Regulation of V(D)J recombination by nucleosome positioning at recombination signal sequences

A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non-consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination.     

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.     

An updated definition of V(D)J recombination signal sequences revealed by high-throughput recombination assays

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.     

A functional analysis of the spacer of V(D)J recombination signal sequences

During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jβ2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jβ2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a “digital” requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an “analog” manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for “RSS information content.” The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein–DNA interactions in various biological systems.     

Footprint analysis of recombination signal sequences in the 12/23 synaptic complex of V(D)J recombination

In V(D)J joining of antigen receptor genes, two recombination signal sequences (RSSs), 12-RSS and 23-RSS, are paired and complexed with the protein products of recombination-activating genes RAG1 and RAG2. Using magnetic beads, we purified the pre- and postcleavage complexes of V(D)J joining and analyzed them by DNase I footprinting. In the precleavage synaptic complex, strong protection was seen not only in the 9-mer and spacer regions but also near the coding border of the 7-mer. This is a sharp contrast to the single RSS-RAG complex where the 9-mer plays a major role in the interaction. We also analyzed the postcleavage signal end complex by footprinting. Unlike what was seen with the precleavage complex, the entire 7-mer and its neighboring spacer regions were protected. The present study indicates that the RAG-RSS interaction in the 7-mer region drastically changes once the synaptic complex is formed for cleavage.     

RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons

The V(D)J recombination reaction in jawed vertebrates is catalyzed by the RAG1 and RAG2 proteins, which are believed to have emerged approximately 500 million years ago from transposon-encoded proteins. Yet no transposase sequence similar to RAG1 or RAG2 has been found. Here we show that the approximately 600-amino acid “core” region of RAG1 required for its catalytic activity is significantly similar to the transposase encoded by DNA transposons that belong to the Transib superfamily. This superfamily was discovered recently based on computational analysis of the fruit fly and African malaria mosquito genomes. Transib transposons also are present in the genomes of sea urchin, yellow fever mosquito, silkworm, dog hookworm, hydra, and soybean rust. We demonstrate that recombination signal sequences (RSSs) were derived from terminal inverted repeats of an ancient Transib transposon. Furthermore, the critical DDE catalytic triad of RAG1 is shared with the Transib transposase as part of conserved motifs. We also studied several divergent proteins encoded by the sea urchin and lancelet genomes that are 25%−30% identical to the RAG1 N-terminal domain and the RAG1 core. Our results provide the first direct evidence linking RAG1 and RSSs to a specific superfamily of DNA transposons and indicate that the V(D)J machinery evolved from transposons. We propose that only the RAG1 core was derived from the Transib transposase, whereas the N-terminal domain was assembled from separate proteins of unknown function that may still be active in sea urchin, lancelet, hydra, and starlet sea anemone. We also suggest that the RAG2 protein was not encoded by ancient Transib transposons but emerged in jawed vertebrates as a counterpart of RAG1 necessary for the V(D)J recombination reaction.     

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.     

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.     

Initiation of V(D)J recombination in vivo: role of recombination signal sequences in formation of single and paired double-strand breaks.

In V(D)J recombination, double-strand breaks (DSBs) are introduced at recombination signal sequences (RSSs) which consist of three distinct elements: a heptamer, a 12 or 23 nucleotide spacer and a nonamer. Efficient DSB formation requires a 12/23 RSS pair and occurs at both RSS in a temporally coupled fashion (coupled cleavage). It remains unknown which RSS elements are important for coupled cleavage. Furthermore, it has not been established whether some RSS components are critical only for cleavage in cis, with others mainly promoting cleavage in trans at the partner RSS. We investigated these questions by analyzing the effects of RSS mutations on the formation of DSBs in vivo. The abundance of DSBs in cis (at the mutant RSS) and in trans (at the consensus RSS) was determined using an established ligation-mediated PCR assay. We also developed a Southern blotting approach that allows the first direct measurement of dual and single RSS cleavage in vivo. Our results demonstrate that the heptamer, spacer and nonamer elements are all required for coupled cleavage in vivo. These studies also provide evidence for cleavage events involving a single RSS both in mutant substrates and in substrates containing a consensus 12/23 RSS pair.     

Unequal signal and coding joint formation in human V(D)J recombination.

