Antigen receptor genes are assembled by a site-specific DNA rearrangement process called V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at antigen receptor gene segments, and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1, providing a biochemical link between the two phases of V(D)J recombination.
The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin RING E3 ligase complex VprBP/DDB1/Cul4A/Roc1 associates with full-length RAG1 through VprBP. This complex is assembled into RAG protein-DNA complexes, and supports in-vitro ubiquitylation activity that is insensitive to RAG1 RING domain mutations. Conditional B lineagespecific VprBP disruption arrests B-cell development at the pro-B-to-pre-B cell transition, but this block is bypassed by expressing rearranged immunoglobulin transgenes. Mice with a conditional VprBP disruption show modest reduction of D-J H rearrangement, whereas V H -DJ H and V j -J j rearrangements are severely impaired. D-J H coding joints from VprBP-insufficent mice show longer junctional nucleotide insertions and a higher mutation frequency in D and J segments than normal. These data suggest full-length RAG1 recruits a cullin RING E3 ligase complex to ubiquitylate an unknown protein(s) to limit error-prone repair during V(D)J recombination. The EMBO Journal (2012) 31, 945-958.
Intercellular adhesion molecules (ICAMs) belong to the immunoglobulin superfamily and participate in diverse cellular processes including host-pathogen interactions. ICAM-1 is expressed on various cell types including macrophages, whereas ICAM-4 is restricted to red blood cells. Here we report the identification of an 11-kDa synthetic protein, M5, that binds to human ICAM-1 and ICAM-4, as shown by in vitro interaction studies, surface plasmon resonance and immunolocalization. M5 greatly inhibits the invasion of macrophages and erythrocytes by Mycobacterium tuberculosis and Plasmodium falciparum, respectively. Pharmacological and siRNA-mediated inhibition of ICAM-1 expression also results in reduced M. tuberculosis invasion of macrophages. ICAM-4 binds to P. falciparum merozoites, and the addition of recombinant ICAM-4 to parasite cultures blocks invasion of erythrocytes by newly released merozoites. Our results indicate that ICAM-1 and ICAM-4 play roles in host cell invasion by M. tuberculosis and P. falciparum, respectively, either as receptors or as crucial accessory molecules.
Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a sideby-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg 2؉ cations with Ca 2؉ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.To generate diverse surface antigen receptor molecules, developing lymphocytes undergo a series of site-specific DNA rearrangements to assemble functional antigen receptor genes from component gene segments (1). This DNA rearrangement process, known as V(D)J recombination, is initiated when two lymphoid cell-specific proteins, called RAG1 and RAG2, assemble a multiprotein synaptic complex with a pair of antigen receptor gene segments and subsequently introduce a DNA double strand break at the end of each gene segment (2). A recombination signal sequence (RSS) 3 that abuts each participating gene segment serves as the binding site of the RAG proteins and directs the location of DNA cleavage. Each RSS contains conserved heptamer and nonamer sequences that are separated by either 12 or 23 bp of DNA of more varied sequence (12RSS and 23RSS, respectively); efficient V(D)J recombination generally only occurs between two RSSs in which the lengths of DNA separating the heptamer and nonamer differ (the 12/23 rule). The RAG proteins mediate DNA cleavage via a nick-hairpin mechanism, breaking the DNA between the RSS heptamer and the coding segment; these reaction products are subsequently processed and repaired by the non-homologous end-joining pathway (1, 3).Previous studies suggest that RAG synaptic complexes are assembled through the stepwise binding of a 12RSS followed by the capture of a 23RSS (4 -6). In vitro biochemical studies suggest synapsis is mediated by a RAG1/2 heterotetramer, but there remains disagreement over the stoichiometry of RAG1 in these complexes (7). In addition, fluorescence resonance energy transfer techniques have recently been applied to examine the orientat...
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