X-ray repair cross-complementing protein 1 (XRCC1) protects cells from the effects of genotoxic stress by coordinating base excision repair (BER) and DNA single-strand break repair. XRCC1 phosphorylation and recruitment to sites of DNA damage are dependent on poly(ADP-ribose) polymerase-1 (PARP1) activity. As a central scaffolding protein, XRCC1 interacts with multiple components of BER pathway and also contributes to DNA double-strand break repair through the alternative nonhomologous end-joining pathway. In this regard, both established and recent studies have highlighted its critical role in spermatogenesis, extending beyond its classical association with PARP1. By integrating expression profiles with functional evidence, this review summarizes current insights into XRCC1 expression and subnuclear localization in spermatogenic cells, as well as its cooperative interaction with PARP1 in maintaining genomic integrity through efficient recombination and repair of genotoxic damage.

Base excision repair (BER) is the predominant pathway for repairing non‑bulky oxidized and alkylated DNA base lesions, and its fidelity depends on the coordinated action of lesion‑specific DNA glycosylases and downstream repair enzymes. This review aims to summarize recent structural, biochemical, and genomic insights into three base excision repair enzymes, MUTYH DNA glycosylase, NTHL1 DNA glycosylase, and DNA polymerase β. This review outlines how MUTYH, NTHL1 and DNA polymerase β protect the genome from mutagenesis, highlights major germline variants associated with disease, and synthesizes the current knowledge on the characteristic single base substitution (SBS) mutational signatures that occur when these repair enzymes are dysfunctional.

Proliferative diabetic retinopathy (PDR) is one of the leading causes of blindness in working-age adults. We have previously shown that the risk of PDR is significantly elevated in individuals with intrauterine exposure to famine. However, the genetic mechanisms mediating this association remain unknown. The aim of the current study was to investigate the molecular underpinnings of famine-related PDR by performing genome-wide association (GWAS) and interaction studies (GWIS). We analysed n = 2925 patients with type 2 diabetes from the DOLCE cohort of Northern Ukraine, of whom n = 1364 were born during historical famine periods (1929-1949, including the Holodomor and World War II). PDR cases were defined as individuals with either diagnosed proliferative retinopathy, laser-treated diabetic retinopathy (DR) or blindness in either eye. GWAS and GWIS were performed using linear mixed model (LMM) adjusted for established risk factors and genetic relationship matrix. GWAS identified rs3795299 in IL22RA1 as the top signal (pLMM = 1.05 × 10-6), which was also the strongest gene in the gene-based analysis (p = 3.19 × 10-5), with suggestive enrichment of response to ketones (GO) and base excision repair (KEGG) pathways. In the GWIS, the strongest signal was rs1506783 in PAPPA2 (pLMM = 1.29 × 10-7). A second biologically credible candidate was rs2230805 in ABCA1 (pLMM = 4.44 × 10-6), reaching borderline genome-wide significance in gene-based analysis (p = 4.31 × 10-6). Interaction analyses showed suggestive enrichment for nucleosomal DNA binding (GO), tryptophan metabolism and glycerolipid metabolism (KEGG) pathways. Furthermore, at nominal significance, we validated variants in previously reported diabetic retinopathy-associated genes, including TCF7L2, SLC2A1, SLC2A11 and VDR in the GWAS, as well as 13 variants in genes including VEGF, VEGFR1, ANGPT1, PLXDC2, SELP and PON2 in the GWIS. Our findings suggest that famine-related PDR susceptibility involves distinct developmental programming mechanisms, including altered insulin-growth signalling (PAPPA2) and lipid metabolism (ABCA1), whereas immune-related pathways (IL22RA1) may contribute to the conventional glycaemia-driven route to PDR through VEGF-mediated angiogenesis. These genes represent potential therapeutic targets and emphasize the importance of the perinatal environment in lifelong vascular health and disease.

