Classical Nonhomologous End-joining Pathway Research Articles

Comparative analysis of basal and etoposide-induced alterations in gene expression by DNA-PKcs kinase activity.

Background: Maintenance of the genome is essential for cell survival, and impairment of the DNA damage response is associated with multiple pathologies including cancer and neurological abnormalities. DNA-PKcs is a DNA repair protein and a core component of the classical nonhomologous end-joining pathway, but it also has roles in modulating gene expression and thus, the overall cellular response to DNA damage. Methods: Using cells producing either wild-type (WT) or kinase-inactive (KR) DNA-PKcs, we assessed global alterations in gene expression in the absence or presence of DNA damage. We evaluated differential gene expression in untreated cells and observed differences in genes associated with cellular adhesion, cell cycle regulation, and inflammation-related pathways. Following exposure to etoposide, we compared how KR versus WT cells responded transcriptionally to DNA damage. Results: Downregulated genes were mostly involved in protein, sugar, and nucleic acid biosynthesis pathways in both genotypes, but enriched biological pathways were divergent, again with KR cells manifesting a more robust inflammatory response compared to WT cells. To determine what major transcriptional regulators are controlling the differences in gene expression noted, we used pathway analysis and found that many master regulators of histone modifications, proinflammatory pathways, cell cycle regulation, Wnt/β-catenin signaling, and cellular development and differentiation were impacted by DNA-PKcs status. Finally, we have used qPCR to validate selected genes among the differentially regulated pathways to validate RNA sequence data. Conclusion: Overall, our results indicate that DNA-PKcs, in a kinase-dependent fashion, decreases proinflammatory signaling following genotoxic insult. As multiple DNA-PK kinase inhibitors are in clinical trials as cancer therapeutics utilized in combination with DNA damaging agents, understanding the transcriptional response when DNA-PKcs cannot phosphorylate downstream targets will inform the overall patient response to combined treatment.

Cancer Cells Upregulate Tau to Gain Resistance to DNA Damaging Agents

Recent reports suggested a role for microtubules in double-strand-DNA break repair. We herein investigated the role of the microtubule-associated protein Tau in radio- and chemotherapy. Noticeably, a lowered expression of Tau in breast cancer cell lines resulted in a significant decrease in mouse-xenograft breast tumor volume after doxorubicin or X-ray treatments. Furthermore, the knockdown of Tau impaired the classical nonhomologous end-joining pathway and led to an improved cellular response to both bleomycin and X-rays. Investigating the mechanism of Tau's protective effect, we found that one of the main mediators of response to double-stranded breaks in DNA, the tumor suppressor p53-binding protein 1 (53BP1), is sequestered in the cytoplasm as a consequence of Tau downregulation. We demonstrated that Tau allows 53BP1 to translocate to the nucleus in response to DNA damage by chaperoning microtubule protein trafficking. Moreover, Tau knockdown chemo-sensitized cancer cells to drugs forming DNA adducts, such as cisplatin and oxaliplatin, and further suggested a general role of Tau in regulating the nuclear trafficking of DNA repair proteins. Altogether, these results suggest that Tau expression in cancer cells may be of interest as a molecular marker for response to DNA-damaging anti-cancer agents. Clinically targeting Tau could sensitize tumors to DNA-damaging treatments.

MicroRNA-145 Impairs Classical Non-Homologous End-Joining in Response to Ionizing Radiation-Induced DNA Double-Strand Breaks via Targeting DNA-PKcs.

DNA double-strand breaks (DSBs) are one of the most lethal types of DNA damage due to the fact that unrepaired or mis-repaired DSBs lead to genomic instability or chromosomal aberrations, thereby causing cell death or tumorigenesis. The classical non-homologous end-joining pathway (c-NHEJ) is the major repair mechanism for rejoining DSBs, and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical factor in this pathway; however, regulation of DNA-PKcs expression remains unknown. In this study, we demonstrate that miR-145 directly suppresses DNA-PKcs by binding to the 3′-UTR and inhibiting translation, thereby causing an accumulation of DNA damage, impairing c-NHEJ, and rendering cells hypersensitive to ionizing radiation (IR). Of note, miR-145-mediated suppression of DNA damage repair and enhanced IR sensitivity were both reversed by either inhibiting miR-145 or overexpressing DNA-PKcs. In addition, we show that the levels of Akt1 phosphorylation in cancer cells are correlated with miR-145 suppression and DNA-PKcs upregulation. Furthermore, the overexpression of miR-145 in Akt1-suppressed cells inhibited c-NHEJ by downregulating DNA-PKcs. These results reveal a novel miRNA-mediated regulation of DNA repair and identify miR-145 as an important regulator of c-NHEJ.

