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 apurinic/apyrimidinic endonuclease, increases spontaneous mutation by up to approximately 600-fold in S. cerevisiae and approximately 200-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 REV1/REV3/REV7 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 (approximately 2-fold) but statistically significant increase in spontaneous mutation, a result which could have important implications for carcinogenesis.

Figure 1

3MeA DNA glycosylase activities were determined for cell-free extracts after galactose induction for the apn1Δ mag1Δ yeast strain BGY149 containing the control plasmid pYES or expressing MAG1, tag or AAG from plasmids pYES-MAG, pYES-tag or pYES-AAG, expressed as fmol 3MeA released per μg of protein extract. Values reported are the mean ± standard deviation (n = 6).

Figure 2

Spontaneous mutation rates determined by fluctuation analysis (or method of the median for strains containing pYES-MAG) after galactose induction for the apn1Δ strain BGY113 (A), the wild-type strain BGY111 (B), and the apn1Δ mag1Δ strain BGY149 (C) of yeast containing the control plasmid pYES or expressing the genes indicated (from plasmids pYES-MAG, pYES-tag, or pYES-AAG). Values reported are the mean ± standard deviation, with the number of independent determinations (n) shown. Note the change in the abscissa scale among A, B, and C. (A, B, and C Inset) Replotted data for pYES, pYES-tag, and pYES-AAG. (C Inset) Spontaneous mutation rate after AAG expression is significantly different (P < 0.02) from the pYES control value, as determined by the Wilcoxon rank-sum test.

Figure 3

Spontaneous mutation rates determined by the method of the median after galactose induction of the apn1Δ strain BGY113 containing plasmid pYES, pYES plus YEpAPN1 (which expresses APN1), pYES-MAG, or pYES-MAG plus YEpAPN1. The values reported are the mean ± standard deviation. (Inset) Replotted data for pYES, pYES plus YEpAPN1, and pYES-MAG plus YEpAPN1.

Figure 4

MMS killing after induction of the GAL1 promoter with galactose (A) or repression with glucose (B) for the Mag⁺ wild-type yeast strain BGY111 containing the control plasmid pYES (○) or its isogenic mag1Δ derivative strain BGY148 containing the plasmid pYES (•), pYES-tag (▿), pYES-MAG (▾) or pYES-AAG (□). A representative experiment is shown in each case; the relative survival between the various strains was always consistent. In addition, for the galactose-induced cells, qualitatively similar results were obtained in apn1Δ (BGY113) and apn1Δ mag1Δ (BGY149) strain backgrounds (data not shown). (C) MMS killing after galactose induction for the Mag⁺ wild-type strain BGY111 containing the control plasmid pYES (○) or its isogenic apn1Δ derivative strain BGY113 containing the plasmids pYES(•), pYES-MAG (▿), YEpAPN1 (▾), or pYES-MAG and YEpAPN1 (□).