Structure and Function of DNA Polymerase I of E.coli

描述(由研究人员提供)：这个项目的总体目标是在分子水平上充分了解DNA聚合酶催化的反应，特别强调聚合酶如何确保复制DNA的底物专一性和准确性。聚合酶的准确性问题具有重要的健康意义，因为DNA聚合酶的错误可能导致突变，导致人类疾病。此外，DNA聚合酶经常作为化疗和抗病毒策略的靶点，在各种诊断生物技术应用中也是重要的，因此了解它们的反应机制是至关重要的。实验是在两种具有不同酶学性质的模型DNA聚合酶上进行的：高精度的DNA聚合酶I(Klenow片段)o E.coli.和来自考古子S.solfararicus的精度低得多的胸径旁路聚合酶。这些酶和几个密切的同源物的结构数据都是可用的，并作为许多计划中的实验的基础。此外，由于聚合酶活性部位的重要特征和反应机制在整个聚合酶家族中都是保守的，用这些简单的模型系统得到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。这些步骤包括底物识别和将活性中心重排为适合化学催化的形式的早期步骤，以及为下一轮加成腾出活性中心所必须发生的移位步骤。将使用各种荧光分析，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的特异性中所起的作用。将使用Klenow片段的突变体来探索聚合酶活性部位的保真度机制。核苷酸类似物将被用来研究碱基对形状和氢键相互作用在Klenow片段和DBH聚合酶的特异性中的作用。为Klenow片段开发的机械探针将应用于胸径的研究，以了解更多关于反应机制和活性位点侧链在这个最近发现的容易出错的聚合酶中的作用。这种酶具有显著的移码倾向，这可能为其在体内的功能提供线索，或许表明有能力绕过某些类型的大型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.