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PubMed2026

BRCA1-A and LIG4 complexes mediate ecDNA biogenesis and cancer drug resistance.

Extrachromosomal circular DNA (ecDNA) is frequently generated within the nucleus, contributing to genome dynamics and heterogeneity, thereby promoting cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here, using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes as key drivers of ecDNA production. Following DNA segmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively mediates ecDNA production by joining the free ends of the DNA fragments. Furthermore, ecDNA from patient tumors harbors junction sites with a LIG4 signature. Notably, disruption of either LIG4 or the BRCA1-A complex in cancer cells impairs ecDNA-mediated adaptation, hindering the development of resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

Humans↗
PubMed2026

Non-syndromic premature ovarian insufficiency associated with monoallelic LIG4 mutation via haploinsufficiency.

BACKGROUND: Premature ovarian insufficiency (POI) is a heterogeneous reproductive disorder, with genetic factors, particularly defects in DNA damage response pathways, increasingly implicated in its pathogenesis. DNA ligase IV (LIG4) is a key enzyme in the non-homologous end joining (NHEJ) pathway responsible for repairing DNA double-strand breaks (DSBs). However, its role in non-syndromic POI remains unclear. This study aimed to investigate the potential contribution of LIG4 variants to non-syndromic POI. RESULTS: Whole-exome sequencing identified a heterozygous frameshift variant in LIG4 (c.1271_1275del) in a three-generation Han Chinese family with non-syndromic POI, which co-segregated with affected individuals. AlphaFold-based structural modeling predicted truncation of the C-terminal XRCC4 interaction region. Functional experiments demonstrated that the mutant LIG4 protein showed reduced stability and was predominantly mislocalized to the cytoplasm of cells. In ovarian KGN cells, LIG4 depletion reduced cell viability, induced stress-associated cellular senescence, and impaired DNA damage repair capacity. In LIG4 knockout 293T cells, co-transfection of wild-type and mutant constructs revealed dose-dependent functional impairment, resulting in increased apoptosis under basal conditions and after phleomycin induced DNA damage, together with delayed repair of DSBs. Reanalysis of public single-cell RNA sequencing data further showed stage specific upregulation of LIG4 during oocyte maturation. Co-expression network analysis revealed enrichment in the Fanconi anemia pathway, phosphatidylinositol 3-kinase signaling pathway, and glycan metabolism. CONCLUSIONS: Our findings suggest that monoallelic LIG4 mutations may represent a potential genetic etiology for non-syndromic POI with sex-limited penetrance. While further validation in more physiologically relevant models is warranted, our data indicate that LIG4 haploinsufficiency may impair DSB repair and disrupt molecular pathways crucial for oocyte maturation and survival, highlighting a potential role of the NHEJ pathway in maintaining human ovarian function.

