PUMA facilitates EMI1-promoted cytoplasmic Rad51 ubiquitination and inhibits DNA repair in stem and progenitor cells.

Maintenance of genetic stability via proper DNA repair in stem and progenitor cells is essential for the tissue repair and regeneration, while preventing cell transformation after damage. Loss of PUMA dramatically increases the survival of mice after exposure to a lethal dose of ionizing radiation (IR), while without promoting tumorigenesis in the long-term survivors. This finding suggests that PUMA (p53 upregulated modulator of apoptosis) may have a function other than regulates apoptosis. Here, we identify a novel role of PUMA in regulation of DNA repair in embryonic or induced pluripotent stem cells (PSCs) and immortalized hematopoietic progenitor cells (HPCs) after IR. We found that PUMA-deficient PSCs and HPCs exhibited a significant higher double-strand break (DSB) DNA repair activity via Rad51-mediated homologous recombination (HR). This is because PUMA can be associated with early mitotic inhibitor 1 (EMI1) and Rad51 in the cytoplasm to facilitate EMI1-mediated cytoplasmic Rad51 ubiquitination and degradation, thereby inhibiting Rad51 nuclear translocation and HR DNA repair. Our data demonstrate that PUMA acts as a repressor for DSB DNA repair and thus offers a new rationale for therapeutic targeting of PUMA in regenerative cells in the context of DNA damage.

Elevated CXorf67 Expression in PFA Ependymomas Suppresses DNA Repair and Sensitizes to PARP Inhibitors.

Ependymoma is the third most common pediatric tumor with posterior fossa group A (PFA) being its most aggressive subtype. Ependymomas are generally refractory to chemotherapies and thus lack any effective treatment. Here, we report that elevated expression of CXorf67 (chromosome X open reading frame 67), which frequently occurs in PFA ependymomas, suppresses homologous recombination (HR)-mediated DNA repair. Mechanistically, CXorf67 interacts with PALB2 and inhibits PALB2-BRCA2 interaction, thereby inhibiting HR repair. Concordantly, tumor cells with high CXorf67 expression levels show increased sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, especially when combined with radiotherapy. Thus, our findings have revealed a role of CXorf67 in HR repair and suggest that combination of PARP inhibitors with radiotherapy could be an effective treatment option for PFA ependymomas.

Benzo[a]pyrene represses DNA repair through altered E2F1/E2F4 function marking an early event in DNA damage-induced cellular senescence.

Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.

NRF2 preserves genomic integrity by facilitating ATR activation and G2 cell cycle arrest.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a well-characterized transcription factor that protects cells against oxidative and electrophilic stresses. Emerging evidence has suggested that NRF2 protects cells against DNA damage by mechanisms other than antioxidation, yet the mechanism remains poorly understood. Here, we demonstrate that knockout of NRF2 in cells results in hypersensitivity to ionizing radiation (IR) in the presence or absence of reactive oxygen species (ROS). Under ROS scavenging conditions, induction of DNA double-strand breaks (DSBs) increases the NRF2 protein level and recruits NRF2 to DNA damage sites where it interacts with ATR, resulting in activation of the ATR-CHK1-CDC2 signaling pathway. In turn, this leads to G2 cell cycle arrest and the promotion of homologous recombination repair of DSBs, thereby preserving genome stability. The inhibition of NRF2 by brusatol increased the radiosensitivity of tumor cells in xenografts by perturbing ATR and CHK1 activation. Collectively, our results reveal a novel function of NRF2 as an ATR activator in the regulation of the cellular response to DSBs. This shift in perspective should help furnish a more complete understanding of the function of NRF2 and the DNA damage response.

Proton Irradiation Increases the Necessity for Homologous Recombination Repair Along with the Indispensability of Non-Homologous End Joining.

Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.

Therapeutic Application of PARP Inhibitors in Neuro-Oncology.

