Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination

V(D)J recombination and immunoglobulin class switch recombination (CSR) are two somatic rearrangement mechanisms that proceed through the introduction of double-strand breaks (DSBs) in DNA. Although the DNA repair factor XRCC4 is essential for the resolution of DNA DSB during V(D)J recombination, its role in CSR has not been established. To bypass the embryonic lethality of XRCC4 deletion in mice, we developed a conditional XRCC4 knockout (KO) using LoxP-flanked XRCC4 cDNA lentiviral transgenesis. B lymphocyte restricted deletion of XRCC4 in these mice lead to an average two-fold reduction in CSR in vivo and in vitro. Our results connect XRCC4 and the nonhomologous end joining DNA repair pathway to CSR while reflecting the possible use of an alternative pathway in the repair of CSR DSB in the absence of XRCC4. In addition, this new conditional KO approach should be useful in studying other lethal mutations in mice.     

Fixing DNA breaks during class switch recombination

Immunoglobulin (Ig) class switch recombination (CSR) involves the breakage and subsequent repair of two DNA sequences, known as switch (S) regions, which flank IgH constant region exons. The resolution of CSR-associated breaks is thought to require the nonhomologous end-joining (NHEJ) DNA repair pathway, but the role of the NHEJ factor DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in this process has been unclear. A new study, in which broken IgH-containing chromosomes in switching B cells were visualized directly, clearly demonstrated that DNA-PKcs and, unexpectedly, the nuclease Artemis are involved in the resolution of switch breaks.     

DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Artemis are classical nonhomologous DNA end-joining (C-NHEJ) factors required for joining a subset of DNA double-strand breaks (DSB), particularly those requiring end processing. In mature B cells, activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by introducing lesions into S regions upstream of two recombining C(H) exons, which are processed into DSBs and rejoined by C-NHEJ to complete CSR. The function of DNA-PKcs in CSR has been controversial with some reports but not others showing that DNA-PKcs-deficient mice are significantly impaired for CSR. Artemis-deficient B cells reportedly undergo CSR at normal levels. Overall, it is still not known whether there are any CSR-associated DSBs that require DNA-PKcs and/or Artemis to be joined. Here, we have used an immunoglobulin (Ig)H locus-specific fluorescent in situ hybridization assay to unequivocally demonstrate that both DNA-PKcs and, unexpectedly, Artemis are necessary for joining a subset of AID-dependent DSBs. In the absence of either factor, B cells activated for CSR frequently generate AID-dependent IgH locus chromosomal breaks and translocations. We also find that under specific activation conditions, DNA-PKcs(-/-) B cells with chromosomal breaks are eliminated or at least prevented from progressing to metaphase via a p53-dependent response.     

The ATPase activity of MLH1 is required to orchestrate DNA double-strand breaks and end processing during class switch recombination

Antibody diversification through somatic hypermutation (SHM) and class switch recombination (CSR) are similarly initiated in B cells with the generation of U:G mismatches by activation-induced cytidine deaminase but differ in their subsequent mutagenic consequences. Although SHM relies on the generation of nondeleterious point mutations, CSR depends on the production of DNA double-strand breaks (DSBs) and their adequate recombination through nonhomologous end joining (NHEJ). MLH1, an ATPase member of the mismatch repair (MMR) machinery, is emerging as a likely regulator of whether a U:G mismatch progresses toward mutation or DSB formation. We conducted experiments on cancer modeled ATPase-deficient MLH1G67R knockin mice to determine the function that the ATPase domain of MLH1 mediates in SHM and CSR. Mlh1(GR/GR) mice displayed a significant decrease in CSR, mainly attributed to a reduction in the generation of DSBs and diminished accumulation of 53BP1 at the immunoglobulin switch regions. However, SHM was normal in these mice, which distinguishes MLH1 from upstream members of the MMR pathway and suggests a very specific role of its ATPase-dependent functions during CSR. In addition, we show that the residual switching events still taking place in Mlh1(GR/GR) mice display unique features, suggesting a role for the ATPase activity of MLH1 beyond the activation of the endonuclease functions of its MMR partner PMS2. A preference for switch junctions with longer microhomologies in Mlh1(GR/GR) mice suggests that through its ATPase activity, MLH1 also has an impact in DNA end processing, favoring canonical NHEJ downstream of the DSB. Collectively, our study shows that the ATPase domain of MLH1 is important to transmit the CSR signaling cascade both upstream and downstream of the generation of DSBs.     

XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes

Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are repaired either faithfully by DNA base excision repair (BER) or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis, we decreased the level of x-ray cross-complementing 1 (XRCC1), a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of SHM in Igh variable regions in Peyer's patch cells, and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1(+/-) splenic B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative nonhomologous end joining. Furthermore, Xrcc1(+/-) B cells had reduced Igh/c-myc translocations during CSR, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways.     

Cernunnos influences human immunoglobulin class switch recombination and may be associated with B cell lymphomagenesis

Cernunnos is involved in the nonhomologous end-joining (NHEJ) process during DNA double-strand break (DSB) repair. Here, we studied immunoglobulin (Ig) class switch recombination (CSR), a physiological process which relies on proper repair of the DSBs, in B cells from Cernunnos-deficient patients. The pattern of in vivo generated CSR junctions is altered in these cells, with unusually long microhomologies and a lack of direct end-joining. The CSR junctions from Cernunnos-deficient patients largely resemble those from patients lacking DNA ligase IV, Artemis, or ATM, suggesting that these factors are involved in the same end-joining pathway during CSR. By screening 269 mature B cell lymphoma biopsies, we also identified a somatic missense Cernunnos mutation in a diffuse large B cell lymphoma sample. This mutation has a dominant-negative effect on joining of a subset of DNA ends in an in vitro NHEJ assay. Translocations involving both Ig heavy chain loci and clonal-like, dynamic IgA switching activities were observed in this tumor. Collectively, our results suggest a link between defects in the Cernunnos-dependent NHEJ pathway and aberrant CSR or switch translocations during the development of B cell malignancies.     

Altered kinetics of nonhomologous end joining and class switch recombination in ligase IV–deficient B cells

