SPECIFICITY OF INTERCALATOR DIRECTED GENOTOXICITY

拟议研究的广泛目标是确定通过哪些机制 嵌入剂在DNA水平上介导突变和致死性。 这些知识最终可能被利用来增强选择性 抗肿瘤作用或降低这些化合物的非特异性遗传毒性 探员们。 最近的研究表明，阿霉素类药物会导致基因缺失突变。 切除修复熟练株和缺失株LacI基因 Coli.缺失终点发生在表观药物部位附近 在DNA中嵌入。阿霉素性缺失的一个焦点 Lac操作员建议与此进行药物特异性相互作用 结构。调节这些突变的机制尚不清楚，但 可能涉及拓扑异构酶II和切除修复酶。 该研究项目的目标是通过以下方式确定机制 哪些嵌入剂在体内介导DNA损伤。具体来说， 拓扑异构酶II和切除修复酶在细胞外的作用 本课程将介绍序列特定的嵌入物与DNA的相互作用。 突变谱将在DNA序列水平上进行分析 与放线菌素D不同，放线菌素D具有已证实的序列特异性 来自阿霉素。LACI中的特定DNA序列，其中阿霉素和 放线菌素D单独与DNA、拓扑异构酶II或切除相互作用 修复酶将在体外被定义。这将通过DNase I完成 足迹、损伤分布和可裂解复合体分析。这个 定义的序列将合并到包含以下内容的lac操作符中 用于整合到哺乳动物细胞系基因组中的结构。 然后，包含细胞系的这些构建物将包括严格的 删除检测系统。靶向体外效应的序列 插层剂将与它们的促进能力相关 哺乳动物细胞中的缺失。这将允许识别 体内导致缺失的机制。

The broad goal of the proposed studies is to define mechanisms by which intercalating agents mediate mutation and lethality at the DNA level. This knowledge may ultimately be exploited to enhance the selective anti-tumor effect or reduce the non-specific genotoxicity of these agents. Recent work suggests that doxorubicin causes deletion mutations in the lacI gene of excision repair proficient and deficient strains of E. coli. Deletion endpoints occur adjacent to sites of apparent drug intercalation in the DNA. A focus of doxorubicin induced deletions in the lac operator suggests a drug specific interaction with this structure. The mechanisms mediating these mutations are not clear but may involve topoisomerase II and excision repair enzymes. The objective of this research project is to identify mechanisms by which intercalating agents mediate DNA damage in vivo. Specifically, the roles of topoisomerase II and excision repair enzymes in addition to sequence specific intercalator interactions with DNA will be addressed. Mutational spectra will be analyzed at the DNA sequence level in E. coli with actinomycin D which has a proven sequence specificity which differs from doxorubicin. Specific DNA sequences in lacI where doxorubicin and actinomycin D interact with DNA alone, topoisomerase II or excision repair enzymes will be defined in vitro. This will be done with DNase I footprinting, damage distribution and cleavable complex assays. The defined sequences will be incorporated into lac operator containing constructs for integration into the genome of mammalian cell lines. These construct containing cell lines will then comprise a stringent deletion detection system. Sequences which target in vitro effects of intercalating agents will be correlated with their ability to promote deletion in mammalian cells. This will allow identification of mechanisms leading to deletion in vivo.