Mechanistic Biology of Topoisomerase 1B

本项目的长期目标是了解IB型DNA拓扑异构酶（topo）如何催化 可逆的DNA链切割和重新连接，以及如何化学和动态性质的 磷酸酪氨酸-DNA共价复合物促进了DNA链形成等重要的生物学过程 转移、重组、超螺旋解旋和抗癌药物结合。在这项工作中，我们将使用 I小的、序列特异性的1B型拓扑异构酶来自牛痘病毒，因为它是唯一适合于 详细的核磁共振、荧光、动力学和热力学研究。具体目标如下： 确定DNA中CCCTT位点特异性识别的基础。基地的重要性， 特异性磷酸二酯相互作用将使用新的碱基类似物和非桥连 甲基膦酸酯取代。(ii)阐明亲核催化反应的机理。的 易断裂的磷酸二酯的催化相互作用将使用以下结合的方法进行剖析： 诱变，非桥接硫代磷酸酯取代。新型固态REDOR NMR结构 方法将用于确认这些相互作用。(iii)了解Topo I如何去除超螺旋 DNA. Topo I必须释放对DNA的控制，才能让超螺旋松弛发生。为了阐明这些关键的 运动，19 F标记的DNA分子和NMR光谱将被用来测量动力学的 共价复合物中的DNA。此外，我们将使用一个小的超螺旋质粒与一个单一的切割 站点，以评估三个关键变量对超螺旋松弛机制的作用：DNA超螺旋 密度、共价复合物的寿命以及酶对旋转DNA的“紧密性”。我们 预计这些测量将指导我们的努力，设计抑制剂的拓扑异构酶I和工程师的 酶进行其他有用的DNA转化。

The long-term objective of this project is to understand how type IB DNA topoisomerases (topo) catalyze reversible DNA strand cleavage and religation, and how the chemical and dynamic nature of the I phosphotyrosyI-DNA covalent complex promotes such important biological processes as DNA strand Itransfer, recombination, supercoil unwinding, and anticancer drug binding. In this work, we will employ the I small, sequence specific type 1B topo from vaccinia virus because it is the only type IB enzyme amenable to Idetailed NMR, fluorescence, kinetic and thermodynamic studies. The specific aims are as follows: (i) I Determine the basis for specific recognition of CCCTT sites in DNA. The importance of base and phosphodiester interactions in specificity will be evaluated using novel base analogs and nonbridging methylphosphonate substitutions, respectively. (ii) Elucidate the mechanism of nucleophilic catalysis. The catalytic interactions of the scissile phosphodiester will be dissected using the combined approach of mutagenesis, nonbridging phosphorothioate substitutions. Novel solid-state REDOR NMR structural methods will be used to confirm these interactions. (iii) Understand how Topo I removes supercoils from DNA. Topo I must release its grip on DNA to allow supercoil relaxation to occur. To elucidate these critical motions, 19F-labeled DNA molecules and NMR spectroscopy will be used to measure the dynamics of the DNA within the covalent complex. In addition, we will use a small supercoiled plasmid with a single cleavage site, to evaluate the role of three key variables on the supercoil relaxation mechanism: the DNA superhelical density, the lifetime of the covalent complex, and the "tightness" of the enzyme grip on the rotating DNA. We anticipate these measurements will guide our efforts to design inhibitors of Topo I and to engineer the enzyme to perform other useful DNA transformations.