Targeted transposition by the V(D)J recombinase

Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by--and specifically targeted to--hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected.     

Extent to which hairpin opening by the Artemis:DNA-PKcs complex can contribute to junctional diversity in V(D)J recombination

V(D)J recombination events are initiated by cleavage at gene segments by the RAG1:RAG2 complex, which results in hairpin formation at the coding ends. The hairpins are opened by the Artemis:DNA-PKcs complex, and then joined via the nonhomologous DNA end joining (NHEJ) process. Here we examine the opening of the hairpinned coding ends from all of the 39 functional human V_H elements. We find that there is some sequence-dependent variation in the efficiency and even the position of hairpin opening by Artemis:DNA-PKcs. The hairpin opening efficiency varies over a 7-fold range. The hairpin opening position varies over the region from 1 to 4 nt 3′ of the hairpin tip, leading to a 2–8 nt single-stranded 3′ overhang at each coding end. This information provides greater clarity on the extent to which the hairpin opening position contributes to junctional diversification 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.     

Increased accumulation of hybrid V(D)J joins in cells expressing truncated versus full-length RAGs.

RAG1 and RAG2 (RAGs) initiate V(D)J recombination by introducing breaks between two coding segments and flanking recombination signals (RSs). Nonhomologous end-joining (NHEJ) proteins then join the coding segments and join the RSs. In wild-type cells, both full-length and truncated ("core") RAGs lead to accumulation of "hybrid" V(D)J joins, in which an RS is appended to a different coding sequence. We now show that while hybrid joins do not accumulate in NHEJ-deficient cells that express full-length RAGs, they do accumulate in NHEJ-deficient cells that express the core RAGS; like those catalyzed by core RAGs in vitro, however, they are sealed on just one DNA strand. These results suggest a potential role for the non-core regions in repressing potentially harmful transposition events.     

Roles of nonhomologous DNA end joining, V(D)J recombination, and class switch recombination in chromosomal translocations

When a single double-strand break arises in the genome, nonhomologous DNA end joining (NHEJ) is a major pathway for its repair. When double-strand breaks arise at two nonhomologous sites in the genome, NHEJ also appears to be a major pathway by which the translocated ends are joined. The mechanism of NHEJ is briefly summarized, and alternative enzymes are also discussed. V(D)J recombination and class switch recombination are specialized processes designed to create double-strand DNA breaks at specific locations in the genomes of lymphoid cells. Sporadic Burkitt's lymphoma and myelomas can arise due to translocation of the c-myc gene into the Ig heavy chain locus during class switch recombination. In other lymphoid neoplasms, the RAG complex can create double-strand breaks that result in a translocation. Such RAG-generated breaks occur at very specific nucleotides that are directly adjacent to sequences that resemble canonical heptamer/nonamer sequences characteristic of normal V(D)J recombination. This occurs in some T cell leukemias and lymphomas. The RAG complex also appears capable of recognizing regions for their altered DNA structure rather than their primary sequence, and this may account for the action by RAGs at some chromosomal translocation sites, such as at the bcl-2 major breakpoint region in the follicular lymphomas that arise in B lymphocytes.     

Non-consensus heptamer sequences destabilize the RAG post-cleavage complex,making ends available to alternative DNA repair pathways

V(D)J recombination entails double-stranded DNA cleavage at the antigen receptor loci by the RAG1/2 proteins, which recognize conserved recombination signal sequences (RSSs) adjoining variable (V), diversity (D) and joining (J) gene segments. After cleavage, RAG1/2 remain associated with the coding and signal ends (SE) in a post-cleavage complex (PCC), which is critical for their proper joining by classical non-homologous end joining (NHEJ). Certain mutations in RAG1/2 destabilize the PCC, allowing DNA ends to access inappropriate repair pathways such as alternative NHEJ, an error-prone pathway implicated in chromosomal translocations. The PCC is thus thought to discourage aberrant rearrangements by controlling repair pathway choice. Since interactions between RAG1/2 and the RSS heptamer element are especially important in forming the RAG-SE complex, we hypothesized that non-consensus heptamer sequences might affect PCC stability. We find that certain non-consensus heptamers, including a cryptic heptamer implicated in oncogenic chromosomal rearrangements, destabilize the PCC, allowing coding and SEs to be repaired by non-standard pathways, including alternative NHEJ. These data suggest that some non-consensus RSS, frequently present at chromosomal translocations in lymphoid neoplasms, may promote genomic instability by a novel mechanism, disabling the PCC’s ability to restrict repair pathway choice.     

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.     

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.     

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.     

Aberrant V(D)J recombination in ataxia telangiectasia mutated-deficient lymphocytes is dependent on nonhomologous DNA end joining

During lymphocyte Ag receptor gene assembly, DNA cleavage by the Rag proteins generates pairs of coding and signal ends that are normally joined into coding joints and signal joints, respectively, by the classical nonhomologous end-joining (NHEJ) pathway of DNA double strand break repair. Coding and signal ends can also be aberrantly joined to each other, generating hybrid joints, through NHEJ or through NHEJ-independent pathways, such as Rag-mediated transposition. Hybrid joints do not participate in the formation of functional Ag receptor genes and can alter the configuration of Ag receptor loci in ways that limit subsequent productive rearrangements. The formation of these nonfunctional hybrid joints occurs rarely in wild type lymphocytes, demonstrating that mechanisms exist to limit both the NHEJ-dependent and the NHEJ-independent joining of a signal end to a coding end. In contrast to wild-type cells, hybrid joint formation occurs at high levels in ataxia telangiectasia mutated (Atm)-deficient lymphocytes, suggesting that Atm functions to limit the formation of these aberrant joints. In this study, we show that hybrid joint formation in Atm-deficient cells requires the NHEJ proteins Artemis, DNA-PKcs, and Ku70, demonstrating that Atm functions primarily by modulating the NHEJ-dependent, and not the NHEJ-independent, joining of coding ends to signal ends.     

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.     

Increased frequency of aberrant V(D)J recombination products in core RAG-expressing mice

RAG1 and RAG2 play a central role in V(D)J recombination, a process for antigen receptor gene assembly. The truncated ‘core’ regions of RAGs are sufficient to catalyze the recombination reaction, although with lower joining efficiency than full-length proteins. To investigate the role of the non-core regions of RAGs in the end-joining phase of antigen receptor rearrangement, we analyzed recombination products isolated from core RAG1 and core RAG2 knock-in mice. Here, we report that the truncation of RAGs increases the frequency of aberrant recombination in vivo. Signal joints (SJs) associated with V-to-D recombination of core RAG1 knock-in mice were normal, whereas those of core RAG2 knock-in mice were highly imprecise, containing large deletions and additions, and in some cases coding sequences. In contrast, we found an elevated level of imprecise D-to-J associated SJs for both core RAG1- and RAG2-expressing mice. Likewise, sequences of coding joints (CJs) were also affected by the expression of core RAGs. Finally, sequences found at the junctions of rearranged T-cell receptor loci were highly influenced by differences in rearranging recombination signal sequence pairs. We provide the first evidence that the non-core regions of RAGs have critical functions in the proper assembly and resolution of recombination intermediates in endogenous antigen receptor loci.     

V(D)J recombination: site-specific cleavage and repair

V(D)J recombination is a site-specific gene rearrangement process that contributes to the diversity of antigen receptor repertoires. Two lymphoid-specific proteins, RAG1 and RAG2, initiate this process at two recombination signal sequences. Due to the recent development of an in vitro assay for V(D)J cleavage, the mechanism of cleavage has been elucidated clearly. The RAG complex recognizes a recombination signal sequence, makes a nick at the border between signal and coding sequence, and carries out a transesterification reaction, resulting in the production of a hairpin structure at the coding sequence and DNA double-strand breaks at the signal ends. RAG1 possesses the active site of the V(D)J recombinase although RAG2 is essential for signal binding and cleavage. After DNA cleavage by the RAG complex, the broken DNA ends are rejoined by the coordinated action of DNA double-strand break repair proteins as well as the RAG complex. The junctional variability resulting from imprecise joining of the coding sequences contributes additional diversity to the antigen receptors.     

Accessibility of chromosomal recombination breaks in nuclei of wild-type and DNA-PKcs-deficient cells

V(D)J recombination is a highly regulated process, proceeding from a site-specific cleavage to an imprecise end joining. After the DNA excision catalyzed by the recombinase encoded by recombination activating genes 1 and 2 (RAG1/2), newly generated recombination ends are believed held by a post-cleavage complex (PC) consisting of RAG1/2 proteins, and are subsequently resolved by non-homologous end joining (NHEJ) machinery. The relay of these ends from PC to NHEJ remains elusive. It has been speculated that NHEJ factors modify the RAG1/2-PC to gain access to the ends or act on free ends after the disassembly of the PC. Thus, recombination ends may either be retained in a complex throughout the recombination process or left as unprotected free ends after cleavage, a condition that may permit an alternative, non-classical NHEJ end joining pathway. To directly test these scenarios on recombination induced chromosomal breaks, we have developed a recombination end protection assay to monitor the accessibility of recombination ends to exonuclease-V in intact nuclei. We demonstrate that these ends are well protected in the nuclei of wild-type cells, suggesting a seamless cleavage-joining reaction. However, divergent end protection of coding versus signal ends was found in cells derived from severe combined immunodeficient (scid) mice that are defective in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While signal ends are resistant, opened coding ends are susceptible to enzymatic modification. Our data suggests a role of DNA-PKcs in protecting chromosomal coding ends. Furthermore, using recombination inducible scid cell lines, we demonstrate that conditional protection of coding ends is inversely correlated with the level of their resolution, i.e., the greater the accessibility of the coding ends, the higher level of coding joints formed. Taken together, our findings provide important insights into the resolution of recombination ends by error-prone alternative NHEJ pathways.     

