Abstract
BackgroundA myriad of new chemicals has been introduced into our environment and exposure to these agents can damage cells and induce cytotoxicity through different mechanisms, including damaging DNA directly. Analysis of global transcriptional and phenotypic responses in the yeast S. cerevisiae provides means to identify pathways of damage recovery upon toxic exposure.ResultsHere we present a phenotypic screen of S. cerevisiae in liquid culture in a microtiter format. Detailed growth measurements were analyzed to reveal effects on ~5,500 different haploid strains that have either non-essential genes deleted or essential genes modified to generate unstable transcripts. The pattern of yeast mutants that are growth-inhibited (compared to WT cells) reveals the mechanisms ordinarily used to recover after damage. In addition to identifying previously-described DNA repair and cell cycle checkpoint deficient strains, we also identified new functional groups that profoundly affect MMS sensitivity, including RNA processing and telomere maintenance.ConclusionsWe present here a data-driven method to reveal modes of toxicity of different agents that impair cellular growth. The results from this study complement previous genomic phenotyping studies as we have expanded the data to include essential genes and to provide detailed mutant growth analysis for each individual strain. This eukaryotic testing system could potentially be used to screen compounds for toxicity, to identify mechanisms of toxicity, and to reduce the need for animal testing.
Highlights
The DNA damage response in budding yeast S. cerevisiae is well characterized, especially regarding its response to the alkylating agent methyl methanesulfonate (MMS) [1,2,3,4,5,6,7,8]
We have queried the essential genes in the Decreased Abundance by mRNA Perturbation (DAmP) library of haploid strains [19,21]; transcript levels in the DAmP library were reduced by tagging the 3’ UTR of the transcripts with a sequence that elicits nonsense-mediated decay [22]
Growth kinetics were recorded in the presence of the DNA damaging agent by optical density measurements at room temperature every 4 hours between 12 and 48 h after transfer to MMS-containing medium (Figure 1), and every strain was queried in at least three independent experiments
Summary
The DNA damage response in budding yeast S. cerevisiae is well characterized, especially regarding its response to the alkylating agent methyl methanesulfonate (MMS) [1,2,3,4,5,6,7,8]. In addition to the ~150 yeast proteins directly involved in DNA repair [9], a plethora of proteins with other biological functions are necessary for recovery after damage [1,2]. As a eukaryotic model system, serves as an eminent tool to develop new methods to unravel pathways for modulating the toxicity of remaining are essential genes that cannot be deleted and are more difficult to study. Essential genes can be studied in hemizygous diploid strains [18] and in haploid strains with either conditional expression of genes or with decreased levels of transcripts [19,20]. Analysis of global transcriptional and phenotypic responses in the yeast S. cerevisiae provides means to identify pathways of damage recovery upon toxic exposure
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