Mechanistic Biology of Topoisomerase 1B

这个项目的长期目标是了解IB型DNA拓扑异构酶(TOPO)是如何催化 可逆的DNA链切割和宗教，以及如何化学和动态的性质 I磷酸酪氨酸I-DNA共价复合体促进DNA链等重要生物学过程 ITransfer、重组、超级线圈解卷和抗癌药物结合。在这项工作中，我们将使用 我来自痘苗病毒的小的、序列特异性的1B型TOPO，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：(一) 我确定了在DNA中特异性识别CCCTT位点的基础。基础和基础的重要性 将使用新的碱基类似物和非桥联来评估磷酸二酯相互作用的特异性 甲基膦酸根的取代反应。(2)阐明亲核催化的机理。这个 剪切型磷酸二酯的催化相互作用将使用以下两种组合方法进行剖析 突变，非桥联硫代磷代换。新型固态Redor核磁共振结构 将使用方法来确认这些交互。(Iii)了解Topo I如何从 DNATOPO I必须释放它对DNA的控制，才能允许超级线圈松弛发生。为了阐明这些关键的 运动，19F标记的DNA分子和核磁共振光谱将被用来测量动力学 共价复合体中的DNA。此外，我们还将使用带有单链切割的超螺旋小质粒 来评估三个关键变量在超螺旋松弛机制中的作用：DNA超螺旋 密度、共价复合体的寿命，以及酶对旋转的DNA的“紧密性”。我们 预期这些测量将指导我们设计TOPO I的抑制剂和设计 酶来执行其他有用的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.