SPECIFICITY OF INTERCALATOR DIRECTED GENOTOXICITY

拟议研究的广泛目标是确定机制， 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用来提高选择性 抗肿瘤作用或降低这些非特异性遗传毒性 剂. 最近的研究表明，阿霉素引起的缺失突变，在 切除修复基因缺失株和缺失株的lacI基因。 杆菌 缺失终点发生在明显的药物位点附近 插入DNA中。 多柔比星诱导缺失的一个焦点 lac操作符表明与此药物特异性相互作用 结构 介导这些突变的机制尚不清楚， 可能涉及拓扑异构酶II和切除修复酶。 本研究项目的目标是通过以下方式确定机制： 所述嵌入剂在体内介导DNA损伤。 具体地说， 拓扑异构酶II和切除修复酶的作用 序列特异性嵌入剂与DNA的相互作用将得到解决。 突变谱将在E.杆菌 与放线菌素D，其具有已证实的序列特异性， 从阿霉素。 lacI中的特异性DNA序列，其中阿霉素和 放线菌素D单独与DNA相互作用，拓扑异构酶II或切除 将在体外定义修复酶。 这将用DNase I完成 足迹法、损伤分布和可裂解复合物测定。 的 定义的序列将被并入lac运算符， 用于整合到哺乳动物细胞系的基因组中的构建体。 然后，这些含有构建体的细胞系将包含严格的 删除检测系统 靶向以下物质的体外作用的序列 插层剂将与它们促进 哺乳动物细胞中的缺失。 这将允许识别 导致体内缺失的机制。

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.