Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The synthesis of several nucleic acid block polymers of the general type dGn.rCidCk is described. The key steps in this procedure were the joining of dCk oligomers, protected at the 3'-OH with an acetyl group, to rCi oligomers by T4 DNA ligase and the purification of the products by RPC-5 column chromatography. The block polymers were characterized by 20% polyacrylamide gel electrophoresis, UV and CD spectra, analytical Cs2SO4 buoyant density analyses, helix-coil transitions and S1 nuclease studies. NMR studies on one member of this series, dGn.rC11dC16, were reported recently (Selsing, E., Wells, R.D., Early, T.A., and Kearns, D.R. (1978) Nature 275, 249-250). The NMR studies and the results described herein indicate that these block polymers are linear duplexes with two adjoining conformations yet are hydrogen-bonded and base-stacked throughout with minimal disruption of the helix at the junction of the two conformations. Computer model building studies described in the following paper (Selsing, E., Wells, R.D., Alden, C.J., and Arnott, S. (1979) J. Biol. Chem. 254, 5417-5422) predict that these nucleic acids contain a bend at the junction region.
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PMID:Polynucleotide block polymers consisting of a DNA.RNA hybrid joined to a DNA.DNA duplex. Synthesis and characterization of dGn.rCidCk duplexes. 44 59

A simple and efficient method for synthesizing long, site-specifically modified RNA molecules was developed whereby segments of RNA were joined with the use of bacteriophage T4 DNA ligase. A single hydrogen or O-methyl group was substituted for the 2'-hydroxyl group at either splice site of a nuclear pre-messenger RNA substrate. Splicing of the modified pre-messenger RNA's in vitro revealed that, although a 2'-hydroxyl is not absolutely required at either splice site, the 2'-hydroxyl at the 3' splice site is important for the second step of splicing. These results are compared to previous studies of analogous 2'-hydroxyl groups in the self-splicing Tetrahymena group I intron.
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PMID:Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. 158 82

Ferric nitrilotriacetate (Fe(3+)-NTA) catalyzes hydrogen peroxide-derived production of hydroxyl radicals, which are known to cause DNA damage. In the present work, Fe(3+)-NTA plus hydrogen peroxide-induced single-strand DNA breaks and repair of the DNA damage were studied in vitro by monitoring DNA damage- and DNA repair-dependent conformational changes of pUC18 plasmid DNA. Single-strand DNA breaks were induced in the pUC18 DNA by Fe(3+)-NTA plus hydrogen peroxide in a dose-dependent fashion. Induction of the DNA damage was inhibited by deferoxamine mesylate (an iron chelator) and by hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO), D-mannitol and ethanol indicating that the DNA damage was caused by hydroxyl radicals which were generated by reaction of Fe(3+)-NTA with hydrogen peroxide. The oxygen radical-induced single-strand DNA breaks were repaired partly (more than 50%) by incubating the damaged DNA at 37 degrees C for 3 h with a partially purified preparation of APEX nuclease (a multifunctional DNA repair enzyme), DNA polymerase beta, four deoxyribonucleoside triphosphates, T4 DNA ligase and ATP. Analyses of the partially purified preparation of APEX nuclease revealed that a 45-kDa protein as well as APEX nuclease in the preparation were involved in the repair of the single-strand DNA breaks. APEX nuclease was suggested to initiate the repair by removing 3' termini blocked by the nucleotide fragments and also by incising the 5' side of AP sites. The 45-kDa protein was suggested to be required for removal of the 5' tags such as 5'-terminal deoxyribose phosphate residues produced by the action of APEX nuclease on AP sites.
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PMID:Oxygen radical-induced single-strand DNA breaks and repair of the damage in a cell-free system. 756 64

