DNA CONFORMATION AND ENEDIYNE TARGET RECOGNITION

这项研究的目的是确定药物的作用 结构和DNA构象在生物识别中的作用 相关的DNA靶点。研究将集中在 核小体DNA与四种烯二炔类抗癌药物相互作用 抗生素：新制癌素（NCS），埃斯帕霉素A1（ESP A1）， 加利车霉素γ 1 [I]（CAL）和C-1027。我们已经确定超感知觉 A1通过邻氨基苯甲酸酯部分的嵌入与DNA结合， 在C-1027中存在类似的结构表明，它也应该 进行嵌入。然而，CAL可以选择弯曲的DNA结构， 并且可以诱导质粒DNA中的螺旋缠绕的等价物。这些 假设将在前两个具体目标的测试， 提议C-1027的嵌入问题将通过以下方式解决： 表征染色质中的DNA损伤和结合药物对 裸DNA的超螺旋和粘度特性。的 CAL结合和DNA弯曲或柔性之间的关系将 通过凝胶电泳和DNA环闭合分析进行研究， 其中CAL/ESP衍生物用于鉴定关键药物结构。 这种关系表明CAL损伤位点可能是非随机的， 分布在基因组中，这一假设将由 核小体DNA中损伤位点的统计测序， 分析确定的长区域内损坏地点的周期性， DNA序列。在第三个具体目标中，烯二炔目标识别 过程将在模型核小体系统中进行研究。CAL损坏是 发现在核小体中DNA急剧弯曲的部位增强 在5S rDNA上重组。这种观察的普遍性将 在分离的核小体和在 E.杆菌为了研究这些因素 参与这项增强，CAL损坏网站将被放置在 在核小体DNA中的不同位置。最后，CAL、ESP A1和 NCS在核小体的不同区域产生损伤，但它们是 都是强效细胞毒性剂根据观察到的NCS偏好 对于破坏转录活性基因，假设， 烯二炔类化合物相似的细胞毒性可能部分与 存在于活性基因中的核小体结构改变。为了验证这一 假设，烯二炔介导的DNA损伤将在 重组和分离的核小体变异体， 转录活性研究结果将与 经典核小体 拟议研究的结果应具有广泛的影响， 设计烯二炔试剂，用于确定生物机制 烯二炔类和其他DNA导向的抗癌药物的作用，和 用于开发体内DNA结构的分子探针。 的 制定生物相关模型目标的重要性 药物-DNA相互作用的研究是清楚的，因为它们揭示了 在其他DNA模型中没有观察到的分子识别特征。

The objective of the proposed research is to define the roles of drug structure and DNA conformation in the recognition of biologically- relevant DNA targets by anticancer drugs. The research will focus on the interactions between nucleosomal DNA and four enediyne antitumor antibiotics: neocarzinostatin (NCS), esperamicin A1 (ESP A1), calicheamicin gamma1[I] (CAL), and C-1027. We have established that ESP A1 binds to DNA by intercalation of an anthranilate moiety, and the presence of a similar structure in C-1027 suggests that it too should undergo intercalation. CAL, however, may select curved DNA structures, and may induce the equivalent of helical winding in plasmid DNA. These hypotheses will be tested in the first two specific aims of the proposal. The question of intercalation by C-1027 will be addressed by characterizing DNA damage in chromatin and the effect of bound drug on the supercoiling and viscometric properties of naked DNA. The relationship between CAL binding and DNA bending or flexibility will be investigated by gel electrophoresis and DNA circle-closure assays, with CAL/ESP derivatives employed to identify critical drug structures. Such a relationship suggests that CAL damage sites may be nonrandomly distributed in the genome, an hypothesis that will be tested by statistical sequencing of damage sites in nucleosomal DNA and by analysis of the periodicity of damage sites in long tracts of defined- sequence DNA. In the third specific aim, enediyne target recognition processes will be studied in model nucleosome systems. CAL damage was found to be enhanced at a site of sharp DNA bending in nucleosomes reconstituted on 5S rDNA. The universal nature of this observation will be tested in isolated nucleosomes and nucleosomes reconstituted on a fragment of the tyrT gene of E. coli. To investigate the factors involved in this enhancement, CAL damage sites will be placed in different locations in the nucleosomal DNA. Finally, CAL, ESP A1 and NCS produce damage in different regions of the nucleosome, yet they are all potent cytotoxic agents. Based on the observed preference of NCS for damaging transcriptionally-active genes, it is hypothesized that the similar cytotoxicities of the enediynes may relate in part to the altered structure of nucleosomes present in active genes. To test this hypothesis, enediyne-mediated DNA damage will be studied in reconstituted and isolated nucleosome variants associated with transcriptional activity. The results will be compared to studies in classical nucleosomes. The results of the proposed studies should have broad implications for the design of enediyne agents, for identifying the biologic mechanisms of action of the enediynes and other DNA-directed anticancer drugs, and for the development of molecular probes of DNA structure in vivo. The importance of developing biologically-relevant model targets for the study of drug-DNA interactions is clear, in as much as they reveal features of molecular recognition not observed in other DNA models.