MOLECULAR EFFECTORS OF ENZYMATIC SYNTHESIS OF DNA

描述(摘自申请者的摘要)：DNA的酶合成 是需要衬底顺序结合的多步骤工艺， 伴随着酶蛋白内的几个构象变化。这个 这些步骤涉及的分子机制还不是很清楚。这个 最近这类酶的许多晶体结构的可获得性， 然而，已经取得了重大进展，基础分子 DNA聚合机理及其与结构的关系 酶的组成可以在原子水平上阐明。的主要目标是 这项建议是继续进行生化、酶学研究 和原型酶的结构性质，即大肠杆菌聚合酶1。 在拟议的研究中选择这种酶是基于这样一个事实：a) PolI大片段(Klenow酶)的三维解剖 与底物络合的家族已得到解决，b)意义重大 关于底物和模板-引物结合过程的信息 Pol I是由动力学分析得到的，c)一些位置(氨基酸 残基)参与底物和模板结合 确定，d)一些具有某些功能含义的催化残留物 都是通过定点突变来鉴定的，并且e)这种酶作为 所有DNA聚合酶机理研究的模型系统。为了 确定并关联执行特定任务的重要结构域 在DNA合成催化中的作用，以下三方方法 将用于：i)保守区中氨基酸残基的定点突变 域或3D模型结构检查所隐含的。深入分析 突变酶的性质将阐明所需氨基酸的作用。 特定结构域中的酸，II)酶的光亲和标记 带模板的蛋白质及其酶切位点的鉴定 模板-模板 关于DNA聚合酶在突变结果解释中的作用 以构建提供详细功能的结构模型 各种域结构在原子细节中的参与和许可 催化反应过渡态的结构解析。这个 阐明了DNA聚合酶的分子机制和功能解剖 方式将导致更好地理解DNA复制、DNA修复和 化学物质的诱变作用和致癌作用。

DESCRIPTION (adapted from applicant's abstract): The enzymatic synthesis of DNA is a multistep process that requires sequential binding of substrate, accompanied by several conformational changes within the enzyme protein. The molecular mechanisms involved in these steps are not well understood. The recent availability of a number of crystal structures of this class of enzymes, however, has made a significant advancement whereby the basic molecular mechanisms of DNA polymerization and their relationship to the structural makeup of enzyme may be clarified at the atomic level. The major objective of this proposal is to continue investigations on the biochemical, enzymological and structural properties of the prototype enzyme, namely E. coli polymerase 1. The choice of this enzyme in the proposed study is based on the fact that a) the three dimensional anatomy of large fragment (Klenow enzyme) of pol I family, complexed with substrates, has been resolved, b) significant information regarding the process of substrate and template-primer binding by pol I has been obtained from kinetic analysis, c) some of the sites (amino acid residues) participating in the substrates and template binding have been identified, d) a number of catalytic residues with some functional implication have been identified by site-directed mutagenesis, and e) this enzyme serves as the model system for mechanistic study of all DNA polymerases. In order to identify and relate important structural domains that carry out specific function in the catalysis of DNA synthesis, the following tripartite approach will be used: i) site directed mutagenesis of amino acid residues in conserved domains or implied by 3-D model structure examinations. An in depth analysis of the properties of mutant enzyme will clarify the role for the desired amino acid in specific domain structure, ii) photo-affinity labeling of enzyme proteins with template-primers and identification of sites of enzyme and template-primer contact and, iii) utilize all available structural information concerning DNA polymerases in the interpretation of mutagenesis results as well as to construct structural models which provide the detailed functional participation of various domain structures in atomic details and permit structural elucidation of the transition state, of the catalytic reaction. The molecular mechanism and functional anatomy of DNA polymerase clarified in this manner will lead to a better understanding of DNA replication, DNA repair and mutagenic effects of chemicals and carcinogenesis.