Immunoglobulin heavy chain class switch recombination (CSR) is believed to occur through the generation and repair of DNA double-strand breaks (DSBs) in the long and repetitive switch regions. Although implied, the role of the major vertebrate DSB repair pathway, nonhomologous end joining (NHEJ), in CSR has been controversial. By somatic gene targeting of DNA ligase IV (Lig4; a key component of NHEJ) in a B cell line (CH12F3) capable of highly efficient CSR in vitro, we found that NHEJ is required for efficient CSR. Disruption of the Lig4 gene in CH12F3 cells severely inhibits the initial rate of CSR and causes a late cell proliferation defect under cytokine stimulation. However, unlike V(D)J recombination, which absolutely requires NHEJ, CSR accumulates to a substantial level in Lig4-null cells. The data revealed a fast-acting NHEJ and a slow-acting alterative end joining of switch region breaks during CSR.DNA double-strand breaks (DSBs) are considered by many to be the most severe form of DNA damage, and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). NHEJ is the primary DSB repair pathway in mammalian cells (1). A characteristic feature of NHEJ is the lack of long stretches of homology at the site of joining (junction), although a few base pairs of microhomology (nucleotides that can be assigned to either of the two DNA ends) are seen at 10–40% of junctions (2). The core components of the NHEJ pathway have been well characterized over the last decade. These include DNA end-binding complex Ku70–Ku80, DNA-dependent protein kinase (DNA-PKcs), Artemis, and the ligase complex x-ray cross complementation (XRCC) 4–DNA ligase IV (Lig4). Several accessory factors, including an XRCC4-like factor (known as XLF or Cernunnos) and polymerases μ and λ, were identified in the following years. However, when NHEJ is disrupted by mutations to its core components, many forms of DSBs can still be joined, suggesting the existence of an alternative route for end joining.During lymphocyte development, physiological DSBs arise as critical intermediates in V(D)J and class switch recombination (CSR). V(D)J recombination is a site-specific DNA recombination that assembles the antigen-binding domain of Ig and T cell receptor genes. The RAG protein complex recognizes recombination signal sequences (RSS) next to the V, D, or J coding segments and cleaves the DNA at the RSS-coding sequence junction. A concerted cleavage of a pair of RSS generates four DNA ends that are joined to form a coding joint and a signal joint, respectively. CSR changes the constant domain of the Ig heavy chain, which allows a B cell to switch from expressing IgM to another isotype (IgG, IgA, or IgE). CSR is directed by kilobase-long repetitive switch regions (Sμ, Sγ, Sα, and Sε) lying upstream of each constant region and requires activation-induced cytidine deaminase (AID). The preponderant experimental evidence is consistent with a cut-and-paste mechanism in a way similar to that of V(D)J recombination (3). First, the deleted region is circularized before its disposal. Second, DSBs were detected in switching B cells by ligation-mediated PCR. Third, phosphorylated histone H2AX (a marker for chromosomal breaks) foci were detected in switching B cells at the IgH locus in an AID-dependent manner. Finally, switch junctions show little or no homology, consistent with direct joining of switch region breaks through NHEJ. Although the exact mechanism for switch region breaks remains unclear, the current view is that AID catalyzes cytidine deamination (converts cytidines to uracils) in the switch regions, followed by the repair of uracils that ultimately results in DSBs (3).In contrast to V(D)J recombination, which absolutely requires NHEJ, direct testing of the role of NHEJ in CSR has met with considerable challenge. One obvious obstacle is that NHEJ-deficient animals cannot generate mature lymphocytes necessary for a CSR assay because the V(D)J recombination defect blocks T and B cell development. To circumvent the developmental block on B cells, Ku- or DNA-PKcs–deficient mice have been crossed with mice harboring preassembled Ig heavy and light chain genes to generate monoclonal NHEJ-deficient B cells (4–7). By this method, CSR was found completely abolished in the absence of Ku70 or Ku80 (4, 5). However, this conclusion must be interpreted with caution, because Ku may have non-NHEJ–related functions (e.g., telomere maintenance) and Ku deficiency causes defective cell proliferation (4, 5). As for DNA-PKcs, controversial results were obtained between a point mutation that inactivates DNA-PKcs and a complete deletion of the gene (6, 7). Although catalytically inactive DNA-PKcs allows normal CSR (7), deletion of the DNA-PKcs gene inhibited CSR to all isotypes except IgG1 (6). A caveat of this approach is that there are no T cells in the B cell–reconstituted mice. As a result, these mice have greatly reduced B cell numbers (4–6) and the B cells are inactive (8). The importance of T cell regulation was demonstrated by a later study showing restored CSR in DNA-PKcs–null mice upon T cell transplantation from a B cell–deficient host (8).In contrast to Ku and DNA-PKcs, XRCC4 and Lig4 have no identified function outside NHEJ and their inactivation results in the most severe form of NHEJ deficiency. Therefore, XRCC4 and Lig4 are the most suitable and specific targets for abolishing NHEJ. However, the B cell reconstitution strategy is not feasible for XRCC4 or Lig4 because deletion of either gene in mice results in embryonic lethality (9, 10). Although p53 deficiency can extend the life of XRCC4 and Lig4 knockout mice, they all succumb to pro–B cell lymphomas shortly after birth (11, 12). Recently, two independent studies used conditional knockout methods to delete XRCC4 in mature B cells (13, 14). Both studies found that CSR was reduced but not abolished in the absence of XRCC4. It is known that XRCC4-deficient cells have a very low level of Lig4. However, it was also reported that even a very low level of Lig4 is still sufficient for NHEJ (15).To unequivocally determine the role of NHEJ in CSR while avoiding the complications associated with some of the animal models, we disrupted the Lig4 gene in a B cell line (CH12F3) capable of highly efficient cytokine-induced CSR in vitro (16). Like its parental line CH12.LX (17, 18), CH12F3 has a mostly stable diploid genome and is thus suitable for gene targeting. We chose to disrupt the Lig4 gene not only because of its central role in NHEJ but also because it is dispensable for somatic cell growth (19). We found that deletion of Lig4 in CH12F3 cells mildly reduced CSR after 3 d of cytokine stimulation, which is qualitatively and quantitatively similar to what was observed with XRCC4-deficient B cells, as well as the Lig4^−/− p53^−/− B cells (13, 14). However, we also found a proliferation defect of Lig4-null CH12F3 cells in the presence of cytokine stimulation. This led us to another important finding that the initial rate of CSR is severely inhibited in Lig4-null cells. Thus, the initially observed mild reduction appears to be an underestimation of a marked CSR defect, which is masked by an enrichment of switched cells. These observations reveal the kinetics of a fast-acting NHEJ and a slow-acting alternative end joining during CSR.     

