Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes |
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Authors: | Mikalai Lapkouski Watchalee Chuenchor Min-Sung Kim Martin Gellert Wei Yang |
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Affiliation: | From the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 |
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Abstract: | 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 Mg2+, 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. |
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Keywords: | DNA endonuclease DNA enzyme immunodeficiency mammal protein complex protein crystallization V(D)J recombination mammalian expression metal ions mammalian expression DNA cleavage |
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