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.     

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.     

Stimulation of V(D)J recombination by histone acetylation

V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.     

Full-length RAG1 promotes contact with coding and intersignal sequences in RAG protein complexes bound to recombination signals paired in cis

The RAG proteins initiate V(D)J recombination by mediating synapsis and cleavage of two different antigen receptor gene segments through interactions with their flanking recombination signal sequences (RSS). The protein–DNA complexes that support this process have mainly been studied using RAG–RSS complexes assembled using oligonucleotide substrates containing a single RSS that are paired in trans to promote synapsis. How closely these complexes model those formed on longer, more physiologically relevant substrates containing RSSs on the same DNA molecule (in cis) remains unclear. To address this issue, we characterized discrete core and full-length RAG protein complexes bound to RSSs paired in cis. We find these complexes support cleavage activity regulated by V(D)J recombination's ‘12/23 rule’ and exhibit plasticity in RSS usage dependent on partner RSS composition. DNA footprinting studies suggest that the RAG proteins in these complexes mediate more extensive contact with sequences flanking the RSS than previously observed, some of which are enhanced by full-length RAG1, and associated with synapsis and efficient RSS cleavage. Finally, we demonstrate that the RAG1 C-terminus facilitates hairpin formation on long DNA substrates, and full-length RAG1 promotes hairpin retention in the postcleavage RAG complex. These results provide new insights into the mechanism of physiological V(D)J recombination.     

Mechanism of fragility at BCL2 gene minor breakpoint cluster region during t(14;18) chromosomal translocation

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Breaks in chromosome 18 are localized at the 3'-UTR of BCL2 gene or downstream and are mainly clustered in either the major breakpoint region or the minor breakpoint cluster region (mcr). The recombination activating gene (RAG) complex induces breaks at IgH locus of chromosome 14, whereas the mechanism of fragility at BCL2 mcr remains unclear. Here, for the first time, we show that RAGs can nick mcr; however, the mechanism is unique. Three independent nicks of equal efficiency are generated, when both Mg(2+) and Mn(2+) are present, unlike a single nick during V(D)J recombination. Further, we demonstrate that RAG binding and nicking at the mcr are independent of nonamer, whereas a CCACCTCT motif plays a critical role in its fragility, as shown by sequential mutagenesis. More importantly, we recapitulate the BCL2 mcr translocation and find that mcr can undergo synapsis with a standard recombination signal sequence within the cells, in a RAG-dependent manner. Further, mutation to the CCACCTCT motif abolishes recombination within the cells, indicating its vital role. Hence, our data suggest a novel, physiologically relevant, nonamer-independent mechanism of RAG nicking at mcr, which may be important for generation of chromosomal translocations in humans.     

HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex

Background  

V(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG) and nonamer (consensus: 5'-ACAAAAACC) motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2) are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element.     

Inverse transposition by the RAG1 and RAG2 proteins: role reversal of donor and target DNA

The lymphoid-specific proteins RAG1 and RAG2 initiate V(D)J recombination by introducing DNA double-strand breaks at the recombination signal sequences (RSSs). In addition to DNA cleavage, the versatile RAG1/2 complex is capable of catalyzing several other reactions, including hybrid joint formation and the transposition of signal ends into a second DNA. Here we show that the RAG1/2 complex also mediates an unusual strand transfer reaction, inverse transposition, in which non-RSS DNA is cleaved and subsequently transferred to an RSS sequence by direct transesterification. Characterization of the reaction products and requirements suggests that inverse transposition is related to both hybrid joint formation and signal-end transposition. This aberrant activity provides another possible mechanism for some chromosomal translocations present in lymphoid tumors.     

The recombination activating gene 1 (RAG1) of rainbow trout (Oncorhynchus mykiss): cloning,expression, and phylogenetic analysis

The characterization of genes involved in the generation of the immune repertoire is an active area of research in lower vertebrate taxa. The recombination activating genes (RAG) have been shown to be essential for V (D) J recombination of T-cell antigen receptor (TCR) and immunoglobulin (Ig) genes, leading to the generation of the primary repertoire. As RAG1 is critical to the differentiation of pre-B and-T cells, its expression within an associated primary lymphoid organ can serve as a developmental marker. To examine the ontogeny of lymphocytes in Oncorhynchus mykiss, we cloned RAG1 from trout and examined its tissue-and lymphocyte-specific expression. The polymerase chain reaction, coupled with degenerate oligonucleotide primers, was used to amplify a homologous probe [(633 base pairs) (bp)] from rainbow trout genomic DNA, which in turn was used to isolate a lambda genomic clone. Sequence analysis of this genomic clone confirmed the RAG1 nature of this gene (3888 bp) and revealed an internal intron of 666 bp. When compared with other previously reported RAG1 sequences, the predicted amino acid translation (1073 aa) displayed a minimum of 78% similarity for the complete sequence and 89% similarity in the conserved region (aa 417-1042). Using northern blot analysis, we found the expression of RAG1 to be limited to surface Ig-n lymphocytes within the thymus. This data forms the basis for a proposal that the thymus of teleost species plays an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.     

