Mechanistic Biology of Topoisomerase 1B

拓扑异构酶 1B 的机制生物学

• 批准号: 6890397
• 负责人: JAMES T. STIVERS
• 金额: $ 27.8万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6890397
• 关键词: DNA topoisomerases; catalyst; chemical cleavage; chemical kinetics; fluorine; intermolecular interaction; molecular dynamics; molecular genetics; nuclear magnetic resonance spectroscopy; nucleic acid structure; phosphoric ester; phosphorus compounds; stable isotope; thermodynamics; vaccinia virus

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.

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.在这项工作中，我们将采用谷谷病毒的小型序列特异性1B型托普（Topo），因为它是唯一可与详细的NMR，荧光以及动力学和热力学研究的IB型酶。 具体目的如下：（i）确定对DNA中CCCTT位点的特异性识别的基础。将分别使用新型的碱类似物和非桥接甲基膦酸酯取代碱和磷酸化酯相互作用在特异性中的重要性。 （ii）阐明亲核催化的机制。将使用诱变，非桥接磷酸盐剂取代的综合方法来解剖剪刀磷酸酯的催化相互作用。新型的固态重复NMR结构方法将用于确认这些相互作用。 （iii）了解我如何从DNA中去除超级螺旋。 TOPO我必须释放其对DNA的握持，以使超级侧侧放松发生。为了阐明这些关键运动，将使用19F标记的DNA分子和NMR光谱法来测量共价复合物中DNA的动力学。 此外，我们将使用带有单个切割位点的小型超螺旋质粒来评估三个关键变量在超侧侧弛豫机制上的作用：DNA超螺旋密度，价值复合物的寿命，以及酶在旋转DNA上的“紧密性”。我们预计这些测量结果将指导我们为设计Topo I抑制剂的努力，并设计酶以执行其他有用的DNA转换。