The endogenous Mtv8 locus and the immunoglobulin repertoire.

The vast diversity of mammalian adaptive antigen receptors allows for robust and efficient immune responses against a wide number of pathogens. The antigen receptor repertoire is built during the recombination of B and T cell receptor (BCR, TCR) loci and hypermutation of BCR loci. V(D)J recombination rearranges these antigen receptor loci, which are organized as an array of separate V, (D), and J gene segments. Transcription activation at the recombining locus leads to changes in the local three-dimensional architecture, which subsequently contributes to which gene segments are utilized for recombination. The endogenous retrovirus (ERV) mouse mammary tumor provirus 8 (Mtv8) resides on mouse chromosome 6 interposed within the large array of light chain kappa V gene segments. As ERVs contribute to changes in genomic architecture by driving high levels of transcription of neighboring genes, it was suggested that Mtv8 could influence the BCR repertoire. We generated Mtv8-deficient mice to determine if the ERV influences V(D)J recombination to test this possibility. We find that Mtv8 does not influence the BCR repertoire.

MicroRNA, miR-501 regulate the V(D)J recombination in B cells.

The stringent regulation of RAGs (Recombination activating genes), the site-specific endonuclease responsible for V(D)J recombination, is important to prevent genomic rearrangements and chromosomal translocations in lymphoid cells. In the present study, we identify a microRNA, miR-501, which can regulate the expression of RAG1 in lymphoid cells. Overexpression of the pre-miRNA construct led to the generation of mature miRNAs and a concomitant reduction in RAG1 expression, whereas inhibition using anti-miRs resulted in its enhanced expression. The direct interaction of the 3'UTR of miR-501 with RAG1 was confirmed by the reporter assay. Importantly, overexpression of miRNAs led to inhibition of V(D)J recombination in B cells, revealing their impact on the physiological function of RAGs. Of interest is the inverse correlation observed for miR-501 with RAG1 in various leukemia patients and lymphoid cell lines, suggesting its possible use in cancer therapy. Thus, our results reveal the regulation of RAG1 by miR-501-3p in B cells and thus V(D)J recombination and its possible implications on immunoglobulin leukemogenesis.

Intra- and interchromosomal contact mapping reveals the Igh locus has extensive conformational heterogeneity and interacts with B-lineage genes.

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.

Three-way contact analysis characterizes the higher order organization of the Tcra locus.

The generation of highly diverse antigen receptors in T and B lymphocytes relies on V(D)J recombination. The enhancer Eα has been implicated in regulating the accessibility of Vα and Jα genes through long-range interactions during rearrangements of the T-cell antigen receptor gene Tcra. However, direct evidence for Eα physically mediating the interaction of Vα and Jα genes is still lacking. In this study, we utilized the 3C-HTGTS assay, a chromatin interaction technique based on 3C, to analyze the higher order chromatin structure of the Tcra locus. Our analysis revealed the presence of sufficient information in the 3C-HTGTS data to detect multiway contacts. Three-way contact analysis of the Tcra locus demonstrated the co-occurrence of the proximal Jα genes, Vα genes and Eα in CD4+CD8+ double-positive thymocytes. Notably, the INT2-TEAp loop emerged as a prominent structure likely to be responsible for bringing the proximal Jα genes and the Vα genes into proximity. Moreover, the enhancer Eα utilizes this loop to establish physical proximity with the proximal Vα gene region. This study provides insights into the higher order chromatin structure of the Tcra locus, shedding light on the spatial organization of chromatin and its impact on V(D)J recombination.

E protein binding at the Tcra enhancer promotes Tcra repertoire diversity.

V(D)J recombination of antigen receptor loci is a highly developmentally regulated process. During T lymphocyte development, recombination of the Tcra gene occurs in CD4+CD8+ double positive (DP) thymocytes and requires the Tcra enhancer (Eα). E proteins are known regulators of DP thymocyte development and have three identified binding sites in Eα. To understand the contribution of E proteins to Eα function, mutants lacking one or two of the respective binding sites were generated. The double-binding site mutant displayed a partial block at the positive selection stage of αß T cell development. Further investigation revealed loss of germline transcription within the Tcra locus at the Jα array, along with dysregulated primary and impaired secondary Vα-Jα rearrangement. Eα E protein binding increases Tcra locus accessibility and regulates TCRα recombination, thus directly promoting Tcra repertoire diversity.

Origin and evolutionary malleability of T cell receptor α diversity.

