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.

真核复制性DNA聚合酶（POLS），POLSδ和ε属于Pols的B家族，它们复制 DNA具有很高的保真度。尽管DNA聚合酶ζ（POLζ）也是B家族的成员，但它有所不同 从复制pol中，它以较低的保真度合成DNA，并且在促进中起着关键作用 通过多种DNA病变复制。在这方面，波尔术是独一无二的。催化活性的polζ是 Rev3催化和Rev7附件亚基的积累；但是，我们证明了聚合酶 在体内包含两个其他亚基，POL31和POL32，我们将此四亚基复合物称为Polζ-D。 此外，我们最近表明，pol的Y家庭成员Rev1也是化学计量学 我们纯化polζ-d和polζ-d1的能力。 D1打开了这些多工厂复合物，以进行详细的机械和结构分析。我们能够 询问与Polζ-D和Polζ-D1的整体架构以及Pol31，Pol32， Rev1潜力POLζ对未分离和受损的DNA底物的催化活性。在AIM 1中，我们 将在DNA存在下对POLζ-D和POLζ-D1进行冷冻电子显微镜（Cryo-EM）分析， 直接检测装置摄像机中最新发展的优势用于成像单分子和 高分辨率的3D重建软件。这些研究将以我们较早的低分辨率模型为基础 波尔ζ-d并首次揭示了酶如何实际与DNA相互作用以及蛋白质的性质 各个亚基之间的蛋白质接触。在AIM 2中，我们将进行稳态的态动力学分析 确定POLζ-D和POLζ-D1的作用机理。这些研究将与 结构研究（目标1）获得了反应途径的动力学图片。我们还将测试结构 通过生化和遗传方法，Rev3活动部位的残留物以及介导的残留物 亚基蛋白 - 蛋白质相互作用将被突变并分配，以便其在体外对DNA合成的影响和 在体内DNA损伤反应上。总之，拟议的研究对于理解如何 真核细胞与由外源性和内源性基因毒素诱导的DNA病变的潜水阵列应对 代理商。