Aamodt Randi M, Falnes Pål Ø, Johansen Rune F, Seeberg Erling, Bjørås Magnar
Department of Molecular Biology, Institute of Medical Microbiology, University of Oslo, National Hospital, N-0027 Oslo, Norway.
J Biol Chem. 2004 Apr 2;279(14):13601-6. doi: 10.1074/jbc.M314277200. Epub 2004 Jan 16.
The AAG family of 3-methyladenine DNA glycosylases was initially thought to be limited to mammalian cells, but genome sequencing efforts have revealed the presence of homologous proteins in certain prokaryotic species as well. Here, we report the first molecular characterization of a functional prokaryotic AAG homologue, i.e. YxlJ, termed bAag, from Bacillus subtilis. The B. subtilis aag gene was expressed in Escherichia coli, and the protein was purified to homogeneity. As expected, B. subtilis Aag was found to be a DNA glycosylase, which releases 3-alkylated purines and hypoxanthine, as well as the cyclic etheno adduct 1,N(6)-ethenoadenine from DNA. However, kinetic analysis showed that bAag removed hypoxanthine much faster than human AAG with a 10-fold higher value for k(cat), whereas the rate of excision of 1, N(6)-ethenoadenine was found to be similar. In contrast, it was found that bAag removes 3-methyladenine and 3-methylguanine approximately 10-20 times more slowly than human AAG, and there was hardly any detectable excision of 7-methylguanine. It thus appears that bAag has a minor role in the repair of DNA alkylation damage and an important role in preventing the mutagenic effects of deaminated purines and cyclic etheno adducts in Bacillus subtilis.
3-甲基腺嘌呤DNA糖基化酶的AAG家族最初被认为仅限于哺乳动物细胞，但基因组测序工作表明某些原核生物物种中也存在同源蛋白。在此，我们报道了来自枯草芽孢杆菌的功能性原核AAG同源物（即YxlJ，命名为bAag）的首次分子特征。枯草芽孢杆菌的aag基因在大肠杆菌中表达，该蛋白被纯化至同质。正如预期的那样，发现枯草芽孢杆菌Aag是一种DNA糖基化酶，可从DNA中释放3-烷基化嘌呤和次黄嘌呤，以及环状乙烯基加合物1,N(6)-乙烯基腺嘌呤。然而，动力学分析表明，bAag去除次黄嘌呤的速度比人AAG快得多，k(cat)值高10倍，而1,N(6)-乙烯基腺嘌呤的切除率相似。相比之下，发现bAag去除3-甲基腺嘌呤和3-甲基鸟嘌呤的速度比人AAG慢约10-20倍，几乎没有检测到7-甲基鸟嘌呤的切除。因此，bAag在枯草芽孢杆菌中对DNA烷基化损伤的修复作用较小，而在防止脱氨基嘌呤和环状乙烯基加合物的诱变作用方面起重要作用。
