Purification and characterization of mammalian DNA methyltransferases by use of monoclonal antibodies.
Previously, we have derived murine hybridomas producing monoclonal antibodies against DNA methyltransferase from human placenta (Kaul, S., Pfeifer, G. P., and Drahovsky, D. (1984) Eur. J. Cell Biol. 34, 330-335). One of these monoclonal antibodies, M2B10, which undergoes immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells, was used for the immunoaffinity purification of mouse and human DNA methyltransferases. In sodium dodecyl sulfate-polyacrylamide gels and in immunoblotting studies, the immunoaffinity-purified mouse DNA methyltransferase revealed 5-6 polypeptides of molecular masses 150-190 kDa. The immunoaffinity-purified human placental DNA methyltransferase was characterized by a polypeptide of 158 kDa, presumably representing the native enzyme molecule and by polypeptides of 105-108 kDa and 50-68 kDa, probably generated by a limited proteolysis of the native enzyme molecule. The immunoaffinity-purified DNA methyltransferases preferred hemimethylated DNA substrates over unmethylated ones, and among all unmethylated substrates tested, poly[(dG-dC).(dG-dC)] had the highest methyl-accepting activity. DNA polymers of at least 90 base pairs in length were required for the binding reaction of the immunoaffinity-purified human DNA methyltransferase, and this initial binding was apparently independent of the nucleotide composition of the DNA polymer and of the presence of S-adenosyl-L-methionine.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003429:Cross Reactions; D004247:DNA / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D012515:Mast-Cell Sarcoma / Q000201:enzymology; D008745:Methylation; D051379:Mice; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases