Strand breaks without DNA rearrangement in V (D)J recombination.

Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.     

Defective signal joint recombination in fanconi anemia fibroblasts reveals a role for Rad50 in V(D)J recombination

V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2 proteins as well as a number of ubiquitously expressed cellular DNA repair proteins that catalyze non-homologous end-joining of DNA double-strand breaks. The evolutionarily conserved Rad50/Mre11/Nibrin protein complex has a role in DNA double-strand break-repair, suggesting that these proteins, too, may participate in V(D)J recombination. Recent findings demonstrating that Rad50 function is defective in cells from patients afflicted with Fanconi anemia provide a possible mechanistic explanation for previous findings that lymphoblasts derived from these patients exhibit subtle defects in V(D)J recombination of extrachromosomal plasmid molecules. Here, we describe a series of findings that provide convincing evidence for a role of the Rad50 protein complex in V(D)J recombination. We found that the fidelity of V(D)J signal joint recombination in fibroblasts from patients afflicted with Fanconi anemia was reduced by nearly tenfold, compared to that observed in fibroblasts from normal donors. Second, we observed that antibody-mediated inhibition of the Rad50, Mre11, or Nibrin proteins reduced the fidelity of signal joint recombination significantly in wild-type cells. The latter finding was somewhat unexpected, because signal joint rejoining in cells from patients with Nijmegen breakage syndrome, which results from mutations in the Nibrin gene, occurs with normal fidelity. However, introduction of anti-Nibrin antibodies into these cells reduced the fidelity of signal joint recombination dramatically. These data reveal for the first time a role for the Rad50 complex in V(D)J recombination, and demonstrate that the protein product of the disease-causing allele responsible for Nijmegen breakage syndrome encodes a protein with residual DNA double-strand break repair activity.     

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.     

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.     

Unequal VH gene rearrangement frequency within the large VH7183 gene family is not due to recombination signal sequence variation, and mapping of the genes shows a bias of rearrangement based on chromosomal location.

Much of the nonrandom usage of V, D, and J genes in the Ab repertoire is due to different frequencies with which gene segments undergo V(D)J rearrangement. The recombination signal sequences flanking each segment are seldom identical with consensus sequences, and this natural variation in recombination signal sequence (RSS) accounts for some differences in rearrangement frequencies in vivo. Here, we have sequenced the RSS of 19 individual V(H)7183 genes, revealing that the majority have one of two closely related RSS. One group has a consensus heptamer, and the other has a nonconsensus heptamer. In vitro recombination substrate studies show that the RSS with the nonconsensus heptamer, which include the frequently rearranging 81X, rearrange less well than the RSS with the consensus heptamer. Although 81X differs from the other 7183-I genes at three positions in the spacer, this does not significantly increase its recombination potency in vitro. The rearrangement frequency of all members of the family was determined in microMT mice, and there was no correlation between the in vitro recombination potential and V(H) gene rearrangement frequency in vivo. Furthermore, genes with identical RSS rearrange at different frequencies in vivo. This demonstrates that other factors can override differences in RSS potency in vivo. We have also determined the gene order of all V(H)7183 genes in a bacterial artificial chromosome contig and show that most of the frequently rearranging genes are in the 3' half of the region. This suggests that chromosomal location plays an important role in nonrandom rearrangement of the V(H)7183 genes.     

Prevalent involvement of illegitimate V(D)J recombination in chromosome 9p21 deletions in lymphoid leukemia

To understand molecular pathways underlying 9p21 deletions, which lead to inactivation of the p16/CDKN2A, p14/ARF, and/or p15/CDKN2B genes, in lymphoid leukemia, 30 breakpoints were cloned from 15 lymphoid leukemia cell lines. Seventeen (57%) breakpoints were mapped at five breakpoint cluster sites, BCS-LL1 to LL5, each of <15 bp. Two breakpoint cluster sites were located within the ARF and CDKN2B loci, respectively, whereas the remaining three were located >100 kb distal to the CDKN2A, ARF, and CDKN2B loci. The sequences of breakpoint junctions indicated that deletions in the 11 (73%) cell lines were mediated by illegitimate V(D)J recombination targeted at the five BCS-LL and six other sites, which contain sequences similar to recombination signal sequences for V(D)J recombination. An extrachromosomal V(D)J recombination assay indicated that BCS-LL3, at which the largest number of breakpoints (i.e. five breakpoints) was clustered, has a V(D)J recombination potential 150-fold less than the consensus recombination signal sequence. Three other BCS-LLs tested also showed V(D)J recombination potential, although it was lower than that of BCS-LL3. These results indicated that illegitimate V(D)J recombination, which was targeted at several ectopic recombination signal sequences widely distributed in 9p21, caused a large fraction of 9p21 deletions in lymphoid leukemia.     

Joining mutants of RAG1 and RAG2 that demonstrate impaired interactions with the coding-end DNA

In V(D)J joining of antigen receptor genes, two recombination signal sequences (RSSs), 12- and 23-RSSs, form a complex with the protein products of recombination activating genes, RAG1 and RAG2. DNaseI footprinting demonstrates that the interaction of RAG proteins with substrate RSS DNA is not just limited to the signal region but involves the coding sequence as well. Joining mutants of RAG1 and RAG2 demonstrate impaired interactions with the coding region in both pre- and postcleavage type complexes. A possible role of this RAG coding region interaction is discussed in the context of V(D)J recombination.     

V(D)J recombinase binding and cleavage of cryptic recombination signal sequences identified from lymphoid malignancies

V(D)J recombination is a process integral to lymphocyte development. However, this process is not always benign, since certain lymphoid malignancies exhibit recurrent chromosomal abnormalities, such as translocations and deletions, that harbor molecular signatures suggesting an origin from aberrant V(D)J recombination. Translocations involving LMO2, TAL1, Ttg-1, and Hox11, as well as a recurrent interstitial deletion at 1p32 involving SIL/SCL, are cited examples of illegitimate V(D)J recombination. Previous studies using extrachromosomal substrates reveal that cryptic recombination signal sequences (cRSSs) identified near the translocation breakpoint in these examples support V(D)J recombination with efficiencies ranging from about 30- to 20,000-fold less than bona fide V(D)J recombination signals. To understand the molecular basis for these large differences, we investigated the binding and cleavage of these cRSSs by the RAG1/2 proteins that initiate V(D)J recombination. We find that the RAG proteins comparably bind all cRSSs tested, albeit more poorly than a consensus RSS. We show that four cRSSs that support levels of V(D)J recombination above background levels in cell culture (LMO2, TAL1, Ttg-1, and SIL) are also cleaved by the RAG proteins in vitro with efficiencies ranging from 18 to 70% of a consensus RSS. Cleavage of LMO2 and Ttg-1 by the RAG proteins can also be detected in cell culture using ligation-mediated PCR. In contrast, Hox11 and SCL are nicked but not cleaved efficiently in vitro, and cleavage at other adventitious sites in plasmid substrates may also limit the ability to detect recombination activity at these cRSSs in cell culture.     

Recognition sequence of a highly conserved DNA binding protein RBP-J kappa.

DNA binding specificity of the RBP-J kappa protein was extensively examined. The mouse RBP-J kappa protein was originally isolated as a nuclear protein binding to the J kappa type V(D)J recombination signal sequence which consisted of the conserved heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences separated by a 23-base pair spacer. Electrophoretic mobility shift assay using DNA probes with mutations in various parts of the J kappa recombination signal sequence showed that the RBP-J kappa protein recognized the sequence outside the recombination signal in addition to the heptamer but did not recognize the nonamer sequence and the spacer length at all. Database search identified the best naturally occurring binding motif (CACTGTGGGAACGG) for the RBP-J kappa protein in the promoter region of the m8 gene in the Enhancer of split gene cluster of Drosophila. The binding assay with a series of m8 motif mutants indicated that the protein recognized mostly the GTGGGAA sequence and also interacted weakly with ACT and CG sequences flanking this hepta-nucleotide. Oligonucleotides binding to the RBP-J kappa protein were enriched from a pool of synthetic oligonucleotides containing 20-base random sequences by the repeated electrophoretic mobility shift assay. The enriched oligomer shared a common sequence of CGTGGGAA. All these data indicate that the RBP-J kappa protein recognizes a unique core sequence of CGTGGGAA and does not bind to the V(D)J recombination signal without the flanking sequence.     

Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

The V(D)J recombinase recognizes a pair of immunoglobulin or T-cell receptor gene segments flanked by recombination signal sequences and introduces double-strand breaks, generating two signal ends and two coding ends. Broken coding ends were initially identified as covalently closed hairpin DNA molecules. Before recombination, however, the hairpins must be opened and the ends must be modified by nuclease digestion and N-region addition. We have now analyzed nonhairpin coding ends associated with various immunoglobulin gene segments in cells undergoing V(D)J recombination. We found that these broken DNA ends have different nonrandom 5′-strand deletions which were characteristic for each locus examined. These deletions correlate well with the sequence characteristics of coding joints involving these gene segments. In addition, unlike broken signal ends, these nonhairpin coding-end V(D)J recombination reaction intermediates have 3′ overhanging ends. We discuss the implications of these results for models of how sequence modifications occur during coding-joint formation.     

Mechanistic basis for coding end sequence effects in the initiation of V(D)J recombination

V(D)J recombination is directed by recombination signal sequences. However, the flanking coding end sequence can markedly affect the frequency of the initiation of V(D)J recombination in vivo. Here we demonstrate that the coding end sequence effect can be qualitatively and quantitatively recapitulated in vitro with purified RAG proteins. We find that coding end sequence specifically affects the nicking step, which is the first biochemical step in RAG-mediated cleavage. The subsequent hairpin formation step is not affected by the coding end sequence. Furthermore, the coding end sequence effect can be ablated by prenicking the substrate, indicating that the coding end effect is specific to the nicking step. In reactions in which both 12- and 23-substrates are present, a suboptimal coding end sequence on one signal can slow down hairpin formation at the partner signal, a result consistent with models in which coordination between the signals occurs at the hairpin formation step. The coding end sequence effect on nicking and the coupling of the 12- and 23-substrates explains how hairpin formation can be rate limiting for some 12/23 pairs, whereas nicking can be rate limiting when low-efficiency coding end sequences are involved.     

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.     

DNA sequence and structure requirements for cleavage of V(D)J recombination signal sequences.

Purified RAG1 and RAG2 proteins can cleave DNA at V(D)J recombination signals. In dissecting the DNA sequence and structural requirements for cleavage, we find that the heptamer and nonamer motifs of the recombination signal sequence can independently direct both steps of the cleavage reaction. Proper helical spacing between these two elements greatly enhances the efficiency of cleavage, whereas improper spacing can lead to interference between the two elements. The signal sequences are surprisingly tolerant of structural variation and function efficiently when nicks, gaps, and mismatched bases are introduced or even when the signal sequence is completely single stranded. Sequence alterations that facilitate unpairing of the bases at the signal/coding border activate the cleavage reaction, suggesting that DNA distortion is critical for V(D)J recombination.     

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.     

The composition of coding joints formed in V(D)J recombination is strongly affected by the nucleotide sequence of the coding ends and their relationship to the recombination signal sequences.

