Structural studies of the post-cleavage complex in V(D)J recombination

V(D)J 重组中裂解后复合物的结构研究

• 批准号: 7734112
• 负责人: MARTIN F. GELLERT
• 金额: $ 67.15万
• 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/7734112
• 关键词: Animals; Biochemical; Catalytic Domain; Complex; Cryoelectron Microscopy; Crystallization; DNA; Data; Development; Disputes; Electron Microscopy; Genes; Genetic Recombination; Immune system; Immunoelectron Microscopy; Immunoglobulins; Literature; Lymphoid; Measurement; Methods; Molecular Weight; Preparation; Process; Proteins; Resolution; Sampling; Scanning Transmission Electron Microscopy Procedures; Site; Staining method; Stains; System; T-Cell Receptor Genes; Time; V(D)J Recombination; Work; Yang; catalyst; image reconstruction; scale up; stoichiometry

Although there is a great deal of biochemical information about the RAG1and RAG2 proteins, no correlated structural data exist so far. As a result of previous work from this group, we identified the best-defined and most stable complex in this system as being the complex RAG1 and RAG2 make with two DNA ends after cutting DNA at the recombination sites. This complex has now been prepared in sufficient quantity for high-resolution electron microscopy. A collaborative arrangement with the groups of Dr. Wei Yang (LMB) and Dr. Alasdair Steven (NIAMS) has produced for the first time pictures of negatively stained samples good enough for image reconstruction, which shows evidence of a hollow core and other defined features. Present efforts focus on higher resolution that can be obtained with cryo-electron microscopy and immuno-electron microscopy, to be combined with molecular weight estimation by scanning transmission electron microscopy. Together with biophysical measurements, the results have already clarified the issue of the protein stoichiometry of the complex, which has been in dispute in the literature. Further scaling up of the protein preparation method may also permit crystallization of the complex.

虽然有大量关于RAG1和RAG2蛋白的生化信息，但到目前为止还没有相关的结构数据。作为该小组先前工作的结果，我们确定该系统中定义最好、最稳定的复合体是在重组位点切割DNA后形成的具有两个DNA末端的复合体RAG1和RAG2。这种络合物现在已经制备了足够数量的高分辨率电子显微镜。与杨伟博士(LMB)和阿拉斯代尔·史蒂文博士(NIAMS)的团队合作，首次制作出了足以用于图像重建的负染样本的图片，其中显示了中空核心和其他明确特征的证据。目前的努力集中在用低温电子显微镜和免疫电子显微镜可以获得更高的分辨率，并结合扫描电子显微镜估计分子量。结合生物物理测量，结果已经澄清了该复合体的蛋白质化学计量问题，这一问题在文献中一直存在争议。蛋白质制备方法的进一步放大也可以允许复合体的结晶。