Each antigen receptor produced through recombination in B cells will contain a heavy (IgH) and light [IgL, (Igκ or Igλ)] chain consisting of VDJ segments for IgH and VJ segments for IgL. These gene clusters extend over 3 Mb, consisting of approximately 140 Vκ, 4 Jκ, 38 Vλ, and 5 Jλ loci for use in the IgL and approximately 150 V_H, 9 D_H, and 4 J_H loci for use in the IgH (Jung et al., 2006; Ji et al., 2010; Collins and Watson, 2018).

Due to expression and degradation mechanisms of the RAG proteins discussed below V(D)J recombination is restricted to G0/G1 phase of the cell cycle. At this point the IgH locus can undergo recombination, first between D_H-J_H segments before V_H-DJ_H joining. Successful rearrangement of the three segments allows for production of a pre-B cell receptor (pre-BCR) and further differentiation to the small pre-B stage where IgL rearrangement can begin. Gene usage during this recombination step is skewed toward Igκ segments over Igλ (2:1 up to 95:5) (Woloschak and Krco, 1987; Lycke et al., 2015). Surface expression of the BCR in the immature B cell activates a checkpoint to determine whether the receptor is autoreactive or non-functional. If either condition occurs, secondary recombination of the Igλ gene segments is used to substitute light chains until the autoreactivity is diminished. Molecular signatures of this recombination event are the usage of more upstream V regions and more downstream J regions (Villa and Notarangelo, 2019). Following proper reactivity, the mature B cell is released from the bone marrow.

The heterotetrameric recombination complex that binds the antigen receptor loci at RSSs is composed of two RAG1 subunits and two RAG2 subunits (Kim et al., 2015). Two discrete RSSs, a heptamer and a nonamer, are required for efficient binding and cleavage. Heptamer sequences follow the pattern of CACAGTG, where only the first three nucleotides are highly conserved and required for cleavage. The stronger binding nonamer sequence, ACAAAAACC, contains several conserved positions required for initial protein complex interaction. RSSs are separated by a 12 or 23 base pair spacer, which exhibits low conservation, but has the potential to introduce a significant effect on recombination efficiency (Hirokawa et al., 2020; Lee et al., 2003). Binding must occur at a pair of RSSs following the 12/23 rule, forming the paired complex (PC), which can be mediated by random collision or locus contraction (see below) (Eastman et al., 1996). Discussed later in the clinical manifestation section, cryptic RSSs (cRSSs) are common throughout the genome and, due to the sequence variation allowed by the RAG complex, may induce off-target effects. For example, frequent RAG-mediated DSBs in c-Myc rely only on the presence of the CAC motif of an RSS heptamer (Hu et al., 2015). Upon binding to DNA, the RAG complex induces a conformational change to the 12- and 23-RSS sites to enable efficient cleavage by RAG1. The recombination complex also utilizes high mobility group box 1/2 (HMGB1/2) to promote DNA bending, enhancing synapsis and cleavage. Once the PC is established, cleavage first occurs on a single strand via a 5’ nick at the heptamer-coding flank junction. This allows for a direct transesterification reaction where the 3’ hydroxyl group attacks the phosphate of the bottom strand. Two cleavage events in the PC generate four broken DNA ends, where two are covalently sealed coding ends (CEs) and two are blunt signal ends (SEs). This reaction takes place without a required external energy source, as the hairpin formation energy is derived from the DNA breakage. The RAG-DNA complex does not form a covalent intermediate making it distinct from other site-specific recombinases and is more similar to bacterial transposases and HIV integrase than its mammalian counterparts (Little et al., 2015). The nicking reaction can occur within minutes but the hairpinning may require hours potentially indicating simultaneous nick locations within the locus (Yu et al., 2004).

