Defective Non-homologous End Joining Research Articles

Abstract PR06: Ovarian cancers harbor defects in nonhomologous end joining resulting in error prone repair and resistance to rucaparib

Abstract Introduction: The most toxic forms of DNA damage, double strand breaks (DSBs), are repaired by two main pathways - homologous recombination repair (HRR) and non-homologous end joining (NHEJ). Fifty percent of ovarian cancers have defects in HRR resulting in hypersensitivity to platinum and PARP inhibitors. When NHEJ is defective, DSBs with mismatched ends can be joined by an error-prone mechanism, involving joining between regions of nucleotide microhomology, but this is highly mutagenic. Defects in the NHEJ pathway may therefore contribute to genomic instability and have been associated with resistance development. In this study we demonstrate that NHEJ is defective, resulting in error prone repair, in ~50% of ovarian cancers. Methods: In a panel of ovarian cancer cell lines (OVCAR3, SKOV3, IGROV-1, A2780, CP70 and MDAH) and 30 primary ovarian cancer cultures generated from ascites collected at the time of surgery, we assessed NHEJ using an assay measuring the ability of cell extracts to end-join linearized plasmid monomers into multimers. We determined the effect of inhibition of DNA-PK by concurrent treatment with a DNA-PK inhibitor (NU7441) on the cytotoxicity of the PARP inhibitor, rucaparib, using the sulforhodamine B (SRB) assay. mRNA expression of DNA-PK, XRCC4, LIG4, KU70 and KU80 was determined using qRT-PCR normalised to GAPDH expression. DNA-PK function was determined by immunofluorescence assessment of auto-phosphorylation of DNA-PK one hour after 2Gy irradiation. Furthermore, we assessed the effect of NU7441 on the homologous recombination function using the γH2AX / RAD51 foci formation assay in a BRCA1 defective cell line. Results: We demonstrated that many cell lines and primary ovarian cancer cultures were able to rejoin compatible DNA-ends accurately, however the amount of rejoining of mismatched ends varied significantly between cultures and four out of six cell lines and 14 of 30 primary cultures assessed were unable to accurately rejoin DSBs with mismatched ends resulting in large deletions and error prone repair. This process was not dependent on expression of DNA-PK, XRCC4 or LIG4. Also, whilst in cell lines the autophosphorylation of DNA-PK correlated with error free rejoining, accurate rejoining in primary cultures was independent of DNA-PK auto-phosphorylation. Error-prone repair in ovarian cancer extracts correlated with reduced expression of KU70 (p=0.006) and KU80 (p=0.009). We found that defects in the NHEJ pathway are associated with resistance to rucaparib in primary ovarian cultures (p=0.02). Furthermore concurrent treatment with the DNA-PK inhibitor was cytostatic and induced resistance to rucaparib in the majority of ovarian cancer cultures. These findings are supported by the finding of HR function recovery in a BRCA1 defective cell line treated with DNA-PK inhibitor. Conclusion: This study has shown, for the first time, that some ovarian cancers are unable to perform accurate end joining, such that error-prone joining predominates. Error prone repair is associated with resistance to PARP inhibitors in vitro. Error prone rejoining is highly mutagenic and may contribute to the genomic instability and development of resistance that is observed in ovarian cancer. This abstract is also presented as Poster A31. Citation Format: Aiste McCormick, Michelle Dixon, Rachel ODonnell, Nicola J. Curtin, Richard J. Edmondson. Ovarian cancers harbor defects in nonhomologous end joining resulting in error prone repair and resistance to rucaparib. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr PR06.