A DNA-PKcs mutation in a radiosensitive T–B– SCID patient inhibits Artemis activation and nonhomologous end-joining

Radiosensitive T^–B^– severe combined immunodeficiency (RS-SCID) is caused by defects in the nonhomologous end-joining (NHEJ) DNA repair pathway, which results in failure of functional V(D)J recombination. Here we have identified the first human RS-SCID patient to our knowledge with a DNA-PKcs missense mutation (L3062R). The causative mutation did not affect the kinase activity or DNA end-binding capacity of DNA-PKcs itself; rather, the presence of long P-nucleotide stretches in the immunoglobulin coding joints indicated that it caused insufficient Artemis activation, something that is dependent on Artemis interaction with autophosphorylated DNA-PKcs. Moreover, overall end-joining activity was hampered, suggesting that Artemis-independent DNA-PKcs functions were also inhibited. This study demonstrates that the presence of DNA-PKcs kinase activity is not sufficient to rule out a defect in this gene during diagnosis and treatment of RS-SCID patients. Further, the data suggest that residual DNA-PKcs activity is indispensable in humans.     

Inducible DNA breaks in Ig S regions are dependent on AID and UNG

Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cmu) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination. Uracil DNA glycosylase (UNG) is required for most CSR activity although its role is disputed. Here we use ligation-mediated PCR to detect DSBs in S regions in splenic B cells undergoing CSR. We find that the kinetics of DSB induction corresponds with AID expression, and that DSBs are AID- and UNG-dependent and occur preferentially at G:C basepairs in WRC/GYW AID hotspots. Our results indicate that AID attacks cytosines on both DNA strands, and staggered breaks are processed to blunt DSBs at the initiating ss break sites. We propose a model to explain the types of end-processing events observed.     

Deletion of the nucleotide excision repair gene Ercc1 reduces immunoglobulin class switching and alters mutations near switch recombination junctions

The structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3' single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a mismatch repair (MMR) protein involved in class switch recombination (CSR). Therefore, ERCC1-XPF has abilities that might be useful for antibody CSR. We tested whether ERCC1 is involved in CSR and found that Ercc1(-)(/)(-) splenic B cells show moderately reduced CSR in vitro, demonstrating that ERCC1-XPF participates in, but is not required for, CSR. To investigate the role of ERCC1 in CSR, the nucleotide sequences of switch (S) regions were determined. The mutation frequency in germline Smicro segments and recombined Smicro-Sgamma3 segments cloned from Ercc1(-)(/)(-) splenic B cells induced to switch in culture was identical to that of wild-type (WT) littermates. However, Ercc1(-)(/)(-) cells show increased targeting of the mutations to G:C bp in RGYW/WRCY hotspots and mutations occur at sites more distant from the S-S junctions compared with WT mice. The results indicate that ERCC1 is not epistatic with MMR and suggest that ERCC1 might be involved in processing or repair of DNA lesions in S regions during CSR.     

DNA polymerase beta is able to repair breaks in switch regions and plays an inhibitory role during immunoglobulin class switch recombination

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), which converts cytosines to uracils in switch (S) regions. Subsequent excision of dU by uracil DNA glycosylase (UNG) of the base excision repair (BER) pathway is required to obtain double-strand break (DSB) intermediates for CSR. Since UNG normally initiates faithful repair, it is unclear how the AID-instigated S region lesions are converted into DSBs rather than correctly repaired by BER. Normally, DNA polymerase beta (Polbeta) would replace the dC deaminated by AID, leading to correct repair of the single-strand break, thereby preventing CSR. We address the question of whether Polbeta might be specifically down-regulated during CSR or inhibited from accessing the AID-instigated lesions, or whether the numerous AID-initiated S region lesions might simply overwhelm the BER capacity. We find that nuclear Polbeta levels are induced upon activation of splenic B cells to undergo CSR. When Polbeta(-/-) B cells are activated to switch in culture, they switch slightly better to IgG2a, IgG2b, and IgG3 and have more S region DSBs and mutations than wild-type controls. We conclude that Polbeta attempts to faithfully repair S region lesions but fails to repair them all.     

