PMID:2201947
| Citation |
Bergerat, A and Guschlbauer, W (1990) The double role of methyl donor and allosteric effector of S-adenosyl-methionine for Dam methylase of E. coli. Nucleic Acids Res. 18:4369-75 |
|---|---|
| Abstract |
The turnover of DNA-adenine-methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC. |
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| Keywords |
Adenosine/analogs & derivatives; Adenosine/pharmacology; Allosteric Regulation; Base Sequence; Binding Sites; Binding, Competitive; DNA, Bacterial/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins; Kinetics; Methylation; Methyltransferases/metabolism; Models, Molecular; Molecular Sequence Data; Protein Conformation; S-Adenosylhomocysteine/pharmacology; S-Adenosylmethionine/metabolism; S-Adenosylmethionine/pharmacology; Site-Specific DNA-Methyltransferase (Adenine-Specific); Sodium Chloride/pharmacology; Temperature; Thermodynamics |
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