Lymphocytes of vertebrate adaptive immune systems acquired the capability to assemble, from split genes in the germline, billions of functional antigen receptors1-3. These receptors show specificity; unlike the broadly tuned receptors of the innate system, antibodies (Ig) expressed by B cells, for instance, can accurately distinguish between the two enantiomers of organic acids4, whereas T cell receptors (TCRs) reliably recognize single amino acid replacements in their peptide antigens5. In developing lymphocytes, antigen receptor genes are assembled from a comparatively small set of germline-encoded genetic elements in a process referred to as V(D)J recombination6,7. Potential self-reactivity of some antigen receptors arising from the quasi-random somatic diversification is suppressed by several robust control mechanisms8-12. For decades, scientists have puzzled over the evolutionary origin of somatically diversifying antigen receptors13-16. It has remained unclear how, at the inception of this mechanism, immunologically beneficial expanded receptor diversity was traded against the emerging risk of destructive self-recognition. Here we explore the hypothesis that in early vertebrates, sequence microhomologies marking the ends of recombining elements became the crucial targets of selection determining the outcome of non-homologous end joining-based repair of DNA double-strand breaks generated during RAG-mediated recombination. We find that, across the main clades of jawed vertebrates, TCRα repertoire diversity is best explained by species-specific extents of such sequence microhomologies. Thus, selection of germline sequence composition of rearranging elements emerges as a major factor determining the degree of diversity of somatically generated antigen receptors.

Contribution of the IGCR1 regulatory element and the 3'Igh CTCF-binding elements to regulation of Igh V(D)J recombination.

Immunoglobulin heavy chain variable region exons are assembled in progenitor-B cells, from VH, D, and JH gene segments located in separate clusters across the Igh locus. RAG endonuclease initiates V(D)J recombination from a JH-based recombination center (RC). Cohesin-mediated extrusion of upstream chromatin past RC-bound RAG presents Ds for joining to JHs to form a DJH-RC. Igh has a provocative number and organization of CTCF-binding elements (CBEs) that can impede loop extrusion. Thus, Igh has two divergently oriented CBEs (CBE1 and CBE2) in the IGCR1 element between the VH and D/JH domains, over 100 CBEs across the VH domain convergent to CBE1, and 10 clustered 3'Igh-CBEs convergent to CBE2 and VH CBEs. IGCR1 CBEs segregate D/JH and VH domains by impeding loop extrusion-mediated RAG-scanning. Downregulation of WAPL, a cohesin unloader, in progenitor-B cells neutralizes CBEs, allowing DJH-RC-bound RAG to scan the VH domain and perform VH-to-DJH rearrangements. To elucidate potential roles of IGCR1-based CBEs and 3'Igh-CBEs in regulating RAG-scanning and elucidate the mechanism of the ordered transition from D-to-JH to VH-to-DJH recombination, we tested effects of inverting and/or deleting IGCR1 or 3'Igh-CBEs in mice and/or progenitor-B cell lines. These studies revealed that normal IGCR1 CBE orientation augments RAG-scanning impediment activity and suggest that 3'Igh-CBEs reinforce ability of the RC to function as a dynamic loop extrusion impediment to promote optimal RAG scanning activity. Finally, our findings indicate that ordered V(D)J recombination can be explained by a gradual WAPL downregulation mechanism in progenitor-B cells as opposed to a strict developmental switch.

Igh and Igk loci use different folding principles for V gene recombination due to distinct chromosomal architectures of pro-B and pre-B cells.

Extended loop extrusion across the immunoglobulin heavy-chain (Igh) locus facilitates VH-DJH recombination following downregulation of the cohesin-release factor Wapl by Pax5, resulting in global changes in the chromosomal architecture of pro-B cells. Here, we demonstrate that chromatin looping and VK-JK recombination at the Igk locus were insensitive to Wapl upregulation in pre-B cells. Notably, the Wapl protein was expressed at a 2.2-fold higher level in pre-B cells compared with pro-B cells, which resulted in a distinct chromosomal architecture with normal loop sizes in pre-B cells. High-resolution chromosomal contact analysis of the Igk locus identified multiple internal loops, which likely juxtapose VK and JK elements to facilitate VK-JK recombination. The higher Wapl expression in Igµ-transgenic pre-B cells prevented extended loop extrusion at the Igh locus, leading to recombination of only the 6 most 3' proximal VH genes and likely to allelic exclusion of all other VH genes in pre-B cells. These results suggest that pro-B and pre-B cells with their distinct chromosomal architectures use different chromatin folding principles for V gene recombination, thereby enabling allelic exclusion at the Igh locus, when the Igk locus is recombined.

