Release of 7-methylguanine residues from alkylated DNA by extracts of Micrococcus luteus and Escherichia coli.

Abstract

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 (...

7-Methylguanine adducts in DNA are normally present at high levels and increase on aging: analysis by HPLC with electrochemical detection.

“…The major adduct is m7Gua, since the N-7 position of the base guanine lies exposed in the major groove of the DNA helix (11)(12)(13). This DNA adduct persists for a long time in cellular DNA since it is repaired enzymatically with low efficiency (14)(15)(16). The presence of m7Gua does not interfere with DNA replication (17) and appears to be of minor harm to the cell in comparison to some of the other methylated adducts.…”

Signals from the Stressed Endoplasmic Reticulum Induce C/EBP-Homologous Protein (CHOP/GADD153)

“…on April 1, 2019 by guest http://mcb.asm.org/ the creation of an apurinic site (8) similar to the one remaining after excision repair of bases methylated by MMS (27). Either because of reduced incorporation of the agent into DNA (7) or because of failure to excise the modified base once incorporation has occurred (6), not all cells are responsive to the DNAdamaging effects of HmdUrd.…”

Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates

“…This begs the question: what is the difference between bolus and fractionated regimens using TMZ? The relatively inefficient in vivo selection of MGMT P140K gene-modified cells, using a fractionated drug treatment regimen, is potentially due to the repair kinetics of a variety of DNA adducts following O 6 BG and TMZ treatment (42)(43)(44)(45)(46)(47). Importantly, in both cases in which we efficiently increased relatively low levels of MGMT P140K gene marking in pigtailed macaques with a provirus copy number of approximately 0.04 (Figure 6), we used O 6 BG-2X and BCNU without prior O 6 BG and TMZ treatment.…”