Hemimethylated duplex DNAs prepared from 5-azacytidine-treated cells.
Duplex heavy-light (HL) DNAs synthesized in the presence of brdUrd and methylation inhibitors were separated from bulk cellular DNA by CsCl density gradient centrifugation and analysed for 5-methylcytosine (5mC) contents by HPLC. DNAs synthesized in the presence of 5 mM ethionine or 2 mg/ml cycloleucine were not detectably hypomethylated, was undermethylated with respect to control DNA. The heavy, or H-strand, in which up to 5% of the cytosine residues were replaced by intact 5-azacytosine, was undermethylated and the HL duplex DNA was therefore strand asymmetrically methylated. This duplex DNA served as an efficient substrate for a crude DNA methyltransferase preparation which transferred the methyl group from S-adenosylmethionine specifically into cytosine residues within the hypomethylated H strand. Increasing levels of incorporated 5-azacytosine inhibited the action of the methyltransferase suggesting that incorporation of 5-azacytosine into DNA may be responsible for the inhibitory effect of 5-azacytidine on DNA methylation.
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D001972:Bromodeoxycytidine / Q000494:pharmacology; D002460:Cell Line; D003515:Cycloleucine / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis* / Q000302:isolation & purification; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D004622:Embryo, Mammalian; D007700:Kinetics; D051379:Mice; D010640:Phenothiazines / Q000494:pharmacology
D010640:Phenothiazines; D003515:Cycloleucine; D001972:Bromodeoxycytidine; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine; C009469:thionine