Lapkouski Mikalai, Chuenchor Watchalee, Kim Min-Sung, Gellert Martin, Yang Wei
From the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
From the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
J Biol Chem. 2015 Jun 5;290(23):14618-25. doi: 10.1074/jbc.M115.641787. Epub 2015 Apr 22.
Mammalian immune receptor diversity is established via a unique restricted set of site-specific DNA rearrangements in lymphoid cells, known as V(D)J recombination. The lymphoid-specific RAG1-RAG2 protein complex (RAG1/2) initiates this process by binding to two types of recombination signal sequences (RSS), 12RSS and 23RSS, and cleaving at the boundaries of RSS and V, D, or J gene segments, which are to be assembled into immunoglobulins and T-cell receptors. Here we dissect the ordered assembly of the RAG1/2 heterotetramer with 12RSS and 23RSS DNAs. We find that RAG1/2 binds only a single 12RSS or 23RSS and reserves the second DNA-binding site specifically for the complementary RSS, to form a paired complex that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 paired complex is active in the presence of Mg(2+), the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex. We report here the purification and initial crystallization of the RAG1/2 signal-end complex for atomic-resolution structure elucidation. Strict pairing of the 12RSS and 23RSS at the binding step, together with information from the crystal structure of RAG1/2, leads to a molecular explanation of the 12/23 rule.
哺乳动物的免疫受体多样性是通过淋巴细胞中一组独特的、受限制的位点特异性DNA重排建立的，这一过程被称为V(D)J重组。淋巴细胞特异性的RAG1-RAG2蛋白复合物（RAG1/2）通过结合两种重组信号序列（RSS），即12RSS和23RSS，并在RSS与V、D或J基因片段的边界处进行切割来启动这一过程，这些基因片段将被组装成免疫球蛋白和T细胞受体。在这里，我们剖析了RAG1/2异源四聚体与12RSS和23RSS DNA的有序组装过程。我们发现，RAG1/2仅结合单个12RSS或23RSS，并专门为互补的RSS保留第二个DNA结合位点，以形成一个配对复合物，这反映了已知的V(D)J重组的12/23规则。组装好的RAG1/2配对复合物在生理相关的金属离子Mg(2+)存在下，对两种RSS DNA进行切口和双链切割，从而产生信号末端复合物，具有活性。我们在此报告了用于原子分辨率结构解析的RAG1/2信号末端复合物的纯化和初步结晶情况。在结合步骤中12RSS和23RSS的严格配对，以及来自RAG1/2晶体结构的信息，为12/23规则提供了分子解释。
