Mitotic viability and metabolic competence in UV-irradiated yeast cells.
Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.
Conconi
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Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164-4660, USA.
Jager-Vottero
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Zhang
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Beard
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Smerdon
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MJ
D015169:Colony Count, Microbial; D004396:Coloring Agents; D004260:DNA Repair; D004271:DNA, Fungal / Q000528:radiation effects*; D004268:DNA-Binding Proteins; D005562:Formazans / Q000032:analysis; D005656:Fungal Proteins / Q000378:metabolism; D008938:Mitosis / Q000528:radiation effects*; D011740:Pyrimidine Dimers / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism / Q000528:radiation effects*; D029701:Saccharomyces cerevisiae Proteins; D013778:Tetrazolium Salts; D014466:Ultraviolet Rays
D004396:Coloring Agents; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005562:Formazans; D005656:Fungal Proteins; D011740:Pyrimidine Dimers; C063915:RAD7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013778:Tetrazolium Salts; C009591:triphenyltetrazolium