PD-L1 deglycosylation promotes its nuclear translocation and accelerates DNA double-strand-break repair in cancer.

Resistance to radiotherapy is a major barrier during cancer treatment. Here using genome-scale CRISPR/Cas9 screening, we identify CD274 gene, which encodes PD-L1, to confer lung cancer cell resistance to ionizing radiation (IR). Depletion of endogenous PD-L1 delays the repair of IR-induced DNA double-strand breaks (DSBs) and PD-L1 loss downregulates non-homologous end joining (NHEJ) while overexpression of PD-L1 upregulates NHEJ. IR induces translocation of PD-L1 from the membrane into nucleus dependent on deglycosylation of PD-L1 at N219 and CMTM6 and leads to PD-L1 recruitment to DSBs foci. PD-L1 interacts with Ku in the nucleus and enhances Ku binding to DSB DNA. The interaction between the IgC domain of PD-L1 and the core domain of Ku is required for PD-L1 to accelerate NHEJ-mediated DSB repair and produce radioresistance. Thus, PD-L1, in addition to its immune inhibitory activity, acts as mechanistic driver for NHEJ-mediated DSB repair in cancer.

Current status and prospect of the DNA double-strand break repair pathway in colorectal cancer development and treatment.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways.

Anti-Ku + myositis: an acquired inflammatory protein-aggregate myopathy.

Myositis with anti-Ku-autoantibodies is a rare inflammatory myopathy associated with various connective tissue diseases. Histopathological studies have identified inflammatory and necrotizing aspects, but a precise morphological analysis and pathomechanistic disease model are lacking. We therefore aimed to carry out an in-depth morpho-molecular analysis to uncover possible pathomechanisms. Muscle biopsy specimens from 26 patients with anti-Ku-antibodies and unequivocal myositis were analyzed by immunohistochemistry, immunofluorescence, transcriptomics, and proteomics and compared to biopsy specimens of non-disease controls, immune-mediated necrotizing myopathy (IMNM), and inclusion body myositis (IBM). Clinical findings and laboratory parameters were evaluated retrospectively and correlated with morphological and molecular features. Patients were mainly female (92%) with a median age of 56.5 years. Isolated myositis and overlap with systemic sclerosis were reported in 31%, respectively. Isolated myositis presented with higher creatine kinase levels and cardiac involvement (83%), whereas systemic sclerosis-overlap patients often had interstitial lung disease (57%). Histopathology showed a wide spectrum from mild to pronounced myositis with diffuse sarcolemmal MHC-class I (100%) and -II (69%) immunoreactivity, myofiber necrosis (88%), endomysial inflammation (85%), thickened capillaries (84%), and vacuoles (60%). Conspicuous sarcoplasmic protein aggregates were p62, BAG3, myotilin, or immunoproteasomal beta5i-positive. Proteomic and transcriptomic analysis identified prominent up-regulation of autophagy, proteasome, and hnRNP-related cell stress. To conclude, Ku + myositis is morphologically characterized by myofiber necrosis, MHC-class I and II positivity, variable endomysial inflammation, and distinct protein aggregation varying from IBM and IMNM, and it can be placed in the spectrum of scleromyositis and overlap myositis. It features characteristic sarcoplasmic protein aggregation on an acquired basis being functionally associated with altered chaperone, proteasome, and autophagy function indicating that Ku + myositis exhibit aspects of an acquired inflammatory protein-aggregate myopathy.

LINC-PINT plays an anti-tumor role in nasopharyngeal carcinoma by binding to XRCC6 and affecting its function.

