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

基本信息

批准号：
10018049
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
项目类别：
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/10018049
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

项目摘要

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 DNA 聚合酶 (Pols)、Pols δ 和 ε 属于 Pols B 家族，它们进行复制保真度非常高的 DNA 尽管 DNA 聚合酶 z (Pol z) 也是 B 家族的成员，但它有所不同。与复制 Pols 不同，它合成保真度较低的 DNA，并在促进通过多种 DNA 损伤进行复制在这方面具有独特的催化活性。由 Rev3 催化亚基和 Rev7 辅助亚基组成；然而，我们表明聚合酶在体内包含两个额外的亚基，Pol31 和 Pol32，我们将这种四亚基复合物称为 Pol z-d。此外，我们最近证明Rev1是Pols Y家族的成员，也是化学计量的 Pol z-d 的组成部分，我们将这种五亚基复合物称为 Pol z-d1，我们纯化 Pol z-d 和 Pol z- 的能力。 d1 打开这些多亚基复合物进行详细的机械和结构分析。第一次提出与 Pol z-d 和 Pol z−d1 的整体架构相关的问题，以及 Pol31、Pol32、在目标 1 中，Rev1 和 Rev1 增强了 Polze 对未受损和受损 DNA 底物的催化活性。将在 DNA 存在的情况下对 Pol z-d 和 Pol z-d1 进行冷冻电子显微镜 (cryo-EM) 分析，利用直接检测设备相机的最新发展来成像单分子和这些研究将建立在我们早期的低分辨率模型的基础上。 Pol z-d 的研究，并首次揭示了酶实际上如何与 DNA 相互作用以及蛋白质的性质 - 在目标 2 中，我们将进行预稳态动力学分析。确定 Pol z-d 和 Pol z-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我们还将测试结构。通过生化和遗传方法，因此 Rev3 活性位点和介导的残基亚基蛋白质-蛋白质相互作用将被突变并分析其对体外 DNA 合成的影响，总之，所提出的研究对于理解体内 DNA 损伤反应非常重要。真核细胞应对由外源性和内源性基因毒性引起的多种 DNA 损伤代理。