cDNA for mouse O6-methylguanine-DNA methyltransfer-ase was expressed in methyltransferase-deficient Escher-ichia coli mutant cells, and the overproduced mouse enzyme was purified to a homogeneous state. Using this purified product, polyclonal antibodies were prepared and used to estimate amounts of the methyltransferase protein in cells. A single cell of NIH3T3 contained 1.8 ×104molecules of the methyltransferase protein. When mouse fibroblasts were immunostained, it was shown that most of the methyltransferase protein exists in the cytoplasm rather than in the nucleus. Using double-stranded oligomers containing a single O6-methylguanine or O4-methylthymine at predetermined sites, the mouse enzyme repaired O6-methylguanine and O4-methylthymine, at an almost 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 suppressed by the function of mouse methyltransferase, in vivo.
