DNA Polymerase Fidelity Mechanisms: Theory and Experiment

DNA 聚合酶保真机制：理论与实验

• 批准号: 9125787
• 负责人: MYRON GOODMAN
• 金额: $ 110.51万
• 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/9125787
• 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 Analysis; 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; Play; 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; atomic interactions; 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; translational approach; 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、稳态前动力学和理论计算分析以及新的化学探针来阐明DNA聚合酶催化结合碱基对区分的分子基础，这是与癌症相关的突变的基本问题。该计划项目包括三个研究项目，结构(项目1)、理论计算(项目2)和动力学与翻译路径的方法(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联的氧分子替换为各种含有截然不同的静电电荷和空间性质的卤代亚甲基衍生物，使我们能够从过渡态(T)的角度来探测保真度。这些底物类似物的使用是我们PPG的一个独特而强大的方面，将使我们能够首次使用立体异构体探针研究TS效应，同时提供一种可行的方法，在通向癌细胞抑制的道路上靶向抑制Polp(项目3)。项目1的目标是获得高分辨率的结构数据 使用在项目3中设计并在核心B中合成的dNTP类似物，Pol？的正常和异常形式。项目2的目标是应用理论和计算机模拟来执行对在基态和TS中支配碱基选择的催化机制的结构/功能分析。这些计算的目的是计算自由能，这些自由能被用来预测氨基酸侧链对精确度的个体贡献， 包括底物结合和POL活性部位的催化作用。我们PPG的核心是，理论(项目2)作为将结构分析(项目1)与运动力学分析(项目3)相结合的智力框架。这是非典型的实验者t检验先验计算预测。这种PPG的一个定义方面是它的双向交织，其中结构数据作为计算预测的起点，并通过实验进行测试，并使用实验数据来完善理论。