fold lower affinity for 5fU. Residues Phe292, Tyr217 and Arg190 play critical roles in substrate binding and catalysis, and the interactions of the C5 modification group of substrates with the cosubstrate and enzyme contribute to the slightly varied binding affinity and activity towards different substrates. After the catalysis, the products are released and new cosubstrate and substrate are reloaded to conduct the next oxidation reaction. Our data reveal the molecular basis for substrate specificity and catalytic mechanism of T7H and provide new insights into the molecular mechanism of substrate recognition and catalysis of TET proteins. PMID:26429971

  14. Substrate flexibility regulates growth and apoptosis of normal but not transformed cells

    NASA Technical Reports Server (NTRS)

    Wang, H. B.; Dembo, M.; Wang, Y. L.

    2000-01-01

    One of the hallmarks of oncogenic transformation is anchorage-independent growth (27). Here we demonstrate that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells. We cultured normal or H-ras-transformed NIH 3T3 cells on flexible collagen-coated polyacrylamide substrates with similar chemical properties but different rigidity. Compared with cells cultured on stiff substrates, nontransformed cells on flexible substrates showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis. These responses on flexible substrates are coupled to decreases in cell spreading area and traction forces. In contrast, transformed cells maintained their growth and apoptotic characteristics regardless of substrate flexibility. The responses in cell spreading area and traction forces to substrate flexibility were similarly diminished. Our results suggest that normal cells are capable of probing substrate rigidity and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival. The loss of this response can explain the unregulated growth of transformed cells.

  15. Molecular basis for the substrate specificity and catalytic mechanism of thymine-7-hydroxylase in fungi

    PubMed Central

    Li, Wenjing; Zhang, Tianlong; Ding, Jianping

    2015-01-01

    TET proteins play a vital role in active DNA demethylation in mammals and thus have important functions in many essential cellular processes. The chemistry for the conversion of 5mC to 5hmC, 5fC and 5caC catalysed by TET proteins is similar to that of T to 5hmU, 5fU and 5caU catalysed by thymine-7-hydroxylase (T7H) in the nucleotide anabolism in fungi. Here, we report the crystal structures and biochemical properties of Neurospora crassa T7H. T7H can bind the substrates only in the presence of cosubstrate, and binding of different substrates does not induce notable conformational changes. T7H exhibits comparable binding affinity for T and 5hmU, but 3-fold lower affinity for 5fU. Residues Phe292, Tyr217 and Arg190 play critical roles in substrate binding and catalysis, and the interactions of the C5 modification group of substrates with the cosubstrate and enzyme contribute to the slightly varied binding affinity and activity towards different substrates. After the catalysis, the products are released and new cosubstrate and substrate are reloaded to conduct the next oxidation reaction. Our data reveal the molecular basis for substrate specificity and catalytic mechanism of T7H and provide new insights into the molecular mechanism of substrate recognition and catalysis of TET proteins. PMID:26429971

  16. Carbon Nanotube Patterning on a Metal Substrate

    NASA Technical Reports Server (NTRS)

    Nguyen, Cattien V. (Inventor)

    2016-01-01

    A CNT electron source, a method of manufacturing a CNT electron source, and a solar cell utilizing a CNT patterned sculptured substrate are disclosed. Embodiments utilize a metal substrate which enables CNTs to be grown directly from the substrate. An inhibitor may be applied to the metal substrate to inhibit growth of CNTs from the metal substrate. The inhibitor may be precisely applied to the metal substrate in any pattern, thereby enabling the positioning of the CNT groupings to be more precisely controlled. The surface roughness of the metal substrate may be varied to control the density of the CNTs within each CNT grouping. Further, an absorber layer and an acceptor layer may be applied to the CNT electron source to form a solar cell, where a voltage potential may be generated between the acceptor layer and the metal substrate in response to sunlight exposure.

  17. Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex

    PubMed Central

    Millet, Armêl; Strauss, François; Delagoutte, Emmanuelle

    2015-01-01

    The enzymatic DNA relaxation requires the DNA to be transiently nicked and rejoined, the covalent topoisomerase-DNA complex being a key intermediate of the nicking-joining reaction. Practically, this reaction is most often characterized by oligonucleotides. However, the incision-religation of an oligonucleotide does not fully recapitulate the incision-religation occuring during relaxation and the preferred substrate for such reaction characterization is supercoiled DNA. We therefore developed a method that used radiolabeled supercoiled DNA mini-circles to characterize the covalent enzyme-DNA complex formed during a relaxation reaction. Resolution of the relaxation products under different conditions permitted to quantify the proportion of covalent complex formed during the relaxation catalyzed by two topoisomerase models, the Escherichia coli topoisomerase I and the calf thymus topoisomerase I. As expected, the covalent complex formed with the calf thymus topoisomerase I was significantly enriched by camptothecin, a widely-used inhibitor of this topoisomerase, and a salt jump permitted the multiple topoisomerases trapped per mini-circle to complete the reaction cycle. The identified positions of the camptothecin-induced incision sites were shown to be independent of the linking number and the substrate circular nature Overall, our results demonstrate that supercoiled mini-circles constitute a powerful and polyvalent substrate to characterize the mechanism of action of novel topoisomerases and inhibitors, including the incision-religation reaction. PMID:26300432

  18. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    PubMed

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d. PMID:26766115

  19. Patterned substrates and methods for nerve regeneration

    DOEpatents

    Mallapragada, Surya K.; Heath, Carole; Shanks, Howard; Miller, Cheryl A.; Jeftinija, Srdija

    2004-01-13

    Micropatterned substrates and methods for fabrication of artificial nerve regeneration conduits and methods for regenerating nerves are provided. Guidance compounds or cells are seeded in grooves formed on the patterned substrate. The substrates may also be provided with electrodes to provide electrical guidance cues to the regenerating nerve. The micropatterned substrates give physical, chemical, cellular and/or electrical guidance cues to promote nerve regeneration at the cellular level.

  20. Substrate for thin silicon solar cells

    DOEpatents

    Ciszek, T.F.

    1998-07-28

    A substrate is described for a photovoltaic device wherein the substrate is the base upon which photosensitive material is to be grown and the substrate comprises an alloy having boron in a range from 0.1 atomic % of the alloy to 1.3 atomic % of the alloy and the substrate has a resistivity less than 3{times}10{sup {minus}3} ohm-cm. 4 figs.

  1. Interactive paper as security substrate

    NASA Astrophysics Data System (ADS)

    Robillard, Jean J.

    1997-08-01

    The threat of counterfeiting Bank Notes and other security documents is steadily increasing with the continuous improvement of printing and copying technologies. The perfect reproduction of a document is indeed reachable with the right combination of technique and economics. Our only protection is to make it difficult by introducing artifacts which appear only through the counterfeiting process or reduce significantly the efficiency of that process. A number of methods are available to defeat counterfeiting none of which are completely satisfactory. The difficulty is not as much the prevention of the duplication than doing it while safeguarding the intrinsic properties (durability) of the documents as far as permanent readability, resistance to wear, etc. Basic approaches are: (1) The light switch: a transparent coating which becomes opaque during any attempt of duplication. (2) An alternative to this first approach is an ink which becomes transparent at the time of duplication. (3) The use of a substrate (paper stock) whose optical density will rise to that of the printed graphic on the bill at the time of copying. This would be a true interactive paper. (4) Luminescent materials that would blind the photoreceptor during exposure. Such material could be in the substrate or in the printing ink. (5) Introduction of an artefact in the printed copy through a hologram hidden in a transparent coating or embossed in the substrate. All approaches have been investigated to a certain extent with variable success. We report hereafter the results of investigations made by the author on alternatives (1) and (2) in this laboratory and at the University of Texas at El Paso under contract from the National Bank of Belgium and with the collaboration of the CNRS in Paris and the University of Marseille France.

  2. Nanobiodevices for fast DNA separation and detection toward nanopore-based DNA sequencing

    NASA Astrophysics Data System (ADS)

    Kaji, Noritada; Yasui, Takao; Baba, Yoshinobu

    2014-03-01

    There is an increasing demand for using micro- and nanofabricated structures as tools for separation, manipulation, detection and analysis of biomolecules such as DNA and proteins. So far, we have developed fabrication techniques for constructing several types of nanostructures on quartz substrate for biomolecules separation, e.g., nanopillar and nanowall array structures, and demonstrated their analytical performances. Some important findings were that the nanopillar array pattern could control the DNA separation mode and electroosmotic flows in the nanopillar array structures were reduced according to the nanopillar spacing. Since these small confined nanospaces are suitable for manipulating biomolecules at a single molecule level, several approaches have been tried to analyze DNA denaturation and DNA-protein interactions in parallel. However, it is difficult to say that the observed phenomena reflect an intrinsic DNA property or DNA-protein interaction manner because all these approaches requires fluorescently labeled DNA molecules for observation. To address these issues, we are trying to develop a novel nanostructure-based and label-free detection system to integrate a biomolecule separation media and a detection system on a single chip.

  3. Replication by a single DNA polymerase of a stretched single-stranded DNA

    PubMed Central

    Maier, Berenike; Bensimon, David; Croquette, Vincent

    2000-01-01

    A new approach to the study of DNA/protein interactions has been opened through the recent advances in the manipulation of single DNA molecules. These allow the behavior of individual molecular motors to be studied under load and compared with bulk measurements. One example of such a motor is the DNA polymerase, which replicates DNA. We measured the replication rate by a single enzyme of a stretched single strand of DNA. The marked difference between the elasticity of single- and double-stranded DNA allows for the monitoring of replication in real time. We have found that the rate of replication depends strongly on the stretching force applied to the template. In particular, by varying the load we determined that the biochemical steps limiting replication are coupled to movement. The replication rate increases at low forces, decreases at forces greater than 4 pN, and ceases when the single-stranded DNA substrate is under a load greater than ≈20 pN. The decay of the replication rate follows an Arrhenius law and indicates that multiple bases on the template strand are involved in the rate-limiting step of each cycle. This observation is consistent with the induced-fit mechanism for error detection during replication. PMID:11050232

  4. Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

    PubMed

    Schellenberg, Matthew J; Perera, Lalith; Strom, Christina N; Waters, Crystal A; Monian, Brinda; Appel, C Denise; Vilas, Caroline K; Williams, Jason G; Ramsden, Dale A; Williams, R Scott

    2016-05-01

    Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. PMID:27060144

  5. Single pore translocation of folded, double-stranded, and tetra-stranded DNA through channel of bacteriophage phi29 DNA packaging motor.

    PubMed

    Haque, Farzin; Wang, Shaoying; Stites, Chris; Chen, Li; Wang, Chi; Guo, Peixuan

    2015-01-01

    The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ∼35-fold slower speed of translocation per base-pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ∼32% of channel current blockage and with ∼64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine. PMID:25890769

  6. Single Pore Translocation of Folded, Double-Stranded, and Tetra-stranded DNA through Channel of Bacteriophage Phi29 DNA Packaging Motor

    PubMed Central

    Haque, Farzin; Wang, Shaoying; Stites, Chris; Chen, Li; Wang, Chi; Guo, Peixuan

    2015-01-01

    The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ~35-fold slower speed of translocation per base pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ~32% of channel current blockage and ~64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine. PMID:25890769

  7. Small satellite solar array substrate

    NASA Technical Reports Server (NTRS)

    Fiore, John N.; Rosanova, Giulio

    1994-01-01

    The SMall EXplorer (SMEX) Fast Auroral SnapshoT (FAST) spacecraft was developed to investigate plasma physics of auroral phenomena at high orbital altitude. The FAST satellite comprises a variety of deployable booms with sensors on the ends, and instruments that protrude from the main body of the spacecraft to obtain the plasma and electromagnetic fields data. This required the plasma disturbance around the satellite to be kept to a minimum. A non deployable, body mounted solar array was implemented. This led to the design of a light weight solar array substrate with a high degree of structural integrity.

  8. LCAT DNA shearing.

    PubMed

    Okabe, Yuka; Lee, Abraham P

    2014-04-01

    We present a novel method to fragment DNA by using lateral cavity acoustic transducers (LCATs). DNA solution is placed within a microfluidic device containing LCATs. The LCATs cause microstreaming, which fragments DNA within the solution without any need for purification or downstream processing. The LCAT-based DNA fragmentation method offers an easy-to-use, low-cost, low-energy way to fragment DNA that is amenable to integration on microfluidic platforms to further automate DNA processing. Furthermore, the LCAT microdevice requires less than 10 µL of sample, and no external equipment is needed besides a piezoelectric transducer. PMID:23850863

  9. Amending pine bark with alternative substrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Due to a number of factors, pine bark supplies have significantly decreased over the past few years. While alternative substrates are being evaluated, many growers are asking if these alternative substrates can be used to stretch existing PB supplies. In this study, two alternative substrates, “Cl...

  10. Synthetic surfaces as models for biomineralization substrates

    SciTech Connect

    Rieke, P.C.; Tarasevich, B.J.; Bentjen, S.B.; Autrey, T.S.; Nelson, D.A.

    1990-01-01

    Polyethylene and oxide substrates were derivatized with functional groups commonly associated with biomineralization substrates. These groups include carboxylate, phosphate, hydroxy, sulfonate, thiol, and amine. Fourier transform infrared spectroscopy and contact angle wetting were used to identify and characterize the products at each step. The efficacy of these groups toward inducing mineralization will be compared with naturally occurring substrates. 10 refs., 5 figs.

  11. Combined plasma/liquid cleaning of substrates

    DOEpatents

    Selwyn, Gary S.; Henins, Ivars

    2003-04-15

    Apparatus and method for cleaning substrates. A substrate is held and rotated by a chuck and an atmospheric pressure plasma jet places a plasma onto predetermined areas of the substrate. Subsequently liquid rinse is sprayed onto the predetermined areas. In one embodiment, a nozzle sprays a gas onto the predetermined areas to assist in drying the predetermined areas when needed.

  12. Formation of phosphonester bonds catalyzed by DNA polymerase.

    PubMed Central

    Victorova, L S; Dyatkina, N B; Mozzherin DJu; Atrazhev, A M; Krayevsky, A A; Kukhanova, M K

    1992-01-01

    3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma- diphosphate and 2'-deoxythymidine-5'-(alpha-methylphosphonyl)-beta, gamma- diphosphate have been synthesized. Both compounds are incorporated into DNA chains during catalysis by reverse transcriptases of human immunodeficiency (HIV) and avian myeloblastosis (AMV) viruses, DNA polymerase beta from rat liver, terminal deoxynucleotidyl transferase from calf thymus and (at a very low rate) is by E. coli DNA polymerase I, Klenow fragment. The first compound is a termination substrate while the second is capable of multiple incorporation into the DNA chains. For instance, reverse transcriptase catalysis resulted in the appearance of 8 residues of second compound. DNA polymerases alpha and epsilon from human placenta incorporated none of the above compounds into DNA chains, although an inhibition of DNA synthesis by both compounds was observed with all enzymes mentioned. The 3'----5'-exonuclease activity of DNA polymerase I, Klenow fragment, hydrolyzed DNA fragments containing phosphonomethyl internucleoside groups, while such DNA fragments were resistant to the E. coli exonuclease III. Images PMID:1371865

  13. Repair of gaps in retroviral DNA integration intermediates.

    PubMed

    Yoder, K E; Bushman, F D

    2000-12-01

    Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3' DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5' two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex. PMID:11070016

  14. Polynucleotide 3'-terminal phosphate modifications by RNA and DNA ligases.

    PubMed

    Zhelkovsky, Alexander M; McReynolds, Larry A

    2014-11-28

    RNA and DNA ligases catalyze the formation of a phosphodiester bond between the 5'-phosphate and 3'-hydroxyl ends of nucleic acids. In this work, we describe the ability of the thermophilic RNA ligase MthRnl from Methanobacterium thermoautotrophicum to recognize and modify the 3'-terminal phosphate of RNA and single-stranded DNA (ssDNA). This ligase can use an RNA 3'p substrate to generate an RNA 2',3'-cyclic phosphate or convert DNA3'p to ssDNA(3')pp(5')A. An RNA ligase from the Thermus scotoductus bacteriophage TS2126 and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate ssDNA 3'p. These modifications of RNA and DNA 3'-phosphates are similar to the activities of RtcA, an RNA 3'-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3'p or DNA 3'p to generate the adenylated intermediate. For RNA (3')pp(5')A, the third step involves attack of the adjacent 2' hydroxyl to generate the RNA 2',3'-cyclic phosphate. These steps are analogous to those in classical 5' phosphate ligation. MthRnl and TS2126 RNA ligases were not able to modify a 3'p in nicked double-stranded DNA. However, T4 DNA ligase and RtcA can use 3'-phosphorylated nicks in double-stranded DNA to produce a 3'-adenylated product. These 3'-terminal phosphate-adenylated intermediates are substrates for deadenylation by yeast 5'Deadenylase. Our findings that classic ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3'-terminal phosphates. PMID:25324547

  15. One-step large-scale deposition of salt-free DNA origami nanostructures

    PubMed Central

    Linko, Veikko; Shen, Boxuan; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.; Tuukkanen, Sampo

    2015-01-01

    DNA origami nanostructures have tremendous potential to serve as versatile platforms in self-assembly -based nanofabrication and in highly parallel nanoscale patterning. However, uniform deposition and reliable anchoring of DNA nanostructures often requires specific conditions, such as pre-treatment of the chosen substrate or a fine-tuned salt concentration for the deposition buffer. In addition, currently available deposition techniques are suitable merely for small scales. In this article, we exploit a spray-coating technique in order to resolve the aforementioned issues in the deposition of different 2D and 3D DNA origami nanostructures. We show that purified DNA origamis can be controllably deposited on silicon and glass substrates by the proposed method. The results are verified using either atomic force microscopy or fluorescence microscopy depending on the shape of the DNA origami. DNA origamis are successfully deposited onto untreated substrates with surface coverage of about 4 objects/mm2. Further, the DNA nanostructures maintain their shape even if the salt residues are removed from the DNA origami fabrication buffer after the folding procedure. We believe that the presented one-step spray-coating method will find use in various fields of material sciences, especially in the development of DNA biochips and in the fabrication of metamaterials and plasmonic devices through DNA metallisation. PMID:26492833

  16. One-step large-scale deposition of salt-free DNA origami nanostructures

    NASA Astrophysics Data System (ADS)

    Linko, Veikko; Shen, Boxuan; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.; Tuukkanen, Sampo

    2015-10-01

    DNA origami nanostructures have tremendous potential to serve as versatile platforms in self-assembly -based nanofabrication and in highly parallel nanoscale patterning. However, uniform deposition and reliable anchoring of DNA nanostructures often requires specific conditions, such as pre-treatment of the chosen substrate or a fine-tuned salt concentration for the deposition buffer. In addition, currently available deposition techniques are suitable merely for small scales. In this article, we exploit a spray-coating technique in order to resolve the aforementioned issues in the deposition of different 2D and 3D DNA origami nanostructures. We show that purified DNA origamis can be controllably deposited on silicon and glass substrates by the proposed method. The results are verified using either atomic force microscopy or fluorescence microscopy depending on the shape of the DNA origami. DNA origamis are successfully deposited onto untreated substrates with surface coverage of about 4 objects/mm2. Further, the DNA nanostructures maintain their shape even if the salt residues are removed from the DNA origami fabrication buffer after the folding procedure. We believe that the presented one-step spray-coating method will find use in various fields of material sciences, especially in the development of DNA biochips and in the fabrication of metamaterials and plasmonic devices through DNA metallisation.

  17. DNA transfer by examination tools--a risk for forensic casework?

    PubMed

    Szkuta, Bianca; Harvey, Michelle L; Ballantyne, Kaye N; van Oorschot, Roland A H

    2015-05-01

    The introduction of profiling systems with increased sensitivity has led to a concurrent increase in the risk of detecting contaminating DNA in forensic casework. To evaluate the contamination risk of tools used during exhibit examination we have assessed the occurrence and level of DNA transferred between mock casework exhibits, comprised of cotton or glass substrates, and high-risk vectors (scissors, forceps, and gloves). The subsequent impact of such transfer in the profiling of a target sample was also investigated. Dried blood or touch DNA, deposited on the primary substrate, was transferred via the vector to the secondary substrate, which was either DNA-free or contained a target sample (dried blood or touch DNA). Pairwise combinations of both heavy and light contact were applied by each vector in order to simulate various levels of contamination. The transfer of dried blood to DNA-free cotton was observed for all vectors and transfer scenarios, with transfer substantially lower when glass was the substrate. Overall touch DNA transferred less efficiently, with significantly lower transfer rates than blood when transferred to DNA-free cotton; the greatest transfer of touch DNA occurred between cotton and glass substrates. In the presence of a target sample, the detectability of transferred DNA decreased due to the presence of background DNA. Transfer had no impact on the detectability of the target profile, however, in casework scenarios where the suspect profiles are not known, profile interpretation becomes complicated by the addition of contaminating alleles and the probative value of the evidence may be affected. The results of this study reiterate the need for examiners to adhere to stringent laboratory cleaning protocols, particularly in the interest of contamination minimisation, and to reduce the handling of items to prevent intra-item transfer. PMID:25735003

  18. Substrate Mediated Enzyme Prodrug Therapy

    PubMed Central

    Fejerskov, Betina; Zelikin, Alexander N.

    2012-01-01

    In this report, we detail Substrate Mediated Enzyme Prodrug Therapy (SMEPT) as a novel approach in drug delivery which relies on enzyme-functionalized cell culture substrates to achieve a localized conversion of benign prodrug(s) into active therapeutics with subsequent delivery to adhering cells or adjacent tissues. For proof-of-concept SMEPT, we use surface adhered micro-structured physical hydrogels based on poly(vinyl alcohol), β-glucuronidase enzyme and glucuronide prodrugs. We demonstrate enzymatic activity mediated by the assembled hydrogel samples and illustrate arms of control over rate of release of model fluorescent cargo. SMEPT was not impaired by adhering cells and afforded facile time - and dose – dependent uptake of the in situ generated fluorescent cargo by hepatic cells, HepG2. With the use of a glucuronide derivative of an anticancer drug, SN-38, SMEPT afforded a decrease in cell viability to a level similar to that achieved using parent drug. Finally, dose response was achieved using SMEPT and administration of judiciously chosen concentration of SN-38 glucuronide prodrug thus revealing external control over drug delivery using drug eluting surface. We believe that this highly adaptable concept will find use in diverse biomedical applications, specifically surface mediated drug delivery and tissue engineering. PMID:23152927

  19. Polycrystalline silicon on tungsten substrates

    NASA Technical Reports Server (NTRS)

    Bevolo, A. J.; Schmidt, F. A.; Shanks, H. R.; Campisi, G. J.

    1979-01-01

    Thin films of electron-beam-vaporized silicon were deposited on fine-grained tungsten substrates under a pressure of about 1 x 10 to the -10th torr. Mass spectra from a quadrupole residual-gas analyzer were used to determine the partial pressure of 13 residual gases during each processing step. During separate silicon depositions, the atomically clean substrates were maintained at various temperatures between 400 and 780 C, and deposition rates were between 20 and 630 A min. Surface contamination and interdiffusion were monitored by in situ Auger electron spectrometry before and after cleaning, deposition, and annealing. Auger depth profiling, X-ray analysis, and SEM in the topographic and channeling modes were utilized to characterize the samples with respect to silicon-metal interface, interdiffusion, silicide formation, and grain size of silicon. The onset of silicide formation was found to occur at approximately 625 C. Above this temperature tungsten silicides were formed at a rate faster than the silicon deposition. Fine-grain silicon films were obtained at lower temperatures.

  20. Crack patterns over uneven substrates.

    PubMed

    Nandakishore, Pawan; Goehring, Lucas

    2016-02-28

    Cracks in thin layers are influenced by what lies beneath them. From buried craters to crocodile skin, crack patterns are found over an enormous range of length scales. Regardless of absolute size, their substrates can dramatically influence how cracks form, guiding them in some cases, or shielding regions from them in others. Here we investigate how a substrate's shape affects the appearance of cracks above it, by preparing mud cracks over sinusoidally varying surfaces. We find that as the thickness of the cracking layer increases, the observed crack patterns change from wavy to ladder-like to isotropic. Two order parameters are introduced to measure the relative alignment of these crack networks, and, along with Fourier methods, are used to characterise the transitions between crack pattern types. Finally, we explain these results with a model, based on the Griffith criteria of fracture, that identifies the conditions for which straight or wavy cracks will be seen, and predicts how well-ordered the cracks will be. Our metrics and results can be applied to any situation where connected networks of cracks are expected, or found. PMID:26762761

  1. A novel in vitro assay to study the mechanism by which DNA polymerases bypass blocking lesions to DNA replication

    SciTech Connect

    Randall, S.K.

    1989-01-01

    We devised a simple gel assay to measure insertion kinetics for any dNTP substrate opposite a target site. Our ability to synthesize an abasic lesion and place it at a single site in synthetic oligonucleotides allows for an in vitro analysis of the mechanism by which DNA polymerases bypass blocking lesions to DNA replication and to identify E. coli polymerases and accessory proteins that allow for insertion and bypass of such lesions. Using this assay we examine the preferred insertion of dATP by Drosophila DNA polymerase {alpha} opposite the abasic lesion compared to dGTP, dCTP, and dTTP for all different nearest-neighbors. The preferred insertion of dATP is governed by a V{sub max} discrimination little affected by nearest-neighbors. A DNA polymerase activity was purified from E coli, deleted for DNA polymerase I, that appears to be part of the SOS response of E. coli since it cannot be induced in lexA(Ind{sup {minus}}) strains. This inducible polymerase is DNA polymerase II. In contrast to DNA polymerase III, DNA polymerase II efficiently incorporates nucleotides opposite the abasic lesion and continues DNA synthesis. We addressed the role of E. coli DNA polymerase I targeted SOS mutagenesis.

  2. The Fabrication of Sub-5-nm Nanochannels in Insulating Substrates using Focused Ion Beam Milling

    PubMed Central

    Menard, Laurent D.; Ramsey, J. Michael

    2011-01-01

    The use of focused ion beam (FIB) milling to fabricate nanochannels with critical dimensions extending below 5 nm is described. FIB milled lines have narrowing widths as they are milled deeper into a substrate. This focusing characteristic is coupled with a two-layered architecture consisting of a relatively thick (>100 nm) metal film deposited onto a substrate. A channel is milled through the metal layer until it penetrates a prescribed depth into the substrate material. The metal is then removed, leaving a nanochannel with smooth surfaces and lateral dimensions as small as sub-5 nm. These open nanochannels can be sealed with a cover plate and the resulting devices are well-suited for single-molecule DNA transport studies. This methodology is used with quartz, single-crystal silicon, and polydimethylsiloxane substrates to demonstrate its general utility. PMID:21171628

  3. New Insights on the Mechanism of Quinoline-based DNA Methyltransferase Inhibitors*

    PubMed Central

    Gros, Christina; Fleury, Laurence; Nahoum, Virginie; Faux, Céline; Valente, Sergio; Labella, Donatella; Cantagrel, Frédéric; Rilova, Elodie; Bouhlel, Mohamed Amine; David-Cordonnier, Marie-Hélène; Dufau, Isabelle; Ausseil, Frédéric; Mai, Antonello; Mourey, Lionel; Lacroix, Laurent; Arimondo, Paola B.

    2015-01-01

    Among the epigenetic marks, DNA methylation is one of the most studied. It is highly deregulated in numerous diseases, including cancer. Indeed, it has been shown that hypermethylation of tumor suppressor genes promoters is a common feature of cancer cells. Because DNA methylation is reversible, the DNA methyltransferases (DNMTs), responsible for this epigenetic mark, are considered promising therapeutic targets. Several molecules have been identified as DNMT inhibitors and, among the non-nucleoside inhibitors, 4-aminoquinoline-based inhibitors, such as SGI-1027 and its analogs, showed potent inhibitory activity. Here we characterized the in vitro mechanism of action of SGI-1027 and two analogs. Enzymatic competition studies with the DNA substrate and the methyl donor cofactor, S-adenosyl-l-methionine (AdoMet), displayed AdoMet non-competitive and DNA competitive behavior. In addition, deviations from the Michaelis-Menten model in DNA competition experiments suggested an interaction with DNA. Thus their ability to interact with DNA was established; although SGI-1027 was a weak DNA ligand, analog 5, the most potent inhibitor, strongly interacted with DNA. Finally, as 5 interacted with DNMT only when the DNA duplex was present, we hypothesize that this class of chemical compounds inhibit DNMTs by interacting with the DNA substrate. PMID:25525263

  4. A Simple and Inexpensive Method for Sending Binary Vector Plasmid DNA by Mail

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe a simple cost-effective technique for the transport of plasmid DNA by mail. Our results demonstrate that common multipurpose printing paper is a satisfactory substrate and superior to the more absorbent 3MM chromatography paper for the transport of plasmid DNA through the U.S. first clas...

  5. Phonon scattering in graphene over substrate steps

    SciTech Connect

    Sevinçli, H.; Brandbyge, M.

    2014-10-13

    We calculate the effect on phonon transport of substrate-induced bends in graphene. We consider bending induced by an abrupt kink in the substrate, and provide results for different step-heights and substrate interaction strengths. We find that individual substrate steps reduce thermal conductance in the range between 5% and 47%. We also consider the transmission across linear kinks formed by adsorption of atomic hydrogen at the bends and find that individual kinks suppress thermal conduction substantially, especially at high temperatures. Our analysis show that substrate irregularities can be detrimental for thermal conduction even for small step heights.

  6. Textured substrate tape and devices thereof

    DOEpatents

    Goyal, Amit

    2006-08-08

    A method for forming a sharply biaxially textured substrate, such as a single crystal substrate, includes the steps of providing a deformed metal substrate, followed by heating above the secondary recrystallization temperature of the deformed substrate, and controlling the secondary recrystallization texture by either using thermal gradients and/or seeding. The seed is selected to shave a stable texture below a predetermined temperature. The sharply biaxially textured substrate can be formed as a tape having a length of 1 km, or more. Epitaxial articles can be formed from the tapes to include an epitaxial electromagnetically active layer. The electromagnetically active layer can be a superconducting layer.

  7. Structural Organization of DNA.

    ERIC Educational Resources Information Center

    Banfalvi, Gaspar

    1986-01-01

    Explains the structural organization of DNA by providing information on the primary, secondary, tertiary, and higher organization levels of the molecule. Also includes illustrations and descriptions of sign-inversion and rotating models for supercoiling of DNA. (ML)

  8. Modeling DNA Replication.

    ERIC Educational Resources Information Center

    Bennett, Joan

    1998-01-01

    Recommends the use of a model of DNA made out of Velcro to help students visualize the steps of DNA replication. Includes a materials list, construction directions, and details of the demonstration using the model parts. (DDR)

  9. HPV DNA test

    MedlinePlus

    The HPV DNA test is used to check for high-risk HPV infection in women. HPV infection around the genitals is ... warts spread when you have sex. The HPV-DNA test is generally not recommended for detecting low- ...

  10. DNA tagged microparticles

    DOEpatents

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth

    2015-05-05

    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  11. Automated cassette-to-cassette substrate handling system

    DOEpatents

    Kraus, Joseph Arthur; Boyer, Jeremy James; Mack, Joseph; DeChellis, Michael; Koo, Michael

    2014-03-18

    An automated cassette-to-cassette substrate handling system includes a cassette storage module for storing a plurality of substrates in cassettes before and after processing. A substrate carrier storage module stores a plurality of substrate carriers. A substrate carrier loading/unloading module loads substrates from the cassette storage module onto the plurality of substrate carriers and unloads substrates from the plurality of substrate carriers to the cassette storage module. A transport mechanism transports the plurality of substrates between the cassette storage module and the plurality of substrate carriers and transports the plurality of substrate carriers between the substrate carrier loading/unloading module and a processing chamber. A vision system recognizes recesses in the plurality of substrate carriers corresponding to empty substrate positions in the substrate carrier. A processor receives data from the vision system and instructs the transport mechanism to transport substrates to positions on the substrate carrier in response to the received data.

  12. Extraction of DNA from Forensic Biological Samples for Genotyping.

    PubMed

    Stray, J E; Liu, J Y; Brevnov, M G; Shewale, J G

    2010-07-01

    Biological forensic samples constitute evidence with probative organic matter. Evidence believed to contain DNA is typically processed for extraction and purification of its nucleic acid content. Forensic DNA samples are composed of two things, a tissue and the substrate it resides on. Compositionally, a sample may contain almost anything and for each, the type, integrity, and content of both tissue and substrate will vary, as will the contaminant levels. This fact makes the success of extraction one of the most unpredictable steps in genotypic analysis. The development of robust genotyping systems and analysis platforms for short tandem repeat (STR) and mitochondrial DNA sequencing and the acceptance of results generated by these methods in the court system, resulted in a high demand for DNA testing. The increasing variety of sample submissions created a need to isolate DNA from forensic samples that may be compromised or contain low levels of biological material. In the past decade, several robust chemistries and isolation methods have been developed to safely and reliably recover DNA from a wide array of sample types in high yield and free of PCR inhibitors. In addition, high-throughput automated workflows have been developed to meet the demand for processing increasing numbers of samples. This review summarizes a number of the most widely adopted methods and the best practices for DNA isolation from forensic biological samples, including manual, semiautomated, and fully automated platforms. PMID:26242594

  13. Quantitative analysis and characterization of self-assembled DNA on a silver surface.

    PubMed

    Kumar, Karuppannan Senthil; Naaman, Ron

    2012-10-16

    Self-assembled monolayers of DNA on a silver surface were prepared and characterized by polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), fluorescence imaging, and (32)P radioactive labeling. The buffer concentration of the DNA solution and the surface roughness of the silver substrate were found to affect the surface coverage of DNA and its hybridization. At low buffer concentrations, surface coverage and hybridization were greatly reduced. Ethidium bromide intercalated into the adsorbed dsDNA clearly indicates the presence of dsDNA. PMID:23025347

  14. The Chromodomains of the Chd1 Chromatin Remodeler Regulate DNA Access to the ATPase Motor

    SciTech Connect

    Hauk, G.; McKnight, J; Nodelman, I; Bowman, G

    2010-01-01

    Chromatin remodelers are ATP-driven machines that assemble, slide, and remove nucleosomes from DNA, but how the ATPase motors of remodelers are regulated is poorly understood. Here we show that the double chromodomain unit of the Chd1 remodeler blocks DNA binding and activation of the ATPase motor in the absence of nucleosome substrates. The Chd1 crystal structure reveals that an acidic helix joining the chromodomains can pack against a DNA-binding surface of the ATPase motor. Disruption of the chromodomain-ATPase interface prevents discrimination between nucleosomes and naked DNA and reduces the reliance on the histone H4 tail for nucleosome sliding. We propose that the chromodomains allow Chd1 to distinguish between nucleosomes and naked DNA by physically gating access to the ATPase motor, and we hypothesize that related ATPase motors may employ a similar strategy to discriminate among DNA-containing substrates.

  15. Surface Modification of Gel-Free Microchannel Surface Electrophoresis Device for DNA Identification

    NASA Astrophysics Data System (ADS)

    Lee, Hyun Ho; Kuo, Yue

    2008-04-01

    A gel-free microchannel electrophoresis device for DNA separation and identification was studied. DNA fragments ranging from 3.5 to 21.2 kbp were effectively separated and identified. The channel's bottom surface was composed of silicon wafer or glass, and the channel wall was composed of SU-8 photoresist. The channel surface was modified with various solutions or plasmas. The separated DNA on the detection electrode was confirmed by electron spectroscopy for chemical analysis. DNA mobility on the glass substrate was higher than that on the Si substrate. In addition, the increase of the ionic strength of the solution on the device decreased the mobility of DNA. In summary, this new device is applicable to large DNA samples.