53BP1 Is Limiting for NHEJ Repair in ATM-deficient Model Systems That Are Subjected to Oncogenic Stress or Radiation

The DNA damage response (DDR) factors ataxia telangiectasia mutated (ATM) and p53 binding protein 1 (53BP1) function as tumor suppressors in humans and mice, but the significance of their mutual interaction to the suppression of oncogenic translocations in vivo has not been investigated. To address this question, the phenotypes of compound mutant mice lacking 53BP1 and ATM (Trp53bp1(-/-)/Atm(-/-)), relative to single mutants, were examined. These analyses revealed that loss of 53BP1 markedly decreased the latency of T-lineage lymphomas driven by RAG-dependent oncogenic translocations in Atm(-/-) mice (average survival, 14 and 23 weeks for Trp53bp1(-/-)/Atm(-/-) and Atm(-/-) mice, respectively). Mechanistically, 53BP1 deficiency aggravated the deleterious effect of ATM deficiency on nonhomologous end-joining (NHEJ)-mediated double-strand break repair. Analysis of V(D)J recombinase-mediated coding joints and signal joints in Trp53bp1(-/-)/Atm(-/-) primary thymocytes is, however, consistent with canonical NHEJ-mediated repair. Together, these findings indicate that the greater NHEJ defect in the double mutant mice resulted from decreased efficiency of rejoining rather than switching to an alternative NHEJ-mediated repair mechanism. Complementary analyses of irradiated primary cells indicated that defects in cell-cycle checkpoints subsequently function to amplify the NHEJ defect, resulting in more frequent chromosomal breaks and translocations in double mutant cells throughout the cell cycle. Finally, it was determined that 53BP1 is dispensable for the formation of RAG-mediated hybrid joints in Atm(-/-) thymocytes but is required to suppress large deletions in a subset of hybrid joints. The current study uncovers novel ATM-independent functions for 53BP1 in the suppression of oncogenic translocations and in radioprotection.

Msl2 Is a Novel Component of the Vertebrate DNA Damage Response

hMSL2 (male-specific lethal 2, human) is a RING finger protein with ubiquitin ligase activity. Although it has been shown to target histone H2B at lysine 34 and p53 at lysine 351, suggesting roles in transcription regulation and apoptosis, its function in these and other processes remains poorly defined. To further characterize this protein, we have disrupted the Msl2 gene in chicken DT40 cells. Msl2−/− cells are viable, with minor growth defects. Biochemical analysis of the chromatin in these cells revealed aberrations in the levels of several histone modifications involved in DNA damage response pathways. DNA repair assays show that both Msl2−/− chicken cells and hMSL2-depleted human cells have defects in non-homologous end joining (NHEJ) repair. DNA damage assays also demonstrate that both Msl2 and hMSL2 proteins are modified and stabilized shortly after induction of DNA damage. Moreover, hMSL2 mediates modification, presumably ubiquitylation, of a key DNA repair mediator 53BP1 at lysine 1690. Similarly, hMSL1 and hMOF (males absent on the first) are modified in the presence of hMSL2 shortly after DNA damage. These data identify a novel role for Msl2/hMSL2 in the cellular response to DNA damage. The kinetics of its stabilization suggests a function early in the NHEJ repair pathway. Moreover, Msl2 plays a role in maintaining normal histone modification profiles, which may also contribute to the DNA damage response.

BCLAF1 is a radiation-induced H2AX-interacting partner involved in γH2AX-mediated regulation of apoptosis and DNA repair

H2AX, a histone H2A variant, has a key role in the cellular response to DNA double-strand breaks (DSBs). H2AX senses DSBs through rapid serine 139 phosphorylation, concurrently leading to the formation of phospho-(γ)H2AX foci with various proteins. However, in the cells with different sensitivity to ionizing radiation (IR)-induced DSBs, still incomplete are those specific proteins selectively recruited by γH2AX to decide different cell fates. Because the abundance of γH2AX indicates the extent of DSBs, we first identified IR-induced dose-dependent H2AX-interacting partners and found that Bcl-2-associated transcription factor 1 (BCLAF1/Btf) showed enhanced association with γH2AX only under high-dose radiation. In acutely irradiated cells, BCLAF1 promoted apoptosis of irreparable cells through disturbing p21-mediated inhibition of Caspase/cyclin E-dependent, mitochondrial-mediated pathways. Meanwhile, BCLAF1 co-localized with γH2AX foci in nuclei and stabilized the Ku70/DNA-PKcs complex therein, facilitating non-homologous end joining (NHEJ)-based DSB repair in surviving cells. In tumor cells, BCLAF1 was intrinsically suppressed, leading to formation of anti-apoptotic Ku70–Bax complexes and disruption of Ku70/DNA-PKcs complexes, all of which contribute to tumor-associated apoptotic resistance and cell survival with defective NHEJ DNA repair. For the first time, our studies reveal that, based on the extent of DNA damage, BCLAF1 is involved in the γH2AX-mediated regulation of apoptosis and DNA repair, and is a γH2AX-interacting tumor suppressor.

