Definition of a core region of RAG-2 that is functional in V(D)J recombination.

The products of the RAG-1 and RAG-2 genes cooperate to allow V(D)J recombination in lymphoid and non-lymphoid cells. As one step toward understanding the role of RAG-2, we have constructed mutated RAG-2 genes and examined their ability to support recombination of plasmid substrates in a fibroblast cell line. The mutations define essential and dispensable parts of the RAG-2 gene. Mutations in the N-terminal part eliminate almost all activity. In the central region of the protein, some but not all local alterations still allow recombination. On the other hand, proteins with large deletions from the C-terminal end, including one truncated by 25%, still retain activity, even though this part of the protein is highly conserved between species. Similar results were obtained with substrates that retain either a signal joint or a coding joint, or perform an inversion. Thus all basic features of V(D)J joining are retained in a RAG-2 protein with only the first 75% of the sequence.     

ATM prevents the persistence and propagation of chromosome breaks in lymphocytes

DNA double-strand breaks (DSBs) induce a signal transmitted by the ataxia-telangiectasia mutated (ATM) kinase, which suppresses illegitimate joining of DSBs and activates cell-cycle checkpoints. Here we show that a significant fraction of mature ATM-deficient lymphocytes contain telomere-deleted ends produced by failed end joining during V(D)J recombination. These RAG-1/2 endonuclease-dependent, terminally deleted chromosomes persist in peripheral lymphocytes for at least 2 weeks in vivo and are stable over several generations in vitro. Restoration of ATM kinase activity in mature lymphocytes that have transiently lost ATM function leads to loss of cells with terminally deleted chromosomes. Thus, maintenance of genomic stability in lymphocytes requires faithful end joining as well a checkpoint that prevents the long-term persistence and transmission of DSBs. Silencing this checkpoint permits DNA ends produced by V(D)J recombination in a lymphoid precursor to serve as substrates for translocations with chromosomes subsequently damaged by other means in mature cells.     

The DDE motif in RAG-1 is contributed in trans to a single active site that catalyzes the nicking and transesterification steps of V(D)J recombination

The process of assembling immunoglobulin and T-cell receptor genes from variable (V), diversity (D), and joining (J) gene segments, called V(D)J recombination, involves the introduction of DNA breaks at recombination signals. DNA cleavage is catalyzed by RAG-1 and RAG-2 in two chemical steps: first-strand nicking, followed by hairpin formation via direct transesterification. In vitro, these reactions minimally proceed in discrete protein-DNA complexes containing dimeric RAG-1 and one or two RAG-2 monomers bound to a single recombination signal sequence. Recently, a DDE triad of carboxylate residues essential for catalysis was identified in RAG-1. This catalytic triad resembles the DDE motif often associated with transposase and retroviral integrase active sites. To investigate which RAG-1 subunit contributes the residues of the DDE triad to the recombinase active site, cleavage of intact or prenicked DNA substrates was analyzed in situ in complexes containing RAG-2 and a RAG-1 heterodimer that carried an active-site mutation targeted to the same or opposite RAG-1 subunit mutated to be incompetent for DNA binding. The results show that the DDE triad is contributed to a single recombinase active site, which catalyzes the nicking and transesterification steps of V(D)J recombination by a single RAG-1 subunit opposite the one bound to the nonamer of the recombination signal undergoing cleavage (cleavage in trans). The implications of a trans cleavage mode observed in these complexes on the organization of the V(D)J synaptic complex are discussed.     