BACKGROUND: LINC-PINT was downregulated in nasopharyngeal carcinoma (NPC) and correlated with treatment efficiency of NPC. However, the underlying mechanism of LINC-PINT in NPC has not yet been fully explored. METHOD: We used CellTiter luminescent assay, clone formation assay, Hoechst staining, and SYTO-9/PI staining to examine cell viability and cell apoptosis regulated by LINC-PINT in NPC cells. Xenograft tumor model, HE staining, Ki67 staining, and TUNEL assay were conducted to assess the role of LINC-PINT in vivo. Bioinformatics and RNA immunoprecipitation assay was performed to identify the binding protein of LINC-PINT. Fluorescence in situ hybridization and immunofluorescence were utilized to measure the colocalization of XRCC6 with LINC-PINT and DNA-PKcs. Mito-Tracker red CMXRos staining was used to label mitochondria in cells specifically. RESULT: We found LINC-PINT was downregulated in many tumors (including NPC) and associated with poor prognosis. The cell viability was significantly inhibited and cell apoptosis was remarkably promoted in LINC-PINT overexpressed cells in contrast to control cells. The growth of tumor xenografts was significantly suppressed and the tumor weight was significantly decreased in LINC-PINT overexpression group compared to the control group. Correspondingly, the positive Ki67 foci was decreased while TUNEL foci was increased in LINC-PINT overexpression group. Mechanically, we verified XRCC6 as a new binding protein of LINC-PINT through RNA binding domains prediction, RIP and colocalization of LINC-PINT and XRCC6. By binding to XRCC6, LINC-PINT interfered the formation of DNA-PK complex, regulated mitochondria accumulation status and affected the modification of apoptosis proteins, leading to more cell apoptosis. CONCLUSION: Our study provided the first evidence that LINC-PINT promotes cell apoptosis in NPC by binding to XRCC6 and affecting its function.

m6A-mediated lnc-OXAR promotes oxaliplatin resistance by enhancing Ku70 stability in non-alcoholic steatohepatitis-related hepatocellular carcinoma.

BACKGROUND: The escalating prevalence of metabolic diseases has led to a rapid increase in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (NASH-HCC). While oxaliplatin (OXA)-based hepatic arterial infusion chemotherapy (HAIC) has shown promise in advanced-stage HCC patients, its efficacy in NASH-HCC remains uncertain. This study aims to assess the effectiveness of OXA-based HAIC and elucidate the mechanisms underlying OXA resistance in NASH-HCC. METHODS: The key lncRNAs were screened through RNA-seq analysis of NASH/non-NASH and OXA-sensitive/OXA-resistant (OXA-S/R) HCC tissues. The biological functions of the lnc-OXAR (OXA resistance-related lncRNA in NASH-HCC) in NASH-HCC were verified through a series of in vitro and in vivo experiments. The molecular mechanism of lnc-OXAR was elucidated by fluorescence in situ hybridization, immunoprecipitation-mass spectrometry (FISH), Immunoprecipitation-Mass Spectrometry (IP-MS), RNA pulldown, RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and a dual-luciferase reporter assay. RESULTS: NASH-HCC exhibited reduced responsiveness to OXA-based HAIC compared to non-NASH HCC. We identified and validated a novel transcript namedlnc-OXAR, which played a crucial role in conferring OXA resistance to NASH-HCC. Inhibition of lnc-OXAR suppressed HCC cell growth and restored OXA sensitivity both in NASH-HCC mouse models and in vitro. Mechanistically, lnc-OXAR recruited Ku70 and cystatin A (CSTA), preventing Ku70 degradation and facilitating DNA double-strand break (DSB) repair, thereby promoting OXA resistance in NASH-HCC. Additionally, WTAP-mediated m6A modification enhanced the stability of lnc-OXAR in an IGF2BP2-dependent manner. Notably, silencing lnc-OXAR significantly enhanced the response to OXA in patient-derived xenograft (PDX) models derived from NASH-HCC. CONCLUSIONS: The reduced responsiveness of NASH-HCC to OXA treatment can be attributed to the upregulation of lnc-OXAR. Our findings provide a rationale for stratifying HCC patients undergoing OXA-based HAIC based on etiology. Lnc-OXAR holds promise as a novel target for overcoming OXA resistance in NASH-HCC and improving prognosis.

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

KDM6A-SND1 interaction maintains genomic stability by protecting the nascent DNA and contributes to cancer chemoresistance.

