Redesigning the substrate specificity of human O(6)-alkylguanine-DNA alkyltransferase. Mutants with enhanced repair of O(4)-methylthymine.
Human O(6)-alkylguanine-DNA alkyltransferase (MGMT) repairs potentially cytotoxic and mutagenic alkylation damage at the O(6)-position of guanine and the O(4)-position of thymine in DNA. We have used random sequence mutagenesis and functional complementation to obtain human MGMT mutants that are resistant to the MGMT inhibitor, O(6)-benzylguanine [Encell, L. P., Coates, M. M., and Loeb, L. A. (1998) Cancer Res. 58, 1013-1020]. Here we describe screening of O(6)-benzylguanine-resistant mutants for altered substrate specificity, i.e., for an increased level of utilization of O(4)-methylthymine (m(4)T) relative to that of O(6)-methylguanine (m(6)G). One mutant identified by the screen, 56-8, containing eight substitutions near the active site (C150Y, S152R, A154S, V155G, N157T, V164M, E166Q, and A170T), was purified and characterized kinetically. The second-order rate constant for repair of m(4)T by the mutant was up to 11.5-fold greater than that of WT MGMT, and the relative m(4)T specificity, k(m(4)T)/k(m(6)G), was as much as 75-fold greater. In competition experiments with both substrates present, the mutant was 277-fold more sensitive to inhibition by m(4)T than WT MGMT. This mutant, and others like it, could help elucidate the complex relationship between adduction at specific sites in DNA and the cytotoxicity and mutagenicity of alkylating agents.
Encell
L P
LP
The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington School of Medicine, Seattle 98195-7705, USA.
Loeb
L A
LA
D019943:Amino Acid Substitution / Q000235:genetics; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism*; D009841:Oligonucleotides / Q000138:chemical synthesis; D015202:Protein Engineering / Q000379:methods; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism
D009841:Oligonucleotides; C021223:O-4-methylthymine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D013941:Thymine