A RAG-1/RAG-2 tetramer supports 12/23-regulated synapsis,cleavage, and transposition of V(D)J recombination signals

Initiation of V(D)J recombination involves the synapsis and cleavage of a 12/23 pair of recombination signal sequences by RAG-1 and RAG-2. Ubiquitous nonspecific DNA-bending factors of the HMG box family, such as HMG-1, are known to assist in these processes. After cleavage, the RAG proteins remain bound to the cut signal ends and, at least in vitro, support the integration of these ends into unrelated target DNA via a transposition-like mechanism. To investigate whether the protein complex supporting synapsis, cleavage, and transposition of V(D)J recombination signals utilized the same complement of RAG and HMG proteins, I compared the RAG protein stoichiometries and activities of discrete protein-DNA complexes assembled on intact, prenicked, or precleaved recombination signal sequence (RSS) substrates in the absence and presence of HMG-1. In the absence of HMG-1, I found that two discrete RAG-1/RAG-2 complexes are detected by mobility shift assay on all RSS substrates tested. Both contain dimeric RAG-1 and either one or two RAG-2 subunits. The addition of HMG-1 supershifts both complexes without altering the RAG protein stoichiometry. I find that 12/23-regulated recombination signal synapsis and cleavage are only supported in a protein-DNA complex containing HMG-1 and a RAG-1/RAG-2 tetramer. Interestingly, the RAG-1/RAG-2 tetramer also supports transposition, but HMG-1 is dispensable for its activity.     

Roles of the "dispensable" portions of RAG-1 and RAG-2 in V(D)J recombination

V(D)J recombination is initiated by introduction of site-specific double-stranded DNA breaks by the RAG-1 and RAG-2 proteins. The broken DNA ends are then joined by the cellular double-strand break repair machinery. Previous work has shown that truncated (core) versions of the RAG proteins can catalyze V(D)J recombination, although less efficiently than their full-length counterparts. It is not known whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the joining step of recombination. Here we examine the effects of truncated RAG proteins on recombination intermediates and products. We found that while truncated RAG proteins generate lower levels of recombination products than their full-length counterparts, they consistently generate 10-fold higher levels of one class of recombination intermediates, termed signal ends. Our results suggest that this increase in signal ends does not result from increased cleavage, since levels of the corresponding intermediates, coding ends, are not elevated. Thus, removal of the "dispensable" regions of the RAG proteins impairs proper processing of recombination intermediates. Furthermore, we found that removal of portions of the dispensable regions of RAG-1 and RAG-2 affects the efficiency of product formation without altering the levels of recombination intermediates. Thus, these evolutionarily conserved sequences play multiple, important roles in V(D)J recombination.     

V(D)J recombination intermediates and non-standard products in XRCC4-deficient cells.

V(D)J recombination assembles immunoglobulin (Ig) and T cell receptor (TCR) gene segments during lymphocyte development. Recombination is initiated by the RAG-1 and RAG-2 proteins, which introduce double-stranded DNA breaks (DSB) adjacent to the Ig and TCR gene segments. The broken ends are joined by the DSB repair machinery, which includes the XRCC4 protein. While XRCC4 is essential for both DSB repair and V(D)J recombination, the functions of this protein remain enigmatic. Because the rare V(D)J recombination products isolated from XRCC4-deficient cells generally show evidence of excessive nucleotide loss, it was hypothesized that XRCC4 may function to protect broken DNA ends. Here we report the first examination of V(D)J recombination intermediates in XRCC4-deficient cells. We found that both types of intermediates, signal ends and coding ends, are abundant in the absence of XRCC4. Furthermore, the signal ends are full length. We also showed that alternative V(D)J recombination products, hybrid joints, form with normal efficiency and without excessive deletion in XRCC4-deficient cells. These data indicate that impaired formation of V(D)J recombination products in XRCC4-deficient cells does not result from excessive degradation of recombination intermediates. Potential roles of XRCC4 in the joining reaction are discussed.     