  16. Direct observation of DNA threading in flap endonuclease complexes.

    PubMed

    AlMalki, Faizah A; Flemming, Claudia S; Zhang, Jing; Feng, Min; Sedelnikova, Svetlana E; Ceska, Tom; Rafferty, John B; Sayers, Jon R; Artymiuk, Peter J

    2016-07-01

    Maintenance of genome integrity requires that branched nucleic acid molecules be accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki-fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates and products, at resolutions of 1.9-2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate's single-stranded branch approaches, threads through and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual 'fly-casting, thread, bend and barb' mechanism. PMID:27273516

  17. Conformal, macroscopic crystalline nanoparticle sheets assembled with DNA.

    PubMed

    Ku, Jessie C; Ross, Michael B; Schatz, George C; Mirkin, Chad A

    2015-05-27

    A novel method for preparing conformal silica-embedded crystalline nanoparticle sheets via DNA programmable assembly provides independent control over nanoparticle size, nanoparticle spacing, and film thickness. The conformal materials retain the nanoparticle crystallinity and spacing after being transferred to flat or highly curved substrates even after being subjected to various mechanical, physical, and chemical stimuli. PMID:25864411

  18. Thermally Stable, Piezoelectric and Pyroelectric Polymeric Substrates

    NASA Technical Reports Server (NTRS)

    Simpson, Joycely O. (Inventor); St.Clair, Terry L. (Inventor)

    1999-01-01

    A thermally stable, piezoelectric and pyroelectric polymeric substrate was prepared. This thermally stable, piezoelectric and pyroelectric polymeric substrate may be used to prepare electromechanical transducers, thermomechanical transducers, accelerometers. acoustic sensors, infrared sensors, pressure sensors, vibration sensors, impact sensors, in-situ temperature sensors, in-situ stress/strain sensors, micro actuators, switches, adjustable fresnel lenses, speakers, tactile sensors. weather sensors, micro positioners, ultrasonic devices, power generators, tunable reflectors, microphones, and hydrophones. The process for preparing these polymeric substrates includes: providing a polymeric substrate having a softening temperature greater than 1000 C; depositing a metal electrode material onto the polymer film; attaching a plurality of electrical leads to the metal electrode coated polymeric substrate; heating the metal electrode coated polymeric substrate in a low dielectric medium; applying a voltage to the heated metal electrode coated polymeric substrate to induce polarization; and cooling the polarized metal electrode coated polymeric electrode while maintaining a constant voltage.

  19. Substrate heater for thin film deposition

    DOEpatents

    Foltyn, Steve R.

    1996-01-01

    A substrate heater for thin film deposition of metallic oxides upon a target substrate configured as a disk including means for supporting in a predetermined location a target substrate configured as a disk, means for rotating the target substrate within the support means, means for heating the target substrate within the support means, the heating means about the support means and including a pair of heating elements with one heater element situated on each side of the predetermined location for the target substrate, with one heater element defining an opening through which desired coating material can enter for thin film deposition and with the heating means including an opening slot through which the target substrate can be entered into the support means, and, optionally a means for thermal shielding of the heating means from surrounding environment is disclosed.

  20. Is DNA a language?

    PubMed

    Tsonis, A A; Elsner, J B; Tsonis, P A

    1997-01-01

    DNA sequences usually involve local construction rules that affect different scales. As such their "dictionary" may not follow Zipf's law (a power law) which is followed in every natural language. Indeed, analysis of many DNA sequences suggests that no linguistics connections to DNA exist and that even though it has structure DNA is not a language. Computer simulations and a biological approach to this problem further support these results. PMID:9039397

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

    PubMed

    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

  2. A clamp-like biohybrid catalyst for DNA oxidation

    NASA Astrophysics Data System (ADS)

    van Dongen, Stijn F. M.; Clerx, Joost; Nørgaard, Kasper; Bloemberg, Tom G.; Cornelissen, Jeroen J. L. M.; Trakselis, Michael A.; Nelson, Scott W.; Benkovic, Stephen J.; Rowan, Alan E.; Nolte, Roeland J. M.

    2013-11-01

    In processive catalysis, a catalyst binds to a substrate and remains bound as it performs several consecutive reactions, as exemplified by DNA polymerases. Processivity is essential in nature and is often mediated by a clamp-like structure that physically tethers the catalyst to its (polymeric) template. In the case of the bacteriophage T4 replisome, a dedicated clamp protein acts as a processivity mediator by encircling DNA and subsequently recruiting its polymerase. Here we use this DNA-binding protein to construct a biohybrid catalyst. Conjugation of the clamp protein to a chemical catalyst with sequence-specific oxidation behaviour formed a catalytic clamp that can be loaded onto a DNA plasmid. The catalytic activity of the biohybrid catalyst was visualized using a procedure based on an atomic force microscopy method that detects and spatially locates oxidized sites in DNA. Varying the experimental conditions enabled switching between processive and distributive catalysis and influencing the sliding direction of this rotaxane-like catalyst.

  3. Roles of DNA helicases in the maintenance of genome integrity

    PubMed Central

    Bochman, Matthew L

    2014-01-01

    Genome integrity is achieved and maintained by the sum of all of the processes in the cell that ensure the faithful duplication and repair of DNA, as well as its genetic transmission from one cell division to the next. As central players in virtually all of the DNA transactions that occur in vivo, DNA helicases (molecular motors that unwind double-stranded DNA to produce single-stranded substrates) represent a crucial enzyme family that is necessary for genomic stability. Indeed, mutations in many human helicase genes are linked to a variety of diseases with symptoms that can be generally described as genomic instability, such as predispositions to cancers. This review focuses on the roles of both DNA replication helicases and recombination/repair helicases in maintaining genome integrity and provides a brief overview of the diseases related to defects in these enzymes. PMID:27308340

  4. High-speed detection of DNA translocation in nanopipettes.

    PubMed

    Fraccari, Raquel L; Ciccarella, Pietro; Bahrami, Azadeh; Carminati, Marco; Ferrari, Giorgio; Albrecht, Tim

    2016-04-14

    We present a high-speed electrical detection scheme based on a custom-designed CMOS amplifier which allows the analysis of DNA translocation in glass nanopipettes on a microsecond timescale. Translocation of different DNA lengths in KCl electrolyte provides a scaling factor of the DNA translocation time equal to p = 1.22, which is different from values observed previously with nanopipettes in LiCl electrolyte or with nanopores. Based on a theoretical model involving electrophoresis, hydrodynamics and surface friction, we show that the experimentally observed range of p-values may be the result of, or at least be affected by DNA adsorption and friction between the DNA and the substrate surface. PMID:26985713

  5. Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging

    PubMed Central

    Rezgui, Rachid; Lestini, Roxane; Kühn, Joëlle; Fave, Xenia; McLeod, Lauren; Myllykallio, Hannu; Alexandrou, Antigoni; Bouzigues, Cedric

    2014-01-01

    As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5′ and 3′-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5′ or 3′ extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases. PMID:25412080

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

    SciTech Connect

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

    2004-02-01

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

  7. Supramolecular Assembly of DNA on Graphene Nanoribbons

    PubMed Central

    Reuven, Darkeyah G.; Shashikala, H. B. Mihiri; Mandal, Sanjay; Williams, Myron N. V.; Chaudhary, Jaideep; Wang, Xiao-Qian

    2013-01-01

    Graphene’s adhesive and charge delocalization properties offer the opportunity for the direct study of biological molecule in the nanoscale regime. The inherent charge on DNA base pairs and the associated phosphate backbone can be probed by non-covalent interactions with graphene, which is a useful platform for the creation of anisotropic nanopatterned biological assemblies. Here, we report the graphene nanoribbon (GNR) supported anisotropic supramolecular self-assembly of single stranded adenine (A), cytosine (C), guanine (G), thymine (T), AT, and GC 20mer oligonucleotides, as well as the unique ordering of double stranded plasmid (circular) and Herring sperm (linear) DNA. The GNRs serve as a double sided adhesive platform for attachment to the SiO2 substrate, as well as DNA oligomers and polymers. The self-assembly is attributed to donor-acceptor interactions between DNA and graphene. These findings demonstrate that the DNA-GNR assembly yields a prospective route to novel bio-relevant nanostructures. PMID:24032074

  8. Mosaic of Commemorative Microscope Substrate

    NASA Technical Reports Server (NTRS)

    2008-01-01

    Written by electron beam lithography in the Microdevices Laboratory of NASA's Jet Propulsion Laboratory, this Optical Microscope substrate helps the Phoenix Mars Mission science team learn how to assemble individual microscope images into a mosaic by aligning rows of text.

    Each line is about 0.1 millimeter tall, the average thickness of a human hair. Except for the Mogensen twins, the names are of babies born and team members lost during the original development of MECA (the Microscopy, Electrochemistry and Conductivity Analyzer) for the canceled 2001 Mars lander mission. The plaque also acknowledges the MECA 2001 principal investigator, now retired.

    This image was taken by the MECA Optical Microscope on Sol 111, or the 111th day of the Phoenix mission (Sept. 16, 2008).

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by JPL, Pasadena, Calif. Spacecraft development was by Lockheed Martin Space Systems, Denver.

  9. Human DHX9 helicase unwinds triple-helical DNA structures.

    PubMed

    Jain, Aklank; Bacolla, Albino; Chakraborty, Prasun; Grosse, Frank; Vasquez, Karen M

    2010-08-24

    Naturally occurring poly(purine.pyrimidine) rich regions in the human genome are prone to adopting non-canonical DNA structures such as intramolecular triplexes (i.e., H-DNA). Such structure-forming sequences are abundant and can regulate the expression of several disease-linked genes. In addition, the use of triplex-forming oligonucleotides (TFOs) to modulate gene structure and function has potential as an approach to targeted gene therapy. Previously, we found that endogenous H-DNA structures can induce DNA double-strand breaks and promote genomic rearrangements. Herein, we find that the DHX9 helicase co-immunoprecipitates with triplex DNA structures in mammalian cells, suggesting a role in the maintenance of genome stability. We tested this postulate by assessing the helicase activity of purified human DHX9 on various duplex and triplex DNA substrates in vitro. DHX9 displaced the third strand from a specific triplex DNA structure and catalyzed the unwinding with a 3' --> 5' polarity with respect to the displaced third strand. Helicase activity required a 3'-single-stranded overhang on the third strand and was dependent on ATP hydrolysis. The reaction kinetics consisted of a pre-steady-state burst phase followed by a linear, steady-state pseudo-zero-order reaction. In contrast, very little if any helicase activity was detected on blunt triplexes, triplexes with 5'-overhangs, blunt duplexes, duplexes with overhangs, or forked duplex substrates. Thus, triplex structures containing a 3'-overhang represent preferred substrates for DHX9, where it removes the strand with Hoogsteen hydrogen-bonded bases. Our results suggest the involvement of DHX9 in maintaining genome integrity by unwinding mutagenic triplex DNA structures. PMID:20669935

  10. HUMAN DHX9 HELICASE UNWINDS TRIPLE HELICAL DNA STRUCTURES☟

    PubMed Central

    Jain, Aklank; Bacolla, Albino; Chakraborty, Prasun; Grosse, Frank; Vasquez, Karen M.

    2010-01-01

    Naturally occurring poly(purine·pyrimidine) rich regions in the human genome are prone to adopt non-canonical DNA structures such as intramolecular triplexes (i.e. H-DNA). Such structure-forming sequences are abundant and can regulate the expression of several diseases-linked genes. In addition, the use of triplex-forming oligonucleotides (TFOs) to modulate gene structure and function has potential as an approach to targeted gene therapy. Previously, we found that endogenous H-DNA structures can induce DNA double-strand breaks and promote genomic rearrangements. Herein, we find that the DHX9 helicase co-immunoprecipitates with triplex DNA structures in mammalian cells, suggesting a role in the maintenance of genome stability. We tested this postulate by assessing the helicase activity of purified human DHX9 on various duplex and triplex DNA substrates in vitro. DHX9 displaced the third strand from a specific triplex DNA structure and catalyzed the unwinding with a 3′→5′ polarity with respect to the displaced third strand. Helicase activity required a 3′-single-stranded overhang on the third strand and was dependent on ATP hydrolysis. The reaction kinetics consisted of a pre-steady-state burst phase followed by a linear, steady-state pseudo-zero-order-reaction. In contrast, very little, if any helicase activity was detected on blunt triplexes, triplexes with 5′-overhangs, blunt duplexes, duplexes with overhangs, or forked duplex substrates. Thus, triplex structures containing a 3′-overhang represent preferred substrates for DHX9, where it removes the strand with Hoogsteen hydrogen-bonded bases. Our results suggest the involvement of DHX9 in maintaining genome integrity by unwinding mutagenic triplex DNA structures. PMID:20669935

  11. Phototoxicity mechanisms: chlorpromazine photosensitized damage to DNA and cell membranes

    SciTech Connect

    Kochevar, K.E.

    1981-07-01

    Photosensitized damage to biological molecules is the initial process in phototoxic responses. It is now recognized that many phototoxic compounds can photosensitize damage to more than one type of biological substrate. The in vitro light-initiated reactions of phototoxic compounds with DNA, soluble proteins and membrane components can be classified by their molecular mechanisms: (1) those in which an excited state of the phototoxic compound (or an unstable species derived from it) reacts directly with the biological substrate and (2) those in which a molecule derived from the phototoxic compound (a photoproduct or an activated oxygen species) reacts with the biological substrate. This paper describes the mechanisms by which chlorpromazine photosensitizes damage to membranes, protein and DNA and compares them to the mechanisms of photosensitization by psoralens, porphyrins, dyes, and other molecules.

  12. Foreign DNA capture during CRISPR–Cas adaptive immunity

    PubMed Central

    Nuñez, James K.; Harrington, Lucas B.; Kranzusch, Philip J.; Engelman, Alan N.; Doudna, Jennifer A.

    2015-01-01

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40 base pair (bp) lengths into clustered regularly interspaced short palindromic repeats (CRISPR) loci as spacer segments1–6. The universally conserved Cas1–Cas2 integrase complex catalyzes spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7–13. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33 nucleotide (nt) protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′–OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo2–4. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci. PMID:26503043

  13. Foreign DNA capture during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Harrington, Lucas B; Kranzusch, Philip J; Engelman, Alan N; Doudna, Jennifer A

    2015-11-26

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci. PMID:26503043

  14. The Many Sides of DNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C.

    1997-01-01

    Explores the meaning of DNA. Discusses histories of DNA, literature on DNA, the contributions of Max Delbruck and Barbara McClintock, life, views of control, current research, and the language of DNA. Contains 24 references. (JRH)

  15. Structural Features of the Interaction between Human 8-Oxoguanine DNA Glycosylase hOGG1 and DNA

    PubMed Central

    Koval, V. V.; Knorre, D. G.; Fedorova, O. S.

    2014-01-01

    The purpose of the present review is to summarize the data related with the structural features of interaction between the human repair enzyme 8-oxoguanine DNA glycosylase (hOGG1) and DNA. The review covers the questions concerning the role of individual amino acids of hOGG1 in the specific recognition of the oxidized DNA bases, formation of the enzyme–substrate complex, and excision of the lesion bases from DNA. Attention is also focused upon conformational changes in the enzyme active site and disruption of enzyme activity as a result of amino acid mutations. The mechanism of damaged bases release from DNA induced by hOGG1 is discussed in the context of structural dynamics. PMID:25349714

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  17. DNA Sequencing apparatus

    DOEpatents

    Tabor, Stanley; Richardson, Charles C.

    1992-01-01

    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  18. Scanning electrochemical microscopy of genomic DNA microarrays--study of adsorption and subsequent interactions.

    PubMed

    Roberts, William S; Davis, Frank; Higson, Séamus P J

    2009-07-01

    The adsorption of genomic DNA and subsequent interactions between adsorbed and solvated DNA have been studied using scanning electrochemical microscopy (SECM). Microarrays of polyethylenimine (PEI) films could be deposited on screen-printed carbon substrates using the SECM. Single stranded herring DNA was electrostatically adsorbed at the surface of the polyethylenimine. The further adsorption of complementary single stranded DNA on the surface was observed to give rise to substantial decreases in interfacial impedance at the surface as measured by increases of tip current of the order of 1-2 nA (6%). Conversely adsorption of DNA from alternate species, i.e. salmon ssDNA on herring ssDNA, yielded much smaller changes in tip current of 0.2 nA. The significance of this work is that the approach opens up the possibility for direct label-free electrochemical interrogation of DNA microarrays as an alternative to other existing optical techniques. PMID:19562194

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

    PubMed Central

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

    2016-01-01

    Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein–DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein–DNA interactions. PMID:26952553

  20. Force regulated dynamics of RPA on a DNA fork

    PubMed Central

    Kemmerich, Felix E.; Daldrop, Peter; Pinto, Cosimo; Levikova, Maryna; Cejka, Petr; Seidel, Ralf

    2016-01-01

    Replication protein A (RPA) is a single-stranded DNA binding protein, involved in most aspects of eukaryotic DNA metabolism. Here, we study the behavior of RPA on a DNA substrate that mimics a replication fork. Using magnetic tweezers we show that both yeast and human RPA can open forked DNA when sufficient external tension is applied. In contrast, at low force, RPA becomes rapidly displaced by the rehybridization of the DNA fork. This process appears to be governed by the binding or the release of an RPA microdomain (toehold) of only few base-pairs length. This gives rise to an extremely rapid exchange dynamics of RPA at the fork. Fork rezipping rates reach up to hundreds of base-pairs per second, being orders of magnitude faster than RPA dissociation from ssDNA alone. Additionally, we show that RPA undergoes diffusive motion on ssDNA, such that it can be pushed over long distances by a rezipping fork. Generally the behavior of both human and yeast RPA homologs is very similar. However, in contrast to yeast RPA, the dissociation of human RPA from ssDNA is greatly reduced at low Mg2+ concentrations, such that human RPA can melt DNA in absence of force. PMID:27016742

  1. DNA in Nanoscale Electronics

    NASA Astrophysics Data System (ADS)

    Slinker, Jason

    2012-10-01

    DNA, the quintessential molecule of life, possesses a number of attractive properties for use in nanoscale circuits. Charge transport (CT) through DNA itself is of both fundamental and practical interest. Fundamentally, DNA has a unique configuration of π-stacked bases in a well ordered, double helical structure. Given its unparalleled importance to life processes and its arrangement of conjugated subunits, DNA has been a compelling target of conductivity studies. In addition, further understanding of DNA CT will elucidate the biological implications of this process and advance its use in sensing technologies. We have investigated the fundamentals of DNA CT by measuring the electrochemistry of DNA monolayers under biologically-relevant conditions. We have uncovered both fundamental kinetic parameters to distinguish between competing models of operation as well as the practical implications of DNA CT for sensing. Furthermore, we are leveraging our studies of DNA conductivity for the manufacture of nanoscale circuits. We are investigating the electrical properties and self-assembly of DNA nanowires containing artificial base pair surrogates, which can be prepared through low cost and high throughput automated DNA synthesis. This unique and economically viable approach will establish a new paradigm for the scalable manufacture of nanoscale semiconductor devices.

