title: Assembly Pathway and Characterization of the RAG1/2-DNA Paired and Signal-end Complexes.
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 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.
authors.affiliation: From the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Mikalai
authors.initials: M
authors.lastname: Lapkouski
authors.affiliation: From the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Watchalee
authors.initials: W
authors.lastname: Chuenchor
authors.affiliation: From the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Min-Sung
authors.initials: MS
authors.lastname: Kim
authors.affiliation: From the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Martin
authors.initials: M
authors.lastname: Gellert
authors.affiliation: From the Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 wei.yang@nih.gov.
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003460:Crystallization; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D055503:Protein Multimerization; D060152:V(D)J Recombination
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C499308:Rag2 protein, mouse; C064658:RAG-1 protein; D004247:DNA
keywords: DNA cleavage; DNA endonuclease; DNA enzyme; V(D)J recombination; immunodeficiency; mammal; mammalian expression; metal ions; protein complex; protein crystallization