DNA Polymerase Fidelity Mechanisms: Theory & Experiment

描述（由申请人提供）：根据NCI的使命，本计划项目的一般意义在于，它旨在解决诱变中的基本问题，并且这样做将与癌症的根本原因具有重要的相关性。 我们建议调查的DNA聚合酶的准确性，相关的理论实验，反之亦然，使用正常和突变的DNA聚合酶，包括易错聚合酶的分子和原子的基础。 我们的主要目标集中在理解聚合酶保真度的原则，动力学与热力学的关系，自由能的来源，使聚合酶能够区分对错，和活性位点的几何约束所定义的特定氨基酸侧链，引物/模板碱基和dNTP底物之间的详细相互作用在Pol活性位点。 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），动力学（项目3）和三个核心设施，生化分析核心（核心B），计算核心（核心C）和行政核心（核心A）。 项目1的目标是获得正常和突变形式的Pol beta的高分辨率结构数据，以研究这种至关重要的人类修复聚合酶的特异性和催化效率的机制，使用将在项目2中进行理论评估和项目3中进行实验的信息。 PPG的一个独特而及时的方面是在聚合酶活性位点的催化效率的结构/功能分析中应用理论和计算机建模方法，如项目2中所提出的。 建模分析计算用于预测氨基酸侧链对保真度的个体贡献的自由能，包括聚合酶活性位点中的底物结合和催化。 该理论作为知识框架，将结构分析与项目3中描述的动力学机理分析结合起来。 分子计算和实验之间通常的相互作用通常是单向的--文献中已有的实验数据被用来“拟合”理论，无论是好是坏。 对于实验主义者来说，检验先验的计算预测通常是非典型的。因此，这个PPG的一个定义方面是它的双向相互作用，其中计算预测实验测试和新的实验数据用于完善理论。

DESCRIPTION (provided by applicant): The general significance of this Program Project is that it is designed to address fundamental issues in mutagenesis and in so doing will have important relevance to the root causes of cancer, in accordance with the mission of NCI. We propose to investigate the molecular and atomic basis for DNA polymerase accuracy, relating theory to experiment and vice versa, using normal and mutant DNA polymerases including error-prone polymerases. Our primary goals are focused on understanding the principles of polymerase fidelity, the relationship of dynamics to thermodynamics, the source of free energy enabling the polymerase to distinguish right from wrong, and active site geometrical constraints as 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 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 beta to investigate the mechanism of specificity and catalytic efficiency of this critically important human repair polymerase, using information that will be evaluated theoretically in Project 2 and experimentally in Project 3. A unique and timely aspect of the PPG is the application of theoretical and compute modeling approaches in structure/function analysis of catalytic efficiencies in polymerase active sites, as proposed in Project 2. The modeling analysis calculates free energies 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 analysis described in Project 3. The usual interplay between molecular computations and experiment is usually unidirectional - experimental data already in the literature are used to "fit" the theory, either for better or for worse. 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.