Uracil-DNA glycosylase inhibitor (Ugi) is a B. subtilis bacteriophage protein that protects the uracil-containing phage DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase (UDG). The 1.9 A crystal structure of Ugi complexed to human UDG reveals that the Ugi structure, consisting of a twisted five-stranded antiparallel beta sheet and two alpha helices, binds by inserting a beta strand into the conserved DNA-binding groove of the enzyme without contacting the uracil specificity pocket. The resulting interface, which buries over 1200 A2 on Ugi and involves the entire beta sheet and an alpha helix, is polar and contains 22 water molecules. Ugi binds the sequence-conserved DNA-binding groove of UDG via shape and electrostatic complementarity, specific charged hydrogen bonds, and hydrophobic packing enveloping Leu-272 from a protruding UDG loop. The apparent mimicry by Ugi of DNA interactions with UDG provides both a structural mechanism for UDG binding to DNA, including the enzyme-assisted expulsion of uracil from the DNA helix, and a crystallographic basis for the design of inhibitors with scientific and therapeutic applications.
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PMID:Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA. 767

Crystal structures of the DNA repair enzyme human uracil-DNA glycosylase (UDG), combined with mutational analysis, reveal the structural basis for the specificity of the enzyme. Within the classic alpha/beta fold of UDG, sequence-conserved residues form a positively charged, active-site groove the width of duplex DNA, at the C-terminal edge of the central four-stranded parallel beta sheet. In the UDG-6-aminouracil complex, uracil binds at the base of the groove within a rigid preformed pocket that confers selectivity for uracil over other bases by shape complementary and by main chain and Asn-204 side chain hydrogen bonds. Main chain nitrogen atoms are positioned to stabilize the oxyanion intermediate generated by His-268 acting via nucleophilic attack or general base mechanisms. Specific binding of uracil flipped out from a DNA duplex provides a structural mechanism for damaged base recognition.
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PMID:Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. 769 13

Exposure of human nasal ciliated epithelium to reactive oxidants generated by the enzymatic xanthine-xanthine oxidase superoxide/hydrogen peroxide (H2O2) and glucose-glucose oxidase H2O2-generating systems, or to reagent H2O2 or hypochlorous acid (HOCl) resulted in significant alterations in ciliary beating. The earliest change noted was the presence of ciliary slowing, progressing eventually to complete ciliary stasis in some areas. Ciliary dyskinesia was seen within the first hour, often from as early as 15 min after exposure of the cells to reactive oxidants. Using peroxidases, various antioxidant enzymes, and oxidant scavengers, we confirmed that these detrimental effects on ciliary function were mediated primarily by H2O2 and HOCl. Moreover, 3-aminobenzamide (3-ABA), an inhibitor of the DNA repair enzyme poly ADP ribose polymerase, prevented H2O2-mediated inhibition of ciliary function, indicating that oxidant-mediated damage to DNA may well be the basis of the effects of H2O2 on ciliated epithelium. Acute and chronic inflammatory responses may therefore present the possible threat of H2O2- or HOCl-inflicted injury on bystander respiratory epithelium, leading to ciliary dyskinesia and slowing.
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PMID:Oxidant-mediated ciliary dysfunction in human respiratory epithelium. 795 61

High level expression of RNA complementary to either neo(r) or src sequences located near the 3' end of recombinant retroviral vectors derived from Rous sarcoma virus inhibited viral replication. Stable integration of proviral DNA was not detected in the presence of antisense RNA. We investigated the mechanism of this inhibition by determining the structure of unintegrated viral DNA (vDNA) intermediates accumulating in the presence of the anti-sense RNA. The major vDNA intermediate detected was a full-length duplex linear molecule with complementary single-stranded long terminal repeats (LTRs). These vDNA linears could be joined directly by T4 DNA ligase to form junctions which contained a single normal LTR. These results can be explained by arrest of linear vDNA formation before strand displacement results in completion of the LTRs. Isolation of these sticky-ended intermediates as linear rather than nicked circular molecules suggests that these complementary vDNA LTR segments were not hydrogen bonded in the infected cell and further implies that completion of LTR synthesis is an ordered, controlled process.
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PMID:Completion of avian retroviral DNA replication intermediates inhibited by antisense RNA. 817 25

