Mechanistic Biology of Topoisomerase 1B

描述（由申请人提供）：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链切割和再连接，以及磷酸酪氨酸-DNA共价复合物的化学和动力学性质如何促进DNA链转移、重组、超螺旋解旋和抗癌药物结合等重要生物学过程。在这项工作中，我们将采用小，序列特异性1B型拓扑从牛痘病毒，因为它是唯一的IB型酶服从详细的NMR，荧光，动力学和热力学研究。 具体目标如下：（i）确定DNA中CCCTT位点特异性识别的基础。碱基和磷酸二酯相互作用在特异性中的重要性将分别使用新型碱基类似物和非桥接甲基膦酸酯取代进行评估。(ii)阐明亲核催化反应的机理。易分裂的磷酸二酯的催化相互作用将使用诱变，非桥接硫代磷酸酯取代的组合方法进行解剖。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如何从DNA中去除超螺旋。Topo I必须释放对DNA的控制，才能让超螺旋松弛发生。为了阐明这些临界运动，19 F标记的DNA分子和NMR光谱将用于测量共价复合物内DNA的动力学。 此外，我们将使用一个小的超螺旋质粒与一个单一的切割位点，以评估超螺旋松弛机制的三个关键变量的作用：DNA超螺旋密度，共价复合物的寿命，和“紧密性”的酶抓地力的旋转DNA。我们预计这些测量将指导我们设计Topo I抑制剂的努力，并设计酶以进行其他有用的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.