ANTHRACYCLINE-INDUCED DNA SEQUENCE SPECIFIC MUTATION

蒽环类药物诱导的 DNA 序列特异性突变

• 批准号: 3197519
• 负责人: W DAVID SEDWICK
• 金额: $ 16万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3197519
• 关键词: DNA; DNA footprinting; DNA gyrase; DNA repair; DNA replication; DNA topoisomerases; Escherichia coli; activation analysis; anthracyclines; bacterial DNA; chemical binding; cytogenetics; cytotoxicity; doxorubicin; drug interactions; enzyme complex; exonuclease; gene deletion mutation; gene mutation; genetic mapping; genetic models; lac operon; model design /development; nucleic acid sequence; oncogenes; operon; polymerase chain reaction; site directed mutagenesis

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.

阿霉素和相关的蒽环类类似物仍然是一线药物 尽管存在相关毒性，但癌症化疗中的药物 限制了其长期临床使用。 然而，机制之间的相互作用 导致其毒性仍不清楚。 我们实验室最近的工作 已证明阿霉素与操作者有选择性相互作用 uvrB 大肠杆菌菌株中的 lacI 区域，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 在这种药物的致突变性。 本提案旨在调查 蒽环类药物对 lacO 区域的诱变特异性的基础 紫胶操纵子，在体外以及在原核和真核系统中。 在 原核生物，将确定蒽环类药物的突变特异性 对于阻遏物诱导和非诱导下的 lacO 和 lacI 的 i-d 区域 状况。 lacO 基因靶标作为单拷贝整合体的反应 在 lacI 控制下，我们将同时在哺乳动物细胞中进行研究。 拟议的研究将把位点特异性诱变方法纳入 正向突变分析，其中特别强调 导致遗传物质缺失的过程。 推定删除 起始序列将通过体内诱导的分析来鉴定 大肠杆菌 lacI/lacO 基因靶标的突变和体外研究。 体外实验将包括足迹以确定结合 阿霉素对线性和回文单链 DNA 的特异性 构建体，阿霉素诱导的 DNA 修复识别位点 酶、核酸酶 SP 和 uvrABC 核酸外切酶，以及阿霉素诱导的 可切割的复杂位点。 这项工作的总体目标是提供一个 为更好地理解 DNA 序列依赖性药物的作用奠定基础 细胞对阿霉素的毒性和突变反应中的相互作用 作为研究嵌入剂 DNA 定向机制的模型 多效性 DNA 损伤潜力。