Engelward B P, Boosalis M S, Chen B J, Deng Z, Siciliano M J, Samson L D
Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, MA 02115.
Carcinogenesis. 1993 Feb;14(2):175-81. doi: 10.1093/carcin/14.2.175.
In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions by DNA glycosylases prevents alkylation induced cell death. We described previously the isolation of a human 3MeA DNA glycosylase (AAG) cDNA that maps to chromosome 16 and hybridizes to specific genomic DNA fragments from a number of mammals, including mouse. As a first step in the generation of a 3MeA DNA glycosylase deficient mouse by homologous replacement in embryonic stem cells, we have cloned the mouse 3MeA DNA glycosylase cDNA. The cloned 1095 base pair cDNA contains a complete 333 amino acid open reading frame that predicts a 36.5 kDa protein and hybridizes to a 1.5 kb mRNA transcript. Mouse 3MeA DNA glycosylase (Aag) transcript levels vary by up to 21 fold among tissues, being highest in the testes and lowest in the heart. The Aag cDNA encodes a glycosylase able to release 3MeA, 7-methylguanine (7MeG) and 3-methylguanine (3MeG) from alkylated DNA. The expression of Aag in E. coli provides substantial resistance against killing by methylating agents, but, unlike its E. coli counterparts, the Aag glycosylase fails to protect against killing by ethylating and propylating agents. A 232 amino acid stretch of the predicted mouse protein shares extensive amino acid identity with rat (93%) and human (83%) 3MeA DNA glycosylases and we observe that all three mammalian glycosylases have a bipartite nuclear localization signal. The Aag gene maps to mouse chromosome 11, suggesting a segment of conserved synteny between mouse chromosome 11 and human chromosome 16, which bears the human 3MeA DNA glycosylase gene. Cloning the mouse 3MeA DNA glycosylase cDNA is a step toward understanding the role of this DNA repair enzyme in mammals.
在大肠杆菌中，DNA糖基化酶对3 - 甲基腺嘌呤（3MeA）DNA损伤的修复可防止烷基化诱导的细胞死亡。我们之前描述了一种人类3MeA DNA糖基化酶（AAG）cDNA的分离，该cDNA定位于16号染色体，并与包括小鼠在内的多种哺乳动物的特定基因组DNA片段杂交。作为通过胚胎干细胞中的同源替换产生3MeA DNA糖基化酶缺陷小鼠的第一步，我们克隆了小鼠3MeA DNA糖基化酶cDNA。克隆的1095个碱基对的cDNA包含一个完整的333个氨基酸的开放阅读框，预测编码一个36.5 kDa的蛋白质，并与一个1.5 kb的mRNA转录本杂交。小鼠3MeA DNA糖基化酶（Aag）的转录水平在不同组织中相差高达21倍，在睾丸中最高，在心脏中最低。Aag cDNA编码一种能够从烷基化DNA中释放3MeA、7 - 甲基鸟嘌呤（7MeG）和3 - 甲基鸟嘌呤（3MeG）的糖基化酶。Aag在大肠杆菌中的表达赋予了对甲基化剂杀伤的显著抗性，但与大肠杆菌中的对应物不同，Aag糖基化酶不能保护细胞免受乙基化剂和丙基化剂的杀伤。预测的小鼠蛋白质的一段232个氨基酸的序列与大鼠（93%）和人类（83%）的3MeA DNA糖基化酶具有广泛的氨基酸同一性，并且我们观察到所有三种哺乳动物糖基化酶都有一个双分核定位信号。Aag基因定位于小鼠11号染色体，这表明小鼠11号染色体和携带人类3MeA DNA糖基化酶基因的人类16号染色体之间存在一段保守的同线性。克隆小鼠3MeA DNA糖基化酶cDNA是迈向了解这种DNA修复酶在哺乳动物中作用的一步。
