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 在DNA的酶促合成过程中。 催化DNA的酶 合成需要多个组分，且呈现出几种常见和几种 独特的属性。 然而，负责这些的基础和/或组件 财产尚未确定。 在这一建议中，我们的意图是 严格评价底物和模板-引物结合的过程 通过鉴定和结构分析， 功能结构域，使用E. coli pol. I作为模型 酵素 大量关于催化性能的信息， DNA合成的要求，以及这种结构的一级结构， 酶是已知的。 所获得的结果不仅将澄清基本的 DNA聚合酶反应的机制，但可以提供洞察 这些特性可能具有重要的实际应用。 我们已经开发了试剂共价连接的程序， 并计划开发新的方案， 用于有效标记结合位点的现有方案。 我们的目标 是分离带有底物和模板-引物结合的肽 来自E. coli Pol. I，并确定它们的主要 顺序 因此，结构研究与功能分析， 单个DNA聚合酶内的各种结构域将阐明 DNA合成的复杂多组分过程的机制。

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.