Genomic instability is one of the hallmarks of cancer. While loss of histone demethylase KDM6A increases the risk of tumorigenesis, its specific role in maintaining genomic stability remains poorly understood. Here, we propose a mechanism in which KDM6A maintains genomic stability independently on its demethylase activity. This occurs through its interaction with SND1, resulting in the establishment of a protective chromatin state that prevents replication fork collapse by recruiting of RPA and Ku70 to nascent DNA strand. Notably, KDM6A-SND1 interaction is up-regulated by KDM6A SUMOylation, while KDM6AK90A mutation almost abolish the interaction. Loss of KDM6A or SND1 leads to increased enrichment of H3K9ac and H4K8ac but attenuates the enrichment of Ku70 and H3K4me3 at nascent DNA strand. This subsequently results in enhanced cellular sensitivity to genotoxins and genomic instability. Consistent with these findings, knockdown of KDM6A and SND1 in esophageal squamous cell carcinoma (ESCC) cells increases genotoxin sensitivity. Intriguingly, KDM6A H101D & P110S, N1156T and D1216N mutations identified in ESCC patients promote genotoxin resistance via increased SND1 association. Our finding provides novel insights into the pivotal role of KDM6A-SND1 in genomic stability and chemoresistance, implying that targeting KDM6A and/or its interaction with SND1 may be a promising strategy to overcome the chemoresistance.

KBM-mediated interactions with KU80 promote cellular resistance to DNA replication stress in CHO cells.

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.

Discovery of UMI-77 as a novel Ku70/80 inhibitor sensitizing cancer cells to DNA damaging agents in vitro and in vivo.

The emergence of chemoresistance poses a significant challenge to the efficacy of DNA-damaging agents in cancer treatment, in part due to the inherent DNA repair capabilities of cancer cells. The Ku70/80 protein complex (Ku) plays a central role in double-strand breaks (DSBs) repair through the classical non-homologous end joining (c-NHEJ) pathway, and has proven to be one of the most promising drug target for cancer treatment when combined with radiotherapy or chemotherapy. In this study, we conducted a high-throughput screening of small-molecule inhibitors targeting the Ku complex by using a fluorescence polarization-based DNA binding assay. From a library of 11,745 small molecules, UMI-77 was identified as a potent Ku inhibitor, with an IC50 value of 2.3 µM. Surface plasmon resonance and molecular docking analyses revealed that UMI-77 directly bound the inner side of Ku ring, thereby disrupting Ku binding with DNA. In addition, UMI-77 also displayed potent inhibition against MUS81-EME1, a key player in homologous recombination (HR), demonstrating its potential for blocking both NHEJ- and HR-mediated DSB repair pathways. Further cell-based studies showed that UMI-77 could impair bleomycin-induced DNA damage repair, and significantly sensitized multiple cancer cell lines to the DNA-damaging agents. Finally, in a mouse xenograft tumor model, UMI-77 significantly enhanced the chemotherapeutic efficacy of etoposide with little adverse physiological effects. Our work offers a new avenue to combat chemoresistance in cancer treatment, and suggests that UMI-77 could be further developed as a promising candidate in cancer treatment.

Modulating DNA damage response in uveal melanoma through embryonic stem cell microenvironment.

BACKGROUND: Uveal melanoma (UVM) is the most common primary intraocular tumor in adults, with a median survival of 4-5 months following metastasis. DNA damage response (DDR) upregulation in UVM, which could be linked to its frequent activation of the PI3K/AKT pathway, contributes to its treatment resistance. We have reported that embryonic stem cell microenvironments (ESCMe) can revert cancer cells to less aggressive states through downregulation of the PI3K signaling, showing promise in modulating the DDR of UVM. METHODS: Since nonhomologous end joining (NHEJ) is the main DNA repair mechanism in UVM, this study utilized gene expression analysis and survival prognosis analysis to investigate the role of NHEJ-related genes in UVM based on public databases. Xenograft mouse models were established to assess the therapeutic potential of ESC transplantation and exposure to ESC-conditioned medium (ESC-CM) on key DNA repair pathways in UVM. Quantitative PCR and immunohistochemistry were used to analyze NHEJ pathway-related gene expression in UVM and surrounding normal tissues. Apoptosis in UVM tissues was evaluated using the TUNEL assay. RESULTS: PRKDC, KU70, XRCC5, LIG4 and PARP1 showed significant correlations with UM progression. High expression of PRKDC and XRCC5 predicted poorer overall survival, while low PARP1 and XRCC6 expression predicted better disease-free survival in UVM patients. ESCMe treatment significantly inhibited the NHEJ pathway transcriptionally and translationally and promoted apoptosis in tumor tissues in mice bearing UVM. Furthermore, ESC transplantation enhanced DDR activities in surrounding normal cells, potentially mitigating the side effects of cancer therapy. Notably, direct cell-to-cell contact with ESCs was more effective than their secreted factors in regulating the NHEJ pathway. CONCLUSIONS: Our results suggest that NHEJ-related genes might serve as prognostic markers and therapeutic targets in UVM. These findings support the therapeutic potential of ESC-based therapy in enhancing UVM sensitivity to radiochemotherapy and improving treatment outcomes while minimizing damage to healthy cells.

