The Molecular Mechanism of Somatic Hypermutation

描述（由申请人提供）：免疫系统已经进化出特定机制，从有限数量的遗传物质中产生不同的抗体特异性。抗体基因是通过少量基因片段的体细胞重组产生的。体细胞重组抗体对许多抗原具有低亲和力。特定抗体对特定抗原的亲和力的微调由体细胞超突变驱动，体细胞超突变是一种将点突变引入免疫球蛋白（IG）基因的可变（V）区的B细胞特异性反应。目前，我们对超突变的机理理解非常有限。我们和其他人已经获得了涉及DNA双链断裂（DSB）作为反应中间体的结果。我们不知道DSB是如何产生的，但它们的产生与最近发现的激活诱导的胞苷脱氨酶无关。我们的工作假设是B细胞特异性核酸酶靶向IG基因的V区，在DNA中产生损伤，该损伤是DSB或分解成DSB。该研究的长期目标是阐明超突变反应的分子机制。拟议项目的第一部分将是确定已知结构的位点特异性断裂是否可以超突变。为此目的，我们将创建小鼠品系，其V区被改变以包含稀有核酸内切酶I-Scel（一种DSB核酸酶）和切口酶N.BstNB1的识别序列。我们将评估这些与诱导表达任一酶的品系杂交后的突变性。超突变（和相关的DSB）完全依赖于IG增强子。在第二部分中，我们将使用生物化学方法来确定控制超突变的增强子内的精确序列元件。然后，我们将通过比较保留或缺乏这些序列的IG κ转基因来验证它们在功能上的重要性。最后，我们将定义结合这些元素的因子，并将超突变机制特异性地靶向IG基因。由于超突变的异常靶向与各种B细胞淋巴瘤有关，这些研究对理解此类恶性肿瘤的产生具有重要意义。

DESCRIPTION (provided by applicant): The immune system has evolved specific mechanisms to generate diverse antibody specificities from limited amounts of genetic material. Antibody genes are generated by the somatic recombination of a small number of gene segments. Somatically recombined antibodies have low affinities for many antigens. Fine-tuning of the affinity of a particular antibody to a particular antigen is driven by somatic hypermutation, a B cell specific reaction which introduces point mutations into the variable (V) regions of immunoglobulin (Ig) genes. Currently, our mechanistic understanding of hypermutation is very limited. We, and others, have obtained results implicating DNA double strand breaks (DSBs) as reaction intermediates. We do not know how the DSBs arise, but their generation is independent of the recently discovered activation-induced cytidine deaminase. Our working hypothesis is that a B cell specific nuclease is targeted to the V regions of Ig genes creating a lesion in the DNA which is, or resolves into, a DSB. The long range goal of the proposed research is to elucidate the molecular mechanism of the hypermutation reaction. The first part of the proposed project will be to establish whether site-specific breaks of known structure can hypermutate. For this purpose, we will create lines of mice with V regions altered to contain the recognition sequence for the rare endonuclease I-Scel (a DSB nuclease) and for the nicking enzyme N.BstNB1. We will assess mutability after crossing these with lines that inducibly express either enzyme. Hypermutation (and the associated DSBs) absolutely depend on the Ig enhancers. In the second part, we will use biochemical methods to define the precise sequence elements within the enhancers which control hypermutation. We will then verify that they are functionally important by comparing Ig kappa transgenes which either retain or lack such sequences. Finally, we will define the factors which bind these elements and target the hypermutation machinery specifically to Ig genes. As aberrant targeting of hypermutation has been linked to a variety of B cell lymphomas, these studies have significant implications for understanding the generation of such malignancies.