Search Results (262)

Background: Non-homologous end joining (NHEJ) is a crucial pathway for repairing DNA double-strand breaks and a key contributor to chemoresistance in cancer. The assembly of the DNA Ligase IV (LIG4)–XRCC4 complex is essential for NHEJ fidelity, however, the regulatory mechanisms governing this complex in cancer remain poorly understood. This study aims to investigate whether and how lactate, a key metabolic byproduct of the Warburg effect, regulates the XRCC4–LIG4 complex and influences chemoresistance. Methods: The functional role of lactate in NHEJ was assessed using DNA repair reporter assays in ovarian cancer cells. Protein–protein interactions were examined through co-immunoprecipitation and pull-down assays. The molecular mechanism of lactate’s action was delineated using a combination of site-directed mutagenesis, in vitro binding assays, and molecular docking. Finally, the physiological relevance of lactate-mediated NHEJ was validated in a preclinical ovarian cancer mouse model treated with cisplatin. Results: We demonstrated that lactate enhances NHEJ repair efficiency and confers resistance to DNA-damaging chemotherapeutics. Mechanistically, lactate directly binds to XRCC4 at key residues, including Y66, E55, and S110, thereby strengthening the XRCC4–LIG4 association. This interaction is independent of protein lactylation. In vivo studies confirmed that lactate-driven NHEJ promotes chemoresistance in ovarian cancer. Conclusions: Our findings reveal lactate as a novel metabolic regulator of the NHEJ pathway by directly allosterically modulating the XRCC4–LIG4 complex. This work establishes a direct molecular link between the Warburg effect and DNA repair-driven chemoresistance, offering new insights into potential therapeutic strategies for ovarian cancer. Full article

DNA damage is a hallmark of the fatal process of neurodegeneration in the central nervous system (CNS). As neurons are terminally differentiated, they accumulate metabolic and oxidative burdens over their whole life span. Unrepaired DNA develops into DNA double-strand breaks (DSBs), which are repaired through homologous recombination (HR) or non-homologous end joining (NHEJ). Being post-mitotic and unable to normally undergo HR, damage and defective repair is especially burdensome to CNS neurons. Current research has not produced treatment to prevent and halt progression of neurodegeneration. Hence, novel targeting strategies are desperately needed. Recent investigations in histone post-translational modifications (PTMs) reveal new mechanistic insight and highlight unexplored targets to ameliorate neurodegeneration. As various histone PTMs dictate and facilitate DSB repair, they represent an underexploited area in investigating DNA damage and incorrect repair aiding neurodegeneration. Here, we review the histone PTM alterations in several neurodegenerative diseases: Amyotrophic Lateral Sclerosis/Frontotemporal Dementia, Parkinson’s Disease, Alzheimer’s Diseases, Multiple Sclerosis, and Huntington’s Disease. These findings emphasize that histone PTM alterations can enable an aberrant DNA damage response (DDR) leading to neurodegeneration. Further research into the connections between histone PTMs and DNA damage in decaying neurons will illuminate novel targets to dampen the aberrant DDR and promote neuronal survival. Full article

Cold-adapted species display remarkable genomic resilience under prolonged freezing and thawing cycles that would be lethal to most organisms. This review synthesizes current knowledge on the molecular mechanisms of DNA damage response (DDR) and epigenetic regulation that collectively safeguard genome integrity in these organisms. We highlight key DNA repair pathways, including base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR), and non-homologous end joining (NHEJ), that are activated during freeze–thaw stress to repair oxidative and strand break damage. Epigenetic regulators such as DNA methyltransferases (DNMTs), histone methyltransferases, and histone deacetylases (HDACs) dynamically remodel chromatin and modulate DDR signaling, facilitating efficient repair and transcriptional reprogramming during recovery. Comparative evidence from freeze-tolerant vertebrates, hibernating mammals, and polar fish underscores the conservation of these adaptive pathways across taxa. Integrating these insights provides a molecular network perspective (MNP) linking DDR and epigenetic mechanisms to environmental resilience, with potential applications in crop improvement and biotechnological adaptation strategies for extreme environments. Full article

CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, Cas9 expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving Cas9 with promoters of AtKu70 and AtKu80, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of AtKu70 and AtKu80 were cloned and used to drive Cas9 in the CRISPR/Cas9 system. Four different genes, GmRj7, GmNNL1, AtPDS3, and AtBRI1, were designed for soybean hairy root transformation and Arabidopsis transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of AtKu70 and AtKu80 achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by Rhizobium rhizogenes and stable genetic transformation with Rhizobium tumefaciens. Full article

Ovarian cancer (OvCa) is one of the most life-threatening female malignancies that affects 300,000 women annually worldwide. Impaired mechanisms of DNA repair are the leading cause of mutations underlying the OvCa development. microRNAs are short non-coding RNAs that regulate the expression of genes by binding to their transcripts and inducing mRNA degradation or inhibition of translation. Here, we review the miRNA-mediated dysregulation of genes involved in DNA damage response (DDR) and DNA repair pathways in OvCa. Apparently, miRNAs are capable of targeting the crucial mediators of DDR (e.g., miR-203a-3p targeting ATM (Ataxia Telangiectasia Mutated)), homologous repair (such as BRCA1 targeted by miR-9, miR-1255b, miR-193b, and miR-148b), non-homologous end joining (with RNF8 being regulated by miR-214), nucleotide excision repair (involving DDB2 targeted by miR-328-3p), or translesion DNA synthesis (involving RAD18, participating also in homologous repair and targeted by miR-379-5p). We also discuss miRNAs (such as miR-519a-3p, let-7e, miR-216b), which affect responses to OvCa therapy by targeting PARP1 (Poly(ADP-Ribose) Polymerase-1). Finally, we also discuss why, despite the identification of multiple miRNAs capable of regulating DNA repair genes, as well as those involved in the response to therapy, no miRNA-based drugs have been approved for OvCa treatment in clinics. Full article

The global population constantly breathes particulate matter with an aerodynamic diameter of ≤10 µm (PM₁₀)—a human carcinogen linked to lung cancer. Previous studies have indicated that PM₁₀ causes DNA damage, including double-strand breaks (DSBs). In particular, DSBs are primarily repaired by the non-homologous end joining (NHEJ) pathway, which is essential for maintaining genomic stability; however, the effects of PM₁₀ exposure on this pathway are unknown. To address this, A549 lung epithelial cells were exposed to 10 µg/cm² of PM₁₀ for 6, 12, and 24 h. We determined that DSBs increased with prolonged exposure, and an increase in the frequency of micronuclei was found. Despite the accumulated DNA damage, no changes in the cell cycle were observed. Reductions in the levels of the Ku80 gene and protein, as well as the Ku70–Ku80 heterodimer—which is essential for initiating NHEJ-mediated repair—were observed. Levels of Artemis (which is responsible for processing DNA damage) remained stable, while levels of the XRCC4 gene and protein (responsible for completing repair) decreased. We conclude that PM₁₀ disrupts two key proteins in the NHEJ pathway, impairing the capacity for DSB repair. This could promote the accumulation of DNA damage and induce genomic instability, contributing to the development of cancer. Full article

