Hypermutation in Bacteria and Humans

总结 对所有生物体的潜在挑战来自环境中天然存在的DNA损伤剂， 例如，在一个实施例中，紫外线辐射和氧气，以及有毒的工业化学品。诱导“超突变”，而 也许违反直觉，是必不可少的，以对抗暴露于环境压力，通过确保细胞和 器官健康超突变，突变发生的频率约为10-2 - 10-3每碱基对，跨越一个 在细菌和人类的死亡和健康之间的范围。健身的关键在于精心调理 超突变我们的资助计划是阐明两种重要的超变酶DNA的调控 大肠杆菌中的聚合酶V突变体（pol V Mut），催化受损细胞上跨损伤DNA合成 DNA模板和激活诱导的脱氧胞苷脱氨酶（AID）是强大免疫应答所需的 在人类身上。Pol V Mut具有多亚基结构，包括RecA分子，E.大肠杆菌重组酶，和 ATP分子沿着其聚合酶活性，pol V Mut还具有内在的DNA依赖性ATP酶 它不同于其他所有的ATP酶。Pol V Mut以两种构象不同的状态存在，活化和 取决于RecA的位置我们假设体内的ATP酶提供了能量 源以在构造状态之间切换，类似于“开-关”拨动开关。我们打算测试一下 假设使用TIRF-FRET显微镜来可视化每个构象之间的切换动态 单分子分辨率下pol V Mut的状态，并使用Cryo-EM确定每个pol V的位置 亚基，最重要的是RecA，以激活和失活形式存在。艾滋病在免疫系统中起着至关重要的作用， 通过启动B细胞中的体细胞超突变（SHM）和类别转换重组（CSR）， 在免疫球蛋白可变区（IgV）和开关区（IgS）DNA转录过程中脱氨基C→U。我们 建议重建第一个生化系统，以研究IgV期间的AID靶向和催化作用， 通过人RNA聚合酶II的IgS转录。本研究旨在建立一个生化基础， 抗体（Ab）多样性产生所需的超突变反应。我们建议使用TIRF-FRET 显微镜观察AID在IgV和IgS转录过程中的作用，包括蛋白质的影响 据信参与将AID靶向停滞的转录气泡。2016年，我们获得了一个晶体 援助的结构。我们现在提出一种获得AID-ssDNA共晶体结构的策略。环境 过敏原会引起哮喘。我们将利用共晶结构设计出抑制IgE的AID抑制剂 治疗哮喘的药物一个新的高风险高回报的项目，旨在实现亲和力成熟， 用AID和易错的人DNA聚合酶η在试管中制备单克隆抗体 任何抗原。作为原理的证明，我们提出了针对三个关键离子通道产生抗体的方法， 感受器与疼痛、热和冷有关。离子通道受体抗体的可用性将提供一个主要的 医学突破，以减轻疼痛和瘙痒的过敏个人。

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.