V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor delta rearrangement signals.

In the murine T cell receptor delta locus, V(D)J recombination events frequently involve the D2 and J1 elements. Here we report the presence of double-strand breaks at recombination signals flanking D2 in approximately 2% of thymus DNA. An excised linear species containing the sequences between D2 and J1 and a circular product of the joining of D2 and J1 recombination signals were also found. Although broken molecules with signal ends were detected, no species with coding ends could be identified. Observation of these broken molecules in thymus, but not in liver or spleen, provides the first direct evidence for an association between specific cleavage of chromosomal DNA and recombination in mammalian cells, and supports a breakage-reunion model of V(D)J recombination.     

The RAG-HMG1 Complex Enforces the 12/23 Rule of V(D)J Recombination Specifically at the Double-Hairpin Formation Step

A central unanswered question concerning the initial phases of V(D)J recombination has been at which step the 12/23 rule applies. This rule, which governs which variable (V), diversity (D), and joining (J) segments are able to pair during recombination, could operate at the level of signal sequence synapsis after RAG-HMG1 complex binding, signal nicking, or signal hairpin formation. It has also been unclear whether additional proteins are required to achieve adherence to the 12/23 rule. We developed a novel system for the detailed biochemical analysis of the 12/23 rule by using an oligonucleotide-based substrate that can include two signals. Under physiologic conditions, we found that the complex of RAG1, RAG2, and HMG1 can successfully recapitulate the 12/23 rule with the same specificity as that seen intracellularly and in crude extracts. The cleavage complex can bind and nick 12×12 and 23×23 substrates as well as 12×23 substrates. However, hairpin formation occurs at both of the signals only on 12×23 substrates. Moreover, under physiologic conditions, the presence of a partner 23-bp spacer suppresses single-site hairpin formation at a 12-bp spacer and vice versa. Hence, this study illustrates that synapsis suppresses single-site reactions, thereby explaining the high physiologic ratio of paired versus unpaired V(D)J recombination events in lymphoid cells.     

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.     

Activation of V(D)J recombination induces the formation of interlocus joints and hybrid joints in scid pre-B-cell lines

V(D)J recombination is the mechanism by which antigen receptor genes are assembled. The site-specific cleavage mediated by RAG1 and RAG2 proteins generates two types of double-strand DNA breaks: blunt signal ends and covalently sealed hairpin coding ends. Although these DNA breaks are mainly resolved into coding joints and signal joints, they can participate in a nonstandard joining process, forming hybrid and open/shut joints that link coding ends to signal ends. In addition, the broken DNA molecules excised from different receptor gene loci could potentially be joined to generate interlocus joints. The interlocus recombination process may contribute to the translocation between antigen receptor genes and oncogenes, leading to malignant transformation of lymphocytes. To investigate the underlying mechanisms of these nonstandard recombination events, we took advantage of recombination-inducible cell lines derived from scid homozygous (s/s) and scid heterozygous (s/+) mice by transforming B-cell precursors with a temperature-sensitive Abelson murine leukemia virus mutant (ts-Ab-MLV). We can manipulate the level of recombination cleavage and end resolution by altering the cell culture temperature. By analyzing various recombination products in scid and s/+ ts-Ab-MLV transformants, we report in this study that scid cells make higher levels of interlocus and hybrid joints than their normal counterparts. These joints arise concurrently with the formation of intralocus joints, as well as with the appearance of opened coding ends. The junctions of these joining products exhibit excessive nucleotide deletions, a characteristic of scid coding joints. These data suggest that an inability of scid cells to promptly resolve their recombination ends exposes the ends to a random joining process, which can conceivably lead to chromosomal translocations.     

Hybrid joint formation in human V(D)J recombination requires nonhomologous DNA end joining

In V(D)J recombination, the RAG proteins bind at a pair of signal sequences adjacent to the V, D, or J coding regions and cleave the DNA, resulting in two signal ends and two hairpinned coding ends. The two coding ends are joined to form a coding joint, and the two signal ends are joined to form a signal joint; this joining is done by the nonhomologous DNA end joining (NHEJ) pathway. A recombinational alternative in which a signal end is recombined with a coding end can also occur in a small percentage of the V(D)J recombination events in murine and human cells, and these are called hybrids (or hybrid joints). Two mechanisms have been proposed for the formation of these hybrids. One mechanism is via NHEJ, after initial cutting by RAGs. The second mechanism does not rely on NHEJ, but rather invokes that the RAGs can catalyze joining of the signal to the hairpinned coding end, by using the 3'OH of the signal end as a nucleophile to attack the phosphodiester bonds of the hairpinned coding end. In the present study, we addressed the question of which type of hybrid joining occurs in a physiological environment, where standard V(D)J recombination presumably occurs and normal RAG proteins are endogenously expressed. We find that all hybrids in vivo require DNA ligase IV in human cells, which is the final component of the NHEJ pathway. Hence, hybrid joints rely on NHEJ rather than on the RAG complex for joining.     

Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events

V(D)J recombination and class switch recombination are the two DNA rearrangement events used to diversify the mouse and human antibody repertoires. While their double strand breaks (DSBs) are initiated by different mechanisms, both processes use non-homologous end joining (NHEJ) in the repair phase. DNA mismatch repair elements (MSH2/MSH6) have been implicated in the repair of class switch junctions as well as other DNA DSBs that proceed through NHEJ. MSH2 has also been implicated in the regulation of factors such as ATM and the MRN (Mre11, Rad50, Nbs1) complex, which are involved in V(D)J recombination. These findings led us to examine the role of MSH2 in V(D)J repair. Using MSH2-/- and MSH2+/+ mice and cell lines, we show here that all pathways involving MSH2 are dispensable for the generation of an intact pre-immune repertoire by V(D)J recombination. In contrast to switch junctions and other DSBs, the usage of terminal homology in V(D)J junctions is not influenced by MSH2. Thus, whether the repair complex for V(D)J recombination is of a canonical NHEJ type or a separate microhomology-mediated-end joining (MMEJ) type, it does not involve MSH2. This highlights a distinction between the repair of V(D)J recombination and other NHEJ reactions.     

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.     

Analysis of regions of RAG-2 important for V(D)J recombination.

The recombinase activating genes RAG-1 and RAG-2 operate together to activate V(D)J recombination, and thus play an essential role in the generation of immune system diversity. As a first step in understanding the function of the RAG-2 protein, we have tested a series of deletion and insertion mutations for their ability to induce V(D)J joining of a variety of model substrates. Mutants were assayed for their ability to induce deletional and inversional V(D)J joining, thereby testing their proficiency at forming both signal and coding joints, and, in some cases, for their ability to carry out recombination of both extrachromosomal and integrated recombination substrates. All these reactions were affected similarly by any one mutation. Although the RAG-2 protein shows extensive evolutionary conservation across its length, we found that the carboxy-terminal portion of RAG-2, including an acidic region, is dispensable for all forms of recombination tested. In contrast, all mutations we created in the N-terminal region severely decreased recombination. Thus, the core active region required for V(D)J recombination is confined to the first three-quarters of the RAG-2 protein.     

Ku86 is not required for protection of signal ends or for formation of nonstandard V(D)J recombination products.

Ku, a heterodimer of 70- and 86-kDa subunits, serves as the DNA binding component of the DNA-dependent protein kinase (DNA-PK). Cells deficient for the 86-kDa subunit of Ku (Ku86-deficient cells) lack Ku DNA end-binding activity and are severely defective for formation of the standard V(D)J recombination products, i.e., signal and coding joints. It has been widely hypothesized that Ku is required for protection of broken DNA ends generated during V(D)J recombination. Here we report the first analysis of V(D)J recombination intermediates in a Ku-deficient cell line. We find that full-length, ligatable signal ends are abundant in these cells. These data show that Ku86 is not required for the protection or stabilization of signal ends, suggesting that other proteins may perform this function. The presence of high levels of signal ends in Ku-deficient cells prompted us to investigate whether these ends could participate in joining reactions. We show that nonstandard V(D)J recombination products (hybrid joints), which involve joining a signal end to a coding end, form with similar efficiencies in Ku-deficient and wild-type fibroblasts. These data support the surprising conclusion that Ku is not required for some types of V(D)J joining events. We propose a novel RAG-mediated joining mechanism, analogous to disintegration reactions performed by retroviral integrases, to explain how formation of hybrid joints can bypass the requirement for Ku and DNA-PK.     

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.     

Intermolecular V(D)J recombination is prohibited specifically at the joining step

V(D)J recombination, normally an intramolecular process, assembles immunoglobulin and T cell receptor genes from V, D, and J coding segments. Oncogenic chromosome translocations can result from aberrant rearrangements, such as occur in intermolecular V(D)J recombination. How this is normally prevented remains unclear; DNA cleavage, joining, or both could be impaired when the recombination signal sequences (RSS) are located in trans, on separate DNA molecules. Here, we show that both trans cleavage and joining of signal ends occur efficiently in vivo. Unexpectedly, trans joining of coding ends is severely impaired (100-to 1000-fold), indicating that protection against intermolecular V(D)J recombination is established at the joining step. These findings suggest a novel surveillance mechanism for eliminating cells containing aberrant V(D)J rearrangements.     