AIRR-C IG Reference Sets: curated sets of immunoglobulin heavy and light chain germline genes.

Introduction: Analysis of an individual's immunoglobulin (IG) gene repertoire requires the use of high-quality germline gene reference sets. When sets only contain alleles supported by strong evidence, AIRR sequencing (AIRR-seq) data analysis is more accurate and studies of the evolution of IG genes, their allelic variants and the expressed immune repertoire is therefore facilitated. Methods: The Adaptive Immune Receptor Repertoire Community (AIRR-C) IG Reference Sets have been developed by including only human IG heavy and light chain alleles that have been confirmed by evidence from multiple high-quality sources. To further improve AIRR-seq analysis, some alleles have been extended to deal with short 3' or 5' truncations that can lead them to be overlooked by alignment utilities. To avoid other challenges for analysis programs, exact paralogs (e.g. IGHV1-69*01 and IGHV1-69D*01) are only represented once in each set, though alternative sequence names are noted in accompanying metadata. Results and discussion: The Reference Sets include less than half the previously recognised IG alleles (e.g. just 198 IGHV sequences), and also include a number of novel alleles: 8 IGHV alleles, 2 IGKV alleles and 5 IGLV alleles. Despite their smaller sizes, erroneous calls were eliminated, and excellent coverage was achieved when a set of repertoires comprising over 4 million V(D)J rearrangements from 99 individuals were analyzed using the Sets. The version-tracked AIRR-C IG Reference Sets are freely available at the OGRDB website (https://ogrdb.airr-community.org/germline_sets/Human) and will be regularly updated to include newly observed and previously reported sequences that can be confirmed by new high-quality data.

The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRß and TCRα Genes and Influences Development of αß T Cell Lineages.

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRß and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRß and/or TCRα genes. These Rag1 mutant mice present diminished positive selection and superantigen-mediated negative selection of conventional αß T cells, decreased genesis of invariant NK T lineage αß T cells, and mature CD4+ αß T cells with elevated autoimmune potential. Our findings reveal that the Rag1 ubiquitin ligase domain functions in vivo to stimulate TCRß and TCRα gene recombination and influence differentiation of αß T lineage cells, thereby establishing replete diversity of αß TCRs and populations of αß T cells while restraining generation of potentially autoreactive conventional αß T cells.

RNA exosome drives early B cell development via noncoding RNA processing mechanisms.

B cell development is linked to successful V(D)J recombination, allowing B cell receptor expression and ultimately antibody secretion for adaptive immunity. Germline noncoding RNAs (ncRNAs) are produced at immunoglobulin (Ig) loci during V(D)J recombination, but their function and posttranscriptional regulation are incompletely understood. Patients with trichohepatoenteric syndrome, characterized by RNA exosome pathway component mutations, exhibit lymphopenia, thus demonstrating the importance of ncRNA surveillance in B cell development in humans. To understand the role of RNA exosome in early B cell development in greater detail, we generated mouse models harboring a B cell-specific cre allele (Mb1cre), coupled to conditional inversion-deletion alleles of one RNA exosome core component (Exosc3) or RNase catalytic subunits (Exosc10 or Dis3). We noticed increased expression of RNA exosome subunits during V(D)J recombination, whereas a B cell developmental blockade at the pro-B cell stage was observed in the different knockout mice, overlapping with a lack of productive rearrangements of VDJ genes at the Ig heavy chain (Igh). This unsuccessful recombination prevented differentiation into pre-B cells, with accumulation of ncRNAs and up-regulation of the p53 pathway. Introduction of a prearranged Igh VDJ allele partly rescued the pre-B cell population in Dis3-deficient cells, although V-J recombination defects were observed at Ig light chain kappa (Igκ), preventing subsequent B cell development. These observations demonstrated that the RNA exosome complex is important for Igh and Igκ recombination and establish the relevance of RNA processing for optimal diversification at these loci during B cell development.

SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination.

SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF. By contrast, SHLD1 is essential for restricting resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR by NHEJ and alternative end-joining. Finally, we show that this SHLD1 function is required for orientation-specific joining of AID-initiated DSBs. Our data thus suggest that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms: SHLD-independent synapsis of V(D)J segments and switch regions within chromatin, and SHLD-dependent protection of AID-DSB ends against resection.

Poor-Quality Vß Recombination Signal Sequences and the DNA Damage Response ATM Kinase Collaborate to Establish TCRß Gene Repertoire and Allelic Exclusion.

