AID in somatic mutation of immunoglobulin genes

描述(由申请人提供)：免疫球蛋白基因的体细胞超突变(SHM)过程是由胞苷脱氨酶AID产生胞苷(C)到尿苷(U)的转换突变。在SHM之外，U在碱基切除修复(BER)和错配修复(MMR)的帮助下被忠实地修复为C。矛盾的是，在SHM过程中，这些修复机制与跨损伤DNA聚合酶一起被招募，通过容易出错的修复在U处和附近产生突变。SHM的一个重要特征是将突变限制在Ig基因的5‘端，包括V(D)J区，但不包括恒定区。通过这种方式，在保护抗体恒定区的生物学功能的同时，创建了抗原结合可变区的巨大多样性。我们考虑了两种可能实现这种V区限制的方式。首先，AID可能只在5‘区活跃，其次，AID可能在整个基因中起作用，但容易出错的修复只在5’区起作用。在对BER或MMR缺陷或两者都有缺陷的小鼠进行的广泛实验中，我们发现了与野生型小鼠相同的突变模式，即限制在可变区及其紧邻侧翼。这导致了AID不作用于Ig基因的3‘部分的结论。我们现在希望确定AID与其脱氨基Cs的序列究竟是如何相互作用的。基于Ig基因启动子在恒定区前面复制时恒定区发生突变的实验，我们提出了如下模型：AID与启动子附近的转录复合体结合，在转录延伸过程中与RNA聚合酶一起移动，在聚合酶后面的负DNA超螺旋中脱氨基单链DNA中的Cs，由于亲和力有限，在距离启动子~2kbp的范围内从转录复合体中解离。我们提出了一种新颖且未经尝试的方法来测试该模型。这些实验将在改良的鸡B细胞系DT40假V-del上进行，该细胞株遭受胞苷脱氨和随后的高比率SHM。该修饰系由J.M.Buerstedde通过删除亲本DT40系的伪V基因而产生，从而使修饰细胞不能进行Ig基因转化。作为对AID的反应，修改的伪V-del DT40细胞只经历SHM，并且这样做的速度很高，因为AID诱导的尿苷不是针对基因转换的。因此，它们是研究SHM的理想工具。如果这些探索性实验的结果是明确的，无论是支持还是拒绝上述SHM模型，它们将提供一个平台和方法来解开剩余的问题，例如AID如何只针对Ig和其他一些基因，包括一些与癌症和其他疾病相关的已知基因。与公共卫生相关：这是一项研究抗体基因过度突变的建议。这一过程是有益的，因为它可以产生针对病原体和癌症的高度特异和有效的抗体。然而，它也是危险的，因为它会导致淋巴细胞癌和自身免疫。

DESCRIPTION (provided by applicant): The process of somatic hypermutation (SHM) of immunoglobulin genes is initiated by the cytidine deaminase AID creating cytidine (C) to uridine (U) transition mutations. Outside of SHM, U is faithfully repaired to C with the help of base excision repair (BER) and mismatch repair (MMR). During SHM, paradoxically, these repair mechanisms are recruited together with translesion DNA polymerases to create mutations at and near the U by error-prone repair. A critical hallmark of SHM is the restriction of the mutations to the 5' end of Ig genes, including the V(D)J region, but sparing the constant region. In this way, an enormous diversity of antigen-binding variable regions is created while protecting the biological functions of the antibody constant regions. We have considered two ways in which this V-region restriction may come about. First, AID may only be active in the 5' region, and second, AID may act throughout the gene but error-prone repair would only operate in the 5' region. In extensive experiments with mice that are defective in BER or MMR or both, we found the same pattern of mutations as in wild type mice, namely restriction to the variable region and its immediate flanks. This leads to the conclusion that AID does not act at the 3' portion of the Ig gene. We now wish to determine how exactly AID interacts with the sequences in which it deaminates Cs. Based on experiments where the constant region was mutated when the Ig gene promoter was duplicated in front of the constant region, we proposed the following model: AID associates with the transcription complex near the promoter, travels with the RNA polymerase during transcript elongation, deaminates Cs in single- stranded DNA arising in negative DNA supercoils behind the polymerase, and dissociates from the transcription complex within ~2kbp from the promoter due to limited affinity. We propose a novel and untried approach to test this model. The experiments will be carried out with a modified chicken B cell line, DT40 PseudoV-del, that suffers cytydine deaminations and subsequent SHM at a high rate. The modified line was produced by J.M. Buerstedde by deleting the pseudo V genes of the parent DT40 line so that the modified cells cannot carry out Ig gene conversion. In response to AID, the modified pseudoV-del DT40 cells undergo only SHM and do so at a high rate since AID- induced uridines are not directed toward gene conversion. They are therefore an ideal tool to study SHM. If the results of these exploratory experiments are clear, either supporting or rejecting the model of SHM described above, they will provide a platform and methods to unravel remaining questions, such as how AID is targeted only to Ig and some other genes, including some of known relevance to cancer and other diseases. PUBLIC HEALTH RELEVANCE: This is a proposal to study the hypermutation of antibody genes. This process is beneficial, because it can result in highly specific and efficient antibodies against pathogens and cancer. However, it is also dangerous, because it can cause cancer of lymphocytes and autoimmunity.