Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.1.1.113 (restriction-modification system)
 350 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Structural studies of the proteins of the BstVI restriction-modification system of Bacillus stearothermophilus V were carried out using intrinsic fluorescence techniques. The exposure and environments of their tryptophanyl residues were determined using collisional quenchers. Quenching of BstVI endonuclease by iodide suggested a heterogeneous class of tryptophan residues, while the results obtained with M.BstVI methylase were consistent with a rather exposed tryptophan population. A comparison of the quenching efficiencies at 20 degrees C and 55 or 60 degrees C showed that their structures are more flexible and open at the temperature at which they exhibit maximal activity. The endonuclease reached its active conformation only after 1 h of incubation at 60 degrees C. Fluorescence changes were observed upon Mn2+ and Mg2+ binding, with Kd values in the range 3-5 microM. The binding of S-adenosyl-L-methionine to the methylase produced conformational changes, which were consistent with binding to a single site of Kd 550 and 680 microM at 20 degrees C and 55 degrees C, respectively. Quenching experiments with iodide showed that the presence of S-adenosyl-L-methionine leads to different conformational states at 20 degrees C and 55 degrees C. These results were interpreted in terms of differences in the structural characteristics of these restriction-modification proteins as well as in terms of differences in the conformational states that these enzymes exhibit at 20 degrees C and at the temperature at which they are most active.
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PMID:Structural studies of the BstVI restriction-modification proteins by fluorescence spectroscopy. 1042 88

RSR:I [N:6-adenine] DNA methyltransferase (M.RSR:I), which recognizes GAATTC and is a member of a restriction-modification system in Rhodobacter sphaeroides, was purified to >95% homogeneity using a simplified procedure involving two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M.RSR:I were performed on unmethylated, hemimethylated, dimethylated or non-specific target DNA duplexes (25 bp) in the presence of sinefungin, a potent inhibitory analog of AdoMet. M. RSR:I binding was affected by the methylation status of the DNA substrate and was enhanced by the presence of the cofactor analog. M. RSR:I bound DNA substrates in the presence of sinefungin with decreasing affinities: hemimethylated > unmethylated > dimethylated >> non-specific DNA. Gel retardation studies with DNA substrates containing an abasic site substituted for the target adenine DNA provided evidence consistent with M.RSR:I extruding the target base from the duplex. Consistent with such base flipping, an approximately 1.7-fold fluorescence intensity increase was observed upon stoichiometric addition of M.RSR:I to hemimethylated DNA containing the fluorescent analog 2-aminopurine in place of the target adenine. Pre-steady-state kinetic and isotope- partitioning experiments revealed that the enzyme displays burst kinetics, confirmed the catalytic competence of the M.RSR:I-AdoMet complex and eliminated the possibility of an ordered mechanism where DNA is required to bind first. The equilibrium dissociation constants for AdoMet, AdoHcy and sinefungin were determined using an intrinsic tryptophan fluorescence-quenching assay.
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PMID:Substrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase. 1102 76

Fluorescence spectroscopy is a technique frequently employed to study protein-nucleic acid interactions. Often, the intrinsic fluorescence emission spectrum of tryptophan residues in a nucleic-acid-binding protein is strongly perturbed upon interaction with a target DNA or RNA. These spectral changes can then be exploited in order to construct binding isotherms and the extract equilibrium association constant together with the stoichiometry of an interaction. However, when a protein contains many tryptophan residues that are not located in the proximity of the nucleic-acid-binding site, changes in the fluorescence emission spectrum may not be apparent or the magnitude too small to be useful. Here, we make use of an extrinsic fluorescence probe, the environmentally sensitive fluorophore 1-anilinonaphthalene-8-sulphonic acid (1,8-ANS). Displacement by DNA of 1,8-ANS molecules from the nucleic-acid-binding site of the Type I modification methylase EcoR124I results in red shifting and an intensity decrease of the 1,8-ANS fluorescence emission spectrum. These spectral changes have been used to investigate the interaction of EcoR124I with DNA target recognition sequences.
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PMID:A competition assay for DNA binding using the fluorescent probe ANS. 1937 88