Structure and mechanism of multisubunit complexes of DNA polymerase zeta

真核生物复制DNA聚合酶(POLS)、POLSδ和POLSε属于POL的B家族，它们可以复制 保真度非常高的DNA。虽然DNA聚合酶ζ(POLζ)也是B家族的成员，但它不同 从复制的POLS中，它以较低的保真度合成DNA，并在促进 通过各种各样的DNA损伤进行复制。在这方面，POLζ是独一无二的。具有催化活性的POLζ是 由Rev3催化亚基和Rev7辅助亚基组成；然而，我们发现聚合酶 在体内含有另外两个亚基，Pol31和Pol32，我们称之为POLζ-d。 此外，我们最近已经证明，Rev1是POLS的Y-家族成员，也是化学计量 POLζ-d的主要成分，我们将这个五亚基复合体称为POLζ-D1。我们提纯POLζ-d和POLζ的能力- D1打开了这些多亚单位复合体，以进行详细的机械和结构分析。我们能够 首次提出与POLζ-d和POLζ−D1的整体架构相关的问题，以及POL31、POL32、 和Rev1增强POLDNA在未损伤和损伤的ζ底物上的催化活性。在目标1中，我们 将在有脱氧核糖核酸存在的情况下对POLζ-d和POLζ-D1进行冷冻电子显微镜(CRYO-EM)分析 用于单分子成像的直接检测设备相机的最新发展优势 用于高分辨率三维重建的软件。这些研究将建立在我们早期的低分辨率模型的基础上 并首次揭示了这种酶是如何与ζ相互作用的，以及蛋白质的性质- 不同亚基之间的蛋白质接触。在目标2中，我们将进行稳态前动力学分析，以 确定POLζ-d和POLζ-d1的作用机制。这些研究将与 结构研究(目标1)以获得反应途径的动力学图像。我们还将测试这些结构 通过生化和遗传方法，使Rev3活性部位的残基和那些介导 亚基蛋白质-蛋白质相互作用将被突变并分析它们对DNA合成的影响。 体内DNA损伤反应的研究。总之，拟议的研究对于了解 真核细胞应对外源性和内源性基因毒性引起的多种DNA损伤 探员们。

Eukaryotic replicative DNA polymerases (Pols), Pols δ and ε belong to the B-family of Pols and they replicate DNA with a very high fidelity. Although DNA polymerase ζ (Polζ) is also a member of the B-family, it differs from the replicative Pols in that it synthesizes DNA with a lower fidelity, and plays a critical role in promoting replication through a wide variety of DNA lesions. Polζ is unique in this regard. Catalytically active Polζ is comprised of the Rev3 catalytic and Rev7 accessory subunits; however, we showed that the polymerase contains two additional subunits in vivo, Pol31 and Pol32, and we refer to this four-subunit complex as Polζ-d. Moreover, we have shown recently that Rev1, a member of the Y-family of Pols, is also a stoichiometric component of Polζ-d, and we refer to this five-subunit complex as Polζ-d1. Our ability to purify Polζ-d and Polζ- d1 opens up these multi-subunit complexes for a detailed mechanistic and structural analysis. We are able to ask for the first time questions related to the overall architecture of Polζ-d and Polζ−d1 and how Pol31, Pol32, and Rev1 potentiate the catalytic activity of Polζ on undamaged and damaged DNA substrates. In Aim 1, we will carry out cryo-electron microscopy (cryo-EM) analysis of Polζ-d and Polζ-d1 in the presence of DNA, taking advantage of the latest developments in direct detection device cameras for imaging single molecules and software for 3-D reconstruction at high-resolution. These studies will build on our earlier low-resolution model of Polζ-d and reveal for the first time how the enzyme actually interacts with DNA and the nature of protein- protein contacts between the various subunits. In Aim 2, we will carry out pre-steady-state kinetic analyses to determine the action mechanisms of Polζ-d, and Polζ-d1. These studies will be performed in conjunction with the structural studies (Aim 1) to obtain a kinetic picture of the reaction pathway. We will also test the structures by biochemical and genetic approaches, whereby residues in the Rev3 active site and those that mediate subunit protein-protein interactions will be mutated and assayed for their effect on DNA synthesis in vitro and on DNA damage response in vivo. Altogether, the proposed studies are important for understanding how eukaryotic cells cope with a diverse array of DNA lesions induced by exogenous and endogenous genotoxic agents.