DNA damage and repair in the hematopoietic system.

Although hematopoietic stem cells (HSCs) in the bone marrow are in a state of quiescence, they harbor the self-renewal capacity and the pluripotency to differentiate into mature blood cells when needed, which is key to maintain hematopoietic homeostasis. Importantly, HSCs are characterized by their long lifespan ( e. g., up to 60 months for mice), display characteristics of aging, and are vulnerable to various endogenous and exogenous genotoxic stresses. Generally, DNA damage in HSCs is endogenous, which is typically induced by reactive oxygen species (ROS), aldehydes, and replication stress. Mammalian cells have evolved a complex and efficient DNA repair system to cope with various DNA lesions to maintain genomic stability. The repair machinery for DNA damage in HSCs has its own characteristics. For instance, the Fanconi anemia (FA)/BRCA pathway is particularly important for the hematopoietic system, as it can limit the damage caused by DNA inter-strand crosslinks, oxidative stress, and replication stress to HSCs to prevent FA occurrence. In addition, HSCs prefer to utilize the classical non-homologous end-joining pathway, which is essential for the V(D)J rearrangement in developing lymphocytes and is involved in double-strand break repair to maintain genomic stability in the long-term quiescent state. In contrast, the base excision repair pathway is less involved in the hematopoietic system. In this review, we summarize the impact of various types of DNA damage on HSC function and review our knowledge of the corresponding repair mechanisms and related human genetic diseases.

Ku70 suppresses alternative end joining in G1-arrested progenitor B cells

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.

CRISPR-Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology has been applied in plant breeding mainly on genes for improving single or multiple traits1-4. Here we show that this technology can also be used to restructure plant chromosomes. Using the Cas9 nuclease from Staphylococcus aureus5, we were able to induce reciprocal translocations in the Mbp range between heterologous chromosomes in Arabidopsis thaliana. Of note, translocation frequency was about five times more efficient in the absence of the classical non-homologous end-joining pathway. Using egg-cell-specific expression of the Cas9 nuclease and consecutive bulk screening, we were able to isolate heritable events and establish lines homozygous for the translocation, reaching frequencies up to 2.5% for individual lines. Using molecular and cytological analysis, we confirmed that the chromosome-arm exchanges we obtained between chromosomes 1 and 2 and between chromosomes 1 and 5 of Arabidopsis were conservative and reciprocal. The induction of chromosomal translocations enables mimicking of genome evolution or modification of chromosomes in a directed manner, fixing or breaking genetic linkages between traits on different chromosomes. Controlled restructuring of plant genomes has the potential to transform plant breeding.

COM1, a factor of alternative non-homologous end joining, lagging behind the classic non-homologous end joining pathway in rice somatic cells.

The role of rice (Oryza sativa) COM1 in meiotic homologous recombination (HR) is well understood, but its part in somatic double-stranded break (DSB) repair remains unclear. Here, we show that for rice plants COM1 conferred tolerance against DNA damage caused by the chemicals bleomycin and mitomycin C, while the COM1 mutation did not compromise HR efficiencies and HR factor (RAD51 and RAD51 paralogues) localization to irradiation-induced DSBs. Similar retarded growth at the post-germination stage was observed in the com1-2 mre11 double mutant and the mre11 single mutant, while combined mutations in COM1 with the HR pathway gene (RAD51C) or classic non-homologous end joining (NHEJ) pathway genes (KU70, KU80, and LIG4) caused more phenotypic defects. In response to γ-irradiation, COM1 was loaded normally onto DSBs in the ku70 mutant, but could not be properly loaded in the MRE11RNAi plant and in the wortmannin-treated wild-type plant. Under non-irradiated conditions, more DSB sites were occupied by factors (MRE11, COM1, and LIG4) than RAD51 paralogues (RAD51B, RAD51C, and XRCC3) in the nucleus of wild-type; protein loading of COM1 and XRCC3 was increased in the ku70 mutant. Therefore, quite differently to its role for HR in meiocytes, rice COM1 specifically acts in an alternative NHEJ pathway in somatic cells, based on the Mre11-Rad50-Nbs1 (MRN) complex and facilitated by PI3K-like kinases. NHEJ factors, not HR factors, preferentially load onto endogenous DSBs, with KU70 restricting DSB localization of COM1 and XRCC3 in plant somatic cells.

