Search Results (177)

Background/Objectives: With the advent of genome sequencing and its widespread use in the clinic, there is a great need to identify mutational biomarkers that predict therapeutic responses. DNA polymerase beta (Polβ) and the base excision repair (BER) pathway have been previously implicated as modulators of response to platinum-based chemotherapies and are mutated in as many as 30% of cancers. Methods: Here, we show in a triple-negative breast cancer (TNBC) model that two classes of mutations in Polβ, reduced catalytic activity (E295K and D256A mutations) and reduced fidelity (I260M), are sufficient to drive cisplatin and carboplatin-specific sensitivity. Cellular response to oxaliplatin in these Polβ mutant models is minimal relative to cisplatin and carboplatin. Results: We show that sensitivity is associated with reduced repair of both platinum-induced DNA intrastrand adducts and interstrand crosslinks (ICLs). Downregulation of the upstream BER factor uracil DNA glycosylase (UNG) reverses drug sensitivity, which is consistent with these Polβ mutations negatively impacting ICL DNA repair to drive drug sensitivity. In addition, the intrastrand adduct repair readout indicates that these lesions also play a role in the sensitivity observed in Polβ mutant models. In vivo studies demonstrate a significant effect on tumor growth delay with cisplatin treatment in tumor xenografts harboring Polβ mutations. Conclusions: These results support the potential for using Polβ mutations as predictive biomarkers for cisplatin and carboplatin therapies in the clinical setting. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a multifactorial metabolic disorder that is strongly associated with mitochondrial dysfunction and oxidative stress, which may potentially compromise the integrity of mitochondrial DNA (mtDNA). However, the role of the base excision repair (BER) pathway—the main mechanism responsible for repairing oxidative lesions in mitochondria—and maintaining mtDNA stability in MASLD remains poorly understood. Here, we analyzed total mRNA expression levels of key BER components in whole-blood samples, along with mitochondrial protein levels of the selected components. Additionally, we assessed the mtDNA copy number and the damage of mtDNA and nuclear DNA in peripheral leukocytes from MASLD patients and healthy controls. We found that MASLD patients differed from controls in mtDNA and nuclear DNA damage, mtDNA copy number, and selected BER-related markers. However, because the MASLD and control groups also differed substantially in age and BMI, these molecular differences should be interpreted as potentially being associated with age- and BMI-related metabolic status rather than attributable to MASLD alone. While several BER-related genes were downregulated at the mRNA level, the corresponding mitochondrial protein levels were not consistently decreased in MASLD (ProteomeXchange: PXD075974), indicating a discordance between transcriptional and protein-level regulation. These results suggest that altered mitochondrial BER and mtDNA instability in peripheral leukocytes may reflect the combined influence of MASLD, aging, obesity, and broader metabolic dysfunction. Full article

Background/Objectives: The base excision repair (BER) pathway repairs oxidative DNA damage, a common and detrimental form of damage to the genome. Although biochemical steps in BER have been well defined, little is understood about how the pathway is regulated. Such regulation is critical, as cells must respond rapidly to DNA damage while avoiding aberrant activation of repair proteins that can produce DNA damage as intermediates in the repair pathway. Indeed, overexpression of the human BER protein NTHL1, a DNA N-glycosylase, can cause genomic instability and early cellular hallmarks of cancer. Methods: We developed a Saccharomyces cerevisiae model to explore how overexpression of NTHL1 may impair cellular function. Results: Overexpression of Ntg1, the budding yeast orthologue of NTHL1, impairs cell growth. To dissect mechanisms underlying this growth defect, we overexpressed either wild-type Ntg1 or a catalytically inactive variant of Ntg1 (ntg1_catdead). Consistent with results obtained for NTHL1, both variants of Ntg1 impair cell growth, but only the wild-type protein causes accumulation of double-strand breaks and chromosome loss. We screened a panel of DNA repair mutants for resistance/sensitivity to overexpression of wild-type Ntg1 or ntg1_catdead. This analysis identified several cellular pathways that protect cells from Ntg1-induced damage, providing insight into the interplay between DNA repair pathways. Finally, we identified a link to SUMOylation and probed into how this post-translational modification could contribute to regulation of Ntg1 function. Conclusions: This study describes a budding yeast system to understand how cells regulate and respond to dysregulation of the BER pathway. Full article