In response to a variety of cellular stresses, poly(ADP-ribose) polymerase 1 (PARP1) has vital roles in orchestrating DNA damage repair and preserving genomic integrity. Clinical activity of PARP inhibitors (PARPis) in BRCA1/2 mutant cancers validated the concept of synthetic lethality between PARP inhibition and deleterious BRCA1/2 mutations, leading to clinical approval of several PARPis. Preclinical and clinical studies aiming to broaden the therapeutic application of PARPis identified sensitivity biomarkers and rationale combination strategies that can target BRCA wild-type and homologous recombination (HR) DNA repair-proficient cancers, including central nervous system (CNS) malignancies. In this review, we summarize recent progress in PARPi therapy in brain tumors, and discuss current opportunities for, and challenges to, the use of PARPis in neuro-oncology.

The absence of the RecN protein suppresses the cellular defects of Deinococcus radiodurans irradiated cells devoid of the PprA protein by limiting recombinational repair of DNA lesions.

The Deinococcus radiodurans bacterium is one of the most radioresistant organisms known. It can repair hundreds of radiation-induced DNA double-strand breaks without loss of viability and reconstitute an intact genome through RecA-dependent and RecA-independent DNA repair pathways. Among the Deinococcus specific proteins required for radioresistance, the PprA protein was shown to play a major role for accurate chromosome segregation and cell division after completion of DNA repair. Here, we analyzed the cellular role of the deinococcal RecN protein belonging to the SMC family and, surprisingly, observed that the absence of the RecN protein suppressed the sensitivity of cells devoid of the PprA protein to γ- and UV-irradiation and to treatment with MMC or DNA gyrase inhibitors. This suppression was not observed when ΔpprA cells were devoid of SMC or SbcC, two other proteins belonging to the SMC family. The absence of RecN also alleviated the DNA segregation defects displayed by ΔpprA cells recovering from γ-irradiation. When exposed to 5 kGy γ-irradiation, ΔpprA, ΔrecN and ΔpprA ΔrecN cells repaired their DNA with a delay of about one hour, as compared to the wild type cells. After irradiation, the absence of RecN reduced recombination between chromosomal and plasmid DNA, indicating that the deinococcal RecN protein is important for recombinational repair of DNA lesions. The transformation efficiency of genomic DNA was also reduced in the absence of the RecN protein. Here, we propose a model in which RecN, via its cohesin activity, might favor recombinational repair of DNA double strand breaks. This might increase, in irradiated cells, DNA constraints with PprA protein being required to resolve them via its ability to recruit DNA gyrase and to stimulate its decatenation activity.

Targeting PFKFB3 radiosensitizes cancer cells and suppresses homologous recombination.

The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.

Recruitment kinetics of the homologous recombination pathway in procyclic forms of Trypanosoma brucei after ionizing radiation treatment.

One of the most important mechanisms for repairing double-strand breaks (DSBs) in model eukaryotes is homologous recombination (HR). Although the genes involved in HR have been found in Trypanosoma brucei and studies have identified some of the proteins that participate in this HR pathway, the recruitment kinetics of the HR machinery onto DNA during DSB repair have not been clearly elucidated in this organism. Using immunofluorescence, protein DNA-bound assays, and DNA content analysis, we established the recruitment kinetics of the HR pathway in response to the DSBs generated by ionizing radiation (IR) in procyclic forms of T. brucei. These kinetics involved the phosphorylation of histone H2A and the sequential recruitment of the essential HR players Exo1, RPA, and Rad51. The process of DSB repair took approximately 5.5 hours. We found that DSBs led to a decline in the G2/M phase after IR treatment, concomitant with cell cycle arrest in the G1/S phase. This finding suggests that HR repairs DSBs faster than the other possible DSB repair processes that act during the G1/S transition. Taken together, these data suggest that the interplay between DNA damage detection and HR machinery recruitment is finely coordinated, allowing these parasites to repair DNA rapidly after DSBs during the late S/G2 proficient phases.

Synthetic Lethality of PARP Inhibition and Ionizing Radiation is p53-dependent.

