MOLECULAR EFFECTORS OF ENZYMATIC SYNTHESIS OF DNA

描述（改编自申请人的摘要）：DNA的酶促合成 是一个需要连续结合底物的多步骤过程， 伴随着酶蛋白内的几种构象变化。的 这些步骤中涉及的分子机制还没有被很好地理解。的 最近可获得这类酶的许多晶体结构， 然而，已经取得了重大进展， DNA聚合的机制及其与结构的关系 酶的组成可以在原子水平上澄清。的主要目标 这项建议是继续研究生物化学，酶学， 以及原型酶E.大肠杆菌聚合酶1。 在拟议研究中选择这种酶是基于以下事实：a） pol Ⅰ大片段（Klenow酶）三维解剖 与底物复合的家族，已被解决，B）显著 关于底物和模板-引物结合过程的信息， pol I已从动力学分析中获得，c）一些位点（氨基酸 残基）参与底物和模板结合， 鉴定，d）具有某些功能含义的许多催化残基 已经通过定点诱变鉴定，和e）该酶用作 用于所有DNA聚合酶的机理研究的模型系统。为了 识别并关联进行特定 在催化DNA合成中的作用，以下三方面的方法 i）在保守的氨基酸序列中的氨基酸残基的定点诱变， 域或隐含的三维模型结构检查。深入分析 突变酶的性质将阐明所需氨基酸的作用 特定结构域结构中的酸，ii）酶的光亲和标记 蛋白质与模板引物和酶的位点的鉴定， 模板-引物接触，和iii）利用所有可用的结构信息 关于DNA聚合酶在解释诱变结果以及 以构建结构模型，提供详细的功能 各种域结构参与原子细节， 催化反应的过渡态的结构解析。的 本文阐明了DNA聚合酶的分子机制和功能解剖， 这种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.