ALKYLPURINE-DNA-N-GLYCOSYLASE KNOCKOUT MICE SHOW INCREASED SUSCEPTIBILITY TO INDUCTION OF MUTATIONS BY METHYL METHANESULFONATE

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells, The null status of t he animals was confirmed at the mRNA level by reverse transcription-PC R nd by the inability of cell extracts of tissues from the knockout (k o) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-me G) from N-3-methylated calf thymus DNA in vitro. Following treatment w ith DNA-methylating agents, increased persistence of 7-meG was found i n liver sections of APNG ko mice in comparison with wild-type (wt) mic e, demonstrating an in vivo phenotype for the APNG null animals, Unlik e other null mutants of the Base excision repair pathway, tale APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, ar e fertile, and have an apparently normal life span. Neither a differen ce in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when k o 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 mt 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 fr om wt mice, These mutations were predominantly single-base-pair change s; in the ko mice, they consisted primarily of AT --> TA and GC --> TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG i n attenuating the mutagenic effects of N-alkylpurines in vivo.