ANTHRACYCLINE-INDUCED DNA SEQUENCE SPECIFIC MUTATION

多柔比星和相关蒽环类抗生素仍然是一线药物 尽管有相关的毒性， 限制了其长期临床应用。 然而，各种机制的相互作用 导致其毒性的原因尚不清楚。 我们实验室最近的工作 已经证明了阿霉素与操作者的选择性相互作用 uvrB E.大肠杆菌菌株，这强烈暗示了 阿霉素与作为中间体的回文结构的相互作用 这种药的致突变性 这项建议是为了调查 蒽环类药物对大肠杆菌lacO区的诱变特异性的基础 乳糖操纵子，在体外和原核和真核系统。 在 原核细胞，将确定蒽环类药物的突变特异性 对于lacO和lacI的i-d区，在阻遏物诱导和未诱导下， 条件 lacO基因靶标作为单拷贝整合子的响应 将同时在哺乳动物细胞中进行研究。 拟议的研究将纳入位点特异性诱变方法， 正向突变分析，将特别强调 导致遗传物质缺失的过程。 推定缺失 起始序列将从体内诱导的 突变和体外研究中的lacI/lacO基因靶E.杆菌 体外实验将包括足迹法以确定结合 阿霉素对线性和回文单链DNA特异性 构建体，阿霉素诱导的DNA修复识别位点 酶、核酸酶SP和uvrABC外切酶以及阿霉素诱导的 可裂解的复合位点。 这项工作的总体目标是提供一个 更好地理解DNA序列依赖性药物作用的基础 细胞对阿霉素的毒性和突变反应的相互作用 作为研究嵌入剂的DNA导向机制的模型， 多效性DNA损伤潜力。

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.