Ubiquitylation of RAG-2 by Skp2-SCF links destruction of the V(D)J recombinase to the cell cycle

The periodic destruction of RAG-2 at the G1-to-S transition couples V(D)J recombination to the G0 and G1 cell cycle phases and coordinates RAG-mediated DNA cleavage with DNA repair by nonhomologous end joining. To define the mechanism by which this occurs, we reproduced cell cycle-dependent regulation of the V(D)J recombinase in a cell-free system. The ubiquitin-proteasomal pathway carries out destruction of RAG-2 in lysates of S phase cells and during S phase in vivo. Remarkably, the Skp2-SCF ubiquitin ligase, which plays a central role in cell cycle regulation through the destruction of p27, mediates ubiquitylation of RAG-2 in vitro and degradation of RAG-2 in vivo. The regulation of antigen receptor gene assembly by Skp2-SCF provides an unexpected and direct mechanistic link between DNA recombination and the cell cycle.     

Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion

Chickens create their immunoglobulin (Ig) repertoires during B cell development in the bursa of Fabricius by intrachromosomal gene conversion. Recent evidence has suggested that Ig gene conversion may involve cis-acting DNA elements related to those involved in V(D)J recombination. Therefore, we have examined the potential role of the V(D)J recombination activating genes, RAG-1 and RAG-2, in regulating chicken Ig gene conversion. In contrast to the coexpression of RAG-1 and RAG-2 observed in mammalian B cells that undergo V(D)J recombination, chicken B cells isolated from the bursa of Fabricius express high levels of the RAG-2 mRNA but do not express RAG-1 mRNA. The developmental and phenotypic characteristics of the bursal lymphocytes and chicken B cell lines that express RAG-2 mRNA demonstrate that selective RAG-2 expression occurs specifically in B cells undergoing Ig diversification by gene conversion. These data suggest that RAG-2 plays a fundamental role in Ig-specific gene conversion.     

Cell cycle-dependent accumulation in vivo of transposition-competent complexes between recombination signal ends and full-length RAG proteins

V(D)J recombination is initiated by a specialized transposase consisting of RAG-1 and RAG-2. Because full-length RAG proteins are insoluble under physiologic conditions, most previous analyses of RAG activity in vitro have used truncated core RAG-1 and RAG-2 fragments. These studies identified an intermediate in V(D)J recombination, the signal end complex (SEC), in which core RAG proteins remain associated with recombination signal sequences at the cleaved signal ends. From transfected cells expressing affinity-tagged RAG proteins, we have isolated in vivo assembled SECs containing full-length RAG proteins and cleaved recombination substrates. SEC formation in vivo did not require the repair proteins DNA-dependent protein kinase, Ku80, or XRCC4. In the presence of full-length RAG-2, SEC formation in vivo was cell cycle-regulated and restricted to the G(0)/G(1) phases. In contrast, complexes accumulated throughout cell cycle in cells expressing a RAG-2 CDK2 phosphorylation site mutant. Both core and full-length SECs supported transposition in vitro with similar efficiencies. Intracellular SECs, which are likely to persist in the absence of coding ends, represent potential donors whose transposition is not suppressed by the non-core regions of the RAG proteins.     

Identification and characterization of a gain-of-function RAG-1 mutant

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.     

Conditional RAG-1 mutants block the hairpin formation step of V(D)J recombination

Hairpin formation serves an important regulatory role in V(D)J recombination because it requires synapsis of an appropriate pair of recombination sites. How hairpin formation is regulated and which regions of the RAG proteins perform this step remain unknown. We analyzed two conditional RAG-1 mutants that affect residues quite close in the primary sequence to an active site amino acid (D600), and we found that they exhibit severely impaired recombination in the presence of certain cleavage site sequences. These mutants are specifically defective for the formation of hairpins, providing the first identification of a region of the V(D)J recombinase necessary for this reaction. Substrates containing mismatched bases at the cleavage site rescued hairpin formation by both mutants, which suggests that the mutations affect the generation of a distorted or unwound DNA intermediate that has been implicated in hairpin formation. Our results also indicate that this region of RAG-1 may be important for coupling hairpin formation to synapsis.     

