Autophosphorylation transforms DNA-PK from protecting to processing DNA ends.

The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.

Structure of an activated DNA-PK and its implications for NHEJ.

DNA-dependent protein kinase (DNA-PK), like all phosphatidylinositol 3-kinase-related kinases (PIKKs), is composed of conserved FAT and kinase domains (FATKINs) along with solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-PK, all existing structures represent inactive states with resolution limited to 4.3 Å at best. Here, we report the cryoelectron microscopy (cryo-EM) structures of DNA-PKcs (DNA-PK catalytic subunit) bound to a DNA end or complexed with Ku70/80 and DNA in both inactive and activated forms at resolutions of 3.7 Å overall and 3.2 Å for FATKINs. These structures reveal the sequential transition of DNA-PK from inactive to activated forms. Most notably, activation of the kinase involves previously unknown stretching and twisting within individual solenoid segments and loosens DNA-end binding. This unprecedented structural plasticity of helical repeats may be a general regulatory mechanism of HEAT-repeat proteins.

ER-associated degradation ligase HRD1 links ER stress to DNA damage repair by modulating the activity of DNA-PKcs.

Proteostasis and genomic integrity are respectively regulated by the endoplasmic reticulum-associated protein degradation (ERAD) and DNA damage repair signaling pathways, with both pathways essential for carcinogenesis and drug resistance. How these signaling pathways coordinate with each other remains unexplored. We found that ER stress specifically induces the DNA-PKcs-regulated nonhomologous end joining (NHEJ) pathway to amend DNA damage and impede cell death. Intriguingly, sustained ER stress rapidly decreased the activity of DNA-PKcs and DNA damage accumulated, facilitating a switch from adaptation to cell death. This DNA-PKcs inactivation was caused by increased KU70/KU80 protein degradation. Unexpectedly, the ERAD ligase HRD1 was found to efficiently destabilize the classic nuclear protein HDAC1 in the cytoplasm, by catalyzing HDAC1's polyubiquitination at lysine 74, at a late stage of ER stress. By abolishing HDAC1-mediated KU70/KU80 deacetylation, HRD1 transmits ER signals to the nucleus. The resulting enhanced KU70/KU80 acetylation provides binding sites for the nuclear E3 ligase TRIM25, resulting in the promotion of polyubiquitination and the degradation of KU70/KU80 proteins. Both in vitro and in vivo cancer models showed that genetic or pharmacological inhibition of HADC1 or DNA-PKcs sensitizes colon cancer cells to ER stress inducers, including the Food and Drug Administration-approved drug celecoxib. The antitumor effects of the combined approach were also observed in patient-derived xenograft models. These findings identify a mechanistic link between ER stress (ERAD) in the cytoplasm and DNA damage (NHEJ) pathways in the nucleus, indicating that combined anticancer strategies may be developed that induce severe ER stress while simultaneously inhibiting KU70/KU80/DNA-PKcs-mediated NHEJ signaling.

Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements.

Chromosomal deletion rearrangements mediated by repetitive elements often involve repeats separated by several kilobases and sequences that are divergent. While such rearrangements are likely induced by DNA double-strand breaks (DSBs), it has been unclear how the proximity of DSBs relative to repeat sequences affects the frequency of such events. We generated a reporter assay in mouse cells for a deletion rearrangement involving repeats separated by 0.4 Mb. We induced this repeat-mediated deletion (RMD) rearrangement with two DSBs: the 5' DSB that is just downstream from the first repeat and the 3' DSB that is varying distances upstream of the second repeat. Strikingly, we found that increasing the 3' DSB/repeat distance from 3.3 kb to 28.4 kb causes only a modest decrease in rearrangement frequency. We also found that RMDs are suppressed by KU70 and RAD51 and promoted by RAD52, CtIP, and BRCA1. In addition, we found that 1%-3% sequence divergence substantially suppresses these rearrangements in a manner dependent on the mismatch repair factor MSH2, which is dominant over the suppressive role of KU70. We suggest that a DSB far from a repeat can stimulate repeat-mediated rearrangements, but multiple pathways suppress these events.

KU70 and CAF-1 in Arabidopsis: Divergent roles in rDNA stability and telomere homeostasis.

Deficiency in chromatin assembly factor-1 (CAF-1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication-dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild-type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single-strand annealing pathway of homology-dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF-1 dysfunction.

