MECHANISTIC STUDIES OF REPLICATIVE POLYMERASES

DNA聚合酶Ⅲ全酶是大肠杆菌的主要复制聚合酶。杆菌 对它的研究揭示了所有细胞（真核和原核）共有的复制复合体的许多显著特征。 这些特征包括包围引物-模板并束缚聚合酶的酸性“滑动夹”或持续合成因子，识别引物末端并将滑动夹转移到DNA上的5-蛋白ATP酶，以及能够协调前导链和滞后链合成以及在复制叉处起作用的解旋酶/引发酶复合物的作用的复合物聚合酶。 大多数特征性DNA聚合酶的聚合酶结构域包含约35，000道尔顿，但复制复合物的催化亚基通常大4-5倍。 这种额外的结构参与了蛋白质-蛋白质相互作用和聚合酶与复制叉上其他蛋白质协调行动所必需的通讯。 在本申请中，提出研究集中于理解参与以下的催化α亚基的结构域：（i）聚合，（ii）与tau的相互作用，允许复制复合物的二聚化，（iii）与β的相互作用，实现高持续合成能力，以及（iv）与β的相互作用，实现校对。 实验方法利用了我们创建的一系列包含一组嵌套缺失的α融合蛋白的可用性。 这些缺失蛋白的特征在于它们的催化活性、功能性通讯和它们结合其它亚基的能力。 我们将确定聚合酶活性位点的关键催化残基，以及参与蛋白质-蛋白质相互作用的关键残基。

DNA polymerase III holoenzyme is the major replicative polymerase of E. coli. Studies of it have revealed many of the salient features of replicative complexes common to all cells, eukaryotic and prokaryotic. These features include an acidic "sliding clamp" or processivity factor that encircles the primer-template and tethers the polymerase, a 5-protein ATPase that recognizes the primer terminus and transfers the sliding clamp onto DNA and a complex polymerase, capable of coordinating leading and lagging strand synthesis and the actions of the helicase/primase complex that function at the replication fork. The polymerase domain of most characterized DNA polymerases encompasses approximately 35,000 daltons, yet the catalytic subunits of replicative complexes are generally 4-5-times larger. This extra structure is involved in the protein-protein interactions and communications necessary for the polymerase to coordinate its actions with the other proteins at the replication fork. In this application, studies are proposed to focus on understanding the domains of the catalytic alpha subunit involved in (i) polymerization, (ii) interactions with tau, permitting dimerization of the replicative complex, (iii) interactions with beta, enabling high processivity, and (iv) interactions with epsilon, enabling proofreading. The experimental approach exploits the availability of a series of alpha fusion proteins we have created that contain a set of nested deletions. These deletion proteins will be characterized in terms of their catalytic activities, functional communication and their ability to bind other subunits. We will identify the key catalytic residues of the polymerase active site, and the key residues involved in protein-protein interactions.