MOUSE METHYLTRANSFERASE FOR REPAIR OF O-6-METHYLGUANINE AND O-4-METHYLTHYMINE IN DNA

cDNA for mouse O-6-methylguanine-DNA methyltransferase was expressed i n methyltransferase-deficient Escherichia coli mutant cells, and the o verproduced mouse enzyme was purified to a homogeneous state. Using th is purified product, polyclonal antibodies were prepared and used to e stimate amounts of the methyltransferase protein in cells. A single ce ll of NIH3T3 contained 1.8x10(4) molecules of the methyltransferase pr otein, When mouse fibroblasts were immunostained, it was shown that mo st of the methyltransferase protein exists in the cytoplasm rather tha n in the nucleus, Using double-stranded oligomers containing a single O-6-methylguanine or O-4-methylthymine at predetermined sites, the mou se enzyme repaired O-6-methylguanine and O-4-methylthymine, at an almo st equal efficiency. In the LacZ reversion assay, MNNG-induced A:T to G:C as well as G:C to A:T transition mutations were efficiently suppre ssed by the function of mouse methyltransferase, in vivo.