Understanding Functional Coordination in a Multifunctional DNA Polymerase

描述（由申请人提供）：DNA聚合酶的多种酶活性必须精心编排和调节，以确保在DNA复制和修复过程中准确有效地合成DNA，同时防止形成致突变或不稳定的DNA中间体。在理解整个DNA聚合酶家族中存在的功能协调方面存在根本性的差距。长期目标是了解DNA聚合酶在DNA复制和修复过程中的分子细节。本应用程序的目的是确定大肠杆菌DNA聚合酶I （Pol I）的多种酶活性是如何协调和调节的，这是追求这一目标的下一步。Pol I是一种易于操作的模型酶，体现了来自更复杂生物体的多功能DNA聚合酶的基本特征。核心假设是，功能配位与Pol i的构象动力学有关。特别是，有人提出，灵活连接的5'核酸酶结构域，通过其动态移动位置的能力，既协调又增强了Pol i的不同酶活性。这一假设是根据申请人实验室的初步数据制定的。这项研究的基本原理是，一旦了解了5'核酸酶结构域的构象状态及其对其他结构域的影响，就有可能了解Pol I如何协调其不同的活动，以及为什么这种协调有时会失败，导致突变、链断裂或其他有害结果。在强有力的初步数据的指导下，该假设将通过追求三个具体目标来验证：(1)剖析Pol I的核苷酸结合周期并揭示5'核酸酶结构域的作用。(2)剖析Pol I的3′-5′外切酶途径，揭示5′核酸酶结构域的作用。(3)确定5′核酸酶活性如何与Pol I的其他酶活性进行物理协调。为了可视化介导Pol I酶功能协调的构象变化，将开发一系列新的单分子荧光光谱方法，并将在平行生化实验中证实单分子数据。该方法是创新的，因为它将采用一种全新的实验方法，基于单个聚合酶分子中酶构象变化的直接可视化，因为它在与DNA底物的单次接触中在不同的活动模式之间切换。这项研究具有重要意义，因为它将建立一个前所未有的水平，了解在DNA复制和修复过程中，多功能DNA聚合酶的不同酶活性是如何协同工作的。

DESCRIPTION (provided by applicant): The multiple enzymatic activities of DNA polymerases must be carefully orchestrated and regulated to ensure accurate and efficient DNA synthesis during DNA replication and repair, while preventing the formation of mutagenic or unstable DNA intermediates. There is a fundamental gap in understanding functional coordination, which exists across the entire DNA polymerase family. The long-term goal is to understand in molecular detail how DNA polymerases function during DNA replication and repair. The objective of this application, which is the next step in the pursuit of this goal, is to determine ow the multiple enzymatic activities of E. coli DNA polymerase I (Pol I) are coordinated and regulated. Pol I is a readily manipulated model enzyme that exemplifies the essential features of multifunctional DNA polymerases from more complex organisms. The central hypothesis is that functional coordination is linked to the conformational dynamics of Pol I. In particular, it is proposed that the flexibly tethered 5' nuclease domain, through its ability to dynamically shift position, both orchestrates and enhances the different enzymatic activities of Pol I. This hypothesis has been formulated on the basis of preliminary data from the applicant's laboratory. The rationale for the proposed research is that once the conformational states of the 5' nuclease domain and its influence on the other domains are known, it will be possible to understand how Pol I coordinates it's different activities and why this coordination sometimes fails, leading to mutations, strand breaks or other deleterious outcomes. Guided by strong preliminary data, the hypothesis will be tested by pursuing three specific aims: (1) Dissect the nucleotide incorporation cycle of Pol I and reveal the role of the 5' nuclease domain. (2) Dissect the 3'-5' exonuclease pathway of Pol I and reveal the role of the 5' nuclease domain. (3) Determine how the 5' nuclease activity is physically coordinated with the other enzymatic activities of Pol I. A range of novel single- molecule fluorescence spectroscopic methods will be developed in order to visualize conformational changes that mediate functional coordination in the Pol I enzyme, and the single-molecule data will be corroborated in parallel biochemical experiments. The approach is innovative, because it will employ a fundamentally new experimental approach, based on direct visualization of enzyme conformational changes within a single polymerase molecule as it switches between different modes of activity during a single encounter with a DNA substrate. The proposed research is significant, because it will establish an unprecedented level of understanding of how the different enzymatic activities of a multifunctional DNA polymerase work together during DNA replication and repair.