ENZYMATIC MECHANISMS OF DNA SYNTHESIS

这项工作的目标是了解DNA 聚合酶复制染色体DNA。 这需要聚合酶 过程是蛋白质的多亚基复合物。 两个这样的酶复合物 正在接受调查E大肠杆菌DNA聚合酶III全酶和小牛DNA α聚合酶 实验方法是确定函数 复合物中每个亚基在体外DNA合成过程中的贡献。 获得了DNA聚合酶Ⅲ全酶的纯化制剂 其由至少七种不同的蛋白质亚基组成。 此外，本发明还 三种稳定的DNA聚合酶III全酶也被 分离的，其缺乏在所述细胞中发现的一个或多个蛋白质亚基。 全酶 在这些DNA聚合酶III的每一个片段中， 全酶a在单个亚基的缺失 以及与全酶相关的一种或多种性质的损失。 这些特性包括ATP依赖性形成稳定的起始 与DNA的复合物，使用多胺包被的DNA和结合的能力 蛋白质的高度进行性合成，以及适当的解离， 天然合成终止位点。 我们正在研究 亚基的特定组合的存在对这些特性的影响，以及 这种方法希望能确定每个亚基的功能作用。 最近 有证据表明全酶在体内可能是功能性二聚体。 我们用电子显微镜检测一种聚合酶 分子与两个底物，并测量每个底物上的合成。 小牛DNA α聚合酶同样复杂， 难以净化。 然而，我们最近开发了一种方法， 高分子量形式免疫亲和层析纯化 这种酶。 提出实验来表征该亚基 酶的结构，并建立功能特性， 单独的亚单位。 ATP、小牛DNA结合蛋白UP1和 小牛DNA依赖性ATP酶对该聚合酶合成特性的影响 将被确定。 预计通过这种方法， 聚合酶亚基和复制相关蛋白， vivo可以在体外组装。

The goal of this work is to understand the mechanisms by which DNA polymerases replicate chromosomal DNA. Polymerases required for this process are multisubunit complexes of proteins. Two such enzyme complexes are under investigation, E. coli DNA polymerase III holoenzyme and calf DNA alpha polymerase. The experimental approach is to determine the functional contribution of each subunit in the complexes during DNA synthesis in vitro. Purified preparations of DNA polymerase III holoenzyme have been obtained which consist of at least seven different protein subunits. Additionally, three stable subassemblies of DNA polymerase III holoenzyme have also been isolated which lack one or more of the protein subunits found in the holoenzyme. In each of these subassemblies of DNA polymerase III holoenzyme a correlation exists between the absence of individual subunits and the loss of one or more properties associated with the holoenzyme. These properties include ATP dependent formation of stable initiation complexes with DNA, ability to use DNA coated with polyamines and binding proteins for highly processive synthesis, and proper dissociation at natural synthesis termination sites. We are examining the effects of the presence of particular combinations of subunits on these properties, and in this way hope to identify the functional role of each subunit. Recent evidence suggests that the holoenzyme may be a functional dimer in vivo. We are using electron microscopy to detect interaction of one polymerase molecule with two substrates, and measure synthesis on each substrate. The calf DNA alpha polymerase is similarly intricate and also has been difficult to purify. However, we have recently developed a method for immunoaffinity chromatography purification of a high molecular weight form of this enzyme. Experiments are proposed to characterize the subunit structure of the enzyme, and to establish functional properties of individual subunits. The effects of ATP, calf DNA binding protein UP1 and calf DNA dependent ATPases on the synthetic properties of this polymerase will be determined. It is expected that by this method the combination of polymerase subunits and replication associated proteins which operate in vivo can be assembled in vitro.