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）过程由胞苷脱氨酶AID启动，产生胞苷(C)到尿苷(U)的过渡突变。在SHM之外，U在碱基切除修复（BER）和错配修复（MMR）的帮助下忠实地修复到C。在SHM期间，矛盾的是，这些修复机制与翻译DNA聚合酶一起被招募，通过容易出错的修复在U和U附近产生突变。SHM的一个关键特征是突变限制在Ig基因的5'端，包括V(D)J区，但保留了恒定区。这样，在保护抗体恒定区生物学功能的同时，抗原结合可变区产生了巨大的多样性。我们已经考虑了v区限制可能产生的两种方式。首先，AID可能只在5′区有活性，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直接侧翼。由此得出结论，AID不作用于Ig基因的3'部分。我们现在希望确定AID如何准确地与它脱氨c的序列相互作用。基于Ig基因启动子在恒定区前复制时恒定区发生突变的实验，我们提出了以下模型：AID与启动子附近的转录复合体结合，在转录延伸期间与RNA聚合酶一起移动，在聚合酶后面的负DNA超圈中产生单链DNA中的Cs脱氨，并在距离启动子约2kbp的范围内由于有限的亲和力而与转录复合体分离。我们提出了一种新颖的、未经尝试的方法来测试这个模型。该实验将在一种改良的鸡B细胞系DT40 PseudoV-del上进行，该细胞系遭受胞苷脱胺和随后的高速率SHM。该修饰系是由J.M. Buerstedde通过删除亲本DT40系的伪V基因，使修饰细胞不能进行Ig基因转化而产生的。在对AID的反应中，经过修饰的pseudoV-del DT40细胞仅发生SHM，并且发生SHM的速率很高，因为AID诱导的尿苷不直接用于基因转化。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，无论是支持还是反对上述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如何仅针对Ig和其他一些基因，包括一些已知的与癌症和其他疾病相关的基因。公共卫生相关性：这是一项研究抗体基因高突变的建议。这个过程是有益的，因为它可以产生针对病原体和癌症的高度特异性和高效的抗体。然而，它也很危险，因为它可以引起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.