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Proceedings of the National Academy of... Jun 1976Ribosomal 30S protein S1 causes disruption of the secondary structure of certain pyrimidine-containing polynucleotides. Helical poly(U), poly(C, U), and neutral and...
Ribosomal 30S protein S1 causes disruption of the secondary structure of certain pyrimidine-containing polynucleotides. Helical poly(U), poly(C, U), and neutral and acidic poly(C) are stoichiometrically converted by S1 to structures indistinguishable from their partially or completely thermally denatured forms, as revealed by circular dichroism. Of the several double- and triple-stranded helical polynucleotides tested that contain one polypurine strand and at least one polypyrimidine strand, only the conformation of the DNA.RNA hybrid, poly(A)-poly(dT), is perturbed. In the presence of S1, this hybrid undergoes a transition to a new structure that has a circular dichroism spectrum unlike either the native or thermally denatured forms. Intercalated ethidium bromide is released from poly(A)-poly(dT) by S1, confirming the occurrence of a conformational rearrangement. The translation inhibitor, autintricarboxylic acid, completely inhibits the action of S1 on polypyrimidines, but has no effect on the conformational perturbation of poly(A(-poly(dT). The possible relation between these observations and the biological function of protein S1 is discussed.
Topics: Circular Dichroism; Escherichia coli; Ethidium; Nucleic Acid Conformation; Poly C; Poly T; Poly U; Polynucleotides; Ribosomal Proteins
PubMed: 778845
DOI: 10.1073/pnas.73.6.1824 -
Biochimie Aug 2012Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of...
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of the phosphodiester linkage between the DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' damaged termini. Recently we have shown that Tdp1 can liberate the 3' DNA phosphate termini from apurinic/apyrimidinic (AP) sites. Here, we found that Tdp1 is more active in the cleavage of the AP sites inside bubble-DNA structure in comparison to ssDNA containing AP site. Furthermore, Tdp1 hydrolyzes AP sites opposite to bulky fluorescein adduct faster than AP sites located in dsDNA. Whilst the Tdp1 H493R (SCAN1) and H263A mutants retain the ability to bind an AP site-containing DNA, both mutants do not reveal endonuclease activity, further suggesting the specificity of the AP cleavage activity. We suggest that this Tdp1 activity can contribute to the repair of AP sites particularly in DNA structures containing ssDNA region or AP sites in the context of clustered DNA lesions.
Topics: Apurinic Acid; Binding Sites; Catalysis; DNA; DNA Repair; DNA-Binding Proteins; Humans; Hydrolysis; Nucleic Acid Conformation; Phosphoric Diester Hydrolases; Polynucleotides; Substrate Specificity
PubMed: 22522093
DOI: 10.1016/j.biochi.2012.04.004 -
Nucleic Acids Research Jun 1983The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer... (Comparative Study)
Comparative Study
The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.
Topics: DNA Polymerase I; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Escherichia coli; Kinetics; Poly A; Poly A-U; Poly dA-dT; Polydeoxyribonucleotides; Structure-Activity Relationship; Templates, Genetic
PubMed: 6344014
DOI: 10.1093/nar/11.11.3753 -
Nucleic Acids Research Jul 1984High performance liquid chromatography analysis of imidazole open ring 7-methylguanine, 2-6 diamino-4-hydroxy-5N-methyl-formamidopyrimidine (rom7G), showed two...
High performance liquid chromatography analysis of imidazole open ring 7-methylguanine, 2-6 diamino-4-hydroxy-5N-methyl-formamidopyrimidine (rom7G), showed two well-separated peaks (fI and fII) of the same magnitude. Rechromatography of each isolated component indicated that they are slowly interconverted to give a 1:1 mixture. NMR analysis demonstrated that the two species observed on reversed phase HPLC are rotational isomers. Thermodynamic measurements strongly suggested that the equilibrium can be assigned to rotation around the N-methyl formamido bond. The two species, fI and fII, separated by HPLC were identified as rotamers E and Z, respectively. The structures of fI and fII were also determined. A polynucleotide containing rom7G was obtained by alkaline treatment of poly (dGC) containing 7-methylguanine. In order to study its structure within the polynucleotide, rom7G was enzymatically excized by E.coli rom7G-DNA glycosylase. The analysis of the products released by the enzyme showed a 1:4 mixture of the two rotamers favoring the Z form (fII).
