RAG2's non-core domain contributes to the ordered regulation of V(D)J recombination

Variable (diversity) joining [V(D)J] recombination of immune gene loci proceeds in an ordered manner with D to J portions recombining first and then an upstream V joins that recombinant. We present evidence that the non-core domain of recombination activating gene (RAG) protein 2 is involved in the regulation of recombinatorial order. In mice lacking the non-core domain of RAG2 the ordered rearrangement is disturbed and direct V to D rearrangements are 10- to 1000-times increased in tri-partite immune gene loci. Some forms of inter-chromosomal translocations between TCRbeta and TCRdelta D gene segments are also increased in the core RAG2 animals as compared with their wild-type (WT) counterparts. In addition, the concise use of proper recombination signal sequences (RSSs) appears to be disturbed in the core RAG2 mice as compared with WT RAG2 animals.     

RAG蛋白在V（D）J重组中的作用

V（D）J重组分为两步,第一步是对特定DNA序列的识别和切割,第二步是断理解末端的解离和重接。V（D）J重组过程中的切割是由RAG蛋白介导的,RAG蛋白在第二阶段起什么样的作用还是一个模糊的问题,但目前已有实验显示RAG蛋白在末端重接反应中亦起着重要的结构性（或许还有催化性）作用。RAG蛋白激活V（D）J重组的活性还受其它一些因素的调节。所有这些都揭示RAG蛋白在其他因素辅助下参与了V（D）J重组的全过程。     

Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination

Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.     

DNA-dependent protein kinase mediates V(D)J recombination via RAG2 phosphorylation

V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the 365(th) serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.     

Unraveling V(D)J recombination; insights into gene regulation

V(D)J recombination assembles antigen receptor genes from component gene segments. We review findings that have shaped our current understanding of this remarkable mechanism, with a focus on two major reports--the first detailed comparison of germline and rearranged antigen receptor loci and the discovery of the recombination activating gene-1.     

The RAG proteins in V(D)J recombination: more than just a nuclease

V(D)J recombination is the process that generates the diversity among T cell receptors and is one of three mechanisms that contribute to the diversity of antibodies in the vertebrate immune system. The mechanism requires precise cutting of the DNA at segment boundaries followed by rejoining of particular pairs of the resulting termini. The imprecision of aspects of the joining reaction contributes significantly to increasing the variability of the resulting functional genes. Signal sequences target DNA recombination and must participate in a highly ordered protein-DNA complex in order to limit recombination to appropriate partners. Two proteins, RAG1 and RAG2, together form the nuclease that cleaves the DNA at the border of the signal sequences. Additional roles of these proteins in organizing the reaction complex for subsequent steps are explored.     

Chromatin remodeling at the Ig loci prior to V(D)J recombination.

Rearrangement of Ig H and L chain genes is highly regulated and takes place sequentially during B cell development. Several lines of evidence indicate that chromatin may modulate accessibility of the Ig loci for V(D)J recombination. In this study, we show that remodeling of V and J segment chromatin occurs before V(D)J recombination at the endogenous H and kappa L chain loci. In recombination-activating gene-deficient pro-B cells, there is a reorganization of nucleosomal structure over the H chain J(H) cluster and increased DNase I sensitivity of V(H) and J(H) segments. The pro-B/pre-B cell transition is marked by a decrease in the DNase I sensitivity of V(H) segments and a reciprocal increase in the nuclease sensitivity of Vkappa and Jkappa segments. In contrast, J(H) segments remain DNase I sensitive, and their nucleosomal organization is maintained in mu(+) recombination-activating gene-deficient pre-B cells. These results indicate that initiation of rearrangement is associated with changes in the chromatin structure of both V and J segments, whereas stopping recombination involves changes in only V segment chromatin. We further find an increase in histone H4 acetylation at both the H and kappa L chain loci at the pro-B cell stage. Although histone H4 acetylation appears to be an early change associated with B cell commitment, acetylation alone is not sufficient to promote subsequent modifications in Ig chromatin.     

Amino acid residues in RAG1 responsible for the interaction with RAG2 during the V(D)J recombination process

The V(D)J recombinase, a complex of RAG1 and RAG2, carries out a gene rearrangement process that is required for the achievement of diverse antigen receptor repertoires during the early developmental stage of lymphocytes. It recognizes a specific site spanning the coding DNA region of antigen receptor genes and produces double-stranded DNA breaks at the board between coding and signal sequences. Two broken DNA ends are joined by a double-stranded break repair system. Both RAG (recombination activation gene) 1 and RAG2 proteins are absolutely required for this process although the catalytic residues of V(D)J recombinase are exclusively located at RAG1 according to recent mutational analyses. In this study we identified some acidic amino acid residues in RAG1 responsible for the interaction with RAG2. Mutation on these residues caused a decrease of cleavage activity in vitro and failure of RAG-RSS DNA synaptic complex formation. This result is complementary to previous reports in which positively charged amino acids in RAG2 play an important role in RAG1 binding.     

