Role of codon 160 in the sensitivity of human O6-alkylguanine-DNA alkyltransferase to O6-benzylguanine

摘要: O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that provides protection from alkylating agents such as dacarbazine, temozolomide, and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which are used for cancer chemotherapy. O6-Benzylguanine (BG) an inhibitor of AGT sensitizes tumors to these agents. BG currently in clinical trials. It possible the presence resistant forms may limit effectiveness this strategy. Previous studies have shown mutant G160R, occur naturally result polymorphism gene, BG, whereas mutants G160W G160A actually more sensitive inhibitor. To examine other mutations at site, random sequence was placed codon 160 cDNA, plasmid library constructed express sequences Escherichia coli. After selection with N-methyl-N'-nitro-N-nitrosoguanidine, BG-resistant were obtained analyzed. Eleven different amino acid substitutions found impart resistance by assay. The most contained histidine or arginine, had EC50 values 12 4.7 microM, respectively, compared wild-type 0.08 but nine alterations led least 10-fold rise value. Three additional site-directed mutagenesis, 6- 11-fold increases BG. Comparisons properties G160R G160E showed enhanced reaction much strongly when acidic residue present position. This account lack mutation even though it did These results indicate many position can lead significant