DNA repair genes are selectively mutated in diffuse large B cell lymphomas

DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis.Normal lymphocyte development and function relies on the successful rearrangement and modification of antigen receptor genes. Diversity of the B cell receptor is largely provided by V(D)J recombination where the variable (V), diversity (D), and joining (J) segments of the immunoglobulin (IG) loci are joined in a combinatorial manner (Jung et al., 2006). After antigen experience, the IG loci are further modified by somatic hypermutation (SHM) and class switch recombination (CSR). The recombination-activating proteins 1 and 2 (RAG1 and RAG2) introduce double-strand breaks (DSBs) at recombination signal sequences located around the V, D, and J genes during V(D)J recombination, whereas activation-induced cytidine deaminase (AID) initiates SHM and CSR by deaminating cytosines to uracils at the V and switch (S) regions of the IG loci (Jung et al., 2006; Di Noia and Neuberger, 2007).A myriad of DNA damage response (DDR) and repair proteins mediate and regulate IG diversification processes. AID activity provokes guanosine/uracil mismatches that are processed by proteins of the base-excision repair (BER) pathway (UNG, APEX1) and mismatch repair (MMR) pathway (MSH2/MSH6, MLH1/PMS2, and EXO1; Di Noia and Neuberger, 2007; Stavnezer et al., 2010). While in the context of CSR such mismatches lead to the generation of DNA DSBs, during SHM, AID activity preferentially results in the establishment of point mutations, although small duplications and deletions may also occur. The resolution of DSBs in V(D)J recombination and CSR is primarily mediated by the nonhomologous end-joining (NHEJ) pathway that becomes activated by DDR proteins such as ATM and Nibrin (NBN; Kotnis et al., 2009). The x-ray repair cross-complementing proteins 4 (XRCC4), XRCC5 (Ku80), and XRCC6 (Ku70) and DNA ligase 4, Artemis, DNA-PKcs, and Cernunnos (XLF) proteins are considered to be the core members of NHEJ (Lieber, 2010).Most B cell neoplasms are thought to originate from antigen-experienced B cells, as tumor cells display SHM at the IG V genes (Klein and Dalla-Favera, 2008). Furthermore, a role for IG diversification mechanisms in the propagation of genomic instability in mature B cell lymphomas is supported by numerous observations. Non-IG genes, including proto-oncogenes such as MYC, BCL6, PIM1, RHOH, or PAX5 are often targeted by SHM, especially in diffuse large B cell lymphomas (DLBCLs; Pasqualucci et al., 2001), one of the most common and aggressive mature B cell lymphoma subtypes. Chromosomal translocations involving the IG loci, with breakpoints in S regions and SHM targets, are also a hallmark of mature B cell lymphomas (Küppers and Dalla-Favera, 2001; Lenz et al., 2007). Furthermore, AID has been shown to be essential for the occurrence of c-myc/IgH translocations and oncogene-driven induction of germinal center–derived lymphomas in mice (Ramiro et al., 2004; Pasqualucci et al., 2008). Of note, the t(14;18) translocation, involving the IGH and the BCL2 loci, characteristic of follicular lymphomas (FLs), is considered to be derived from defective V(D)J recombination processes (Küppers and Dalla-Favera, 2001).Despite the crucial role of DDR and repair proteins during antibody diversification processes, there is lack of evidence that supports their direct involvement in the propagation of genomic instability in human B cell lymphomas. In contrast, individuals with biallelic germline mutations in some of the DNA repair genes that encode proteins involved in IG diversification processes often display an increased risk for development of lymphoid malignancies in addition to immunodeficiency (de Miranda et al., 2011). In this study, we systematically analyzed the coding regions of key DDR and repair genes that have been associated, or could potentially be associated, with IG gene diversification processes in a set of mature B cell lymphomas, with a focus on DLBCL. The defects in a subset of DDR and repair genes identified here, and their association with genomic instability phenotypes, support their role in the tumorigenesis of DLBCL.     