Humans↗
PubMed2026

Comprehensive In Silico Analysis Identifies MSTO1 and LIG1 as Candidate Biomarkers With Diagnostic and Prognostic Relevance in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and remains a major cause of cancer-related mortality worldwide. Its poor clinical outcomes are largely attributed to late-stage diagnosis and the limited accuracy of currently available diagnostic and prognostic biomarkers. Therefore, identifying novel molecular markers with improved sensitivity, specificity, and therapeutic relevance is essential for enhancing early detection and guiding personalized treatment strategies. AIMS: To identify and prioritize novel candidate HCC biomarkers with diagnostic and prognostic value and potential therapeutic vulnerability using integrated multi-omics, survival, functional dependency, and tumor microenvironment analyses. METHODS AND RESULTS: We examined the mRNA and protein expression levels of 8 DEGs in HCC tissues in the TCGA and CPTAC datasets using UALCAN, which showed that MSTO1 and LIG1 were overexpressed consistently in HCC relative to normal liver tissues. Moreover, elevated expression levels of these genes were significantly associated with higher tumor grade and advanced stage. Kaplan-Meier plotter survival data confirmed that increased expression of MSTO1 and LIG1 was associated with poorer overall survival. The DepMap CRISPR knockout data confirmed a functional dependency of both genes in HCC cell lines. CBioPortal analyses provided characterization of genomic alterations and enabled enrichment analysis of co-expressed genes, and the TCGA-UALCAN pan-cancer analyses supported the assessment of tissue specificity across tumor types. TIMER3 analyses linked candidate gene expression with immune cell infiltration patterns. Diagnostic performance by ROC analysis showed excellent discrimination for MSTO1 (AUC = 0.987) and good discrimination for LIG1 (AUC = 0.897). Multivariate Cox regression with Benjamini-Hochberg FDR correction across the eight genes supported MSTO1 as a candidate independent prognostic factor after adjustment for tumor stage, grade, etiology, age, and sex (HR = 1.29, p = 0.035), whilst LIG1 showed no independent prognostic value. Promoter methylation of MSTO1 and ADH4, assessed via UALCAN, showed that both genes were significantly differentially methylated in the promoter region of primary HCC tissues compared with normal liver tissues. Our study also confirmed the biological and clinical relevance of established HCC biomarkers: TERT, IRAK1, and ADH4. CONCLUSION: MSTO1 and LIG1 emerged as candidate diagnostic biomarkers in HCC. Additionally, MSTO1 showed a candidate prognostic association with overall survival that remained significant after adjusting for tumor stage, grade, and etiology, as well as patients' age, but not after further adjustment for AFP status. Functional data also highlighted MSTO1 as a candidate therapeutic dependency. On the other hand, LIG1 showed no independent prognostic association in either multivariate model. Their differential expression and functional essentiality in HCC cell lines highlighted their value for further experimental and independent-cohort validation before potential integration into biomarker development pipelines aimed at improving early detection and targeted therapy in HCC.

Humans↗
PubMed2025

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Humans↗
PubMed2026

Revealing the Association of LIG1 Genetic Variants With Pterygium Susceptibility and Demographic Characteristics in a Taiwanese Population.

BACKGROUND/AIM: Pterygium is a common ocular surface disorder associated with long-term ultraviolet exposure and/or oxidative DNA damage. Accumulated evidence suggests that defects in DNA repair pathways may contribute to pterygium risk. DNA ligase I (LIG1), a key enzyme involved in DNA replication and base excision repair, has been involved in the etiology of several human diseases, including cancers. However, its role in pterygium has never been examined before. This study aimed at exploring the association between the LIG1 genotypes and pterygium risk in a Taiwanese population. PATIENTS AND METHODS: The hospital-based case-control study was conducted including 165 patients with pterygium and 320 age- and sex-matched non-pterygium controls. LIG1 rs20579 genotypes were accessed utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Stratified analysis and the calculation of odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were used for evaluating the associations between genotypes and pterygium risk. RESULTS: Individuals carrying the homozygous variant AA genotype exhibited a significantly elevated risk of pterygium compared with those carrying the GG genotype (OR=2.74, 95% CI=1.17-6.43, p=0.0305). Under the recessive model, the AA genotype conferred a 2.65-fold elevated risk (95% CI=1.14-6.18, p=0.0350). The variant A allele was also associated with increased susceptibility (OR=1.46, 95% CI=1.03-2.07, p=0.0413). Stratified analyses revealed significant associations specifically among individuals aged ≥60 years (OR=5.64, 95% CI=1.67-19.04, p=0.0039) and males (OR=5.23, 95% CI=1.74-15.73, p=0.0034), but not those of younger ages or females. CONCLUSION: The LIG1 rs20579 genotype is significantly associated with pterygium susceptibility in Taiwanese individuals, particularly among elderly and male subjects. These findings support the involvement of impaired DNA repair machinery in pterygium pathogenesis and suggest that LIG1 rs20579 may serve as a novel genetic biomarker for risk assessment and early detection of pterygium.

Humans↗