Mouse methyltransferase for repair of O6-methylguanine and O4-methylthymine in DNA

cDNA for mouse O⁶-methylguanine-DNA methyltransfer-ase was expressedin methyltransferase-deficient Escher-ichia coli mutant cells,and the overproduced mouse enzyme was purified to a homogeneousstate. Using this purified product, polyclonal antibodies wereprepared and used to estimate amounts of the methyltransferaseprotein in cells. A single cell of NIH3T3 contained 1.8 x10⁴moleculesof the methyltransferase protein. When mouse fibroblasts wereimmunostained, it was shown that most of the methyltransferaseprotein exists in the cytoplasm rather than in the nucleus.Using double-stranded oligomers containing a single O⁶-methylguanineor O⁴-methylthymine at predetermined sites, the mouse enzymerepaired O⁶-methylguanine and O⁴-methylthymine, at an almostequal efficiency. In the LacZ reversion assay, MNNG-inducedA: T to G: C as well as G: C to A: T transition mutations wereefficiently suppressed by the function of mouse methyltransferase,in vivo.