The Experts below are selected from a list of 963 Experts worldwide ranked by ideXlab platform
Megan E. Núñez - One of the best experts on this subject based on the ideXlab platform.
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thymine dimer induced structural changes to the dna duplex examined with reactive probes
Biochemistry, 2008Co-Authors: Amy E Rumora, Katarzyna M Kolodziejczak, Anne Wagner, Megan E. NúñezAbstract:Despite significant progress in the past decade, questions still remain about the complete structural, dynamic, and thermodynamic effect of the cis-syn cyclobutane pyrimidine dimer lesion (hereafter called the thymine dimer) on double-stranded genomic DNA. We examined a 19-mer oligodeoxynucleotide duplex containing a thymine dimer lesion using several small, base-selective reactive chemical probes. These molecules probe whether the presence of the dimer causes the base pairs to be more accessible to the solution, either globally or adjacent to the dimer. Though all of the probes confirm that the overall structure of the dimer-containing duplex is conserved compared to that of the undamaged parent duplex, reactions with both Diethyl Pyrocarbonate and Rh(bpy)2(chrysi)3+ indicate that the duplex is locally destabilized near the lesion. Reactions with potassium permanganate and DEPC hint that the dimer-containing duplex may also be globally more accessible to the solution through a subtle shift in the double-...
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thymine dimer induced structural changes to the dna duplex examined with reactive probes
Biochemistry, 2008Co-Authors: Amy E Rumora, Katarzyna M Kolodziejczak, Anne Wagner, Megan E. NúñezAbstract:Despite significant progress in the past decade, questions still remain about the complete structural, dynamic, and thermodynamic effect of the cis-syn cyclobutane pyrimidine dimer lesion (hereafter called the thymine dimer) on double-stranded genomic DNA. We examined a 19-mer oligodeoxynucleotide duplex containing a thymine dimer lesion using several small, base-selective reactive chemical probes. These molecules probe whether the presence of the dimer causes the base pairs to be more accessible to the solution, either globally or adjacent to the dimer. Though all of the probes confirm that the overall structure of the dimer-containing duplex is conserved compared to that of the undamaged parent duplex, reactions with both Diethyl Pyrocarbonate and Rh(bpy)2(chrysi)3+ indicate that the duplex is locally destabilized near the lesion. Reactions with potassium permanganate and DEPC hint that the dimer-containing duplex may also be globally more accessible to the solution through a subtle shift in the double-...
Ursula Liebl - One of the best experts on this subject based on the ideXlab platform.
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Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX.
Journal of Biological Chemistry, 2006Co-Authors: Sébastien Graziani, Julie Bernauer, Stéphane Skouloubris, Marc Graille, Cong-zhao Zhou, Christophe Marchand, Paulette Decottignies, Herman Van Tilbeurgh, Hannu Myllykallio, Ursula LieblAbstract:By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by Diethyl Pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.
Michael J Waring - One of the best experts on this subject based on the ideXlab platform.
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high resolution footprinting studies of drug dna complexes using chemical and enzymatic probes
Methods in Enzymology, 2001Co-Authors: Michael J WaringAbstract:Publisher Summary This chapter discusses high-resolution footprinting techniques of drug–DNA complexes using chemical and enzymatic probes. Footprinting is a method that has been developed for determining the sequence-specific binding of small molecules, oligonucleotides, or proteins to DNA. The technique, which was originally designed for analyzing protein–DNA interactions, is based on the ability of ligands to protect DNA from enzymatic or chemical cleavage at their binding sites. Several enzymatic and chemical agents have been developed as footprinting probes, and each has its own characteristic advantages and disadvantages. Enzymes, such as DNase I, are often easy to use but typically overestimate the size of the footprint (on account of their size) and generate uneven cleavage patterns in the absence of added ligand. Chemical agents, such as dimethyl sulfate, osmium tetroxide, or Diethyl Pyrocarbonate (DEPC), are of limited usefulness because they react only with specific DNA bases. Other chemical probes such as methidiumpropyl–EDTA–Fe(II) [MPE–Fe(II)] act by intercalation and so perturb the DNA structure. Arguably the best cleavage agent for accurately mapping small molecule binding sites on DNA is the hydroxyl radical.