  2. DNA structure and function.

    PubMed

    Travers, Andrew; Muskhelishvili, Georgi

    2015-06-01

    The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase. PMID:25903461

  3. Human DNA repair genes.

    PubMed

    Wood, R D; Mitchell, M; Sgouros, J; Lindahl, T

    2001-02-16

    Cellular DNA is subjected to continual attack, both by reactive species inside cells and by environmental agents. Toxic and mutagenic consequences are minimized by distinct pathways of repair, and 130 known human DNA repair genes are described here. Notable features presently include four enzymes that can remove uracil from DNA, seven recombination genes related to RAD51, and many recently discovered DNA polymerases that bypass damage, but only one system to remove the main DNA lesions induced by ultraviolet light. More human DNA repair genes will be found by comparison with model organisms and as common folds in three-dimensional protein structures are determined. Modulation of DNA repair should lead to clinical applications including improvement of radiotherapy and treatment with anticancer drugs and an advanced understanding of the cellular aging process. PMID:11181991

  4. Hairpin DNA Switch for Ultrasensitive Spectrophotometric Detection of DNA Hybridization Based on Gold Nanoparticles and Enzyme Signal Amplification

    SciTech Connect

    Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong; Maham, Aihui; Lin, Yuehe

    2010-08-01

    A novel DNA detection platform based on a hairpin-DNA switch, nanoparticles, and enzyme signal amplification for ultrasensitive detection of DNA hybridization has been developed in this work. In this DNA assay, a “stem-loop” DNA probe dually labeled with a thiol at its 5’ end and a biotin at its 3’ end, respectively, was used. This probe was immobilized on the gold nanoparticles (AuNPs) anchored by a protein, globulin, on a 96-well microplate. In the absence of target DNA, the immobilized probe with the stem-loop structure shields the biotin from being approached by a bulky horseradish peroxidase linked-avidin (avidin-HRP) conjugate due to the steric hindrance. However, in the presence of target DNA, the hybridization between the hairpin DNA probe and the target DNA causes significant conformational change of the probe, which forces biotin away from the surface of AuNPs. As a result, the biotin becomes accessible by the avidin-HRP, and the target hybridization event can be sensitively detected via the HRP catalyzed substrate 3, 3', 5, 5'-tetramethylbenzidine using spectrophometric method. Some experimental parameters governing the performance of the assay have been optimized. At optimal conditions, this DNA assay can detect DNA at the concentration of femtomolar level by means of a signal amplification strategy based on the combination of enzymes and nanoparticles. This approach also has shown excellent specificity to distinguish single-base mismatches of DNA targets because of the intrinsic high selectivity of the hairpin DNA probe.

  5. Thymine DNA glycosylase exhibits negligible affinity for nucleobases that it removes from DNA

    PubMed Central

    Malik, Shuja S.; Coey, Christopher T.; Varney, Kristen M.; Pozharski, Edwin; Drohat, Alexander C.

    2015-01-01

    Thymine DNA Glycosylase (TDG) performs essential functions in maintaining genetic integrity and epigenetic regulation. Initiating base excision repair, TDG removes thymine from mutagenic G·T mispairs caused by 5-methylcytosine (mC) deamination and other lesions including uracil (U) and 5-hydroxymethyluracil (hmU). In DNA demethylation, TDG excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which are generated from mC by Tet (ten–eleven translocation) enzymes. Using improved crystallization conditions, we solved high-resolution (up to 1.45 Å) structures of TDG enzyme–product complexes generated from substrates including G·U, G·T, G·hmU, G·fC and G·caC. The structures reveal many new features, including key water-mediated enzyme–substrate interactions. Together with nuclear magnetic resonance experiments, the structures demonstrate that TDG releases the excised base from its tight product complex with abasic DNA, contrary to previous reports. Moreover, DNA-free TDG exhibits no significant binding to free nucleobases (U, T, hmU), indicating a Kd >> 10 mM. The structures reveal a solvent-filled channel to the active site, which might facilitate dissociation of the excised base and enable caC excision, which involves solvent-mediated acid catalysis. Dissociation of the excised base allows TDG to bind the beta rather than the alpha anomer of the abasic sugar, which might stabilize the enzyme–product complex. PMID:26358812

  6. DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2

    PubMed Central

    Cejka, Petr; Cannavo, Elda; Polaczek, Piotr; Masuda-Sasa, Taro; Pokharel, Subhash; Campbell, Judith L.; Kowalczykowski, Stephen C.

    2011-01-01

    The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires processing of broken ends. For repair to commence, the DSB must first be resected to generate a 3'-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad511. Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases, and topoisomerases2–4. Here we have biochemically reconstituted elements of the resection process and reveal that it requires the nuclease, Dna2, the RecQ-family helicase, Sgs1, and the ssDNA-binding protein, Replication protein-A (RPA). We establish that Dna2, Sgs1, and RPA comprise a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5'-terminated strand of the DNA break and to inhibit 3'→5' degradation by Dna2, actions which generate and protect the 3'-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11-Rad50-Xrs2 complex (MRX) play important roles as stimulatory components. Stimulation of end resection by the Top3-Rmi1 heterodimer and the MRX proteins is via complex formation with Sgs15,6 that unexpectedly stimulates DNA unwinding. We suggest that Top3-Rmi1 and MRX are important for recruitment of the Sgs1-Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding initial steps of recombinational DNA repair in eukaryotes. PMID:20811461

  7. Kinetic mechanism for formation of the active, dimeric UvrD helicase-DNA complex.

    PubMed

    Maluf, Nasib K; Ali, Janid A; Lohman, Timothy M

    2003-08-22

    Escherichia coli UvrD protein is a 3' to 5' SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase. PMID:12788954

  8. Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells.

    PubMed

    Canitrot, Y; Hoffmann, J S; Calsou, P; Hayakawa, H; Salles, B; Cazaux, C

    2000-09-01

    The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases delta and/or epsilon participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase beta exhibited a five- to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol beta in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol beta is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol beta in cancer cells could perturb the well-defined specific functions of DNA polymerases during error-free DNA transactions. PMID:10973926

  9. Insights into protein -- DNA interactions, stability and allosteric communications: A computational study of MutS-DNA recognition complexes

    NASA Astrophysics Data System (ADS)

    Negureanu, Lacramioara; Salsbury, Freddie

    2012-02-01

    DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. The most abundant MMR mismatch-binding factor in eukaryotes, MutS, recognizes and initiates the repair of base-base mismatches and small insertion/deletions. We performed molecular dynamics simulations on mismatched and damaged MutS-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutS proteins recognize the mismatched and platinum cross-linked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutS binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. A large number of known MMR cancer causing mutations among the residues were found.

  10. HDAC8 Substrates: Histones and Beyond

    PubMed Central

    Wolfson, Noah A.; Pitcairn, Carol Ann; Fierke, Carol A.

    2012-01-01

    The lysine deacetylase family of enzymes (HDACs) was first demonstrated to catalyze deacetylation of acetyllysine residues on histones. In subsequent years, HDACs have been shown to recognize a large pool of acetylated non-histone proteins as substrates. Recently, thousands of acetylated proteins have been discovered, yet in most cases, the HDAC that catalyzes deacetylation in vivo has not been identified. This gap has created the need for better in vivo, in vitro, and in silico approaches for determining HDAC substrates. While HDAC8 is the best kinetically and structurally characterized HDAC, few efficient substrates have yet been substantiated in vivo. In this review we delineate factors that may be important for determining HDAC8 substrate recognition and catalytic activity, including structure, complex formation, and post-translational modifications. This summary provides insight into the challenges of identifying in vivo substrates for HDAC8, and provides a good vantage point for understanding the variables important for predicting HDAC substrate recognition. PMID:23175386

  11. Non-permeable substrate carrier for electroplating

    DOEpatents

    Abas, Emmanuel Chua; Chen, Chen-An; Ma, Diana Xiaobing; Ganti, Kalyana Bhargava

    2012-11-27

    One embodiment relates to a substrate carrier for use in electroplating a plurality of substrates. The substrate carrier comprises a non-conductive carrier body on which the substrates are to be held. Electrically-conductive lines are embedded within the carrier body, and a plurality of contact clips are coupled to the electrically-conductive lines embedded within the carrier body. The contact clips hold the substrates in place and electrically couple the substrates to the electrically-conductive lines. The non-conductive carrier body is continuous so as to be impermeable to flow of electroplating solution through the non-conductive carrier body. Other embodiments, aspects and features are also disclosed.

  12. Non-permeable substrate carrier for electroplating

    SciTech Connect

    Abas, Emmanuel Chua; Chen, Chen-an; Ma, Diana Xiaobing; Ganti, Kalyana; Divino, Edmundo Anida; Ermita, Jake Randal G.; Capulong, Jose Francisco S.; Castillo, Arnold Villamor

    2015-12-29

    One embodiment relates to a substrate carrier for use in electroplating a plurality of substrates. The substrate carrier comprises a non-conductive carrier body on which the substrates are to be held. Electrically-conductive lines are embedded within the carrier body, and a plurality of contact clips are coupled to the electrically-conductive lines embedded within the carrier body. The contact clips hold the substrates in place and electrically couple the substrates to the electrically-conductive lines. The non-conductive carrier body is continuous so as to be impermeable to flow of electroplating solution through the non-conductive carrier body. Other embodiments, aspects and features are also disclosed.

  13. Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA

    PubMed Central

    Salceda, Javier; Fernández, Xavier; Roca, Joaquim

    2006-01-01

    Eukaryotic topoisomerases I and II efficiently remove helical tension in naked DNA molecules. However, this activity has not been examined in nucleosomal DNA, their natural substrate. Here, we obtained yeast minichromosomes holding DNA under (+) helical tension, and incubated them with topoisomerases. We show that DNA supercoiling density can rise above +0.04 without displacement of the histones and that the typical nucleosome topology is restored upon DNA relaxation. However, in contrast to what is observed in naked DNA, topoisomerase II relaxes nucleosomal DNA much faster than topoisomerase I. The same effect occurs in cell extracts containing physiological dosages of topoisomeraseI and II. Apparently, the DNA strand-rotation mechanism of topoisomerase I does not efficiently relax chromatin, which imposes barriers for DNA twist diffusion. Conversely, the DNA cross-inversion mechanism of topoisomerase II is facilitated in chromatin, which favor the juxtaposition of DNA segments. We conclude that topoisomerase II is the main modulator of DNA topology in chromatin fibers. The nonessential topoisomerase I then assists DNA relaxation where chromatin structure impairs DNA juxtaposition but allows twist diffusion. PMID:16710299

  14. Removing bonded skin from a substrate

    NASA Technical Reports Server (NTRS)

    Chartier, E. N.

    1980-01-01

    Metal skin is peeled off like sardine-can cover with key. Method is useful in removing bonded skins from any substrate where substrate is strong enough not to buckle or tear when bonded skin is rolled free. Also, it is useful for removing sections of damaged skin where bladders of other equipment below substrate might be damaged if saw or router were used to cut completely through skin.

  15. Substrate for thin silicon solar cells

    DOEpatents

    Ciszek, T.F.

    1995-03-28

    A photovoltaic device for converting solar energy into electrical signals comprises a substrate, a layer of photoconductive semiconductor material grown on said substrate, wherein the substrate comprises an alloy of boron and silicon, the boron being present in a range of from 0.1 to 1.3 atomic percent, the alloy having a lattice constant substantially matched to that of the photoconductive semiconductor material and a resistivity of less than 1{times}10{sup {minus}3} ohm-cm. 4 figures.

  16. Plasmons in graphene on uniaxial substrates

    SciTech Connect

    Arrazola, I.; CIC nanoGUNE Consolider, 20018 Donostia-San Sebastián ; Hillenbrand, R.; Nikitin, A. Yu.

    2014-01-06

    Placing graphene on uniaxial substrates may have interesting application potential for graphene-based photonic and optoelectronic devices. Here, we analytically derive the dispersion relation for graphene plasmons on uniaxial substrates and discuss their momentum, propagation length, and polarization as a function of frequency, propagation direction, and both ordinary and extraordinary dielectric permittivities of the substrate. We find that the plasmons exhibit an anisotropic propagation, yielding radially asymmetric field patterns when a point emitter launches plasmons in the graphene layer.

  17. Substrate for thin silicon solar cells

    DOEpatents

    Ciszek, Theodore F.

    1995-01-01

    A photovoltaic device for converting solar energy into electrical signals comprises a substrate, a layer of photoconductive semiconductor material grown on said substrate, wherein the substrate comprises an alloy of boron and silicon, the boron being present in a range of from 0.1 to 1.3 atomic percent, the alloy having a lattice constant substantially matched to that of the photoconductive semiconductor material and a resistivity of less than 1.times.10.sup.-3 ohm-cm.

  18. High quality oxide films on substrates

    DOEpatents

    Ruckman, Mark W.; Strongin, Myron; Gao, Yong L.

    1994-01-01

    A method for providing an oxide film of a material on the surface of a substrate using a reactive deposition of the material onto the substrate surface in the presence of a solid or liquid layer of an oxidizing gas. The oxidizing gas is provided on the substrate surface in an amount sufficient to dissipate the latent heat of condensation occurring during deposition as well as creating a favorable oxidizing environment for the material.

  19. High quality oxide films on substrates

    DOEpatents

    Ruckman, M.W.; Strongin, M.; Gao, Y.L.

    1994-02-01

    A method is described for providing an oxide film of a material on the surface of a substrate using a reactive deposition of the material onto the substrate surface in the presence of a solid or liquid layer of an oxidizing gas. The oxidizing gas is provided on the substrate surface in an amount sufficient to dissipate the latent heat of condensation occurring during deposition as well as creating a favorable oxidizing environment for the material. 4 figures.

  20. Gold nanoparticle wires for sensing DNA and DNA/protein interactions

    NASA Astrophysics Data System (ADS)

    Shao, Liqin; Diao, J. J.; Tang, Zhipeng; Liu, Song; Shen, Sophie C.; Liu, Jiankang; Rui, Xianfeng; Yu, Dapeng; Zhao, Qing

    2014-03-01

    The discontinuous Vertical Evaporation-driven Colloidal Deposition (dVECD) method has been used as a green technique for formatting nanoparticle wires by the direct deposition of nanoparticles from colloid suspensions onto hydrophilic substrates, without any lithographic procedures. Gold nanoparticles of different sizes are deposited into wire arrays for electronic detection of biological molecules. A sensitive detection of DNA molecules as low as ~1 pM is achieved due to a high surface to volume ratio of the porous structures. The effects of the gold nanoparticles' size, DNA concentration, and DNA length on detection sensitivity of these gold nanoparticle wire sensors are discussed. Moreover, we can also detect the interaction between DNAs and proteins. Gold nanoparticle wires prepared by the nontoxic and simple dVECD are promising for detecting viruses involved in diseases.The discontinuous Vertical Evaporation-driven Colloidal Deposition (dVECD) method has been used as a green technique for formatting nanoparticle wires by the direct deposition of nanoparticles from colloid suspensions onto hydrophilic substrates, without any lithographic procedures. Gold nanoparticles of different sizes are deposited into wire arrays for electronic detection of biological molecules. A sensitive detection of DNA molecules as low as ~1 pM is achieved due to a high surface to volume ratio of the porous structures. The effects of the gold nanoparticles' size, DNA concentration, and DNA length on detection sensitivity of these gold nanoparticle wire sensors are discussed. Moreover, we can also detect the interaction between DNAs and proteins. Gold nanoparticle wires prepared by the nontoxic and simple dVECD are promising for detecting viruses involved in diseases. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr06560j

  1. Substrate recognition and specificity of double-stranded RNA binding proteins.

    PubMed

    Vuković, Lela; Koh, Hye Ran; Myong, Sua; Schulten, Klaus

    2014-06-01

    Recognition of double-stranded (ds) RNA is an important part of many cellular pathways, including RNA silencing, viral recognition, RNA editing, processing, and transport. dsRNA recognition is often achieved by dsRNA binding domains (dsRBDs). We use atomistic molecular dynamics simulations to examine the binding interface of the transactivation response RNA binding protein (TRBP) dsRBDs to dsRNA substrates. Our results explain the exclusive selectivity of dsRBDs toward dsRNA and against DNA-RNA hybrid and dsDNA duplexes. We also provide corresponding experimental evidence. The dsRNA duplex is recognized by dsRBDs through the A-form of three duplex grooves and by the chemical properties of RNA bases, which have 2'-hydroxyl groups on their sugar rings. Our simulations show that TRBP dsRBD discriminates dsRNA- from DNA-containing duplexes primarily through interactions at two duplex grooves. The simulations also reveal that the conformation of the DNA-RNA duplex can be altered by dsRBD proteins, resulting in a weak binding of dsRBDs to DNA-RNA hybrids. Our study reveals the structural and molecular basis of protein-RNA interaction that gives rise to the observed substrate specificity of dsRNA binding proteins. PMID:24801449

  2. Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA

    PubMed Central

    Eddy, Sarah; Maddukuri, Leena; Ketkar, Amit; Zafar, Maroof K.; Henninger, Erin E.; Pursell, Zachary F.; Eoff, Robert L.