The three guanosines of the central core of a hammerhead ribozyme were replaced by 2-aminopurine ribonucleoside, xanthosine, isoguanosine, inosine, and deoxyguanosine. These analogues were incorporated by automated solid-phase synthesis, with the exception of isoguanosine. This was introduced by ligating a donor, which carried the isoguanosine at its 5'-end, and an acceptor oligoribonucleotide by a T4 DNA ligase-catalyzed reaction. Most of these modifications lowered the rate constant of cleavage by the hammerhead ribozyme drastically. Inspection of the possible hydrogen-bonding interactions disturbed by these modifications suggests that there is no G12A9 or A13G8 mismatched base pair in the central region. Increasing the Mg2+ concentration from 10 to 50 mM did not enhance these rates appreciably. This makes it improbable that the guanosines, including their 2'-hydroxyl groups, are involved in the binding of the catalytically active Mg2+. Transition-state destabilizing energies of 0.6-4.7 kcal mol-1 suggest that essentially all guanosines are involved in a hydrogen-bonding network.
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PMID:Importance of exocyclic base functional groups of central core guanosines for hammerhead ribozyme activity. 821 33

The three key challenges addressed in our development of SPECITOPE, a tool for screening large structural databases for potential ligands to a protein, are to eliminate infeasible candidates early in the search, incorporate ligand and protein side-chain flexibility upon docking, and provide an appropriate rank for potential new ligands. The protein ligand-binding site is modeled by a shell of surface atoms and by hydrogen-bonding template points for the ligand to match, conferring specificity to the interaction. SPECITOPE combinatorially matches all hydrogen-bond donors and acceptors of the screened molecules to the template points. By eliminating molecules that cannot match distance or hydrogen-bond constraints, the transformation of potential docking candidates into the ligand-binding site and the shape and hydrophobic complementarity evaluations are only required for a small subset of the database. SPECITOPE screens 140,000 peptide fragments in about an hour and has identified and docked known inhibitors and potential new ligands to the free structures of four distinct targets: a serine protease, a DNA repair enzyme, an aspartic proteinase, and a glycosyltransferase. For all four, protein side-chain rotations were critical for successful docking, emphasizing the importance of inducible complementarity for accurately modeling ligand interactions. SPECITOPE has a range of potential applications for understanding and engineering protein recognition, from inhibitor and linker design to protein docking and macromolecular assembly.
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PMID:Screening a peptidyl database for potential ligands to proteins with side-chain flexibility. 974 46

Postischemic endothelial dysfunction may occur as a result of the effects of endogenous oxidants like hydrogen peroxide. Since endothelium-dependent vasodilator function may be affected by pHi, the effect of hydrogen peroxide on endothelial pHi was examined. Hydrogen peroxide (100 micromol/L for 10 minutes) decreased pHi from 7.24+/-0.01 to 7.02+/-0.02 and inhibited recovery from an ammonium chloride-induced intracellular acid load in carboxy SNARF 1 (c-SNARF 1)-loaded human aortic endothelial cells in bicarbonate-free solution. Prior inhibition of Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride (10 micromol/L), by removal of extracellular Na+, or by glycolytic inhibition with iodoacetic acid blocked the subsequent effect of hydrogen peroxide on pHi. A 2-minute exposure to 100 micromol/L H2O2 decreased intracellular ATP levels by approximately 40%; this was prevented by 3-aminobenzamide and nicotinamide (1 mmol/L each), inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase. Both 3-aminobenzamide and nicotinamide significantly inhibited the hydrogen peroxide-induced intracellular acidification and the effect of hydrogen peroxide on recovery from an intracellular acid load. Hydrogen peroxide decreases pHi in human endothelial cells by inhibiting Na+/H+ exchange. This appears to be mediated by activation of the DNA repair enzyme poly(ADP-ribose) polymerase and subsequent depletion of intracellular ATP. Since a decrease in pHi in this range may alter the activity of NO synthase or affect the synthesis of vasodilator prostaglandins, the effect of hydrogen peroxide on the endothelial Na+/H+ exchanger may be important in the pathogenesis of postischemic endothelial dysfunction.
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PMID:Hydrogen peroxide decreases pHi in human aortic endothelial cells by inhibiting Na+/H+ exchange. 974 60


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