Base excision repair (BER) and nucleotide excision repair (NER) are traditionally regarded as independent pathways; however, accumulating evidence indicates that ultraviolet (UV)-damaged DNA-binding protein (UV-DDB), a core NER factor, stimulates BER DNA glycosylases, including alkyladenine DNA glycosylase (AAG). Despite this functional link, the molecular basis of the UV-DDB/AAG interaction and its regulation by DNA remain unclear. This study investigated the direct interaction between AAG and UV-DDB using electrophoretic mobility shift assays (EMSA), surface plasmon resonance (SPR), biolayer interferometry (BLI) and AlphaFold3-based structural modeling under DNA-free and DNA-bound conditions. SPR analysis revealed that AAG and UV-DDB form a high-affinity complex in the absence of DNA ($K_D$ ≈ 17.5 nM), which is maintained but reduced approximately 2.6-fold upon binding to apurinic/apyrimidinic site (AP site)-containing dsDNA ($K_D$ ≈ 46.2 nM). BLI analysis independently confirmed this interaction under both DNA-free and DNA-bound conditions, with inter-platform differences consistent with previously reported BLI/SPR variability. EMSA showed UV-DDB-mediated ternary complex formation accompanied by redistribution of binary AAG/DNA species. AlphaFold3 modeling predicted that AAG associates with DDB1 in the DNA-free state, whereas under DNA-bound conditions, DDB2 recognizes the AP site while AAG repositions toward the lesion with multiple active site residues placed in close proximity. These findings support a model in which DNA binding acts as a molecular switch that reconfigures the UV-DDB/AAG interaction, potentially enabling UV-DDB to function as a recruitment platform that facilitates directional progression of AAG through the BER cycle, and providing a structural basis for coordinated integration of BER and NER. Full article

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. Full article

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. Full article

Non-alcoholic fatty liver disease (NAFLD) is one of the most common chronic liver disorders and has been linked to oxidative stress. Therefore, it can be hypothesized that NAFLD may be associated with genes encoding proteins involved in the base-excision repair (BER) pathway. Moreover, mitochondrial dysfunction plays a significant role in the development of NAFLD. In light of these observations, we suggested that fatty liver may be associated with genes that encode proteins responsible for mitochondrial DNA (mtDNA) degradation. This study evaluates single-nucleotide polymorphisms (SNPs) within the EXOG, ENDOG, POLG, FEN1, PARP1, and XRCC1 genes in 99 patients and 104 controls. SNP genotyping was performed using TaqMan probes and the findings were presented as odds ratios with corresponding 95% confidence intervals. Each of the eight investigated SNPs was found to modulate the risk of NAFLD occurrence. The analysis revealed that the studied haplotypes of EXOG and XRCC1 significantly affected the frequency of NAFLD in patients. The findings allow us to assume that there is a link between FEN1, PARP1, XRCC1, POLG, EXOG, and ENDOG and liver steatosis. We believe that the impaired repair and degradation of damaged mtDNA may have a significant impact on the development of NAFLD. Full article

DNA damage requires repair via the endonuclease IV-mediated base excision repair (BER) pathway, which corrects apurinic/apyrimidinic (AP) sites. Chlamydophila pneumoniae AP endonuclease IV (CpEndoIV), the sole AP endonuclease in this pathogen, is crucial for genomic integrity. As humans lack a homologous protein, it represents a potential therapeutic target. In this study, we report the first crystal structure of CpEndoIV at 1.97 Å resolution. The structure reveals two Zn²⁺, one Mg²⁺, and a malonate molecule bound in the active site, marking the first observation of Mg²⁺ coordination in the EndoIV family. Compared to the three-Zn²⁺ model with a narrow, deep pocket for precise AP-site cleavage, the Zn²⁺/Mg²⁺-bound state has a wider, shallower pocket that might promote diverse catalytic activities. Combined with enzymatic assays, we suggest that the mixed Zn²⁺/Mg²⁺ model is better adapted for CpEndoIV to operate under host oxidative stress. Malonate binds to the metal ions, occupying the positions normally coordinated by water molecules. This binding mode may mimic the coordination of the substrate to the metal ions, and the protein conformation resembles that of the enzyme upon substrate binding at the active site. This study provides a structural basis for the functional characterization of CpEndoIV and offers a reference for the development of targeted inhibitors against diseases caused by Chlamydophila pneumoniae. Full article