DNA damage-induced cellular senescence is sufficient to suppress tumorigenesis: a mouse model

Tumor suppressor p53-dependent apoptosis is critical in suppressing tumorigenesis. Previously, we reported that DNA double-strand breaks (DSBs) at the V(D)J recombination loci induced genomic instability in the developing lymphocytes of nonhomologous end-joining (NHEJ)-deficient, p53-deficient mice, which led to rapid lymphomagenesis. To test the ability of p53-dependent cell cycle arrest to suppress tumorigenesis in the absence of apoptosis in vivo, we crossbred NHEJ-deficient mice into a mutant p53R172P background; these mice have defects in apoptosis induction, but not cell cycle arrest. These double-mutant mice survived longer than NHEJ/p53 double-null mice and, remarkably, were completely tumor free. We detected accumulation of aberrant V(D)J recombination-related DSBs at the T cell receptor (TCR) locus, and high expression levels of both mutant p53 and cell cycle checkpoint protein p21, but not the apoptotic protein p53-upregulated modulator of apoptosis. In addition, a substantial number of senescent cells were observed among both thymocytes and bone marrow cells. Cytogenetic studies revealed euploidy and limited chromosomal breaks in these lymphoid cells. The results indicate that precursor lymphocytes, which normally possess a high proliferation potential, are able to withdraw from the cell cycle and undergo senescence in response to the persistence of DSBs in a p53-p21-dependent pathway; this is sufficient to inhibit oncogenic chromosomal abnormality and suppress tumorigenesis.     

A new type of radiosensitive T-B-NK+ severe combined immunodeficiency caused by a LIG4 mutation

V(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of mature B and T cells. A subset of T-B-NK+ SCID patients is sensitive to ionizing radiation, and the majority of these patients have mutations in Artemis. We present a patient with a new type of radiosensitive T-B-NK+ SCID with a defect in DNA ligase IV (LIG4). To date, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. The patient described here shows that a LIG4 mutation can also cause T-B-NK+ SCID without developmental defects. The LIG4-deficient SCID patient had an incomplete but severe block in precursor B cell differentiation, resulting in extremely low levels of blood B cells. The residual D(H)-J(H) junctions showed extensive nucleotide deletions, apparently caused by prolonged exonuclease activity during the delayed D(H)-J(H) ligation process. In conclusion, different LIG4 mutations can result in either a developmental defect with minor immunological abnormalities or a SCID picture with normal development.     

Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development

The process of V(D)J recombination is crucial for regulating the development of B cells and for determining their eventual antigen specificity. Here we assess the developmental regulation of the V(D)J recombinase directly, by monitoring the double-stranded DNA breaks produced in the process of V(D)J recombination. This analysis provides a measure of recombinase activity at immunoglobulin heavy and light chain loci across defined developmental stages spanning the process of B cell development. We find that expression of a complete immunoglobulin heavy chain protein is accompanied by a drastic change in the targeting of V(D)J recombinase activity, from being predominantly active at the heavy chain locus in pro-B cells to being exclusively restricted to the light chain loci in pre-B cells. This switch in locus-specific recombinase activity results in allelic exclusion at the immunoglobulin heavy chain locus. Allelic exclusion is maintained by a different mechanism at the light chain locus. We find that immature, but not mature, B cells that already express a functional light chain protein can undergo continued light chain gene rearrangement, by replacement of the original rearrangement on the same allele. Finally, we find that the developmentally regulated targeting of V(D)J recombination is unaffected by enforced rapid transit through the cell cycle induced by an Eμ-myc transgene.     

High frequency of normal DJH joints in B cell progenitors in severe combined immunodeficiency mice

The severe combined immunodeficiency (scid) mouse has a defective V(D)J recombinase activity that results in arrested lymphoid development at the pro-B cell stage in the B lineage. The defect is not absolute and scid mice do attempt gene rearrangement. Indeed, approximately 15% of all scid mice develop detectable levels of oligoclonal serum immunoglobulin and T cell activity. To gain more insight into the scid defect and its effect on V(D)J rearrangement, we analyzed DJH recombination in scid bone marrow. We determined that DJH structures are present in scid bone marrow and occur at a frequency only 10-100 times less than C.B-17+/+. The scid DJH repertoire is limited and resembles fetal liver DJH junctions, with few N insertions and predominant usage of reading frame 1. Moreover, 70% of the DJH structures were potentially productive, indicating that normal V(D)J recombinants should be arising continually.