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.

抽象的免疫系统的多样化需要两个主要的DNA重组途径：V（d）J和类开关重组（CSR）。 V（d）J和CSR都有几个中间步骤的了解较少。这些中间步骤是这些途径最相关的方面的基础，因为它们是天生不稳定。仅静态结构生物学方法就不足以了解这些中间体。这里描述的动态方法使我们能够理解这些不稳定 V（d）J和CSR途径的遗传和获取（肿瘤）疾病的关键的中间体。从应用的角度来看，该提案中获得的理解使我们最终使用生化系统可改善针对病原病毒和细菌的抗体。很重要当前的提案超过85％的人类淋巴恶性肿瘤本质上是B细胞，我们已经表明两个染色体处的断裂阶段是由于V（d）J和Ig CSR中的失败汇合而产生的机制。免疫球蛋白基因座的染色体断裂通常是由于在突触会随着抹布的过早释放末端时，突触得出。抹布手也可能发生故障 - 前往NHEJ途径（以加入末端）。我们在该提案中研究了抹布功能的所有这些方面。另一个染色体断裂是由于CSR酶的脱靶行为，称为激活诱导脱氨酶（AID），我们在该提案的第二个项目中研究。 Lieber实验室完成了钥匙上述所有酶的生物化学。我们是第一个也是唯一重新构成整个的实验室 V（d）J途径使用完全纯化的酶。尽管最近有美丽的抹布和援助原子结构蛋白质，这些酶的动态作用及其失败是剩下的差距。此外肿瘤，由抹布引起的疾病和辅助酶负责超过三分之一的遗传人类免疫缺陷称为SCID。我的实验室已经使用当前的资金期来开发使用内部功能我们在高分辨率的单分子测定中，特别是Cryo-EM的V（d）J和CSR的独特纯化蛋白和sm-fret。在2019年和2020年，我们发布了第一个SM-Fret，不仅是蛋白质，而且还发表了动态sm-fret是在我的实验室中完成的。我的实验室现在也具有完整的冷冻EM能力，这与当前提案的后期阶段。我们还可以在此提案中执行相关的生化步骤除裸DNA外，在单核和多核底物上。在项目1中，我们剖析了关键易受伤害抹布突触中的点及其移交给NHEJ途径。在项目2中，我们研究 IG类开关重组（CSR）的独立过程。 Lieber实验室是第一个发现千目标的人开关序列处的长度染色体R环。我们是唯一能够重建整个CSR的实验室使用纯化的底物和蛋白质的途径。我们运用累积技术来提出关键问题关于企业社会责任的发生以及疾病状态的失败方式。