BQ323636.1 (BQ) is a splice variant of NCOR2. Its overexpression is associated with endocrine therapy and chemoresistance in estrogen receptor-positive (ER+ve) breast cancer. This study investigates how BQ overexpression drives doxorubicin (DOX) resistance by enhancing androgen receptor (AR) signaling and non-homologous end joining (NHEJ). BQ overexpressed breast cancer cell lines (MCF-7, T-47D, BT-549, MDA-MB-453), showed increased AR activity (ARE-luciferase assay) and demonstrated DOX resistance (EC50 > 10-fold with DHT, p < 0.05), as assessed via cell viability, TUNEL, and comet assays. RNA-sequencing (GSE295979, GSE2048) revealed the involvement of AR signaling. BQ upregulated cell cycle-related kinase (CCRK), stabilizing KU70, a key NHEJ protein, resulting in enhanced NHEJ activity (EJ5-GFP assay, p < 0.01). Co-immunoprecipitation confirmed the interaction between CCRK and KU70, and CCRK was found to modulate the protein stability of KU70. AR inhibition with bicalutamide in BQ overexpressing cells reversed DOX resistance. Xenograft models validated AR-dependent DOX resistance. In ER+ve breast cancer patient samples, high CCRK expression correlated with DOX resistance (p = 0.002) and metastasis (p = 0.001). Kaplan–Meier analysis showed poorer overall survival (p < 0.001) and disease-specific survival (p < 0.001) in cancers with high CCRK. Cox-regression analysis showed that high CCRK was a poorer prognostic factor of overall survival (p < 0.001; RR 3.056, 95% CI 1.661, 5.621, AR (p < 0.001; RR 3.420, 95% CI 1.783, 6.562), and disease-specific survival (p < 0.001; RR 2.731, 95% CI 1.472, 5.067). The BQ-AR-CCRK-KU70 axis represents a novel mechanism of DOX resistance in ER+ve breast cancer, suggesting AR or CCRK inhibition as a potential therapeutic strategy. Full article

The CRISPR/Cas9 genome editing system has emerged as an effective platform to generate loss-of-function gene edits through non-homologous end joining (NHEJ) without a repair template. To verify whether small molecules can enhance the efficiency of CRISPR/ Cas9-mediated NHEJ gene editing in porcine cells, this experiment investigated the effects of six small-molecule compounds, namely Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, on the efficiency of CRISPR/Cas9-mediated NHEJ gene editing. The results showed the optimal concentrations of the small molecules, including Repsox, Zidovudine, IOX1, GSK-J4, YU238259, and GW843682X, for in vitro-cultured PK15 viability. Compared with the control group, the single small molecules Repsox, Zidovudine, GSK-J4, and IOX1 increased the efficiency of NHEJ-mediated gene editing 3.16-fold, 1.17-fold, 1.16-fold, and 1.120-fold, respectively, in the Cas9-sgRNA RNP delivery system. There were no benefits when using YU238259 and GW843682X compared with the control group. In the CRISPR/Cas9 plasmid delivery system, the Repsox, Zidovudine, IOX1, and GSK-J4 treatments increased the efficiency of NHEJ-mediated gene editing 1.47-fold, 1.15-fold, 1.21-fold, and 1.23-fold, respectively, compared with the control group. Repsox can also improve the efficiency of NHEJ-mediated multi-gene editing based on a CRISPR sgRNA-tRNA array. We also explored the mechanism of Repsox’s effect on the efficiency of NHEJ-mediated gene editing. The results showed that Repsox reduces the expression levels of SMAD2, SMAD3, and SMAD4 in the TGF-β pathway, indicating that Repsox can increase the efficiency of CRISPR NHEJ-mediated gene editing in porcine cells through the TGF-β pathway. Full article

Breast cancer remains one of the most prevalent malignancies worldwide, and radiation therapy is a central component of its management. However, intrinsic or acquired resistance to radiation significantly compromises therapeutic efficacy. This systematic review aimed to identify and evaluate molecular mechanisms and interventions that influence radiation sensitivity in breast cancer models. A comprehensive PubMed search was conducted using the terms “breast cancer” and “radiation resistance” for studies published between 2002 and 2024. Seventy-nine eligible studies were included. The most frequently investigated mechanisms included the dysregulation of the PI3K/AKT/mTOR and MAPK signaling pathways, enhanced DNA damage repair via non-homologous end joining (NHEJ), and the overexpression of cancer stem cell markers such as CD44⁺/CD24⁻/low and ALDH1. Several studies highlighted the role of non-coding RNAs, particularly the lncRNA DUXAP8 and microRNAs such as miR-21, miR-144, miR-33a, and miR-634, in modulating radiation response. Components of the tumor microenvironment, including cancer-associated fibroblasts and immune regulators, also contributed to radiation resistance. By synthesizing current evidence, this review provides a consolidated resource to guide future mechanistic studies and therapeutic development. This review highlights promising molecular targets and emerging strategies to enhance radiosensitivity and offers a foundation for translational research aimed at improving outcomes in radiation-refractory breast cancer. Full article