Modulation of RAG/DNA complex by HSP70 in V(D)J recombination

V(D)J recombination, a site-specific gene rearrangement process, requires two RAG1 and RAG2 proteins specifically recognizing recombination signal sequences and forming DNA double-strand breaks. The broken DNA ends tightly bound to RAG proteins are joined by repair proteins. Here, we found that heat shock protein 70 was associated with RAG2 following two-step affinity chromatography purification. It was also co-immunoprecipitated with RAG2 in pro-B cells. Purified HSP70 protein disrupted RAG/DNA complexes assembled in vitro and also inhibited the V(D)J cleavage (both nick and hairpin formation) in a dose-dependent manner. This HSP70 action required ATP energy. These data suggest that HSP70 might play a crucial role in disassembling RAG/DNA complexes stably formed during V(D)J recombination.     

DNA cleavage activity of the V(D)J recombination protein RAG1 is autoregulated

RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore, the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions that may lead to genomic instability in B and T lymphocytes.     

A basic motif in the N-terminal region of RAG1 enhances V(D)J recombination activity.

The variable portions of antigen receptor genes are assembled from component gene segments by a site-specific recombination reaction known as V(D)J recombination. The RAG1 and RAG2 proteins are the critical lymphoid cell-specific components of the recombination enzymatic machinery and are responsible for site-specific DNA recognition and cleavage. Previous studies had defined a minimal, recombinationally active core region of murine RAG1 consisting of amino acids 384 to 1008 of the 1,040-residue RAG1 protein. No recombination function has heretofore been ascribed to any portion of the 383-amino-acid N-terminal region that is missing from the core, but it seems likely to be of functional significance, based on its evolutionary conservation. Using extrachromosomal recombination substrates, we demonstrate here that the N-terminal region enhances the recombination activity of RAG1 by up to an order of magnitude in a variety of cell lines. Deletion analysis localized a region of the N terminus critical for this effect to amino acids 216 to 238, and further mutagenesis demonstrated that a small basic amino acid motif (BIIa) in this region is essential for enhancing the activity of RAG1. Despite the fact that BIIa is important for the interaction of RAG1 with the nuclear localization factor Srp-1, it does not appear to enhance recombination by facilitating nuclear transport of RAG1. A variety of models for how this region stimulates the recombination activity of RAG1 are considered.     

Joining-deficient RAG1 mutants block V(D)J recombination in vivo and hairpin opening in vitro

The RAG proteins cleave at V(D)J recombination signal sequences then form a postcleavage complex with the broken ends. The role of this complex in end processing and joining, if any, is undefined. We have identified two RAG1 mutants proficient for DNA cleavage but severely defective for coding and signal joint formation, providing direct evidence that RAG1 is critical for joining in vivo and strongly suggesting that the postcleavage complex is important in end joining. We have also identified a RAG1 mutant that is severely defective for both hairpin opening in vitro and coding joint formation in vivo. These data suggest that the hairpin opening activity of the RAG proteins plays an important physiological role in V(D)J recombination.     

RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons

The V(D)J recombination reaction in jawed vertebrates is catalyzed by the RAG1 and RAG2 proteins, which are believed to have emerged approximately 500 million years ago from transposon-encoded proteins. Yet no transposase sequence similar to RAG1 or RAG2 has been found. Here we show that the approximately 600-amino acid “core” region of RAG1 required for its catalytic activity is significantly similar to the transposase encoded by DNA transposons that belong to the Transib superfamily. This superfamily was discovered recently based on computational analysis of the fruit fly and African malaria mosquito genomes. Transib transposons also are present in the genomes of sea urchin, yellow fever mosquito, silkworm, dog hookworm, hydra, and soybean rust. We demonstrate that recombination signal sequences (RSSs) were derived from terminal inverted repeats of an ancient Transib transposon. Furthermore, the critical DDE catalytic triad of RAG1 is shared with the Transib transposase as part of conserved motifs. We also studied several divergent proteins encoded by the sea urchin and lancelet genomes that are 25%−30% identical to the RAG1 N-terminal domain and the RAG1 core. Our results provide the first direct evidence linking RAG1 and RSSs to a specific superfamily of DNA transposons and indicate that the V(D)J machinery evolved from transposons. We propose that only the RAG1 core was derived from the Transib transposase, whereas the N-terminal domain was assembled from separate proteins of unknown function that may still be active in sea urchin, lancelet, hydra, and starlet sea anemone. We also suggest that the RAG2 protein was not encoded by ancient Transib transposons but emerged in jawed vertebrates as a counterpart of RAG1 necessary for the V(D)J recombination reaction.     

