Structural studies of sequential DNA cleavage by RAG1/RAG2 proteins in V(D)J recombination

V(D)J 重组中 RAG1/RAG2 蛋白连续 DNA 切割的结构研究

• 批准号: 9771218
• 负责人: MARTIN F. GELLERT
• 金额: $ 271.81万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9771218
• 关键词: Animals; Base Pairing; Biochemical; Catalytic Domain; Complex; Cryoelectron Microscopy; Crystallization; DNA; DNA Binding; Enzymes; Ephrin-A5; Genes; Genetic Recombination; Human; Immune system; Immunoglobulins; Immunologic Deficiency Syndromes; Left; Malignant lymphoid neoplasm; Mediating; Molecular; Mutation; Peptide Signal Sequences; Process; Proteins; Publishing; Reaction; Regulatory Element; Reporting; Research; Site; Structure; T-Cell Receptor Genes; Techniques; Time; Transposase; V(D)J Recombination; Work; catalyst; leukemia/lymphoma; programs; protein complex; protein function; protein structure

Further progress has been made in the past year in our research program aimed at understanding the functions of the RAG1-RAG2 protein complex in molecular detail. In 2015, we reported the first crystal structure of the RAG1/RAG2 complex. This was a major advance, making it possible for the first time to give an explanation of the so called 12/23 rule in V(D)J recombination, which forces it to work on two different DNA sites. But this was a structure of the protein complex alone, without DNA, which left many questions about mechanism unanswered. It was necessary to obtain detailed information on structures containing DNA, and to study them at all stages of the cleavage reaction. As the DNA cleavage occurs in several defined steps, at least four distinct species of the RAG-DNA complex can be obtained. Crystals of most of these species were obtained and their structures were refined. In addition, we used the technique of cryo-electron microscopy to obtain further information. Combining these techniques, we found that an essential step in RAG-mediated DNA cleavage is the unwinding of several base pairs of DNA adjacent to the recombination signal sequence. This unexpected step may also be found in related enzymes of the transposase class. This work has been published. We are also continuing our work on longer versions of the RAG proteins, which contain regulatory elements controlling RAG activity.

在过去的一年里，我们的研究项目取得了进一步的进展，旨在从分子细节上了解RAG1-RAG2蛋白复合体的功能。2015年，我们首次报道了RAG1/RAG2复合体的晶体结构。这是一项重大的进步，使人们第一次能够解释V(D)J重组中的所谓12/23规则，该规则迫使它在两个不同的DNA位点上工作。但这是一个单独的蛋白质复合体的结构，没有DNA，这留下了许多关于机制的问题没有答案。有必要获得有关含有DNA的结构的详细信息，并在切割反应的所有阶段对其进行研究。当DNA在几个确定的步骤中发生切割时，至少可以获得四种不同的RAG-DNA复合体。得到了大部分这类化合物的晶体，并对它们的结构进行了优化。此外，我们还利用低温电子显微镜技术获得了进一步的信息。结合这些技术，我们发现RAG介导的DNA切割的一个重要步骤是解开重组信号序列附近的几个DNA碱基对。这一意想不到的步骤也可能在转座酶类的相关酶中发现。这部作品已经出版了。我们还在继续研究更长版本的RAG蛋白，它包含控制RAG活性的调控元件。