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 inactivated by incubation with the pseudosubstrate, O6-benzylguanine, but the equivalent protein from the Escherichia coli ogt gene is much less sensitive and the Saccharomyces cerevisiae and E. coli ada gene product AGTs are completely resistant to this compound. We have expressed the normal human AGT and various point mutations (C145A, W100A, and P140A) in an ada- ogt- strain of E. coli and tested these proteins against DNA substrates containing O6-methylguanine, for inactivation by O6-benzylguanine and for the ability to produce guanine from O6-benzylguanine. The C145A mutation was inactive as expected since this residue forms the methyl acceptor site. Mutants W100A and P140A were fully active against methylated DNA substrates but the P140A mutant was much less sensitive to inactivation by O6-benzylguanine and failed to form significant amounts of [3H]guanine when incubated with O6-benzyl[8-3H]-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. These results suggest that this proline residue affects the configuration of the active site allowing the O6-benzylguanine to enter and react with the mammalian AGT. The production of resistance to O6-benzylguanine by a single base change raises the possibility that such resistance may arise quite readily in cells of tumors treated therapeutically with the combination of O6-benzylguanine and an alkylating agent.
Crone
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TM
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.
Pegg
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AE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C064976:O(6)-benzylguanine; D008769:Methylnitronitrosoguanidine; D006147:Guanine; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase