RAG1-DNA binding in V(D)J recombination - Specificity and DNA-induced conformational changes revealed by fluorescence and CD spectroscopy
RAG1-DNA binding in V(D)J recombination - Specificity and DNA-induced conformational changes revealed by fluorescence and CD spectroscopy
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DOI：
10.1074/jbc.m209758200
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发表时间：
2003-02-21
影响因子：
4.8
通讯作者：
Schatz, DG
中科院分区：
文献类型：
--
作者：
Ciubotaru, M;Ptaszek, LM;Schatz, DG
RAG1和RAG2蛋白共同构成核酸酶，启动免疫球蛋白和T细胞受体基因的组装，这一反应被称为V(D)J重组。RAG1。在RAG1/2复合体识别重组信号序列（RSS）中起核心作用。为了研究控制RAG1- rss相互作用的参数，我们从细菌中纯化了融合到短Strep标签上的小鼠核心RAG1蛋白（氨基酸377-1008）。Strep-RAG1 （StrRAG1）蛋白在没有DNA的情况下以二聚体的形式存在于较宽的蛋白浓度范围内（25-500 nm），并以相当高的亲和力和特异性（表观K-D = 41 nm）与RSS结合。电泳迁移率和极化各向异性实验均表明溶液中只存在一种StrRAG1-DNA。频域光谱测量的各向异性衰减表明，该复合物含有StrRAG1二聚体与单个DNA分子结合。利用蛋白质固有荧光和圆二色性的测量，我们证明StrRAG1在与RSS结合后发生了主要的构象变化。稳态荧光和丙烯酰胺猝灭研究表明，这种构象变化与内在蛋白质荧光团从疏水环境到溶剂暴露环境的重新定位有关。rss诱导的StrRAG1构象变化可能影响RAG1与RAG2的相互作用和突触复合体的形成。
The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1. plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nm) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K-D = 41 nm) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.