V(D)J recombination proceeds in two stages. Precise cleavage at the border of the conserved recombination signal sequences (RSSs) and the coding ends results in flush double-stranded signal ends and coding ends terminating in hairpins. In the second stage, the signal and coding ends are processed into signal and coding joints. Coding ends containing certain nucleotide homopolymers affect the efficiency of V(D)J recombination. In this study, we have tested the effect of small changes in coding-end nucleotide composition on the frequency of coding- and signal joint formation. Furthermore, we have determined the sequences of coding joints resulting from recombination of coding ends with different compositions. We found that the presence of two T nucleotides 5' of both RSSs, but not a single T, reduces the frequency of signal joint formation, i.e., interferes with the cleavage stage of V(D)J recombination. However, coding-joint processing is sensitive even to a single T. Both the sequence of the coding ends and the particular RSS (12-mer or 23-mer) with which the coding end is associated affect the final composition of the coding joints. Thus, the presence of P nucleotides, the conservation of one undeleted coding end, the formation of joints without any deletions, and the template-dependent insertion of nucleotides are strongly influenced by the coding-end nucleotide composition and/or RSS association. The implications of these results with respect to the processing of coding ends are discussed.     

Distinct DNA sequence and structure requirements for the two steps of V(D)J recombination signal cleavage.

Cleavage of V(D)J recombination signals by purified RAG1 and RAG2 proteins permits the dissection of DNA structure and sequence requirements. The two recognition elements of a signal (nonamer and heptamer) are used differently, and their cooperation depends on correct helical phasing. The nonamer is most important for initial binding, while efficient nicking and hairpin formation require the heptamer sequence. Both nicking and hairpin formation are remarkably tolerant of variations in DNA structure. Certain flanking sequences inhibit hairpin formation, but this can be bypassed by base unpairing, and even a completely single-stranded signal sequence is well utilized. We suggest that DNA unpairing around the signal-coding border is essential for the initiation of V(D)J combination.     

Gene conversion in human rearranged immunoglobulin genes

Over the past 20 years, many DNA sequences have been published suggesting that all or part of the V(H) segment of a rearranged immunoglobulin gene may be replaced in vivo. Two different mechanisms appear to be operating. One of these is very similar to primary V(D)J recombination, involving the RAG proteins acting upon recombination signal sequences, and this has recently been proven to occur. Other sequences, many of which show partial V(H) replacements with no addition of untemplated nucleotides at the V(H)-V(H) joint, have been proposed to occur by an unusual RAG-mediated recombination with the formation of hybrid (coding-to-signal) joints. These appear to occur in cells already undergoing somatic hypermutation in which, some authors are convinced, RAG genes are silenced. We recently proposed that the latter type of V(H) replacement might occur by homologous recombination initiated by the activity of AID (activation-induced cytidine deaminase), which is essential for somatic hypermutation and gene conversion. The latter has been observed in other species, but not in human Ig genes, so far. In this paper, we present a new analysis of sequences published as examples of the second type of rearrangement. This not only shows that AID recognition motifs occur in recombination regions but also that some sequences show replacement of central sections by a sequence from another gene, similar to gene conversion in the immunoglobulin genes of other species. These observations support the proposal that this type of rearrangement is likely to be AID-mediated rather than RAG-mediated and is consistent with gene conversion.     

Recombination activating gene 1 product alone possesses endonucleolytic activity

Two lymphoid-specific proteins, RAG1 and RAG2, are required for the initiation of the V(D)J recombination in vitro. The V(D)J cleavage that is mediated by RAG proteins at the border between the coding and signal sequences results in the production of a hairpin at the coding end and a double-stranded break at the signal end. Two hairpin coding ends are re-opened, modified, and sealed; whereas, the signal ends are directly ligated. Here I report that only RAG1 can carry out a distinct endonucleolytic activity in vitro using an oligonucleotide substrate that is tethered by a short single-stranded DNA. The purified RAG1 protein alone formed a nick at the near position to the recombination signal sequence. This endonucleolytic activity was eliminated by immunoprecipitation using the RAG1-specific antibody, and required the 3'-hydroxy group. All of the RAG1 mutants that were incapable of the nick and hairpin formation in the V(D)J cleavage analysis also showed this new endonucleolytic activity. This suggests that the nicking activity that was observed might be functionally different from the nick formation in the V(D)J cleavage.