MOLECULAR EFFECTORS OF ENZYMATIC SYNTHESIS OF DNA

模板导向基底中涉及的基本机制 酶促合成DNA的方法的选择还不是很好 明白 催化这些反应的酶在它们的 属性和结构组成。 这项建议的主要目的是 继续我们对生物化学，酶学和 原型酶，即E.杆菌DNA 聚合酶I 在拟议的研究中选择这种酶是基于 a）一个大碎片的三维解剖 pol I的（Klenow酶）已得到解决，B）重要信息 关于底物和模板-引物通过pol I结合的过程， 通过动力学分析获得，c）一些位点（氨基酸 残基）参与底物和模板的结合， 已被确定，d）这种酶继续作为模型， 所有DNA聚合酶的机制研究。 为了把重要的 功能结构域在DNA催化中的特定功能作用 合成，我们将采用位点特异性抑制剂，底物 类似物和UV介导的底物和模板的共价键合 核苷酸转化为酶蛋白。 含有活性位点的肽 将被分离并测序 整体结构功能分析 将通过类似的酶修饰分析来证实和补充 通过pol I结构基因的位点特异性诱变。 目标 用于诱变研究的残基将是那些通过化学方法显示的残基。 修饰或亲和标记研究以及模型所暗示的那些 DNA聚合反应。 分子机制和功能 以这种方式澄清的DNA聚合酶的解剖将导致 更好地理解DNA复制，DNA修复和 化学物质和致癌物质的诱变作用。 此外，新 可以合成抗微生物剂和抗病毒剂， 感染原聚合酶的特定结构域。

The basic mechanism(s) involved in the template directed substrate selection for the process of enzymatic synthesis of DNA have not been well understood. The enzymes catalyzing these reactions differ in their properties and structural makeup. The major objective of this proposal is to continue our investigations on the biochemical, enzymological and structural properties of the prototype enzyme, namely E. coli DNA polymerase I. The choice of this enzyme in the proposed studies is based on the facts that a) the three-dimensional anatomy of a large fragment (Klenow enzyme) of pol I has been resolved, b) significant information regarding the process of substrate and template-primer binding by pol I has been obtained through kinetic analyses, c) some of the sites (amino acid residues) participating in the binding of substrates and templates have been identified and, d) this enzyme continues to serve as a model for the mechanistic study of all DNA polymerases. In order to relate the important functional domains to a specific functional role(s) in the catalysis of DNA synthesis, we shall employ the site specific inhibitors, substrate analogues and UV mediated covalent bonding of substrate and template nucleotides to enzyme protein. The peptides containing the active sites will be isolated and sequenced. The overall structure-function analyses will be confirmed and complemented by similar analysis of enzymes modified by site specific mutagenesis of the pol I structural gene. The target residues for mutagenesis studies will be those revealed by chemical modification or affinity labeling studies as well as those implied by model of DNA polymerization reaction. The molecular mechanisms and functional anatomy of the DNA polymerase clarified in this manner will lead to a better understanding of the processes of DNA replication, DNA repair, and the mutagenic effects of chemicals and carcinogens. In addition, newer antimicrobial and antiviral agents can be synthesized which will target specific structural domains of the polymerase of the infectious agents.