Crosslinking of Dam methyltransferase with S-adenosyl-methionine

• Published in: FEBS letters 1991-03, Vol.280 (1), p.147-151
• Main Authors: Wenzel, Caroline, Moulard, Maxime, Lobner-Olesen, Anders, Guschlbauer, Wilhelm
• Format: Article
• Language: English
• Subjects: 3H3C-S-adenosyl-methionine; [14C]AdoMet; [3H]AdoMet; AdoHcy; AdoMet; Analytical, structural and metabolic biochemistry; Binding, Competitive; Biological and medical sciences; Cross-Linking Reagents - metabolism; Dam methylase; dithiothreitol; DNA-adenine-methyltransferase; DTT; Endopeptidases - pharmacology; Enzyme Activation; Enzyme Stability - drug effects; Enzyme Stability - radiation effects; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hot Temperature; Methyltransferases - metabolism; Photocrosslinking; Proteolysis; S-adenosyl-homocysteine; S-Adenosyl-methionine; S-adenosyl[3,414C]methionine; S-Adenosylmethionine - metabolism; S-isobutyl-adenosine; SIBA; Sinefungin; Site-Specific DNA-Methyltransferase (Adenine-Specific); Substrate Specificity - drug effects; Substrate Specificity - radiation effects; Transferases; Ultraviolet Rays
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(91)80224-Q

Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 μM S-adenosyl-methionine; it was inhibited in the presence of substates which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins which do not bind S-adenosyl-methionine, as well as heat activated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.