Cloning and characterization of a mouse 3-methyladenine/7-methyl-guanine/3-methylguanine DNA glycosylase cDNA whose gene maps to chromosome 11

Abstract

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.