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: 10000711
• 负责人: MARTIN F. GELLERT
• 金额: $ 80.07万
• 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/10000711
• 关键词: Animals; Base Pairing; Biochemical; Catalytic Domain; Cells; DNA; DNA Binding; Enzymes; Ephrin-A5; Genes; Genetic Recombination; Human; Immune system; Immunoglobulins; Immunologic Deficiency Syndromes; Malignant lymphoid neoplasm; Mutation; Paper; Peptide Signal Sequences; Process; Proteins; Publications; Regulatory Element; Reporting; Research; Structure; T-Cell Receptor Genes; Transposase; V(D)J Recombination; Work; base; catalyst; leukemia/lymphoma; novel; programs; protein function

Advances in our research program on the structural analysis of the RAG1-RAG2 protein have provided a fuller understanding of its functions in the past year. We now have obtained detailed structures representing all stages of the RAG1/2 cleavage of DNA, and find some unexpected features. The previously reported unwinding of several base pairs of DNA adjacent to the recombination signal sequence, found to be necessary to activate the DNA for cleavage, is accompanied by the unwound strands meshing together in a DNA zipper with alternating bases from opposite strand interleaved and stabilized by stacking forces. This novel structure is clearly more stable in the non-aqueous protein interior than a strand-separated one. As RAG1/2 has a secondary transposase activity, we have also determined the structure of its strand-transfer intermediate in which it has captured a target DNA but not yet released the product. With RAG, this intermediate requires much more distortion of target DNA than in related transposases, probably explaining why RAG is an inefficient transposase, as is compatible with its primary activity as a DNA-cutting enzyme. Transposition in the context of immune system cells would most likely be harmful. Two papers describing these results have been submitted for publication. As these results were obtained with truncated but active forms of the RAG proteins, we are now pursuing structures of longer forms, which contain regulatory elements of the RAG1 N-terminus and the RAG2 C-terminus that control RAG activity.

在过去的一年里，我们对RAG 1-RAG 2蛋白结构分析的研究计划取得了进展，使人们对其功能有了更全面的了解。我们现在已经获得了代表RAG 1/2切割DNA的所有阶段的详细结构，并发现了一些意想不到的特征。先前报道的与重组信号序列相邻的DNA的几个碱基对的解旋，发现是激活DNA进行切割所必需的，伴随着解旋的链在DNA拉链中啮合在一起，其中来自相反链的交替碱基交错并通过堆叠力稳定。这种新的结构显然是更稳定的非水蛋白质内部比链分离的。 由于RAG 1/2具有二级转座酶活性，我们还确定了其链转移中间体的结构，其中它已捕获靶DNA但尚未释放产物。对于RAG，该中间体需要比相关转座酶更多的靶DNA的扭曲，这可能解释了为什么RAG是一种低效的转座酶，因为这与其作为DNA切割酶的主要活性是相容的。在免疫系统细胞的背景下，易位很可能是有害的。描述这些结果的两篇论文已提交出版。 由于这些结果是用RAG蛋白的截短但活性形式获得的，我们现在正在追求更长形式的结构，其包含控制RAG活性的RAG 1 N-末端和RAG 2 C-末端的调节元件。