Upon cleavage the RAG complex stays associated with the broken ends forming a post-cleavage complex (PCC). This structure permits CEs to dissociate first, under the correct conditions to enter the NHEJ pathway. SEs are retained in the complex until physical disassembly can occur due to RAG2 degradation, however, this process is only speculative (Mizuta et al., 2002). Joined SEs ultimately create a non-replicative episome which is routinely lost during cell division (Smith et al., 2019). As discussed in the clinical manifestation section regulation of this component is necessary as well, due to the potential for translocation or other off-target effects if the complex is retained.

NHEJ proceeds through a couple of seemingly simple steps. The exposed DNA ends are first recognized by the Ku heterodimer, a ring-shaped protein (Fell and Schild-Poulter, 2015). Together with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku forms the DNA-PK holoenzyme (Yue et al., 2020). This complex binds to the break site and acts as a scaffold for other repair proteins. XRCC4 (together with its binding partner Ligase IV) and XLF are recruited to the break site (Mcelhinny et al., 2000) and aid the DNA ends in coming together, a transient process called synapsis (Reid et al., 2015; Graham et al., 2017; Zhao et al., 2019). Structural studies and super-resolution microscopy have shown that XRCC4 and XLF can accomplish this by forming filaments along the DNA, which helps bridge the two ends (Hammel et al., 2010; Ropars et al., 2011; Mahaney et al., 2013; Reid et al., 2015; Chen et al., 2021). Once the DNA ends are aligned, Ligase IV seals the backbones to complete repair (Conlin et al., 2017). Over the years, it has become clear that a host of accessory factors are implicated in NHEJ, some of which are functionally redundant. We will discuss the best studied accessory factors and the implications of their functional redundancies later in this review.

If NHEJ is unavailable, repair can proceed through Alternative End Joining (alt-EJ). Alt-EJ is a less well-defined process that involves a different set of proteins, most prominently DNA Polymerase Theta (PolQ), that mediate microhomology-based annealing of resected DNA ends (Sallmyr and Tomkinson, 2018). Alt-EJ is exceptionally error-prone and usually only serves as a backup pathway. It does not typically occur during V(D)J recombination, since the hand-off of the break sites to the NHEJ machinery is tightly arranged. Indeed, deficiency in core NHEJ factors often leads to cell death (Wang et al., 2020). If certain key proteins in the hand-off fail, however, alt-EJ may be employed and lead to genome instability and disease.

With this three step process established there is still information lacking on direct influences for the recruitment of a RAG complex to RSS regions as well as subsequent pairing to the partner RSS, regulation by non-catalytic regions of RAG proteins, and certain redundant features of NHEJ factors during the repair phase, each of which will be highlighted by the following sections.

RAG1 is a 1,040 aa protein consisting of a large N-terminal non-core domain (aa 1–384), the core region (384–1,008), and a short C-terminal non-core domain (1,008–1,040) (Schatz and Swanson, 2011; Little et al., 2015; Lescale and Deriano, 2017; Villa and Notarangelo, 2019). The functional core region contains the essential sites for DNA/RSS binding, homo- and hetero-dimerization and DNA cleavage (facilitated by D600, D708, and E962). The nonamer binding domain (NBD) interacts with the nonamer RSS, with the downstream dimerization and DNA binding domain (DDBD) providing a site for RAG1 homo-dimerization (Villa and Notarangelo, 2019). Regulation of contact with the heptamer RSS, ssDNA, and RAG2 is controlled by motifs within the central region. The C-terminal core region contains nonspecific DNA binding activity to mediate contact with the coding sequence flanking the RSS. A catalytic triad within the central and C-terminal domains coordinates metal ions (Mg²⁺) to activate water for ssDNA nicking activity. A zinc binding domain (ZBD) also spanning the central and C-terminal regions (722–965) is important for interaction with RAG2.

