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、前稳态动力学和理论计算分析以及新颖的化学探针来阐明 DNA 聚合酶催化的分子基础，其中碱基对辨别是与癌症相关的突变的基本问题。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）和动力学以及转化路径方法（项目 3）。我们通过用含有广泛不同静电荷和空间特性的多种卤代亚甲基衍生物替换一个或两个磷酸桥接氧分子，成功合成了 dNTP 底物类似物，这使我们能够从过渡态 (T) 角度探测保真度。这些底物类似物的使用是我们 PPG 的一个独特而强大的方面，将使我们能够首次使用立体异构探针研究 TS 效应，同时为靶向抑制 Pol p 提供可行的方法，从而实现癌细胞抑制（项目 3）。项目1的目标是获取高分辨率结构数据 Pol ¿ 的正常和异常形式，使用项目 3 中设计并在核心 B 中合成的 dNTP 类似物。项目 2 的目标是应用理论和计算机建模来对控制基态和 TS 中碱基选择的催化机制进行结构/功能分析。计算的目的是计算自由能，用于预测氨基酸侧链对保真度的单独贡献， 包括 pol 活性位点的底物结合和催化。我们 PPG 的核心是理论（项目 2）作为将结构分析（项目 1）与动力学机制分析（项目 3）结合起来的知识框架。对于实验者来说，测试先验的计算预测是不典型的。该 PPG 的一个决定性方面是其双向插层，其中结构数据作为计算预测的起点，通过实验进行测试，并使用实验数据来完善理论。