Plasticity of the Mre11-Rad50-Xrs2-Sae2 nuclease ensemble in the processing of DNA-bound obstacles.

The budding yeast Mre11-Rad50-Xrs2 (MRX) complex and Sae2 function together in DNA end resection during homologous recombination. Here we show that the Ku complex shields DNA ends from exonucleolytic digestion but facilitates endonucleolytic scission by MRX with a dependence on ATP and Sae2. The incision site is enlarged into a DNA gap via the exonuclease activity of MRX, which is stimulated by Sae2 without ATP being present. RPA renders a partially resected or palindromic DNA structure susceptible to MRX-Sae2, and internal protein blocks also trigger DNA cleavage. We present models for how MRX-Sae2 creates entry sites for the long-range resection machinery.

Physiological protein blocks direct the Mre11-Rad50-Xrs2 and Sae2 nuclease complex to initiate DNA end resection.

DNA double-strand break repair by homologous recombination is initiated by DNA end resection, which is commenced by the Mre11-Rad50-Xrs2 complex and Sae2 in yeast. Here we report that the nonhomologous end joining factor Ku limits the exonuclease activity of Mre11 and promotes its endonuclease to cleave 5'-terminated DNA strands at break sites. Following initial endonucleolytic cleavage past the obstacle, Exo1 specifically extends the resection track, leading to the generation of long 3' overhangs that are required for homologous recombination. These experiments provide mechanistic insights into how short-range and long-range DNA end resection enzymes overcome obstacles near broken DNA ends to initiate recombination.

Keeping it real: MRX-Sae2 clipping of natural substrates.

The yeast Mre11-Rad50-Xrs2 (MRX) complex and Sae2 function together to initiate DNA end resection, an essential early step in homology-dependent repair of DNA double-strand breaks (DSBs). In this issue of Genes & Development, Wang and colleagues (pp. 2331-2336) and Reginato and colleagues (pp. 2325-2330) report that a variety of physiological protein blocks, including Ku, RPA, and nucleosomes, stimulate MRX-Sae2 endonuclease cleavage in vitro. These studies have important implications for how cells deal with a range of barriers to end resection and highlight the crucial role of Sae2 in activating MRX cleavage at the correct cell cycle stage.

Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii.

Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bioproduction of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K. phaffii, but improvements in gene-editing methods are desirable to further accelerate engineering of this yeast. In this study, we have developed a versatile vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences including single-stranded oligonucleotides. Notably, we performed site-specific point mutations and full gene deletions using short (90 nt) single-stranded oligonucleotides at very high efficiencies. Lastly, we present a strategy for transient inactivation of nonhomologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). This system enables precise CRISPR-Cas9-based editing (including multiplexing) and facilitates simple reversion to NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and are compatible with high-throughput automated workflows.

Transplantation of Neural Stem Cells-Overexpressed Ku70 Improves Neurological Deficits in a Mice Model of Cerebral Ischemia Stroke.

Cerebral ischemic stroke is a cerebrovascular disease, which is related to DNA damage. Many researches have shown that Ku70 is a key regulator for DNA damage. Here, we aimed to explore Ku70 roles in cerebral ischemic stroke and its potential molecular mechanism. In our study, neural stem cells (NSCs) were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) for constructing cerebral ischemic stroke cell model. CCK8 assay, Brdu/GFP staining, flow cytometry and TUNEL staining were performed to examine cell proliferation, cell cycle and apoptosis, respectively. Relative mRNA and protein levels were detected by quantitative real-time PCR and western blot analysis, respectively. Ku70 positive cells were examined by immunofluorescence staining. Comet assay was employed to determine DNA damage. Animal experiments were performed to assess the effect of transplanting NSCs and Ku70-overexpressed NSCs on neurological deficits, infarct volume, brain edema and blood‒brain barrier (BBB) integrity in middle cerebral artery occlusion (MCAO) model. Our data found that Ku70 expression was decreased in NSCs after OGD/R. Overexpression of Ku70 reduced DNA damage and apoptosis of OGD/R-induced NSCs. Knockdown of Ku70 promoted the activity of ATM/p53. Moreover, KU60019 (ATM-specific inhibitor) reversed the promoting effects of Ku70 silencing on DNA damage and apoptosis in OGD/R-induced NSCs. In animal experiments, transplantation of NSCs-overexpressed Ku70 enhanced cell survival, improved motor function, reduced infarct volume, relieved brain edema and alleviated BBB dysfunction in MCAO mice models. In conclusion, Ku70 overexpression repressed the DNA damage and apoptosis in OGD/R-induced NSCs by regulating ATM/p53 pathway, and transplantation of NSCs-overexpressed Ku70 played neuroprotective effects in MCAO mice models.

