DNA Polymerase Fidelity Mechanisms: Theory and Experiment

描述(由申请者提供)：该计划项目旨在根据NCI的使命，解决与癌症根本原因相关的突变的基本问题。我们建议以人DNA聚合酶β作为模型系统，研究DNA聚合酶准确性的分子基础，将理论与实验联系起来，反之亦然。POLβ在避免癌症方面起着关键作用，因为它的调控缺失或突变破坏会导致染色体不稳定和肿瘤发生。我们的主要目标是了解聚合酶保真度的原理，该原理由特定氨基酸侧链、引物/模板碱基和Pol活性部位的dNTP底物之间的详细相互作用所定义。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，一个生化合成和分析核心(核心B)，一个计算核心(核心C)和一个管理核心(核心A)。项目1的目标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中设计并在Core B中合成的一类新的核苷酸类似物。这些类似物将用于药物设计和给药策略，以确定它们在小鼠和培养细胞模型系统中作为抗癌药物的潜在用途，并以翻译的方法靶向骨肿瘤。PPG的一个独特和及时的方面是应用理论和计算机模拟方法来分析聚合酶活性部位的催化效率，如项目2所建议的。建模分析计算自由能，用于预测氨基酸侧链对保真度的个体贡献，包括底物结合和聚合酶活性部位的催化作用。该理论是将结构分析与运动力学分析结合起来的智力框架，如项目3所述。对实验者来说，检验先验的计算预测通常是不典型的。因此，这种PPG的一个定义方面是它的双向相互作用，其中计算预测通过实验进行测试，并使用新的实验数据来完善理论。

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.