The RAG1/RAG2 complex constitutes a 3' flap endonuclease: implications for junctional diversity in V(D)J and transpositional recombination

During V(D)J recombination, processing of branched coding end intermediates is essential for generating junctional diversity. Here, we report that the RAG1/ RAG2 recombinase is a 3' flap endonuclease. Substrates of this nuclease activity include various coding end intermediates, suggesting a direct role for RAG1/ RAG2 in generating junctional diversity during V(D)J recombination. Evidence is also provided indicating that site-specific RSS nicking involves RAG1/RAG2-mediated processing of a localized flap-like structure, implying 3' flap nicking in multiple DNA processing reactions. We have also demonstrated that the bacterial transposase Tn10 contains a 3' flap endonuclease activity, suggesting a mechanistic parallel between RAG1/RAG2 and other transposases. Based on these data, we propose that numerous transposases may facilitate genomic evolution by removing single-stranded extensions during the processing of excision site junctions.     

Intermolecular V(D)J recombination

V(D)J recombination plays a prominent role in the generation of the antigen receptor repertoires of B and T lymphocytes. It is also likely to be involved in the formation of chromosomal translocations, some of which may result from interchromosomal recombination. We have investigated the potential of the V(D)J recombination machinery to perform intermolecular recombination between two plasmids, either unlinked or linked by catenation. In either case, recombination occurs in trans to yield signal and coding joints, and the results do not support the existence of a mechanistic block to the formation of coding joints in trans. Instead, we observe that linearization of the substrate, which does not alter the cis or trans status of the recombination signals, causes a specific and dramatic reduction in coding joint formation. This unexpected result leads us to propose a "release and recapture" model for V(D)J recombination in which coding ends are frequently released from the postcleavage complex and the efficiency of coding joint formation is influenced by the efficiency with which such ends are recaptured by the complex. This implies the existence of mechanisms, operative during recombination of chromosomal substrates, that act to prevent coding end release or to facilitate coding end recapture.     

A RAG1 and RAG2 Tetramer Complex Is Active in Cleavage in V(D)J Recombination

During V(D)J recombination two proteins, RAG1 and RAG2, assemble as a protein-DNA complex with the appropriate DNA targets containing recombination signal sequences (RSSs). The properties of this complex require a fairly elaborate set of protein-protein and protein-DNA contacts. Here we show that a purified derivative of RAG1, without DNA, exists predominantly as a homodimer. A RAG2 derivative alone has monomer, dimer, and larger forms. The coexpressed RAG1 and RAG2 proteins form a mixed tetramer in solution which contains two molecules of each protein. The same tetramer of RAG1 and RAG2 plus one DNA molecule is the form active in cleavage. Additionally, we show that both DNA products following cleavage can still be held together in a stable protein-DNA complex.     

Distinct Roles of RAG1 and RAG2 in Binding the V(D)J Recombination Signal Sequences

The RAG1 and RAG2 proteins initiate V(D)J recombination by introducing double-strand breaks at the border between a recombination signal sequence (RSS) and a coding segment. To understand the distinct functions of RAG1 and RAG2 in signal recognition, we have compared the DNA binding activities of RAG1 alone and RAG1 plus RAG2 by gel retardation and footprinting analyses. RAG1 exhibits only a three- to fivefold preference for binding DNA containing an RSS over random sequence DNA. Although direct binding of RAG2 by itself was not detected, the presence of both RAG1 and RAG2 results in the formation of a RAG1-RAG2-DNA complex which is more stable and more specific than the RAG1-DNA complex and is active in V(D)J cleavage. These results suggest that biologically effective discrimination between an RSS and nonspecific sequences requires both RAG1 and RAG2. Unlike the binding of RAG1 plus RAG2, RAG1 can bind to DNA in the absence of a divalent metal ion and does not require the presence of coding flank sequence. Footprinting of the RAG1-RAG2 complex with 1,10-phenanthroline-copper and dimethyl sulfate protection reveal that both the heptamer and the nonamer are involved. The nonamer is protected, with extensive protein contacts within the minor groove. Conversely, the heptamer is rendered more accessible to chemical attack, suggesting that binding of RAG1 plus RAG2 distorts the DNA near the coding/signal border.     

Identification of two topologically independent domains in RAG1 and their role in macromolecular interactions relevant to V(D)J recombination

V(D)J recombination is instigated by the recombination-activating proteins RAG1 and RAG2, which catalyze site-specific DNA cleavage at the border of the recombination signal sequence (RSS). Although both proteins are required for activity, core RAG1 (the catalytically active region containing residues 384-1008 of 1040) alone displays binding specificity for the conserved heptamer and nonamer sequences of the RSS. The nonamer-binding region lies near the N terminus of core RAG1, whereas the heptamer-binding region has not been identified. Here, potential domains within core RAG1 were identified using limited proteolysis studies. An iterative procedure of DNA cloning, protein expression, and characterization revealed the presence of two topologically independent domains within core RAG1, referred to as the central domain (residues 528-760) and the C-terminal domain (residues 761-980). The domains do not include the nonamer-binding region but rather largely span the remaining relatively uncharacterized region of core RAG1. Characterization of macromolecular interactions revealed that the central domain bound to the RSS with specificity for the heptamer and contained the predominant binding site for RAG2. The C-terminal domain bound DNA cooperatively but did not show specificity for either conserved RSS element. This domain was also found to self-associate, implicating it as a dimerization domain within RAG1.