    2015-01-01

    We have investigated the action of the human DNA polymerase epsilon (hpol ε) and eta (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA relative to non-G4 DNA, while hpol ε binds both G4 and non-G4 substrates with near equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain greater than 25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol η fidelity increases ~15-fold when copying G22. By way of comparison, hpol ε retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol η is ~100-fold more accurate than hpol ε when comparing the mis-insertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model where a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication. PMID:25903680

  3. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA

  4. Foam drainage placed on a porous substrate.

    PubMed

    Arjmandi-Tash, O; Kovalchuk, N; Trybala, A; Starov, V

    2015-05-14

    A model for drainage/imbibition of a foam placed on the top of a porous substrate is presented. The equation of liquid imbibition into the porous substrate is coupled with a foam drainage equation at the foam/porous substrate interface. The deduced dimensionless equations are solved using a finite element method. It was found that the kinetics of foam drainage/imbibition depends on three dimensionless numbers and the initial liquid volume fraction. The result shows that there are three different regimes of the process. Each regime starts after initial rapid decrease of a liquid volume fraction at the foam/porous substrate interface: (i) rapid imbibition: the liquid volume fraction inside the foam at the foam/porous substrate interface remains constant close to a final liquid volume fraction; (ii) intermediate imbibition: the liquid volume fraction at the interface with the porous substrate experiences a peak point and imbibition into the porous substrate is slower as compared with the drainage; (iii) slow imbibition: the liquid volume fraction at the foam/porous substrate interface increases to a maximum limiting value and a free liquid layer is formed between the foam and the porous substrate. However, the free liquid layer disappears after some time. The transition points between these three different drainage/imbibition regimes were delineated by introducing two dimensionless numbers. PMID:25811970

  5. Substrate tolerant direct block copolymer nanolithography.

    PubMed

    Li, Tao; Wang, Zhongli; Schulte, Lars; Ndoni, Sokol

    2016-01-01

    Block copolymer (BC) self-assembly constitutes a powerful platform for nanolithography. However, there is a need for a general approach to BC lithography that critically considers all the steps from substrate preparation to the final pattern transfer. We present a procedure that significantly simplifies the main stream BC lithography process, showing a broad substrate tolerance and allowing for efficient pattern transfer over wafer scale. PDMS-rich poly(styrene-b-dimethylsiloxane) (PS-b-PDMS) copolymers are directly applied on substrates including polymers, silicon and graphene. A single oxygen plasma treatment enables formation of the oxidized PDMS hard mask, PS block removal and polymer or graphene substrate patterning. PMID:26606904

  6. Forensic DNA analysis.

    PubMed

    McDonald, Jessica; Lehman, Donald C

    2012-01-01

    Before the routine use of DNA profiling, blood typing was an important forensic tool. However, blood typing was not very discriminating. For example, roughly 30% of the United States population has type A-positive blood. Therefore, if A-positive blood were found at a crime scene, it could have come from 30% of the population. DNA profiling has a much better ability for discrimination. Forensic laboratories no longer routinely determine blood type. If blood is found at a crime scene, DNA profiling is performed. From Jeffrey's discovery of DNA fingerprinting to the development of PCR of STRs to the formation of DNA databases, our knowledge of DNA and DNA profiling have expanded greatly. Also, the applications for which we use DNA profiling have increased. DNA profiling is not just used for criminal case work, but it has expanded to encompass paternity testing, disaster victim identification, monitoring bone marrow transplants, detecting fetal cells in a mother's blood, tracing human history, and a multitude of other areas. The future of DNA profiling looks expansive with the development of newer instrumentation and techniques. PMID:22693781

  7. Neural substrates underlying intentional empathy

    PubMed Central

    Wang, Gang; Yang, Xuedong; Wang, Xiaoying; Northoff, Georg; Han, Shihui

    2012-01-01

    Although empathic responses to stimuli with emotional contents may occur automatically, humans are capable to intentionally empathize with other individuals. Intentional empathy for others is even possible when they do not show emotional expressions. However, little is known about the neuronal mechanisms of this intentionally controlled empathic process. To investigate the neuronal substrates underlying intentional empathy, we scanned 20 healthy Chinese subjects, using fMRI, when they tried to feel inside the emotional states of neutral or angry faces of familiar (Asian) and unfamiliar (Caucasian) models. Skin color evaluation of the same stimuli served as a control task. Compared to a baseline condition, the empathy task revealed a network of established empathy regions, including the anterior cingulate cortex, bilateral inferior frontal cortex and bilateral anterior insula. The contrast of intentional empathy vs skin color evaluation, however, revealed three regions: the bilateral inferior frontal cortex, whose hemodynamic responses were independent of perceived emotion and familiarity and the right-middle temporal gyrus, whose activity was modulated by emotion but not by familiarity. These findings extend our understanding of the role of the inferior frontal cortex and the middle temporal gyrus in empathy by demonstrating their involvement in intentional empathy. PMID:21511824

  8. Comet assay to measure DNA repair: approach and applications

    PubMed Central

    Azqueta, Amaya; Slyskova, Jana; Langie, Sabine A. S.; O’Neill Gaivão, Isabel; Collins, Andrew

    2014-01-01

    Cellular repair enzymes remove virtually all DNA damage before it is fixed; repair therefore plays a crucial role in preventing cancer. Repair studied at the level of transcription correlates poorly with enzyme activity, and so assays of phenotype are needed. In a biochemical approach, substrate nucleoids containing specific DNA lesions are incubated with cell extract; repair enzymes in the extract induce breaks at damage sites; and the breaks are measured with the comet assay. The nature of the substrate lesions defines the repair pathway to be studied. This in vitro DNA repair assay has been modified for use in animal tissues, specifically to study the effects of aging and nutritional intervention on repair. Recently, the assay was applied to different strains of Drosophila melanogaster proficient and deficient in DNA repair. Most applications of the repair assay have been in human biomonitoring. Individual DNA repair activity may be a marker of cancer susceptibility; alternatively, high repair activity may result from induction of repair enzymes by exposure to DNA-damaging agents. Studies to date have examined effects of environment, nutrition, lifestyle, and occupation, in addition to clinical investigations. PMID:25202323

  9. Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III*

    PubMed Central

    Kuznetsov, Nikita A.; Kladova, Olga A.; Kuznetsova, Alexandra A.; Ishchenko, Alexander A.; Saparbaev, Murat K.; Zharkov, Dmitry O.; Fedorova, Olga S.

    2015-01-01

    Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3′-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln41 and Leu81 as DNA lesion sensors. PMID:25869130

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

    NASA Technical Reports Server (NTRS)

    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.

  11. Thioredoxin from Streptomyces aureofaciens controls coiling of plasmid DNA.

    PubMed

    Golubnitchaya-Labudova, O; Horecka, T; Kapalla, M; Perecko, D; Kutejova, E; Lubec, G

    1998-01-01

    A number of potential functions of thioredoxin have been proposed in literature, including a role for DNA replication. The aim of our study was to investigate the effects of thioredoxin from Streptomyces aureofaciens (Trx S.a.) on plasmid DNA. Trx S.a. was incubated with plasmid forms and the incubation product(s) characterized on agarose gels. To compare Trx activity with enzymes with known DNA modifying activities, topoisomerase I, II (gyrase) and T4 DNA ligase were incubated with plasmid DNA in parallel. For the demonstration of nick removal a PCR technique was used. Trx S.a. bound non-specifically to plasmid DNA relaxing supercoiled circle closed form (CCC form) with subsequent formation of the circle closed form (CC form) as a major product. The amplification of a specific DNA template, possible only after nick removal, took place following incubation with Trx. The effect of topoisomerase I on plasmid DNA resembled Trx S.a. activity. We propose the following mechanism for CCC relaxation: Binding of Trx leads to a break of one strand and CC is formed by stepwise relaxation, ending with nick removal. The concomitant finding of open circle form (OC form) generation after incubation with Trx may indicate the generation of an intermediate due to the postulated strand break at initiation. This control of coiling may play a role in the DNA replication machinery, providing CC as a readily available substrate for DNA polymerases. In addition, Trx may serve in DNA repair mechanisms by its nonspecific binding to DNA and nick removing activity. PMID:9449230

  12. Fluorescence Detection of Single DNA Molecules.

    PubMed

    Huang, Weidong; Wang, Yue; Wang, Zhimin

    2015-09-01

    Single-molecule detection (SMD) and single-molecule fluorescence resonance energy transfer (smFRET) were conducted using Cy3- and Cy5-labeled single-strand DNAs (ssDNAs) either immobilized on substrates or encapsulated in microdroplets. High-quality fluorescent images were obtained using a total internal reflection fluorescence microscope (TIRFM). In the substrate system, deposition of a low concentration of fluorescence molecules on substrates through electrostatic adsorption showed that most of the fluorescence spots were single molecules, and the mean value of signal to noise ratio (S/N) reached 6.9 ± 0.34. smFRET analysis was conducted through immobilization of donor- and acceptor-labeled oligonucleotides on substrates. In the droplet system, fluorophor-labeled oligonucleotides were injected into T-type microfluidics. Single and double fluorophor-labeled DNA molecules encapsulated in droplets were detected, the FRET efficiency and inter-dye distance of a single donor-acceptor pair were measured accurately. smFRET was conducted detailedly in the tortuous channel for the first time. PMID:26215080

  13. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

    DOEpatents

    McCutchen-Maloney, Sandra L.

    2002-01-01

    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  14. DNA profiles from fingermarks.

    PubMed

    Templeton, Jennifer E L; Linacre, Adrian

    2014-11-01

    Criminal investigations would be considerably improved if DNA profiles could be routinely generated from single fingermarks. Here we report a direct DNA profiling method that was able to generate interpretable profiles from 71% of 170 fingermarks. The data are based on fingermarks from all 5 digits of 34 individuals. DNA was obtained from the fingermarks using a swab moistened with Triton-X, and the fibers were added directly to one of two commercial DNA profiling kits. All profiles were obtained without increasing the number of amplification cycles; therefore, our method is ideally suited for adoption by the forensic science community. We indicate the use of the technique in a criminal case in which a DNA profile was generated from a fingermark on tape that was wrapped around a drug seizure. Our direct DNA profiling approach is rapid and able to generate profiles from touched items when current forensic practices have little chance of success. PMID:25391915

  15. Electrocatalysis in DNA Sensors.

    PubMed

    Furst, Ariel; Hill, Michael G; Barton, Jacqueline K

    2014-12-14

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  16. DNA Media Storage

    PubMed Central

    Bogard, Christy M.; Rouchka, Eric C.

    2010-01-01

    In 1994, University of Southern California computer scientist Dr. Leonard Adelman solved the Hamiltonian Path Problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA Computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of Multiple Sequence Alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors. PMID:20622994

  17. DNA Media Storage.

    PubMed

    Bogard, Christy M; Rouchka, Eric C

    2007-09-01

    In 1994, University of Southern California computer scientist Dr. Leonard Adelman solved the Hamiltonian Path Problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA Computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of Multiple Sequence Alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors. PMID:20622994

  18. Ribonucleotides in Bacterial DNA

    PubMed Central

    Schroeder, Jeremy W.; Randall, Justin R.; Matthews, Lindsay A.; Simmons, Lyle A.

    2014-01-01

    In all living cells, DNA is the storage medium for genetic information. Being quite stable, DNA is well-suited for its role in storage and propagation of information, but RNA is also covalently included in DNA through various mechanisms. Recent studies also demonstrate useful aspects of including ribonucleotides in the genome during repair. Therefore, our understanding of the consequences of RNA inclusion into bacterial genomic DNA is just beginning, but with its high frequency of occurrence the consequences and potential benefits are likely to be numerous and diverse. In this review, we discuss the processes that cause ribonucleotide inclusion in genomic DNA, the pathways important for ribonucleotide removal and the consequences that arise should ribonucleotides remain nested in genomic DNA. PMID:25387798

  19. Electrocatalysis in DNA Sensors

    PubMed Central

    Furst, Ariel; Hill, Michael G.; Barton, Jacqueline K.

    2014-01-01

    Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

  20. Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication

    PubMed Central

    Narita, Yohei; Ryo, Akihide; Kawashima, Daisuke; Sugimoto, Atsuko; Kanda, Teru; Kimura, Hiroshi

    2013-01-01

    Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5, as a Pin1 substrate in glutathione S-transferase (GST) pulldown and immunoprecipitation assays. Lambda protein phosphatase treatment abolished the binding of BALF5 to Pin1, and mutation analysis of BALF5 revealed that replacement of the Thr178 residue by Ala (BALF5 T178A) disrupted the interaction with Pin1. To further test the effects of Pin1 in the context of virus infection, we constructed a BALF5-deficient recombinant virus. Exogenous supply of wild-type BALF5 in HEK293 cells with knockout recombinant EBV allowed efficient synthesis of viral genome DNA, but BALF5 T178A could not provide support as efficiently as wild-type BALF5. In conclusion, we found that EBV DNA polymerase BALF5 subunit interacts with Pin1 through BALF5 Thr178 in a phosphorylation-dependent manner. Pin1 might modulate EBV DNA polymerase conformation for efficient, productive viral DNA replication. PMID:23221557

  1. Integrated microfluidic systems for DNA analysis.

    PubMed

    Njoroge, Samuel K; Chen, Hui-Wen; Witek, Małgorzata A; Soper, Steven A

    2011-01-01

    The potential utility of genome-related research in terms of evolving basic discoveries in biology has generated widespread use of DNA diagnostics and DNA forensics and driven the accelerated development of fully integrated microfluidic systems for genome processing. To produce a microsystem with favorable performance characteristics for genetic-based analyses, several key operational elements must be strategically chosen, including device substrate material, temperature control, fluidic control, and reaction product readout. As a matter of definition, a microdevice is a chip that performs a single processing step, for example microchip electrophoresis. Several microdevices can be integrated to a single wafer, or combined on a control board as separate devices to form a microsystem. A microsystem is defined as a chip composed of at least two microdevices. Among the many documented analytical microdevices, those focused on the ability to perform the polymerase chain reaction (PCR) have been reported extensively due to the importance of this processing step in most genetic-based assays. Other microdevices that have been detailed in the literature include those for solid-phase extractions, microchip electrophoresis, and devices composed of DNA microarrays used for interrogating DNA primary structure. Great progress has also been made in the areas of chip fabrication, bonding and sealing to enclose fluidic networks, evaluation of different chip substrate materials, surface chemistries, and the architecture of reaction conduits for basic processing steps such as mixing. Other important elements that have been developed to realize functional systems include miniaturized readout formats comprising optical or electrochemical transduction and interconnect technologies. These discoveries have led to the development of fully autonomous and functional integrated systems for genome processing that can supply "sample in/answer out" capabilities. In this chapter, we focus on

  2. DNA-based machines.

    PubMed

    Wang, Fuan; Willner, Bilha; Willner, Itamar

    2014-01-01

    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications. PMID:24647836

  3. Pursuing DNA Catalysts for Protein Modification

    PubMed Central

    Silverman, Scott K.

    2015-01-01

    Conspectus Catalysis is a fundamental chemical concept, and many kinds of catalysts have considerable practical value. Developing entirely new catalysts is an exciting challenge. Rational design and screening has provided many new small-molecule catalysts, and directed evolution has been used to optimize or redefine the function of many protein enzymes. However, these approaches have inherent limitations that prompt the pursuit of different kinds of catalysts using other experimental methods. Nature evolved RNA enzymes, or ribozymes, for key catalytic roles that in modern biology are limited to phosphodiester cleavage/ligation and amide bond formation. Artificial DNA enzymes, or deoxyribozymes, have great promise for a broad range of catalytic activities. They can be identified from unbiased (random) sequence populations, as long as the appropriate in vitro selection strategies can be implemented for their identification. Notably, in vitro selection is different in key conceptual and practical ways from rational design, screening, and directed evolution. This Account describes the development by in vitro selection of DNA catalysts for many different kinds of covalent modification reactions of peptide and protein substrates, inspired in part by our earlier work with DNA-catalyzed RNA ligation reactions. In one set of studies, we have sought DNA-catalyzed peptide backbone cleavage, with the long-term goal of artificial DNA-based proteases. We originally anticipated that amide hydrolysis should be readily achieved, but in vitro selection instead led surprisingly to deoxyribozymes for DNA phosphodiester hydrolysis; this was unexpected because uncatalyzed amide bond hydrolysis is 105-fold faster. After developing a suitable selection approach that actively avoids DNA hydrolysis, deoxyribozymes were identified for hydrolysis of esters and aromatic amides (anilides). Aliphatic amide cleavage remains an ongoing focus, including via inclusion in the catalyst of chemically

  4. DNA damage and L1 retrotransposition.

    PubMed

    Farkash, Evan A; Luning Prak, Eline T

    2006-01-01

    Barbara McClintock was the first to suggest that transposons are a source of genome instability and that genotoxic stress assisted in their mobilization. The generation of double-stranded DNA breaks (DSBs) is a severe form of genotoxic stress that threatens the integrity of the genome, activates cell cycle checkpoints, and, in some cases, causes cell death. Applying McClintock's stress hypothesis to humans, are L1 retrotransposons, the most active autonomous mobile elements in the modern day human genome, mobilized by DSBs? Here, evidence that transposable elements, particularly retrotransposons, are mobilized by genotoxic stress is reviewed. In the setting of DSB formation, L1 mobility may be affected by changes in the substrate for L1 integration, the DNA repair machinery, or the L1 element itself. The review concludes with a discussion of the potential consequences of L1 mobilization in the setting of genotoxic stress. PMID:16877815

  5. The DNA-Binding Domain of Yeast Rap1 Interacts with Double-Stranded DNA in Multiple Binding Modes

    PubMed Central

    2015-01-01

    Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1DBD) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1DBD forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1DBD to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1DBD likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1DBD makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1DBD does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation. PMID:25382181

  6. Surface Modification of Nanocellulose Substrates

    NASA Astrophysics Data System (ADS)

    Zoppe, Justin Orazio

    Cellulose fibers constitute an important renewable raw material that is utilized in many commercial applications in non-food, paper, textiles and composite materials. Chemical functionalization is an important approach for improving the properties of cellulose based materials. Different approaches are used to graft polymeric chains onto cellulose substrates, which can be classified by two principal routes, namely 'grafting onto' or 'grafting from' methods. Never-dried cellulose nanocrystals (CNCs) or nanowhiskers produced from sulfuric acid hydrolysis of ramie fibers were used as substrates for surface chemical functionalization with various macromolecules. In addition, the use of cellulose nanocrystals to reinforce poly(epsilon-caprolactone) (PCL) nanofibers was studied. Chemical grafting with low molecular weight polycaprolactone diol onto cellulose nanocrystals was carried out in an attempt to improve the interfacial adhesion with the fiber matrix. Significant improvements in the mechanical properties of the nanofibers after reinforcement with unmodified cellulose nanocrystals were confirmed. Fiber webs from PCL reinforced with 2.5% unmodified CNCs showed ca. 1.5-fold increase in Young's modulus and ultimate strength compared to PCL webs. The CNCs were also grafted with poly(N-isopropylacrylamide) (poly(NiPAAm)) brushes via surface-initiated single-electron transfer living radical polymerization (SI-SETLRP) under various conditions at room temperature. The grafting process depended on the initiator and/or monomer concentrations used. No observable damage occurred to the CNCs after grafting, as determined by X-ray diffraction. Size exclusion chromatography analyses of polymer chains cleaved from the cellulose nanocrystals indicated that a higher degree of polymerization was achieved by increasing initiator or monomer loading, most likely caused by local heterogeneities yielding higher rates of polymerization. In addition, the colloidal stability and thermo

  7. Tiny telomere DNA

    PubMed Central

    Ren, Jinsong; Qu, Xiaogang; Trent, John O.; Chaires, Jonathan B.

