DNA Polymerase Fidelity Mechanisms: Theory and Experiment

DNA 聚合酶保真机制：理论与实验

• 批准号: 8549424
• 负责人: MYRON GOODMAN
• 金额: $ 114.68万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-03 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8549424
• 关键词: Active Sites; Affinity; Amino Acids; Area; Base Excision Repairs; Base Pairing; Binding; Biochemistry; Biological Sciences; Catalysis; Cations; Cells; Cereals; Charge; Chemicals; Chemistry; Chicago; Computer Simulation; Coupled; DNA; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Development; Discipline; Discrimination; Drug Design; Electronics; Electrostatics; Enzymes; Event; Family; Free Energy; Future; Goals; Grant; Individual; Investigation; Kinetics; Left; Life; Link; Malignant Neoplasms; Measures; Mitochondria; Modeling; Molecular; Mutagenesis; Myron; Nitrogen; Nucleotides; Oxygen; Polymerase; Principal Investigator; Property; Proteins; Research Personnel; Research Project Grants; Resolution; Roentgen Rays; Role; Side; Specificity; Stereoisomer; Structure; Testing; Theoretical model; Time; United States National Institutes of Health; Universities; Variant; analog; base; bisphosphonate; cancer cell; carbene; computer studies; design; divalent metal; human DNA; inhibitor/antagonist; innovation; inorganic phosphate; medical schools; mutant; novel; programs; research study; scaffold; simulation; success; theories; tumorigenesis

This PPG integrates multiple disciplines to apply X-ray structural studies, presteady state kinetic and theoretical computational analyses and novel chemical probes to elucidate the molecular basis of DNA polymerase catalysis incorporating base-pair discrimination, a fundamental issue in mutagenesis relevant to cancer. The Program Project contains three research projects, structural (Project 1), theoretical computational (Project 2), and kinetics coupled with an approach toward translational paths (Project 3). Our success at synthesizing dNTP substrate analogs, by replacing one or both phosphate bridging oxygen molecules with a large variety of halo-methylene derivatives containing widely differing electrostatic charge and steric properties, allows us to probe fidelity from a transitions state (T) perspective. The use of these substrate analogs is a uniquely powerful aspect of our PPG, and will allow us for the first time to investigate TS effects using stereoisomeric probes, while offering a feasible approach for targeted inhibition of Pol p, on a path toward cancer cell inhibition (Project 3). The objective of Project 1 is to obtain high-resolution structural data for normal and aberrant forms of Pol ¿, using the dNTP analogs designed in Project 3 and synthesized in Core B. The goal of Project 2 is the application of theoretical and computer modeling to perform structure/function analyses of catalytic mechanisms that govern base selection both in the ground-state and TS. The computations are aimed at calculating free energies, which are used to predict individual contributions of amino acid side chains to fidelity, including substrate binding and catalysis in the pol active site. Central to our PPG is that the theory (Project 2) serves as the intellectual framework with which to marry structural analysis (Project 1) with kinetic mechanistic analysis (Project 3). It is atypical for the experimentalist t test a priori computational predictions. A defining aspect of this PPG is its bidirectional interply, where structural data serve as a starting point for computational predictions, which are tested experimentally, and where the experimental data are used to refine the theory.

该PPG整合了多个学科，应用x射线结构研究，预稳态动力学和理论计算分析以及新型化学探针来阐明DNA聚合酶催化的分子基础，包括碱基对识别，这是与癌症相关的诱变的基本问题。该计划项目包含三个研究项目，结构（项目1），理论计算（项目2），动力学与方法对平移路径（项目3）。我们成功地合成了dNTP底物类似物，通过将一个或两个磷酸桥接氧分子替换为含有多种不同静电电荷和位阻性质的halo-亚甲基衍生物，使我们能够从过渡态(T)的角度探测保真度。这些底物类似物的使用是我们PPG的一个独特强大的方面，并将使我们首次使用立体异构体探针研究TS的作用，同时为靶向抑制Pol p提供一种可行的方法，在通往癌细胞抑制的道路上（项目3）。项目1的目标是获得高分辨率的结构数据