Biochemical Basis of Somatic Hypermutation

描述（由申请人提供）：一项与健康相关的基本科学挑战，涉及免疫系统如何抵御各种感染因子。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）和类开关重组（CSR）。Ab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过程中启动SHM和CSR。APOBEC3G (A3G)，它转换C？U在逆转录病毒cDNA上，在限制艾滋病病毒（HIV-1）在T细胞中的感染中起重要作用。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Ab多样化和对逆转录病毒感染施加先天抗性方面的程序化作用至关重要。从机制的角度来看，了解AID和A3G的生化特性需要破译用于脱胺DNA链中C碱基的过程酶的随机特性。AID-和A3G催化的脱氨以“偶然”的方式发生，导致不同的突变分布在它们的DNA靶点、AID的Ig变量和开关区以及A3G的HIV-1 cDNA中。深入的体外分析旨在揭示突变多样性分布的生化基础是本提案的关键目标。这两种酶都采用了一种渐进的扫描过程，包括沿着ssDNA滑动和跳跃。具体目标1和3分别分析AID和A3G的扫描和解析机制。特异性目的2研究了WT AID与人类高igm -2综合征相关的AID突变体的脱胺特性，后者不能发生Ab多样化。特异性Aim 4使用激光单分子显微镜观察AID和A3G扫描，并测试Aim 1-3衍生的3d扫描机制。AID引发一系列突变事件，包括易出错的DNA聚合酶、碱基切除修复（BER）和错配修复（MMR）酶，最终形成大量高度突变的抗体基因。在Specific Aim 5中，我们拓宽了我们的视角，从AID的“下游”来看，研究易于出错的错配修复和碱基切除修复的体外系统。

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.