Hypermutation in Bacteria and Humans

细菌和人类的超突变

• 批准号: 9924572
• 负责人: MYRON GOODMAN
• 金额: $ 54.87万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9924572
• 关键词: ATP phosphohydrolase; Affinity; Antibodies; Antibody Diversity; Antigens; Applications Grants; Asthma; B-Lymphocytes; Bacteria; Base Pairing; Biochemical; Catalysis; Cell Death; Cells; Cessation of life; Cryoelectron Microscopy; Crystallization; DNA; DNA Damage; DNA biosynthesis; DNA polymerase V; DNA-dependent ATPase; Energy-Generating Resources; Ensure; Environment; Enzymes; Escherichia coli; Exposure to; Fc Receptor; Fluorescence Resonance Energy Transfer; Frequencies; Generation of Antibody Diversity; Genetic; Genetic Transcription; Human; Hypersensitivity; IgE; Immune response; Immunoglobulin Class Switching; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; Individual; Ion Channel; Lesion; Location; Medical; Microscopy; Molecular Conformation; Monoclonal Antibodies; Mutation; Organism; Oxygen; Pain; Play; Polymerase; Production; Proteins; Pruritus; RNA Polymerase II; Rad30 protein; Reaction; Regulation; Resolution; Role; Stress; Structure; System; Testing; Tube; Ultraviolet Rays; deoxycytidine deaminase; design; environmental allergen; fitness; high reward; high risk; human DNA; inhibitor/antagonist; novel; pain relief; receptor; recombinase; reconstitution; research study; response; single molecule; toxic industrial chemical

Summary Existential challenges to all organisms result from DNA damaging agents present naturally in the environment, e.g., UV radiation and oxygen, and from toxic industrial chemicals. The induction of “hypermutation”, while perhaps counterintuitive, is essential to counter exposure to environmental stress by ensuring cell and organismic fitness. Hypermutations, mutations occurring at frequencies ~ 10-2 – 10-3 per base pair, straddle a range between death and fitness in bacteria and humans. The key to fitness is to carefully regulate hypermutation. Our grant proposal is to elucidate the regulation of two essential hypermutator enzymes, DNA polymerase V mutasome (pol V Mut) in Escherichia coli that catalyzes translesion DNA synthesis on damaged DNA templates, and activation-induced deoxycytidine deaminase (AID) required for a robust immune response in humans. Pol V Mut has a multisubunit structure that includes a RecA molecule, the E. coli recombinase, and a molecule of ATP. Along with its polymerase activity, pol V Mut also has an intrinsic DNA-dependent ATPase activity different from all other ATPases. Pol V Mut exists in two conformationally distinct states, activated and deactivated depending on the location of RecA. We hypothesize that the internal ATPase provides an energy source to switch between conformation states, akin to an “on-off” toggle switch. We propose to test this hypothesis using TIRF-FRET microscopy to visualize the dynamics of switching between each conformational state of pol V Mut at single-molecule resolution, and to use Cryo-EM to determine the location of each pol V subunit, most importantly RecA, in activated and deactivated forms. AID plays an essential role in the immune response by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) in B-cells by deaminating C→U during transcription of immunoglobulin variable (IgV) and switch (IgS) region DNA. We propose to reconstitute the first biochemical system to investigate AID targeting and catalysis during IgV and IgS transcription by human RNA polymerase II. This study is intended to establish a biochemical basis for the hypermutation reactions required in the generation of antibody (Ab) diversity. We propose to use TIRF-FRET microscopy to visualize the action of AID during IgV and IgS transcription, including the influence of proteins believed to be involved in targeting AID to stalled transcription bubbles. In 2016, we obtained a crystal structure for AID. We now propose a strategy to obtain an AID-ssDNA co-crystal structure. Environmental allergens can cause asthma. We will use the co-crystal structure to design AID inhibitors that suppress IgE production to treat asthma. A novel high-risk high-reward project, designed to achieve affinity maturation in a test tube, using AID and error-prone human DNA polymerase η, is aimed at generating monoclonal Abs against any antigen. As a proof of principal, we propose to generate Abs against three critical ion channel receptors involved in pain, heat and cold. The availability of ion channel receptor Abs would provide a major medical breakthrough to relieve pain and itching in hypersensitive individuals.

摘要 对所有生物的生存挑战源于环境中自然存在的DNA损伤剂， 例如，紫外线辐射和氧气，以及有毒的工业化学品。“超突变”的诱导，而 也许是违反直觉的，这对于通过确保细胞和 身体健康。超突变，每一碱基对发生频率约为10-2-10-3的突变，跨越 在细菌和人类中，死亡和健康之间的范围。健身的关键是仔细调节 超突变。我们的拨款建议是为了阐明两种重要的超变种酶DNA的调节。 大肠杆菌中催化跨损伤DNA合成的聚合酶V突变酶体(PolV Mut) DNA模板和激活诱导的脱氧胞苷脱氨酶(AID)是强大的免疫反应所必需的 在人类身上。Pol V Mut具有多亚基结构，包括RecA分子、大肠杆菌重组酶和 一种三磷酸腺苷分子。在其聚合酶活性的同时，PolV Mut还具有一种内在的DNA依赖的ATPase 活性不同于所有其他ATPase。Pol V Mut以两种不同的构象状态存在，激活状态和 根据RecA的位置停用。我们假设体内的ATPase提供一种能量 在构象状态之间切换的源，类似于“开-关”切换开关。我们建议对此进行测试 使用TIRF-FRET显微镜观察不同构象之间转换动力学的假说 在单分子分辨率下获得PolV Mut的状态，并使用Cryo-EM确定每个PolV的位置 亚基，最重要的是RecA，以激活和失活的形式存在。援助在免疫系统中起着至关重要的作用 在B细胞中启动体细胞高突变(SHM)和类切换重组(CSR)的反应 免疫球蛋白可变区和转换区转录过程中C-→U的脱氨基。我们 建议重建第一个生化系统来研究免疫球蛋白和免疫球蛋白的靶向和催化作用 人RNA聚合酶II转录免疫球蛋白。本研究旨在为免疫球蛋白的生物化学研究奠定基础 产生抗体(Ab)多样性所需的高度突变反应。我们建议使用TIRF-FRET 显微镜观察免疫球蛋白在免疫球蛋白和免疫球蛋白转录过程中的作用，包括蛋白质的影响 据信参与了针对陷入停滞的转录泡泡的AID。2016年，我们获得了一颗水晶 用于AID的结构。我们现在提出了一种获得AID-ssDNA共晶结构的策略。环境 过敏原会导致哮喘。我们将使用共晶体结构来设计抑制IgE的艾滋病抑制剂 用于治疗哮喘的产品。一个新的高风险高回报项目，旨在实现亲和力成熟 使用艾滋病和容易出错的人类DNA聚合酶η的试管旨在产生单克隆抗体 对抗任何抗原。作为原理的证明，我们建议生成针对三个关键离子通道的抗体 感受器与疼痛、高温和寒冷有关。离子通道受体抗体的可获得性将提供主要的 医学上的突破，可缓解过敏症患者的疼痛和瘙痒。