    2002-01-01

    We describe the design, synthesis and biophysical characterization of a novel DNA construct in which a folded quadruplex structure is joined to a standard double helix. Circular dichroism, gel electrophoresis, three-dimensional UV melting and differential scanning calorimetry were all used to characterize the structure. Rigorous molecular dynamics simulations were used to build a plausible atomic-level structural model of the DNA construct. This novel DNA construct provides a model for the duplex–quadruplex junction region at the end of chromosomal DNA and offers a system for the study of structure-selective ligand binding. PMID:12034817

  8. DNA Damage Response

    PubMed Central

    Giglia-Mari, Giuseppina; Zotter, Angelika; Vermeulen, Wim

    2011-01-01

    Structural changes to DNA severely affect its functions, such as replication and transcription, and play a major role in age-related diseases and cancer. A complicated and entangled network of DNA damage response (DDR) mechanisms, including multiple DNA repair pathways, damage tolerance processes, and cell-cycle checkpoints safeguard genomic integrity. Like transcription and replication, DDR is a chromatin-associated process that is generally tightly controlled in time and space. As DNA damage can occur at any time on any genomic location, a specialized spatio-temporal orchestration of this defense apparatus is required. PMID:20980439

  9. Electrochemical probe for the monitoring of DNA-protein interactions.

    PubMed

    Meunier-Prest, Rita; Bouyon, Alice; Rampazzi, Eve; Raveau, Suzanne; Andreoletti, Pierre; Cherkaoui-Malki, Mustapha

    2010-08-15

    Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient way for DNA-functionalized surfaces. Here we describe the development of an electrochemical assay for the detection of DNA-protein interactions based on the modification of the electrochemical response of methylene blue (MB) intercalated in the DNA strands. Using a functionalized electrode with double stranded DNA carrying T3 RNA polymerase binding sequence, we show a substantial attenuation of the current upon the DNA-protein interaction. Moreover, a Langmuir binding isotherm for T3 RNA polymerase (T3 Pol) gives a dissociation constant K(D) equal to 0.46+/-0.23 microM. Such value is 100 times lower than the calculated K(D) for the non-specific interaction of bovine serum albumin (BSA) with T3 Pol promoter. In addition, the use of the T7 RNA polymerase (T7 Pol) promoter instead of the T3 Pol promoter induces an increase of K(D) from 0.46 microM to more than 25 microM. Accordingly, this strong decrease in the affinity of T3 Pol towards an off-target DNA promoter reveals an electrochemical sequence-specific discrimination of DNA-protein interactions. In conclusion, our results show that the developed electrochemical test allows the monitoring of DNA-protein interactions with high specificity and with an in situ protein detection threshold at a nanomolar range. PMID:20447818

  10. Integrating prokaryotes and eukaryotes: DNA transposases in light of structure.

    PubMed

    Hickman, Alison Burgess; Chandler, Michael; Dyda, Fred

    2010-02-01

    DNA rearrangements are important in genome function and evolution. Genetic material can be rearranged inadvertently during processes such as DNA repair, or can be moved in a controlled manner by enzymes specifically dedicated to the task. DNA transposases comprise one class of such enzymes. These move DNA segments known as transposons to new locations, without the need for sequence homology between transposon and target site. Several biochemically distinct pathways have evolved for DNA transposition, and genetic and biochemical studies have provided valuable insights into many of these. However, structural information on transposases - particularly with DNA substrates - has proven elusive in most cases. On the other hand, large-scale genome sequencing projects have led to an explosion in the number of annotated prokaryotic and eukaryotic mobile elements. Here, we briefly review biochemical and mechanistic aspects of DNA transposition, and propose that integrating sequence information with structural information using bioinformatics tools such as secondary structure prediction and protein threading can lead not only to an additional level of understanding but possibly also to testable hypotheses regarding transposition mechanisms. Detailed understanding of transposition pathways is a prerequisite for the long-term goal of exploiting DNA transposons as genetic tools and as a basis for genetic medical applications. PMID:20067338

  11. Mapping Transcription Factors on Extended DNA: A Single Molecule Approach

    NASA Astrophysics Data System (ADS)

    Ebenstein, Yuval; Gassman, Natalie; Weiss, Shimon

    The ability to determine the precise loci and distribution of nucleic acid binding proteins is instrumental to our detailed understanding of cellular processes such as transcription, replication, and chromatin reorganization. Traditional molecular biology approaches and above all Chromatin immunoprecipitation (ChIP) based methods have provided a wealth of information regarding protein-DNA interactions. Nevertheless, existing techniques can only provide average properties of these interactions, since they are based on the accumulation of data from numerous protein-DNA complexes analyzed at the ensemble level. We propose a single molecule approach for direct visualization of DNA binding proteins bound specifically to their recognition sites along a long stretch of DNA such as genomic DNA. Fluorescent Quantum dots are used to tag proteins bound to DNA, and the complex is deposited on a glass substrate by extending the DNA to a linear form. The sample is then imaged optically to determine the precise location of the protein binding site. The method is demonstrated by detecting individual, Quantum dot tagged T7-RNA polymerase enzymes on the bacteriophage T7 genomic DNA and assessing the relative occupancy of the different promoters.

  12. Designing new strategy for controlling DNA orientation in biosensors.

    PubMed

    Feng, Chao; Ding, Hong-ming; Ren, Chun-lai; Ma, Yu-qiang

    2015-01-01

    Orientation controllable DNA biosensors hold great application potentials in recognizing small molecules and detecting DNA hybridization. Though electric field is usually used to control the orientation of DNA molecules, it is also of great importance and significance to seek for other triggered methods to control the DNA orientation. Here, we design a new strategy for controlling DNA orientation in biosensors. The main idea is to copolymerize DNA molecules with responsive polymers that can show swelling/deswelling transitions due to the change of external stimuli, and then graft the copolymers onto an uncharged substrate. In order to highlight the responsive characteristic, we take thermo-responsive polymers as an example, and reveal multi-responsive behavior and the underlying molecular mechanism of the DNA orientation by combining dissipative particle dynamics simulation and molecular theory. Since swelling/deswelling transitions can be also realized by using other stimuli-responsive (like pH and light) polymers, the present strategy is universal, which can enrich the methods of controlling DNA orientation and may assist with the design of the next generation of biosensors. PMID:26400770

  13. Designing new strategy for controlling DNA orientation in biosensors

    PubMed Central

    Feng, Chao; Ding, Hong-ming; Ren, Chun-lai; Ma, Yu-qiang

    2015-01-01

    Orientation controllable DNA biosensors hold great application potentials in recognizing small molecules and detecting DNA hybridization. Though electric field is usually used to control the orientation of DNA molecules, it is also of great importance and significance to seek for other triggered methods to control the DNA orientation. Here, we design a new strategy for controlling DNA orientation in biosensors. The main idea is to copolymerize DNA molecules with responsive polymers that can show swelling/deswelling transitions due to the change of external stimuli, and then graft the copolymers onto an uncharged substrate. In order to highlight the responsive characteristic, we take thermo-responsive polymers as an example, and reveal multi-responsive behavior and the underlying molecular mechanism of the DNA orientation by combining dissipative particle dynamics simulation and molecular theory. Since swelling/deswelling transitions can be also realized by using other stimuli-responsive (like pH and light) polymers, the present strategy is universal, which can enrich the methods of controlling DNA orientation and may assist with the design of the next generation of biosensors. PMID:26400770

  14. Single Molecule Measurements of Protelomerase TelK-DNA Complexes

    NASA Astrophysics Data System (ADS)

    Landry, Markita; Khafizov, Rustem; Huang, Wai Mun; Chemla, Yann

    2008-10-01

    Protein-DNA interactions lie at the heart of many essential cellular processes such as replication, recombination, and repair. Recent advances in optical ``tweezers'' have made it possible to resolve motions on the scale of a single base pair of DNA, 3.4å. High-resolution optical traps have the potential to reveal these interactions at their fundamental length scales and should reveal how certain proteins bind to DNA or recognize target sequences. Telomerases are enzymes that have been actively studied in various organisms because of their fundamental involvement with both cancer and aging^1. Protelomerase TelK is an enzyme responsible for forming closed DNA hairpin ends in linear DNA. TelK is not an ATP dependant enzyme, which is surprising given the degree of DNA distortion accomplished by the enzyme, and the large energy barrier intrinsic in DNA hairpin formation. Therefore, our focus is on TelK mutants lacking their c-terminal domain, and TelK YF mutants lacking their tyrosine active site amino acid. Preliminary data have shown remarkable differences in protein binding and unbinding forces caused by the removal of a single oxygen atom from a 73 kDa protein. Further measurements using high-resolution optical tweezers should provide fundamental insights into the nature and importance of the electrostatic interactions between TelK and its DNA substrate. 1. Shay, J. et al. Rad. Res. 155, 188 (2001) [1] Huang, W. et al. Mol. Cell. 27, 901 (2007).

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

    PubMed

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

    2014-04-01

    Bacterial resistance against common antibiotics is an increasing health problem. New pharmaceuticals for the treatment of infections caused by resistant pathogens are needed. Small proline-rich antimicrobial peptides (PrAMPs) from insects are known to bind intracellularly to the conventional substrate binding cleft of the E. coli Hsp70 chaperone DnaK. Furthermore, bactenecins from mammals, members of the cathelicidin family, also contain potential DnaK binding sites. Crystal structures of bovine and sheep Bac7 in complex with the DnaK substrate binding domain show that the peptides bind in the forward binding mode with a leucine positioned in the central hydrophobic pocket. In most structures, proline and arginine residues preceding leucine occupy the hydrophobic DnaK binding sites -1 and -2. Within bovine Bac7, four potential DnaK binding sites were identified. PMID:24164259

  16. Substrate optimization for integrated circuit antennas

    NASA Astrophysics Data System (ADS)

    Alexopoulos, N. G.; Katehi, P. B.; Rutledge, D. B.

    1983-07-01

    The reciprocity theorem and integral equation techniques are employed to determine the properties of integrated-circuit antennas. The effect of surface waves is considered for dipole and slot elements on substrates. The radiation and bandwidth of microstrip dipoles are optimized in terms of substrate thickness and permittivity.

  17. Phytotoxic evaluation of whole pine tree substrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Decreased availability and increased cost of quality substrates are issues facing many horticulture crop producers. Peat moss and pine bark are the most widely used substrate components, yet producers have become more aware of acceptable alternative components. Processed whole pine trees have been i...

  18. High-emittance coatings on metal substrates

    NASA Technical Reports Server (NTRS)

    Emanuelson, R. C.; Luoma, W. L.; Walek, W. J.

    1968-01-01

    High-emittance coatings of iron, calcium, and zirconium titanates thermally sprayed on stainless steel, columbium-1 percent zirconium, and beryllium substrates promote and control radiative heat transfer from the metal substrates. Adherence, compatibility and emittance stability at elevated temperature and high vacuum were evaluated.

  19. Direct transfer of graphene onto flexible substrates.

    PubMed

    Martins, Luiz G P; Song, Yi; Zeng, Tingying; Dresselhaus, Mildred S; Kong, Jing; Araujo, Paulo T

    2013-10-29

    In this paper we explore the direct transfer via lamination of chemical vapor deposition graphene onto different flexible substrates. The transfer method investigated here is fast, simple, and does not require an intermediate transfer membrane, such as polymethylmethacrylate, which needs to be removed afterward. Various substrates of general interest in research and industry were studied in this work, including polytetrafluoroethylene filter membranes, PVC, cellulose nitrate/cellulose acetate filter membranes, polycarbonate, paraffin, polyethylene terephthalate, paper, and cloth. By comparing the properties of these substrates, two critical factors to ensure a successful transfer on bare substrates were identified: the substrate's hydrophobicity and good contact between the substrate and graphene. For substrates that do not satisfy those requirements, polymethylmethacrylate can be used as a surface modifier or glue to ensure successful transfer. Our results can be applied to facilitate current processes and open up directions for applications of chemical vapor deposition graphene on flexible substrates. A broad range of applications can be envisioned, including fabrication of graphene devices for opto/organic electronics, graphene membranes for gas/liquid separation, and ubiquitous electronics with graphene. PMID:24127582

  20. Biaxially textured metal substrate with palladium layer

    DOEpatents

    Robbins, William B [Maplewood, MN

    2002-12-31

    Described is an article comprising a biaxially textured metal substrate and a layer of palladium deposited on at least one major surface of the metal substrate; wherein the palladium layer has desired in-plane and out-of-plane crystallographic orientations, which allow subsequent layers that are applied on the article to also have the desired orientations.

  1. Buffer layers on biaxially textured metal substrates

    DOEpatents

    Shoup, Shara S.; Paranthamam, Mariappan; Beach, David B.; Kroeger, Donald M.; Goyal, Amit

    2001-01-01

    A method is disclosed for forming a biaxially textured buffer layer on a biaxially oriented metal substrate by using a sol-gel coating technique followed by pyrolyzing/annealing in a reducing atmosphere. This method is advantageous for providing substrates for depositing electronically active materials thereon.

  2. The Silver Nanorod Array SERS Substrates

    NASA Astrophysics Data System (ADS)

    Zhao, Yiping; Liu, Yongjun

    2010-08-01

    The fabrication of large area, uniform and high enhancement substrates for surface enhanced Raman scattering (SERS) based sensing is a bottle-neck for practical applications of SERS. Recently using oblique angle deposition (OAD) method, we have fabricated silver nanorod arrays with SERS enhancement factor >108, and SERS intensity variation <14%. The SERS spectra from those substrates have been demonstrated to be able to distinguish different viruses and virus strains, bacteria, microRNAs, or other chemical and biological molecules. We have performed a detailed characterization on those Ag nanorod substrates. The SERS enhancement factor depends strongly on the nanorod length and the fabrication conditions. For different deposition angle, there is an optimal nanorod length that gives the maximum enhancement. The SERS enhancement seems to directly depend on the reflectivity of the Ag nanorod substrates at the excitation wavelength regardless of the deposition angles and rod length. The SERS performance also depends strongly on the configurations of the excitation laser beam: the incident angle, the polarization, and the reflectance of the underlayer substrates. A simple modified Greenler's model is proposed to qualitatively explain those effects. The possible origin for the high enhancement of the Ag nanorod substrates has been studied by placing the Raman probe molecules on different locations of the substrates, and we have found that the side surfaces of the nanorod arrays contributes more to the SERS enhancement compared to the ends. We propose that this is due to the anisotropic absorbance nature of the Ag nanorod substrates.

  3. Direct transfer of graphene onto flexible substrates

    NASA Astrophysics Data System (ADS)

    Pimenta, Luiz Gustavo; Song, Yi; Zeng, Tingying; Dresselhaus, Mildred; Kong, Jing; Araujo, Paulo

    2014-03-01

    We explore the direct transfer via lamination of chemical vapor deposition graphene onto different flexible substrates. The transfer method investigated here is fast, simple, and does not require an intermediate transfer membrane, such as polymethylmethacrylate. Various substrates of general interest in research and industry were studied including polytetrafluoroethylene filter membranes, PVC, cellulose nitrate/cellulose acetate filter membranes, polycarbonate, paraffin, polyethylene terephthalate, paper, and cloth. By comparing the properties of these substrates, two critical factors to ensure a successful transfer on bare substrates were identified: the substrate's hydrophobicity and good contact between the substrate and graphene. For substrates that do not satisfy those requirements, polymethylmethacrylate can be used as a surface modifier or glue to ensure successful transfer. Our results can be applied to facilitate present processes and open up directions for applications of chemical vapor deposition (CVD) graphene on flexible substrates. A broad range of applications of CVD graphene can be envisioned, including fabrication of graphene devices for opto/organic electronics, graphene membranes for gas/liquid separation, and ubiquitous electronics with graphene.

