Site-Specific Recombination in Human Health & Disease

人类健康中的位点特异性重组

基本信息

批准号：
10618161
负责人：
MICHAEL R LIEBER
金额：
$ 42.9万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10618161
关键词：
Accounting Antibodies B lymphoid malignancy B-Lymphocytes Behavior Biochemical Biochemistry Biological Assay Blood Cells Child Chromosomal Breaks Chromosome Pairing Chromosomes Complex Cryoelectron Microscopy DNA Defect Disease Enzymes Failure Funding Gene Rearrangement Genetic Recombination Health Human Immune Immune system Immunoglobulin Class Switching Immunoglobulin Switch Recombination Immunoglobulins Inherited Knowledge Length Malignant Neoplasms Nature Neoplasms Nonhomologous DNA End Joining Pathway interactions Phase Polynucleosome Positioning Attribute Process Proteins Publishing Resolution Site Structure System Technology V(D)J Recombination activation-induced cytidine deaminase clinical predictors congenital immunodeficiency improved leukemia/lymphoma neoplastic pathogenic bacteria pathogenic virus premature protein purification reconstitution single molecule single-molecule FRET structural biology

项目摘要

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.

抽象的我们的免疫系统的多样化需要两条主要的 DNA 重组途径：V(D)J 和 class 开关重组（CSR）。 V(D)J 和 CSR 都有几个鲜为人知的中间步骤。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。仅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对 V(D)J 和 CSR 途径的遗传性和获得性（肿瘤）疾病至关重要的中间体。从应用的角度来看，在该提案中获得的理解使我们最终能够使用生化系统产生针对致病病毒和细菌的改进抗体。重要的是目前的建议是，超过 85% 的人类淋巴恶性肿瘤本质上是 B 细胞，并且我们已经证明两条染色体的断裂阶段是由 V(D)J 和 Ig CSR 的故障汇合引起的机制。免疫球蛋白位点处的染色体断裂通常是由于免疫球蛋白免疫过程中的失败造成的。当 RAG 复合体过早释放末端时，突触会发生步骤。故障也可能发生在 RAG 手动中到 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（称为激活诱导）引起的脱氨酶（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关键工作上述所有酶的生物化学。我们是第一个也是唯一一个重建整个系统的实验室 V(D)J 途径使用完全纯化的酶。尽管 RAG 和 AID 最近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空白。此外由 RAG 和 AID 酶引起的肿瘤、疾病是导致人类遗传性遗传病的三分之一以上的原因称为 SCID 的免疫缺陷。我的实验室利用当前的资助期限来开发实验室内使用的能力我们在高分辨率单分子检测（特别是冷冻电镜）中针对 V(D)J 和 CSR 的独特纯化蛋白和 sm-FRET。 2019年和2020年，我们发表了第一个sm-FRET，其中不仅包括蛋白质，还包括动态 sm-FRET 是在我的实验室完成的。我的实验室现在也拥有完整的冷冻电镜能力，这与当前提案的后期阶段。我们也可以进行本提案中的相关生化步骤除裸露 DNA 外，还可用于单核小体和多核小体底物。在项目1中，我们剖析了关键的漏洞 RAG 突触步骤中的点及其向 NHEJ 通路的传递。在项目2中，我们研究了 Ig 类开关重组 (CSR) 的独立过程。利伯实验室是第一个发现千碱基的实验室开关序列处染色体 R 环的长度。我们是唯一能够重构整个 CSR 的实验室使用纯化的底物和蛋白质的途径。我们运用累积的技术来提出关键问题关于企业社会责任如何发生以及它如何在疾病状态下失败。