STUDIES OF ACTINOMYCIN D INDUCED DNA STRUCTURE

放线菌素D是一种嵌入型抗生素/抗肿瘤药物 它以平衡的方式与DNA结合。这种药物表现出一种 5‘-GC-3’序列具有明显的特异性。活体内 这种药物的疗效一直被认为是DNA的作用 有约束力的。插层剂如放线菌素D也有 已经被证明强加了明确的DNA二级结构 它们的嵌入处发生了变化。在中概述的研究中 这一建议提出了改变DNA二级结构的性质 放线菌素，被视为模型嵌入器，将在 一系列酶促合成的DNA双链。初步 实验表明，核酸内切酶I是理想的 适合于这项任务具有定位放线菌素的能力 对DNA分子的影响以及对DNA继发性改变的反应 由配基结合诱导的结构。通过考虑已知的 结构：序列基序将有可能在单个 碱基对解析结构信息的传播方式 通过DNA双螺旋。具体地说，DNA酶I的裂解率 将在四个B型DNA双链上进行检查以产生 关于酶的准确报告能力的信息 局部螺旋扭转值。这些信息将用于 在没有DNA的情况下使用“改变的”DNA序列的实验 放线菌素D评价其基本结构决定 所采用的序列。然后，相同的序列将被 在放线菌素D结合的情况下进行分析 与改变的DNA结构并列。这样一来，就是 可以评估插层的二级结构效应 DNA结合。对拓扑异构酶敏感的事实是 DNA二级结构的改变对插层的响应 体内的分子将这种结构反应指向为 在未来的设计中很重要，临床上重要的，抗 抗癌药物。

Actinomycin D is an intercalating antibiotic/antitumor agent which binds in an equilibrium fashion to DNA. The drug exhibits a pronounced specificity for 5'-GC-3' sequencies. The in vivo efficacy of the drug has been reputed to be a function of DNA binding. Intercalating agents such as actinomycin D have also been shown to impose well defined DNA secondary structure changes at their intercalation site. In the research outlined in this proposal the DNA secondary structure altering properties of actinomycin, viewed as a model intercalater, will be examined on a series of synthetic DNA duplexes enzymatically. Preliminary experiments have shown that the endonuclease DNAse I is ideally suited for this task having the ability to both locate actinomycin on DNA molecules and to respond to alteration in DNA secondary structure induced by ligand binding. By taking into account known structure: sequence motifs it will be possible to assess at single base-pair resolution how structural information is propagated through a DNA double helix. Specifically, DNAse I cleavage rates will be examined on four model B DNA duplexes to generate information on the ability of the enzyme to accurately report local helical twist values. This information will be utilized in experiments employing 'altered' DNA sequences in the absence of actinomycin D to evaluate the basal structural determinates of the sequences employed. The same sequences will then be analyzed in the presence of an actinomycin D bound in juxtaposition to the altered DNA structures. In this way it is possible to assess the secondary structure effects of intercalative DNA binding. The fact that topoisomerases which are sensitive to alterations in DNA secondary structure respond to intercalating molecules in vivo points to this type of structural response as important in the future design of new, clinically important, anti- cancer agents.