  4. Positional scanning substrate combinatorial library (PS-SCL) approach to define caspase substrate specificity.

    PubMed

    Poręba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Drąg, Marcin

    2014-01-01

    Positional scanning substrate combinatorial library (PS-SCL) is a powerful tool for studying substrate specificity of proteolytic enzymes. Here, we describe the protocol for analyzing S4-S2 pockets preferences of caspases using PS-SCL. Additionally, we describe procedures for the identification of optimal substrates sequence after PS-SCL, solid phase synthesis, and purification of selected fluorogenic substrates, as well as their kinetic analysis. PMID:24567093

  5. Large microchannel array fabrication and results for DNA sequencing

    SciTech Connect

    Pastrone, R L; Balch, J W; Brewer, L R; Copeland, A C; Davidson , J C; Fitch, J P; Kimbrough, J R; Madabhushi, R S; Richardson, P M; Swierkowski, S P; Tarte, L A; Vainer, M

    1999-01-07

    We have developed a process for the production of microchannel arrays on bonded glass substrates up to I4 x 58 cm, for DNA sequencing. Arrays of 96 and 384 microchannels, each 46 cm long have been built. This technology offers significant advantages over discrete capillaries or conventional slab-gel approaches. High throughput DNA sequencing with over 550 base pairs resolution has been achieved. With custom fabrication apparatus, microchannels are etched in a borosilicate substrate, and then fusion bonded to a top substrate 1.1 mm thick that has access holes formed in it. SEM examination shows a typical microchannel to be 40 x 180 micrometers by 46 cm Iong; the etch is approximately isotropic, leaving a key undercut, for forming a rounded channel. The surface roughness at the bottom of the 40 micrometer deep channel has been profilometer measured to be as low as 20 nm; the roughness at the top surface was 2 nm. Etch uniformity of about 5% has been obtained using a 22% vol. HF / 78% Acetic acid solution. The simple lithography, etching, and bonding of these substrates enables efficient production of these arrays and extremely precise replication From master masks and precision machining with a mandrel. Keywords: microchannels, microchannel plates, DNA sequencing, electrophoresis, borosilicate glass

  6. Mechanics of Protein-Mediated DNA Looping

    NASA Astrophysics Data System (ADS)

    Meiners, Jens-Christian

    2009-03-01

    The formation of looped DNA-protein complexes in which a protein or protein assembly binds to multiple distant operator sites on the DNA is a common feature for many regulatory schemes on the transcriptional level. In a living cell, a multitude of mechanical forces and constraints act on these complexes, and it is imperative to understand their effects on biological function. For this aim, we study the lactose repressor as a model system for protein-mediated DNA looping in single-molecule experiments. Using a novel axial constant-force optical trapping scheme that allows us to manipulate sub-micron DNA fragments with well-controlled forces down to the 10 fN range, we show that mechanical tension in the substrate DNA of hundred femtonewton is sufficient to disrupt the loop formation process, which suggests that such mechanical tension may provide a mechanical pathway to controlling gene expression in vivo. From the force sensitivity of the loop formation process, we can also infer the topology of the looped complex; in our case an antiparallel conformation. In addition, we will present new tethered-particle microscopy data that shows lifetimes of the looped complexes that are two to three orders of magnitude shorter than those measured in biochemical competition assays and discuss possible interpretations, including the suggestion that operator binding of the lactose repressor tetramer leads to a destabilization of the dimer-dimer interface and that thus the loop breakdown process is mostly a dissociation of the tetramer into two dimers, instead, as widely assumed, an unbinding of the tetramer from the DNA.

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

    PubMed

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

    2016-01-01

    Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step. PMID:27099790

  8. Substrate solder barriers for semiconductor epilayer growth

    DOEpatents

    Drummond, T.J.; Ginley, D.S.; Zipperian, T.E.

    1989-05-09

    During the growth of compound semiconductors by epitaxial processes, substrates are typically mounted to a support. In modular beam epitaxy, mounting is done using indium as a solder. This method has two drawbacks: the indium reacts with the substrate, and it is difficult to uniformly wet the back of a large diameter substrate. Both of these problems have been successfully overcome by sputter coating the back of the substrate with a thin layer of tungsten carbide or tungsten carbide and gold. In addition to being compatible with the growth of high quality semiconductor epilayers this coating is also inert in all standard substrate cleaning etchants used for compound semiconductors, and provides uniform distribution of energy in radiant heating.

  9. Substrate solder barriers for semiconductor epilayer growth

    DOEpatents

    Drummond, Timothy J.; Ginley, David S.; Zipperian, Thomas E.

    1989-01-01

    During the growth of compound semiconductors by epitaxial processes, substrates are typically mounted to a support. In modular beam epitaxy, mounting is done using indium as a solder. This method has two drawbacks: the indium reacts with the substrate, and it is difficult to uniformly wet the back of a large diameter substrate. Both of these problems have been successfully overcome by sputter coating the back of the substrate with a thin layer of tungsten carbide or tungsten carbide and gold. In addition to being compatible with the growth of high quality semiconductor epilayers this coating is also inert in all standard substrate cleaning etchants used for compound semiconductors, and provides uniform distribution of energy in radiant heating.

  10. Substrate Effects for Atomic Chain Electronics

    NASA Technical Reports Server (NTRS)

    Yamada, Toshishige; Saini, Subhash (Technical Monitor)

    1998-01-01

    A substrate for future atomic chain electronics, where adatoms are placed at designated positions and form atomically precise device components, is studied theoretically. The substrate has to serve as a two-dimensional template for adatom mounting with a reasonable confinement barrier and also provide electronic isolation, preventing unwanted coupling between independent adatom structures. For excellent structural stability, we demand chemical bonding between the adatoms and substrate atoms, but then good electronic isolation may not be guaranteed. Conditions are clarified for good isolation. Because of the chemical bonding, fundamental adatom properties are strongly influenced: a chain with group IV adatoms having two chemical bonds, or a chain with group III adatoms having one chemical bond is semiconducting. Charge transfer from or to the substrate atoms brings about unintentional doping, and the electronic properties have to be considered for the entire combination of the adatom and substrate systems even if the adatom modes are well localized at the surface.

  11. Thin Film Transistors On Plastic Substrates

    DOEpatents

    Carey, Paul G.; Smith, Patrick M.; Sigmon, Thomas W.; Aceves, Randy C.

    2004-01-20

    A process for formation of thin film transistors (TFTs) on plastic substrates replaces standard thin film transistor fabrication techniques, and uses sufficiently lower processing temperatures so that inexpensive plastic substrates may be used in place of standard glass, quartz, and silicon wafer-based substrates. The silicon based thin film transistor produced by the process includes a low temperature substrate incapable of withstanding sustained processing temperatures greater than about 250.degree. C., an insulating layer on the substrate, a layer of silicon on the insulating layer having sections of doped silicon, undoped silicon, and poly-silicon, a gate dielectric layer on the layer of silicon, a layer of gate metal on the dielectric layer, a layer of oxide on sections of the layer of silicon and the layer of gate metal, and metal contacts on sections of the layer of silicon and layer of gate metal defining source, gate, and drain contacts, and interconnects.

  12. Isolation of DNA methyltransferase from plants

    SciTech Connect

    Ehrlich, K.; Malbroue, C.

    1987-05-01

    DNA methyltransferases (DMT) were isolated from nuclei of cauliflower, soybean, and pea by extraction with 0.35 M NaCl. Assays were performed on hemimethylated Micrococcus luteus DNA or on M. luteus DNA to test for maintenance or de novo methylase activity, respectively. Fully methylated DNA was used as a substrate to determine background levels of methylation. Based on these tests, yields of maintenance DMT activity in the crude extract from pea hypocotyl, soybean hypocotyl, and cauliflower inflorescence were 2.8, 0.9, and 1.6 units per g wet tissue (one unit equals 1 pmol of methyl from (/sup 3/H)AdoMet incorporated into acid precipitable material per h at 30/sup 0/). Two peaks of DMT activity were detected in the soybean nuclear extract following phosphocellulose chromatography. One eluted at 0.4 M and the other at 0.8 M KCl. With both fractions maintenance activity was approximately 2 times that of the de novo activity. Using gel filtration the DMT eluted at 220,000 Daltons. The optimal pH for activity was between 6.5 and 7.0, and the optimal temperature was 30/sup 0/.

  13. Kinetic characterization of on-chip DNA ligation on dendron-coated surfaces with nanoscaled lateral spacings

    NASA Astrophysics Data System (ADS)

    Kim, Eung-Sam; Lee, Namgyu; Park, Joon Won; Choi, Kwan Yong

    2013-10-01

    We analyzed the enzymatic profiles of on-chip DNA ligation as we controlled the lateral spacing of surface-immobilized DNA substrates using dendron molecules with different sizes at the nanoscale. Enzymatic on-chip DNA ligation was performed on the dendron-coated surface within 20 min with no need for post-ligation gel electrophoresis. The enzymatic DNA repair was assessed by the fluorescence intensity at the repaired DNA duplex after thermally dissociating the unligated Cy3-labeled DNA from the DNA duplex, in which the Cy3-labeled DNA was hybridized prior to the on-chip DNA ligation. The rate of the nick-sealing reaction on the 27-acid dendron surface was 3-fold higher than that on the 9-acid dendron surface, suggesting that the wider lateral spacing determined by the larger dendron molecule could facilitate the access of DNA ligase to the nick site. The performance of on-chip DNA ligation was dropped to 10% and 3% when the nick was replaced by one- and two-nucleotide-long gaps, respectively. The 5‧ terminal phosphorylation of DNA strands by polynucleotide kinase and the on-chip DNA cleavage by endonucleases were also quantitatively monitored throughout the on-chip DNA ligation on the dendron-coated surface. A better understanding of the enzymatic kinetics of on-chip DNA ligation will contribute to a more reliable performance of various on-chip DNA ligation-based assays.

  14. The Scrunchworm Hypothesis: Transitions Between A-DNA and B-DNA Provide the Driving Force for Genome Packaging in Double-Stranded DNA Bacteriophages

    PubMed Central

    Harvey, Stephen C.

    2015-01-01

    Double-stranded DNA bacteriophages have motors that drive the genome into preformed capsids, using the energy releas ed by hydrolysis of ATP to overcome the forces opposing DNA packaging. Viral packaging motors are the strongest of all biological motors, but it is not known how they generate these forces. Several models for the process of mechanochemical force generation have been put forward, but there is no consensus on which, if any, of these is correct. All the existing models assume that protein-generated forces drive the DNA forward. The scrunchworm hypothesis proposes that the DNA molecule is the active force-generating core of the motor, not simply a substrate on which the motor operates. The protein components of the motor dehydrate a section of the DNA, converting it from the B form to the A form and shortening it by about 23%. The proteins then rehydrate the DNA, which converts back to the B form. Other regions of the motor grip and release the DNA to capture the shortening-lengthening motions of the B→A→B cycle (“scrunching”), so that DNA is pulled into the motor and pushed forward into the capsid. This DNA-centric mechanism provides a quantitative physical explanation for the magnitude of the forces generated by viral packaging motors. It also provides a simple explanation for the fact that each of the steps in the burst cycle advances the DNA by 2.5 base pairs. The scrunchworm hypothesis is consistent with a large body of published data, and it makes four experimentally testable predictions. PMID:25486612

  15. Quantitative analysis of trace Pb(II) by a DNAzyme cracking-rhodamine 6G SERRS probe on AucoreAgshell nanosol substrate

    NASA Astrophysics Data System (ADS)

    Liu, Qingye; Wei, Yanyan; Luo, Yanghe; Liang, Aihui; Jiang, Zhiliang

    2014-07-01

    In pH 7.2 Tris-HCl buffer solution containing 0.09 mol/L NaCl at 80 °C, the single-stranded substrate DNA hybrids with the enzyme DNA to form double-stranded DNA (dsDNA). The substrate chain of dsDNA could be cracked catalytically by Pb2+ to produce a short single-stranded DNA (ssDNA) that adsorbed on the AucoreAgshell nanoparticle (Au/AgNP) surface to form stable Au/AgNP-ssDNA conjugate to prevent aggregation by NaCl, and it combined with rhodamine 6G (RhG) to form RhG-Au/AgNP-ssDNA probe that exhibited a strong surface-enhanced resonance Raman scattering (SERRS) peak at 1510 cm-1. With the increase of Pb2+ concentration, the SERRS peak increased linearly due to the more RhG-Au/AgNP-ssDNA probe forming. Under the selected conditions, the increased SERRS intensity ΔI was linear to Pb2+ concentration in the range of 5.0 × 10-8-7.0 × 10-7 mol/L, with a detection limit of 7 × 10-9 mol/L Pb2+.

  16. Single-Molecule Investigation of Response to Oxidative DNA Damage by a Y-Family DNA Polymerase.

    PubMed

    Raper, Austin T; Gadkari, Varun V; Maxwell, Brian A; Suo, Zucai

    2016-04-12

    Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo and rescue stalled DNA replication machinery. Dpo4, a well-characterized model Y-family DNA polymerase, is known to catalyze translesion synthesis across a variety of DNA lesions including 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxo-dG). Our previous X-ray crystallographic, stopped-flow Förster resonance energy transfer (FRET), and computational simulation studies have revealed that Dpo4 samples a variety of global conformations as it recognizes and binds DNA. Here we employed single-molecule FRET (smFRET) techniques to investigate the kinetics and conformational dynamics of Dpo4 when it encountered 8-oxo-dG, a major oxidative lesion with high mutagenic potential. Our smFRET data indicated that Dpo4 bound the DNA substrate in multiple conformations, as suggested by three observed FRET states. An incoming correct or incorrect nucleotide affected the distribution and stability of these states with the correct nucleotide completely shifting the equilibrium toward a catalytically competent complex. Furthermore, the presence of the 8-oxo-dG lesion in the DNA stabilized both the binary and ternary complexes of Dpo4. Thus, our smFRET analysis provided a basis for the enhanced efficiency which Dpo4 is known to exhibit when replicating across from 8-oxo-dG. PMID:27002236

  17. Replicative DNA polymerases.

    PubMed

    Johansson, Erik; Dixon, Nicholas

    2013-06-01

    In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3'-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein-DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact. PMID:23732474

  18. Many Ways to Loop DNA

    PubMed Central

    Griffith, Jack D.

    2013-01-01

    In the 1960s, I developed methods for directly visualizing DNA and DNA-protein complexes using an electron microscope. This made it possible to examine the shape of DNA and to visualize proteins as they fold and loop DNA. Early applications included the first visualization of true nucleosomes and linkers and the demonstration that repeating tracts of adenines can cause a curvature in DNA. The binding of DNA repair proteins, including p53 and BRCA2, has been visualized at three- and four-way junctions in DNA. The trombone model of DNA replication was directly verified, and the looping of DNA at telomeres was discovered. PMID:24005675

  19. Long-range charge transport in single G-quadruplex DNA molecules

    NASA Astrophysics Data System (ADS)

    Livshits, Gideon I.; Stern, Avigail; Rotem, Dvir; Borovok, Natalia; Eidelshtein, Gennady; Migliore, Agostino; Penzo, Erika; Wind, Shalom J.; di Felice, Rosa; Skourtis, Spiros S.; Cuevas, Juan Carlos; Gurevich, Leonid; Kotlyar, Alexander B.; Porath, Danny

    2014-12-01

    DNA and DNA-based polymers are of interest in molecular electronics because of their versatile and programmable structures. However, transport measurements have produced a range of seemingly contradictory results due to differences in the measured molecules and experimental set-ups, and transporting significant current through individual DNA-based molecules remains a considerable challenge. Here, we report reproducible charge transport in guanine-quadruplex (G4) DNA molecules adsorbed on a mica substrate. Currents ranging from tens of picoamperes to more than 100 pA were measured in the G4-DNA over distances ranging from tens of nanometres to more than 100 nm. Our experimental results, combined with theoretical modelling, suggest that transport occurs via a thermally activated long-range hopping between multi-tetrad segments of DNA. These results could re-ignite interest in DNA-based wires and devices, and in the use of such systems in the development of programmable circuits.

  20. DNA Origami Mask for Sub-Ten-Nanometer Lithography.

    PubMed

    Diagne, Cheikh Tidiane; Brun, Christophe; Gasparutto, Didier; Baillin, Xavier; Tiron, Raluca

    2016-07-26

    DNA nanotechnology is currently widely explored and especially shows promises for advanced lithography due to its ability to define nanometer scale features. We demonstrate a 9 × 14 nm(2) hole pattern transfer from DNA origami into an SiO2 layer with a sub-10-nm resolution using anhydrous HF vapor in a semiconductor etching machine. We show that the resulting SiO2 pattern inherits its shape from the DNA structure within a process time ranging from 30 to 60 s at an etching rate of 0.2 nm/s. At 600 s of etching, the SiO2 pattern meets corrosion and the overall etching reaction is blocked. These results, in addition to the entire surface coverage by magnesium occurring on the substrate at a density of 1.1 × 10(15) atom/cm(2), define a process window, fabrication rules, and limits for DNA-based lithography. PMID:27281227

  1. Multiple cellular mechanisms prevent chromosomal rearrangements involving repetitive DNA

    PubMed Central

    George, Carolyn M.; Alani, Eric

    2012-01-01

    Repetitive DNA is present in the eukaryotic genome in the form of segmental duplications, tandem and interspersed repeats, and satellites. Repetitive sequences can be beneficial by serving specific cellular functions (e.g. centromeric and telomeric DNA) and by providing a rapid means for adaptive evolution. However, such elements are also substrates for deleterious chromosomal rearrangements that affect fitness and promote human disease. Recent studies analyzing the role of nuclear organization in DNA repair and factors that suppress non-allelic homologous recombination have provided insights into how genome stability is maintained in eukaryotes. In this review we outline the types of repetitive sequences seen in eukaryotic genomes and how recombination mechanisms are regulated at the DNA sequence, cell organization, chromatin structure, and cell cycle control levels to prevent chromosomal rearrangements involving these sequences. PMID:22494239

  2. Influence of Substrate Complexity on the Diastereoselective Formation of Spiroiminodihydantoin and Guanidinohydantoin from Chromate Oxidation

    PubMed Central

    Gremaud, Julia N.; Martin, Brooke D.; Sugden, Kent D.