Topics: Alkylation; Chromatography, High Pressure Liquid; Guanine; Magnetic Resonance Spectroscopy; Nucleic Acid Conformation; Polydeoxyribonucleotides; Stereoisomerism
PubMed: 6462910
DOI: 10.1093/nar/12.13.5429 -
The Biochemical Journal Aug 1980A technique is described in which the incorporation of a polynucleotide substrate into the matrix of a polyacrylamide gel allows the use of electrophoresis for the...
A technique is described in which the incorporation of a polynucleotide substrate into the matrix of a polyacrylamide gel allows the use of electrophoresis for the detection of polycationic ribonuclease activity rather than simply the presence of protein. Because use is made of the catalytic properties of ribonucleases, polynucleotide/polyacrylamide-gel electrophoresis is apparoximately 10(5) times more sensitive for the detection of these enzymes than conventional gel electrophoresis with the use of protein-staining dyes. Initial studies showed that the poor migration, in the gels, of highly charged polycationic ribonucleases in the presence of negatively charged synthetic polynucleotides could be overcome by high concentrations of spermine. The positively charged polyamine, by neutralizing the polyanionic polynucleotide, enabled these basic enzymes to migrate considerable distances in the gel. Electrophoresis of the RNAases under conditions of low pH, and incubation of the gel at neutral pH followed by staining for polynucleotide, resulted in coloured gels containing clear bands that define regions of enzyme activity. Alterations in spermine concentration or substrate identity caused changes in the positions of these bands, suggesting a dynamic interaction among the enzyme, polyamine and polynucleotide. Because of the advantages, in terms of selectivity and sensitivity of polynucleotide/polyacrylamide-gel electrophoresis, this technique was used to demonstrate the nuclease homogenity of three purified bovine muscle enzymes, and to compare these enzymes with each other, as well as with bovine pancreatic ribonuclease A.
Topics: Animals; Cattle; Electrophoresis, Polyacrylamide Gel; Models, Chemical; Muscles; Pancreas; Polynucleotides; Ribonucleases; Spermine; Staining and Labeling
PubMed: 6161609
DOI: 10.1042/bj1890277 -
Nucleic Acids Research Jun 19782'-Deoxy-2'-fluoroadenosine was chemically transformed to its 5'-diphosphate and polymerized with polynucleotide phosphorylase to give poly(2'-deoxy-2'-fluoroadenylic...
2'-Deoxy-2'-fluoroadenosine was chemically transformed to its 5'-diphosphate and polymerized with polynucleotide phosphorylase to give poly(2'-deoxy-2'-fluoroadenylic acid) [poly(Af)]. Polymerization proceeded smoothly as in the case of poly(A) and the yield of the polymerization was 55%. The UV absorption spectra of poly(Af) closely resembled those of poly(A) and the hypochromicity was 32% at pH 7.0. The CD profile at 25 degrees and neutrality showed similar pattern to that of other poly(2'-deoxy-2'-halogenoadenylic acids) with somewhat larger [theta] values both in the positive and negative maxima. Acid titration of poly(Af) showed a transition point at pH 5.2 and the Tm of the acid form was 37 degrees which was significantly lower than that of poly(A), but similar to that of poly(2'-azido-2'-deoxyadenylic acid). Poly(Af) formed 1:1 and 1:2 complexes with poly-(U) having Tm of 49 degrees and 62 degrees at 0.04M and 0.15M Na(+) concentration, respectively. Poly(Af) also formed a 1:2 complex with poly(I) and its Tm was 36 degrees at 0.05M Na(+) concentration. These data showed that poly(Af) has rather similar properties to those of poly(A), but not to poly(dA).
Topics: Circular Dichroism; Nucleic Acid Conformation; Poly A; Polydeoxyribonucleotides; Polyribonucleotide Nucleotidyltransferase; Spectrophotometry, Ultraviolet; Structure-Activity Relationship
PubMed: 673838
DOI: 10.1093/nar/5.6.1877 -
Proceedings of the National Academy of... Nov 1972Linear double-stranded molecules of the circularly permuted and terminally redundant DNA of Salmonella bacteriophage P22 have been converted to oligomeric products in...
Linear double-stranded molecules of the circularly permuted and terminally redundant DNA of Salmonella bacteriophage P22 have been converted to oligomeric products in the presence of polynucleotide ligase coded for by the coliphage T4. The reaction has been monitored by sucrose density-gradient centrifugation and electron microscopy. It goes slowly and gives yields of 30-40%. The products are mainly dimers and trimers, but higher oligomers are also present.DNA ligase extracted from uninfected Escherichia coli seems unable to perform a similar reaction, which is concluded to involve the fully base-paired termini. Linear double-stranded molecules of simian virus(SV) 40 DNA, produced by the action of the bacterial restriction endonuclease R(1), are oligomerized by either ligase; therefore, this reaction seems to involve single-stranded cohesive ends. No mixed products could be found when P22 DNA and linear SV 40 DNA were exposed together to the T4 ligase.