Identification and Quantification of Radiotherapy-related Protein Expression in Cancer Tissues Using the Qupath Software and Prediction of Treatment Response.

BACKGROUND/AIM: Automated measurement of immunostained samples can enable more convenient and objective prediction of treatment outcome from radiotherapy. We aimed to validate the performance of the QuPath image analysis software in immune cell markers detection by comparing QuPath cell counting results with those of physician manual cell counting. PATIENTS AND METHODS: CD8- and FoxP3-stained cervical, CD8-stained oropharyngeal, and Ku70-stained prostate cancer tumor sections were analyzed in 104 cervical, 92 oropharyngeal, and 58 prostate cancer patients undergoing radiotherapy at our Institution. RESULTS: QuPath and manual counts were highly correlated. When divided into two groups using ROC curves, the agreement between QuPath and manual counts was 89.4% for CD8 and 88.5% for FoxP3 in cervical cancer, 87.0% for CD8 in oropharyngeal cancer and 80.7% for Ku70 in prostate cancer. In cervical cancer, the high CD8 group based on QuPath counts had a better prognosis and the low CD8 group had a significantly worse prognosis [p=0.0003; 5-year overall survival (OS), 65.9% vs. 34.7%]. QuPath counts were more predictive than manual counts. Similar results were observed for FoxP3 in cervical cancer (p=0.002; 5-year OS, 62.1% vs. 33.6%) and CD8 in oropharyngeal cancer (p=0.013; 5-year OS, 80.2% vs. 47.2%). In prostate cancer, high Ku70 group had worse and low group significantly better outcome [p=0.007; 10-year progression-free survival (PFS), 56.0% vs. 93.8%]. CONCLUSION: QuPath showed a strong correlation with manual counting, confirming its utility and accuracy and potential applicability in clinical practice.

DNA-PK participates in pre-rRNA biogenesis independent of DNA double-strand break repair.

Although DNA-PK inhibitors (DNA-PK-i) have been applied in clinical trials for cancer treatment, the biomarkers and mechanism of action of DNA-PK-i in tumor cell suppression remain unclear. Here, we observed that a low dose of DNA-PK-i and PARP inhibitor (PARP-i) synthetically suppresses BRCA-deficient tumor cells without inducing DNA double-strand breaks (DSBs). Instead, we found that a fraction of DNA-PK localized inside of nucleoli, where we did not observe obvious DSBs. Moreover, the Ku proteins recognize pre-rRNA that facilitates DNA-PKcs autophosphorylation independent of DNA damage. Ribosomal proteins are also phosphorylated by DNA-PK, which regulates pre-rRNA biogenesis. In addition, DNA-PK-i acts together with PARP-i to suppress pre-rRNA biogenesis and tumor cell growth. Collectively, our studies reveal a DNA damage repair-independent role of DNA-PK-i in tumor suppression.

High expression of PPP1CC promotes NHEJ-mediated DNA repair leading to radioresistance and poor prognosis in nasopharyngeal carcinoma.

