A SINGLE AMINO-ACID CHANGE IN HUMAN O6-ALKYLGUANINE-DNA ALKYLTRANSFERASE DECREASING SENSITIVITY TO INACTIVATION BY O6-BENZYLGUANINE

Mammalian O6-alkylguanine-DNA alkyltransferases (AGTs) are readily ina ctivated by incubation with the pseudosubstrate, O6-benzylguanine, but the equivalent protein from the Escherichia coli ogt gene is much les s sensitive and the Saccharomyces cerevisiae and E. coli ada gene prod uct AGTs are completely resistant to this compound. We have expressed the normal human AGT and various point mutations (C145A, W100A, and P1 40A) in an ada- ogt- strain of E. coli and tested these proteins again st DNA substrates containing O6-methylguanine, for inactivation by O6- benzylguanine and for the ability to produce guanine from O6-benzylgua nine. The C145A mutation was inactive as expected since this residue a ctive against methylated DNA substrates but the P140A mutant was much less sensitive to inactivation by O6-benzylguanine and failed to form significant amounts of [H-3]guanine when incubated with O6-benzyl[8-H- 3]-guanine. The proline at position 140 in mammalian AGTs is replaced by alanine in the Ada and yeast AGTs and by serine in the Ogt AGT. The se results suggest that this proline residue affects the configuration of the active site allowing the O6-benzylguanine to enter and react w ith the mammalian AGT. The production of resistance to O6-benzylguanin e by a single base change raises the possibility that such resistance may arise quite readily in cells of tumors treated therapeutically wit h the combination of O6-benzylguanine and an alkylating agent.