The human DEK oncogene regulates DNA damage response signaling and repair

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)-dependent protein kinase catalytic subunit (DNA-PKcs)-null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice.

Yeast Nej1 Is a Key Participant in the Initial End Binding and Final Ligation Steps of Nonhomologous End Joining

In Saccharomyces cerevisiae, the key components of the nonhomologous end joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs) are yeast Ku (yKu), Mre11-Rad50-Xrs2, Dnl4-Lif1, and Nej1. Here, we examined the role of Nej1 in NHEJ by a combination of molecular genetic and biochemical approaches. As expected, the recruitment of Nej1 to in vivo DSBs is dependent upon yKu. Surprisingly, Nej1 is required for the stable binding of yKu to in vivo DSBs, in addition to Dnl4-Lif1. Thus, Nej1 and Dnl4-Lif1 are independently recruited by yKu to in vivo DSBs, forming a stable ternary complex that channels DSBs into the NHEJ pathway. In accord with these results, purified Nej1 interacts with yKu and preferentially binds to DNA ends bound by yKu. Furthermore, the binding of a mixture of Nej1 and Dnl4-Lif1 to DNA ends bound by yKu is greater than the sum of the binding of the individual proteins, indicating that pairwise interactions among yKu, Nej1, and Dnl4-Lif1 contribute to complex assembly at DNA ends. Nej1 stimulates intermolecular ligation by Dnl4-Lif1, but, more interestingly, the addition of Nej1 results in more than one intermolecular ligation per Dnl4 molecule. Thus, Nej1 not only plays an important role in determining repair pathway choice by participating in the initial NHEJ complex formed at DSBs but also contributes to the reactivation of Dnl4-Lif1 after repair is complete, thereby increasing the capacity of the NHEJ repair pathway.

ARTEMIS stabilizes the genome and modulates proliferative responses in multipotent mesenchymal cells

BackgroundUnrepaired DNA double-stranded breaks (DSBs) cause chromosomal rearrangements, loss of genetic information, neoplastic transformation or cell death. The nonhomologous end joining (NHEJ) pathway, catalyzing sequence-independent direct rejoining of DSBs, is a crucial mechanism for repairing both stochastically occurring and developmentally programmed DSBs. In lymphocytes, NHEJ is critical for both development and genome stability. NHEJ defects lead to severe combined immunodeficiency (SCID) and lymphoid cancer predisposition in both mice and humans. While NHEJ has been thoroughly investigated in lymphocytes, the importance of NHEJ in other cell types, especially with regard to tumor suppression, is less well documented. We previously reported evidence that the NHEJ pathway functions to suppress a range of nonlymphoid tumor types, including various classes of sarcomas, by unknown mechanisms.ResultsHere we investigate roles for the NHEJ factor ARTEMIS in multipotent mesenchymal stem/progenitor cells (MSCs), as putative sarcomagenic cells of origin. We demonstrate a key role for ARTEMIS in sarcoma suppression in a sensitized mouse tumor model. In this context, we found that ARTEMIS deficiency led to chromosomal damage but, paradoxically, enhanced resistance and proliferative potential in primary MSCs subjected to various stresses. Gene expression analysis revealed abnormally regulated stress response, cell proliferation, and signal transduction pathways in ARTEMIS-defective MSCs. Finally, we identified candidate regulatory genes that may, in part, mediate a stress-resistant, hyperproliferative phenotype in preneoplastic ARTEMIS-deficient MSCs.ConclusionsOur discoveries suggest that Art prevents genome damage and restrains proliferation in MSCs exposed to various stress stimuli. We propose that deficiency leads to a preneoplastic state in primary MSCs and is associated with aberrant proliferative control and cellular stress resistance. Thus, our data reveal surprising new roles for ARTEMIS and the NHEJ pathway in normal MSC function and fitness relevant to tumor suppression in mesenchymal tissues.

