Abstract: ABSTRACT: BACKGROUND: Upon exposure to agents that damage DNA, Saccharomyces cerevisiae undergo widespread reprogramming of gene expression. Such a vast response may be due not only to damage to DNA but also damage to proteins, RNA, and lipids. Here the transcriptional response of S. cerevisiae specifically induced by DNA damage was discerned by exposing S. cerevisiae to a panel of three radiomimetic enediyne antibiotics (calicheamicin, esperamicin A1 and neocarzinostatin) that bind specifically to DNA and generate varying proportions of single- and double-strand DNA breaks. The genome-wide responses were compared to those induced by the non-selective oxidant gamma-radiation. RESULTS: Given well-controlled exposures that resulted in similar and minimal cell death (~20-25%) across all conditions, the extent of gene expression modulation was markedly different depending on treatment with the enediynes or gamma-radiation. Exposure to gamma-radiation resulted in more extensive transcriptional changes classified both by the number of genes modulated and the magnitude of change. Common biological responses were identified between the enediynes and gamma-radiation, with the induction of DNA repair and stress response genes, and the repression of ribosomal biogenesis genes. Despite these common responses, a fraction of the response induced by gamma radiation was repressed by the enediynes and vise versa, suggesting that the enediyne response is not entirely radiomimetic. Regression analysis identified 55 transcripts with gene expression induction associated both with double- or single-strand break formation. The S. cerevisae DNA damage signature genes as defined by Gasch et al. [1] were enriched among regulated transcripts associated with single-strand breaks, while genes involved in cell cycle regulation were associated with double-strand breaks. CONCLUSIONS: Dissection of the transcriptional response in yeast that is specifically signaled by DNA strand breaks has identified that single-strand breaks provide the signal for activation of transcripts encoding proteins involved in the DNA damage signature in S. cerevisiae, and double-strand breaks signal changes in cell cycle regulation genes.