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

cDNA for mouse O-6-methylguanine-DNA methyltransferase was expressed in methyltransferase-deficient Escherichia 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 1.8x10(4) molecules 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 O-6-methylguanine or O-4-methylthymine at predetermined sites, the mouse enzyme repaired O-6-methylguanine and O-4-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.