Delineation of the Xrcc4-interacting Region in the Globular Head Domain of Cernunnos/XLF

In mammals, the majority of DNA double-strand breaks are processed by the nonhomologous end-joining (NHEJ) pathway, composed of seven factors: Ku70, Ku80, DNA-PKcs, Artemis, Xrcc4 (X4), DNA-ligase IV (L4), and Cernunnos/XLF. Cernunnos is part of the ligation complex, constituted by X4 and L4. To improve our knowledge on the structure and function of Cernunnos, we performed a systematic mutagenesis study on positions selected from an analysis of the recent three-dimensional structures of this factor. Ten of 27 screened mutants were nonfunctional in several DNA repair assays. Outside amino acids critical for the expression and stability of Cernunnos, we identified three amino acids (Arg(64), Leu(65), and Leu(115)) essential for the interaction with X4 and the proper function of Cernunnos. Docking the crystal structures of the two factors further validated this probable interaction surface of Cernunnos with X4.

DNA-PKcs deficiency in human: long predicted, finally found

To describe new immunological and molecular findings of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a new candidate gene for radiosensitive T-B-severe combined immunodeficiency (SCID), which has implications for the diagnostic strategy of T-B-SCID. The first human mutation in the gene encoding DNA-PKcs (PRKDC) has been identified in a radiosensitive T-B-SCID patient. A mutation in the DNA-PKcs gene has been predicted for a long time, but spontaneous mutations had only been identified in mouse, horse and dog models. DNA-PKcs is a key player in the nonhomologous end joining (NHEJ) pathway of DNA double strand break repair. Correct V(D)J recombination of T cell receptor and immunoglobulin genes is fully dependent on NHEJ, as it is involved in the formation of coding and signal joints. Therefore, a NHEJ defect results in absence of T and B cells. The DNA-PKcs deficient patient presented as a classical SCID patient, not different from a recombination activating gene or Artemis deficiency. The mutation concerned a hypomorphic missense mutation (L3062R) that did not result in absence of protein expression nor in deficient in vivo or in vitro (auto)phosphorylation. Although mutated DNA-PKcs was still able to recruit Artemis to the site of DNA damage, it was probably defective in Artemis activation. In the spontaneous animal models, however, the kinase activity was completely lost, which is essentially different from the human mutation. This observation suggests that some aspects of the DNA-PKcs function are unique to humans.

Characterization of a Natural Mutator Variant of Human DNA Polymerase λ which Promotes Chromosomal Instability by Compromising NHEJ

BackgroundDNA polymerase lambda (Polλ) is a DNA repair polymerase, which likely plays a role in base excision repair (BER) and in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSB).Principal FindingsHere, we described a novel natural allelic variant of human Polλ (hPolλ) characterized by a single nucleotide polymorphism (SNP), C/T variation in the first base of codon 438, resulting in the amino acid change Arg to Trp. In vitro enzyme activity assays of the purified W438 Polλ variant revealed that it retained both DNA polymerization and deoxyribose phosphate (dRP) lyase activities, but had reduced base substitution fidelity. Ectopic expression of the W438 hPolλ variant in mammalian cells increases mutation frequency, affects the DSB repair NHEJ pathway, and generates chromosome aberrations. All these phenotypes are dependent upon the catalytic activity of the W438 hPolλ.ConclusionsThe expression of a cancer-related natural variant of one specialized DNA polymerase can be associated to generic instability at the cromosomal level, probably due a defective NHEJ. These results establish that chromosomal aberrations can result from mutations in specialized DNA repair polymerases.