In order to understand potential interaction partners of RAG1, Brecht et al. examined association via a proximity-dependent biotin identification screening (Brecht et al., 2020). Results here indicated interaction with multiple nucleolar factors suggesting localization to this region of the nucleus outside of G1 cell cycle phase. By sequestering the protein to the nucleolus, off-target recombination events were dampened and genome stability was promoted. Upon induced recombination and G1 cell cycle arrest, RAG1 was observed to be released from the nucleolar regions and allowed to bind partner RAG2 and RSSs. Truncation of the full length protein determined that two sequences within the N-terminal non-core region were responsible for nucleolar entry (residues 243–249) and export (1–215) (Brecht et al., 2020).

In addition to nucleolar localization, the N-terminal non-core domains of RAG1 are responsible for regulation of cellular protein levels, mediation of interaction with other factors, and coordination of zinc ions, all of which act to enhance recombination activity. The zinc dimerization domain in this region (265–380) acts as a counterpart to the core ZBD, but here facilitates homo-dimerization (Lescale and Deriano, 2017). Overlapping with this domain is a RING motif, at residues 264–389, which has the capability to act as an E3 ubiquitin ligase for both autoubiquitylation and modification of other proteins (Grazini et al., 2010). RAG1 autoubiquitylation at K233 has been shown to stimulate cleavage activity in a cell free assay and exhibit post-transcriptional regulation in mice studies (Singh and Gellert, 2015; Beilinson et al., 2021). Ubiquitin modification of other proteins occurs at different stages of recombination, such as polyubiquitylation of KPNA1 or the monoubiquitylation of histones (discussed below). Sequestering RAG1 after nuclear import may be achieved by KPNA1 interaction with the basic motif BIIa within residues 218–263, only relieved by KPNA1 ubiquitylation for sub-nuclear localization (Simkus et al., 2009). However, follow up reports have discussed ubiquitylation activity mediated by additional complexes rather than the isolated RING region (discussed below) (Kassmeier et al., 2012). At the C-terminus of RAG1 the non-core region is only 32 residues, but inhibition of hairpin formation is controlled by this motif (Grundy et al., 2010). Interaction of the RAG complex with modified histones overcomes the inhibition possibly due to RAG2-mediated conformation changes associated with RAG2 C-terminal regions.

RAG2 is a 527 aa protein consisting of a core domain (1–351) and a C-terminal non-core domain (352–527). The core region is comprised of six Kelch-like motifs which form a six bladed ß-propeller responsible for efficient DNA cleavage (Schatz and Swanson, 2011; Little et al., 2015; Lescale and Deriano, 2017; Villa and Notarangelo, 2019). The second and sixth ß-strands are responsible for making contact with RAG1 (Little et al., 2015; Villa and Notarangelo, 2019). On its own RAG2 is monomeric, forming a 2:2 heterotetramer with RAG1 to form the RAG recombination complex (Bailin et al., 1999). As with RAG1, the non-core domain is not required for recombination activity, but regulates various parts of the recombination mechanism.

The RAG2 C-terminal non-core region is composed of two main components, an acidic hinge (351–408) and a plant homeodomain (PHD, 414–487). Although many studies have reported the RAG2 non-core region spans residues 387–527, assays with further truncations of the RAG2 core region have displayed efficient recombination with only residues 1–351 (Coussens et al., 2013; Kim et al., 2015). The acidic hinge, linking core RAG2 and the PHD, contains a high concentration of acidic residues contributing to flexibility of the region. Neutralization of the residues severely reduces the flexibility and leads to increased genomic instability as aberrant repair begins to occur (Coussens et al., 2013). Two separate regions within the acidic hinge are necessary to regulate recombination activity. Serial truncations of the hinge by Wu et al. lead to the discovery that the Igκ locus was hypermethylated upon deletion of residues 350–383 and occurred in a RAG1-independent manner (Wu et al., 2017). Demethylation of the chromosome in this context may assist in facilitating allelic exclusion, preventing further recombination on the locus. Coupled with this in the acidic hinge is an autoinihibitory function by residues 388–405, where relief is required to promote activity. As discussed below, histone recognition mediates this inhibition, with mutations bypassing the necessity for this interaction (Lu et al., 2015). The PHD component is responsible for interactions with chromatin, specifically at modified histones, based on full-length and truncated constructs submitted to ChIP-seq experiments (see below) (Teng et al., 2015). Mutations to this site, such as W453A, result in overall loss of genome localization and reduced recombination activity (Liu et al., 2007; Teng et al., 2015). At the far C-terminus phosphorylation of T490 promotes cell cycle-regulated degradation at the G1-S transition (Zhang et al., 2011). RAG2 T490A mutation can lead to persistent accumulation throughout the cell cycle as degradation is reduced. This overexpression results in continuous opportunities for RSS/cRSS target cleavage in the presence of RAG1 and recombination intermediates. The mutation also plays a role in stabilizing genomic interaction displayed by Rodgers et al. where slowed diffusion, measured in live cells via fluorescence recovery after photobleaching, was indicative of stronger interactions with modified histones (Rodgers et al., 2019).

