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.

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.

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.

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-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.

Molecular cloning, subcellular localization, and rapid recruitment to DNA damage sites of chicken Ku70.

Ku70 is a multifunctional protein with pivotal roles in DNA repair via non-homologous end-joining, V(D)J recombination, telomere maintenance, and neuronal apoptosis control. Nonetheless, its regulatory mechanisms remain elusive. Chicken Ku70 (GdKu70) cDNA has been previously cloned, and DT40 cells expressing it have significantly contributed to critical biological discoveries. GdKu70 features an additional 18 amino acids at its N-terminus compared to mammalian Ku70, the biological significance of which remains uncertain. Here, we show that the 5' flanking sequence of GdKu70 cDNA is not nearly encoded in the chicken genome. Notably, these 18 amino acids result from fusion events involving the NFE2L1 gene on chromosome 27 and the Ku70 gene on chromosome 1. Through experiments using newly cloned chicken Ku70 cDNA and specific antibodies, we demonstrated that Ku70 localizes within the cell nucleus as a heterodimer with Ku80 and promptly accumulates at DNA damage sites following injury. This suggests that the functions and spatiotemporal regulatory mechanisms of Ku70 in chickens closely resemble those in mammals. The insights and resources acquired will contribute to elucidate the various mechanisms by which Ku functions. Meanwhile, caution is advised when interpreting the previous numerous key studies that relied on GdKu70 cDNA and its expressing cells.

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.

CALCRL induces resistance to daunorubicin in acute myeloid leukemia cells through upregulation of XRCC5/TYK2/JAK1 pathway.

Chemotherapy is the main treatment option for acute myeloid leukemia (AML), but acquired resistance of leukemic cells to chemotherapeutic agents often leads to difficulties in AML treatment and disease relapse. High calcitonin receptor-like (CALCRL) expression is closely associated with poorer prognosis in AML patients. Therefore, this study was performed by performing CALCRL overexpression constructs in AML cell lines HL-60 and Molm-13 with low CALCRL expression. The results showed that overexpression of CALCRL in HL-60 and Molm-13 could confer resistance properties to AML cells and reduce the DNA damage and cell cycle G0/G1 phase blocking effects caused by daunorubicin (DNR) and others. Overexpression of CALCRL also reduced DNR-induced apoptosis. Mechanistically, the Cancer Clinical Research Database analyzed a significant positive correlation between XRCC5 and CALCRL in AML patients. Therefore, the combination of RT-PCR and Western blot studies further confirmed that the expression levels of XRCC5 and PDK1 genes and proteins were significantly upregulated after overexpression of CALCRL. In contrast, the phosphorylation levels of AKT/PKCε protein, a downstream pathway of XRCC5/PDK1, were significantly upregulated. In the response study, transfection of overexpressed CALCRL cells with XRCC5 siRNA significantly upregulated the drug sensitivity of AML to DNR. The expression levels of PDK1 protein and AKT/PKCε phosphorylated protein in the downstream pathway were inhibited considerably, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were upregulated. Animal experiments showed that the inhibitory effect of DNR on the growth of HL-60 cells and the number of bone marrow invasions were significantly reversed after overexpression of CALCRL in nude mice. However, infection of XCRR5 shRNA lentivirus in HL-60 cells with CALCRL overexpression attenuated the effect of CALCRL overexpression and upregulated the expression of apoptosis-related proteins induced by DNR. This study provides a preliminary explanation for the relationship between high CALCRL expression and poor prognosis of chemotherapy in AML patients. It offers a more experimental basis for DNR combined with molecular targets for precise treatment in subsequent studies.

Mycobacterium tuberculosis Ku Stimulates Multi-round DNA Unwinding by UvrD1 Monomers.

Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to infection, the bacterium is exposed to both reactive oxygen species and nitrogen intermediates that can cause DNA damage. It is becoming clear that the DNA damage response in Mtb and related actinobacteria function via distinct pathways as compared to well-studied model bacteria. For example, we have previously shown that the DNA repair helicase UvrD1 is activated for processive unwinding via redox-dependent dimerization. In addition, mycobacteria contain a homo-dimeric Ku protein, homologous to the eukaryotic Ku70/Ku80 dimer, that plays roles in double-stranded break repair via non-homologous end-joining. Kuhas been shown to stimulate the helicase activity of UvrD1, but the molecular mechanism, as well as which redox form of UvrD1 is activated, is unknown. We show here that Ku specifically stimulates multi-round unwinding by UvrD1 monomers which are able to slowly unwind DNA, but at rates 100-fold slower than the dimer. We also demonstrate that the UvrD1 C-terminal Tudor domain is required for the formation of a Ku-UvrD1 protein complex and activation. We show that Mtb Ku dimers bind with high nearest neighbor cooperativity to duplex DNA and that UvrD1 activation is observed when the DNA substrate is bound with two or three Ku dimers. Our observations reveal aspects of the interactions between DNA, Mtb Ku, and UvrD1 and highlight the potential role of UvrD1 in multiple DNA repair pathways through different mechanisms of activation.