V(D)J recombination and RAG-mediated transposition in yeast

Antigen receptor genes are assembled during lymphoid development by a specialized recombination reaction normally observed only in cells of the vertebrate immune system. Here, we show that expression in Saccharomyces cerevisiae of murine RAG1 and RAG2, the lymphoid-specific components of the V(D)J recombinase, is sufficient to induce V(D)J cleavage and rejoining in this lower eukaryote. The RAG proteins cleave recombination substrates introduced into yeast cells, generating signal ends that can be joined to form signal joints. These signal joints are precise, as in mammalian cells, and their formation is dependent on a yeast nonhomologous end-joining protein, the XRCC4 homolog LIF1. Moreover, joining of SmaI-generated blunt ends is generally imprecise in the yeast strain used here, suggesting that the RAG proteins influence signal-end joining. Cleaved signal ends are also transposed into new sites in DNA, allowing RAG-induced transposition to be studied in vivo.     

Cutting and closing without recombination in V(D)J joining.

Open and shut junctions are rare (V(D)J joining products in which site-specific recognition, cleavage and re-ligation of joining signals has been uncoupled from recombination. Here, we investigate the relationship of opening and shutting to recombination in two ways. First, we have tested a series of substrates containing one or two joining signals in an in vivo assay. Opening and shutting can be readily observed in substrates that have only one consensus joining signal. Thus, unlike recombination, the majority of open and shut events do not require interactions between two canonical joining signals. Next we examined two-signal substrates to investigate the effect of signal proximity on the frequency of dual open and shut events. These experiments indicate that at least some of the time opening and shutting can be a two-signal transaction. Together these results point to two mechanistically related, but distinct origins for open and shut joining events. In one case, cutting and closing may occur without interaction between two signals. In the other, we suggest that interaction of a canonical signal with 'cryptic' signal-like elements whose sequence is extensively diverged from canonical signals, may bias the V(D)J recombination machinery towards opening and shutting rather than recombination. Open and shut operations could in this way provide a means whereby mistakes in target recognition by the V(D)J recombination machinery produce a non-recombinant outcome, avoiding deleterious chromosomal rearrangements in lymphoid tissues.     

Irradiation promotes V(D)J joining and RAG-dependent neoplastic transformation in SCID T-cell precursors

Defects in the nonhomologous end-joining (NHEJ) pathway of double-stranded DNA break repair severely impair V(D)J joining and selectively predispose mice to the development of lymphoid neoplasia. This connection was first noted in mice with the severe combined immune deficient (SCID) mutation in the DNA-dependent protein kinase (DNA-PK). SCID mice spontaneously develop thymic lymphoma with low incidence and long latency. However, we and others showed that low-dose irradiation of SCID mice dramatically increases the frequency and decreases the latency of thymic lymphomagenesis, but irradiation does not promote the development of other tumors. We have used this model to explore the mechanistic basis by which defects in NHEJ confer selective and profound susceptibility to lymphoid oncogenesis. Here, we show that radiation quantitatively and qualitatively improves V(D)J joining in SCID cells, in the absence of T-cell receptor-mediated cellular selection. Furthermore, we show that the lymphocyte-specific endonuclease encoded by the recombinase-activating genes (RAG-1 and RAG-2) is required for radiation-induced thymic lymphomagenesis in SCID mice. Collectively, these data suggest that irradiation induces a DNA-PK-independent NHEJ pathway that facilitates V(D)J joining, but also promotes oncogenic misjoining of RAG-1/2-induced breaks in SCID T-cell precursors.     

Paleo-Immunology: Evidence Consistent with Insertion of a Primordial Herpes Virus-Like Element in the Origins of Acquired Immunity

Background

The RAG encoded proteins, RAG-1 and RAG-2 regulate site-specific recombination events in somatic immune B- and T-lymphocytes to generate the acquired immune repertoire. Catalytic activities of the RAG proteins are related to the recombinase functions of a pre-existing mobile DNA element in the DDE recombinase/RNAse H family, sometimes termed the “RAG transposon”.

