A Chemical and Genetic Approach Together Define the Biological Consequences of 3-Methyladenine Lesions in the Mammalian Genome

Abstract

DNA-damaging agents produce a plethora of cellular responses that include p53 induction, cell cycle arrest, and apoptosis. It is generally assumed that it is the DNA damage produced by these agents that triggers such responses, but there is limited direct evidence to support this assumption. Here, we used DNA alkylation repair proficient and deficient isogenic mouse cell lines to demonstrate that the signal to trigger p53 induction, cell cycle arrest, and apoptosis in response to alkylating agents does emanate from DNA damage. Moreover, we established that 3-methyladenine, a relatively minor DNA lesion produced by most methylating agents (which form mainly 7-methylguanine), can specifically induce sister chromatid exchange, chromatid and chromosome gaps and breaks, S phase arrest, the accumulation of p53, and apoptosis. This study was made possible by the generation of 3-methyladenine DNA glycosylase null mutant cells by targeted homologous recombination and by the chemical synthesis of a methylating agent that almost exclusively produces 3-methyladenine DNA lesions. The combined use of these two experimental tools has defined the biological consequences of 3-methyladenine, a DNA lesion produced by endogenous cellular metabolites, environmental carcinogens, and chemotherapeutic alkylating agents.

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

“…In sharp contrast, the t 1/2 of 3MeA in Aag -/-cells is >5 h, thus demonstrating that Aag provides a substantial kinetic advantage, so that 3MeA lesions are cleared at least 10 times faster in Aag +/+ cells than in Aag -/-cells. This difference in repair rates is consistent with the model that unrepaired 3MeA lesions are potentially toxic (10), since the vast majority of the 3MeA lesions would be cleared from the genome of wild-type cells well within the time frame of a single cell cycle (∼10 h), whereas Aag -/-cells may be forced to divide in the presence of a substantial number of adducts.…”

“…Alkylating agents are abundant in our environment and endogenously produced in cells and these agents can react with DNA to create a broad spectrum of alkylated bases (2,10,38). Of particular interest are non-coding potentially toxic lesions, such as 3MeA (10,11,39).…”

“…Indeed, Aag -/-cells are susceptible to 3MeA-induced sister chromatid exchange, chromosome aberrations, S phase arrest and apoptosis (10). 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.…”

“…Previous studies of Aag -/-cells have shown that 3MeA lesions inhibit progression through S phase (10). To determine if Aag -/-cells suffered delayed cell cycle progression under the conditions used to study in vivo repair, we measured the amount of DNA present in populations of cells at various times post-exposure to cold MNU (or solvent control) under identical conditions as those used for exposure to [ 3 H]MNU (see Materials and Methods).…”

“…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).…”