Substrates for studying V(D)J recombination in human cells and two human pre-B-cell lines that have active V(D)J recombination activity are described. Using these substrates, we have been able to analyze the relative efficiency of signal joint and coding joint formation. Coding joint formation was five- to sixfold less efficient than signal joint formation in both cell lines. This imbalance between the two halves of the reaction was demonstrated on deletional substrates, where each joint is assayed individually. In both cell lines, the inversional reaction (which requires formation of both a signal and a coding joint) was more than 20-fold less efficient than signal joint formation alone. The signal and coding sequences are identical in all of these substrates. Hence, the basis for these differential reaction ratios appears to be that coding joint and signal joint formation are both inefficient and their combined effects are such that inversions (two-joint reactions) reflect the product of these inefficiencies. Physiologically, these results have two implications. First, they show how signal and coding joint formation efficiencies can affect the ratio of deletional to inversional products at endogenous loci. Second, the fact that not all signal and coding joints go to completion implies that the recombinase is generating numerous broken ends. Such unresolved ends may participate in pathologic chromosomal rearrangements even when the other half of the same reaction may have proceeded to resolution.     

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.     

A complex of RAG-1 and RAG-2 proteins persists on DNA after single-strand cleavage at V(D)J recombination signal sequences.

The recombination activating gene (RAG) 1 and 2 proteins are required for initiation of V(D)J recombination in vivo and have been shown to be sufficient to introduce DNA double-strand breaks at recombination signal sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly conserved palindromic heptamer that is separated from a slightly less conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random sequence. Despite the high sequence specificity of RAG-mediated cleavage at RSSs, direct binding of the RAG proteins to these sequences has been difficult to demonstrate by standard methods. Even when this can be demonstrated, questions about the order of events for an individual RAG-RSS complex will require methods that monitor aspects of the complex during transitions from one step of the reaction to the next. Here we have used template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) in order to assess occupancy of the reaction intermediates by the RAG complex during the reaction. In addition, this approach allows analysis of the accessibility of end products of a RAG-catalyzed cleavage reaction for N nucleotide addition. The results indicate that RAG proteins form a long-lived complex with the RSS once the initial nick is generated, because the 3'-OH group at the nick remains obstructed for TdT-catalyzed N nucleotide addition. In contrast, the 3'-OH group generated at the signal end after completion of the cleavage reaction can be efficiently tailed by TdT, suggesting that the RAG proteins disassemble from the signal end after DNA double-strand cleavage has been completed. Therefore, a single RAG complex maintains occupancy from the first step (nick formation) to the second step (cleavage). In addition, the results suggest that N region diversity at V(D)J junctions within rearranged immunoglobulin and T cell receptor gene loci can only be introduced after the generation of RAG-catalyzed DNA double-strand breaks, i.e. during the DNA end joining phase of the V(D)J recombination reaction.     

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.     

Novel strand exchanges in V(D)J recombination

We describe novel products of V(D)J recombination in which signal sequences become joined to coding elements, in contrast to the standard reaction whose products are junctions of two signal sequences or two coding elements. In this variant reaction, the recombination machinery evidently recognizes signal sequences and introduces strand breaks at the normal positions, but then connects the elements in unusual combinations. The lack of fixed directionality indicates that recombination sites are not uniquely aligned when strand exchange occurs. The discovery of these variant junctions suggests a model for the evolution of the antigen receptor loci.     

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.     

V(D)J recombinase-mediated processing of coding junctions at cryptic recombination signal sequences in peripheral T cells during human development

V(D)J recombinase mediates rearrangements at immune loci and cryptic recombination signal sequences (cRSS), resulting in a variety of genomic rearrangements in normal lymphocytes and leukemic cells from children and adults. The frequency at which these rearrangements occur and their potential pathologic consequences are developmentally dependent. To gain insight into V(D)J recombinase-mediated events during human development, we investigated 265 coding junctions associated with cRSS sites at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in peripheral T cells from 111 children during the late stages of fetal development through early adolescence. We observed a number of specific V(D)J recombinase processing features that were both age and gender dependent. In particular, TdT-mediated nucleotide insertions varied depending on age and gender, including percentage of coding junctions containing N-nucleotide inserts, predominance of GC nucleotides, and presence of inverted repeats (Pr-nucleotides) at processed coding ends. In addition, the extent of exonucleolytic processing of coding ends was inversely related to age. We also observed a coding-partner-dependent difference in exonucleolytic processing and an age-specific difference in the subtypes of V(D)J-mediated events. We investigated these age- and gender-specific differences with recombination signal information content analysis of the cRSS sites in the human HPRT locus to gain insight into the mechanisms mediating these developmentally specific V(D)J recombinase-mediated rearrangements in humans.     

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.