MOLECULAR EFFECTORS OF ENZYMATIC SYNTHESIS OF DNA

模板导向底物涉及的基本机制(S) 酶法合成DNA过程的选择一直不是很好 明白了。催化这些反应的酶在它们的 属性和结构组成。这项提议的主要目标是 继续我们对生物化学、酶学和 大肠杆菌DNA原型酶的结构性质 聚合酶I。在拟议的研究中选择这种酶是基于 关于a)大碎片的三维解剖 (Klenow酶)的PolI已被解析，b)重要信息 关于PolI与底物和模板-引物结合的过程， 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 和，d)该酶继续作为模型用于 所有DNA聚合酶的机理研究。为了将重要的 功能结构域在脱氧核糖核酸催化中的特定功能作用(S) 合成时，我们将使用部位特异的抑制剂，底物 类似物与紫外光介导的底物和模板的共价键 将核苷酸转化为酶蛋白。含有活性部位的多肽 将被分离并测序。总体结构--功能分析 将通过对修饰的酶进行类似的分析来确认和补充 通过polI结构基因的定点突变。目标是 用于突变研究的残留物将是由化学物质揭示的残留物。 修饰或亲和标记研究以及模型所暗示的研究 DNA聚合反应的结果。分子机制及其功能 以这种方式阐明的DNA聚合酶的解剖将导致 更好地了解DNA复制、DNA修复和 化学物质和致癌物的诱变效应。此外，较新的 可以合成抗微生物和抗病毒药物，这些药物将针对 感染性病原体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.