Targeted radionuclide therapy (TRT) utilizes radiopharmaceuticals to deliver radiation directly to cancer cells while sparing healthy tissues. Beyond the absorbed dose of ablative radiation, TRT induces non-targeted effects (NTEs) that significantly enhance its therapeutic efficacy. These effects include radiation-induced bystander effects (RIBEs), abscopal effects (AEs), radiation-induced genomic instability (RIGI), and adaptive responses, which collectively influence the behavior of cancer cells and the tumor microenvironment (TME). TRT also modulates immune responses, promoting immune-mediated cell death and enhancing the efficacy of combination therapies, such as the use of immune checkpoint inhibitors. The molecular mechanisms underlying TRT involve DNA damage, oxidative stress, and apoptosis, with repair pathways like homologous recombination (HR) and non-homologous end joining (NHEJ) playing critical roles. However, challenges such as tumor heterogeneity, hypoxia, and radioresistance limit the effectiveness of this approach. Advances in theranostics, which integrate diagnostic imaging with TRT, have enabled personalized treatment approaches, while artificial intelligence and improved dosimetry offer potential for treatment optimization. Despite the significant survival benefits of TRT in prostate cancer and neuroendocrine tumors, 30–40% of patients remain unresponsive, which highlights the need for further research into molecular pathways, long-term effects, and combined therapies. This review outlines the dual mechanisms of TRT, direct toxicity and NTEs, and discusses strategies to enhance its efficacy and expand its use in oncology. Full article

The “milky disease” in Chinese mitten crabs (Eriocheir sinensis), caused by Metschnikowia bicuspidata, poses significant threats to aquaculture, though its pathogenic mechanisms remain poorly understood. This study employs transcriptomic sequencing to analyze gene expression changes in Metschnikowia bicuspidata under hemocyte challenge, iron overload (1 mmol/mL), and combined stress, with functional validation through Common in Fungal Extracellular Membrane (CFEMgene) overexpression strains. Key findings reveal that (1) hemocyte challenge activated base excision repair (−log₁₀[P] = 7.58) and ribosome biogenesis pathways, indicating fungal adaptation through DNA repair and enhanced protein synthesis to counter host immune attacks (e.g., ROS-mediated damage). (2) Iron overload induced glutathione metabolism and pentose phosphate pathway enrichment, demonstrating mitigation of ferroptosis through NADPH/GSH antioxidant systems and autophagy/proteasome coordination. (3) Under combined stress, ribosome biogenesis (−log₁₀[P] = 1.3) and non-homologous end-joining pathways coordinated DNA repair with stress protein synthesis, complemented by vacuolar V-ATPase-mediated iron compartmentalization. (4) CFEM genes showed significant upregulation under hemocyte stress, with overexpression strains exhibiting enhanced biofilm formation (35% increased MTT cytotoxicity) and infectivity (40% higher infection rate), confirming CFEM domains mediate pathogenesis through iron homeostasis and virulence factor production. This work elucidates how M. bicuspidata employs metabolic reprogramming, oxidative stress responses, and CFEM-mediated iron regulation to establish infection, providing critical insights for developing targeted control strategies against milky disease. Full article