Methodology/Principal Findings

Novel to this work is the suggestion that the DDE recombinase responsible for the origins of acquired immunity was encoded by a primordial herpes virus, rather than a “RAG transposon.” A subsequent “arms race” between immunity to herpes infection and the immune system obscured primary amino acid similarities between herpes and immune system proteins but preserved regulatory, structural and functional similarities between the respective recombinase proteins. In support of this hypothesis, evidence is reviewed from previous published data that a modern herpes virus protein family with properties of a viral recombinase is co-regulated with both RAG-1 and RAG-2 by closely linked cis-acting co-regulatory sequences. Structural and functional similarity is also reviewed between the putative herpes recombinase and both DDE site of the RAG-1 protein and another DDE/RNAse H family nuclease, the Argonaute protein component of RISC (RNA induced silencing complex).

Conclusions/Significance

A “co-regulatory” model of the origins of V(D)J recombination and the acquired immune system can account for the observed linked genomic structure of RAG-1 and RAG-2 in non-vertebrate organisms such as the sea urchin that lack an acquired immune system and V(D)J recombination. Initially the regulated expression of a viral recombinase in immune cells may have been positively selected by its ability to stimulate innate immunity to herpes virus infection rather than V(D)J recombination Unlike the “RAG-transposon” hypothesis, the proposed model can be readily tested by comparative functional analysis of herpes virus replication and V(D)J recombination.     

RAG-1-deficient mice have no mature B and T lymphocytes.

The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.     

RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.

We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.     

Both high mobility group (HMG)-boxes and the acidic tail of HMGB1 regulate recombination-activating gene (RAG)-mediated recombination signal synapsis and cleavage in vitro

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.     

The V(D)J recombination activating gene, RAG-1

The RAG-1 (recombination activating gene-1) genomic locus, which activates V(D)J recombination when introduced into NIH 3T3 fibroblasts, was isolated by serial genomic transfections of oligonucleotide-tagged DNA. A genomic walk spanning 55 kb yielded a RAG-1 genomic probe that detects a single 6.6-7.0 kb mRNA species in transfectants and pre-B and pre-T cells. RAG-1 genomic and cDNA clones were biologically active when introduced into NIH 3T3 cells. Nucleotide sequencing of human and mouse RAG-1 cDNA clones predicts 119 kd proteins of 1043 and 1040 amino acids, respectively, with 90% sequence identity. RAG-1 has been conserved between species that carry out V(D)J recombination, and its pattern of expression correlates exactly with the pattern of expression of V(D)J recombinase activity. RAG-1 may activate V(D)J recombination indirectly, or it may encode the V(D)J recombinase itself.     

The MRE11-RAD50-XRS2 complex, in addition to other non-homologous end-joining factors, is required for V(D)J joining in yeast

Lymphoid cells of the vertebrate immune system rely on factors in the non-homologous end-joining (NHEJ) DNA repair pathway to form signal joints during V(D)J recombination. Unlike other end-joining reactions, signal joint formation is a specialized case of NHEJ that also requires the lymphoid-specific RAG proteins. Whether V(D)J recombination requires the Mre11-Rad50-Nbs1 complex remains an open question, as null mutations in any member of the complex are lethal in mammals. However, Saccharomyces cerevisiae strains carrying null mutations in components of the homologous Mre11p-Rad50p-Xrs2p (MRX) complex are viable. We therefore took advantage of a recently developed V(D)J recombination assay in yeast to assess the role of MRX in V(D)J joining. Here we confirmed that signal joint formation in yeast is dependent on the same NHEJ factors known to be required in mammalian cells. In addition, we showed an absolute requirement for the MRX complex in signal joining, suggesting that the Mre11-Rad50-Nbs1 complex may be required for signal joint formation in mammalian cells as well.