Structure and mechanism of multisubunit complexes of DNA polymerase zeta

DNA聚合酶zeta多亚基复合物的结构和机制

• 批准号: 10249252
• 负责人: ANEEL K. AGGARWAL
• 金额: $ 46.36万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10249252
• 关键词: 3-Dimensional; Active Sites; Address; Architecture; Binding; Biochemical Genetics; Biological Assay; Bypass; Catalytic Domain; Chemicals; Complex; Computer software; Cryoelectron Microscopy; DNA; DNA Damage; DNA biosynthesis; DNA lesion; DNA polymerase zeta; DNA-Directed DNA Polymerase; Detection; Development; Devices; Discrimination; Electron Microscopy; Enzymatic Biochemistry; Enzymes; Eukaryota; Eukaryotic Cell; Family; Genetic study; Genome Stability; Genotoxic Stress; Image; In Vitro; Ionizing radiation; Kinetics; Learning; Left; Lesion; Mediating; Modeling; Molecular Conformation; Mutagens; Mutate; Mutation; Nature; Negative Staining; Nucleotides; Pathway interactions; Play; Polymerase; Positioning Attribute; Protein Subunits; Proteins; Reaction; Resolution; Role; Site; Specimen; Structural Models; Structure; Sunlight; Testing; Time; UV induced; base; cell injury; dimer; genetic approach; in vivo; insight; member; microscopic imaging; pollutant; protein protein interaction; reconstruction; recruit; response; single molecule; yeast genetics

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.

真核复制型DNA聚合酶（Pos），Pos δ和ε属于Pos的B家族，它们复制 DNA具有很高的保真度。虽然DNA聚合酶β（Polymerase β，Polymerase β）也是B家族的一员，但它不同于 与复制型Pos不同，它以较低的保真度合成DNA，并在促进DNA合成中起着关键作用。 通过各种各样的DNA损伤进行复制。波尔布特在这方面是独一无二的。催化活性聚合物 由Rev 3催化亚基和Rev 7辅助亚基组成;然而，我们发现聚合酶 在体内含有两个额外的亚基，Pol 31和Pol 32，我们将这种四亚基复合物称为Pol 31-d。 此外，我们最近已经表明，Rev 1，一个成员的Y-家庭的Pos，也是一个化学计量的 Pol E-d的组成部分，我们将这个五亚基复合物称为Pol E-d 1。我们净化波尔丹和波尔丹的能力- D1打开了这些多亚基复合物用于详细的机理和结构分析。我们能够 第一次提出与Pol C-d和Pol C-d1的整体架构有关的问题，以及Pol 31，Pol 32， 和Rev 1增强了Pol β对未损伤和损伤DNA底物的催化活性。目标1： 将在存在DNA的情况下对Pol E10-d和Pol E10-d1进行低温电子显微镜（cryo-EM）分析， 最新发展的直接检测设备相机成像单分子的优势， 高分辨率三维重建软件。这些研究将建立在我们早期的低分辨率模型上 首次揭示了这种酶实际上是如何与DNA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，我们将进行预稳态动力学分析， 确定了Pol β-d和Pol β-d1的作用机制。这些研究将与 结构研究（目的1），以获得反应途径的动力学图。我们还将测试 通过生物化学和遗传学方法，其中Rev 3活性位点中的残基和介导 将突变亚基蛋白质-蛋白质相互作用，并测定它们对体外DNA合成的影响， 对体内DNA损伤反应的影响总而言之，拟议的研究对于理解如何 真核细胞科普外源性和内源性遗传毒性诱导的各种DNA损伤 剂.