The SAP domain of Ku facilitates its efficient loading onto DNA ends.

The evolutionarily conserved DNA repair complex Ku serves as the primary sensor of free DNA ends in eukaryotic cells. Its rapid association with DNA ends is crucial for several cellular processes, including non-homologous end joining (NHEJ) DNA repair and telomere protection. In this study, we conducted a transient kinetic analysis to investigate the impact of the SAP domain on individual phases of the Ku-DNA interaction. Specifically, we examined the initial binding, the subsequent docking of Ku onto DNA, and sliding of Ku along DNA. Our findings revealed that the C-terminal SAP domain of Ku70 facilitates the initial phases of the Ku-DNA interaction but does not affect the sliding process. This suggests that the SAP domain may either establish the first interactions with DNA, or stabilize these initial interactions during loading. To assess the biological role of the SAP domain, we generated Arabidopsis plants expressing Ku lacking the SAP domain. Intriguingly, despite the decreased efficiency of the ΔSAP Ku complex in loading onto DNA, the mutant plants exhibited full proficiency in classical NHEJ and telomere maintenance. This indicates that the speed with which Ku loads onto telomeres or DNA double-strand breaks is not the decisive factor in stabilizing these DNA structures.

Structural and functional basis of inositol hexaphosphate stimulation of NHEJ through stabilization of Ku-XLF interaction.

The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.

DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

PC4-mediated Ku complex PARylation facilitates NHEJ-dependent DNA damage repair.

Radiotherapy is one of the mainstay treatments for hepatocellular carcinoma (HCC). However, a substantial number of patients with HCC develop radioresistance and eventually suffer from tumor progression or relapse, which is a major impediment to the use of radiotherapy. Therefore, elucidating the mechanisms underlying radioresistance and identifying novel therapeutic targets to improve patient prognosis are important in HCC management. In this study, using in vitro and in vivo models, laser microirradiation and live cell imaging methods, and coimmunoprecipitation assays, we report that a DNA repair enhancer, human positive cofactor 4 (PC4), promotes nonhomologous end joining-based DNA repair and renders HCC cells resistant to radiation. Mechanistically, PC4 interacts with poly (ADP-ribose) polymerase 1 and directs Ku complex PARylation, resulting in the successful recruitment of the Ku complex to damaged chromatin and increasing the efficiency of nonhomologous end joining repair. Clinically, PC4 is highly expressed in tumor tissues and is correlated with poor prognosis in patients with HCC. Taken together, our data suggest that PC4 is a DNA repair driver that can be targeted to radiosensitize HCC cells.

RUSC1-AS1 promotes the malignant progression of breast cancer depending on the regulation of the miR-326/XRCC5 pathway.

BACKGROUND: Many long noncoding RNAs (lncRNAs) are the key regulators for cancer progression, including breast cancer (BC). RUSC1 antisense 1 (RUSC1-AS1) has been found to be highly expressed in BC, but its role and potential molecular mechanism in BC remain to be further elucidated. METHODS: Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was utilized to measure RUSC1-AS1, microRNA (miR)-326 and X-ray repair cross-complementing group 5 (XRCC5) expression. Cell proliferation, metastasis, cell cycle, apoptosis and angiogenesis were determined by cell counting kit-8, colony formation, transwell, flow cytometry and tube formation assays. Protein expression was detected by western blot analysis. The targeted relationship between miR-326 and RUSC1-AS1 or XRCC5 was validated using dual-luciferase reporter assay and RIP assay. Xenograft models were constructed to uncover the effect of RUSC1-AS1 on BC tumorigenesis. RESULTS: RUSC1-AS1 was upregulated in BC, and its downregulation suppressed BC proliferation, metastasis, cell cycle, angiogenesis, and tumor growth. MiR-326 was confirmed to be sponged by RUSC1-AS1, and its inhibitor reversed the regulation of RUSC1-AS1 silencing on BC progression. XRCC5 could be targeted by miR-326. Overexpression of XRCC5 reversed the inhibitory impacts of miR-326 on BC progression. CONCLUSION: RUSC1-AS1 could serve as a sponge of miR-326 to promote BC progression by targeting XRCC5, suggesting that RUSC1-AS1 might be a target for BC treatment.

Noncanonical functions of Ku may underlie essentiality in human cells.

