Repair-deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing.

Abstract

In Escherichia coli, the repair of 3‐methyladenine (3MeA) DNA lesions prevents alkylation‐induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair‐deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag‐deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate‐induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation‐induced chromosome damage and cytotoxicity.

Contribution of Base Excision Repair, Nucleotide Excision Repair, and DNA Recombination to Alkylation Resistance of the Fission Yeast Schizosaccharomyces pombe

“…The contribution of 3MeA DNA glycosylase activity to MMS sensitivity of mammalian cells was more modest but nevertheless significant; MMS doses that allowed approximately 10% survival of the wild type caused less than 1% survival of 3MeA DNA glycosylase-deficient Aag Ϫ/Ϫ null cells (12). Note that there was no detectable 3MeA DNA glycosylase activity in cell extracts made from Aag Ϫ/Ϫ embryonic stem cells, suggesting that the presence of another 3MeA DNA glycosylase with redundant activity is unlikely (12).…”

True

“…13 It has been assumed that N 3 -methyladenine might kill mammalian cells by blocking DNA replication. 13 Conversely, other authors reported that overexpression of MPG was followed by enhanced chemosensitivity to methylating agents, suggesting that apurinic sites, formed as a result of MPG action, might induce sister chromatid exchanges and chromosomal aberrations. 34 In our model system, represented by a leukemic cell line unable to process O 6 -methylguanine, it is reasonable to hypothesize that the cytotoxic and apoptotic effects of the combination of TZM and PADPRP inhibitors might be the consequence of strand interruptions derived from the removal of N-methylpurines and of the resulting apurinic residues.…”

In vivo repair of methylation damage in Aag 3-methyladenine DNA glycosylase null mouse cells

“…Aag +/+ and Aag -/-embryonic stem (ES) cells were described previously (21). For these studies, AB1 and clone 65 were used to study wild-type activity (clone 65 was electroporated simultaneously with Aag -/-clones and is of similar passage number).…”

“…The potential biological relevance of NER activity on methylated bases (20) has not yet been established in vivo. In mammals, the main repair pathway for 3MeA is thought to be BER initiated by the Aag 3MeA DNA glycosylase (alkyladenine DNA glycosylase) (12,21). In addition to 3MeA, Aag is able to remove a broad spectrum of lesions, including 7MeG, hypoxanthine and 1,N 6 -ethenoadenine (22)(23)(24).…”

“…In previous studies, no residual repair of 3MeA was detected in extracts from Aag -/-cells in vitro, suggesting that Aag -/-cells *To whom correspondence should be addressed. Tel: +1 617 258 0260; Fax: +1 617 258 0499; Email: bevin@mit.edu lack the ability to remove toxic 3MeA lesions from the genome (21). However, the conditions for this assay were optimized for detecting bases released by Aag and, therefore, might not detect release by another glycosylase or by NER.…”