The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair

Purpose Squamous cell carcinomas (SCCs) are characterized by moderate radiosensitivity. We have established the human head & neck SCC cell line SKX, which shows an exceptionally high radiosensitivity. It was the aim of this study to understand the underlying mechanisms. Materials & methods Experiments were performed with SKX and FaDu, the latter taken as a control of moderate radiosensitivity. Cell lines were grown as xenografts as well as cell cultures. For xenografts, radiosensitivity was determined via local tumour control assay, and for cell cultures using colony assay. For cell cultures, apoptosis was determined by Annexin V staining and G1-arrest by BrdU labelling. Double-strand breaks (DSBs) were detected by both constant-field gel electrophoresis (CFGE) and γH2AX-foci technique; DSB rejoining was also assessed by in vitro rejoining assay; chromosomal damage was determined by G01-assay. Results Compared to FaDu, SKX cells are extremely radiosensitive as found for both xenografts (TCD 50 for 10 fractions 46.0 Gy [95% C.I.: 39; 54 Gy] vs. 18.9 Gy [95% C.I.: 13; 25 Gy]) and cell cultures (D 0.01; 7.1 vs. 3.5 Gy). Both cell lines showed neither radiation-induced apoptosis nor radiation-induced permanent G1-arrest. For DSBs, there was no difference in the induction but for repair with SKX cells showing a higher level of both, slowly repaired DSBs and residual DSBs. The in vitro DSB repair assay revealed that SKX cells are defective in nonhomologous endjoining (NHEJ), and that more than 40% of DSBs are rejoined by single-strand annealing (SSA). SKX cells also depicted a two-fold higher number of lethal chromosomal aberrations when compared to FaDu cells. Conclusions The extreme radiosensitivity of the SCC SKX seen both in vivo and in vitro can be ascribed to a reduced DNA double-strand break repair, resulting from a defect in NHEJ. This defect might be due to preferred usage of other pathways, such as SSA, which prevents efficient endjoining.

A novel missense RAG-1 mutation results in T−B−NK+ SCID in Athabascan-speaking Dine Indians from the Canadian Northwest Territories

DNA double-strand repair factors in the non-homologous end joining (NHEJ) pathway resolve DNA double-strand breaks introduced by the recombination-activating gene (RAG) proteins during V(D)J recombination of T and B lymphocyte receptor genes. Defective NHEJ and subsequent failure of V(D)J recombination leads to severe combined immunodeficiency disease (SCID). We originally linked T(-)B(-)NK(+) SCID in Athabascan-speaking Native Americans in the Southwestern US and Northwest Territories of Canada to chromosome 10. However, despite a common ancestry, the null mutation in the Artemis gene that we found to be causal in the SCID among the Navajo and Apache Indians was not present in the Dine Indians in the Northwest Territories. We now report a novel homozygous missense mutation (R776W) in RAG-1 in three children with T(-)B(-)NK(+) SCID from two related families of Athabascan-speaking Dine Indians in the Canadian Northwest Territories. As expected, we found no increased sensitivity to ionizing radiation in patient fibroblasts. The impaired activity of this RAG-1 mutant in V(D)J recombination was confirmed by the EGFP-based V(D)J recombination assays. Overexpression of wild type RAG-1 in patient fibroblasts complemented V(D)J recombination, with recovery of both coding and signal joint formation. Our results indicate that the novel R776W missense mutation in RAG-1 is causal in the T(-)B(-)NK(+) SCID phenotype in Athabascan-speaking Dine Indians from the Canadian Northwest Territories.

