Site-Specific Recombination in Human Health & Disease

摘要 我们免疫系统的多样化需要两个主要的DNA重组途径：V（D）J和class 开关重组（CSR）。V（D）J和CSR都有几个知之甚少的中间步骤。这些 中间步骤是这些途径中与疾病最相关的方面的基础，因为它们是 本质上不稳定单靠静态结构生物学方法不足以理解 这些中间体。这里描述的动态方法使我们能够理解这些不稳定的 中间体是V（D）J和CSR途径的遗传性和获得性（肿瘤）疾病的关键。 从应用的角度来看，在这一建议中获得的理解使我们最终能够使用 生物化学系统，以产生针对病原性病毒和细菌的改进的抗体。重要 根据目前的建议，超过85%的人类淋巴恶性肿瘤本质上是B细胞，并且我们已经表明， 两条染色体上的断裂期是由V（D）J和IG CSR中的失败汇合而产生的 机制等在免疫球蛋白基因座处的染色体断裂通常是由于在免疫球蛋白基因座的扩增过程中的失败。 当RAG复合体过早地释放末端时，突触步骤。故障也可能发生在RAG手- 到NHEJ通路（用于连接末端）。我们在本提案中研究了RAG功能的所有这些方面。 另一种染色体断裂是由于CSR酶的脱靶行为引起的，称为激活诱导的染色体断裂。 脱氨酶（AID），我们在本提案的第二个项目中研究。利伯实验室已经完成了 生物化学对上述所有酶的作用。我们是第一个也是唯一一个能重建 使用完全纯化的酶的V（D）J途径。尽管最近RAG和AID的原子结构很漂亮， 蛋白质，这些酶的动态作用以及它们如何失效是仍然存在的差距。除了 肿瘤，由RAG和AID酶引起的疾病是导致超过三分之一的遗传性人类疾病的原因。 免疫缺陷称为SCID。我的实验室利用当前的资助期开发了实验室内的能力， 我们在高分辨率单分子测定中用于V（D）J和CSR的独特纯化蛋白，特别是冷冻EM 和sm-FRET。在2019年和2020年，我们发表了第一个sm-FRET，其中不仅蛋白质，而且 在我的实验室里做了动态的sm-FRET。我的实验室现在也有了完整的低温电磁能力，这将是相关的， 目前提案的后期阶段。我们也可以在这个建议中进行相关的生化步骤 除了裸DNA之外，还可以在单核体和多核体底物上进行。在项目1中，我们剖析了关键的脆弱性， 点的RAG突触步骤和他们的手关闭NHEJ途径。在项目2中，我们研究了 独立的IG类转换重组（CSR）过程。Lieber实验室是第一个发现这种酶的 长度染色体R-环在开关序列。我们是唯一一个能重建整个CSR的实验室 途径使用纯化的底物和蛋白质。我们运用累积的技术提出关键问题， 关于CSR是如何发生的以及它在疾病状态下是如何失败的。

ABSTRACT Diversification of our immune system requires two primary DNA recombination pathways: V(D)J and class switch recombination (CSR). Both V(D)J and CSR have several poorly understood intermediate steps. These intermediate steps are the basis for the most disease-relevant aspects of these pathways because they are inherently unstable. Static structural biology approaches alone are not sufficient to understand the instability of these intermediates. The dynamic approaches described here permit us to understand these unstable intermediates that are key to both inherited and acquired (neoplastic) diseases of the V(D)J and CSR pathways. From an applied standpoint, the understanding gained in this proposal positions us to eventually use biochemical systems to generate improved antibodies against pathogenic viruses and bacteria. Important for the current proposal, over 85% of human lymphoid malignancies are B cell in nature, and we have shown that the breakage phase at the two chromosomes arises by a confluence of failures in the V(D)J and Ig CSR mechanisms. The chromosome break at the immunoglobulin locus is typically due to failures during the synapsis steps as the RAG complex prematurely releases the ends. Failures can also occur in the RAG hand- off to the NHEJ pathway (for joining the ends). We study all of these aspects of RAG function in this proposal. The other chromosome break arises due to the off-target behavior of the CSR enzyme called activation-induced deaminase (AID), which we study in the second Project of this proposal. The Lieber lab has done key biochemistry on all of the enzymes mentioned above. We are the first and only lab to reconstitute the entire V(D)J pathway using fully purified enzymes. Despite beautiful recent atomic structures of RAG and AID proteins, the dynamic action of these enzymes and how they fail is the gap that remains. In addition to neoplasms, diseases caused by RAG and AID enzymes are responsible for over one-third of inherited human immune deficiencies called SCID. My lab has used the current funding period to develop in-lab capability to use our unique purified proteins for V(D)J and CSR in high resolution single molecule assays, specifically cryo-EM and sm-FRET. In 2019 and 2020, we published the first sm-FRET in which not only the proteins but also the dynamic sm-FRET were done in my lab. My lab also now has full cryo-EM abilities, which would be relevant at the later stages of the current proposal. We also can carry out the relevant biochemical steps in this proposal on mono- and polynucleosomal substrates in addition to naked DNA. In Project 1, we dissect the key vulnerable points in the RAG synapsis steps and their hand-off to the NHEJ pathway. In Project 2, we study the independent process of Ig class switch recombination (CSR). The Lieber lab was the first to discover kilobase length chromosomal R-loops at switch sequences. We are the only lab able to reconstitute the entire CSR pathway using purified substrates and proteins. We apply our cumulative technologies to ask key questions about how CSR occurs and how it fails in disease states.