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Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand breaks (DSBs) throughout interphase but predominates in G1 phase when homologous recombination is unavailable. Complexes containing the Ku70/80 (“Ku”) and XRCC4/ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Lig4 are absolutely required for joining RAG1/2 endonuclease (“RAG”)-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Lig4-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA, and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially in association with G1-phase down-regulation of Lig1. Finally, we suggest that differential impacts of Ku deficiency versus Lig4 deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-deficient developing lymphocyte and neuronal cells.

Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but predominates in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 (“Ku”) and XRCC4/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Ligase IV are absolutely required for joining RAG1/2-endonucease (“RAG”)-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Ligase IV also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Ligase IV-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially, in association with G1-phase down-regulation of Ligase1. Finally, we suggest that differential impacts of Ku-deficiency versus Lig4-deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-defcient developing lymphocyte and neuronal cells.Significance StatementAlternative end-joining (A-EJ) is implicated in oncogenic translocations and mediating DNA double-strand break (DSB) repair in cycling cells when classical nonhomologous endjoining (C-NHEJ) factors of the C-NHEJ Ligase complex are absent. However, V(D)J recombination-associated DSBs that occur in G1 cell cycle-phase progenitor lymphocytes are joined exclusively by the C-NHEJ pathway. Until now, however, the overall mechanisms that join general DSBs in G1-phase progenitor B cells had not been fully elucidated. Here, we report that Ku, a core C-NHEJ double-strand break recognition complex, directs repair of a variety of different targeted DSBs towards C-NHEJ and suppresses A-EJ in G1-phase cells. We suggest this Ku activity explains how Ku-deficiency can rescue the neuronal development and embryonic lethality phenotype of Ligase 4-deficient mice.

SUMMARYThe representation of odor in olfactory cortex (piriform) is distributive and unstructured and can only be afforded behavioral significance upon learning. We performed 2-photon imaging to examine the representation of odors in piriform and in two downstream stations, the orbitofrontal cortex (OFC) and medial prefrontal cortex (mPFC), as mice learned olfactory associations. In piriform we observed minor changes in neural activity unrelated to learning. In OFC, 30% of the neurons acquired robust responses to conditioned stimuli (CS+) after learning, and these responses were gated by context and internal state. The representation in OFC, however, diminished after learning and persistent representations of CS+ and CS− odors emerged in mPFC. Optogenetic silencing indicates that these two brain structures function sequentially to consolidate the learning of appetitive associations. These data demonstrate the transformation of a representation of odor identity in piriform into transient and persistent representations of value in the prefrontal cortex.

The representation of odor in olfactory cortex (piriform) is distributive and unstructured and can only be afforded behavioral significance upon learning. We performed 2-photon imaging to examine the representation of odors in piriform and in two downstream areas, the orbitofrontal cortex (OFC) and the medial prefrontal cortex (mPFC), as mice learned olfactory associations. In piriform, we observed that odor responses were largely unchanged during learning. In OFC, 30% of the neurons acquired robust responses to conditioned stimuli (CS+) after learning, and these responses were gated by internal state and task context. Moreover, direct projections from piriform to OFC can be entrained to elicit learned olfactory behavior. CS+ responses in OFC diminished with continued training, whereas persistent representations of both CS+ and CS- odors emerged in mPFC. Optogenetic silencing indicates that these two brain structures function sequentially to consolidate the learning of appetitive associations.

Classical non-homologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but is thought to predominate in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/Ligase4 core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While such factors are exclusively required for joining RAG-initiated DSBs during V(D)J recombination in G1-phase lymphocyte progenitors, cycling cells deficient for core C-NHEJ factors join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination to C-NHEJ has been attributed to RAG-mediated exclusion of A-EJ; however, it remains unclear whether A-EJ is similarly excluded from more general DSBs in G1. Here, we report that Ku actively and robustly suppresses A-EJ of both RAG- and Cas9-mediated DSBs in G1-arrested progenitor B cell lines. Thus, while Cas9/gRNA DSBs remain as free broken ends in Ligase4-deficient G1-arrested progenitor B cells, deletion of Ku70 in Ligase4-deficient cells restores DSB re-joining and translocation to levels observed in Ku70-deficient counterparts. Correspondingly, while V(D)J recombination is abrogated in Ligase4-deficient lines, V(D)J-like joining occurs in Ku70-deficient and Ku70/Ligase4-double-deficient lines through a translocation-based A-EJ mechanism. We conclude that in G1, Ligase4-deficient progenitor B cells are functionally end-joining deficient due to a complete Ku-dependent block in A-EJ. Thus, the differential impacts of Ku deficiency versus XRCC4/Ligase4 deficiency on V(D)J recombination, severity of neuronal apoptosis, and embryonic development, including ability of Ku-deficiency to rescue Ligase4-deficient embryonic lethality, may be explained by Ku-mediated inhibition of A-EJ in the G1 cell cycle phase. Citation Format: Richard L. Frock, Vipul Kumar, Zhouyi Liang, Jeff Zurita, Zhou Du, Sherry G. Lin, Cristian Boboila, Frederick W. Alt. Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1745.

