Structure and Function of DNA Polymerase I of E.coli

描述（由研究者提供）：该项目的总体目标是在分子水平上全面了解DNA聚合酶催化的反应，特别强调聚合酶如何确保底物特异性和复制DNA的准确性。聚合酶的准确性问题具有重要的健康意义，因为DNA聚合酶产生的错误可能导致突变，导致人类疾病。此外，DNA聚合酶经常在化疗和抗病毒策略中被靶向，并且在各种诊断生物技术应用中很重要，因此了解它们的反应机制至关重要。实验提出了两个模型DNA聚合酶，具有对比的酶的性质：高度准确的DNA聚合酶I（克列诺片段）或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。这些包括参与底物识别和活性位点重排为化学催化形式的早期步骤，以及为下一个添加循环腾出活性位点而必须发生的易位步骤。将使用各种荧光测定，结合快速单周转动力学，目的是确定所涉及的物理过程，并了解它们在反应特异性中的作用。聚合酶活性位点的保真度机制将使用Klenow片段的突变体进行探索。核苷酸类似物将被用来调查的作用，碱基对的形状和氢键相互作用的特异性Klenow片段和Dbh聚合酶。针对Klenow片段开发的机制探针将应用于Dbh的研究，以了解更多关于这种最近发现的易错聚合酶的反应机制和活性位点侧链的作用。这种酶具有显著的移码倾向，这可能为其体内功能提供线索，可能表明它有能力绕过某些类型的大体积DNA损伤。

DESCRIPTION (provided by investigator): The overall goal of this project is a full understanding, at the molecular level, of the reactions catalyzed by DNA polymerases, with particular emphasis on how polymerases ensure substrate specificity and accuracy in copying DNA. The question of polymerase accuracy has important health implications because the errors made by DNA polymerases can result in mutations leading to human disease. Moreover, DNA 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. Experiments are proposed on two model DNA polymerases that have contrasting enzymatic properties: the highly accurate DNA polymerase I (Klenow fragment) o 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. These include the early steps involved in substrate recognition and rearrangement of the active site into a form poised for chemical catalysis, and the translocation step that must occur to vacate the active site for the next cycle of addition. A variety of fluorescence assays will be used, in combination with rapid single-turnover kinetics, with the goals of identifying the physical processes involved and understanding their roles in the specificity of the reaction. Fidelity mechanisms at the polymerase active site will be explored using mutants of Klenow fragment. Nucleotide analogues will be used to investigate the role of base pair shape and hydrogen-bonding interactions in the specificity of both Klenow fragment and Dbh polymerase. Mechanistic probes developed for Klenow fragment will be applied to studies of Dbh, in order to learn more about the reaction mechanism and the role of active site side chains in this recently discovered error-prone polymerase. This enzyme has a remarkable propensity for frameshifting, which may provide clues as to its in vivo function, perhaps indicating an ability to bypass certain types of bulky DNA lesions.