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，以应用X射线结构研究，质疑状态动力学和理论计算分析和新的化学问题，以阐明融合了基础对歧视的DNA聚合酶催化的分子基础，这是碱基对歧视，这是与癌症相关的诱变中的基本问题。该计划项目包含三个研究项目，结构化（项目1），理论计算（项目2）和动力学以及转化路径的方法（项目3）。我们通过用一个或两个磷酸盐桥接氧分子用大量含有广泛不同静电电荷和空间特性的大量卤代甲基衍生物代替一个或两个磷酸盐桥接氧分子来成功合成DNTP底物类似物的成功，使我们能够从过渡状态（t）的角度探测。这些底物类似物的使用是我们PPG的独特功能，它将首次使用立体异构体问题研究TS效应，同时为靶向靶向POL P提供可行的方法，以实现癌细胞抑制的路径（项目3）。项目1的目的是获得高分辨率结构数据 使用项目3中设计的DNTP类似物并在核心B中合成的dNTP类似物的正常和异常形式。项目2的目标是将理论和计算机建模的应用用于对催化机制进行结构/功能分析，这些催化机制控制基础选择中的基础选择。这些计算旨在计算自由能，这些能量用于预测氨基酸侧链对富达性的个体贡献， 包括POL活性位点中的底物结合和催化。我们PPG的核心是，该理论（项目2）是将结构分析（项目1）与动力学机械分析（项目3）结合的智力框架。实验者t检验的先验计算预测是非典型的。该PPG的一个定义方面是其双向相互，其中结构数据是计算预测的起点，这些预测是通过实验测试的，以及使用实验数据来完善理论的起点。