Classical nonhomologous DNA end-joining (C-NHEJ), which is a major DNA double-strand break (DSB) repair pathway in mammalian cells, plays a dominant role in joining DSBs during Ig heavy chain (IgH) class switch recombination (CSR) in activated B lymphocytes. However, in B cells deficient for one or more requisite C-NHEJ factors, such as DNA ligase 4 (Lig4) or XRCC4, end-joining during CSR occurs by a distinct alternative end-joining (A-EJ) pathway. A-EJ also has been implicated in joining DSBs found in oncogenic chromosomal translocations. DNA ligase 3 (Lig3) and its cofactor XRCC1 are widely considered to be requisite A-EJ factors, based on biochemical studies or extrachromosomal substrate end-joining studies. However, potential roles for these factors in A-EJ of endogenous chromosomal DSBs have not been tested. Here, we report that Xrcc1 inactivation via conditional gene-targeted deletion in WT or XRCC4-deficient primary B cells does not have an impact on either CSR or IgH/c-myc translocations in activated B lymphocytes. Indeed, homozygous deletion of Xrcc1 does not impair A-EJ of I-SceI–induced DSBs in XRCC4-deficient pro–B-cell lines. Correspondingly, substantial depletion of Lig3 in Lig4-deficient primary B cells or B-cell lines does not impair A-EJ of CSR-mediated DSBs or formation of IgH/c-myc translocations. Our findings firmly demonstrate that XRCC1 is not a requisite factor for A-EJ of chromosomal DSBs and raise the possibility that DNA ligase 1 (Lig1) may contribute more to A-EJ than previously considered.

To be added., Immunoglobulin heavy chain (IgH) class-switch recombination (CSR) replaces initially expressed Cμ (IgM) constant regions (CH) exons with downstream CH exons. Stimulation of B cells with anti-CD40 plus interleukin-4 induces CSR from Cμ to Cγ1 (IgG1) and Cε (IgE), the latter of which contributes to the pathogenesis of atopic diseases. Although Cε CSR can occur directly from Cμ, most mature peripheral B cells undergo CSR to Cε indirectly, namely from Cμ to Cγ1, and subsequently to Cε. Physiological mechanisms that influence CSR to Cγ1 versus Cε are incompletely understood. In this study, we report a role for B cell developmental maturity in IgE CSR. Based in part on a novel flow cytometric IgE CSR assay, we show that immature B cells preferentially switch to IgE versus IgG1 through a mechanism involving increased direct CSR from Cμ to Cε. Our findings suggest that IgE dysregulation in certain immunodeficiencies may be related to impaired B cell maturation.