DNA polymerase beta (Polβ) is a 39 kDa, single polypeptide enzyme that possesses both gap tailoring and nucleotidyl transferase activity and is the key polymerase involved in base excision repair (BER) and the final steps of active gene demethylation. We demonstrated that residues in the mouse Polβ protein, L301 and V303, are critical for Polβ’s interaction with the BER scaffolding protein X-ray repair cross-complementing 1 (XRCC1), and mutation of these residues impairs Polβ’s ability to bind to XRCC1, negatively impacting BER complex assembly. We developed Polβ^{L301R-V303R/L301R-V303R} knock-in mice to explore how defects with this essential protein complex impact genome stability in the mouse. We found these mice to be viable and fertile yet exhibited a modest reduction in body weight. Here, we examined the protein and mRNA levels in tissues from wild-type (WT), heterozygous (HET), and homozygous (HOM) Polβ^{L301R-V303R/L301R-V303R} mice and the derived fibroblast cell lines. We show that HOM mice have significantly diminished Polβ protein levels, as compared to WT mice, in several tissues, yet Polβ mRNA levels were not significantly different, suggesting the decreased levels of Polβ protein could not be attributed to lower gene expression. Upon examination of Polβ stability in mouse ear fibroblasts derived from WT and HOM mice, results are consistent with human cell studies that the Polβ^L301R-V303R protein is unstable and undergoes proteasome-mediated degradation. Finally, we evaluated WT, and HOM, liver and brain genomic DNA samples for 5-methylcytosine/5-hydroxymethylcytosine (5mC/5hmC) levels by nanopore sequencing to investigate the impact of suppressed Polβ protein levels on active gene demethylation. As expected, we found tissue-specific trends in methylation, when comparing the brain and liver. However, we were unable to discern substantial differences in methylation levels between WT and HOM mice, suggesting that in the absence of external stressors, low Polβ levels do not impact methylation patterns. Full article

In this work, we analyzed the ability to incorporate 8-oxo-dATP by several human DNA polymerases: replicative Pol ε (exo-) from Family B; base excision repair (BER) enzymes Pol β and Pol λ from Family X; and translesion Pol η, Pol ι, and Pol κ from Family Y. We demonstrated that human DNA polymerases differ in their abilities to discriminate against 8-oxo-dATP. Among the tested DNA polymerases, Pol λ exhibited the worst ability to discriminate against 8-oxo-dATP opposite template T on DNA substrates with a protruding single-stranded 5′-end and a double-stranded DNA with a 1 nt gap. Pol β and DNA polymerases of Family Y showed relatively high accuracy. Pol η demonstrated the most effective discrimination against 8-oxo-dATP on templates T and G. Pol ι exclusively incorporated 8-oxo-dATP opposite template G but not T. Unexpectedly, the catalytic subunit of high-fidelity Pol ε (exo-) incorporated 8-oxo-dATP opposite templates T and G with higher efficiency compared with the error-prone polymerases of Family Y and Pol β. While the structures of human polymerases with incoming 8-oxo-dATP are not available, we speculate on a possible mechanism of 8-oxo-dATP discrimination. Full article

OGG1 and MUTYH are base excision repair (BER) DNA glycosylases (DGs) from the Helix–hairpin–Helix superfamily responsible for initiating and coordinating the repair of 8-oxo-7,8-dihydroguanine (OG), and its replication-derived mispair with adenine (OG:A), respectively. The DNA repair activities of these DGs are pivotal to safeguarding nuclear and mitochondrial genomes. Indeed, DG functional impairment is associated with numerous pathologies, including neurodegenerative diseases, metabolic syndromes, and cancer. The timely and precise localization and processing of oxidized nucleobases carried out by these DGs are modulated by a complex regulatory network at both transcriptional and posttranslational levels, as well as intricate protein–protein interaction networks. In the absence of regulation, inappropriate and imbalanced DG activity may trigger telomeric instability, changes in transcriptional profiles and cell death. This review focuses on summarizing key features of OGG1 and MUTYH function, with a special emphasis on structure, regulation, and novel emerging roles. Full article

