Hypermutation in Bacteria and Humans

总结 所有生物体的生存挑战均来自于环境中自然存在的 DNA 损伤剂， 例如，紫外线辐射和氧气，以及有毒工业化学品。诱导“超突变”，同时 也许违反直觉，通过确保细胞和 有机体健康。超突变，每个碱基对发生频率约为 10-2 – 10-3 的突变，跨越 a 细菌和人类的死亡和健康之间的范围。健身关键在于细心调节 超突变。我们的资助提案是阐明两种重要的超变酶 DNA 的调节 大肠杆菌中的聚合酶 V 突变体 (pol V Mut) 可催化受损DNA 的跨损伤合成 强大的免疫反应所需的 DNA 模板和激活诱导的脱氧胞苷脱氨酶 (AID) 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。除了聚合酶活性外，pol V Mut 还具有内在的 DNA 依赖性 ATP 酶 活性不同于所有其他 ATP 酶。 Pol V Mut 以两种构象不同的状态存在，激活状态和 根据 RecA 的位置停用。我们假设内部 ATP 酶提供能量 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 假设使用 TIRF-FRET 显微镜可视化每种构象之间切换的动态 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，有激活和失活的形式。 AID在免疫中发挥重要作用 通过在 B 细胞中启动体细胞超突变 (SHM) 和类别转换重组 (CSR) 来响应 在免疫球蛋白可变区 (IgV) 和开关区 (IgS) DNA 转录过程中使 C→U 脱氨基。我们 提议重建第一个生化系统来研究 IgV 和 IgV 期间的 AID 靶向和催化 人 RNA 聚合酶 II 转录 IgS。本研究旨在为该研究奠定生化基础。 产生抗体 (Ab) 多样性所需的超突变反应。我们建议使用 TIRF-FRET 显微镜观察 IgV 和 IgS 转录过程中 AID 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，我们获得了晶体 援助的结构。我们现在提出一种获得 AID-ssDNA 共晶结构的策略。环保 过敏原可引起哮喘。我们将利用共晶结构来设计抑制IgE的AID抑制剂 生产治疗哮喘。一个新颖的高风险高回报项目，旨在实现亲和力成熟 试管，使用 AID 和容易出错的人类 DNA 聚合酶 η，旨在产生单克隆抗体 针对任何抗原。作为原理证明，我们建议针对三个关键离子通道生成 Abs 涉及疼痛、热和冷的受体。离子通道受体抗体的可用性将提供主要的 缓解过敏人群疼痛和瘙痒的医学突破。

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.