DIYNENE ANTIBIOTICS AND THEIR DNA CLEAVAGE CHEMISTRY

加利车霉素γ 1 I（CLM），新制癌素发色团， 埃斯波霉素A1、动力霉素、凯达西定、c-1027和马度罗肽素是 结构上前所未有且不断增长的二炔家族成员 抗肿瘤抗生素通过硫醇和/或简单的还原活化 热重排产生双自由基物质， 电环化过程是该类的特征。当 结合在DNA的小沟中，这些自由基引发螺旋断裂 通过从一股或两股中提取氢来实现。所得DNA 自由基与分子氧反应并碎裂增强。而 单链断裂通常是观察到的主要裂解事件，CLM 它既具有明显的序列选择性， 卡特使用本更新中概述的各种实验 我们试图了解这些属性的起源以及如何 CLM可用作蛋白质/核酸结构的探针，并可用于 分子生物学中有用工具的设计。假定的致命性 DNA损伤、细胞凋亡诱导和细胞毒性 以及这些化合物对细胞周期的影响， 通过优先摄取或选择性递送至癌细胞， 有效的治疗是可能的。测试后者的临床试验 原则正在进行中。 与CLM相关的低动力学同位素效应的研究- 从DNA中诱导氢提取和明显缺乏 将完成同位素诱导的分支。这两个事件 将区分以同时或逐步方式发生。的 A束的异常行为将被检查为动力学加速度 作为小沟变窄的函数。氢的效率 将对提取进行监测， 裂解级联将作为温度的函数进行评价。的 CLM与超螺旋质粒DNA的相互作用将被研究， 插入的A/T富集片段的功能导致不同程度的 曲率计划用核小体进行广泛的实验来研究 “热点”的出现，以了解CLM裂解是否是 与核小体中的物理位置或 特定的DNA序列将使用自动化方法来评估 在混合序列核小体中产生的大量数据。一 CLM的降解产物基本上没有序列选择性， 保留双链切割特性将与同源结构域连接， 以实现用于高效切割DNA的特异性结合。成功 在这项努力中，将在已经存在的DNA序列测试中进行评估。 并将为最终的实验奠定基础， 用于基因鉴定，例如在果蝇中。最后，CLM 与转移RNA、核糖体RNA和信使RNA的反应将是 研究了在真核细胞中，RNA是比RNA更容易接近的靶点。 DNA.这些实验将监测RNA对反应的敏感性 与CLM，特别是核糖体中的rRNA进行比较， 核小体和染色质。

Calicheamicin gamma1I (CLM), the neocarzinostatin chromophore, esperamicin A1, dynemicin, kedarcidin, c-1027 and maduropeptin are members of the structurally unprecedented and growing family of diynene antitumor antibiotics. Reductive activation by thiols and/or simple thermal rearrangement generates diradical species by an electrocyclization process that is characteristic of this class. When bound in the minor groove of DNA these radicals initiate helix cleavage by hydrogen abstraction from one or both strands. The resulting DNA radicals react with molecular oxygen and fragmentation ensues. While single-strand breaks are typically the major cleavage event observed, CLM is both notably sequence-selective and very largely a double-strand cutter. Using a diverse array of experiments outlined in this renewal proposal we seek to understand the origins of these properties and how CLM can be used as a probe of protein/nucleic acid structure and in the design of useful tools in molecular biology. The presumed lethality of DNA damage, the induction of apoptosis and the demonstrated cytotoxicity and cell cycle effects of these compounds have animated hope that, through preferential uptake or selective delivery to cancer cells, effective therapies might be possible. Clinical trials testing the latter principle are underway. Investigation of the low kinetic isotope effects associated with CLM- induced hydrogen abstraction from DNA and the apparent absence of isotope-induced branching will be completed. Whether these two events occur in a simultaneous or stepwise manner will be distinguished. The unusual behavior of A-tracts will be examined for kinetic acceleration as a function of minor groove narrowing. The efficiencies of hydrogen abstraction will be monitored and the distribution of cuts in the cleavage cascade will be evaluated as a function of temperature. The interaction of CLM with supercoiled plasmid DNA will be investigated as a function of inserted A/T-rich segments causing varying degrees of curvature. Extensive experiments are planned with nucleosomes to study the occurrence of "hot spots" to understand whether CLM cleavage is associated with physical location in the nucleosome or features of particular DNA sequences. Automated methods will be used to evaluate large amounts of data generated in mixed sequence nucleosomes. A degradation product of CLM having essentially no sequence selectivity but retaining double-strand cutting properties will be linked to homeodomains to achieve specific binding for high efficiency cleavage of DNA. Success in this effort will be evaluated in tests with DNA sequences already in hand and will lay the groundwork for eventual experiments to be undertaken for gene identification e.g. in Drosophila. Finally, CLM reaction with transfer-, ribosomal- and messenger-RNA will be investigated. In eukaryotic cells, RNA is a more accessible target than DNA. These experiments will monitor the susceptibility of RNA to reaction with CLM and particularly with rRNA in the ribosome for comparison to nucleosomes and chromatin.