To begin the process of recombination, RAG proteins must first associate with an RSS within the Mb chromosomal antigen receptor locus. The limiting of initial RAG binding can be considered a regulatory mechanism to prevent DNA nicks at random sites within the genome and may be facilitated solely by 3D diffusion to scan for sites rich in modified histones which indicate active chromatin (Lovely et al., 2020). ChIP-seq experiments by Teng et al. and Ji et al. determined the binding pattern of RAG1 and RAG2 across V(D)J segments and the entire genome revealing chromatin features which may influence the recruitment of these proteins (Ji et al., 2010; Teng et al., 2015). Within the antigen receptor loci both RAG1 and RAG2 were observed to bind at J segments in the Igκ locus and both D and J segments in the IgH locus (Ji et al., 2010). In this region, an RSS is necessary for strong binding with mutation to the nonamer sequence reducing overall recruitment. Outside of these loci, however, RAG1 localization is poorly indicated by RSS presence alone, along with cRSSs and heptamers depletion from observed binding sites suggesting that other chromatin features may play a role in RAG complex recruitment. The genomic localization of RAG2 is significantly broader with binding sites dependent on regions with high levels of methylated histone 3 (H3K4me3) and physical association determined by co-immunoprecipitation (Teng et al., 2015; Rodgers et al., 2019). As noted above, interaction of RAG2 and H3K4me3 is facilitated by the PHD of RAG2 (Matthews et al., 2007; Teng et al., 2015) The necessity of H3K4me3 binding was then determined to be due to autoinhibition of the RAG complex by RAG2 (Grundy et al., 2010). Stimulation with exogenous H3K4me3 relieved the reduced binding and catalysis, with truncation of this site uncoupling the necessity for histone recognition. Studies by Lu et al. and Bettridge et al. determined allosteric conformational changes occur to both RAG1, at the DDBD and catalytic region, and RAG2 at the autoinhibitory region allowing for increased accessibility (Lu et al., 2015; Bettridge et al., 2017). Mutations to this region can bypass the need for histone recognition to promote activity but will likely increase off-target effects (Lu et al., 2015). While RAG2 and H3K4me3 display a linear correlation of interaction, the non-linear correlation of RAG1 and H3K4me3 suggests additional features. Maman et al. used additional ChIP-seq experiments to determine possible factors for RAG1 interaction (Maman et al., 2016). H3K4me3 overlap with RAG1 was determined to be RAG2-histone dependent making it insufficient to determine RAG1 binding throughout the genome and the role methylation plays in off-target binding. Another histone modification, H3K27Ac, was instead determined to be RAG2 independent and more so influenced by N-terminal regions of RAG1, but with little direct evidence the significance is unclear (Maman et al., 2016).

In addition to initial binding, the RAG-DNA complex must associate with a second, partner RSS to perform recombination. Regulation at this point is achieved through the physical proximity of the V gene segments, which are spread across over 2 Mb of DNA (Figure 3A). While proximal segments may be paired via random collision, large scale chromosomal conformational changes are utilized to direct pairing at both central and distal regions within the loci, skewing interaction partners and providing a diverse set of antigen receptors (Figure 3B). Various mechanisms of chromosomal looping enables regions to be brought into close proximity during pro- and pre-B stages via proteins such as YY1, Ikaros, Pax5 and CTCF.

