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.     

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.     

Functional Analysis of Coordinated Cleavage in V(D)J Recombination

V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn²⁺, but cleavage is restricted to substrates containing two signals when Mg²⁺ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.     

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。     

染色体重构与免疫球蛋白的V(D)J重组

免疫球蛋白是由B细胞合成、分泌的．它的可变区特异结合抗原,恒定区发挥免疫球蛋白的生物学功能,介导体液免疫反应。编码免疫球蛋白可变区的V、D、J基因片段互相分开、以胚系形式存在。在B细胞发育过程中．胚系基因片段通过DNA重组相连,组装成育转录活性单位。V(D)J重组是严格调控的。其中,B细胞染色体的乙酰化修饰,引起局部染色体的结构改变是一重要机制。     

Recombination centres and the orchestration of V(D)J recombination

The initiation of V(D)J recombination by the recombination activating gene 1 (RAG1) and RAG2 proteins is carefully orchestrated to ensure that antigen receptor gene assembly occurs in the appropriate cell lineage and in the proper developmental order. Here we review recent advances in our understanding of how DNA binding and cleavage by the RAG proteins are regulated by the chromatin structure and architecture of antigen receptor genes. These advances suggest novel mechanisms for both the targeting and the mistargeting of V(D)J recombination, and have implications for how these events contribute to genome instability and lymphoid malignancy.     

抗原受体基因重组的表观遗传学调控研究新进展

淋巴细胞是哺乳动物唯一能发生体细胞基因组变化的一类细胞,淋巴细胞在发育过程中通过V(D)J重组获得成熟的特异的抗原受体基因,实现了免疫细胞抗原识别惊人的多样性.关于V(D)J重组的调控机制一直是免疫学研究的重要问题,然而直到将表观遗传学研究引入这一领域,综合遗传学和表观遗传学的研究才真正揭示V(D)J重组精细的调控机制.综述了新近发现的V(D)J重组过程中重要的表观遗传学调控机制,如CpG甲基化,组蛋白修饰,核小体重塑及核拓扑学变化.     

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.     

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.     

VDJSeq-Solver: In Silico V(D)J Recombination Detection Tool

In this paper we present VDJSeq-Solver, a methodology and tool to identify clonal lymphocyte populations from paired-end RNA Sequencing reads derived from the sequencing of mRNA neoplastic cells. The tool detects the main clone that characterises the tissue of interest by recognizing the most abundant V(D)J rearrangement among the existing ones in the sample under study. The exact sequence of the clone identified is capable of accounting for the modifications introduced by the enzymatic processes. The proposed tool overcomes limitations of currently available lymphocyte rearrangements recognition methods, working on a single sequence at a time, that are not applicable to high-throughput sequencing data. In this work, VDJSeq-Solver has been applied to correctly detect the main clone and identify its sequence on five Mantle Cell Lymphoma samples; then the tool has been tested on twelve Diffuse Large B-Cell Lymphoma samples. In order to comply with the privacy, ethics and intellectual property policies of the University Hospital and the University of Verona, data is available upon request to ti.rvinu.oeneta@itnetu.otroppus after signing a mandatory Materials Transfer Agreement. VDJSeq-Solver JAVA/Perl/Bash software implementation is free and available at http://eda.polito.it/VDJSeq-Solver/.     

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.     

Failure of Hairpin-Ended and Nicked DNA To Activate DNA-Dependent Protein Kinase: Implications for V(D)J Recombination

V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PK_CS or p460). Ku directs DNA-PK_CS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PK_CS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PK_CS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.     

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.     

Self-association and conformational properties of RAG1: implications for formation of the V(D)J recombinase

RAG1 and RAG2 catalyze the initial DNA cleavage steps in V(D)J recombination. Fundamental properties of these proteins remain largely unknown. Here, self-association and conformational properties of murine core RAG1 (residues 384–1008) were examined. As determined by multi-angle laser light scattering measurements, the molecular masses of two predominant core RAG1 species corresponded to dimeric and tetrameric states. Similar results were obtained using a RAG1 fragment containing residues 265–1008, indicating that a non-core portion of RAG1 does not alter the oligomerization states observed for the core region. The fraction of core RAG1 in the tetrameric state increased significantly at lower ionic strengths (0.2 versus 0.5 M NaCl), indicating that this oligomeric form may factor into the physiological function of RAG1. In addition, the secondary structural content of core RAG1, obtained by circular dichroism spectroscopy, demonstrated a significant dependence on ionic strength with a 26% increase in α-helical content from 0.2 to 1.0 M NaCl. Together, these results indicate that structural and oligomerization properties of core RAG1 are strongly dependent on electrostatic interactions. Furthermore, the secondary structure of core RAG1 changes upon binding to DNA, with larger increases in α-helical content upon binding to the recombination signal sequence (RSS) as compared with non-sequence-specific DNA. As shown by electrophoretic mobility shift assays, higher order oligomeric forms of core RAG1 bound to the canonical RSS. Furthermore, core RAG2 (residues 1–387) formed complexes with multimeric RAG1 species bound to a single RSS, providing additional support for the physiological relevance of higher order oligomeric states of RAG1.     

