title: Point mutations at multiple sites including highly conserved amino acids maintain activity, but render O6-alkylguanine-DNA alkyltransferase insensitive to O6-benzylguanine.
abstract: The DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), is inactivated by reaction with the pseudosubstrate, O(6)-benzylguanine (BG). This inactivation sensitizes tumour cells to chemotherapeutic alkylating agents, and BG is aimed at enhancing cancer treatment in clinical trials. Point mutations in a 24 amino acid sequence likely to form the BG-binding pocket were identified using a screening method designed to identify BG-resistant mutants. It was found that alterations in 21 of these residues were able to render AGT resistant to BG. These included mutations at the highly conserved residues Lys(165), Leu(168) and Leu(169). The two positions at which changes led to the largest increase in resistance to BG were Gly(156) and Lys(165). Eleven mutants at Gly(156) were identified, with increases in resistance ranging from 190-fold (G156V) to 4400-fold (G156P). Two mutants at Lys(165) found in the screen (K165S and K165A) showed 620-fold and 100-fold increases in resistance to BG. Two mutants at the Ser(159) position (S159I and S159V) were >80-fold more resistant than wild-type AGT. Eleven active mutants at Leu(169) were also resistant to BG, but with lower increases (5-86-fold). Fourteen BG-resistant mutants were found for position Cys(150), with 3-26-fold increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation. Six BG-resistant mutants at Asn(157) were found with increases of 4-13-fold. These results show that many changes can render human AGT resistant to BG without preventing the ability to protect tumour cells from therapeutic alkylating agents.
authors.affiliation: Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, P.O. Box 850, 500 University Drive, Hershey, PA 17033-0850, U.S.A.
authors.forename: M
authors.initials: M
authors.lastname: Xu-Welliver
authors.forename: A E
authors.initials: AE
authors.lastname: Pegg
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics*; D004260:DNA Repair / Q000187:drug effects; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli; D015723:Gene Library; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology / Q000633:toxicity; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: D000970:Antineoplastic Agents; C064976:O(6)-benzylguanine; D008769:Methylnitronitrosoguanidine; D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase