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Structural and biophysical studies of DNA topoisomerases

DNA 拓扑异构酶的结构和生物物理研究

基本信息

批准号：
9235823
负责人：
Alfonso Mondragon
金额：
$ 32.09万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9235823
关键词：
Active Sites Affect Anti-Bacterial Agents Antibiotics Archaea Bacteria Biochemical Biochemistry Biophysics Cell physiology Cells Chromosome Segregation Complex Cryoelectron Microscopy DNA DNA Binding Domain DNA Gyrase DNA Repair DNA Topoisomerases DNA topoisomerase V DNA-Protein Interaction Data Electron Microscopy Enzymes Family Fluorescence Fluorescence Microscopy Funding Genetic Recombination Genetic Transcription Goals Heterogeneity Laboratories Length Life Lyase Magnetism Malignant Neoplasms Methods Molecular Biology Molecular Conformation Molecular Machines Monitor Movement Positioning Attribute Property Protein Conformation Protein Family Proteins Reaction Relaxation Reporting Research Design Resolution Role Site Structure Superhelical DNA Techniques Testing Therapeutic Therapeutic Agents Time Topoisomerase Topoisomerase III Type I DNA Topoisomerases Work X-Ray Crystallography base biophysical analysis chemotherapeutic agent design design and construction ds-DNA instrument macromolecule nanoscale novel novel therapeutics polypeptide repair enzyme repaired single molecule single-molecule FRET structural biology synergism

项目摘要

Project Summary Topoisomerases are ubiquitous proteins found across all three domains of life (bacteria, archaea, and eukarya). They are involved in several cellular processes and the importance of their cellular role is underscored by the fact that they are the target of several cancer chemotherapeutic agents and antibiotics. Topoisomerases change the topology of DNA by transiently breaking one (type I) or two (type II) DNA strands to allow passage of either a single or double DNA strand through the break or swiveling of one strand around the other. The study of the structure and function of topoisomerases promises not only to further our understanding of proteins that interact with DNA and alter its topological properties, but also to provide important information to aid in the design of new therapeutic agents. This proposal is concerned with biochemical, biophysical, and structural studies of different topoisomerases. The long term goal of the project is to provide a comprehensive understanding of topoisomerase action at many different length and time scales, from the atomic level to the nano scale. In the past period we made substantial progress towards this goal, including using single molecule methods to discover the role of pauses in the mechanism of type IA topoisomerases, characterizing the topoisomerase active site of topoisomerase V, solving the structures of 78 kDa and 97 kDa fragments of topoisomerase V containing one or two of the DNA repair sites, and discovering the presence of a third repair domain. In addition, we continue making progress on our work on a complex of gyrase with a large DNA fragment and also developed a new instrument that is capable of simultaneously manipulating single DNA molecules and reporting on movements of a protein by fluorescence. Our studies are providing important information on topoisomerases and allowing us to relate atomic structures to the wealth of existing functional, biochemical, and biophysical data. For the next project period we propose to continue and expand our studies of topoisomerases. The specific aims for this proposal are: i) to probe the mechanism of type IA topoisomerases by novel single molecule approaches, ii) to continue our structural and biochemical studies of topoisomerase V, and iii) to continue our structural and mechanistic studies of a gyrase/DNA complex. The work is based on a combination of molecular biology and biochemical methods to produce and characterize the macromolecules that we require for our work, X-ray crystallography and electron microscopy methods to solve their structures, and single molecule studies to elucidate their mechanism.

项目摘要拓扑异构酶是在生命的所有三个领域（细菌、古细菌和细菌）中发现的普遍存在的蛋白质。真核生物）。它们参与几种细胞过程，其细胞作用的重要性是事实上，它们是几种癌症化疗剂和抗生素的靶标。拓扑异构酶通过瞬时破坏一条（I型）或两条（II型）DNA链来改变DNA的拓扑结构允许单链或双链DNA通过断裂或旋转一条链，另一对拓扑异构酶结构和功能的研究不仅有助于我们进一步了解拓扑异构酶的结构和功能，了解与DNA相互作用并改变其拓扑性质的蛋白质，但也提供重要的信息，以帮助设计新的治疗剂。这项建议涉及不同的生物化学，生物物理和结构研究。拓扑异构酶该项目的长期目标是全面了解拓扑异构酶在许多不同的长度和时间尺度上起作用，从原子级到纳米级。在在过去的一段时间里，我们朝着这一目标取得了实质性进展，包括使用单分子方法，发现停顿在IA型拓扑异构酶机制中的作用，表征拓扑异构酶拓扑异构酶V的活性位点，解析拓扑异构酶V的78 kDa和97 kDa片段的结构含有一个或两个DNA修复位点，并发现第三个修复结构域的存在。在此外，我们继续在具有大DNA片段的促旋酶复合物方面取得进展，开发了一种新的仪器，能够同时操纵单个DNA分子，通过荧光报告蛋白质的运动。我们的研究提供了重要的信息，拓扑异构酶，使我们能够将原子结构与现有的丰富的功能，生物化学，和生物物理数据。在下一个项目期间，我们建议继续并扩大我们对以下方面的研究：拓扑异构酶本研究的具体目标是：i）探索IA型拓扑异构酶的作用机制通过新的单分子方法，ii）继续我们对拓扑异构酶V的结构和生物化学研究，和iii）继续我们对促旋酶/DNA复合物的结构和机理的研究。这项工作是基于一个结合分子生物学和生物化学方法来生产和表征大分子我们的工作需要X射线晶体学和电子显微镜方法来解决它们的结构，和单分子研究来阐明它们的机制。