Photolabeling of the EcoP15 DNA methyltransferase with S-adenosyl-L-methionine.
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 [methyl-3H]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 autoradiography. 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.
Ahmad
I
I
Department of Biochemistry, Indian Institute of Science, Bangalore.
Rao
D N
DN
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial; D015263:Deoxyribonucleases, Type III Site-Specific / Q000378:metabolism*; D008969:Molecular Sequence Data; D010777:Photochemistry; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology; D013379:Substrate Specificity
D000345:Affinity Labels; D004269:DNA, Bacterial; D012436:S-Adenosylmethionine; C023451:endodeoxyribonuclease EcoP15I; D015263:Deoxyribonucleases, Type III Site-Specific