Quantitation of S-methylcysteine formed in O6-methylguanine-DNA:methyltransferase

The O6-methylguanine-DNA:methyltransferase is known to transfer the methyl group from O6-methylguanine (O6-mG) in DNA to the cysteinyl residue of the methyl acceptor protein which is the methyltransferase itself. We developed a novel method to estimate the amount of S-methylcysteine formed in the acceptor protein, utilizing O6-[methyl-3H]methylguanine-containing DNA as the methyl donor. Following hydrolysis of the methyl-incorporated macromolecules in formic acid--HCl, S-[methyl-3H]methylcysteine is chromatographically isolated on a small column of Dowex-50(H+). Utilizing this method, we measured O6-mG DNA:methyltransferase activity in rat liver during neonatal development; the activity increases from 0.013 pmol methyl transferred/mg protein at 2 days postnatally to 0.06 pmol/mg at 8 weeks, the latter being equal to the adult liver activity.