The loss of a classical non-homologous end-joining (NHEJ) repair factor, XLF, shows strong effects in non-lymphoid cells, but in lymphoid cells its absence surprisingly has only modest effects on V(D)J recombination. Frederick Alt and colleagues show that in lymphoid cells, two other repair factors — ATM kinase and histone protein H2AX — have functional redundancy with XLF. Thus, mice that are deficient in both ATM and XLF have compromised conventional NHEJ, although alternative end-joining is retained. The results hint that the redundant function in end-joining that XLF has with both ATM and H2AX may be related to a role for ATM in chromatin accessibility. Although loss of XLF, a classical non-homologous DNA end-joining (NHEJ) repair factor, shows strong effects in non-lymphoid cells, in lymphoid cells its absence has only modest effects on V(D)J recombination. This study now shows that in lymphoid cells, two other repair factors — ATM kinase and histone protein H2AX — have functional redundancy with XLF. Thus, mice deficient in both ATM and XLF have compromised conventional NHEJ, although alternative end-joining is retained. The results hint that the redundant function in end-joining that XLF has with both ATM and H2AX may have to do with an ATM role in chromatin accessibility. Classical non-homologous DNA end-joining (NHEJ) is a major mammalian DNA double-strand-break (DSB) repair pathway. Deficiencies for classical NHEJ factors, such as XRCC4, abrogate lymphocyte development, owing to a strict requirement for classical NHEJ to join V(D)J recombination DSB intermediates1,2. The XRCC4-like factor (XLF; also called NHEJ1) is mutated in certain immunodeficient human patients and has been implicated in classical NHEJ3,4,5,6; however, XLF-deficient mice have relatively normal lymphocyte development and their lymphocytes support normal V(D)J recombination5. The ataxia telangiectasia-mutated protein (ATM) detects DSBs and activates DSB responses by phosphorylating substrates including histone H2AX7. However, ATM deficiency causes only modest V(D)J recombination and lymphocyte developmental defects, and H2AX deficiency does not have a measurable impact on these processes7,8,9. Here we show that XLF, ATM and H2AX all have fundamental roles in processing and joining DNA ends during V(D)J recombination, but that these roles have been masked by unanticipated functional redundancies. Thus, combined deficiency of ATM and XLF nearly blocks mouse lymphocyte development due to an inability to process and join chromosomal V(D)J recombination DSB intermediates. Combined XLF and ATM deficiency also severely impairs classical NHEJ, but not alternative end-joining, during IgH class switch recombination. Redundant ATM and XLF functions in classical NHEJ are mediated by ATM kinase activity and are not required for extra-chromosomal V(D)J recombination, indicating a role for chromatin-associated ATM substrates. Correspondingly, conditional H2AX inactivation in XLF-deficient pro-B lines leads to V(D)J recombination defects associated with marked degradation of unjoined V(D)J ends, revealing that H2AX has a role in this process.

Ig heavy chain (IgH) class-switch recombination (CSR) replaces the IgH Cμ constant region exons with one of several sets of downstream IgH constant region exons (e.g., Cγ, Cε, or Cα), which affects switching from IgM to another IgH class (e.g., IgG, IgE, or IgA). Activation-induced cytidine deaminase (AID) initiates CSR by promoting DNA double-strand breaks (DSBs) within switch (S) regions flanking the donor Cμ (Sμ) and a downstream acceptor CH(e.g., Sγ, Sε, Sα) that are then joined to complete CSR. DSBs generated in Sμ frequently are joined within Sμ to form internal switch region deletions (ISD). AID-induced ISD and mutations have been considered rare in downstream S regions, suggesting that AID targeting to these S regions requires its prior recruitment to Sμ. We have now assayed for CSR and ISD in B cells lacking Sμ (Sμ−/−B cells). In Sμ−/−B cells activated for CSR to IgG1 and IgE, CSR to IgG1 was greatly reduced; but, surprisingly, CSR to IgE occurred at nearly normal levels. Moreover, normal B cells had substantial Sγ1 ISD and increased mutations in and near Sγ1, and levels of both were greatly increased in Sμ−/−B cells. Finally, Sμ−/−B cells underwent downstream CSR between Sγ1 and Sε on alleles that lacked Sμ CSR to these sequences. Our findings show that AID targets downstream S regions independently of CSR with Sμ and implicate an alternative pathway for IgE class switching that involves generation and joining of DSBs within two different downstream S regions before Sμ joining.

Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain ( IgH ) constant region exons. CSR is completed by joining a DSB in the donor Sμ to a DSB in a downstream acceptor S region (e.g., Sγ1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into Sμ during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both Sμ and Sγ1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

Analytical methods generally use a wide range of statistical mathematical functions, such as the linear function, the hyperbolic function, the higher-order parabolic function and the exponential function to better approximate economic trends. The testing of a wide range of functions is mandatory, in order to choose the ones that offer the possibility of predicting trends more accurately. In the current report, we introduce several functions and compare them on datasets concerning foreign trade of Romania over the 1996 to 2000 period. We conclude that some of the complexities of transitioning from a communist to a capitalist economy, as reflected in the foreign trade, are best captured by complex and logistic functions defined below. Furthermore, we describe a seasonal variation in the volume of trade, which may be characteristic for all transition economies in East-European countries.

Disparate models for the development of peripheral B cells may reflect significant heterogeneity in recirculating long-lived B cells that have not been previously accounted for. We show in this study that the murine recirculating B cell pool contains two distinct, long-lived, posttransitional, follicular B cell populations. Follicular Type I IgMlow B cells require Ag-derived and Btk-dependent signals for their development and make up the majority of cells in the recirculating follicular B cell pool. Follicular type II B cells do not require Btk- or Notch-2-derived signals, make up about a third of the long-lived recirculating B cell pool, and can develop in the absence of Ag. These two follicular populations exhibit differences in basal tyrosine phosphorylation and in BCR-induced proliferation, suggesting that they may represent functionally distinct populations of long-lived recirculating B cells.

