Specificity of O6-alkylguanine-DNA alkyltransferase

Extensive investigations of the specificity of O6-alkylguanine-DNA alkyltransferase (AAT) have been carried out. These studies have shown that: (i) the mammalian protein differs from that of Escherichia coli in lacking the ability to remove methyl groups from O4-methylthymine; (ii) the protein can remove longer alkyl groups from the O6 position but the rate of repair declines as the chain length increases; (iii) O6-methylguanine in RNA is much less active as a substrate for the protein than O6-methylguanine in double-stranded DNA; (iv) the free-base O6-alkylguanine is a very weak substrate for the protein so that reaction with it leads to the loss of alkyltransferase activity. (This property can be used to deplete AAT in cultured cells and in tissues and tumours after administration of O6-methylguanine); and (v) oligodeoxynucleotides containing O6-methylguanine are substrates for AAT. Such oligodeoxynucleotides can be labelled with 32P at very high specific activity and can be used in an ultrasensitive assay for AAT activity.