PARP inhibitors (PARPi) are potentially effective therapeutic agents capable of inducing synthetic lethality in tumors with deficiencies in homologous recombination (HR)-mediated DNA repair such as those carrying BRCA1 mutations. However, BRCA mutations are rare, the majority of tumors are proficient in HR repair, and thus most tumors are resistant to PARPi. Previously, we observed that ionizing radiation (IR) initiates cytoplasmic translocation of BRCA1 leading to suppression of HR-mediated DNA repair and induction of synthetic PARPi lethality in wild-type BRCA1 and HR-proficient tumor cells. The tumor suppressor p53 was identified as a key factor that regulates DNA damage-induced BRCA1 cytoplasmic sequestration following IR. However, the role of p53 in IR-induced PARPi sensitization remains unclear. This study elucidates the role of p53 in IR-induced PARPi cytotoxicity in HR-proficient cancer cells and suggests p53 status may help define a patient population that might benefit from this treatment strategy. Sensitization to PARPi following IR was determined in vitro and in vivo utilizing human breast and glioma tumor cells carrying wild-type BRCA1 and p53, and in associated cells in which p53 function was modified by knockdown or mutation. In breast and glioma cells with proficient HR repair, IR-induced BRCA1 cytoplasmic sequestration, HR repair inhibition, and subsequent PARPi sensitization in vitro and in vivo was dependent upon functional p53.Implications: Implications: p53 status determines PARP inhibitor sensitization by ionizing radiation in multiple BRCA1 and HR-proficient tumor types and may predict which patients are most likely to benefit from combination therapy. Mol Cancer Res; 16(7); 1092-102. ©2018 AACR.

AKT overactivation can suppress DNA repair via p70S6 kinase-dependent downregulation of MRE11.

Deregulated AKT kinase activity due to PTEN deficiency in cancer cells contributes to oncogenesis by incompletely understood mechanisms. Here, we show that PTEN deletion in HCT116 and DLD1 colon carcinoma cells leads to suppression of CHK1 and CHK2 activation in response to irradiation, impaired G2 checkpoint proficiency and radiosensitization. These defects are associated with reduced expression of MRE11, RAD50 and NBS1, components of the apical MRE11/RAD50/NBS1 (MRN) DNA damage response complex. Consistent with reduced MRN complex function, PTEN-deficient cells fail to resect DNA double-strand breaks efficiently after irradiation and show greatly diminished proficiency for DNA repair via the error-free homologous recombination (HR) repair pathway. MRE11 is highly unstable in PTEN-deficient cells but stability can be significantly restored by inhibiting mTORC1 or p70S6 kinase (p70S6K), downstream kinases whose activities are stimulated by AKT, or by mutating a residue in MRE11 that we show is phosphorylated by p70S6K in vitro. In primary human fibroblasts, activated AKT suppresses MRN complex expression to escalate RAS-induced DNA damage and thereby reinforce oncogene-induced senescence. Taken together, our data demonstrate that deregulation of the PI3K-AKT/ mTORC1/ p70S6K pathways, an event frequently observed in cancer, exert profound effects on genome stability via MRE11 with potential implications for tumour initiation and therapy.

CTCF facilitates DNA double-strand break repair by enhancing homologous recombination repair.

The repair of DNA double-strand breaks (DSBs) is mediated via two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. DSB repair is vital for cell survival, genome stability, and tumor suppression. In contrast to NHEJ, HR relies on extensive homology and templated DNA synthesis to restore the sequence surrounding the break site. We report a new role for the multifunctional protein CCCTC-binding factor (CTCF) in facilitating HR-mediated DSB repair. CTCF is recruited to DSB through its zinc finger domain independently of poly(ADP-ribose) polymers, known as PARylation, catalyzed by poly(ADP-ribose) polymerase 1 (PARP-1). CTCF ensures proper DSB repair kinetics in response to γ-irradiation, and the loss of CTCF compromises HR-mediated repair. Consistent with its role in HR, loss of CTCF results in hypersensitivity to DNA damage, inducing agents and inhibitors of PARP. Mechanistically, CTCF acts downstream of BRCA1 in the HR pathway and associates with BRCA2 in a PARylation-dependent manner, enhancing BRCA2 recruitment to DSB. In contrast, CTCF does not influence the recruitment of the NHEJ protein 53BP1 or LIGIV to DSB. Together, our findings establish for the first time that CTCF is an important regulator of the HR pathway.

RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination.

RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.

miR-21-mediated Radioresistance Occurs via Promoting Repair of DNA Double Strand Breaks.

miR-21, as an oncogene that overexpresses in most human tumors, is involved in radioresistance; however, the mechanism remains unclear. Here, we demonstrate that miR-21-mediated radioresistance occurs through promoting repair of DNA double strand breaks, which includes facilitating both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR). The miR-21-promoted NHEJ occurs through targeting GSK3B (a novel target of miR-21), which affects the CRY2/PP5 pathway and in turn increases DNA-PKcs activity. The miR-21-promoted HRR occurs through targeting both GSK3B and CDC25A (a known target of miR-21), which neutralizes the effects of targeting GSK3B-induced CDC25A increase because GSK3B promotes degradation of both CDC25A and cyclin D1, but CDC25A and cyclin D1 have an opposite effect on HRR. A negative correlation of expression levels between miR-21 and GSK3ß exists in a subset of human tumors. Our results not only elucidate miR-21-mediated radioresistance, but also provide potential new targets for improving radiotherapy.

The Use of Laser Microirradiation to Investigate the Roles of Cohesins in DNA Repair.

In addition to their mitotic and transcriptional functions, cohesin plays critical roles in DNA damage response (DDR) and repair. Specifically, cohesin promotes homologous recombination (HR) repair of DNA double-strand breaks (DSBs), which is conserved from yeast to humans, and is a critical effector of ATM/ATR DDR kinase-mediated checkpoint control in mammalian cells. Optical laser microirradiation has been instrumental in revealing the damage site-specific functions of cohesin and, more recently, uncovering the unique role of cohesin-SA2, one of the two cohesin complexes uniquely present in higher eukaryotes, in DNA repair in human cells. In this review, we briefly describe what we know about cohesin function and regulation in response to DNA damage, and discuss the optimized laser microirradiation conditions used to analyze cohesin responses to DNA damage in vivo.

Late DNA Damage Mediated by Homologous Recombination Repair Results in Radiosensitization with Gemcitabine.

Gemcitabine (dFdCyd) shows broad antitumor activity in solid tumors in chemotherapeutic regimens or when combined with ionizing radiation (radiosensitization). While it is known that mismatches in DNA are necessary for dFdCyd radiosensitization, the critical event resulting in radiosensitization has not been identified. Here we hypothesized that late DNA damage (≥24 h after drug washout/irradiation) is a causal event in radiosensitization by dFdCyd, and that homologous recombination repair (HRR) is required for this late DNA damage. Using γ-H2AX as a measurement of DNA damage in MCF-7 breast cancer cells, we demonstrate that 10 or 80 nM dFdCyd alone produced significantly more late DNA damage compared to that observed within 4 h after treatment. The combination of dFdCyd treatment followed by irradiation did not produce a consistent increase in DNA damage in the first 4 h after treatment, however, there was a synergistic increase 24-48 h later relative to treatment with dFdCyd or radiation alone. RNAi suppression of the essential HRR protein, XRCC3, significantly decreased both radiosensitization and late DNA damage. Furthermore, inhibition of HRR with the Rad51 inhibitor B02 prevented radiosensitization when added after, but not during, treatment with dFdCyd and radiation. To our knowledge, this is the first published study to show that radiosensitization with dFdCyd results from a synergistic increase in DNA damage at 24-48 h after drug and radiation treatment, and that this damage and radiosensitization require HRR. These results suggest that tumors that overexpress HRR will be more vulnerable to chemoradiotherapy, and treatments that increase HRR and/or mismatches in DNA will enhance dFdCyd radiosensitization.

DDB2 increases radioresistance of NSCLC cells by enhancing DNA damage responses.

