MOLECULAR EFFECTORS OF ENZYMATIC DNA SYNTHESIS

我们的长期目标是理解和解开一些错综复杂的问题 在酶促合成DNA的过程中。催化DNA的酶 合成需要多种成分，并表现出几种常见和少数几种 独一无二的特性。然而，负责这些的基础和/或组件 属性尚未确定。在这项建议中，我们的意图是 严格评估底物和模板-底物结合的过程 通过对涉事客体的鉴定和结构分析 利用大肠杆菌Pol.的Klenow片段构建功能结构域。我是一名模特 酵素。关于催化性能的大量信息和 对DNA合成的要求，以及这个的一级结构 酶现在是已知的。所获得的结果不仅将澄清基本的 DNA聚合酶反应的机制，但可能为深入了解 可能具有重要实际应用的性质。 我们已经开发了试剂的共价连接程序，以 并计划开发新的方案并改进 用于有效标记结合位点的现有协议。我们的目标 目的是分离带有底物和模板-引物结合的多肽 Klenow片段的位点。并确定它们的主要特征 序列。因此，结构研究与功能分析一起， 各个DNA聚合酶中的不同结构域将阐明 复杂的多组分DNA合成过程的机理。

Our long-range goals are to understand and unravel some of the intricacies in the process of the enzymatic synthesis of DNA. Enzymes catalyzing DNA synthesis require muliple components and exhibit several common and a few unique properties. Yet the basis and/or components responsible for these properties have not been identified. In this proposal, our intention is to critically evaluate the process of substrate and template-primer binding through the identification and structural analysis of the involved functional domains using Klenow fragment of E. coli pol. I as a model enzyme. A large body of information on the catalytic properties and requirements for DNA synthesis, as well as the primary structure of this enzyme are now known. The results obtained will not only clarify the basic mechanisms of the DNA polymerase reaction but may provide insight into properties which may have important practical applications. We have developed procedures for the covalent linkage of reagents to specific enzyme sites and plan to develop new protocols and improve existing protocols for the efficient labeling of binding sites. Our goal is to isolate peptides bearing the substrate and template-primer binding sites from Klenow fragment of E. coli Pol. I and to determine their primary sequence. Thus, structural studies together with functional analyses of various structural domains within individual DNA polymerases will clarify the mechanisms of the complex multicomponent process of DNA synthesis.