Causes and Effects of Loss of Classical Nonhomologous End Joining Pathway in Parasitic Eukaryotes.

We report frequent losses of components of the classical nonhomologous end joining pathway (C-NHEJ), one of the main eukaryotic tools for end joining repair of DNA double-strand breaks, in several lineages of parasitic protists. Moreover, we have identified a single lineage among trypanosomatid flagellates that has lost Ku70 and Ku80, the core C-NHEJ components, and accumulated numerous insertions in many protein-coding genes. We propose a correlation between these two phenomena and discuss the possible impact of the C-NHEJ loss on genome evolution and transition to the parasitic lifestyle.IMPORTANCE Parasites tend to evolve small and compact genomes, generally endowed with a high mutation rate, compared with those of their free-living relatives. However, the mechanisms by which they achieve these features, independently in unrelated lineages, remain largely unknown. We argue that the loss of the classical nonhomologous end joining pathway components may be one of the crucial steps responsible for characteristic features of parasite genomes.

Both the classical and alternative non-homologous end joining pathways contribute to the fusion of drastically shortened telomeres induced by TRF2 overexpression

ABSTRACTThe double-stranded telomeric binding protein TRF2 is expressed in many human cancers at elevated levels. Moreover, experimental overexpression of TRF2 in human cells causes replication stalling in telomeric tracts, which leads to drastic telomere shortening and fusion of deprotected chromosome ends. To understand which end joining pathway is involved in mediating these chromosome fusions, we overexpressed TRF2 in human HCT116 cell lines that were deficient for the DNA Ligase 4 (Lig4)-dependent classical non-homologous end joining (C-NHEJ) or the DNA Ligase 3 (Lig3)-dependent alternative non-homologous end joining (A-NHEJ) pathway. Surprisingly, abrogation of either Lig4 or nuclear Lig3 significantly reduced inter-chromosomal fusion of drastically shortened telomeres, suggesting that both the C-NHEJ and A-NHEJ pathways are involved in mediating this type of fusion. Fusion between deprotected sister chromatids, however, only required the Lig3-dependent A-NHEJ pathway. Interestingly, a previous study reported similar end joining pathway requirements for the fusion of critically shortened telomeres during a telomere attrition-based cellular crisis. We speculate that, as in cellular crisis, the same repair pathway(s) may drive clonal and genomic evolution in human cancers containing elevated TRF2 levels.

The Lysine Histone Methyltransferase SETD2 Is Required for Appropriate Immunoglobulin VDJ Recombination