Characterization of a KU70-disrupted strain of the mannosylerythritol lipid-producing yeast Pseudozyma tsukubaensis constructed by a marker recycling system.

The basidiomycetous yeast Pseudozyma tsukubaensis is known as an industrial mannosylerythritol lipid producer. In this study, the PtURA5 marker gene was deleted by homologous recombination. Using the PtURA5-deleted mutant as a host strain, we obtained a derivative disrupted for the PtKU70 gene, a putative ortholog of the KU70 gene encoding a protein involved in the nonhomologous end-joining pathway of DNA repair. Subsequently, the introduced PtURA5 gene was re-deleted by marker recycling. These results demonstrated that the PtURA5 gene can be used as a recyclable marker gene. Although the frequency of homologous recombination has been shown to be increased by KU70 disruption in other fungi, the PtKU70-disrupted strain of P. tsukubaensis did not demonstrate an elevated frequency of homologous recombination. Furthermore, the PtKU70-disrupted strain did not show increased susceptibility to bleomycin. These results suggested that the function of this KU70 ortholog in P. tsukubaensis is distinct from that in other fungi.

Efficient RNA interference method by feeding in Brachionus plicatilis (Rotifera).

Rotifers are small, ubiquitous invertebrate animals found throughout the world and have emerged as a promising model system for studying molecular mechanisms in the fields of experimental ecology, aquatic toxicology, and geroscience. However, the lack of efficient gene expression manipulation techniques has hindered the study of rotifers. In this study, we used the L4440 plasmid with two reverse-oriented T7 promoters, along with RNase-deficient E. coli HT115, to efficiently produce dsRNA and thereby present an efficient feeding-based RNAi method in Brachionus plicatilis. We targeted Bp-Ku70 & Ku80, key proteins in the DNA double-strand breaks repair pathway, and then subjected rotifers to UV radiation. We found that the mRNA expression, fecundity, as well as survival rate diminished significantly as a result of RNAi. Overall, our results demonstrate that the feeding-based RNAi method is a simple and efficient tool for gene knockdown in B. plicatilis, advancing their use as a model organism for biological research.

(-)-Epigallocatechin-3-gallate induced apoptosis by dissociation of c-FLIP/Ku70 complex in gastric cancer cells.

Anti-cancer properties of (-)-epigallocatechin-3-gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE-inhibitory protein (c-FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti-cancer role of EGCG in gastric cancer (GC) cells through dissociation of c-FLIP/Ku70 complex. MKN-45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT-PCR. Protein expression of c-FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c-FLIP/Ku70 complex as well as Ku70 translocation were studied by sub-cellular fractionation and co-immunoprecipitation. EGCG induced apoptosis in MKN-45 cells with substantial up-regulation of P53 and P21, down-regulation of c-Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c-FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c-FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.

Nuclear expression of Ku70/80 is associated with CHEK2 germline mutations in breast cancer.

Ku70/80 protein inhibitors reduce the repair of DNA double-strand breaks via the Ku70/80 pathway, so they can be used to treat cancers with Ku70/80 overexpression. Since the association of Ku70/80 with germline CHEK2 mutations in breast cancer is unknown, in this study we evaluated the expression of Ku70/80 in breast cancers with germline CHEK2 mutations. Immunohistochemistry with a Ku70/80 antibody on tissue microarrays from 225 CHEK2-associated breast cancers was used and automatically assessed with computerized image analysis. We report that the vast majority of breast cancers expressed high level of nuclear Ku70/80 and a small percentage of tumors (3.5%) were negative for Ku70/80 expression. There was a significant difference between the nuclear Ku70/80 expression in CHEK2-associated vs. CHEK2-non-associated breast cancers in all tumors (p = 0.009), and in the estrogen receptor (ER) positive subgroup of breast cancers (p = 0.03). This study is the first reporting an association of Ku70/80 expression with CHEK2 germline mutations in breast cancer. The results suggest that evaluation of Ku70/80 expression in breast cancer may improve the selection of breast cancer patients for Ku70/80 inhibitor therapy, and point to CHEK2-associated breast cancer and a subset of ER-positive breast cancer as potential suitable targets for such therapy.