NF-κB1 and Notch2 are both required for the development of marginal zone (MZ) B cells. Analysis of B lymphocyte development in mice that are doubly heterozygous at the Notch2 and NF-κB1 loci revealed synergism between Notch2 and NF-κB1 during MZ B cell development. Two known transcriptional targets of the Notch pathway, Hes-5 and Deltex-1, were found to be preferentially expressed in MZ B cells and regulated by NF-κB1. These studies provide in vivo evidence for a genetic interaction between the Notch and NF-κB pathways.

The addition of nontemplated (N) nucleotides to coding ends in V(D)J recombination is the result of the action of a unique DNA polymerase, TdT. Although N-nucleotide addition by TdT plays a critical role in the generation of a diverse repertoire of Ag receptor genes, the mechanism by which TdT acts remains unclear. We conducted a structure-function analysis of the murine TdT protein to determine the roles of individual structural motifs that have been implicated in protein-protein and protein-DNA interactions important for TdT function in vivo. This analysis demonstrates that the N-terminal portion of TdT, including the BRCA-1 C-terminal (BRCT) domain, is not required for TdT activity, although the BRCT domain clearly contributes quantitatively to N-nucleotide addition activity. The second helix-hairpin-helix domain of TdT, but not the first, is required for activity. Deletional analysis also suggested that the entire C-terminal region of TdT is necessary for N-nucleotide addition in vivo. The long isoform of TdT was found to reduce N-nucleotide addition by the short form of TdT, but did not increase nucleotide deletion from coding ends in either human or rodent nonlymphoid cells. We consider these results in light of the recently reported structure of the catalytic region of TdT.

The Mre11 protein has well-documented functions in the repair of DNA double-strand breaks via homologous recombination. Now, several new studies reveal that Mre11 also has a role in mammalian DNA double-strand break repair by nonhomologous end joining.

Classical nonhomologous end joining (C-NHEJ) is one of the two major known pathways for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Our understanding of C-NHEJ has been derived, in significant part, through studies of programmed physiologic DNA DSBs formed during V(D)J recombination in the developing immune system. Studies of immunoglobulin heavy-chain (IgH) class-switch recombination (CSR) also have revealed that there is an "alternative" end-joining process (A-EJ) that can function, relatively robustly, in the repair of DSBs in activated mature B lymphocytes. This A-EJ process has also been implicated in the formation of oncogenic translocations found in lymphoid tumors. In this review, we discuss our current understanding of C-NHEJ and A-EJ in the context of V(D)J recombination, CSR, and the formation of chromosomal translocations.

Classical nonhomologous end joining (C-NHEJ) is one of the two major known pathways for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Our understanding of C-NHEJ has been derived, in significant part, through studies of programmed physiologic DNA DSBs formed during V(D)J recombination in the developing immune system. Studies of immunoglobulin heavy-chain (IgH) class-switch recombination (CSR) also have revealed that there is an "alternative" end-joining process (A-EJ) that can function, relatively robustly, in the repair of DSBs in activated mature B lymphocytes. This A-EJ process has also been implicated in the formation of oncogenic translocations found in lymphoid tumors. In this review, we discuss our current understanding of C-NHEJ and A-EJ in the context of V(D)J recombination, CSR, and the formation of chromosomal translocations.

Recurrent chromosomal abnormalities, especially chromosomal translocations, are strongly associated with certain subtypes of leukemia, lymphoma and solid tumors. The appearance of particular translocations or associated genomic alterations can be important indicators of disease prognosis, and in some cases, certain translocations may indicate appropriate therapy protocols. To date, most of our knowledge about chromosomal translocations has derived from characterization of the highly selected recurrent translocations found in certain cancers. Until recently, mechanisms that promote or suppress chromosomal translocations, in particular, those responsible for their initiation, have not been addressed. For translocations to occur, two distinct chromosomal loci must be broken, brought together (synapsed) and joined. Here, we discuss recent findings on processes and pathways that influence the initiation of chromosomal translocations, including the generation fo DNA double strand breaks (DSBs) by general factors or in the context of the Lymphocyte-specific V(D)J and IgH class-switch recombination processes. We also discuss the role of spatial proximity of DSBs in the interphase nucleus with respect to how DSBs on different chromosomes are justaposed for joining. In addition, we discuss the DNA DSB response and its role in recognizing and tethering chromosomal DSBs to prevent translocations, as well as potential roles of the classical and alternative DSB end-joining pathways in suppressing or promoting translocations. Finally, we discuss the potential roles of long range regulatory elements, such as the 3'IgH enhancer complex, in promoting the expression of certain translocations that are frequent in lymphomas and, thereby, contributing to their frequent appearance in tumors.