Radiotherapy resistance is one of the major factors limiting the efficacy of radiotherapy in lung cancer patients. The extensive investigations indicate the diversity in the mechanisms underlying radioresistance. Here, we revealed that DNA damage binding protein 2 (DDB2) is a potential regulator in the radiosensitivity of non-small cell lung cancer (NSCLC) cells. DDB2, originally identified as a DNA damage recognition factor in the nucleotide excision repair, promotes the survival and inhibits the apoptosis of NSCLC cell lines upon ionizing radiation (IR). Mechanistic investigations demonstrated that DDB2 is able to facilitate IR-induced phosphorylation of Chk1, which plays a critical role in the cell cycle arrest and DNA repair in response to IR-induced DNA double-strand breaks (DSBs). Indeed, knockdown of DDB2 compromised the G2 arrest in the p53-proficient A549 cell line and reduced the efficiency of homologous recombination (HR) repair. Taken together, our data indicate that the expression of DDB2 in NSCLC could be used as a biomarker to predict radiosensitivity of the patients. Targeting Chk1 can be used to increase the efficacy of radiotherapy in patients of NSCLC possessing high levels of DDB2.

Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair.

The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency.

RNF4 regulates DNA double-strand break repair in a cell cycle-dependent manner.

Both RNF4 and KAP1 play critical roles in the response to DNA double-strand breaks (DSBs), but the functional interplay of RNF4 and KAP1 in regulating DNA damage response remains unclear. We have previously demonstrated the recruitment and degradation of KAP1 by RNF4 require the phosphorylation of Ser824 (pS824) and SUMOylation of KAP1. In this report, we show the retention of DSB-induced pS824-KAP1 foci and RNF4 abundance are inversely correlated as cell cycle progresses. Following irradiation, pS824-KAP1 foci predominantly appear in the cyclin A (-) cells, whereas RNF4 level is suppressed in the G0-/G1-phases and then accumulates during S-/G2-phases. Notably, 53BP1 foci, but not BRCA1 foci, co-exist with pS824-KAP1 foci. Depletion of KAP1 yields opposite effect on the dynamics of 53BP1 and BRCA1 loading, favoring homologous recombination repair. In addition, we identify p97 is present in the RNF4-KAP1 interacting complex and the inhibition of p97 renders MCF7 breast cancer cells relatively more sensitive to DNA damage. Collectively, these findings suggest that combined effect of dynamic recruitment of RNF4 to KAP1 regulates the relative occupancy of 53BP1 and BRCA1 at DSB sites to direct DSB repair in a cell cycle-dependent manner.

Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations.

DNA double strand breaks (DSB) are the most deleterious lesions for the integrity of the genome, as their misrepair can lead to the formation of chromosome translocations. Cells have evolved two main repair pathways to suppress the formation of these genotoxic lesions: homology-dependent, error-free homologous recombination repair (HRR), and potentially error-prone, classical, DNA-PK-dependent non-homologous end-joining (c-NHEJ). The most salient feature of c-NHEJ, speed, will largely suppress chromosome translocation formation, while sequence alterations at the junction remain possible. It is now widely accepted that when c-NHEJ is inactivated, globally or locally, an alternative form of end-joining (alt-EJ) removes DSBs. Alt-EJ operates with speed and fidelity markedly lower than c-NHEJ, causing thus with higher probability chromosome translocations, and generating more extensive sequence alterations at the junction. Our working hypothesis is that alt-EJ operates as a backup to c-NHEJ. Recent results show that alt-EJ can also backup abrogated HRR in G2 phase cells, again at the cost of elevated formation of chromosome translocations. These observations raise alt-EJ to a global rescuing mechanism operating on ends that have lost their chromatin context in ways that compromise processing by HRR or c-NHEJ. While responsible for eliminating from the genome highly cytotoxic DNA ends, alt-EJ provides this function at the price of increased translocation formation. Here, we analyze recent literature on the mechanisms of chromosome translocation formation and propose a functional hierarchy among DSB processing pathways that makes alt-EJ the global backup pathway. We discuss possible ramifications of this model in cellular DSB management and pathway choice, and analyze its implications in radiation carcinogenesis and the design of novel therapeutic approaches.