Molecular Mechanisms of Class Switch Recombination

本申请提出了免疫球蛋白重链（IgH）类别转换机制的研究 重组（CSR）和体细胞超突变（SHM）。我们已经证明，激活诱导胞苷 脱氨酶（AID）是一种单链DNA（ssDNA）特异性胞苷脱氨酶，是CSR的起始酶， 采用了一系列新的生化和遗传方法来阐明艾滋病获得的机制， 在转录产生的ssDNA的背景下获得转录的双链（ds）DNA序列 结构和/或某些AID修饰或辅因子。我们还表明，企业社会责任可以雇用一般 艾滋病引发的DNA双链断裂（DSB）的突触过程，即一般的DNA修复因子 在CSR中起作用，并且两个不同的末端连接途径融合S区断裂以完成CSR。我们 目前的建议是在三个具体目标的背景下根据这些意见提出的。我们的第一个目标是利用 生物化学和遗传学的方法来阐明艾滋病的功能和调节的基本机制。在这 在这方面，我们开发了从正常B细胞中纯化AID的方法，在体外测定转录依赖性 双链DNA的AID脱氨基作用和评价体内AID功能的遗传学方法阐明 生物化学我们的第二个目标是解决DNA序列影响AID活性的机制， 其结果。对于这些研究，我们开发了靶向突变检测，以取代内源性IgH类 开关（S）区和外显子编码IgH可变区与测试序列，将允许我们确定 底物序列如何影响AID活性以及CSR和SHM中其他相关因素。统称 目的1和2的互补生物化学和遗传测定提供了阐明 参与IgH CSR和SHM启动和调节的因素和机制。第三个目标是 阐明AID诱导的DSB的修复过程，以完成CSR。对于这些研究，我们再次 已经开发了大量的试剂和新的方法，包括细胞遗传学方法来跟踪CSR 染色体的相关断裂，新的遗传方法来研究参与长距离突触的因素， DSB和遗传模型，以阐明DSB修复途径，完成IgH CSR。我们建议的研究 应该提供新的见解抗体生产的机制，通过IgH CSR，因此， 与理解免疫缺陷、疫苗免疫学和自身免疫性疾病有关。由于CSR是 所需的IgE生产，这项工作也将有关了解过敏性疾病的发病机制 和哮喘。最后，这项工作与B细胞恶性肿瘤有关，因为它们通常涉及染色体 通过异常的CSR将易位的癌基因与IgH S区域连接的易位。

This application proposes studies of the mechanisms of immunoglobulin heavy chain (IgH) class switch recombination (CSR) and Somatic Hypermutation (SHM). We have shown that Activation Induced Cytidine Deaminase (AID), the initiator of CSR, is a single strand DNA (ssDNA) specific cytidine deaminase and we employed a series of novel biochemical and genetic approaches to elucidate mechanisms by which AID gains access to transcribed double strand (ds)DNA sequences in the context transcription-generated ssDNA structures and/or certain AID modifications or co-factors. We also showed that CSR may employ general processes for synapsis of AID-initiated DNA double strand breaks (DSBs), that general DNA repair factors function in CSR, and that two distinct end-joining pathways fuse S region breaks to complete CSR. Our current proposal builds on these observations in the context of three specific Aims. Our first aim proposes use of biochemical and genetic approaches to elucidate basic mechanisms of AID function and regulation. In this regard, we developed methods to purify AID from normal B cells, in vitro assays for transcription-dependent AID deamination of dsDNA DNA, and genetic approaches to evaluate in vivo AID functions elucidated biochemically. Our second aim addresses mechanisms by which DNA sequences influence AID activity and its outcome. For these studies, we developed targeted mutation assays to replace endogenous IgH class switch (S) regions and exons encoding IgH variable regions with test sequences that will allow us to determine how substrate sequences influence activities of AID and other relevant factors in CSR and SHM. Together, the complementary biochemical and genetic assays of Aims 1 and 2 offer a powerful approach for elucidating factors and mechanisms involved in initiation and regulation of IgH CSR and SHM. A third proposed aim is to elucidate processes involved in the repair of AID induced DSBs to complete CSR. For these studies, we again have developed a large array of reagents and novel approaches, including cytogenetic methods to follow CSR related breaks in chromosomes, novel genetic approaches to study factors involved in long range synapsis of DSBs, and genetic models to elucidate DSB repair pathways that complete IgH CSR. Our proposed studies should provide novel insights into the mechanism of antibody production via IgH CSR and, therefore, be relevant to understanding immunodeficiencies, vaccine immunology, and autoimmune diseases. As CSR is required for IgE production, the work will also be relevant to understanding pathogenesis of allergic diseases and asthma. Finally, the work is relevant to B cell malignancies as they often involve chromosomal translocations that link translocated oncogenes to IgH S regions via aberrant CSR.