Specific amino acid sequences required for O6-methylguanine-DNA methyltransferase activity: analyses of three residues at or near the methyl acceptor site.
Specific amino acid sequences required for O6-methylguanine-DNA methyltransferase activity: analyses of three residues at or near the methyl acceptor site.
复制标题
O6-甲基鸟嘌呤-DNA 甲基转移酶活性所需的特定氨基酸序列：分析甲基受体位点处或附近的三个残基。
DOI：
10.1093/carcin/13.5.837
复制
发表时间：
1992
期刊：
影响因子：
4.7
通讯作者：
M. Sekiguchi
中科院分区：
文献类型：
--
作者：
L. Chueh;T. Nakamura;Y. Nakatsu;K. Sakumi;H. Hayakawa;M. Sekiguchi
O 6-甲基鸟嘌呤-DNA甲基转移酶在预防肿瘤诱导中起重要作用。为了阐明甲基转移酶蛋白的高度保守的氨基酸序列的意义，通过定点突变克隆的人甲基转移酶cDNA引入氨基酸取代，并检查突变形式的酶的活性和稳定性。当发生甲基转移的半胱氨酸-145被其他氨基酸取代时，所有分离的突变体均表现出甲基转移酶阴性表型。从其中一个阴性突变体中分离出甲基转移酶阳性回复突变体，所有这些突变体都携带半胱氨酸密码子。因此，半胱氨酸残基对于接受甲基是必需的，并且不能被其他氨基酸取代。使用这种阴性和阳性选择程序，分析扩展到受体位点附近的其他残基。在组氨酸-146位点，四个取代（苯丙氨酸，蛋氨酸，天冬酰胺和谷氨酰胺）表现出积极的表型，但在这些突变体的甲基转移酶活性水平低。缬氨酸-148取代有6种类型的阳性回复突变体，其中突变体携带异亮氨酸，半胱氨酸和丙氨酸表现出显着高水平的甲基转移酶活性。在甲基转移酶缺陷的人细胞中表达了一些突变形式的cDNA，并证实了用大肠杆菌细胞获得的结果。
O6-Methylguanine-DNA methyltransferase plays an important role in preventing tumor induction. To elucidate the significance of a highly conserved amino acid sequence of methyltransferase protein, amino acid substitutions were introduced by site-directed mutagenesis of cloned cDNA for human methyltransferase and the activity and stability of mutant forms of enzyme were examined. When cysteine-145, to which the methyl transfer occurs, was replaced by other amino acids, all of the mutants isolated showed the methyltransferase-negative phenotype. From one of the negative mutants, methyltransferase-positive revertants were isolated, all of which carried codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and cannot be replaced by other amino acids. Using this negative and positive selection procedure, analyses were extended to other residues near the acceptor site. At the histidine-146 site, four substitutions (phenylalanine, methionine, asparagine and glutamine) exhibited the positive phenotype but the levels of methyltransferase activity in these mutants were low. With valine-148 substitutions there were six types of positive revertants, among which mutants carrying isoleucine, cysteine and alanine showed significantly high levels of methyltransferase activity. Some mutant forms of cDNA were expressed in methyltransferase-deficient human cells, and the results obtained with Escherichia coli cells were confirmed.