DNA Polymerase Fidelity Mechanisms: Theory and Experiment

DNA 聚合酶保真机制：理论与实验

• 批准号: 7693819
• 负责人: MYRON GOODMAN
• 金额: $ 111.59万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-16 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7693819
• 关键词: DNA; Polymerase; Fidelity; Mechanisms; Theory

DESCRIPTION (provided by applicant): This Program Project is designed to address fundamental issues in mutagenesis relevant to the root causes of cancer, in accordance with the mission of NCI. We propose to investigate the molecular basis of DNA polymerase accuracy, relating theory to experiment and vice versa, using human DNA polymerase beta as a model system. Pol beta plays a key role in the avoidance of cancer, because its loss of regulation or disruption by mutation induces chromosome instability and tumorigenesis. Our primary goals are focused on understanding the principles of polymerase fidelity defined by the detailed interactions between specific amino acid side chains, primer/template bases and dNTP substrates at the Pol active site. The Program Project contains three research projects, structural (Project 1), theoretical computational (Project 2), kinetics (Project 3) and three core facilities, a Biochemical Synthetic and Analysis Core (Core B), a Computational Core (Core C) and an Administrative Core (Core A). The goal of Project 1 is to obtain high-resolution structural data for normal and mutant forms of pol ? using a new class of nucleotide analogs designed in Project 3 and synthesized in Core B. These analogs will be used in drug design and delivery strategies to establish their potential use as anticancer agents in mouse and cultured cell model systems, in a translational approach to target bone tumors. A unique and timely aspect of the PPG is the application of theoretical and computer-modeling approaches to structure/function analysis of catalytic efficiencies in polymerase active sites, as proposed in Project 2. The modeling analysis calculates free energies, which are used to predict individual contributions of amino acid side chains to fidelity, including substrate binding and catalysis in the polymerase active site. The theory serves as the intellectual framework with which to marry structural analysis with kinetic mechanistic analyses described in Project 3. It is usually atypical for the experimentalist to test a priori computational predictions. Thus, a defining aspect of this PPG is its bidirectional interplay, where computational predictions are tested experimentally and new experimental data are used to refine the theory.

描述（由申请人提供）：该计划项目旨在解决与NCI的任务，解决与癌症根本原因有关的诱变中的基本问题。我们建议使用人DNA聚合酶β作为模型系统，研究DNA聚合酶精度的分子基础，将理论与实验相关，反之亦然。 POL Beta在避免癌症中起关键作用，因为它因突变而导致的调节或破坏会导致染色体不稳定性和肿瘤发生。我们的主要目标集中在理解聚合酶保真度的原理，这些原理是由POL活性位点的特定氨基酸侧链，引物/模板碱和DNTP底物之间的详细相互作用所定义的。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），动力学（项目3）和三个核心设施，一个生化合成和分析核心（核心B），计算核心（核心C）和一个管理核心（核心A）。项目1的目的是获得正常和突变形式的POL的高分辨率结构数据？使用在项目3中设计的一类新的核苷酸类似物并在核心B中合成。这些类似物将在药物设计和输送策略中使用，以在靶向骨肿瘤的转化方法中确定其作为小鼠和培养的细胞模型系统中抗癌药物的潜在用途。 PPG的一个独特而及时的方面是将理论和计算机模型的方法应用于项目2中提出的聚合酶活性位点的催化效率的结构/功能分析。建模分析计算自由能，这些分析用于预测氨基酸侧链对屈服于效率和凝结蛋白蛋白酶的依赖性链接的单个贡献，包括在catalstration catalsys中，这些构成链接构成了catimations canalsissive和cat catalsive in catalsive ins catalsive。该理论是将结构分析与项目3中描述的动力学机械分析结合的智力框架。实验学家测试先验计算预测通常是非典型的。因此，该PPG的一个定义方面是其双向相互作用，其中计算预测经过实验测试，并使用新的实验数据来完善理论。