Transient disruption of non-homologous end-joining facilitates targeted genome manipulations in the filamentous fungus Aspergillus nidulans

We have developed a transiently disrupted nkuA system in Aspergillus nidulans for efficient gene targeting. The nkuA disruption was made by inserting a counter-selectable marker flanked by a direct repeat (DR) composed of nkuA sequences. In the disrupted state, the non-homologous end-joining (NHEJ) activity is abolished and gene targeting can be performed with success rates identical to those obtained with permanent nkuA knock-out strains. When gene targeting is complete, the functional nkuA allele can be re-established via a simple selection step, thereby eliminating the risk that defective NHEJ influences subsequent analyses of the manipulated strain. Our system will facilitate construction of large numbers of defined mutations in A. nidulans. Moreover, as the system can likely be adapted to other filamentous fungi, we expect it will be particularly beneficial in species where NHEJ cannot be restored by sexual crossing.

The Bre5/Ubp3 ubiquitin protease complex from budding yeast contributes to the cellular response to DNA damage

The ubiquitination status of proteins can control numerous aspects of protein function through targeted destruction or by altering protein–protein interactions, subcellular localization, or enzymatic activity. In addition to enzymes that mediate the conjugation of ubiquitin moieties to target proteins, there are enzymes that catalyze the removal of ubiquitin, termed ubiquitin proteases. One such ubiquitin protease, Ubp3, exists in a complex with a partner protein: Bre5. This complex has been implicated in a variety of cellular activities, and was recently identified in large-scale screens for genetic interactions with known components of the DNA damage response pathway. We found that this complex plays a role in the cellular response to the DNA damaging agent phleomycin and strains lacking the complex have a defect in non-homologous end joining. Although this complex is also important for telomeric silencing, maintenance of the cell wall, and global transcriptional regulation, we present evidence suggesting that the role of this complex in DNA damage responses is distinct from these other roles. First, we found that Ubp3/Bre5 functions antagonistically with Bul1 in DNA damage responses, but not in its other cellular functions. Additionally, we have generated mutants of Bre5 that are specifically defective in DNA damage responses.

Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres.

Long interspersed element-1 (LINE-1 or L1) elements are abundant, non-long-terminal-repeat (non-LTR) retrotransposons that comprise approximately 17% of human DNA. The average human genome contains approximately 80-100 retrotransposition-competent L1s (ref. 2), and they mobilize by a process that uses both the L1 endonuclease and reverse transcriptase, termed target-site primed reverse transcription. We have previously reported an efficient, endonuclease-independent L1 retrotransposition pathway (EN(i)) in certain Chinese hamster ovary (CHO) cell lines that are defective in the non-homologous end-joining (NHEJ) pathway of DNA double-strand-break repair. Here we have characterized EN(i) retrotransposition events generated in V3 CHO cells, which are deficient in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and have both dysfunctional telomeres and an NHEJ defect. Notably, approximately 30% of EN(i) retrotransposition events insert in an orientation-specific manner adjacent to a perfect telomere repeat (5'-TTAGGG-3'). Similar insertions were not detected among EN(i) retrotransposition events generated in controls or in XR-1 CHO cells deficient for XRCC4, an NHEJ factor that is required for DNA ligation but has no known function in telomere maintenance. Furthermore, transient expression of a dominant-negative allele of human TRF2 (also called TERF2) in XRCC4-deficient XR-1 cells, which disrupts telomere capping, enables telomere-associated EN(i) retrotransposition events. These data indicate that L1s containing a disabled endonuclease can use dysfunctional telomeres as an integration substrate. The findings highlight similarities between the mechanism of EN(i) retrotransposition and the action of telomerase, because both processes can use a 3' OH for priming reverse transcription at either internal DNA lesions or chromosome ends. Thus, we propose that EN(i) retrotransposition is an ancestral mechanism of RNA-mediated DNA repair associated with non-LTR retrotransposons that may have been used before the acquisition of an endonuclease domain.