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 CH 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.

Immunoglobulin variable region exons are assembled in developing B cells by V(D)J recombination. Once mature, these cells undergo class-switch recombination (CSR) when activated by antigen. CSR changes the heavy chain constant region exons (Ch) expressed with a given variable region exon from Cmu to a downstream Ch (for example, Cgamma, Cepsilon or Calpha), thereby switching expression from IgM to IgG, IgE or IgA. Both V(D)J recombination and CSR involve the introduction of DNA double-strand breaks and their repair by means of end joining. For CSR, double-strand breaks are introduced into switch regions that flank Cmu and a downstream Ch, followed by fusion of the broken switch regions. In mammalian cells, the 'classical' non-homologous end joining (C-NHEJ) pathway repairs both general DNA double-strand breaks and programmed double-strand breaks generated by V(D)J recombination. C-NHEJ, as observed during V(D)J recombination, joins ends that lack homology to form 'direct' joins, and also joins ends with several base-pair homologies to form microhomology joins. CSR joins also display direct and microhomology joins, and CSR has been suggested to use C-NHEJ. Xrcc4 and DNA ligase IV (Lig4), which cooperatively catalyse the ligation step of C-NHEJ, are the most specific C-NHEJ factors; they are absolutely required for V(D)J recombination and have no known functions other than C-NHEJ. Here we assess whether C-NHEJ is also critical for CSR by assaying CSR in Xrcc4- or Lig4-deficient mouse B cells. C-NHEJ indeed catalyses CSR joins, because C-NHEJ-deficient B cells had decreased CSR and substantial levels of IgH locus (immunoglobulin heavy chain, encoded by Igh) chromosomal breaks. However, an alternative end-joining pathway, which is markedly biased towards microhomology joins, supports CSR at unexpectedly robust levels in C-NHEJ-deficient B cells. In the absence of C-NHEJ, this alternative end-joining pathway also frequently joins Igh locus breaks to other chromosomes to generate translocations.

Protein kinase C-associated kinase (PKK; DIK/RIP4) is an ankyrin-repeat containing serine/threonine receptor-interacting protein (RIP)-family kinase that can activate NFkappaB, and is required for keratinocyte development. In earlier studies, the expression of a catalytically inactive mutant of PKK in the B cell lineage resulted in a marked decrease in peripheral B cells in the spleen and a severe reduction of B-1 B cells. Here we explore the consequences of a null mutation in PKK with respect to the generation of peripheral B cell lineages and the activation of NFkappaB. We show that PKK is not required for the production of B cells in the bone marrow or for the development and maintenance of all mature B lymphocyte populations. We also show that PKK is not required for the activation of NFkappaB downstream of the BCR, CD40, or TLR-4 in B cells. Taken together, these data demonstrate that the loss of this RIP-family kinase does not compromise B lymphocyte development and maintenance, but leaves open the possibility that PKK may have a redundant role in these processes.

The classical nonhomologous end-joining (C-NHEJ) DNA double-strand break (DSB) repair pathway employs the Ku70/80 complex (Ku) for DSB recognition and the XRCC4/DNA ligase 4 (Lig4) complex for ligation. During IgH class switch recombination (CSR) in B lymphocytes, switch (S) region DSBs are joined by C-NHEJ to form junctions either with short microhomologies (MHs; “MH-mediated” joins) or no homologies (“direct” joins). In the absence of XRCC4 or Lig4, substantial CSR occurs via “alternative” end-joining (A-EJ) that generates largely MH-mediated joins. Because upstream C-NHEJ components remain in XRCC4- or Lig4-deficient B cells, residual CSR might be catalyzed by C-NHEJ using a different ligase. To address this, we have assayed for CSR in B cells deficient for Ku70, Ku80, or both Ku70 and Lig4. Ku70- or Ku80-deficient B cells have reduced, but still substantial, CSR. Strikingly, B cells deficient for both Ku plus Lig4 undergo CSR similarly to Ku-deficient B cells, firmly demonstrating that an A-EJ pathway distinct from C-NHEJ can catalyze CSR end-joining. Ku-deficient or Ku- plus Lig4-deficient B cells are also biased toward MH-mediated CSR joins; but, in contrast to XRCC4- or Lig4-deficient B cells, generate substantial numbers of direct CSR joins. Our findings suggest that more than one form of A-EJ can function in CSR.