STRUCTURE/FUNCTION OF DNA POLYMERASE I OF E COLI

描述（改编自研究者摘要）：本项目的总体目标是在分子水平上完整描述由大肠杆菌DNA聚合酶I进行的三种反应。大肠杆菌：聚合酶、3 '-5'核酸外切酶和5 '-3'核酸外切酶。聚合酶和3 '-5'核酸外切酶（校对）功能一起确保DNA合成的准确性，而5 '-3'核酸外切酶与DNA修复和滞后链复制中的聚合酶活性协调。聚合酶的酶性质经常在抗病毒和化疗策略中被用于治疗;此外，由DNA聚合酶产生的复制错误是多种人类疾病的可能原因。5 '-3'核酸外切酶是真核生物“瓣状核酸内切酶”的相对物，其在DNA复制、重组和修复的各个方面发挥重要作用，并且其缺失增加基因组不稳定性。对几种不同类型的大量聚合酶的结构研究表明，所有聚合酶都具有相似的活性位点布局和反应机制。因此，相对简单的E.大肠杆菌DNA聚合酶I（Pol I）作为解决与所有聚合酶相关的问题的有价值的模型，其中许多聚合酶作为实验系统将不太容易处理。Pol I及其近亲（包括几种共晶结构）的结构数据提供了大量关于活性位点布局的细节，但也提出了许多额外的问题。以结构信息为出发点，实验计划，这将解决在共晶结构中看到的相互作用的功能意义。光交联将用于确定DNA的未复制模板链的路径，并且诱变和生物化学研究的组合将用于鉴定功能上重要的蛋白质-DNA接触。超越由共晶复合物提供的静态图像，将采用各种荧光方法来探测涉及运动的Pol I酶学的几个方面：聚合酶反应途径内发生的构象转变，进行性DNA合成期间的易位，以及校对期间聚合酶和3 '-5'外切核酸酶位点之间的DNA底物的穿梭。在这个提议中的许多实验将Pol I的生物化学性质与其在体内的功能联系起来：研究聚合酶反应中如何实现保真度，DNA聚合酶与RNA聚合酶的区别，以及聚合酶和5 '-3'核酸外切酶活性如何协调以产生正确的生物学终点。

DESCRIPTION (adapted from investigator's abstract): The overall goal of this project is a complete description, at the molecular level, of the three reactions carried out by DNA polymerase I of E. coli: polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. Together the polymerase and 3'-5' exonuclease (proofreading) functions ensure the accuracy of DNA synthesis, while the 5'-3' exonuclease coordinates with the polymerase activity in DNA repair and lagging strand replication. The enzymatic properties of polymerases are frequently exploited therapeutically in antiviral and chemotherapeutic strategies; moreover, replication errors made by DNA polymerases are the likely causes of a variety of human diseases. The 5'-3' exonuclease is a relative of the eukaryotic "flap endonucleases" which play an essential role in various aspects of DNA replication, recombination and repair, and whose absence increases genome instability. Structural studies of a substantial number of polymerases, of several different types, show that all polymerases share a similar active site layout and reaction mechanism. Therefore, the comparatively simple E. coli DNA polymerase I (Pol I) serves as a valuable model for addressing issues relevant to all polymerases, many of which would be much less tractable as experimental systems. The structural data available for Pol I and its close relatives (including several cocrystal structures) provides a wealth of detail about the layout of the active sites, but poses many additional questions. Taking the structural information as a starting point, experiments are planned which will address the functional significance of the interactions seen in the cocrystal structures. Photo-crosslinking will be used to determine the path of the uncopied template strand of DNA, and a combination of mutagenesis and biochemical studies will be used to identify functionally important protein-DNA contacts. Moving beyond the static picture provided by the cocrystal complexes, a variety of fluorescence methods will be employed to probe several aspects of Pol I enzymology that involve movement: conformational transitions that take place within the polymerase reaction pathway, translocation during processive DNA synthesis, and shuttling of a DNA substrate between polymerase and 3'-5' exonuclease sites during proofreading. Many of the experiments in this proposal relate the biochemical properties of Pol I to its function in vivo: investigating how fidelity is achieved in the polymerase reaction, what distinguishes a DNA polymerase from an RNA polymerase, and how polymerase and 5'-3' exonuclease activities are coordinated so as to produce the correct biological end point.