Alkylpurine–DNA–N-Glycosylase Knockout Mice Show Increased Susceptibility to Induction of Mutations by Methyl Methanesulfonate

Abstract

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3 H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three-to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly singlebase-pair changes; in the ko mice, they consisted primarily of AT3TA and GC3TA transversions, which most likely are caused by 3-meA and 3-or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.Alkylpurine-DNA-N-glycosylase (APNG) is one of a growing list of enzymes responsible for the recognition and excision of altered bases in the first step of the base excision repair pathway (49, 56). In the simplest form of base excision repair, the resulting abasic site is then repaired by the sequential action of an apurinic-apyrimidinic (AP) endonuclease that generates a single-strand break, the removal of the 5Ј-terminal deoxyribose phosphate residue, insertion of a single nucleotide by DNA polymerase ␤, and finally ligation of the repaired patch by DNA ligase I or XRCC1-DNA ligase III (55, 56).Mammalian APNGs have been shown to be active against a wide range of modified bases in vitro, many structurally unrelated to 3-methyladenine (3-meA), the substrate after which the enzyme was first named (32). In particular, APNG appears to be the only glycosylase in mammalian cells that can release hypoxanthine from DNA, a promutagenic base resulting from the spontaneous deamination of adenine (22,47). Likewise, the highly mutagenic adduct 1,N 6 -ethenoadenine (41), which is produced by metabolic products of the environmental hepatocarcinogens vinyl chloride and ethyl carbamate, is released by APNG (22, 48); indeed, according to one report, the recombinant human enzyme reacted 10-to 20-fold more efficiently with this adduct than with 3-meA in an in vitro assay (14). These results, together ...

Repair of exocyclic DNA adducts: rings of complexity

“…(46)(47)(48) Such analysis provides an unambiguous means for the designation of the substrate specificity and for the exploration of backup activities for the missing enzyme. Biologically, however, it is surprising that these knockout mice did not show any overt phenotypic abnormalities (45,46) or significant increase in the spontaneous mutation rate, even increased mutations were observed in the hprt gene of the T lymphocytes of ANPG À/À mice treated with methyl methanesulfonate. (45) When the same mice were challenged with vinyl carbamate (49) or ethyl carbamate, (50) levels of eA were significantly higher and persisted longer in DNA from ANPG À/À mice than wild-type mice, indicating the cellular removal of eA by ANPG.…”

“…Biologically, however, it is surprising that these knockout mice did not show any overt phenotypic abnormalities (45,46) or significant increase in the spontaneous mutation rate, even increased mutations were observed in the hprt gene of the T lymphocytes of ANPG À/À mice treated with methyl methanesulfonate. (45) When the same mice were challenged with vinyl carbamate (49) or ethyl carbamate, (50) levels of eA were significantly higher and persisted longer in DNA from ANPG À/À mice than wild-type mice, indicating the cellular removal of eA by ANPG. It is puzzling, though, that the increased levels of adducts were not paralleled by the increased incidence of liver tumors in these mice.…”

“…(44) Part of the above biochemical results on substrate specificity was verified when ANPG À/À knockout mice were generated independently by the groups of Elder and Samson. (45,46) Using cell-free extracts and synthetic oligonucleotides/modified DNA, ANPG was shown to be the primary glycosylase excising eA, 1,N 2 -eG, 3mA and Hx. (46)(47)(48) Such analysis provides an unambiguous means for the designation of the substrate specificity and for the exploration of backup activities for the missing enzyme.…”

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

“…However, 7MeG lesions may spontaneously depurinate to form potentially mutagenic abasic sites (7)(8)(9). 3MeA is cytotoxic and potentially mutagenic in mammalian cells, presumably because it can block DNA replication (10)(11)(12)(13)(14)(15). O 6 MeG, on the other hand, is highly mutagenic, since it mispairs very efficiently with thymine during DNA replication (16,17).…”

“…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).…”