Hypermutation in Bacteria and Humans

细菌和人类的超突变

基本信息

批准号：
10626889
负责人：
MYRON GOODMAN
金额：
$ 50.52万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
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/10626889
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 DNA DNA Damage DNA biosynthesis DNA polymerase V DNA-dependent ATPase Deamination 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 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 Single-Stranded DNA Stress Structure System Testing Tube Ultraviolet Rays Visualization deoxycytidine deaminase design environmental allergen fitness high reward high risk human DNA inhibitor novel pain relief receptor recombinase reconstitution research study response single molecule toxic industrial chemical

项目摘要

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.

概括 DNA破坏因子在环境中自然存在的DNA破坏药物所造成的所有生物的存在挑战，例如，紫外线辐射和氧气，以及有毒工业化学品。诱导“超名”，而也许违反直觉，对于通过确保细胞和细胞来抵抗环境压力至关重要有机健身。高压，突变发生在频率约10-2 - 10-3，跨A跨A 在细菌和人类的死亡与健身之间的范围。健身的关键是仔细调节超名。我们的赠款提案是阐明对两种必不可少的高压酶，DNA的调节大肠杆菌中的聚合酶v untasome（pol v mut），催化translesion DNA合成在受损 DNA模板和激活诱导的脱氧胞苷脱氨酶（AID）（AID）需要强大的免疫反应在人类中。 pol V mut具有多亚基结构，其中包括RECA分子，大肠杆菌重组酶和 ATP分子。伴随其聚合酶活性，POL V MUT还具有固有的DNA依赖性ATPase 活动与所有其他ATPases不同。 Pol V Mut存在于两个构象上不同的状态，即激活和取决于RECA的位置。我们假设内部ATPase提供了能量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显微镜假设可视化每个构象之间切换的动力学单分子分辨率下的pol V mut状态，并使用冷冻EM确定每个Pol V的位置亚基，最重要的是reca，以激活和停用形式。援助在免疫中起着至关重要的作用 B细胞中启动的体细胞超突变（SHM）和类切换重组（CSR）的响应免疫球蛋白变量（IGV）和开关（IGS）区域DNA转录期间的脱孔C→U。我们提议重建第一个研究IGV期间辅助靶向和催化的生化系统人RNA聚合酶II的IGS转录。这项研究旨在为产生抗体（AB）多样性所需的超数反应。我们建议使用tirf-fret 显微镜以可视化IgV和IgS转录过程中的辅助作用，包括蛋白质的影响据信与靶向辅助有关停滞的转录气泡。 2016年，我们获得了水晶辅助结构。现在，我们提出了一种获得AID-SSDNA共晶体结构的策略。环境的过敏原会引起哮喘。我们将使用共晶体结构来设计抑制IgE的帮助抑制剂生产以治疗哮喘。一个新型的高风险高回报项目，旨在实现一个亲和力成熟使用辅助和容易出错的人DNA聚合酶η的试管旨在产生单克隆ABS 反对任何抗原。作为本金的证明，我们建议针对三个关键离子渠道产生ABS 疼痛，热和寒冷涉及的受体。离子通道受体ABS的可用性将提供主要医疗突破，以挽救过度敏感的人的疼痛和瘙痒。