INTRACELLULAR-LOCALIZATION AND FUNCTION OF DNA-REPAIR METHYLTRANSFERASE IN HUMAN-CELLS

An antibody preparation specific for human O-6-methylguanine-DNA methy ltransferase (EC 2.1.1.63) was obtained by immunoaffinity purification on two types of affinity columns with the purified human and mouse me thyltransferase proteins as ligands. The antibodies were used in Weste rn blotting analysis of fractionated cell extracts. More than 90% of t he methyltransferase protein was recovered in the cytoplasmic fraction s with both human HeLa S3 cells and MR-M cells, the latter overproduci ng the enzyme 36 times as much as the former. Cytoplasmic localization of the methyltransferase in HeLa S3 cells was further confirmed by in situ immunostaining. By Western blotting analysis of fractionated cel l extracts from HeLa S3 cells treated with alkylating agents, we found that amounts of the enzyme decreased more rapidly in the nuclear frac tion than in the cytoplasmic fraction, and recovery of the enzyme leve l in the cytoplasmic fraction was slower than that in the other. These results suggest that the methyltransferase protein is degraded in the nucleus after it commits the repair reaction and that the cytoplasmic enzyme is transported into the nucleus as the nuclear methyltransfera se is used up in this manner.