Cell cycle dependence of ionizing radiation-induced DNA deletions and antioxidant radioprotection in Saccharomyces cerevisiae

Abstract

The yeast DEL assay is an effective method for measuring intrachromosomal recombination events resulting in DNA deletions that when occurring in mammalian cells are often associated with genomic instability and carcinogenesis. Here we used the DEL assay to measure gamma-ray-induced DNA deletions throughout different phases of yeast culture growth. Whereas yeast survival differed by only up to twofold throughout the yeast growth phase, proliferating cells in lag and early exponential growth phases were tenfold more sensitive to ionizing radiation-induced DNA deletions than cells in stationary phase. Radiation-induced DNA deletion potential was found to correlate directly with the fraction of cells in S/G(2) phase. The ability of the antioxidants l-ascorbic acid and DMSO to protect against radiation-induced DNA deletions was also measured within the different phases of yeast culture growth. Yeast cells in lag and early exponential growth phases were uniquely protected by antioxidant treatment, whereas nondividing cells in stationary phase could not be protected against the induction of DNA deletions. These results are compared with those from mammalian cell studies, and the implications for radiation-induced carcinogenesis and radioprotection are discussed.

FIG. 1

Panel A: Yeast cell density measured in cell cultures as a function of time after inoculation. Panel B: The fractions of cells in G₁/G₀ and S/G₂ in the same cultures. G₁/G₀ cells were measured by scoring nonbudding yeast and S/G₂ cells were measured by scoring budding yeast cells. Panel C: Radiation-induced cell killing and homologous DNA deletion (DEL) events per 10⁴ surviving cells in cell cultures irradiated with 1000 Gy at different times after inoculation. For all panels, each point is the mean of four independent experiments ± SEM.

FIG. 2

Representative images of budding and non-budding yeast from 0-, 2-, 8- and 30-h cultures are presented in panels A, B, C and D, respectively.

FIG. 3

Protection against 1000 Gy γ-ray-induced cell killing and DNA deletions in yeast culture grown for 4 to 30 h by DMSO (panel A) and ascorbic acid (panel B). Protection against deletion events was generally present only during stages of yeast exponential growth and not during stationary phase. Each measurement is the mean of three independent experiments ± SEM.

FIG. 4

Radiation-induced DNA deletions (DEL) per 10⁴ surviving cells as a function of the fraction of cells in S/G₂ for 70 independent measurements. Cultures with more cells in S/G₂ were more susceptible to radiation-induced DNA deletions. This correlation was highly significant (P < 0.0001) as calculated by Pearson test.