Despite the great advances by using microorganism-based genotoxicity testing systems to assess environmental genotoxic compounds, most of them respond poorly, particularly to oxidative agents. In this study, we systematically examined the RNR3-lacZ reporter gene expression in Saccharomyces cerevisiae mutant strains defective in the protection against reactive oxygen species and found that only YAP1 deletion resulted in a significant enhancement in the detection of oxidative damage. To our surprise, YAP1 deletion also caused an increased cellular sensitivity to a variety of DNA damage. This altered sensitivity appears to be independent of oxidative damage because under conditions in which vitamin C treatment rescued oxidative damage, it failed to reverse the phenotypes caused by other types of DNA damage. Furthermore, although inactivation of cell permeability genes enhanced the RNR3-lacZ detection sensitivity particularly to large molecular weight compounds, their effects on small molecular oxidative agents are minimal. Taken together, this study helps to create a hypersensitive genotoxicity testing system to a broad range of DNA-damaging agents by deleting a single yeast gene.