Protein phosphatase 1 catalytic subunit gamma (PPP1CC) promotes DNA repair and tumor development and progression, however, its underlying mechanisms remain unclear. This study investigated the molecular mechanism of PPP1CC's involvement in DNA repair and the potential clinical implications. High expression of PPP1CC was significantly correlated with radioresistance and poor prognosis in human nasopharyngeal carcinoma (NPC) patients. The mechanistic study revealed that PPP1CC bound to Ku70/Ku80 heterodimers and activated DNA-PKcs by promoting DNA-PK holoenzyme formation, which enhanced nonhomologous end junction (NHEJ) -mediated DNA repair and led to radioresistance. Importantly, BRCA1-BRCA2-containing complex subunit 3 (BRCC3) interacted with PPP1CC to enhance its stability by removing the K48-linked polyubiquitin chain at Lys234 to prevent PPP1CC degradation. Therefore, BRCC3 helped the overexpressed PPP1CC to maintain its high protein level, thereby sustaining the elevation of DNA repair capacity and radioresistance. Our study identified the molecular mechanism by which PPP1CC promotes NHEJ-mediated DNA repair and radioresistance, suggesting that the BRCC3-PPP1CC-Ku70 axis is a potential therapeutic target to improve the efficacy of radiotherapy.

CircUGGT2 facilitates progression and cisplatin resistance of bladder cancer through nonhomologous end-joining pathway.

The development of resistance to cisplatin (CDDP) in bladder cancer presents a notable obstacle, with indications pointing to the substantial role of circular RNAs (circRNAs) in this resistance. Nevertheless, the precise mechanisms through which circRNAs govern resistance are not yet fully understood. Our findings demonstrate that circUGGT2 is significantly upregulated in bladder cancer, facilitating cancer cell migration and invasion. Additionally, our analysis of eighty patient outcomes revealed a negative correlation between circUGGT2 expression levels and prognosis. Using circRNA pull-down assays, mass spectrometry analyses, and RNA Immunoprecipitation (RIP), it was shown that circUGGT2 interacts with the KU heterodimer, consisting of KU70 and KU80. Both KU70 and KU80 are critical components of the non-homologous end joining (NHEJ) pathway, which plays a role in CDDP resistance. Flow cytometry was utilized in this study to illustrate the impact of circUGGT2 on the sensitivity of bladder cancer cell lines to CDDP through its interaction with KU70 and KU80. Additionally, a reduction in the levels of DNA repair factors associated with the NHEJ pathway, such as KU70, KU80, DNA-PKcs, and XRCC4, was observed in chromatin of bladder cancer cells following circUGGT2 knockdown post-CDDP treatment, while the levels of DNA repair factors in total cellular proteins remained constant. Thus, the promotion of CDDP resistance by circUGGT2 is attributed to its facilitation of repair factor recruitment to DNA breaks via interaction with the KU heterodimer. Furthermore, our study demonstrated that knockdown of circUGGT2 resulted in reduced levels of γH2AX, a marker of DNA damage response, in CDDP-treated bladder cancer cells, implicating circUGGT2 in the NHEJ pathway for DNA repair.

The cGAS-Ku80 complex regulates the balance between two end joining subpathways.

As the major DNA sensor that activates the STING-TBK1 signaling cascade, cGAS is mainly present in the cytosol. A number of recent reports have indicated that cGAS also plays critical roles in the nucleus. Our previous work demonstrated for the first time that cGAS is translocated to the nucleus upon the occurrence of DNA damage and inhibits homologous recombination (HR), one of the two major pathways of DNA double strand break (DSB) repair. However, whether nuclear cGAS regulates the other DSB repair pathway, nonhomologous end joining (NHEJ), which can be further divided into the less error-prone canonical NHEJ (c-NHEJ) and more mutagenic alternative NHEJ (alt-NHEJ) subpathways, has not been characterized. Here, we demonstrated that cGAS tipped the balance of the two NHEJ subpathways toward c-NHEJ. Mechanistically, the cGAS-Ku80 complex enhanced the interaction between DNA-PKcs and the deubiquitinase USP7 to improve DNA-PKcs protein stability, thereby promoting c-NHEJ. In contrast, the cGAS-Ku80 complex suppressed alt-NHEJ by directly binding to the promoter of Polθ to suppress its transcription. Together, these findings reveal a novel function of nuclear cGAS in regulating DSB repair, suggesting that the presence of cGAS in the nucleus is also important in the maintenance of genome integrity.

Uncoupling programmed DNA cleavage and repair scrambles the Paramecium somatic genome.

In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.

A di-acetyl-decorated chromatin signature couples liquid condensation to suppress DNA end synapsis.