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.     

Coding sequence composition flanking either signal element alters V(D)J recombination efficiency.

Lymphoid V(D)J rearrangement is targeted by recombination signal sequences (RSS) bordering V, D or J exons. We demonstrate that the DNA composition of flanking coding positions, particularly poly(A) or poly(T) stretches at one or both RSS, diminishes V(D)J recombination up to 100-fold. Positionally correct cleavages occur in the inhibited reactions, since the junctions formed show the same frequency of precision as uninhibited reactions. Open/shut cleavage/rejoining is not increased at a normal RSS in substrates containing inhibitory A/T homopolymers versus random sequence at a second RSS. Thus recombinase action at both cleavage sites is severely disrupted by modified coding sequences.     

Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals

Sequences encoding immunoglobulin variable domains are known to be assembled from variable (V), diversity (D), and joining (J) segments by site-specific recombination. We present a sensitive and rapid assay for V-(D)-J recombination that uses plasmid DNA transiently introduced into transformed pre-B cells, and demonstrates that the recombination is independent of any unique chromosomal context. Sequences sufficient to constitute recombination sites are contained within the 84 and 42 bp flanking, respectively, the murine J kappa 1 and V kappa L8 segments, which include the known heptamer-nonamer V-(D)-J joining signals. Deletion and inversion occur at comparable frequencies. Thus, V-(D)-J recombination may be relatively insensitive to the topological arrangement of sites, and events at the two novel junctions produced by the reaction may be coupled.     

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.     

DNA-PKcs dependence of Artemis endonucleolytic activity, differences between hairpins and 5' or 3' overhangs

During V(D)J recombination, the RAG proteins create DNA hairpins at the V, D, or J coding ends, and the structure-specific nuclease Artemis is essential to open these hairpins prior to joining. Artemis also is an endonuclease for 5' and 3' overhangs at many DNA double strand breaks caused by ionizing radiation, and Artemis functions as part of the nonhomologous DNA end joining pathway in repairing these. All of these activities require activation of the Artemis protein by interaction with and phosphorylation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In this study, we have identified a region of the Artemis protein involved in the interaction with DNA-PKcs. Furthermore, the biochemical and functional analyses of C-terminally truncated Artemis variants indicate that the hair-pin opening and DNA overhang endonucleolytic features of Artemis are triggered by DNA-PKcs in two modes. First, autoinhibition mediated by the C-terminal tail of Artemis is relieved by phosphorylation of this tail by DNA-PKcs. Thus, C-terminally truncated Artemis derivatives imitate DNA-PKcs-activated wild type Artemis protein and exhibit intrinsic hairpin opening activity. Second, DNA-PKcs may optimally configure 5' and 3' overhang substrates for the endonucleolytic function of Artemis.     

Postcleavage sequence specificity in V(D)J recombination

Unintended DNA rearrangements in a differentiating lymphocyte can have severe, oncogenic consequences, but the mechanisms for avoiding pathogenic outcomes in V(D)J recombination are not well understood. The first level at which fidelity is instituted is in discrimination by the recombination proteins between authentic and inauthentic recombination signal sequences. Nevertheless, this discrimination is not absolute and cannot fully eliminate targeting errors. To learn more about the basis of specificity during V(D)J recombination, we have investigated whether it is possible for the recombination machinery to detect an inaccurately targeted sequence subsequent to cleavage. These studies indicate that even postcleavage steps in V(D)J recombination are sequence specific and that noncanonical sequences will not efficiently support the resolution of recombination intermediates in vivo. Accordingly, interventions after a mistargeting event conceivably occur at a late stage in the joining process and the likelihood may well be crucial to enforcing fidelity during antigen receptor gene rearrangement.     

Cryptic signals and the fidelity of V(D)J joining.

V(D)J recombination is responsible for the de novo creation of antigen receptor genes in T- and B-cell precursors. To the extent that lymphopoiesis takes place throughout an animal's lifetime, recombination errors present an ongoing problem. One type of aberrant rearrangement ensues when DNA sequences resembling a V(D)J joining signal are targeted by mistake. This study investigates the type of sequence likely to be subject to mistargeting, the level of joining-signal function associated with these sequences, and the number of such cryptic joining signals in the genome.     

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.     

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.