No Requirement for V(D)J Recombination in p53-Deficient Thymic Lymphoma

The p53 tumor suppressor is activated in response to a variety of cellular stress signals, although specific in vivo signals that trigger tumor suppression are unknown. In mouse thymocytes, where p53 inactivation leads to tumorigenesis, several observations suggest that V(D)J recombination of T-cell receptor (TCR) loci could provide a DNA damage signal triggering p53-dependent apoptosis and tumor suppression. Inactivation of p53 would allow V(D)J driven mutation of additional cancer genes, facilitating tumorigenesis. Here, we show that mice with a p53 deficiency in thymocytes and unable to carry out V(D)J recombination are not impaired in the development of thymoma. Recombination-activating gene (RAG) deficiencies were introduced into both p53^−/− mice and TgTΔN transgenic mice, a strain in which 100% of the mice develop thymoma due to thymocyte-specific inactivation of p53 by a simian virus 40 T-antigen variant. V(D)J recombination was dispensable for tumorigenesis since thymomas developed with or without the RAG-1 or RAG-2 gene, although some delay was observed. When V(D)J recombination was suppressed by expression of rearranged TCR transgenes, 100% of the TgTΔN mice developed thymoma, surprisingly with reduced latency. Further introduction of a RAG deficiency into these mice had no impact on the timing or frequency of tumorigenesis. Finally, karyotype and chromosome painting analyses showed no evidence for TCR gene translocations in p53-deficient thymomas, although abundant aneuploidy involving frequent duplication of certain chromosomes was present. Thus, contrary to the current hypothesis, these studies indicate that signals other than V(D)J recombination promote p53 tumor suppression in thymocytes and that the mechanism of tumorigenesis is distinct from TCR translocation oncogene activation.     

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.     

A C-terminal region of RAG1 contacts the coding DNA during V(D)J recombination

The site-specific DNA rearrangement process, called V(D)J recombination, creates much of the diversity of immune receptor molecules in the adaptive immune system. Central to this reaction is the organization of the protein-DNA complex containing the proteins RAG1 and RAG2 and their DNA targets. A long-term goal is to appreciate the three-dimensional relationships between the proteins and DNA that allow the assembly of the appropriate reaction intermediates, resulting in concerted cleavage and directed rejoining of the DNA ends. Previous cross-linking approaches have mapped RAG1 contacts on the DNA. RAG1 protein contacts the DNA at the conserved heptamer and nonamer sequences as well as at the coding DNA adjacent to the heptamer. Here we subject RAG1, covalently cross-linked to DNA substrates, to partial cyanogen bromide degradation or trypsin proteolysis in order to map contacts on the protein. We find that coding-sequence contacts occur near the C terminus of RAG1, while contacts made within the recombination signal sequence occur nearer the N terminus of the core region of RAG1. A deletion protein lacking the C-terminal DNA-contacting region is still capable of making the N-terminal contacts. This suggests that the two binding interactions may exist on two separate domains of the protein. A trypsin cleavage pattern of the native protein supports this conclusion. A two-domain model for RAG1 is evaluated with respect to the larger recombination complex.     

A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.     

RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination

RAG1 and RAG2 proteins are key components in V(D)J recombination. The core region of RAG1 is capable of catalyzing the recombination reaction; however, the biological function of non-core RAG1 remains largely unknown. Here, we show that in a murine-model carrying the RAG1 ring-finger conserved cysteine residue mutation (C325Y), V(D)J recombination was abrogated at the cleavage step, and this effect was accompanied by decreased mono-ubiquitylation of histone H3. Further analyses suggest that un-ubiquitylated histone H3 restrains RAG1 to the chromatin by interacting with the N-terminal 218 amino acids of RAG1. Our data provide evidence for a model in which ubiquitylation of histone H3 mediated by the ring-finger domain of RAG1 triggers the release of RAG1, thus allowing its transition into the cleavage phase. Collectively, our findings reveal that the non-core region of RAG1 facilitates chromosomal V(D)J recombination in a ubiquitylation-dependent pathway.