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku's role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function. While we observed loss of cell viability upon Ku depletion, we did not detect significant changes in telomere length, nor did we record lethal levels of DNA damage upon loss of Ku. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study suggests that the driving cause of loss of cell viability in Ku70 knockouts is not linked to the functions of Ku in DNA repair or at telomeres. Moreover, our data shows that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

SMYD2 inhibition-mediated hypomethylation of Ku70 contributes to impaired nonhomologous end joining repair and antitumor immunity.

DNA damage repair (DDR) is a double-edged sword with different roles in cancer susceptibility and drug resistance. Recent studies suggest that DDR inhibitors affect immune surveillance. However, this phenomenon is poorly understood. We report that methyltransferase SMYD2 plays an essential role in nonhomologous end joining repair (NHEJ), driving tumor cells adaptive to radiotherapy. Mechanically, in response to DNA damage, SMYD2 is mobilized onto chromatin and methylates Ku70 at lysine-74, lysine-516, and lysine-539, leading to increased recruitment of Ku70/Ku80/DNA-PKcs complex. Knockdown of SMYD2 or its inhibitor AZ505 results in persistent DNA damage and improper repair, which sequentially leads to accumulation of cytosolic DNA, and activation of cGAS-STING pathway and triggers antitumor immunity via infiltration and activation of cytotoxic CD8+ T cells. Our study reveals an unidentified role of SMYD2 in regulating NHEJ pathway and innate immune responses, suggesting that SMYD2 is a promising therapeutic target for cancer treatment.

The flexible and iterative steps within the NHEJ pathway.

Cellular and biochemical studies of nonhomologous DNA end joining (NHEJ) have long established that nuclease and polymerase action are necessary for the repair of a very large fraction of naturally-arising double-strand breaks (DSBs). This conclusion is derived from NHEJ studies ranging from yeast to humans and all genetically-tractable model organisms. Biochemical models derived from recent real-time and structural studies have yet to incorporate physical space or timing for DNA end processing. In real-time single molecule FRET (smFRET) studies, we analyzed NHEJ synapsis of DNA ends in a defined biochemical system. We described a Flexible Synapsis (FS) state in which the DNA ends were in proximity via only Ku and XRCC4:DNA ligase 4 (X4L4), and in an orientation that would not yet permit ligation until base pairing between one or more nucleotides of microhomology (MH) occurred, thereby allowing an in-line Close Synapsis (CS) state. If no MH was achievable, then XLF was critical for ligation. Neither FS or CS required DNA-PKcs, unless Artemis activation was necessary to permit local resection and subsequent base pairing between the two DNA ends being joined. Here we conjecture on possible 3D configurations for this FS state, which would spatially accommodate the nuclease and polymerase processing steps in an iterative manner. The FS model permits repeated attempts at ligation of at least one strand at the DSB after each round of nuclease or polymerase action. In addition to activation of Artemis, other possible roles for DNA-PKcs are discussed.

Kaempferol inhibits non-homologous end joining repair via regulating Ku80 stability in glioma cancer.

BACKGROUND: Targeting DNA damage response and DNA repair proficiency of cancers is an important anticancer strategy. Kaempferol (Kae), a natural flavonoid, displays potent antitumor properties in some cancers. However, the precise underlying mechanism of Kae regulates DNA repair system are poorly understood. PURPOSE: We aim to evaluate the efficacy of Kae in the treatment of human glioma as well as the molecular mechanism regarding DNA repair. STUDY DESIGN: Effects of Kae on glioma cells were detected using CCK-8 and EdU labeling assays. The molecular mechanism of Kae on glioma was determined using RNAseq. The inhibition effects of Kae on DNA repair were verified using Immunoprecipitation, immunofluorescence, and pimEJ5-GFP report assays. For in vivo study, orthotopic xenograft models were established and treated with Kae or vehicle. Glioma development was monitored by bioluminescence imaging, Magnetic Resonance Imaging (MRI), and brain sections Hematoxylin/Eosin (HE) staining. Immunohistochemical (IHC) analysis was used to detect expression of Ku80, Ki67 and γH2AX in engrafted glioma tissue. RESULTS: We found that Kae remarkably inhibits viability of glioma cells and decreases its proliferation. Mechanistically, Kae regulates multiple functional pathways associated with cancer, including non-homologous end joining (NHEJ) repair. Further studies revealed that Kae inhibits release of Ku80 from the double-strand breaks (DSBs) sites via reducing ubiquitylation and degradation of Ku80. Therefore, Kae significantly suppresses NHEJ repair and induces accumulation of DSBs in glioma cells. Moreover, Kae displays a dramatic inhibition effects on glioma growth in an orthotopic transplantation model. These data demonstrate that Kae can induce deubiquitination of Ku80, suppress NHEJ repair and inhibit glioma growth. CONCLUSION: Our findings indicate that inhibiting release of Ku80 from the DSBs by Kae may be a potential effective approach for glioma treatment.