Topics: Carbon Isotopes; Centrifugation, Density Gradient; Coliphages; DNA, Circular; DNA, Viral; Escherichia coli; Kinetics; Lysogeny; Microscopy, Electron; Oligonucleotides; Polynucleotide Ligases; Polynucleotides; Salmonella Phages; Simian virus 40; Tritium
PubMed: 4343970
DOI: 10.1073/pnas.69.11.3389 -
The Annals of Thoracic Surgery Aug 1994Heparin is normally used for anticoagulation during cardiopulmonary bypass (CPB), but its use is contraindicated in patients with a history of heparin-induced...
Heparin is normally used for anticoagulation during cardiopulmonary bypass (CPB), but its use is contraindicated in patients with a history of heparin-induced thrombocytopenia, heparin-provoked thrombosis, or both. Heparin therapy can also be ineffective due to heparin resistance. A short-acting, oligonucleotide-based thrombin inhibitor (thrombin aptamer) may potentially serve as a substitute for heparin in these and other clinical situations. We tested a novel thrombin aptamer in a canine CPB pilot study to determine its anticoagulant efficacy, the resultant changes in coagulation variables, and the aptamer's clearance mechanisms and pharmacokinetics. Seven dogs were studied initially: Four received varied doses of the aptamer (to establish the pharmacokinetic profile) and 3 received heparin. Subsequently, 4 other dogs underwent CPB, receiving a constant infusion of the aptamer before CPB (to characterize the baseline coagulation status), with partial CPB and hemodilution, during 60 minutes of total CPB, and, finally, after a 2-hour recovery period. At a 0.5 mg.kg-1.min-1 dose, the activated clotting time rose with aptamer infusion from 106 +/- 12 seconds to 187 +/- 8 seconds (+/- 1 standard deviation) (p = 0.014), increased further with hemodilution (to 259 +/- 41 seconds; p = 0.017), and was even more prolonged during total CPB (> 1,500 seconds; p < 0.001). This later increase in the activated clotting time paralleled a rise in the plasma concentration of the thrombin aptamer during total CPB, as determined by high-performance liquid chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Aptamers, Nucleotide; Blood Coagulation; Cardiopulmonary Bypass; Chromatography, High Pressure Liquid; Dogs; Dose-Response Relationship, Drug; Heparin; Infusions, Intravenous; Oligonucleotides; Pilot Projects; Polynucleotides; Prothrombin Time; Thrombin
PubMed: 8067830
DOI: 10.1016/0003-4975(94)92206-3 -
Proceedings of the National Academy of... Feb 2006The nucleotide 5'-dGMP and polynucleotide poly(dGdC).poly(dGdC) have been irradiated by using a 200-fs, 200-nm laser pulses and spectrally characterized by using...
The nucleotide 5'-dGMP and polynucleotide poly(dGdC).poly(dGdC) have been irradiated by using a 200-fs, 200-nm laser pulses and spectrally characterized by using time-resolved infrared spectroscopy. Under the experimental conditions, 200-nm excitation generates both electronic excited states and radical cations through photoionization; the former decay rapidly to vibrationally hot ground state. By using infrared signatures we have been able to follow these processes, and at time scales of >1 ns we observe an infrared marker band at 1,702 cm(-1) within both 5'-dGMP and the polynucleotide assigned to a photoionized product of guanine. This transient has also been reproduced through indirect chemistry through the reaction with photogenerated carbonate radical with 5'-dGMP. The ability to use time-resolved infrared spectroscopy in this way paves the way for developing solution-phase studies to investigate both direct and indirect radiation chemistry of DNA.
Topics: DNA; DNA Damage; Lasers; Polydeoxyribonucleotides; Spectroscopy, Fourier Transform Infrared
PubMed: 16467159
DOI: 10.1073/pnas.0506860103 -
European Journal of Biochemistry Jun 1970
Kinetics of polymerization and phosphorolysis reactions of Escherichia coli polynucleotide phosphorylase. Evidence for multiple binding of polynucleotide in phosphorolysis.
Topics: Adenine Nucleotides; Binding Sites; Chemical Phenomena; Chemistry; Escherichia coli; Kinetics; Polynucleotides; Protein Binding; RNA Nucleotidyltransferases
PubMed: 4918555
DOI: 10.1111/j.1432-1033.1970.tb00281.x