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footprinting titration studies on the binding of echinomycin to dna incapable of forming hoogsteen base pairs
Biochemistry, 1993Co-Authors: Eric W Sayers, Michael J WaringAbstract:: In order to investigate the possible importance of Hoogsteen base pairing to the DNA-binding ability of echinomycin, quantitative DNase I footprinting has been performed. The substrate was the tyrT DNA restriction fragment, either "native" or substituted with one of the purine analogs 2'-deoxy-7-deazaadenosine and 2'-deoxy-7-deazaguanosine in both strands. The modified DNA species were prepared by PCR and selectively labeled at the 5' terminus of one strand (usually the upper "Watson" strand) with [32P]ATP and polynucleotide kinase. Proper incorporation of the analog nucleotides was verified by Maxam-Gilbert G- and C-sequencing reactions as well as exposure to osmium tetroxide and Diethyl Pyrocarbonate. OsO4 was found to react strongly with the 7-deaza nucleotides, providing a good check of faithful incorporation. The previously observed echinomycin-induced hyperreactivity of purines toward Diethyl Pyrocarbonate was eliminated by incorporating the appropriate 7-deazapurine. The DNase I footprinting titration studies greatly refined the existing knowledge of the DNA-binding characteristics of echinomycin, as they revealed five general types of concentration-dependent behavior at single-bond resolution. Estimates of microscopic binding constants at individual DNA binding sites were obtained by measuring the antibiotic concentration which produced a half-maximal effect on the concentration of a given DNase I cleavage product. All binding sites contained one or more CpG steps, and all CpG steps analyzed formed part of a binding site for echinomycin. No consistent differences in the estimated binding constants for these sites were observed by comparing normal and modified DNAs, indicating that the abolition of formal Hoogsteen pairs did not significantly alter the thermodynamics of echinomycin-DNA interaction. The lack of any detectable decrease in binding constants for critical sites in the 7-deazapurine-substituted DNAs argues against any anti-syn conformational transition of purine nucleosides occurring in association with the bis-intercalative complex formation.
Amy E Rumora - One of the best experts on this subject based on the ideXlab platform.
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thymine dimer induced structural changes to the dna duplex examined with reactive probes
Biochemistry, 2008Co-Authors: Amy E Rumora, Katarzyna M Kolodziejczak, Anne Wagner, Megan E. NúñezAbstract:Despite significant progress in the past decade, questions still remain about the complete structural, dynamic, and thermodynamic effect of the cis-syn cyclobutane pyrimidine dimer lesion (hereafter called the thymine dimer) on double-stranded genomic DNA. We examined a 19-mer oligodeoxynucleotide duplex containing a thymine dimer lesion using several small, base-selective reactive chemical probes. These molecules probe whether the presence of the dimer causes the base pairs to be more accessible to the solution, either globally or adjacent to the dimer. Though all of the probes confirm that the overall structure of the dimer-containing duplex is conserved compared to that of the undamaged parent duplex, reactions with both Diethyl Pyrocarbonate and Rh(bpy)2(chrysi)3+ indicate that the duplex is locally destabilized near the lesion. Reactions with potassium permanganate and DEPC hint that the dimer-containing duplex may also be globally more accessible to the solution through a subtle shift in the double-...
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thymine dimer induced structural changes to the dna duplex examined with reactive probes
Biochemistry, 2008Co-Authors: Amy E Rumora, Katarzyna M Kolodziejczak, Anne Wagner, Megan E. NúñezAbstract:Despite significant progress in the past decade, questions still remain about the complete structural, dynamic, and thermodynamic effect of the cis-syn cyclobutane pyrimidine dimer lesion (hereafter called the thymine dimer) on double-stranded genomic DNA. We examined a 19-mer oligodeoxynucleotide duplex containing a thymine dimer lesion using several small, base-selective reactive chemical probes. These molecules probe whether the presence of the dimer causes the base pairs to be more accessible to the solution, either globally or adjacent to the dimer. Though all of the probes confirm that the overall structure of the dimer-containing duplex is conserved compared to that of the undamaged parent duplex, reactions with both Diethyl Pyrocarbonate and Rh(bpy)2(chrysi)3+ indicate that the duplex is locally destabilized near the lesion. Reactions with potassium permanganate and DEPC hint that the dimer-containing duplex may also be globally more accessible to the solution through a subtle shift in the double-...
Paulette Decottignies - One of the best experts on this subject based on the ideXlab platform.
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Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX.
Journal of Biological Chemistry, 2006Co-Authors: Sébastien Graziani, Julie Bernauer, Stéphane Skouloubris, Marc Graille, Cong-zhao Zhou, Christophe Marchand, Paulette Decottignies, Herman Van Tilbeurgh, Hannu Myllykallio, Ursula LieblAbstract:By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by Diethyl Pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.
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direct evidence for the different roles of the n and c terminal regulatory disulfides of sorghum leaf nadp malate dehydrogenase in its activation by reduced thioredoxin
FEBS Letters, 1996Co-Authors: Emmanuelle Issakidis, Paulette Decottignies, Martine Lemaire, Jeanpierre Jacquot, Myroslawa MiginiacmaslowAbstract:Abstract Plant NADP-dependent malate dehydrogenase is activated through thiol/disulfide interchange with reduced thioredoxin. Previous studies showed that this process involves the reduction of two different disulfides per subunit: one N-terminal, the other C-terminal. Substitution of regulatory cysteines at each end by site-directed mutagenesis and comparison of activation kinetics of the mutants led us to propose a model for the activation mechanism where the C-terminal end shielded the access to the catalytic residues, whereas the N-terminal end was involved in the slow conformational change of the active site. In the present study, we took advantage of the previous identification of the catalytic histidine residue which can be specifically derivatized by Diethyl Pyrocarbonate to test the accessibility of the active site. The results clearly show that in the mutants where the C-terminal bridge is open the active site histidine is freely accessible to the reagent, whereas in the mutants where the N-terminal bridge is open, the active site cannot be reached without activation, thus demonstrating the validity of the model.