Rearrangement of the immunoglobulin heavy chain locus (IgH) through non-homologous end-joining (NHEJ)-mediated VDJ recombination is a requisite step in normal lymphocyte development. This process is initiated through cleavage of DNA by RAG1/2 recombinases and repair by classical NHEJ pathway factors. Loss or defects of these factors can impair end-processing (DNA-PKcs, ARTEMIS) or end-ligation (KU70/80, XRCC4, XLF, LIGIV) of rearrangement and are drivers of disease, notably, severe combined immunodeficiency (SCID). In addition to these core NHEJ factors, other proteins, such as ATM kinase, have been shown to be critical for the stabilization of the post-cleavage VDJ intermediates to ensure proper signal joint formation. While epigenetic regulation of this process has been described through the maintenance of accessibility for transcription factors and RAG1/2, very little is known about the role of chromatin modifications on NHEJ-mediated VDJ recombination.Histone H3 lysine-36 tri-methylation (H3K36me3) is a histone modification associated with actively transcribed genes and has been shown to be critical for many biological processes, including transcription, mismatch repair and homologous recombination. Tri-methylation is catalyzed by the non-redundant histone methyltransferase, SETD2. Loss of function mutations in SETD2 or dominant negative onco-histone mutations in the H3K36 residue have been described in a broad array of tumors, including several hematopoietic malignancies. To study the role of Setd2 in hematopoiesis, we generated conditional knockout mice, where loss of Setd2 results in the ablation of the H3K36me3 mark. While heterozygous mice had no overt phenotype, mice with homozygous loss of Setd2(Setd2KO) displayed aberrant hematopoiesis, with a significant depletion of lymphoid primed progenitor population and B/T-cells in hematopoietic tissues. Setd2 loss early in hematopoiesis lead to a profound block of B-cell development at the proB cell stage and an early block in T-cell development, in a compartment where TCRβ rearrangements initiate. In Setd2KO mice, V to DJ rearrangements of the IgH locus were impaired, suggesting that the block at the proB stage of development was due to defective VDJ recombination. To assess this, we crossed our knockout mice with an IgHelMD4 mouse strain, and found that the block at the proB stage could be bypassed by the expression of a transgenic, fully rearranged IgH locus.We confirmed global loss of H3K36me3 in proB cells from our Setd2KO mice by ChIP-Seq and found that the ablation of this mark did not affect germline IgH locus accessibility (by ATAC-seq) or local chromatin architecture (H3K4me3 or H3K9ac) at a well-studied critical regulatory region near the Eμ enhancer. Loss of Setd2 and H3K36me3 only mildly effected sterile transcription of IgH genes and did not effect the overall cellular proliferative rate or the recruitment of NHEJ factors. Furthermore, loss of Setd2 did not cooperate to generate B-cell lymphomas in a p53-/- background. However, sequencing of VDJ recombination products from knockout mice indicated not only decreased clonality of products and variable gene usage but also resulted in alterations of complementarity defining region-3 (CDR3) hypervariable sequences. These results phenocopy defects or loss of NHEJ end-ligation factors and suggest that Setd2 and H3K36me3 could play a pivotal role in the end-ligation steps of NHEJ.Defects in end-ligation and resolution of VDJ intermediates have previously been shown to result in the formation of aberrant coding-signal hybrid joints. To see if this was occurring with loss of Setd2, we generated knockout murine proB v-Abl transformed lines where we could induce κ light chain rearrangement upon addition of an Abl kinase inhibitor. Remarkably, upon induction in Setd2KO v-Abl lines, we detected the formation of hybrid joints by both PCR and by the loss of a GFP-based coding-joint formation signal from a reporter expressed in these cells. We additionally could detect hybrid joint formation in the endogenous κ light chain locus of Setd2KO splenocytes, further indicating a role for Setd2/H3K36me3 in the end-ligation process. These studies demonstrate a novel and critical role for SETD2 and H3K36me3 in preventing aberrant, non-functional VDJ end joining of NHEJ-induced DNA breaks during lymphocyte development. DisclosuresNo relevant conflicts of interest to declare.

TDP1 suppresses mis-joining of radiomimetic DNA double-strand breaks and cooperates with Artemis to promote optimal nonhomologous end joining.

The Artemis nuclease and tyrosyl-DNA phosphodiesterase (TDP1) are each capable of resolving protruding 3′-phosphoglycolate (PG) termini of DNA double-strand breaks (DSBs). Consequently, both a knockout of Artemis and a knockout/knockdown of TDP1 rendered cells sensitive to the radiomimetic agent neocarzinostatin (NCS), which induces 3′-PG-terminated DSBs. Unexpectedly, however, a knockdown or knockout of TDP1 in Artemis-null cells did not confer any greater sensitivity than either deficiency alone, indicating a strict epistasis between TDP1 and Artemis. Moreover, a deficiency in Artemis, but not TDP1, resulted in a fraction of unrepaired DSBs, which were assessed as 53BP1 foci. Conversely, a deficiency in TDP1, but not Artemis, resulted in a dramatic increase in dicentric chromosomes following NCS treatment. An inhibitor of DNA-dependent protein kinase, a key regulator of the classical nonhomologous end joining (C-NHEJ) pathway sensitized cells to NCS, but eliminated the sensitizing effects of both TDP1 and Artemis deficiencies. These results suggest that TDP1 and Artemis perform different functions in the repair of terminally blocked DSBs by the C-NHEJ pathway, and that whereas an Artemis deficiency prevents end joining of some DSBs, a TDP1 deficiency tends to promote DSB mis-joining.