The C-terminal portion of RAG2 protects against transposition in vitro

The assembly of antigen receptor genes by V(D)J recombination is initiated by the RAG1/RAG2 protein complex, which introduces double-strand breaks between recombination signal sequences and their coding DNA. Truncated forms of RAG1 and RAG2 are functional in vivo and have been used to study V(D)J cleavage, hybrid joint formation and transposition in vitro. Here we have characterized the activities of the full-length proteins. Unlike core RAG2, which supports robust transposition in vitro, full-length RAG2 blocks transposition of signal ends following V(D)J cleavage. Thus, one role of this non-catalytic domain may be to prevent transposition in developing lymphoid cells. Although full-length RAG1 and RAG2 proteins rarely form hybrid joints in vivo in the absence of non-homologous end-joining factors, we show that the full-length proteins alone can catalyze this reaction in vitro.     

Repair of chromosomal RAG-mediated DNA breaks by mutant RAG proteins lacking phosphatidylinositol 3-like kinase consensus phosphorylation sites

Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are members of the phosphatidylinositol 3-like family of serine/threonine kinases that phosphorylate serines or threonines when positioned adjacent to a glutamine residue (SQ/TQ). Both kinases are activated rapidly by DNA double-strand breaks (DSBs) and regulate the function of proteins involved in DNA damage responses. In developing lymphocytes, DSBs are generated during V(D)J recombination, which is required to assemble the second exon of all Ag receptor genes. This reaction is initiated through a DNA cleavage step by the RAG1 and RAG2 proteins, which together comprise an endonuclease that generates DSBs at the border of two recombining gene segments and their flanking recombination signals. This DNA cleavage step is followed by a joining step, during which pairs of DNA coding and signal ends are ligated to form a coding joint and a signal joint, respectively. ATM and DNA-PKcs are integrally involved in the repair of both signal and coding ends, but the targets of these kinases involved in the repair process have not been fully elucidated. In this regard, the RAG1 and RAG2 proteins, which each have several SQ/TQ motifs, have been implicated in the repair of RAG-mediated DSBs. In this study, we use a previously developed approach for studying chromosomal V(D)J recombination that has been modified to allow for the analysis of RAG1 and RAG2 function. We show that phosphorylation of RAG1 or RAG2 by ATM or DNA-PKcs at SQ/TQ consensus sites is dispensable for the joining step of V(D)J recombination.     

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.     

Variable diversity joining recombination: nonhairpin coding ends in thymocytes of SCID and wild-type mice

Initiation of V(D)J recombination results in broken DNA molecules with blunt recombination signal ends and covalently sealed (hairpin) coding ends. In SCID mice, coding joint formation is severely impaired and hairpin coding ends accumulate as a result of a deficiency in the catalytic subunit of DNA-dependent protein kinase, an enzyme involved in the repair of DNA double-strand breaks. In this study, we report that not all SCID coding ends are hairpinned. We have detected open Jdelta1 and Ddelta2 coding ends at the TCRdelta locus in SCID thymocytes. Approximately 25% of 5'Ddelta2 coding ends were found to be open. Large deletions and abnormally long P nucleotide additions typical of SCID Ddelta2-Jdelta1 coding joints were not observed. Most Jdelta1 and Ddelta2 coding ends exhibited 3' overhangs, but at least 20% had unique 5' overhangs not previously detected in vivo. We suggest that the SCID DNA-dependent protein kinase deficiency not only reduces the efficiency of hairpin opening, but also may affect the specificity of hairpin nicking, as well as the efficiency of joining open coding ends.     

Evidence for Ku70/Ku80 association with full-length RAG1

Antigen receptor genes are assembled by a site-specific DNA rearrangement process called V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at antigen receptor gene segments, and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1, providing a biochemical link between the two phases of V(D)J recombination.     

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.