DIFFERENTIAL INACTIVATION OF MAMMALIAN AND ESCHERICHIA-COLI O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASES BY O-6-BENZYLGUANINE

The action of O-6-benzylguanine (O-6-BzlG) on recombinant mammalian and Escherichia coli O-6-alkylguanine-DNA alkyltransferases (ATase; EC2.1.1.63; methylated-DNA-protein-cysteine methyltransferase) was compared by preincubation of these proteins with the base, followed by measurement of residual ATase activity using [H-3]methylated substrate DNA. All of the mammalian proteins examined were inactivated by O-6-BzlG (Chinese hamster: I-40, 0.04 mu M; human and rat: I-40, 0.06 mu M); however, the murine ATase was substantially more resistant requiring 4-5 fold higher concentrations of O-6-BzlG to achieve the same levels of inactivation (I-40, 0.28 mu M). A similar differential inactivation was seen with human and murine ATases when extracts of 3T6 (murine) cells and Raji (human) cells were compared. Of the two E. coli ATase proteins, only the ogt-encoded protein was inactivated, but approximately 400 times more O-6-BzlG was required to achieve a level of inactivation similar to that seen with the human protein (I-40, 24.8 mu M). When O-6-BzlG was present in an oligonucleotide, the differential effect on the murine, human and ogt-encoded ATases was not seen and only the ada-encoded ATase remained refractory under the conditions used.