    2009-01-01

    Chromate is a human carcinogen with a poorly defined mechanism of DNA damage. In vitro and prokaryotic studies have shown that DNA damage may occur via the formation of the hydantoin lesions guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) from further oxidation of 8-oxo-7,8-dihydroguanine (8oxoG). The unusual structure of these lesions coupled with their enhanced mutagenicity make them attractive for study with regard to their role in chromate-induced cancer. We have studied the formation of Gh versus Sp and their associated diastereomers following oxidation by model Cr(V) complexes and from in situ chromate reduction by ascorbate and glutathione. Identification of the two optically assigned diastereomers of Sp (R-Sp, S-Sp) as well as the two diastereomers of Gh, (Gh1 and Gh2; not yet optically assigned) were carried out using increasingly sterically hindered substrates (nucleoside → ssDNA → dsDNA). Lesion formation and diastereomeric preference was found to be highly oxidant- and substrate-dependent. The Ir(IV) positive control showed a shift from near equal levels of Gh and Sp, and near equal levels of all four diastereomers in the nucleoside, to all Gh formation in dsDNA, with a 5-fold enhancement in Gh2 over Gh1. The two model Cr(V) complexes used in this study, Cr(V)-salen and Cr(V)-ehba, showed opposite trends going from nucleoside to dsDNA with Cr(V)-salen giving enhanced Sp formation (with mainly R-Sp formed) and the Cr(V)-ehba having an oxidation profile nearly identical to that of Ir(IV). The two chromate reduction systems, Cr6+/ascorbate and Cr6+/glutathione, designed to model the intracellular reduction of chromate, showed lower levels of oxidation in all substrates. Notable in this group was the shift in the formation of the lesions to essentially all Sp for the Cr6+/ascorbate system with the most sterically hindered substrate, dsDNA. These results, when coupled with the known diastereomeric preference for excision of hydantoin lesions by

  3. Unique subunit packing in mycobacterial nanoRNase leads to alternate substrate recognitions in DHH phosphodiesterases

    PubMed Central

    Srivastav, Rajpal; Kumar, Dilip; Grover, Amit; Singh, Ajit; Manjasetty, Babu A.; Sharma, Rakesh; Taneja, Bhupesh

    2014-01-01

    DHH superfamily includes RecJ, nanoRNases (NrnA), cyclic nucleotide phosphodiesterases and pyrophosphatases. In this study, we have carried out in vitro and in vivo investigations on the bifunctional NrnA-homolog from Mycobacterium smegmatis, MSMEG_2630. The crystal structure of MSMEG_2630 was determined to 2.2-Å resolution and reveals a dimer consisting of two identical subunits with each subunit folding into an N-terminal DHH domain and a C-terminal DHHA1 domain. The overall structure and fold of the individual domains is similar to other members of DHH superfamily. However, MSMEG_2630 exhibits a distinct quaternary structure in contrast to other DHH phosphodiesterases. This novel mode of subunit packing and variations in the linker region that enlarge the domain interface are responsible for alternate recognitions of substrates in the bifunctional nanoRNases. MSMEG_2630 exhibits bifunctional 3′-5′ exonuclease [on both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) substrates] as well as CysQ-like phosphatase activity (on pAp) in vitro with a preference for nanoRNA substrates over single-stranded DNA of equivalent lengths. A transposon disruption of MSMEG_2630 in M. smegmatis causes growth impairment in the presence of various DNA-damaging agents. Further phylogenetic analysis and genome organization reveals clustering of bacterial nanoRNases into two distinct subfamilies with possible role in transcriptional and translational events during stress. PMID:24878921

  4. A paper-based inkjet-fabricated substrate for SERS detection and differentiation of PCR products

    NASA Astrophysics Data System (ADS)

    Hoppmann, Eric P.; White, Ian M.

    2013-05-01

    Surface enhanced Raman spectroscopy (SERS) is a highly sensitive sensing technique, offering sensitivity comparable to that of fluorescence while providing structure-dependent analyte information. In recent years, we have developed an innovative optofluidic SERS substrate by inkjet printing metal nanoparticles onto paper. By virtue of generating a SERS substrate on cellulose, we gain a flexible SERS sensing device, as well as the ability to harness the intrinsic wicking properties of paper to enable both separation and concentration of analytes. Here we demonstrate the application of paper-chromatographic separation to allow on-substrate separation, concentration and discrimination. By using inexpensive single-labeled DNA probes in a typical PCR amplification, we obtain a mixture containing whole probes (negative result) and probes which have been hydrolyzed by the Taq polymerase (positive result). Leveraging the solubility differences between the whole and hydrolyzed probes and the cellulose separation matrix, we are able to perform a multiplexed interrogation of the targets. Notably, this does not require the use of dual labeled DNA probes (expensive) or multiple excitation sources and filter sets needed for a multiplexed fluorescence measurement (expensive and bulky). All SERS measurements are performed using a portable spectrometer and diode laser; in combination with a portable low-power DNA amplification system, this technique has the potential to be used for rapid on-site multiplexed genetic detection, without requiring complex optical equipment.

  5. Nanoparticle bridge DNA biosensor

    NASA Astrophysics Data System (ADS)

    Huang, Hong-Wen

    A new DNA sensing method is demonstrated in which DNA hybridization events lead to the formation of nanoparticle satellites that bridge two electrodes and are detected electrically. The hybridization events are exclusively carried out only on specific locations, the surfaces of C-ssDNA modified 50 nm GNPs. The uniqueness of this work is that only a small number of T-ccDNA molecules (<10) is required to form the nanoparticle satellites, allowing ultra-sensitive DNA sensing. The principle of this new DNA sensing technique has been demonstrated using target DNA and three-base-pair-mismatched DNA in 20nM concentrations. Three single-stranded DNA (ssDNA) system is used in our experiment which includes Capture-ssDNA (C-ssDNA), Target-ssDNA (T-ssDNA) and Probe-ssDNA (P-ssDNA). Both C-ssDNA and P-ssDNA are modified by a thiol group and can hybridize with different portions of T-ssDNA. T-ssDNA requires no modification in three ssDNA system, which is beneficial in many applications. C-ssDNA modified 50nm gold nanoparticle (C-50au) and P-ssDNA modified 30nm gold nanoparticle (P-30au) are prepared through the reaction of thiol-gold chemical bonding between thiolated ssDNA and gold nanoparticle (GNP) (C-ssDNA with 50nm GNP, P-ssDNA with 30nm GNP). We controllably place the C-50au only on the SiO2 band surface (˜ 90nm width) between two gold electrodes (source and drain electrodes) by forming positively- and negatively-charged self-assembled monolayers (SAMs) on SiO2 and gold surface, respectively. DNA modified GNP is negatively charged due to ionization of phosphate group on DNA back bone. C-50au therefore is negatively charged and can only be attracted toward SiO2 area (repelled by negatively charged gold electrode surface). The amine group of positively-charged SAMs on SiO2 surface is then passivated by converting to non-polar methyl functional group after C-50au placement. P-30au is first hybridized with T-ssDNA in the solution phase (T-P- 30au formed) and is introduced

  6. Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases.

    PubMed

    Loh, Ern; Loeb, Lawrence A

    2005-12-01

    DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity. PMID:16230053

  7. Structural analysis of isosteviol and related compounds as DNA polymerase and DNA topoisomerase inhibitors.

    PubMed

    Mizushina, Yoshiyuki; Akihisa, Toshihiro; Ukiya, Motohiko; Hamasaki, Yusuke; Murakami-Nakai, Chikako; Kuriyama, Isoko; Takeuchi, Toshifumi; Sugawara, Fumio; Yoshida, Hiromi

    2005-09-01

    Isosteviol (ent-16-ketobeyeran-19-oic acid) is a hydrolysis product of stevioside, which is a natural sweetener produced in the leaves of Stevia rebaudiana (Bertoni) Bertoni. In this report, we prepared isosteviol and related compounds from stevioside by microbial transformation and chemical conversion and assayed the inhibitory activities toward DNA metabolic enzymes and human cancer cell growth. Among twelve compounds obtained, only isosteviol (compound 3) potently inhibited both mammalian DNA polymerases (pols) and human DNA topoisomerase II (topo II), and IC50 value for pol alpha was 64.0 microM. This compound had no inhibitory effect on higher plant (cauliflower) pols, prokaryotic pols, human topo I, and DNA metabolic enzymes such as human telomerase, T7 RNA polymerase, and bovine deoxyribonuclease I. With pol alpha, isosteviol acted non-competitively with the DNA template-primer and nucleotide substrate. Isosteviol prevented the growth of human cancer cells, with LD50 values of 84-167 microM, and 500 microg of the compound caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 53.0%). The relationship between the structure of stevioside-based compounds and these activities were discussed. PMID:15935396

  8. Distinct substrate specificities of Arabidopsis DCL3 and DCL4

    PubMed Central

    Nagano, Hideaki; Fukudome, Akihito; Hiraguri, Akihiro; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2014-01-01

    In Arabidopsis thaliana, Dicer-like 3 (DCL3) and Dicer-like 4 (DCL4) cleave long, perfect double-stranded RNAs (dsRNAs) into 24 and 21 nucleotides (nt) small interfering RNAs, respectively, which in turn function in RNA-directed DNA methylation and RNA interference, respectively. To reveal how DCL3 and DCL4 individually recognize long perfect dsRNAs as substrates, we biochemically characterized DCL3 and DCL4 and compared their enzymatic properties. DCL3 preferentially cleaves short dsRNAs with 5′ phosphorylated adenosine or uridine and a 1 nt 3′ overhang, whereas DCL4 cleaves long dsRNAs with blunt ends or with a 1 or 2 nt 3′ overhang with similar efficiency. DCL3 produces 24 nt RNA duplexes with 2 nt 3′ overhangs by the 5′ counting rule. Inorganic phosphate, NaCl and KCl enhance DCL3 activity but inhibit DCL4 activity. These results indicate that plants use DCLs with distinct catalytic profiles to ensure each dsRNA substrate generates only a specific length of siRNAs that trigger a unique siRNA-mediated response. PMID:24214956

  9. Dynamic Conformational Change Regulates the Protein-DNA Recognition: An Investigation on Binding of a Y-Family Polymerase to Its Target DNA

    PubMed Central

    Chu, Xiakun; Liu, Fei; Maxwell, Brian A.; Wang, Yong; Suo, Zucai; Wang, Haijun; Han, Wei; Wang, Jin

    2014-01-01

    Protein-DNA recognition is a central biological process that governs the life of cells. A protein will often undergo a conformational transition to form the functional complex with its target DNA. The protein conformational dynamics are expected to contribute to the stability and specificity of DNA recognition and therefore may control the functional activity of the protein-DNA complex. Understanding how the conformational dynamics influences the protein-DNA recognition is still challenging. Here, we developed a two-basin structure-based model to explore functional dynamics in Sulfolobus solfataricus DNA Y-family polymerase IV (DPO4) during its binding to DNA. With explicit consideration of non-specific and specific interactions between DPO4 and DNA, we found that DPO4-DNA recognition is comprised of first 3D diffusion, then a short-range adjustment sliding on DNA and finally specific binding. Interestingly, we found that DPO4 is under a conformational equilibrium between multiple states during the binding process and the distributions of the conformations vary at different binding stages. By modulating the strength of the electrostatic interactions, the flexibility of the linker, and the conformational dynamics in DPO4, we drew a clear picture on how DPO4 dynamically regulates the DNA recognition. We argue that the unique features of flexibility and conformational dynamics in DPO4-DNA recognition have direct implications for low-fidelity translesion DNA synthesis, most of which is found to be accomplished by the Y-family DNA polymerases. Our results help complete the description of the DNA synthesis process for the Y-family polymerases. Furthermore, the methods developed here can be widely applied for future investigations on how various proteins recognize and bind specific DNA substrates. PMID:25188490

  10. The Use of Nucleosome Substrates Improves Binding of SAM Analogs to SETD8.

    PubMed

    Strelow, John M; Xiao, Min; Cavitt, Rachel N; Fite, Nathan C; Margolis, Brandon J; Park, Kyu-Jin

    2016-09-01

    SETD8 is the methyltransferase responsible for monomethylation of lysine at position 20 of the N-terminus of histone H4 (H4K20). This activity has been implicated in both DNA damage and cell cycle progression. Existing biochemical assays have utilized truncated enzymes containing the SET domain of SETD8 and peptide substrates. In this report, we present the development of a mechanistically balanced biochemical assay using full-length SETD8 and a recombinant nucleosome substrate. This improves the binding of SAM, SAH, and sinefungin by up to 10,000-fold. A small collection of inhibitors structurally related to SAM were screened and 40 compounds were identified that only inhibit SETD8 when a nucleosome substrate is used. PMID:27369108

  11. Pre-steady-state kinetics shows differences in processing of various DNA lesions by Escherichia coli formamidopyrimidine-DNA glycosylase

    PubMed Central

    Koval, Vladimir V.; Kuznetsov, Nikita A.; Zharkov, Dmitry O.; Ishchenko, Alexander A.; Douglas, Kenneth T.; Nevinsky, Georgy A.; Fedorova, Olga S.

    2004-01-01

    Formamidopyrimidine-DNA-glycosylase (Fpg pro tein, MutM) catalyses excision of 8-oxoguanine (8-oxoG) and other oxidatively damaged purines from DNA in a glycosylase/apurinic/apyrimidinic-lyase reaction. We report pre-steady-state kinetic analysis of Fpg action on oligonucleotide duplexes containing 8-oxo-2′-deoxyguanosine, natural abasic site or tetrahydrofuran (an uncleavable abasic site analogue). Monitoring Fpg intrinsic tryptophan fluorescence in stopped-flow experiments reveals multiple conformational transitions in the protein molecule during the catalytic cycle. At least four and five conformational transitions occur in Fpg during the interaction with abasic and 8-oxoG-containing substrates, respectively, within 2 ms to 10 s time range. These transitions reflect the stages of enzyme binding to DNA and lesion recognition with the mutual adjustment of DNA and enzyme structures to achieve catalytically competent conformation. Unlike these well-defined binding steps, catalytic stages are not associated with discernible fluorescence events. Only a single conformational change is detected for the cleavable substrates at times exceeding 10 s. The data obtained provide evidence that several fast sequential conformational changes occur in Fpg after binding to its substrate, converting the protein into a catalytically active conformation. PMID:14769949

  12. Electrostatic interactions play an essential role in DNA repair and cold-adaptation of uracil DNA glycosylase.

    PubMed

    Olufsen, Magne; Smalås, Arne O; Brandsdal, Bjørn O

    2008-03-01

    Life has adapted to most environments on earth, including low and high temperature niches. The increased catalytic efficiency and thermoliability observed for enzymes from organisms living in constantly cold regions when compared to their mesophilic and thermophilic cousins are poorly understood at the molecular level. Uracil DNA glycosylase (UNG) from cod (cUNG) catalyzes removal of uracil from DNA with an increased k(cat) and reduced K(m) relative to its warm-active human (hUNG) counterpart. Specific issues related to DNA repair and substrate binding/recognition (K(m)) are here investigated by continuum electrostatics calculations, MD simulations and free energy calculations. Continuum electrostatic calculations reveal that cUNG has surface potentials that are more complementary to the DNA potential at and around the catalytic site when compared to hUNG, indicating improved substrate binding. Comparative MD simulations combined with free energy calculations using the molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) method show that large opposing energies are involved when forming the enzyme-substrate complexes. Furthermore, the binding free energies obtained reveal that the Michaelis-Menten complex is more stable for cUNG, primarily due to enhanced electrostatic properties, suggesting that energetic fine-tuning of electrostatics can be utilized for enzymatic temperature adaptation. Energy decomposition pinpoints the residual determinants responsible for this adaptation. PMID:18196298

  13. Curating DNA specimens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA data are used in a variety of ethnobiological disciplines including archaeology, conservation, ecology, medicinal plants and natural products research, taxonomy and systematics, crop evolution and domestication, and genetic diversity. It frequently is convenient to store and share DNA among coop...

  14. Routine DNA testing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Routine DNA testing. It’s done once you’ve Marker-Assisted Breeding Pipelined promising Qantitative Trait Loci within your own breeding program and thereby established the performance-predictive power of each DNA test for your germplasm under your conditions. By then you are ready to screen your par...

  15. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  16. MICROWAVE RESONANCES IN DNA

    EPA Science Inventory

    This report describes spectroscopic studies of DNA which were undertaken to better understand a physical basis for microwave absorption by this molecule. hree types of studies are described. ) The low frequency scattered light spectrum of DNA was studied by two methods. irst, Ram...

  17. Replicating repetitive DNA.

    PubMed

    Tognetti, Silvia; Speck, Christian

    2016-05-27

    The function and regulation of repetitive DNA, the 'dark matter' of the genome, is still only rudimentarily understood. Now a study investigating DNA replication of repetitive centromeric chromosome segments has started to expose a fascinating replication program that involves suppression of ATR signalling, in particular during replication stress. PMID:27230530

  18. Hydrogels: DNA bulks up

    NASA Astrophysics Data System (ADS)

    Labean, Thom

    2006-10-01

    Since the 1940s DNA has been known as the genetic material connected to heredity, and from the early 1980s it has also been considered as a potential structural material for nanoscale construction. Now, a hydrogel made entirely of DNA brings this molecule into the realm of bulk materials.

  19. Translesion DNA synthesis

    PubMed Central

    Vaisman, Alexandra; McDonald, John P.; Woodgate, Roger

    2014-01-01

    All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell’s replicase. Under these situations, cells are forced to choose between recombination-mediated “damage avoidance” pathways, or use a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions, but also downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases. PMID:26442823

  20. Characterization of muntjac DNA

    SciTech Connect

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.