STRUCTURE/FUNCTION OF DNA POLYMERASE I OF E COLI

描述（改编自研究者摘要）：该项目的总体目标是在分子水平上完整描述大肠杆菌DNA聚合酶I进行的三种反应：聚合酶，3‘-5’外切酶和5‘-3’外切酶。聚合酶和3′-5′外切酶（校对）共同保证了DNA合成的准确性，而5′-3′外切酶在DNA修复和滞后链复制中与聚合酶活性协调。聚合酶的酶特性经常被用于抗病毒和化疗策略的治疗；此外，DNA聚合酶产生的复制错误可能是多种人类疾病的原因。5‘-3’外切酶是真核生物“皮瓣内切酶”的近亲，在DNA复制、重组和修复的各个方面起着至关重要的作用，其缺失会增加基因组的不稳定性。对几种不同类型的大量聚合酶的结构研究表明，所有的聚合酶都具有相似的活性位点布局和反应机制。因此，相对简单的大肠杆菌DNA聚合酶I （Pol I）作为一个有价值的模型，用于解决与所有聚合酶相关的问题，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（包括几个共晶结构）的结构数据提供了有关活性位点布局的丰富细节，但提出了许多额外的问题。以结构信息为出发点，计划进行实验，以解决在共晶结构中看到的相互作用的功能意义。光交联将用于确定未复制的DNA模板链的路径，而诱变和生化研究的结合将用于识别功能重要的蛋白质-DNA接触。除了共晶复合物提供的静态图像之外，各种荧光方法将用于探测Pol I酶学中涉及运动的几个方面：聚合酶反应途径内发生的构象转变，DNA合成过程中的易位，以及校对过程中DNA底物在聚合酶和3‘-5’外切酶位点之间的穿梭。本提案中的许多实验将Pol I的生化特性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’外切酶活性如何协调以产生正确的生物终点。

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.