Both ATM and DNA-PK Are the Main Regulators of HIV-1 Post-Integrational DNA Repair.

The integration of a DNA copy of an HIV-1 RNA genome into the host genome, carried out by the viral enzyme integrase, results in the formation of single-stranded gaps in cellular DNA that must be repaired. Here, we have analyzed the involvement of the PI3K kinases, ATM, ATR, and DNA-PKcs, which are important players in the DNA damage response (DDR) in HIV-1 post-integrational DNA repair (PIR). The participation of the DNA-PK complex in HIV-1 PIR has been previously shown, and the formation of a complex between the viral integrase and the DNA-PK subunit, Ku70, has been found to be crucial for efficient PIR. Now, we have shown that the inhibition of both DNA-PKcs and ATM, but not ATR, significantly reduces PIR efficiency. The activation of both kinases is a sequential process, where one kinase, being activated, activates the other, and it occurs simultaneously with the integration of viral DNA. This fact suggests that the activation of both kinases triggers PIR. Most interestingly, the activation of not only DNA-PKcs, but also ATM depends on the complex formation between integrase and Ku70. The elucidation of the interactions between viruses and DDR is important both for understanding the modulation of host cell functions by these pathogens and for developing new approaches to combat viral infections.

Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage.

The Ku heterodimer, composed of subunits Ku70 and Ku80, is known for its essential role in repairing double-stranded DNA breaks via non-homologous end joining (NHEJ). We previously identified Ku70 S155 as a novel phosphorylation site within the von Willebrand A-like (vWA) domain of Ku70 and documented an altered DNA damage response in cells expressing a Ku70 S155D phosphomimetic mutant. Here, we conducted proximity-dependent biotin identification (BioID2) screening using wild-type Ku70, Ku70 S155D mutant, and Ku70 with a phosphoablative substitution (S155A) to identify Ku70 S155D-specific candidate proteins that may rely on this phosphorylation event. Using the BioID2 screen with multiple filtering approaches, we compared the protein interactor candidate lists for Ku70 S155D and S155A. TRIP12 was exclusive to the Ku70 S155D list, considered a high confidence interactor based on SAINTexpress analysis, and appeared in all three biological replicates of the Ku70 S155D-BioID2 mass spectrometry results. Using proximity ligation assays (PLA), we demonstrated a significantly increased association between Ku70 S155D-HA and TRIP12 compared to wild-type Ku70-HA cells. In addition, we were able to demonstrate a robust PLA signal between endogenous Ku70 and TRIP12 in the presence of double-stranded DNA breaks. Finally, co-immunoprecipitation analyses showed an enhanced interaction between TRIP12 and Ku70 upon treatment with ionizing radiation, suggesting a direct or indirect association in response to DNA damage. Altogether, these results suggest an association between Ku70 phospho-S155 and TRIP12.

Sodium Butyrate Enhances the Cytotoxic Effect of Etoposide in HDACi-Sensitive and HDACi-Resistant Transformed Cells.

To overcome the problem of antitumor agent toxicity for normal cells, a combined therapy using drugs with synergistic effects seems to be more effective. We investigated the molecular mechanisms of the sensitization of tumor cells resistant and sensitive to histone deacetylase inhibitors (HDACis) upon etoposide treatment together with the HDACi sodium butyrate (NaBut). We showed that NaBut enhances the cytotoxic effect of etoposide in both HDACi-sensitive and HDACi-resistant cells due to the accumulation of the Bax protein and the dissociation of Ku70-Bax inhibitory complexes. In HDACi-resistant cells, NaBut causes the cytoplasmic accumulation of Bax dissociated from mitochondria in complexes with Ku70 proteins. The increased phosphorylation of the pro-apoptotic Bad protein due to the NaBut-induced activation of Erk and Akt kinases is one of the possible reasons for the accumulation of Bax in the cytoplasm. Despite the inactivation of Bax in HDACi-resistant cells, its accumulation in the cytoplasm upon NaBut treatment makes it possible to enhance the apoptotic response against agents activating the intrinsic pathway of apoptosis. Thus, HDACis involved in combined therapy mediate the sensitization of tumor cells to genotoxic drugs, regardless of the cells' resistance to HDACis.

