Generation of a strong mutator phenotype in yeast by imbalanced base excision repair

Abstract

Increased spontaneous mutation is associated with increased cancer risk. Here, by using a model system, we show that spontaneous mutation can be increased several hundred-fold by a simple imbalance between the first two enzymes involved in DNA base excision repair. The Saccharomyces cerevisiae MAG1 3-methyladenine (3MeA) DNA glycosylase, when expressed at high levels relative to the apurinic͞apyrimidinic endonuclease, increases spontaneous mutation by up to Ϸ600-fold in S. cerevisiae and Ϸ200-fold in Escherichia coli. Genetic evidence suggests that, in yeast, the increased spontaneous mutation requires the generation of abasic sites and the processing of these sites by the REV1͞REV3͞REV7 lesion bypass pathway. Comparison of the mutator activity produced by Mag1, which has a broad substrate range, with that produced by the E. coli Tag 3MeA DNA glycosylase, which has a narrow substrate range, indicates that the removal of endogenously produced 3MeA is unlikely to be responsible for the mutator effect of Mag1. Finally, the human AAG 3-MeA DNA glycosylase also can produce a small (Ϸ2-fold) but statistically significant increase in spontaneous mutation, a result which could have important implications for carcinogenesis.Mutation is a characteristic of all living organisms and can arise from a variety of sources. DNA is chemically unstable and its replication imperfect; such instability and infidelity can generate mutations. In addition, a variety of endogenous metabolites and exogenous physical and chemical agents can generate potentially mutagenic DNA adducts. Despite these threats organisms maintain a relatively low mutation rate, due in part to a diverse group of DNA repair pathways (1). Defects in several kinds of DNA repair are known to elevate spontaneous mutation (2, 3), and certain mammalian DNA repair defects that influence spontaneous mutation result in remarkably increased cancer risk (4).Subtly altered DNA bases, such as deaminated, oxidized, and alkylated bases, are repaired by the highly conserved base excision repair pathway (5, 6). The first step of this multistep pathway involves the release of a modified DNA base by a specific DNA glycosylase. The resulting abasic site represents another form of DNA damage that is potentially cytotoxic and mutagenic (7,8). Abasic sites are processed, in most cases, by apurinic͞ apyrimidinic (AP) endonucleases that cleave DNA 5Ј to the abasic site, generating a single strand break that cannot be ligated. The abasic sugar residue is removed by deoxyribophosphodiesterase, the gap filled by DNA polymerase, and the remaining nick sealed by DNA ligase (5).In S. cerevisiae, the 3MeA DNA glycosylase encoded by MAG1 (9) is the major activity for removing alkylated bases. Mag1 releases a variety of bases from DNA in vitro, including 3MeA, 7-methylguanine, 7-methyladenine, 3-methylguanine (9, 10), 7-hydroxyethylguanine, 7-chloroethylguanine (11), 1,N 6 -ethenoadenine (12), and hypoxanthine (deaminated adenine) (13). The abasic sites generated by Mag1 are processed norm...

Rev1 and Polζ influence toxicity and mutagenicity of Me-lex, a sequence selective N3-adenine methylating agent

“…These results suggest that the AP sites are a major source of spontaneous mutagenesis, and that constitutive Mag1 activity contributes to the sMF. This result is consistent with the observation that Mag1 activity is associated with a weak mutator phenotype, which is even more evident when the protein is over-expressed [23,24]. …”

Low Integrated DNA Repair Score and Lung Cancer Risk

“…In addition, we found that increased enzymatic activity of MPG, a DNA repair enzyme of the DNA glycosylases family, which removes from DNA multiple alkylation and oxidative lesions, is associated with increased lung cancer risk (18). This surprising finding illustrates the notion that imbalances in DNA repair (23)(24)(25), and not necessarily low activity of DNA repair, can be involved in cancer risk.…”

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene

“…For example, in yeast, when BER is imbalanced by an excess of abasic sites (generated by over-expression N-methylpurine-DNA glycosylases, MPG), the rate of spontaneous mutation is increased signi®cantly (Chakravarti et al, 1991;Berdal et al, 1998;Glassner et al, 1998;Lau et al, 2000). Chinese hamster ovary cells over-expressing human MPG (a broad spectrum glycosylase), however, when exposed to methylating agents, did not show increased mutagenesis but showed inhibition of DNA synthesis, chromosomal aberrations and DNA breaks (Ibeanu et al, 1992;Coquerelle et al, 1995).…”