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

 描述(申请人提供)：激活诱导胞苷脱氨酶(AID)启动两个由激活的B细胞进行的程序性突变过程，极大地提高抗体反应的效率，无论是在感染还是在疫苗接种的背景下。在体细胞高度突变(SHM)中，AID通过在免疫球蛋白基因座可变区外显子(编码抗原结合域的外显子)内产生的起始病变转化为点突变来使抗体的抗原结合谱系多样化。在类开关重组(CSR)中，免疫球蛋白重链切换区的AID活性导致DNA双链断裂(DSB)，DSB为表达的IgH恒定区外显子的交换提供中介，决定了病原体消除效应的功能。我们对AID活性的两个关键功能方面缺乏明确的理解：它如何针对特定的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理解上的一个根本差距。最近的研究结果表明，广泛中和抗艾滋病毒抗体(抗艾滋病毒bNAbs)的可变区被广泛且必然地过度突变，这突显了理解这些机制的相关性。我们现在建议检验靶底物的DNA序列促进特异性AID靶向并影响这种靶向的结果的假设，并在此背景下详细阐明免疫球蛋白重链可变区外显子(目标1)和切换区(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等位基因小鼠模型系统和突变图谱工具，可以有效地测量给定DNA序列中的AID突变活性。我们将使用假设驱动的方法，将候选序列基序嵌入到合成序列中，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 将进行数据处理，以发现新的主题。这些研究应该为SHM和CSR的机制以及产生各种有效抗体的机制提供丰富的基本见解。信息也将与我们对脱离目标的理解相关 有助于致癌突变和染色体易位的活动。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，例如那些促进或指导高度突变的抗艾滋病毒bNAb沿着所需途径成熟的方法。

 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.