Characterization of human polymorphic DNA repair methyltransferase

The O-6-methylguanine-DNA methyltransferase (MGMT) is a critical defence against alkylation-induced mutagenesis and carcinogenesis, More than a 20-fold interindividual difference in the MGMT activity is known to exist among human cultured fibroblasts. We previously reported three allelic variants of the human MGMT gene, namely V1, V2, and V3, Both V1 and V2 carry amino acid substitutions, Leu(84)Phe and Trp(65)Cys, respectively, while V3 has a silent mutation. In order to reveal the pharmacogenetic and ecogenetic significance of polymorphism in the human MGMT gene, we investigated the in-vivo characteristics of V1 and V2 methyltransferase enzyme. Escherichia coli strain KT233 (ogt(-), ada(-)) and mer(-) HeLa MR cells carrying a V1 sequence exhibited almost the same level of sensitivity against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as did those with a wild-type sequence. The level of methyltransferase protein in those cells was essentially the same as for the wild-type and V1 samples. On the other hand, E. coli and human cells expressing V2 cDNA showed a significantly reduced level of survival, In these cells, V2 protein was hardly detected, even though mRNA was produced normally. An in-vitro translation experiment revealed that the V2 sequence had the potential to produce methyltransferase protein, as did the wild-type and V1 sequences. There was also evidence for a small amount of V2 protein being produced but rapidly degraded, thus implying that the V2 molecule is unstable in vivo. Using purified recombinant proteins, we estimated the kinetic values of wild-type and variant form of enzymes, which would support these views. From these results, we concluded that the wildtype and V1 protein have similar enzymatic and physicochemical properties, while V2 protein is considered to be unstable and rare. Pharmacogenetics 10:59-66 (C) 2000 Lippincott Williams & Wilkins.