XLF Interacts with the XRCC4-DNA Ligase IV Complex to Promote DNA Nonhomologous End-Joining

DNA nonhomologous end-joining (NHEJ) is a predominant pathway of DNA double-strand break repair in mammalian cells, and defects in it cause radiosensitivity at the cellular and whole-organism levels. Central to NHEJ is the protein complex containing DNA Ligase IV and XRCC4. By searching for additional XRCC4-interacting factors, we identified a previously uncharacterized 33 kDa protein, XRCC4-like factor (XLF, also named Cernunnos), that has weak sequence homology with XRCC4 and is predicted to display structural similarity to XRCC4. We show that XLF directly interacts with the XRCC4-Ligase IV complex in vitro and in vivo and that siRNA-mediated downregulation of XLF in human cell lines leads to radiosensitivity and impaired NHEJ. Furthermore, we establish that NHEJ-deficient 2BN cells derived from a radiosensitive and immune-deficient patient lack XLF due to an inactivating frameshift mutation in its gene, and that reintroduction of wild-type XLF into such cells corrects their radiosensitivity and NHEJ defects. XLF thus constitutes a novel core component of the mammalian NHEJ apparatus.

Repair of DNA Damage Induced by Accelerated Heavy Ions in Mammalian Cells Proficient and Deficient in the Non-homologous End-Joining Pathway

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/microm carbon ions, and 200 keV/microm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = approximately 1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G(1) premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.

Non-homologous End-Joining Defect in Fanconi Anemia Hematopoietic Cells Exposed to Ionizing Radiation

Fanconi anemia is a genetically heterogeneous recessive disease characterized mainly by bone marrow failure and cancer predisposition. Although it is accepted that Fanconi cells are highly sensitive to DNA crosslinking agents, their response to ionizing radiation is still unclear. Using pulsed-field gel electrophoresis, we have observed that radiation generates a similar number of DNA double-strand breaks in normal and Fanconi cells from three (FA-A, FA-C and FA-F) of the 11 complementation groups identified. Nonsynchronized as well as nonproliferating Fanconi anemia cells showed an evident defect in rejoining the double-strand breaks generated by ionizing radiation, indicating defective non-homologous end-joining repair. At the cellular level, no difference in the radiosensitivity of normal and FA-A lymphoblast cells was noted, and a modest increase in the radiosensitivity of Fanca-/- hematopoietic progenitor cells was observed compared to Fanca+/+ cells. Finally, when animals were exposed to a fractionated total-body irradiation of 5 Gy, a similar hematopoietic syndrome was observed in wild-type and Fanca-/- mice. Taken together, our observations suggest that Fanconi cells, in particular those having nonfunctional Fanconi proteins upstream of FANCD2, have a defect in the non-homologous end-joining repair of double-strand breaks produced by ionizing radiation, and that compensatory mechanisms of DNA repair and/or stem cell regeneration should limit the impact of this defect in irradiated organisms.

The role of BRCA1 in non-homologous end-joining

From the genotypic viewpoint, single nucleotide polymorphisms in the genes of the non-homologous end-joining (NHEJ) pathway, which is important in the repair of DNA double-strand breaks, have been shown to be associated with increased breast cancer risk. However, more phenotypic evidence is needed to strengthen the link between defective NHEJ genes and breast cancer development. Recently, BRCA1-deficient mouse embryonic fibroblasts were found to have significantly reduced NHEJ activity, suggesting an accessory role of BRCA1 in NHEJ. Since BRCA1 is a well-documented breast cancer susceptibility gene, this association between NHEJ and BRCA1 not only suggests a role of BRCA1 in NHEJ, but also provides support for the tumorigenic contribution of the NHEJ pathway to breast cancer development. Interestingly, the phenotypic data show that BRCA1 may promote only specific subtypes of NHEJ, e.g. in vivo precise and terminal end-joining capacities, and have either a suppressive or no effect on others. However, these findings have remained inconclusive, and the lack of consistency between these results may be at least partly explained by the use of different assays, which may measure different subtypes of NHEJ, and of different cell lines investigated. Although some insights have been obtained, the whole picture of NHEJ repair in mammalian cells is far from complete, and the questions of how many subpathways are involved or how we can investigate each subpathway have not yet been adequately addressed.