Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes [Protein Structure and Folding]

April 22nd, 2015 by Lapkouski, M., Chuenchor, W., Kim, M.-S., Gellert, M., Yang, W.

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 12 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 (PC) that reflects the known 12/23 rule of V(D)J recombination. The assembled RAG1/2 PC is active in the presence of Mg2+, the physiologically relevant metal ion, in nicking and double-strand cleavage of both RSS DNAs to produce a signal-end complex (SEC). We report here the purification and initial crystallization of the RAG1/2 SEC complex for atomic-resolution structure elucidation. Strict pairing of the 12 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.