(5bj) Systematic Design of Cell-Based Biosensors for Sensitive and Selective Detection of DNA-Damaging Agents

Because DNA damage can lead to cancer or cell death, the ability to detect genotoxicity is crucial in preserving human health, and most current testing methods are insufficient for adequately determining the risks associated with uncharacterized compounds. A thorough understanding of how organisms sense and respond to various types of DNA damage is necessary to improve on current genotoxicity assays. Within the last 10 years, the widespread use of DNA microarrays has enabled the transcriptional profiling of various species in response to multiple genotoxic agents. Although there is still much to learn, these experiments along with traditional deletion strain fitness assays have broadened our knowledge of the cellular pathways involved in responding to DNA damage. In our study oligonucleotide microarrays were used to measure the global transcriptional response of the model organism Saccharomyces cerevisiae after exposure to multiple doses of 2 representative DNA damaging agents, methyl methanesulfonate (MMS) and gamma radiation (g-ray). After thorough statistical filtering and hierarchical clustering, we were able to identify many genes that were sensitive to MMS and g-ray in a dose dependent manner. Once identified, one copy of several of these genes were systematically deleted from the genome of diploid yeast strains and replaced with a gene encoding green fluorescent protein, creating mutant strains that fluoresce in response to genotoxicity. These strains were then exposed to multiple doses of numerous DNA-damaging agents for various lengths of time and their fluorescent response was quantified using flow cytometry. Genotoxic detection was found to be rapid and very sensitive compared to more traditional methods.