KuINins as a New Class of HIV-1 Inhibitors That Block Post-Integration DNA Repair.

Integration of HIV-1 genomic cDNA results in the formation of single-strand breaks in cellular DNA, which must be repaired for efficient viral replication. Post-integration DNA repair mainly depends on the formation of the HIV-1 integrase complex with the Ku70 protein, which promotes DNA-PK assembly at sites of integration and its activation. Here, we have developed a first-class inhibitor of the integrase-Ku70 complex formation that inhibits HIV-1 replication in cell culture by acting at the stage of post-integration DNA repair. This inhibitor, named s17, does not affect the main cellular function of Ku70, namely its participation in the repair of double-strand DNA breaks through the non-homologous end-joining pathway. Using a molecular dynamics approach, we have constructed a model for the interaction of s17 with Ku70. According to this model, the interaction of two phenyl radicals of s17 with the L76 residue of Ku70 is important for this interaction. The requirement of two phenyl radicals in the structure of s17 for its inhibitory properties was confirmed using a set of s17 derivatives. We propose to stimulate compounds that inhibit post-integration repair by disrupting the integrase binding to Ku70 KuINins.

TBX20 inhibits colorectal cancer tumorigenesis by impairing NHEJ-mediated DNA repair.

DNA high methylation is one of driving force for colorectal carcinoma (CRC) pathogenesis. Transcription factors (TFs) can determine cell fate and play fundamental roles in multistep process of tumorigenesis. Dysregulation of DNA methylation of TFs should be vital for the progression of CRC. Here, we demonstrated that TBX20, a T-box TF family protein, was downregulated with hypermethylation of promoter in early-stage CRC tissues and correlated with a poor prognosis for CRC patients. Moreover, we identified PDZRN3 as the E3 ubiquitin ligase of TBX20 protein, which mediated the ubiquitination and degradation of TBX20. Furthermore, we revealed that TBX20 suppressed cell proliferation and tumor growth through impairing non-homologous DNA end joining (NHEJ)-mediated double-stranded break repair by binding the middle domain of both Ku70 and Ku80 and therefore inhibiting their recruitment on chromatin in CRC cells. Altogether, our results reveal the tumor-suppressive role of TBX20 by inhibiting NHEJ-mediated DNA repair in CRC cells, and provide a potential biomarker for predicting the prognosis of patients with early-stage CRC and a therapeutic target for combination therapy.

Neutrophils Alter DNA Repair Landscape to Impact Survival and Shape Distinct Therapeutic Phenotypes of Colorectal Cancer.

BACKGROUND & AIMS: Tumor-infiltrating neutrophils (polymorphonuclear neutrophils [PMNs]) are a prominent feature of colorectal cancer (CRC), where they can promote cytotoxicity or exacerbate disease outcomes. We recently showed that in acute colon injury, PMNs can increase DNA double-strand break (DSB) burden and promote genomic instability via microRNA-dependent inhibition of homologous recombination (HR) repair. In this study, we aimed to establish whether in inflamed colon, neutrophils shape the DSB-repair responses to impact CRC progression and sensitivity/resistance to DNA-repair targeted therapy. METHODS: Human sporadic CRC biopsies, The Cancer Genome Atlas gene expression analyses, tumor xenografts, and murine CRC models, as well as small-molecule inhibition of key DSB-repair factors were leveraged to investigate changes in the DSB-repair landscape and identify unique CRC responses with/without tumor infiltration by PMNs. RESULTS: We reveal that neutrophils exert a functional dualism in cancer cells, driving temporal modulation of the DNA damage landscape and resolution of DSBs. PMNs were found to promote HR deficiency in low-grade CRC by miR-155-dependent downregulation of RAD51, thus attenuating tumor growth. However, neutrophil-mediated genotoxicity due to accumulation of DSBs led to the induction of non-homologous end-joining (NHEJ), allowing for survival and growth of advanced CRC. Our findings identified a PMN-induced HR-deficient CRC phenotype, featuring low RAD51 and low Ku70 levels, rendering it susceptible to synthetic lethality induced by clinically approved PARP1 inhibitor Olaparib. We further identified a distinct PMN-induced HR-deficient CRC phenotype, featuring high Ku70 and heightened NHEJ, which can be therapeutically targeted by specific inhibition of NHEJ. CONCLUSIONS: Our work delineates 2 mechanism-based translatable therapeutic interventions in sporadic CRC.