Apurinic/apyrimidinic endonuclease 1 (APE1) plays a critical role in the base excision repair (BER) pathway, and its aberrant activity is closely associated with various cancers, including cervical cancer, as well as neurodegenerative diseases. Herein, we report a novel "turn-on" fluorescent biosensor for the sensitive and selective detection of APE1 activity based on CdTe quantum dots (QDs) and silver ions (Ag+). The sensing strategy relies on the inner filter effect (IFE)-induced fluorescence quenching of CdTe QDs by Ag+, followed by fluorescence recovery upon specific cleavage of an AP site-containing dsDNA probe by APE1. The released poly-C fragments competitively bind with Ag+, separating Ag+ from the surface of CdTe QDs and restoring the fluorescence. Under optimized conditions, the biosensor exhibits a linear response to APE1 activity in the range of 2-10 U mL-1 with a detection limit as low as 0.09 U mL-1. The sensor demonstrates excellent selectivity against interfering biomolecules, good stability, and reproducibility. Moreover, it was successfully applied to detect APE1 activity in HeLa cell lysates, showing satisfactory recovery rates. This work provides a green, simple, and robust platform for APE1 activity detection in complex biological matrices, with potential applications in cancer-related biomedical research.

Background & ObjectivesCholangiocarcinoma (CCA) is a highly aggressive disease with a 5-year survival of ∼9%. FGFR2 aberrations, including gene fusions and activating point mutations, occur in ∼10% of CCA cases and represent a targetable driver. Although FGFR inhibitors show clinical promise, resistance and metastatic progression remain major challenges. Understanding clonal evolution during metastasis and treatment failure may identify novel therapeutic strategies and biomarkers to improve outcomes. This study investigates clonal evolution across primary and metastatic tumor sites in CCA to identify pathways and biomarkers associated with disease progression.MethodsWe performed a multi-regional analysis of 21 CCA patients (10 FGFR2-fusion positive, 11 wildtype) using whole-exome sequencing (WES) and RNA-sequencing on samples obtained from primary and metastatic sites through rapid research autopsy. Oncogenic SNVs and indels were annotated using OncoKB. Clonality analysis was performed using phylogenetic tools (PyClone, PhylogicNDT, and ASCETIC) to define relationships between primary and metastatic clones.ResultsRapid autopsy yielded 281 samples for WES and 62 samples for RNA-seq (Table 1). Disease progression was associated with an increased burden of oncogenic drivers in both hepatic lesions and distant metastases. Tumor evolution followed a tree/branch model, with distinct patterns between FGFR-driven and non-FGFR-driven CCAs. FGFR-driven CCAs exhibited evolutionary patterns consistent with DNA damage and chromosomal instability, while non-FGFR-driven CCAs showed alterations in chromatin remodelers and the tumor immune microenvironment. RNA-seq analysis revealed increased expression of Base Excision Repair (BER) genes in distant metastases, a pattern not observed in other DNA damage repair pathways. BER enrichment was more pronounced in FGFR2-fusion tumors, suggesting FGFR2 may modulate BER to promote metastasis and chemoresistance.ConclusionsUsing rapid autopsy, this study provides new insights into the molecular drivers of metastatic progression in CCA, highlighting the role of clonal evolution and DNA repair dysregulation. Identification of Base Excision Repair as a potential vulnerability-particularly in FGFR2-fusion tumors-supports the rationale for targeted combination therapies in advanced CCA.26 TableDemographic Table of Rapid Autopsy CohortCharacteristicAll PatientsFGFR DrivenNon-FGFR DrivenAverage Age at Diagnosis (years)59.15761.2Total No. Patients211110Female No. (%)10 (47%)4 (36%)6 (60%)Male No. (%)11 (53%)7 (63%)4 (40%)Median Stage at DiagnosisIVIVIVAverage Age at Death61.159.362.9Total No. Exome Samples281162119Total No. Transcriptome Samples623829Median lines of Therapy (range)3 (1-5)3 (2-5)3(1-4)

