Cell extracts from Micrococcus luteus release both free 3-methyladenine and free 7-methylguanine from alkylated DNA. The glycosylase activity responsible for the liberation of 7-methylguanine is not 3-methyladenine-DNA glycosylase, which, when purified, does not liberate it. Furthermore, the heat inactivation rates of the two enzymatic activities are different. The release of 7-methylguanine by chemical depurination of ethanol-soluble oligonucleotides has been ruled out. A similar activity releasing 7-methylguanine is also found in Escherichia coli Among chemical carcinogens, alkylating agents cause a variety of damage to DNA. The main products are N-7 alkylguanines and N-3 alkyladenines (for review, see refs. 1, 2). It has also been shown (3) that all the base oxygens can be alkylated.The removal of alkylated bases from DNA in vivo is performed enzymatically (4). In Micrococcus luteus, m3A is removed by a specific DNA glycosylase to give the free base and the apurinic site (5). This excision also occurs in Escherichia coli (6) and mammalian cells (7). However, neither purified M. luteus nor E. coli m3A-DNA glycosylases can remove m7G (5-6), although, some data suggest that, in E. coli B, the excision of a relatively small amount of m7G can occur (8). In mammalian cells, the m7G is lost at different rates, depending on the biological system studied. A half-life of 24-26 hr has been reported in hamster (9) and in 1OTL1/2 cells (10), and a much longer halflife (60 hr) has been reported in mouse organs in vivo (11,12). This is a faster rate than expected in the case of chemical depurination, which is about 150 hr at pH 7 (13).In view of these findings, we examined whether in vitro bacterial extracts can remove m7G. We found that extracts of M. luteus and of E. coli are able to excise m7G.
MATERIALS AND METHODSPreparation of DNA Containing m3A and m7G.[3H]Methylated DNA was prepared, as described (5), by treating calf thymus DNA (Choay, Paris) (4 mg in 2 ml of 0.1 M NaClO4/0.05 M Na cacodylate, pH 7.5) with 5 mCi of [methyl-3H]dimethylsulfate (1 Ci = 3.7 X 1010 becquerels; Amersham) for 1 hr at room temperature. The alkylated DNA was purified by three cycles of precipitating it with ethanol and then redissolving it, and then it was dialyzed three times against standard saline citrate for 16 hr and finally against 0.1 M saline citrate. It was kept at -20°C. The specific activity of the labeled methylated DNA was 1.0-2.0 X 106 cpm/,umol. For analysis of the alkylated bases, the methylated DNA was made 0.1 M in HC1, and the purine bases were released by hydrolysis at 37°C for 20 hr.Growth luteus or E. coli cells (4 ml) were placed in an ice bath for 30 min, thawed at room temperature, and immediately placed at 0C. Lysozyme (0. 2 ml of 4 mg/ml solution in 0.25 M Tris HCl, pH 7.5) and 16 A.l of aprotinin at 1 mg/ml (Choay, Paris) were added. After 20 min, the cells were warmed and kept at 370C until they began to lyse (usually <4 min). At this time, phenylmethanesulfonyl fluoride and p-toluenesulfonyl fluoride (...