MOLECULAR EFFECTORS OF ENZYMATIC SYNTHESIS OF DNA

描述：拟议研究的长期目标是了解 模板依赖的DNA合成过程中涉及的分子事件。这个 大肠杆菌DNA聚合酶I的Klenow片段将被用作 原型模型系统，因为广泛表征了机械学 并对该酶的结构性质进行了研究。它是 第一个晶体结构已被解决的DNA聚合酶。域名 该酶中参与结合的氨基酸残基 底物和/或对磷酸二酯键的催化过程有贡献 编队将被定义。为此，亲和标记和 将实施定点突变方法。此外, Klenow碎片的晶体坐标的可用性允许 带侧链的完整三维结构模型的建立 使用分子模拟和建模程序。模型建造了结构 在规划和解释方面是一个极好的指南 结果。这种3D结构也可能暗示氨基酸残基是 有可能参与特定的功能提供很好的实验 诱变的目标。用突变酶获得的结果要么是 确认预测或建议修改现有的 结构模型及其功能含义。由于这些原因， 研究人员希望确定)功能上重要的氨基酸的努力 Poli、b)中的酸残基阐明了它们所起的作用，包括相互作用 与底物，c)推导出不同氨基贡献的功能结构域 酸，以及d)阐明模板依赖底物的作用机制 该基因的选择及其添加到引物末端的机理。

DESCRIPTION: The long term goal of the proposed research is to understand the molecular events involved during template dependent DNA synthesis. The Klenow fragment of the E. coli DNA polymerase I will be utilized as a prototype model system, since extensive characterization of the mechanistic and structural properties of this enzyme have been carried out. It is the first DNA polymerase whose crystal structure has been solved. The domains of amino acid residues in this enzyme which participate in the binding of substrates and/or contribute to the catalytic process of phosphodiester bond formation will be defined. For this purpose, both affinity labeling and site-directed mutagenesis approaches will be implemented. In addition, availability of the crystal coordinates of the Klenow fragment has permitted model building of the complete 3-dimensional structure with side chains using molecular simulation and modeling programs. The model built structure serves as an excellent guide in planning as well as in the interpretation of results. This 3D structure may also suggest amino acid residues which are likely to participate in specific functions providing a good experimental target for mutagenesis. Results obtained with mutant enzymes would either confirm the prediction or would suggest modifications to the existing structural models and their functional implications. As a result of these efforts the investigator hopes to identify a) functionally important amino acid residues in pol I, b) clarify the role they play including interactions with substrates, c) deduce functional domains contributed by different amino acids, and d) clarify the mechanism of template-dependent substrate selection and the mechanisms of its addition onto primer terminus.