The monoallelic expression (allelic exclusion) of diverse lymphocyte Ag receptor genes enables specific immune responses. Allelic exclusion is achieved by asynchronous initiation of V(D)J recombination between alleles and protein encoded by successful rearrangement on the first allele signaling permanent inhibition of V rearrangement on the other allele. The ATM kinase that guides DNA repair and transiently suppresses V(D)J recombination also helps impose allelic exclusion through undetermined mechanisms. At the TCRß locus, one Vß gene segment (V31) rearranges only by inversion, whereas all other Vß segments rearrange by deletion except for rare cases in which they rearrange through inversion following V31 rearrangement. The poor-quality recombination signal sequences (RSSs) of V31 and V2 help establish TCRß gene repertoire and allelic exclusion by stochastically limiting initiation of Vß rearrangements before TCRß protein-signaled permanent silencing of Vß recombination. We show in this study in mice that ATM functions with these RSSs and the weak V1 RSS to shape TCRß gene repertoire by restricting their Vß segments from initiating recombination and hindering aberrant nonfunctional Vß recombination products, especially during inversional V31 rearrangements. We find that ATM collaborates with the V1 and V2 RSSs to help enforce allelic exclusion by facilitating competition between alleles for initiation and functional completion of rearrangements of these Vß segments. Our data demonstrate that the fundamental genetic DNA elements that underlie inefficient Vß recombination cooperate with ATM-mediated rapid DNA damage responses to help establish diversity and allelic exclusion of TCRß genes.

Combining genotypes and T cell receptor distributions to infer genetic loci determining V(D)J recombination probabilities.

Every T cell receptor (TCR) repertoire is shaped by a complex probabilistic tangle of genetically determined biases and immune exposures. T cells combine a random V(D)J recombination process with a selection process to generate highly diverse and functional TCRs. The extent to which an individual's genetic background is associated with their resulting TCR repertoire diversity has yet to be fully explored. Using a previously published repertoire sequencing dataset paired with high-resolution genome-wide genotyping from a large human cohort, we infer specific genetic loci associated with V(D)J recombination probabilities using genome-wide association inference. We show that V(D)J gene usage profiles are associated with variation in the TCRB locus and, specifically for the functional TCR repertoire, variation in the major histocompatibility complex locus. Further, we identify specific variations in the genes encoding the Artemis protein and the TdT protein to be associated with biasing junctional nucleotide deletion and N-insertion, respectively. These results refine our understanding of genetically-determined TCR repertoire biases by confirming and extending previous studies on the genetic determinants of V(D)J gene usage and providing the first examples of trans genetic variants which are associated with modifying junctional diversity. Together, these insights lay the groundwork for further explorations into how immune responses vary between individuals.

Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Igk Feedback Inhibit V κ Recombination to Enforce Igκ Allelic Exclusion.

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms. ATM promotes repair of RAG1/RAG2 (RAG) endonuclease-induced DSBs and transduces signals from RAG DSBs during Igk gene rearrangement on one allele to transiently inhibit RAG1 protein expression, Igk accessibility, and RAG cleavage of the other allele. Yet, the relative contributions of ATM functions in DSB repair versus signaling to enforce AgR allelic exclusion remain undetermined. In this study, we demonstrate that inactivation in mouse pre-B cells of the NF-κB essential modulator (Nemo) protein, an effector of ATM signaling, diminishes RAG DSB-triggered repression of Rag1/Rag2 transcription and Igk accessibility but does not result in aberrant repair of RAG DSBs like ATM inactivation. We show that Nemo deficiency increases simultaneous biallelic Igk cleavage in pre-B cells and raises the frequency of B cells expressing Igκ proteins from both alleles. In contrast, the incidence of biallelic Igκ expression is not elevated by inactivation of the SpiC transcriptional repressor, which is induced by RAG DSBs in an ATM-dependent manner and suppresses Igk accessibility. Thus, we conclude that Nemo-dependent, ATM-mediated DNA damage signals enforce Igκ allelic exclusion by orchestrating transient repression of RAG expression and feedback inhibition of additional Igk rearrangements in response to RAG cleavage on one Igk allele.

huARdb: human Antigen Receptor database for interactive clonotype-transcriptome analysis at the single-cell level.

T-cell receptors (TCRs) and B-cell receptors (BCRs) are critical in recognizing antigens and activating the adaptive immune response. Stochastic V(D)J recombination generates massive TCR/BCR repertoire diversity. Single-cell immune profiling with transcriptome analysis allows the high-throughput study of individual TCR/BCR clonotypes and functions under both normal and pathological settings. However, a comprehensive database linking these data is not yet readily available. Here, we present the human Antigen Receptor database (huARdb), a large-scale human single-cell immune profiling database that contains 444 794 high confidence T or B cells (hcT/B cells) with full-length TCR/BCR sequence and transcriptomes from 215 datasets. All datasets were processed in a uniform workflow, including sequence alignment, cell subtype prediction, unsupervised cell clustering, and clonotype definition. We also developed a multi-functional and user-friendly web interface that provides interactive visualization modules for biologists to analyze the transcriptome and TCR/BCR features at the single-cell level. HuARdb is freely available at https://huarc.net/database with functions for data querying, browsing, downloading, and depositing. In conclusion, huARdb is a comprehensive and multi-perspective atlas for human antigen receptors.

Limitations of lymphoblastoid cell lines for establishing genetic reference datasets in the immunoglobulin loci.

Lymphoblastoid cell lines (LCLs) have been critical to establishing genetic resources for biomedical science. They have been used extensively to study human genetic diversity, genome function, and inform the development of tools and methodologies for augmenting disease genetics research. While the validity of variant callsets from LCLs has been demonstrated for most of the genome, previous work has shown that DNA extracted from LCLs is modified by V(D)J recombination within the immunoglobulin (IG) loci, regions that harbor antibody genes critical to immune system function. However, the impacts of V(D)J on short read sequencing data generated from LCLs has not been extensively investigated. In this study, we used LCL-derived short read sequencing data from the 1000 Genomes Project (n = 2,504) to identify signatures of V(D)J recombination. Our analyses revealed sample-level impacts of V(D)J recombination that varied depending on the degree of inferred monoclonality. We showed that V(D)J associated somatic deletions impacted genotyping accuracy, leading to adulterated population-level estimates of allele frequency and linkage disequilibrium. These findings illuminate limitations of using LCLs and short read data for building genetic resources in the IG loci, with implications for interpreting previous disease association studies in these regions.

RAG1 splicing mutation causes enhanced B cell differentiation and autoantibody production.

Hypomorphic RAG1 or RAG2 mutations cause primary immunodeficiencies and can lead to autoimmunity, but the underlying mechanisms are elusive. We report here a patient carrying a c.116+2T>G homozygous splice site mutation in the first intron of RAG1, which led to aberrant splicing and greatly reduced RAG1 protein expression. B cell development was blocked at both the pro-B to pre-B transition and the pre-B to immature B cell differentiation step. The patient B cells had reduced B cell receptor repertoire diversity and decreased complementarity determining region 3 lengths. Despite B cell lymphopenia, the patient had abundant plasma cells in the BM and produced large quantities of IgM and IgG Abs, including autoantibodies. The proportion of naive B cells was reduced while the frequency of IgD-CD27- double-negative (DN) B cells, which quickly differentiated into Ab-secreting plasma cells upon stimulation, was greatly increased. Immune phenotype analysis of 52 patients with primary immunodeficiency revealed a strong association of the increased proportion of DN B and memory B cells with decreased number and proportion of naive B cells. These results suggest that the lymphopenic environment triggered naive B cell differentiation into DN B and memory B cells, leading to increased Ab production.

SARS-CoV-2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the coronavirus disease 2019 (COVID-19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS-CoV-2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID-19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS-CoV-2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS-CoV-2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines.

DNA End Joining: G0-ing to the Core.

Humans have evolved a series of DNA double-strand break (DSB) repair pathways to efficiently and accurately rejoin nascently formed pairs of double-stranded DNA ends (DSEs). In G0/G1-phase cells, non-homologous end joining (NHEJ) and alternative end joining (A-EJ) operate to support covalent rejoining of DSEs. While NHEJ is predominantly utilized and collaborates extensively with the DNA damage response (DDR) to support pairing of DSEs, much less is known about A-EJ collaboration with DDR factors when NHEJ is absent. Non-cycling lymphocyte progenitor cells use NHEJ to complete V(D)J recombination of antigen receptor genes, initiated by the RAG1/2 endonuclease which holds its pair of targeted DSBs in a synapse until each specified pair of DSEs is handed off to the NHEJ DSB sensor complex, Ku. Similar to designer endonuclease DSBs, the absence of Ku allows for A-EJ to access RAG1/2 DSEs but with random pairing to complete their repair. Here, we describe recent insights into the major phases of DSB end joining, with an emphasis on synapsis and tethering mechanisms, and bring together new and old concepts of NHEJ vs. A-EJ and on RAG2-mediated repair pathway choice.