Iyama A, Sakumi K, Nakabeppu Y, Sekiguchi M
Department of Biochemistry, Kyushu University, Fukuoka, Japan.
Carcinogenesis. 1994 Apr;15(4):627-33. doi: 10.1093/carcin/15.4.627.
cDNA encoding rabbit O6-methylguanine-DNA methyltransferase that repairs DNA damaged by alkylating agents was isolated, using as a probe a fragment of mouse cDNA coding for a region containing the active site of the enzyme. The nucleotide sequence of the cDNA revealed that the rabbit methyltransferase is a 181-amino acid polypeptide with a mol. wt of 19,385. Expression of the cDNA in a methyltransferase-deficient Escherichia coli mutant resulted in appearance of a 23 kDa polypeptide with methyltransferase activity, and this rendered the E.coli cells resistant to N-methyl-N'-nitro-N-nitrosoguanidine, in terms of both cell killing and induction of mutation. The rabbit methyltransferase is highly homologous to this enzyme in human, mouse, rat and hamster, but is 26-30 amino acid residues shorter as compared with methyltransferases in other mammalian species. Based on a comparison of the nucleotide sequences for the C-terminal regions of these proteins, we propose that a single base substitution, which would generate a TGA termination codon, was introduced into the sequence for the rabbit enzyme during the process of evolution. Existence of the naturally occurring truncated form of methyltransferase suggests that the longer C-terminal tails of other mammalian methyltransferases may have no significant role in exerting functions of the enzyme in vivo.
利用编码包含该酶活性位点区域的小鼠cDNA片段作为探针，分离出了编码兔O6-甲基鸟嘌呤-DNA甲基转移酶的cDNA，该酶可修复由烷化剂导致的DNA损伤。该cDNA的核苷酸序列显示，兔甲基转移酶是一种由181个氨基酸组成的多肽，分子量为19,385。在缺乏甲基转移酶的大肠杆菌突变体中表达该cDNA，导致出现了一种具有甲基转移酶活性的23 kDa多肽，这使得大肠杆菌细胞在细胞杀伤和突变诱导方面对N-甲基-N'-硝基-N-亚硝基胍具有抗性。兔甲基转移酶与人类、小鼠、大鼠和仓鼠中的这种酶高度同源，但与其他哺乳动物物种的甲基转移酶相比，其氨基酸残基短26 - 30个。基于对这些蛋白质C末端区域核苷酸序列的比较，我们推测在进化过程中，兔酶的序列中引入了一个单碱基替换，该替换会产生一个TGA终止密码子。天然存在的截短形式的甲基转移酶的存在表明，其他哺乳动物甲基转移酶较长的C末端尾巴在体内发挥该酶的功能方面可能没有显著作用。