Distal V_H utilization is facilitated by each of the proteins under slightly different mechanisms. Within the IgH locus are enhancer regions, intronic enhancer (iEµ) and a 3’ regulatory region which provide sites for contraction protein binding. Yin Yang 1 (YY1) is a zinc finger protein with multiple functions in regards to transcription activation and repression (Liu et al., 2007). Knockouts of this protein during B-cell development yield a block at the pro-B cell stage due to insufficient V_H-DJ_H recombination without influencing expression of additional V(D)J recombination components (Figure 3Ci). Using ChIP and 3D DNA FISH Liu et al. were able to determine that YY1 binds to iEµ sites to provide a node for locus contraction (Liu et al., 2007). Deletion of iEµ does not inhibit YY1 binding to the overall chromosome loci, indicating that additional sequences and factors influence the contraction (Guo et al., 2011). In addition, YY1 may have heterotypic interactions with CTCF, however, changes to rearrangement if CTCF levels are decreased are not as pronounced as the knockdown of YY1 in reducing recombination efficiency (Degner et al., 2011; Weintraub et al., 2017). Ikaros, also a zinc finger protein, contributes various roles in differentiation control and chromosome accessibility (Reynaud et al., 2008). In a similar manner, reduction of Ikaros leads to overall low V_H-DJ_H recombination with heavily skewed usage of proximal V_H segments (Figure 3Ci). Pax5, a B-cell commitment factor for differentiation, controls transcription, but also functions by positioning chromatin towards the central nuclear regions ensuring active chromatin is in an extended state and promotes locus contraction (Ebert et al., 2011; Fuxa et al., 2004). When Pax5 is deleted, cells will have characteristic features of uncommitted progenitors, such as the ability to change differentiation pathway following cytokine stimulation, and exhibit reduced diversity due to a 50-fold reduction of distal V recombination (Figure 3Ci). Contraction by Pax5 is mediated through Pax5-activated intergenic repeats (PAIRs) over 750 kB of the distal V_H gene which allow for interaction with iEµ sites (Verma-Gaur et al., 2012). Via Bio-ChIP-chip Ebert et al. showed that this mechanism is specifically lost in the pre-B cell stage where other mechanisms must be used to promote distal gene usage (Ebert et al., 2011).

CCCTC-binding factor (CTCF) is another zinc finger protein with diverse functions for transcriptional control, but also mediating chromosomal contacts across the genome (Phillips and Corces, 2009; Degner et al., 2011). Deletion of CTCF reduces overall B-cell maturation and arrests development at pre-B stage for those progressing that far. Mediation of contraction here is regulated by the presence of CTCF-binding elements (CBEs), 14 bp conserved targets within the Ig loci (Hu et al., 2015). Association of pairs of convergently oriented CBEs bound by CTCF and the cohesin complex form loop domains of the antigen receptor loci restricting the V segments (Zhao et al., 2016; Zhang et al., 2019). Cohesin, consisting of multiple subunits including Rad21, will form loops of various sizes by extrusion of chromatin in an ATP-dependent manner until reaching a CTCF bound CBE (Ba et al., 2020). The loop extrusion process is dynamic and allows for a subset of CTCF/cohesin formed loops to exist at any given time enhancing genetic diversity (Degner et al., 2011). Large chromosomal structural rearrangements also compete with the short range collisional recombination which is extinguished during CTCF downregulation as RAG proceeds to distal V_H regions without obstruction therefore limiting diversity (Figure 3Cii), yet in the Igκ locus proximal usage is increased (De Almeida et al., 2011; Ba et al., 2020; Zhang et al., 2022). In contrast, deletion of cohesin subunit Rad21 eliminates all recombination at most sites, except for proximal regions which form a synapse due to collisional diffusion (Figure 3Ci). Cohesin unloading is regulated by the expression of WAPL throughout the cell cycle. Pax5 repression of the WAPL promoter during pro- and pre-B cell stages allows for increased cohesin residence time on chromatin extending loop sizes by circumventing CBE obstructions (Hill et al., 2020; Dai et al., 2021). Under these circumstances, there is a general increase of recombination at all sites (Figure 3Ciii). For a comprehensive look into cohesin-mediated loop extrusion, we refer the reader to the recent review by Zhang et al., (2022).

