Photolabeling of the EcoP15 DNA methyltransferase with S-adenosyl-l-methionine

Abstract

Radioactivity from S-adenosyl-l-[methyl-³H]methionine ([methyl-³H]AdoMet) was bound to the EcoP15 DNA methyltransferase (M · EcoP15) following short-wave ultraviolet (UV) irradiation. The labeled protein was subjected to polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and detected by fluorography and autoradiography. Labeling was found to be dependent on the concentration of AdoMet and time of UV irradiation. The photolabeling by [methyl³H]AdoMet was specific and blocked by S-adenosyl-l-homocysteine (AdoHcy) and sinefungin which are known to function as competitive inhibitors. Limited digestion of the M · EcoP15-AdoMet adduct by Staphylococcus aureus protease V8 generated three peptides of approx. 50, 32 and 30 kDa. Interestingly, only the 30-kDa peptide fragment contained radioactivity, as detected by SDS-PAGE, followed by fluorography and autoradiog- raphy. Further, sequencing of a few amino acids at the N-terminus of these peptides showed that the 30-kDa fragment was the N-terminal portion of M · EcoP15. These results suggest that photolabeling is at the AdoMet-binding site and that the N-terminal half of M · EcoP15 may be involved in substrate binding.