Development of RNR3- and RAD54-GUS reporters for testing genotoxicity in Saccharomyces cerevisiae.
S. cerevisiae RNR3 and RAD54 gene transcription becomes strongly activated upon DNA damage. This property was used to construct yeast strains in which DNA damage can be monitored by a very sensitive fluorogenic assay in a convenient 96-well microtiter plate format. These strains carried stably integrated fusions of RNR3 or RAD54 promoters to the E. coli beta-glucuronidase GUS gene. GUS activity was measured by fluorogenic detection, a method that greatly increases the precision and sensitivity of the assay. Detection levels were similar to those of real-time quantitative PCR methods and close to the limits of biological response. The two reporters differed in terms of fold-induction, activation kinetics, sensitivity and specificity upon exposure to a variety of genotoxic compounds. While RNR3-GUS showed the fastest response, RAD54-GUS showed the highest sensitivity: similar to previous reported sensitivities for bacterial and eukaryotic genotoxic detection systems. These reporter strains may complement current genotoxicity tests, but they also have the advantages of higher flexibility, requirement for shorter incubation times, and the capability of being fully automated. In addition, the intrinsic features of the system facilitate its easy improvement by genetic manipulating the yeast strain or by introducing mammalian metabolizing enzymes.
Boronat
Susanna
S
Institut de Biologia Molecular de Barcelona, Consejo Superior de Investigaciones Científicas, Jordi Girona, 18, 08034, Barcelona, Spain.
Piña
Benjamin
B
D004249:DNA Damage / Q000235:genetics; D004265:DNA Helicases; D045643:DNA Repair Enzymes; D017930:Genes, Reporter / Q000235:genetics*; D007700:Kinetics; D009152:Mutagenicity Tests; D012262:Ribonucleoside Diphosphate Reductase / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D029701:Saccharomyces cerevisiae Proteins; C517042:RNR3 protein, S cerevisiae; D012262:Ribonucleoside Diphosphate Reductase; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes