Structure and function of DNA Polymerase I of E.coli

描述（由申请人提供）：本项目的总体目标是在分子水平上全面了解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聚合酶的错误可能导致突变，导致人类疾病。此外，DMA聚合酶经常成为化疗和抗病毒策略的目标，并且在各种诊断生物技术应用中也很重要，因此了解其反应机制至关重要。我们的研究集中在两个模型的DMA聚合酶，具有截然不同的酶性质：高度准确的DMA聚合酶I（Klenow片段）的E。大肠杆菌的Dbh旁路聚合酶，以及古细菌S.太阳神这些酶和几个密切的同系物的结构数据是可用的，并作为许多计划的实验的基础。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在聚合酶反应途径中的非共价步骤的调查，因为这些构象转换可能参与区分正确配对的基板和导致聚合酶错误的错配。我们将使用荧光测定结合快速单周转动力学，以调查的构象变化率和对反应途径的错配底物，活性位点突变和受损的DMA的影响。DMA损伤和DMA合成错误可能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，因此对聚合酶结构和机制的研究与设计有效药物和了解它们如何工作有关。DNA聚合酶也是现代医学中使用的许多诊断工具的关键部分，并且将在用于诊断目的的新DNA测序技术的开发中发挥关键作用。

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.