Eltrombopag Promotes DNA Repair in Human Hematopoietic Stem and Progenitor Cells: Implications for the Treatment of Fanconi Anemia

Fanconi anemia (FA) is an inherited genomic instability syndrome resulting from loss-of-function mutations in any one of at least 21 FANC genes critical for DNA repair. Accumulation of unresolved DNA double strand breaks (DSBs) results in chromosomal rearrangement, accounting in part for the high incidence of bone marrow failure (BMF) and leukemia in patients with FA. Safe, curative options are currently unavailable. Recent investigations have uncovered a specific function of thrombopoietin (TPO), a key regulator of hematopoietic stem/progenitor cell (HSPC) self-renewal and survival, in promoting DNA damage response in these cells. TPO was shown to specifically increase the efficiency of non-homologous end joining (NHEJ), the predominant repair mechanism for DSBs in HSPCs; however, recombinant TPO is no longer approved for clinical applications. The alternative orally bioavailable small molecule TPO receptor agonist, eltrombopag (epag), was recently used in clinical trials to successfully treat life-threatening cytopenias in patients with acquired BMF. In this study, we investigate whether epag can promote NHEJ DSB repair in human HSPCs and thus provide a potential new therapeutic modality for patients with FA. To assess DNA repair activity of epag in FA CD34+ HSPCs, a population of cells that is markedly reduced in these patients, CD34+ HSPCs from 6 healthy individuals were subjected to CRISPR/Cas9-induced knockout mutations in FANCA, the most commonly mutated gene in FA. These FA HSPCs were cultured for 24 hr in the presence of early-acting cytokines, SCF and Flt3-L (designated “SF”), or SF supplemented with epag (“SFE”) or TPO (“SFT”) prior to induction of DSBs by exposure to 2Gy γ-irradiation (IR). Cells were then cultured for an additional 1, 5, or 24 hr to assess the kinetics of DNA repair, as measured by decreases in H2AX phosphorylation (γH2AX), an indicator of IR-induced DSBs. Maximal H2AX phosphorylation was observed 1 hr after IR of FA HSPCs and was similar for all culture conditions (>90% γH2AX+ cells), indicating that epag and TPO do not prevent DSB formation or phosphorylation of H2AX. Five hours after IR, most FA HSPCs cultured with epag (Fig. A) or TPO (Fig. B) had resolved the IR-induced DSBs, while much higher percentages of γH2AX+ cells persisted in the control SF group. The observed effect was specific to epag and TPO; removal of SCF had no significant impact on DNA repair. Regardless of culture condition, the majority of FA HSPCs either resolved DSBs or progressed through apoptosis by 24 hr post-IR. These findings indicate that epag and TPO increase the kinetics of DSB repair in FA HSPCs. To gain insight into the primary mechanism of DNA repair promoted by epag and TPO, we inhibited DNA-PK, an essential component of the classical NHEJ (C-NHEJ) pathway. Addition of a DNA-PK inhibitor (NU7441) had no impact on DSB formation measured at 1 hr or on DNA repair at 24 hr, but completely abrogated the enhanced kinetics of DSB repair observed at 5 hr with epag (Fig. A) and TPO (Fig. B). These data indicate that culturing FA HSPCs with epag or TPO promotes the fast-acting DNA-PK-dependent C-NHEJ DNA repair mechanism, a pathway known to promote genomic stability. In contrast, cells cultured without epag or TPO resolved DSBs using a slower, DNA-PK-independent alternative NHEJ (alt-NHEJ) mechanism, a pathway associated with genomic instability. Shunting of DSB repair in rapid C-NHEJ with epag (Fig. C) or TPO (Fig. D) was associated with substantial increase in survival of γ-irradiated FA HSPCs compared with control (SF) groups. In contrast, when C-NHEJ DNA repair was inhibited with NU7441, the cell survival benefit observed with epag (Fig. C) or TPO (Fig. D) was abolished. In colony forming unit (CFU) progenitor assays, γ-irradiated HSPCs cultured with epag or TPO yielded 4- to 6-fold more CFUs than control SF groups (Fig. E). When γ-irradiated HSPCs were transplanted into immunodeficient NSG mice, a 2-fold increase in human cell engraftment was observed in cultures containing epag or TPO compared to controls (p<0.01), suggesting activation of DNA repair activity by these cytokines in cells with long-term repopulating capacity (Fig. F). Overall, our data indicate that epag and TPO enhance DSB repair in human HSPCs by promoting the fast-operating and faithful C-NHEJ pathway. A phase II clinical trial is in development to assess safety and efficacy of epag in the treatment of hematological manifestations of FA. [Display omitted] DisclosuresGuenther:Agilent Laboratories: Other: Stipend, Research Funding; Norvartis: Research Funding. Cheruku:Novartis: Other: Stipend, Research Funding. Smith:Novartis: Research Funding; Agilent Laboratories: Research Funding. Larochelle:StemCell Technologies: Patents & Royalties: StemDiff Hematopoietic Kit; Novartis: Research Funding; Novartis: Research Funding.

