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

Ahmad, Ishtiyaque and Rao, Desirazu N (1994) Photolabeling of the EcoP15 DNA methyltransferase with S-adenosyl-l-methionine. In: Gene, 142 (1). pp. 67-71.

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Abstract

Radioactivity from S-adenosyl-L-[methyl-H-3] methionine ([methyl-H-3]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-H-3]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.

Item Type:	Editorials/Short Communications
Publication:	Gene
Publisher:	Elsevier science
Additional Information:	Copyright of this artical belongs to Elsevier science.
Keywords:	DNA methylation;sinefungin;restriction-modification;UV crosslinking
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	08 Apr 2011 10:53
Last Modified:	08 Apr 2011 10:53
URI:	http://eprints.iisc.ac.in/id/eprint/36691

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