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.     

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.     

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a side-by-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg²⁺ cations with Ca²⁺ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.To generate diverse surface antigen receptor molecules, developing lymphocytes undergo a series of site-specific DNA rearrangements to assemble functional antigen receptor genes from component gene segments (1). This DNA rearrangement process, known as V(D)J recombination, is initiated when two lymphoid cell-specific proteins, called RAG1 and RAG2, assemble a multiprotein synaptic complex with a pair of antigen receptor gene segments and subsequently introduce a DNA double strand break at the end of each gene segment (2). A recombination signal sequence (RSS)³ that abuts each participating gene segment serves as the binding site of the RAG proteins and directs the location of DNA cleavage. Each RSS contains conserved heptamer and nonamer sequences that are separated by either 12 or 23 bp of DNA of more varied sequence (12RSS and 23RSS, respectively); efficient V(D)J recombination generally only occurs between two RSSs in which the lengths of DNA separating the heptamer and nonamer differ (the 12/23 rule). The RAG proteins mediate DNA cleavage via a nick-hairpin mechanism, breaking the DNA between the RSS heptamer and the coding segment; these reaction products are subsequently processed and repaired by the non-homologous end-joining pathway (1, 3).Previous studies suggest that RAG synaptic complexes are assembled through the stepwise binding of a 12RSS followed by the capture of a 23RSS (4–6). In vitro biochemical studies suggest synapsis is mediated by a RAG1/2 heterotetramer, but there remains disagreement over the stoichiometry of RAG1 in these complexes (7). In addition, fluorescence resonance energy transfer techniques have recently been applied to examine the orientation of DNA strands within the synaptic complex. The data obtained from these experiments led the authors to favor a model in which the RSSs adopt a bent and crossed configuration in the synaptic complex, although an alternative model in which synaptic complexes containing RSSs in parallel and antiparallel configurations assemble with similar frequency could not be formally excluded (8). For most in vitro biochemical studies, the synaptic complex has been assembled by incubating the RAG proteins with a pair of oligonucleotide substrates, one containing a 12RSS, and one containing a 23RSS. Whether the RAG proteins and the RSSs adopt the same configuration in synaptic complexes assembled with oligonucleotide substrates as those assembled with longer, more physiological substrates remains to be verified, but some studies suggest there are DNA length-dependent differences in RAG-mediated RSS binding and cleavage activity (9, 35).To directly observe and analyze RAG-RSS synaptic complexes assembled on long DNA substrates that more closely model an RSS embedded in chromosomal DNA, we used atomic force microscopy (AFM), given its previously demonstrated success for visualizing synaptic complexes in other systems (10–14), and its ability to reveal structural details for synaptic complexes that correlate well with independently obtained crystallographic data (15). AFM has also been recently applied to study the bending of 12RSS substrates by RAG1 and RAG2 (16). We report here the first successful visualization of RAG-RSS synaptic complexes by AFM, and describe their characterization with respect to DNA arrangement and the protein stoichiometry within the complexes. These data provide new and important insight into how RAG-RSS synaptic complexes are assembled and organized.     

Inducible chromosomal translocation of AML1 and ETO genes through Cre/loxP-mediated recombination in the mouse

Transgenic mice have been used to explore the role of chromosomal translocations in the genesis of tumors. But none of these efforts has actually involved induction of a translocation in vivo. Here we report the use of Cre recombinase to replicate in vivo the t(8;21) translocation found in human acute myeloid leukemia (AML). As in the human tumors, the murine translocation fuses the genes AML1 and ETO. We used homologous recombination to place loxP sites at loci that were syntenic with the break points for the human translocation. Cre activity was provided in mice by a transgene under the control of the Nestin promoter, or in cultured B cells by infecting with a retroviral vector encoding Cre. In both instances, Cre activity mediated interchromosomal translocations that fused the AML1 and ETO genes. Thus, reciprocal chromosomal translocations that closely resemble rearrangements found in human cancers can be achieved in mice.     

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.     

Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA

During V(D)J recombination, recombination activating gene (RAG)1 and RAG2 bind and cleave recombination signal sequences (RSSs), aided by the ubiquitous DNA-binding/-bending proteins high-mobility group box protein (HMGB)1 or HMGB2. HMGB1/2 play a critical, although poorly understood, role in vitro in the assembly of functional RAG–RSS complexes, into which HMGB1/2 stably incorporate. The mechanism of HMGB1/2 recruitment is unknown, although an interaction with RAG1 has been suggested. Here, we report data demonstrating only a weak HMGB1–RAG1 interaction in the absence of DNA in several assays, including fluorescence anisotropy experiments using a novel Alexa488-labeled HMGB1 protein. Addition of DNA to RAG1 and HMGB1 in fluorescence anisotropy experiments, however, results in a substantial increase in complex formation, indicating a synergistic binding effect. Pulldown experiments confirmed these results, as HMGB1 was recruited to a RAG1–DNA complex in a RAG1 concentration-dependent manner and, interestingly, without strict RSS sequence specificity. Our finding that HMGB1 binds more tightly to a RAG1–DNA complex over RAG1 or DNA alone provides an explanation for the stable integration of this typically transient architectural protein in the V(D)J recombinase complex throughout recombination. These findings also have implications for the order of events during RAG–DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1–DNA interactions.     