Apurinic/apyrimidinic endonuclease 1 (APE1) is a pivotal biomarker frequently overexpressed in cancer cells, hence in situ monitoring of its dynamic alterations during cell death is crucial for therapeutic evaluation. Herein, we designed highly sensitive and selective fluorescent nanoprobes based on DNA-functionalized gold nanoparticles (AuNPs) for real-time imaging and monitoring of APE1 activity in living cells. The nanoprobes, composed of APE1-responsive double-stranded DNA containing apurinic/apyrimidinic (AP) sites and conjugated with black hole quencher 2 (BHQ2), are immobilized on the surface of AuNPs. Our results demonstrate that electrical stimulation (ES) selectively induces pyroptosis of HeLa cells, and nearly complete suppression of APE1 activity as ES-induced pyroptosis progressed was revealed via confocal microscopy analyses. The ES treatment leads to a marked accumulation of intracellular reactive oxygen species (ROS) and the resulting oxidative stress causes a pronounced up-regulation of γ-H2AX, indicating severe DNA double-strand breaks. We propose that the ROS-mediated down-regulation of APE1 contributes to impaired base excision repair (BER) and exacerbates genomic instability during ES-induced pyroptosis. Notably, this phenomenon did not occur in normal H8 cells. This study establishes a nanoprobe sensing platform for real-time monitoring of APE1 dynamics and identifies the down-regulation of APE1 as an indicator of effective ES-induced pyroptosis in cancer cells, providing new insights into therapeutic assessment in cancer treatment.

XRCC1 deficiency drives telomeric chromatin leakage, inflammatory signalling and senescence.

Rewiring cell death and evading repair: Evolution of temozolomide and emerging strategies against resistant glioblastoma.

DNA damage induced by reactive oxygen species (ROS) can result in mutations that contribute to the development of human diseases such as cancer, neurological disorders, cardiovascular disease, and diabetes. Human 8-oxoguanine DNA glycosylase (hOGG1) is responsible for repairing the major DNA oxidative product, namely 8-oxoguanine (8oG). hOGG1 is a bifunctional DNA glycosylase, which cleaves both the glycosidic and phosphodiester bonds in damaged nucleotides as part of base excision repair (BER). While nucleotide deglycosylation to yield an enzyme-DNA imine cross-link has been well studied both experimentally and computationally for several bifunctional glycosylases, relatively little is known about the subsequent and typically rate-limiting phosphodiester-bond cleavage step. To unveil the atomic-level details of the β-lyase pathway for a bifunctional glycosylase, the present study uses a combination of classical MD and QM/MM MD simulations to characterize the hOGG1 mechanism of action. Our simulations reveal that the cleaved 8oG glycosylation product rapidly leaves the active site, precluding the previously proposed product-assisted elimination and supporting the allosteric nature of 8oG activators. Although QM/MM MD calculations suggest a neutral cross-link prevents lyase activity, a pathway involving initial hydrolysis of a cationic cross-link followed by D268-catalyzed phosphate elimination is catalytically feasible and the first proposed mechanism to unify all existing experimental kinetic, mutagenic, and structural data. Our newly characterized mechanism of action can push forward the development of small-molecule hOGG1 inhibitors and activators as disease therapeutics, while key mechanistic features may be generalizable for understanding the function of other bifunctional glycosylases.

Systemic sclerosis (SSc) is a chronic, autoimmune, fibrotic disorder involving immune dysregulation, vascular abnormalities and progressive fibrosis. Although oxidative stress and defective DNA repair have been implicated in its pathogenesis, the impact of vitamin D on DNA repair pathways remains unclear. This study aimed to investigate the expression of DNA repair enzymes in SSc, explore their relationship with vitamin D status and assess the effects of vitamin D supplementation on the transcriptional expression of these enzymes. Peripheral blood samples were collected from 52 female patients with SSc and 31 age-matched healthy controls (HCs). Gene expression levels of base excision repair (BER) enzymes (APE1 and OGG1) and nucleotide excision repair (NER) enzymes (XPA and XPC) were analyzed. Serum vitamin D levels were measured and correlated with disease activity scores. In a prospective arm of the study, patients received six months of vitamin D supplementation and their DNA repair capacity was evaluated pre- and post-intervention. Baseline expression of APE1 and OGG1 was significantly lower in SSc patients than in HCs, whereas expression of the NER genes remained unchanged, indicating selective impairment of the BER pathway. Vitamin D deficiency was prevalent in SSc and inversely correlated with disease severity. Supplementation significantly increased serum vitamin D levels and up-regulated APE1 and OGG1 expression; while NER genes remained unaffected. These findings are consistent with evidence of elevated oxidative DNA lesions in SSc and support a mechanistic link between BER activity and the repair of oxidative DNA damage. SSc patients exhibit reduced transcription of BER-specific enzymes associated with vitamin D deficiency andrestoration of vitamin D levels partially rescues BER enzyme expression. These findingshighlight a potentially modifiable axis linking micronutrient status, genomic stability and disease activity and provide a rationale for investigating vitamin D optimization as an adjunctive strategy to enhance DNA repair and potentially attenuate inflammatory and fibrotic processes in SSc.