Appropriate DNA end synapsis, regulated by core components of the synaptic complex including KU70-KU80, LIG4, XRCC4, and XLF, is central to non-homologous end joining (NHEJ) repair of chromatinized DNA double-strand breaks (DSBs). However, it remains enigmatic whether chromatin modifications can influence the formation of NHEJ synaptic complex at DNA ends, and if so, how this is achieved. Here, we report that the mitotic deacetylase complex (MiDAC) serves as a key regulator of DNA end synapsis during NHEJ repair in mammalian cells. Mechanistically, MiDAC removes combinatorial acetyl marks on histone H2A (H2AK5acK9ac) around DSB-proximal chromatin, suppressing hyperaccumulation of bromodomain-containing protein BRD4 that would otherwise undergo liquid-liquid phase separation with KU80 and prevent the proper installation of LIG4-XRCC4-XLF onto DSB ends. This study provides mechanistic insight into the control of NHEJ synaptic complex assembly by a specific chromatin signature and highlights the critical role of H2A hypoacetylation in restraining unscheduled compartmentalization of DNA repair machinery.

DNA-PKcs suppresses illegitimate chromosome rearrangements.

Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ involved in humans and A-EJ in mice predominantly. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in humans is not known. We find partial DNA-PKcs inhibition in human cells leads to increased translocations and the continued involvement of a dampened NHEJ. In contrast, complete DNA-PKcs inhibition substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse progenitor B cell lines, significantly impairs V(D)J recombination and generated higher rates of translocations as a consequence of dysregulated coding and signal end joining. Genetic DNA-PKcs inhibition suppresses NHEJ entirely, with repair phenotypically resembling Ku70-deficient A-EJ. In contrast, we find DNA-PKcs necessary in generating the near-exclusive MMEJ associated with Lig4 deficiency. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement while also contributing to MMEJ in both species.

DNA-PK controls Apollo's access to leading-end telomeres.

The complex formed by Ku70/80 and DNA-PKcs (DNA-PK) promotes the synapsis and the joining of double strand breaks (DSBs) during canonical non-homologous end joining (c-NHEJ). In c-NHEJ during V(D)J recombination, DNA-PK promotes the processing of the ends and the opening of the DNA hairpins by recruiting and/or activating the nuclease Artemis/DCLRE1C/SNM1C. Paradoxically, DNA-PK is also required to prevent the fusions of newly replicated leading-end telomeres. Here, we describe the role for DNA-PK in controlling Apollo/DCLRE1B/SNM1B, the nuclease that resects leading-end telomeres. We show that the telomeric function of Apollo requires DNA-PKcs's kinase activity and the binding of Apollo to DNA-PK. Furthermore, AlphaFold-Multimer predicts that Apollo's nuclease domain has extensive additional interactions with DNA-PKcs, and comparison to the cryo-EM structure of Artemis bound to DNA-PK phosphorylated on the ABCDE/Thr2609 cluster suggests that DNA-PK can similarly grant Apollo access to the DNA end. In agreement, the telomeric function of DNA-PK requires the ABCDE/Thr2609 cluster. These data reveal that resection of leading-end telomeres is regulated by DNA-PK through its binding to Apollo and its (auto)phosphorylation-dependent positioning of Apollo at the DNA end, analogous but not identical to DNA-PK dependent regulation of Artemis at hairpins.

Ku80 is indispensable for repairing DNA double-strand breaks at highly methylated sites in human HCT116 cells.

DNA double-strand breaks (DSBs) are harmful to mammalian cells and a few of them can cause cell death. Accumulating DSBs in these cells to analyze their genomic distribution and their potential impact on chromatin structure is difficult. In this study, we used CRISPR to generate Ku80-/- human cells and arrested the cells in G1 phase to accumulate DSBs before conducting END-seq and Nanopore analysis. Our analysis revealed that DNA with high methylation level accumulates DSB hotspots in Ku80-/- human cells. Furthermore, we identified chromosome structural variants (SVs) using Nanopore sequencing and observed a higher number of SVs in Ku80-/- human cells. Based on our findings, we suggest that the high efficiency of Ku80 knockout in human HCT116 cells makes it a promising model for characterizing SVs in the context of 3D chromatin structure and studying the alternative-end joining (Alt-EJ) DSB repair pathway.