Structure and function of DNA Polymerase I of E.coli

描述（由申请人提供）：该项目的总体目标是在分子水平上充分理解由DMA聚合酶催化的反应，特别强调聚合酶如何确保复制DMA的底物特异性和准确性。聚合酶的准确性问题具有重要的健康意义，因为DMA聚合酶产生的错误可能导致导致人类疾病的突变。此外，DMA聚合酶经常是化疗和抗病毒策略的靶点，在各种诊断生物技术应用中也很重要，因此了解它们的反应机制至关重要。我们的研究集中在两种具有不同酶性质的模型DMA聚合酶：大肠杆菌的高精度DMA聚合酶I （Klenow片段）和古细菌S. solfataricus的准确度低得多的Dbh绕过聚合酶。这些酶和一些接近的同源物的结构数据是可用的，并作为许多计划实验的基础。此外，由于聚合酶活性位点和反应机制的重要特征在整个聚合酶家族中都是保守的，因此用这些简单的模型系统获得的结果将具有更广泛的相关性。一个主要的优先事项将是研究聚合酶反应途径中的非共价步骤，因为这些构象转变可能涉及区分正确配对的底物和导致聚合酶错误的错对。我们将使用荧光分析结合快速单次翻转动力学来研究构象变化的速率以及对错配底物、活性位点突变和DMA损伤的反应途径的影响。DMA损伤和DMA合成中的错误可导致导致癌症等疾病的突变，因此了解DMA聚合酶如何发挥作用以避免这些结果非常重要。抗病毒药物经常靶向病毒聚合酶，因此研究聚合酶的结构和机制对于设计有效的药物和了解其作用机制具有重要意义。DNA聚合酶也是现代医学中使用的许多诊断工具的重要组成部分，并将在用于诊断目的的新DNA测序技术的发展中发挥关键作用。

DESCRIPTION (provided by applicant): The overall goal of this project is a full understanding, at the molecular level, of the reactions catalyzed by DMA polymerases, with particular emphasis on how polymerases ensure substrate specificity and accuracy in copying DMA. The question of polymerase accuracy has important health implications because the errors made by DMA polymerases can result in mutations leading to human disease. Moreover, DMA polymerases are frequently targeted in chemotherapeutic and antiviral strategies, as well as being important in a variety of diagnostic biotechnology applications, so an understanding of their reaction mechanisms is crucial. Our investigations focus on two model DMA polymerases that have contrasting enzymatic properties: the highly accurate DMA polymerase I (Klenow fragment) of E. coli, and the much less accurate Dbh bypass polymerase from the archaeon S. solfataricus. Structural data are available for both these enzymes and several close homologues, and serve as the basis for many of the planned experiments. Moreover, because the important features of the polymerase active site and reaction mechanism are conserved throughout the polymerase family, the results obtained with these simple model systems will have much wider relevance. A major priority will be the investigation of noncovalent steps in the polymerase reaction pathway because these conformational transitions are likely to be involved in distinguishing between correctly paired substrates and the mispairs that result in polymerase errors. We will use fluorescence assays in combination with rapid single-turnover kinetics to investigate the rates of conformational changes and the effect on the reaction pathway of mispaired substrates, active site mutations and damaged DMA. DMA damage and errors in DMA synthesis can cause mutations that lead to diseases such as cancer, making it important to understand how DMA polymerases function to avoid these outcomes. Antiviral drugs frequently target viral polymerases so research into polymerase structures and mechanism is relevant in designing effective drugs and understanding how they work. DNA polymerases are also a crucial part of many of the diagnostic tools used in modern medicine and will be pivotal in the development of new DNA sequencing technologies for diagnostic purposes.