Doxorubicin and related anthracycline analogues have remained front-line drugs in chemotherapy of cancer in spite of associated toxicities that limit their long term clinical use. However, the interplay of mechanisms leading to their toxicity remains unclear. Recent work in our laboratory has demonstrated a selective interaction of doxorubicin with the operator region of lacI in a uvrB E. coli strain, which strongly implicates a doxorubicin interaction with this palindromic structure as an intermediate in the mutagenicity of this drug. This proposal is to investigate the basis of the mutagenic specificity of anthracyclines for the lacO region of the lac operon, in vitro and in procaryotic and eukaryotic systems. In procaryotes, mutational specificity of anthracyclines will be determined for lacO and the i-d region of lacI under repressor-induced and uninduced conditions. Response of the lacO gene target as a single copy integrant under lacI control will be simultaneously investigated in mammalian cells. Proposed studies will incorporate site specific mutagenesis approaches into a forward mutational analysis, which will place special emphasis on processes leading to deletions of genetic material. Putative deletion initiating sequences will be identified from analysis of in vivo-induced mutations and in vitro studies in the lacI/lacO gene target of E. coli. Experiments in vitro will include footprinting to determine the binding specificity of doxorubicin for linear and palindromic single stranded DNA constructs, doxorubicin-induced recognition sites for the DNA repair enzymes, nuclease SP and uvrABC exinuclease, as well as doxorubicin-induced cleavable complex sites. The overall aim of this work is to provide a basis for better understanding of the role of DNA sequence dependent drug interactions in the toxic and mutational responses of cells to doxorubicin as a model for study of the DNA directed mechanisms of intercalators with pleiotropic DNA damage potential.
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