Enhancer and intergenic regions of the antigen receptor loci are also important for CTCF mediated loop extrusion as deletion or mutation of these sites provides limited diversity and dysfunction. iEµ in the IgH locus is required for efficient recombination, where deletion increases proximal gene usage and reduced chromosomal relocation to the central nuclear regions to limit total accessibility (Guo et al., 2011). The intergenic control region 1 (IGCR1), between V_H and D_H segments, contains CBEs to suppress V_H usage prior to D_H-J_H rearrangement. Deletion of the IGCR1 region promotes proximal V_H usage, but also induces off-target breaks spreading up to 120 kb upstream of the proximal V_H segments for potential cRSS replacement (Figure 3Civ). CBEs within the V_H region prevent distal usage past these segments in the absence of IGCR1 regulation (Hu et al., 2015). The Vκ-Jκ locus contains additional DNase hypersensitive (HS) regions which influence chromosomal conformation change. Upon deletion of HS3-6 there is only a moderate decrease of middle gene usage, with overall insignificant changes to locus contraction. However, HS1-2 deletion results in at least a 7-fold increase of proximal gene usage with 3D DNA FISH indicating a 50% decreasing in overall contraction of the Ig locus (Xiang et al., 2013).

Deficiency in RAG proteins results in an overall lack of recombination efficiency and diversity, with lower expression leading to a harsher clinical outcome. This deficiency can lead to several phenotypes including severe combined immunodeficiency (SCID), combined immunodeficiency with granulomas or autoimmunity (CID-G/AI) and Omenn Syndrome (OS) (Schwarz et al., 1996; Schuetz et al., 2008). For an extensive analysis of the pathogenesis of these RAG-mediated deficiencies we refer the reader to the recent review from Bosticardo et al. (Bosticardo et al., 2021). SCID, and to the lesser extent CID-G/AI, can cause major vulnerability to minor infections, with current treatment through methods such as bone marrow transplant (Buckley, 2004). Recent large cohort studies for RAG deficiency show occurrence in 12% of SCID cases and 42% of atypical SCID cases (Dvorak et al., 2019). OS patients display a complex pathogenesis with symptoms similar to SCID, except an estimated 90% of cases are due to RAG mutations (Marrella et al., 2011). Over 60 naturally occurring mutations resulting in immunodeficiency have been mapped to just the core regions of RAGs with effects such as destabilized structures between RAGs or other components, decreased DNA binding, and catalytic deficiency (Kim et al., 2015). Two example mutations related to OS, V779M and C328G in RAG1, reduce recombination through different mechanisms, decreased cleavage efficiency and joint formation, respectively (Grazini et al., 2010; Matthews et al., 2015). Lee et al. and Tirosh et al. determined the recombination efficiency of RAG1 and RAG2, respectively, using mutations present in patient samples with varying disease type and severity (Lee et al., 2014; Tirosh et al., 2019). A high density of mutations occur in the NBD and mutations to this region or the heptamer binding motif of RAG1 tend to exhibit significantly lower activity even though the protein is catalytically active (Lee et al., 2014). In RAG2 samples, the overrepresentation occurs in the PHD, affecting histone interaction and the autoinhibitory mechanism. Various mutations in RAGs may also circumvent the checkpoints related to autoreactivity leading to reduced functional circulating B-cells in addition to the reduced repertoire (Villa and Notarangelo, 2019). As noted in Figure 3, interfering with locus contraction leads to decreased antigen receptor diversity and mutations to proteins involved, such as cohesin subunits and Ikaros, have been associated with immune disease (Bjorkman et al., 2018; Kuehn et al., 2021). Recurrence of the same low activity mutations in RAGs or other proteins required for recombination could allow for prediction of disease severity in newly diagnosed patients and potential for personalized medicine for achieving a significant level of recombination based on genotype.

