Biochemical Basis of Somatic Hypermutation

描述(由申请者提供)：一个与健康相关的基本科学挑战解决了免疫系统如何保护免受各种传染病的影响。我们的广泛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需要两个关键事件，即体细胞超突变(SHM)和类切换重组(CSR)。AB的多样化需要一种B细胞特异性酶的作用，即激活诱导胞苷脱氨酶(AID)。AID是APOBEC核酸胞苷脱氨酶家族的成员，在免疫球蛋白基因转录过程中将C？U转化为SHM和CSR。APOBEC3G(A3G)是逆转录病毒上的C？U转换蛋白，在限制艾滋病病毒(HIV-1)在T细胞中的感染中起着重要作用。从生物学的角度来看，了解AID和A3G的生化特性对于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抵抗力方面的编程作用是至关重要的。从机械学的角度来看，要理解AID和A3G的生化特性，就需要破译设计用于脱氨DNA链中C碱基的过程酶的随机特性。A3G和A3G催化的脱氨基反应以一种“随意”的方式发生，导致不同的突变分布在它们的DNA靶标、AID的Ig可变区和切换区以及A3G的HIV-1cDNA中。深入的体外分析旨在揭示突变多样化分布的生化基础，是这项提议的关键目标。这两种酶都采用了一个过程扫描过程，包括沿着单链DNA滑动和跳跃。具体目标1和3分别分析了AID和A3G的扫描和脱氨机制。特定目的2检查WT AID与与人类高IgM-2综合征相关的AID突变体的脱氨特性，在这些突变体中，抗体多样化无法发生。特定目标4使用激光单分子显微镜来可视化由AID和A3G进行的扫描，并测试从AIMS 1-3派生的3-D扫描机制。AID引发一系列突变事件，涉及容易出错的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。在特定的目标5中，我们拓宽了我们的视野，着眼于AID的“下游”，以研究容易出错的错配修复和碱基切除修复的体外系统。 公共卫生评论：在所有生物体中，从微生物到人类，突变几乎总是有害的，这是不言而喻的，它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“容易出错的”DNA修复，故意在极高的水平上引入突变。这些突变途径是有益的，而且在提供免疫多样性、一般健康和避免细胞死亡方面往往是必不可少的。这项拟议的研究探索了两种人类DNA胞苷脱氨酶的作用机制，即激活诱导胞苷脱氨酶(AID)和APOBEC3G(A3G)。援助可确保抗体多样化。A3G对HIV-1逆转录病毒感染具有天生的抵抗力。这些酶受到严格的调控，因为人们知道，如果AID或A3G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A3G催化的脱氨基的生化特性，这种脱氨基以一种“随意”的方式发生，导致不同的突变分布 在他们的DNA靶标中，AID的免疫球蛋白可变区和A3G的HIV-1互补DNA。

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.