The Bacillus subtilis counterpart of the mammalian 3-methyladenine DNA glycosylase has hypoxanthine and 1,N6-ethenoadenine as preferred substrates.
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.
Aamodt
Randi M
RM
Department of Molecular Biology, Institute of Medical Microbiology, University of Oslo, National Hospital, N-0027 Oslo, Norway.
Falnes
Pål Ø
PØ
Johansen
Rune F
RF
Seeberg
Erling
E
Bjørås
Magnar
M
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism*; D000478:Alkylation; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D045647:DNA Glycosylases / Q000235:genetics* / Q000378:metabolism*; D019175:DNA Methylation; D003641:Deamination; D004926:Escherichia coli / Q000235:genetics; D019271:Hypoxanthine / Q000378:metabolism*; D008322:Mammals; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
C004981:1,N(6)-ethenoadenine; D019271:Hypoxanthine; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D000225:Adenine