Abstract
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.
Highlights
Alkylating agents represent one of the most abundant classes of mutagenic and genotoxic agents present in the environment
B. subtilis Homologue of the Mammalian AAG DNA Glycosylase seems to have a more important role in the removal of premutagenic residues induced by deamination and lipid peroxidation rather than for cytotoxic residues induced by alkylating agents
An Open Reading Frame in Bacillus subtilis That Encodes a Protein with Homology to the Mammalian 3-Methyladenine DNA Glycosylase—Mammalian 3mA DNA glycosylases have been characterized from mouse, man, and rat [13,14,15, 18, 23, 24] and are different in structure from those found in E. coli and most other bacteria
Summary
Alkylating agents represent one of the most abundant classes of mutagenic and genotoxic agents present in the environment. B. subtilis possesses two AlkA homologues and a third DNA glycosylase protein with close similarity to AlkC(YhaZ), a recently identified repair gene in Bacillus cereus.. The high number of 3mA DNA glycosylases in Bacillus species probably reflects the heavy exposure of earth bacteria to environmental alkylating agents like methyl chloride. The Ada protein, which is a methyltransferase that becomes activated upon methylation exposure, regulates the adaptive response in E. coli and induces transcription of AlkA and other repair proteins involved in protection against alkylation damage. B. subtilis Homologue of the Mammalian AAG DNA Glycosylase seems to have a more important role in the removal of premutagenic residues induced by deamination and lipid peroxidation rather than for cytotoxic residues induced by alkylating agents
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