Development of a screening system for DNA damage inducible chemicals.

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In 1995, we constructed a fusion plasmid of the 5' promoter region of RNR3 and lacz, and measured the beta-galactosidase activity in order to detect induction of RNR3. The gene RNR3 in Saccharomyces cerevisiae was induced by various DNA-damaging chemicals such as alkylating agents which from bulky DNA adducts, a radical producer, and an intercalator. When yeast expressing rat CYP1A1 was exposed to procarcinogen of 2-aminofluorene, a concentration-dependent induction of RNR3 was observed. Measurement of the induction of DNA damage-responsive genes using yeast expressing various kinds of P450 appears to be sensitive and accurate test system for metabolism-related procarcinogens.To investigate cis-acting regulation, the deletion analysis of the promoter region of the RNR3 gene was performed and we identified two upstream repressing sequences in the RNR3 regulatory region in 1996. A 18-base-pairs fragment located between -211 and -193 in this regulatory region was found to be essential for the induction of RNR3. We demonstrated that enhanced expression of RNR3 in response to DNA damage was essentially regulated by transcriptional repressors and their cognate binding sites of DNA damage responsive element.We also studied new test system using DNA damage responsive genes or repair genes in humans in 1997. We found that gadd153 gene could be an available indicator to detect DNA damage.