Epigenetic modifications are heritable, reversible alterations that causally reshape chromatin architecture and thereby influence DNA repair without changing nucleotide sequence. DNA methylation, histone modifications and non-coding RNAs profoundly influence DNA repair mechanisms and genomic stability. Aberrant epigenetic patterns in cancer compromise DNA damage recognition and repair, therefore impairing homologous recombination (HR), non-homologous end joining (NHEJ), and base excision repair (BER) by suppressing key repair genes and lowering access to repair sites. Then it is dissected how loss-of-function mutations in Switch/Sucrose non-fermentable, imitation switch and CHD (Chromodomain helicase DNA-binding) chromatin-remodeling complexes impair nucleosome repositioning, preventing effective damage sensing and assembly of repair machinery. Non-coding RNAs contribute to epigenetic silencing at DNA break sites, exacerbating repair deficiencies. This review evaluates recent advances concerning epigenetic dysfunction and DNA repair impairment. It is also highlighted that nanoparticle-mediated delivery strategies are designed to overcome pharmacologic resistance. It is presented how epigenetic dysregulation of DNA repair can guide more effective and drug-resistant cancer therapies. Full article

Therapeutic gene editing strategies utilize endogenous DNA repair pathways—nonhomologous end joining (NHEJ) or homology-directed repair (HDR)—to introduce targeted genomic modifications. Because HDR is restricted to dividing cells, whereas NHEJ functions in both dividing and non-dividing cells, NHEJ-based approaches are better suited for in vivo gene editing in the largely post-mitotic airway epithelium. Homology-independent targeted insertion (HITI), an NHEJ-based method, offers a promising strategy for cystic fibrosis (CF) gene therapy. Here, we applied HITI to drive the expression of a promoterless reporter through an exon trap strategy in both proliferating airway basal cells and well-differentiated primary airway epithelial cultures derived from transgenic ROSA^mTmG ferrets. We also established a versatile human gene editing reporter (GER) airway basal cell line capable of multipotent differentiation, enabling real-time visualization of editing outcomes and the quantitative assessment of HDR- and NHEJ-based editing efficiencies. Together, these platforms provide easily accessible tools for optimizing genome editing strategies in the respiratory epithelium and advancing clinically relevant delivery strategies for CF gene therapy. Full article

Background: Defective DNA repair systems result in the accumulation of mutations, loss of genomic integrity, and eventually cancer. Following initial malignant transformation due to specific DNA damage and defective DNA repair, cancer cells become reliant upon other DNA repair pathways for their survival. The co-occurrence of specific repair deficiencies brings catastrophic outcomes such as cell death for cancer cells and thus holds promise as a potential therapeutic strategy. Exploring the co-occurrence and mutual exclusivity of mutational signatures provides valuable knowledge regarding combinations of defective repair pathways that are cooperative and confer selective advantage to cancer cells and those that are detrimental and cannot be tolerated by them. Methods: Taking advantage of mutational signature profiling, we analyzed whole-genome sequences of 1014 breast cancers to reveal the underlying mutational processes and their interrelationships. Results: We found an inverse relationship between deficiencies of homologous recombination (HRd) and non-homologous end joining (NHEJd) with reactive oxygen species (ROS). Moreover, HRd and NHEJd co-occurred with APOBEC but were mutually exclusive with mismatch repair deficiency (MMRd) and ROS. Our analysis revealed that SBS8 and SBS39 signatures of currently unknown etiology correlate with NHEJd. ID1 and ID2 signatures co-occur with ROS and have mutual exclusivity with HRd, SBS8, SBS39 and NHEJd. The ID4 signature, with currently unknown etiology, has mutual exclusivity with HRd and NHEJd and co-occurred with ROS. On the other hand, the ID15 signature, with currently unknown etiology, co-occurred with SBS8, SBS39, HRd, NHEJd and DBS2, while having an inverse relationship with MMRd and ROS. Comparing the mutational signatures of HRd and non-HRd TNBC genomes reveals the unique presence of ROS signatures in non-HRd tumors and the lack of ROS signature in HRd tumors. Conclusion: Taken together, these analyses indicate the possible application of mutation signatures and their interactions in advancing patient stratification and suggest appropriate therapies targeting the make-up of individual tumors’ mutational processes. Ultimately, this information provides the opportunity to discover promising synthetic lethal candidates targeting DNA repair systems. Full article

CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy. Full article