Mechanistic Biology of Topoisomerase 1B

描述(申请人提供)：该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链切割和宗教，以及磷酸酪氨酸I-DNA共价复合体的化学和动态性质如何促进DNA链转移、重组、超级线圈解旋和抗癌药物结合等重要生物过程。在这项工作中，我们将使用来自痘苗病毒的小的、序列特异性的1B型TOPO，因为它是唯一可以进行详细的核磁共振、荧光以及动力学和热力学研究的IB酶。具体目标如下：(I)确定特定识别DNA中CCCTT位点的基础。碱基和磷酸二酯的相互作用在特异性中的重要性将分别使用新的碱基类似物和非桥联的甲基膦酸类取代物来评估。(2)阐明亲核催化的机理。剪切型磷二酯的催化相互作用将使用诱变和非桥联硫代磷代换相结合的方法进行剖析。新的固体红外线核磁共振结构方法将被用来证实这些相互作用。(Iii)了解Topo I如何从DNA中移除超级线圈。TOPO 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分子和核磁共振波谱将被用来测量DNA在共价复合体中的动力学。此外，我们将使用一个具有单一切割位点的小超螺旋质粒，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DNA的抓握能力。我们预计这些测量将指导我们设计Topo I的抑制剂，并设计这种酶来执行其他有用的DNA转化。

DESCRIPTION (provided by applicant): 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 phosphotyrosyI-DNA covalent complex promotes such important biological processes as DNA strand transfer, recombination, supercoil unwinding, and anticancer drug binding. In this work, we will employ the small, sequence specific type 1B topo from vaccinia virus because it is the only type IB enzyme amenable to detailed NMR, fluorescence, and kinetic and thermodynamic studies. The specific aims are as follows: (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.