[Role of O6-methylguanine-DNA methyltransferase in repair of DNA damaged by alkylating agents].
Some human tumor cell lines show the Mer- (Mex-) phenotype characterized by defects in repair of O6-methylguanine. Since the Mer- cells are deficient in O6-methylguanine-DNA methyltransferase activity, we examined whether introduction of the cloned bacterial gene, ada, encoding methyltransferase would make Mer- cells resistant to alkylating agents. When a DNA fragment carrying the ada gene of Escherichia coli on pSV2 neo vector was introduced into HeLa Mer- cells, transformants which are resistant to alkylating agents were obtained. The cells produced a large amount of methyltransferase enzyme. By expressing foreign genes in E. coli cells, it may be possible to clone DNA repair genes of various organisms. In this manner, we cloned the Bacillus subtilis gene that encodes a constitutive O6-methylguanine-DNA methyltransferase. Based on the nucleotide sequence of the gene, it was deduced that the protein comprises 165 amino acids and that the molecular weight is about 18,800. In principle, this method is applicable for cloning of cDNA for methyltransferase from higher organisms. The sequence homology evidenced in the present experiments also provides a useful tool for screening the gene or cDNA for methyltransferase. We developed a new vector which can replicate autonomously in EB-transformed lymphoblasts as well as in fibroblast cell lines such as HeLa. We found that the cloned ada gene on the vector is effective for increasing survival rates of Mer- cells treated with alkylating agents. This system can be used for cloning cDNA for human methyltransferase.
Sekiguchi
M
M
Dept. of Biochemistry, Faculty of Medicine, Kyushu University, Fukuoka.
Hayakawa
H
H
Kodama
K
K
Ishizaki
K
K
Ikenaga
M
M
D000477:Alkylating Agents / Q000633:toxicity*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004249:DNA Damage; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D008780:Methyltransferases / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics; D019853:O(6)-Methylguanine-DNA Methyltransferase
D000477:Alkylating Agents; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase