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.

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...

The assembly of the gene segments coding for the variable portions of lymphocyte antigen receptors (immunoglobulins and T-cell receptors) occurs via a somatic recombination reaction known as V(D)J recombination. This process is mediated by two lymphoid cell-specific factors, RAG1 and RAG2 (recombination-activating gene products), that perform DNA cleavage at a pair of recombination signal sequences (RSSs) flanking the coding segments to be joined. RSSs consist of short palindromic heptamer and A/T-rich nonamer elements separated by a less well conserved spacer of 12 or 23 bp (12-RSS and 23-RSS). In vivo, recombination occurs most efficiently with a 12/23 RSS pair, a phenomenon referred to as the 12/23 rule.Under appropriate conditions in vitro, the RAG proteins together with high-mobility-group protein HMGB1 or HMGB2 perform coupled DNA cleavage of RSS substrates in accordance with the 12/23 rule (16). This process is thought to be initiated by the binding of the RAG recombinase to one RSS, followed by the capture of a second RSS, thus assembling the synaptic, or paired, complex (PC) (22, 28). HMGB1 and HMGB2 proteins play critical roles in vitro in facilitating the binding of the RAG proteins to the 23-RSS and the formation of the PC (15, 16), activities thought to rely on their ability to recognize bent or distorted DNA structures (3, 7). DNA cleavage takes place in two steps: a nick is first introduced between the heptamer and the coding DNA, and the 3Ј hydroxyl group thus liberated then attacks the other strand of the duplex to generate a covalently sealed hairpin coding end and a blunt signal end (16). Nicking can occur before or after synapsis, but hairpin formation occurs coordinately at the two RSSs within the PC (15). The PC is thus a critical intermediate in which the recombining partners are chosen and DNA double-strand breaks are made.Numerous studies have identified functionally important residues and domains within the catalytically essential, or core, region of RAG1 (10): an N-terminal nonamer binding domain that interacts with the RSS nonamer, a central domain that interacts with RAG2 and the heptamer and contains two of three acidic residues thought to contribute to the RAG active site, and a C-terminal domain with dimerization and nonspecific DNA binding activity and the third active-site residue. Less is known about the RAG2 core region.The structure of the PC is poorly understood. The PC is thought to contain two (37) or more than two (28) RAG1 monomers, two monomers of RAG2 (28,37), and an unknown number of HMGB1 or HMGB2 subunits. The nonamer binding and catalytic domains that interact with a particular RSS are contributed by different RAG1 monomers (36). This and the dependence of hairpin formation upon synapsis suggest careful coordination between the catalytic events at the two RSSs, a possibility supported by the finding that nicking at one RSS is required for hairpin formation at the partner RSS (41).Very little is known about the structure of the two DNA molecules in the PC. RAG1/2 an...

RAG-1 and RAG-2 initiate V(D)J recombination by cleaving DNA at recombination signal sequences through sequential nicking and transesterification reactions to yield blunt signal ends and coding ends terminating in a DNA hairpin structure. Ubiquitous DNA repair factors then mediate the rejoining of broken DNA. V(D)J recombination adheres to the 12/23 rule, which limits rearrangement to signal sequences bearing different lengths of DNA (12 or 23 base pairs) between the conserved heptamer and nonamer sequences to which the RAG proteins bind. Both RAG proteins have been subjected to extensive mutagenesis, revealing residues required for one or both cleavage steps or involved in the DNA end-joining process. Gain-of-function RAG mutants remain unidentified. Here, we report a novel RAG-1 mutation, E649A, that supports elevated cleavage activity in vitro by preferentially enhancing hairpin formation. DNA binding activity and the catalysis of other DNA strand transfer reactions, such as transposition, are not substantially affected by the RAG-1 mutation. However, 12/23-regulated synapsis does not strongly stimulate the cleavage activity of a RAG complex containing E649A RAG-1, unlike its wild-type counterpart. Interestingly, wild-type and E649A RAG-1 support similar levels of cleavage and recombination of plasmid substrates containing a 12/23 pair of signal sequences in cell culture; however, E649A RAG-1 supports about threefold more cleavage and recombination than wild-type RAG-1 on 12/12 plasmid substrates. These data suggest that the E649A RAG-1 mutation may interfere with the RAG proteins' ability to sense 12/23-regulated synapsis. V(D)J recombination is the process by which noncontiguous antigen receptor gene coding segments, called variable (V), diversity (D), and joining (J), are assembled during lymphocyte development to produce the variable region exon of a mature antigen receptor gene (3). V(D)J recombination occurs in two distinct phases. In the first phase, two lymphoid cell-specific proteins called RAG-1 and RAG-2 assemble a multiprotein synaptic complex with two different gene segments through interactions with a conserved recombination signal sequence (RSS) that adjoins each gene segment. Each RSS contains a conserved heptamer and nonamer sequence, separated by either 12 or 23 base pairs of intervening DNA of more varied composition (12-RSS and 23-RSS, respectively). Generally, synaptic complexes are assembled with two RSSs whose spacer lengths are different (the 12/23 rule). Subsequently, the RAG proteins catalyze a DNA double-strand break at each RSS (for reviews, see references 10 and 13), yielding a postcleavage complex containing four DNA ends: two blunt, 5Ј phosphorylated recombination signal ends and two coding ends terminating in DNA hairpin structures (41,42,46). The RAG proteins generate these recombination intermediates by nicking the DNA at the junction between the RSS and the coding sequence and then transferring the resulting 3Ј-OH to the opposing DNA strand by direct transesterification (32,...

Background: RAG1 and RAG2 initiate V(D)J recombination by assembling a synaptic complex with a pair of antigen receptor gene segments through interactions with their flanking recombination signal sequence (RSS), and then introducing a DNA double-strand break at each RSS, separating it from the adjacent coding segment. While the RAG proteins are sufficient to mediate RSS binding and cleavage in vitro, these activities are stimulated by the architectural DNA binding and bending factors HMGB1 and HMGB2. Two previous studies (Bergeron et al., 2005, andDai et al., 2005) came to different conclusions regarding whether only one of the two DNA binding domains of HMGB1 is sufficient to stimulate RAG-mediated binding and cleavage of naked DNA in vitro. Here we test whether this apparent discrepancy is attributed to the choice of divalent metal ion and the concentration of HMGB1 used in the cleavage reaction.

Antigen receptor genes are assembled by a form of site-specific DNA rearrangement termed 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 recombination signal sequences (RSSs), 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 biochemical evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1 and can become integrated into a stable RAG-RSS complex assembled with full-length RAG1 and core RAG2, but not core RAG1 (aa 384–1040) and either core or full-length RAG2. Formation of this complex minimally requires residues 211–1040 of RAG1. These results provide a biochemical link between the two phases of V(D)J recombination. This research is supported by National Institutes of Health grant 1R01 AI055599 to P.C.S.