Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186.
DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.
Som
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S
Department of Pharmacology, State University of New York Health Science Center, Brooklyn 11203.
Friedman
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S
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003545:Cysteine / Q000378:metabolism / Q000528:radiation effects*; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008745:Methylation; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010777:Photochemistry; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives* / Q000737:chemistry; D014357:Trypsin
C016207:S-adenosyl-2-methylmethionine; D012436:S-Adenosylmethionine; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; D002918:Chymotrypsin; D014357:Trypsin; D003545:Cysteine