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.     

RAG-heptamer interaction in the synaptic complex is a crucial biochemical checkpoint for the 12/23 recombination rule

In V(D)J recombination, the RAG1 and RAG2 protein complex cleaves the recombination signal sequences (RSSs), generating a hairpin structure at the coding end. The cleavage occurs only between two RSSs with different spacer lengths of 12 and 23 bp. Here we report that in the synaptic complex, recombination-activating gene (RAG) proteins interact with the 7-mer and unstack the adjacent base in the coding region. We generated a RAG1 mutant that exhibits reduced RAG-7-mer interaction, unstacking of the coding base, and hairpin formation. Mutation of the 23-RSS at the first position of the 7-mer, which has been reported to impair the cleavage of the partner 12-RSS, demonstrated phenotypes similar to those of the RAG1 mutant; the RAG interaction and base unstacking in the partner 12-RSS are reduced. We propose that the RAG-7-mer interaction is a critical step for coding DNA distortion and hairpin formation in the context of the 12/23 rule.     

Recombination Activating Genes (RAG) in Lymphoma Development

The recombination activating genes (RAG) play a central role in the generation of a diverse immune repertoire by mediating DNA recombination events at antigen receptor loci in developing B and T lymphocytes. However, inappropriate RAG activity throughout the genome has been implicated in a large variety of human and mouse lymphomas. Mechanisms by which RAG can provoke or perpetuate lymphoma include deregulation of certain genes by translocation to antigen receptor regulatory regions, the formation of chimeric oncogenes, inactivation of tumor suppressor or micro-RNA loci, or activation of oncogenes. Here we present the T cell receptor enhancer (Eb) deficient mouse as a tractable in vivo model system to study the role of RAG activity in the context of lymphoma development, and contrast our system with those of others. We posit a general hypothesis that virtually any mutation that impairs early lymphocyte development at stages when RAG is expressed can constitute a pro-carcinogenic event. Our model system provides a means to assess the roles of RAG activity in human lymphoid malignancies.     

MSH6- or PMS2-deficiency causes re-replication in DT40 B cells,but it has little effect on immunoglobulin gene conversion or on repair of AID-generated uracils

The mammalian antibody repertoire is shaped by somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) loci of B lymphocytes. SHM and CSR are triggered by non-canonical, error-prone processing of G/U mismatches generated by activation-induced deaminase (AID). In birds, AID does not trigger SHM, but it triggers Ig gene conversion (GC), a ‘homeologous’ recombination process involving the Ig variable region and proximal pseudogenes. Because recombination fidelity is controlled by the mismatch repair (MMR) system, we investigated whether MMR affects GC in the chicken B cell line DT40. We show here that Msh6^−/− and Pms2^−/− DT40 cells display cell cycle defects, including genomic re-replication. However, although IgVλ GC tracts in MMR-deficient cells were slightly longer than in normal cells, Ig GC frequency, donor choice or the number of mutations per sequence remained unaltered. The finding that the avian MMR system, unlike that of mammals, does not seem to contribute towards the processing of G/U mismatches in vitro could explain why MMR is unable to initiate Ig GC in this species, despite initiating SHM and CSR in mammalian cells. Moreover, as MMR does not counteract or govern Ig GC, we report a rare example of ‘homeologous’ recombination insensitive to MMR.     

A Non-Sequence-Specific DNA Binding Mode of RAG1 Is Inhibited by RAG2

RAG1 and RAG2 proteins catalyze site-specific DNA cleavage reactions in V(D)J recombination, a process that assembles antigen receptor genes from component gene segments during lymphocyte development. The first step towards the DNA cleavage reaction is the sequence-specific association of the RAG proteins with the conserved recombination signal sequence (RSS), which flanks each gene segment in the antigen receptor loci. Questions remain as to the contribution of each RAG protein to recognition of the RSS. For example, while RAG1 alone is capable of recognizing the conserved elements of the RSS, it is not clear if or how RAG2 may enhance sequence-specific associations with the RSS. To shed light on this issue, we examined the association of RAG1, with and without RAG2, with consensus RSS versus non-RSS substrates using fluorescence anisotropy and gel mobility shift assays. The results indicate that while RAG1 can recognize the RSS, the sequence-specific interaction under physiological conditions is masked by a high-affinity non-sequence-specific DNA binding mode. Significantly, addition of RAG2 effectively suppressed the association of RAG1 with non-sequence-specific DNA, resulting in a large differential in binding affinity for the RSS versus the non-RSS sites. We conclude that this represents a major means by which RAG2 contributes to the initial recognition of the RSS and that, therefore, association of RAG1 with RAG2 is required for effective interactions with the RSS in developing lymphocytes.