Human lymphomas can involve RAG-mediated deletions or potential translocations between the Ig locus V(D)J segments and non-Ig locus. The main area of concern is the presence of cRSSs which mimic the RSS motif, but exist outside of the antigen receptor loci (Onozawa and Aplan, 2012; Hu et al., 2015; Teng et al., 2015). RAG-mediated cleavage at cRSS sites could be detrimental to cell viability as uncontrolled regions are disturbed. Notch-1, a ligand activator transcription factor which transduces signaling information from the cell surface to the nucleus, contains 14 cRSSs within the 30 kb locus (Mijuskovic et al., 2015). N-terminal truncation caused by cRSS-mediated deletion exhibits constitutive ligand independent intracellular activity. Using ChIP-seq data from Ji et al., RAG2-H3K4me-Notch-1 5’ binding and colocalization indicates that RAG-mediated cleavage has a high likelihood of occurring in this region (Onozawa and Aplan, 2012). Multiple sets of cRSSs are also involved in the deletion of Jak1 exons 6–8, leading to activation with multiple roles in cell growth and survival (Mijuskovic et al., 2015). Additional RAG-mediated deletions have occurred at Trat1, Phlda1, Agpat9, CDKN2a/b, Ikaros, and have been attributed to Tal1-Sil fusion (Mullighan et al., 2008; Onozawa and Aplan, 2012; Larmonie et al., 2013; Mijuskovic et al., 2015). Even so, a majority of other oncogenic breakpoints detected in lymphomas do not contain cRSS sites and may be due to event at non-B form DNA structures (Raghavan et al., 2004).

Translocation due to off-target RAG-mediated events would be more detrimental to cell viability, but have eluded direct detection in the genome. Translocations themselves are common and likely due to recombination events, but as of July 2019, there have been no documented cases of leukemia and lymphomas which could be traced directly to a RAG-mediated transposition event (Zhang et al., 2017; Smith et al., 2019). Even translocations which involve the antigen receptor loci, such as Bcl2-IgH or BCR-ABL1, lack substantial evidence of initial RAG-mediated DSBs (Mossadegh-Keller et al., 2021; Yuan et al., 2021). This may be due to a lack of ability to screen for these type of lesions as the limitations of some sequencing methods may overlook certain breakpoint features, however, recent improvements to next generation sequencing and whole genome sequencing will allow for higher discovery rate of these off-target RAG induced breaks (Nordlund et al., 2020; Afkhami et al., 2021; Xiao et al., 2021). The excised signal circle (ESC) complex consisting of the SEs, RAG proteins, and other factors is another source of potential reintegration into the genome (Kirkham et al., 2019). This complex can be extremely dangerous for oncogenic upregulation due to the presence of V region adjacent promoters. More likely are asymmetric cleavage events (“cut and run”), where a closed ESC binds and cleaves at a cRSS before continuing on a series of unchaperoned DNA DSBs. These events have yet to proven in vivo, yet acute lymphoblastic leukemia patients have shown oncogenic activation through translocation events, such as ETV6-RUNX1 gene fusion, which could be facilitated by RAG-mediated ‘cut and run’ events, however, more research is necessary to understand a direct involvement of RAGs in this type of tumorigenesis (Papaemmanuil et al., 2014; Kirkham et al., 2019). In addition, translocation of the DNA fragment of the ESC complex may not be due to new RAG-mediated cleavage events, but instead insertion at independently formed DSBs, leading to further genomic instability (Antoszewska-Smith et al., 2017; Rommel et al., 2017).