DNA repair of mutagenic lesions is imperfect, allowing mutations to accumulate unevenly across the genome. In base excision repair, glycosylases must locate rare damaged bases embedded in diverse sequence contexts, yet how these contexts shape recognition and mutational outcomes remains unresolved. Here, we introduce a high-throughput approach that quantifies glycosylase binding across thousands of lesion-containing sequences. Focusing on the cytosine deamination pathway, we map the recognition landscapes of human UDG, TDG, and MBD4. Binding depends strongly on sequence context, extending several bases beyond the lesion and including non-additive interactions between neighboring positions. Structural analyses and molecular dynamics simulations implicate DNA-shape features, including minor groove width, as determinants of recognition. Nearest-neighbor preferences resemble deamination-related cancer mutational signatures, whereas broader-context preferences track variation in cytosine-thymine balance across matched human genomic contexts. Together, these findings establish a versatile and generalizable platform for decoding glycosylase recognition and linking repair specificity to mutational patterns.

DNA oxidation damage and its repair are essential for maintaining genomic integrity in the human limbal epithelium, which harbors corneal epithelial stem cells. This study investigated the distribution of the DNA base oxidation 8-oxoguanine (8-oxoG) and the base excision repair (BER) enzymes 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease 1 (APE1) in non-cultured and eye-bank organ-cultured human limbal epithelia. Immunohistochemistry was used to assess the localization and staining intensity of 8-oxoG, OGG1, and APE1, evaluated semi-quantitatively and by image analysis. In situ hybridization was performed to detect the distribution of OGG1 and APE1 gene expression in organ-cultured tissue. In non-cultured limbal epithelia, nuclear 8-oxoG staining was more frequently observed in superficial epithelial layers, whereas nuclear OGG1 and APE1 staining predominated in basal layers. In organ-cultured epithelia, a higher proportion of superficial nuclei exhibited 8-oxoG staining, while the basal predominance of OGG1 was reduced and that of APE1 was preserved. Transcripts of OGG1 and APE1 were detected in basal- as well as in suprabasal layers of organ-cultured epithelia. These findings demonstrate the presence of DNA base oxidation and BER-related enzymes in basal and suprabasal human limbal epithelial cells during storage of corneal tissue under commonly used eye-bank organ-cultured conditions prior to transplantation.

Understanding resistance in glioblastoma: insights into personalized and targeted therapeutic strategies.

Chronic inhalation exposure to nanoplastics, specifically polyethylene terephthalate (PET) nanoplastics (PET-NPLs) is an emerging health concern, yet the long-term consequences for genomic stability and DNA damage response (DDR) capacity in bronchial epithelial cells remain poorly characterized. For this study, human bronchial epithelial BEAS-2B cells were continuously exposed to PET-NPLs for over 20 weeks, after which elevated basal DNA genotoxic damage was observed, as assessed by the alkaline comet assay. In addition, a broad transcriptional suppression of the DDR, with 27 of 84 profiled genes involved in DDR showing reduced expression relative to passage-matched control was observed. The suppressed genes span ATM/ATR checkpoint signaling, homologous recombination (HR), base excision repair (BER), nucleotide excision repair (NER), and apoptotic pathways. To determine whether chronic PET-NPL exposure altered susceptibility to acute genotoxic challenge in a damage-type-specific manner, cells were treated with methyl methanesulfonate (MMS), ultraviolet-C (UV-C) radiation, or bleomycin. While MMS and UV-C induced comparable levels of DNA damage in control and PET-exposed cells, bleomycin produced significantly greater damage in PET-exposed cells, indicating selective sensitization to doble-strand breaks (DSB)-type and oxidative genotoxic insults. Transcriptional profiling during bleomycin challenge identified 18 DDR genes with relatively higher expression in PET-exposed cells compared to passage-matched controls, encompassing HR, BER, ATM/ATR signaling, the Fanconi anemia pathway, and apoptosis. Furthermore, PET-exposed cells retained significantly higher residual DNA damage after 3 h of bleomycin challenge, indicating a persistent early repair deficit. Together, these findings suggest that chronic PET-NPL exposure specifically compromises the bronchial epithelial DDR, with potential implications for long-term genomic stability in respiratory epithelia subjected to nanoplastic inhalation.