Deletion of TpKu70 facilitates gene targeting in Talaromyces pinophilus and identification of TpAmyR involvement in amylase production.

Talaromyces pinophilus is a promising filamentous fungus for industrial production of biomass-degrading enzymes used in biorefining, and its genome was recently sequenced and reported. However, functional analysis of genes in T. pinophilus is rather limited owing to lack of genetic tools. In this study, a putative TpKu70 encoding the Ku70 homolog involved in the classic non-homologous end-joining pathway was deleted in T. pinophilus 1-95. ΔTpKu70 displayed no apparent defect in vegetative growth and enzyme production, and presented similar sensitivity to benomyl, bleomycin, and UV, when compared with the wild-type T. pinophilus strain 1-95. Seven genes that encode putative transcription factors, including TpAmyR, were successfully knocked out in ΔTpKu70 at 61.5-100% of homologous recombination frequency, which is significantly higher than that noted in the wild-type. Interestingly, ΔTpAmyR produced approximately 20% of amylase secreted by the parent strain ΔTpKu70 in medium containing soluble starch from corn as the sole carbon source. Real-time quantitative reverse transcription PCR showed that TpAmyR positively regulated the expression of genes encoding α-amylase and glucoamylase. Thus, this study provides a useful tool for genetic analysis of T. pinophilus, and identification of a key role for the transcription factor TpAmyR in amylase production in T. pinophilus.

The ATM Kinase Restrains Joining of Both VDJ Signal and Coding Ends.

The evidence that ATM affects resolution of RAG-induced DNA double-strand breaks is profuse and unequivocal; moreover, it is clear that the RAG complex itself cooperates (in an undetermined way) with ATM to facilitate repair of these double-strand breaks by the classical nonhomologous end-joining pathway. The mechanistic basis for the cooperation between ATM and the RAG complex has not been defined, although proposed models invoke ATM and RAG2's C terminus in maintaining the RAG postcleavage complex. In this study, we show that ATM reduces the rate of both coding and signal joining in a robust episomal assay; we suggest that this is the result of increased stability of the postcleavage complex. ATM's ability to inhibit VDJ joining requires its enzymatic activity. The noncore C termini of both RAG1 and RAG2 are also required for ATM's capacity to limit signal (but not coding) joining. Moreover, potential phosphorylation targets within the C terminus of RAG2 are also required for ATM's capacity to limit signal joining. These data suggest a model whereby the RAG signal end complex is stabilized by phosphorylation of RAG2 by ATM.

Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes

DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome.

Restoration of ATM Expression in DNA-PKcs-Deficient Cells Inhibits Signal End Joining.

Unlike most DNA-dependent protein kinase, catalytic subunit (DNA-PKcs)-deficient mouse cell strains, we show in the present study that targeted deletion of DNA-PKcs in two different human cell lines abrogates VDJ signal end joining in episomal assays. Although the mechanism is not well defined, DNA-PKcs deficiency results in spontaneous reduction of ATM expression in many cultured cell lines (including those examined in this study) and in DNA-PKcs-deficient mice. We considered that varying loss of ATM expression might explain differences in signal end joining in different cell strains and animal models, and we investigated the impact of ATM and/or DNA-PKcs loss on VDJ recombination in cultured human and rodent cell strains. To our surprise, in DNA-PKcs-deficient mouse cell strains that are proficient in signal end joining, restoration of ATM expression markedly inhibits signal end joining. In contrast, in DNA-PKcs-deficient cells that are deficient in signal end joining, complete loss of ATM enhances signal (but not coding) joint formation. We propose that ATM facilitates restriction of signal ends to the classical nonhomologous end-joining pathway.

Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

High-throughput, genome-wide translocation sequencing (HTGTS) studies of activated B cells have revealed that DNA double-strand breaks (DSBs) capable of translocating to defined bait DSBs are enriched around the transcription start sites (TSSs) of active genes. We used the HTGTS approach to investigate whether a similar phenomenon occurs in primary neural stem/progenitor cells (NSPCs). We report that breakpoint junctions indeed are enriched around TSSs that were determined to be active by global run-on sequencing analyses of NSPCs. Comparative analyses of transcription profiles in NSPCs and B cells revealed that the great majority of TSS-proximal junctions occurred in genes commonly expressed in both cell types, possibly because this common set has higher transcription levels on average than genes transcribed in only one or the other cell type. In the latter context, among all actively transcribed genes containing translocation junctions in NSPCs, those with junctions located within 2 kb of the TSS show a significantly higher transcription rate on average than genes with junctions in the gene body located at distances greater than 2 kb from the TSS. Finally, analysis of repair junction signatures of TSS-associated translocations in wild-type versus classical nonhomologous end-joining (C-NHEJ)-deficient NSPCs reveals that both C-NHEJ and alternative end-joining pathways can generate translocations by joining TSS-proximal DSBs to DSBs on other chromosomes. Our studies show that the generation of transcription-associated DSBs is conserved across divergent cell types.

Aberrant recombination and repair during immunoglobulin class switching in BRCA1-deficient human B cells

Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute to genome integrity and tumor suppression. Its participation in the repair of DNA double-strand breaks (DSBs) during homologous recombination (HR) is well recognized, whereas its involvement in the second major DSB repair pathway, nonhomologous end-joining (NHEJ), remains controversial. Here we have studied the role of BRCA1 in the repair of DSBs in switch (S) regions during immunoglobulin class switch recombination, a physiological, deletion/recombination process that relies on the classical NHEJ machinery. A shift to the use of microhomology-based, alternative end-joining (A-EJ) and increased frequencies of intra-S region deletions as well as insertions of inverted S sequences were observed at the recombination junctions amplified from BRCA1-deficient human B cells. Furthermore, increased use of long microhomologies was found at recombination junctions derived from E3 ubiquitin-protein ligase RNF168-deficient, Fanconi anemia group J protein (FACJ, BRIP1)-deficient, or DNA endonuclease RBBP8 (CtIP)-compromised cells, whereas an increased frequency of S-region inversions was observed in breast cancer type 2 susceptibility protein (BRCA2)-deficient cells. Thus, BRCA1, together with its interaction partners, seems to play an important role in repairing DSBs generated during class switch recombination by promoting the classical NHEJ pathway. This may not only provide a general mechanism underlying BRCA1's function in maintaining genome stability and tumor suppression but may also point to a previously unrecognized role of BRCA1 in B-cell lymphomagenesis.

DNA double strand break repair in mitosis is suppressed by phosphorylation of XRCC4.

DNA double strand break repair in mitosis is suppressed by phosphorylation of XRCC4.

Reprint of "Functional overlaps between XLF and the ATM-dependent DNA double strand break response".

Developing B and T lymphocytes generate programmed DNA double strand breaks (DSBs) during the V(D)J recombination process that assembles exons that encode the antigen-binding variable regions of antibodies. In addition, mature B lymphocytes generate programmed DSBs during the immunoglobulin heavy chain (IgH) class switch recombination (CSR) process that allows expression of different antibody heavy chain constant regions that provide different effector functions. During both V(D)J recombination and CSR, DSB intermediates are sensed by the ATM-dependent DSB response (DSBR) pathway, which also contributes to their joining via classical non-homologous end-joining (C-NHEJ). The precise nature of the interplay between the DSBR and C-NHEJ pathways in the context of DSB repair via C-NHEJ remains under investigation. Recent studies have shown that the XLF C-NHEJ factor has functional redundancy with several members of the ATM-dependent DSBR pathway in C-NHEJ, highlighting unappreciated major roles for both XLF as well as the DSBR in V(D)J recombination, CSR and C-NHEJ in general. In this review, we discuss current knowledge of the mechanisms that contribute to the repair of DSBs generated during B lymphocyte development and activation with a focus on potential functionally redundant roles of XLF and ATM-dependent DSBR factors.