Biochemical Basis of Somatic Hypermutation

描述（由申请人提供）：一个基本的健康相关的科学挑战，解决了免疫系统如何保护免受各种各样的传染性病原体。我们的主要目标是研究人类免疫多样性的生化基础。从低特异性抗体产生高亲和力抗体（Ab）需要两个关键事件，即体细胞超突变（SHM）和类别转换重组（CSR）。抗体多样化需要B细胞特异性酶，活化诱导的胞苷脱氨酶（AID）的作用。AID是核酸胞苷脱氨酶APOBEC家族的一员，可将C？U在免疫球蛋白基因转录期间启动SHM和CSR。APOBEC 3G（A3 G），其将C？逆转录病毒cDNA上的U，在限制T细胞中艾滋病病毒（HIV-1）的感染中起着重要作用。从生物学的角度来看，AID和A3 G的生化特性的理解是必不可少的，以掌握这些酶在确保抗体多样化和对逆转录病毒感染施加先天性抗性的程序化作用。从机械的角度来看，AID和A3 G的生化特性的理解需要破译的随机特性的加工酶设计脱氨基的DNA链中的C碱基。AID-和A3 G-催化的脱氨基作用以“偶然”的方式发生，导致分布在其DNA靶、AID的IG可变区和开关区以及A3 G的HIV-1 cDNA中的不同突变。旨在揭示突变多样性分布的生化基础的深入体外分析是本提案的一个关键目标。这两种酶都采用了连续的扫描过程，包括沿着ssDNA的沿着和跳跃。具体目标1和3分别分析了AID和A3 G的扫描和脱氨基机制。具体目标2检查了WT AID与人类中与高IgM-2综合征相关的AID突变体相比的脱氨基性质，其中Ab多样化未能发生。具体目标4使用激光单分子显微镜来可视化AID和A3 G扫描，并测试源自目标1-3的三维扫描机制。AID引发了一系列突变事件，涉及易错DNA聚合酶、碱基切除修复（BER）和错配修复（MMR）酶，最终导致高度突变的抗体基因库。在具体目标5中，我们拓宽了我们的视角，从AID的“下游”看，研究易错配修复和碱基切除修复的体外系统。 公共卫生部门：在所有生物体中，从微生物到人类，突变几乎总是有害的，是许多疾病的根本原因，最突出的是癌症。然而，有一些程序化的途径涉及“易错”DNA修复，故意引入极高水平的突变。这些突变途径是有益的，并且通常在提供免疫多样性、一般适应性和避免细胞死亡方面是必不可少的。该研究探索了两种人类DNA胞苷脱氨酶，激活诱导的胞苷脱氨酶（AID）和APOBEC 3G（A3 G）的作用机制。艾滋病署确保抗体多样化。A3 G对HIV-1逆转录病毒感染具有先天抗性。这些酶受到严格的调控，因为如果AID或A3 G在错误的时间或错误的地点表达，就会发生癌症。这项研究需要破译AID和A3 G催化的脱氨基的生物化学性质，这些脱氨基以“偶然”的方式发生，导致分布在不同区域的不同突变。 在它们的DNA靶标中，免疫球蛋白可变区用于AID，HIV-1互补DNA用于A3 G。

DESCRIPTION (provided by applicant): A fundamental health-related scientific challenge addresses how the immune system protects against a wide variety of infectious agents. Our broad objective is to investigate the biochemical basis of human immunodiversity. Two key events are required to produce high affinity antibodies (Ab) from lower specificity antibodies, namely, somatic hypermutation (SHM) and class switch recombination (CSR). Ab diversification requires the action of a B-cell specific enzyme, activation induced cytidine deaminase (AID). AID, a member of the APOBEC family of nucleic acid cytidine deaminases, converts C?U during transcription of immunoglobulin genes to initiate SHM and CSR. APOBEC3G (A3G), which converts C?U on retroviral cDNA, plays an instrumental role in restricting infection of the AIDS virus (HIV-1) in T cells. From a biological perspective, an understanding of the biochemical properties of AID and A3G is essential to grasp the programmed roles for these enzymes in ensuring Ab diversification and in imposing innate resistance against retroviral infection. From a mechanistic perspective, an understanding of the biochemical properties of AID and A3G entail deciphering the stochastic properties of processive enzymes designed to deaminate C bases in DNA strands. AID- and A3G-catalyzed deaminations occur in a "haphazard" manner resulting in diverse mutations distributed throughout their DNA targets, Ig variable and switch regions for AID, and HIV-1 cDNA for A3G. An in-depth in vitro analysis aimed at revealing the biochemical basis for the diverse distribution of mutations is a key objective of this proposal. Both enzymes employ a processive scanning process, involving sliding and jumping along ssDNA. Specific Aims 1 and 3 analyze scanning and deamination mechanisms for AID and A3G, respectively. Specific Aim 2 examines the deamination properties of WT AID compared to AID mutants associated with hyper-IgM-2 syndrome in humans, in which Ab diversification fails to occur. Specific Aim 4 uses laser single molecule microscopy to visualize scanning by AID and A3G and to test 3-D scanning mechanisms derived from Aims 1-3. AID instigates a cascade of mutational events involving error-prone DNA polymerases, base excision repair (BER) and mismatch repair (MMR) enzymes culminating in a pool of highly mutated antibody genes. In Specific Aim 5, we broaden our perspective and look "downstream" from AID, to investigate in vitro systems for error-prone mismatch repair and base excision repair. PUBLIC HEALTH REVELANCE: In all organisms, from microorganisms to humans, it is axiomatic that mutations are almost always deleterious, serving as a fundamental cause of numerous diseases, most prominently cancer. There are, however, programmed pathways involving "error-prone" DNA repair that deliberately introduce mutations at extremely high levels. These mutational pathways are beneficial, and often essential in providing immunological diversity, general fitness and avoidance of cell death. The proposed research explores the mechanisms used by two human DNA cytidine deaminases, activation-induced cytidine deaminase (AID) and APOBEC3G (A3G). AID ensures antibody diversification. A3G imposes innate resistance against HIV-1 retroviral infection. The enzymes are under tight regulation, because cancer is known to occur if AID or A3G are expressed at the wrong time or in the wrong place. The research entails deciphering the biochemical properties of AID- and A3G-catalyzed deaminations, which occur in a "haphazard" manner resulting in diverse mutations distributed throughout their DNA targets, immunoglobulin variable regions for AID, and HIV-1 complementary DNA for A3G.