Mechanisms by which DNA sequence directs AID-mediated mutagenesis of Ig loci

DNA 序列指导 AID 介导的 Ig 位点突变的机制

• 批准号: 8907409
• 负责人: Joyce Keshin Hwang
• 金额: $ 3.55万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2018-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8907409
• 关键词: Affinity; Alleles; Antibodies; Antibody Repertoire; Antibody Response; Antigens; Area; B-Lymphocytes; Base Composition; Bioinformatics; Biological Assay; Biological Models; Characteristics; Chromosomal translocation; Complementarity Determining Regions; Cytidine; DNA; DNA Double Strand Break; DNA Sequence; Data; Deamination; Enzymes; Exons; Fc Receptor; Generations; Goals; HIV; Heavy-Chain Immunoglobulins; Immune response; Immune system; Immunization; Immunoglobulin Class Switching; Immunoglobulin Constant Region; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; Immunoglobulins; Indium; Infection; Lead; Lesion; Maps; Mature B-Lymphocyte; Measures; Mediating; Mutagenesis; Mutate; Mutation; Oncogenic; Outcome; Pathway interactions; Pattern; Point Mutation; Positioning Attribute; Process; Property; Rana; Relative (related person); Repetitive Sequence; Role; SWI1; Specificity; Structure of germinal center of lymph node; System; Testing; Time; Vaccination; activation-induced cytidine deaminase; antigen antibody binding; antigen binding; base; density; design; insertion/deletion mutation; insight; mouse model; neutralizing antibody; novel; novel strategies; pathogen; programs; public health relevance; receptor; research study; tool

 DESCRIPTION (provided by applicant): Activation-induced cytidine deaminase (AID) initiates two processes of programmed mutagenesis undertaken by activated B cells that vastly increase the efficacy of antibody responses, whether in the context of infection or vaccination. In somatic hypermutation (SHM), AID diversifies the antigen-binding repertoire of antibodies by creating initiating lesions that are converted into point mutations within the immunoglobulin loci variable region exons, the exons that encode the antigen-binding domain. In class switch recombination (CSR), AID activity in immunoglobulin heavy chain switch regions leads to DNA double strand breaks (DSBs) that provide intermediates for exchange of expressed IgH constant region exons, which determine pathogen- elimination effector function. We lack a definitive understanding of two functionally critical aspects of AID activity: how it is targeted to specific DNA regions, and how AID activity leads to different outcomes during SHM and CSR. This is a fundamental gap in our understanding of antibody maturation processes. Recent findings that the variable regions of broadly neutralizing antibodies against HIV (anti-HIV bnAbs) are extensively and necessarily hypermutated underscore the relevance of understanding these mechanisms. We now propose to test the hypothesis that the DNA sequence of target substrates promotes specific AID targeting and influences the outcome of such targeting, and, in this context, to elucidate in detai the roles of particular sequences and motifs of the immunoglobulin heavy chain variable regions exons (Aim 1) and switch regions (Aim 2). To this end, we developed a novel V(D)J passenger allele mouse model system and mutation mapping tools that efficiently measure AID mutation activity within a given DNA sequence. We will use hypotheses-driven approaches in which candidate sequence motifs are embedded in synthetic sequences and tested in our passenger allele system for effects on AID activity. In parallel, bioinformatics analyses of mutation and DSB data will be performed to discover novel motifs. These studies should provide a wealth of basic insights into the mechanism of SHM and CSR and thus into the mechanisms that generate diverse and potent antibodies. Information will also be relevant to our understanding of off-target AID activity that contributes to oncogenic mutations and chromosomal translocations. Beyond that, proposed experiments may contribute to designing approaches to optimize immune responses such as those that promote or direct the maturation of highly mutated anti-HIV bnAbs along desired pathways.

 描述（由申请方提供）：活化诱导的胞苷脱氨酶（AID）启动活化B细胞进行的两个程序性诱变过程，无论是在感染还是疫苗接种的情况下，这都极大地增加了抗体应答的效力。在体细胞超突变（SHM）中，AID通过产生起始损伤使抗体的抗原结合库多样化，所述起始损伤转化为免疫球蛋白基因座可变区外显子（编码抗原结合结构域的外显子）内的点突变。在类别转换重组（CSR）中，免疫球蛋白重链转换区中的AID活性导致DNA双链断裂（DSB），其提供用于交换表达的IgH恒定区外显子的中间体，其决定病原体消除效应子功能。 我们对AID活性的两个功能关键方面缺乏明确的理解：它如何靶向特定的DNA区域，以及AID活性如何在SHM和CSR期间导致不同的结果。这是我们对抗体成熟过程的理解中的一个根本差距。最近的研究发现，广泛中和抗体的可变区抗HIV（抗HIV bnAb）是广泛和必然的高度突变强调了理解这些机制的相关性。 我们现在提出测试的假设，即靶底物的DNA序列促进特定的AID靶向和影响这种靶向的结果，并在这种情况下，详细阐明免疫球蛋白重链可变区外显子（目标1）和开关区（目标2）的特定序列和基序的作用。为此，我们开发了一种新的V（D）J乘客等位基因小鼠模型系统和突变作图工具，可以有效地测量给定DNA序列内的AID突变活性。我们将使用假设驱动的方法，其中候选序列基序嵌入合成序列中，并在我们的乘客等位基因系统中测试对AID活性的影响。同时，突变和DSB的生物信息学分析 数据将被执行以发现新的图案。 这些研究应该为SHM和CSR的机制提供丰富的基本见解，从而产生多样化和有效的抗体的机制。信息也将与我们对脱靶的理解相关 有助于致癌突变和染色体易位的AID活性。除此之外，拟议的实验可能有助于设计优化免疫反应的方法，例如促进或指导高度突变的抗HIV bnAb沿着所需途径成熟的方法。