Base excision repair is a major pathway that repairs single-base DNA damage. We recently demonstrated that human DNA polymerase β (hPolβ) fills single-nucleotide gaps after Schiff base formation but before β-elimination, implying that its dRP lyase domain remains covalently crosslinked to DNA during gap-filling synthesis. Because uncrosslinked Polβ dissociates rapidly from DNA (∼3 s−1), mechanistic investigation has been challenging. To elucidate the kinetic mechanism of correct incorporation by DNA-crosslinked hPolβ, we generated a catalytically active crosslinked hPolβ‒DNA complex and performed pre-steady-state kinetic, thermodynamic, and structural analyses. Sulfur elemental effects of 3.7 ± 0.4 and 24 ± 4 for correct and incorrect nucleotide incorporation, respectively, suggest the chemical step is rate-limiting for incorrect, but not for correct, nucleotide incorporation. Pulse-chase and pulse-quench assays revealed a 33% difference in reaction amplitude, establishing the existence of a ternary intermediate preceding the chemical step. Eyring analysis identified a high activation free energy barrier, while the lack of viscosity dependence rules out large domain motions, indicating that the rate-limiting pre-chemical step involves local active-site rearrangements. Together with structurally characterized intermediates, these findings establish the first minimal kinetic mechanism for correct nucleotide incorporation by a DNA-crosslinked polymerase and identify local active-site rearrangements as the rate-limiting step.

Human single-strand-selective monofunctional uracil DNA glycosylase 1 (hSMUG1) removes uracil, 5-hydroxymethyluracil (5hmU) and 5-fluorouracil (5FU) from DNA, thereby initiating the base excision repair (BER) process. hSMUG1 is important for maintaining genomic integrity and plays a significant role in cancer biology. Here, we present the structures of hSMUG1, including complexes with products (uracil and 5FU) and an enzyme-product complex of hSMUG1 with double-stranded DNA (dsDNA). Analysis of our hSMUG1-dsDNA complex reveals how uracil is flipped out of the dsDNA for excision and identifies key residues that we confirm to be critical for both DNA binding and enzymatic activity. Furthermore, our hSMUG1 substrate complexes, molecular dynamics simulations and neutron diffraction data suggest a mechanism by which the substrate uracil rotates following base excision. The structural and functional information presented here will be highly useful for the future development of inhibitors and/or activators targeting hSMUG1.

Base excision repair hierarchy in eukaryotes: Intrinsically disordered region-mediated regulation of genomic surveillance and assembly dynamics.

Apurinic/apyrimidinic (AP) sites, also known as abasic sites, are among the most frequent DNA lesions, arising spontaneously or as intermediates in base excision repair. Their structural impediment to DNA replication fork progression, lack of coding information, and conversion into strand breaks constitute a threat and can lead to genome instability if not properly managed. This review examines the impact of AP sites on DNA replication, detailing mechanisms of lesion bypass, including translesion synthesis, template switching, and repriming of DNA synthesis. We highlight protective pathways that shield AP sites from nucleolytic attack and explore how endogenous processes such as uracil excision, cytosine methylation, and oxidative damage generate these lesions. By integrating biochemical and cellular perspectives, we present a comprehensive view of how cells replicate their DNA in the presence of AP sites and how their mismanagement contributes to replication stress, mutagenesis, and disease.

The role and therapeutic potential of DNA glycosylases in Alzheimer's disease.

Caulobacter crescentus harbours an evolutionarily distinct UDG (CC2084), UdgC, sharing features with family I and family V UDGs.