Neurons have exceptionally high energy demands, sustained by thousands to millions of mitochondria per cell. Each mitochondrion depends on the integrity of its mitochondrial DNA (mtDNA), which encodes essential electron transport chain (ETC) subunits required for oxidative phosphorylation (OXPHOS). The continuous, high-level ATP production by OXPHOS generates reactive oxygen species (ROS) that pose a significant threat to the nearby mtDNA. To counter these insults, neurons rely on base excision repair (BER), the principal mechanism for removing oxidative and other small, non-bulky base lesions in nuclear and mtDNA. BER involves a coordinated enzymatic pathway that excises damaged bases and restores DNA integrity, helping maintain mitochondrial genome stability, which is vital for neuronal bioenergetics and survival. When mitochondrial BER is impaired, mtDNA becomes unstable, leading to ETC dysfunction and a self-perpetuating cycle of bioenergetic failure, elevated ROS levels, and continued mtDNA damage. Damaged mtDNA fragments can escape into the cytosol or extracellular space, where they act as damage-associated molecular patterns (DAMPs) that activate innate immune pathways and inflammasome complexes. Chronic activation of these pathways drives sustained neuroinflammation, exacerbating mitochondrial dysfunction and neuronal loss, and functionally links genome instability to innate immune signaling in neurodegenerative diseases. This review summarizes recent advancements in understanding how BER preserves mitochondrial genome stability, affects neuronal health when dysfunctional, and contributes to damage-driven neuroinflammation and neurodegenerative disease progression. We also explore emerging therapeutic strategies to enhance mtDNA repair, optimize its mitochondrial environment, and modulate neuroimmune pathways to counteract neurodegeneration. Full article

DNA is continuously exposed to endogenous and exogenous factors that induce oxidative modifications leading to mutations and genomic instability. Oxidative DNA damage plays a dual role, contributing to physiological signaling at low levels while promoting mutagenesis, carcinogenesis and degenerative diseases when unpaired. Among various lesions, an oxidized base, such as 8-oxo-2′-deoxyguanosine (8-oxodG), is one of the major biomarkers of oxidative stress and genomic damage. Cells have evolved sophisticated repair processes, including base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR), to maintain genomic integrity. Dysregulation or polymorphism of these repair genes has been linked with cancer, neurologic, and cardiovascular disorders. This review discusses an overview of what is presently known concerning oxidative DNA damage and repair mechanisms, particularly emphasizing their molecular players, signaling routes, and human disease implications. It further refers to the latest advances in CRISPR-based technologies and multi-omics approaches that are redefining our understanding of DNA damage response (DDR) networks and creating new frontiers for therapeutic interventions. Full article

Poly (ADP-ribose) polymerase 1 (PARP1) is an important enzyme that plays a central role in the DNA damage response, facilitating repair of single-stranded DNA breaks via the base excision repair (BER) pathway and thus genomic integrity. Its therapeutic relevance is compounded in breast cancer, particularly in BRCA1 or BRCA2 mutant cancers, where compromised homologous recombination repair (HRR) leaves a synthetic lethal dependency on PARP1-mediated repair. This review comprehensively discusses the recent advances in computational chemistry for the discovery of PARP1 inhibitors, focusing on their application in breast cancer therapy. Techniques such as molecular docking, molecular dynamics (MD) simulations, quantitative structure–activity relationship (QSAR) modeling, density functional theory (DFT), time-dependent DFT (TD-DFT), and machine learning (ML)-aided virtual screening have revolutionized the discovery of inhibitors. Some of the most prominent examples are Olaparib (IC₅₀ = 5 nM), Rucaparib (IC₅₀ = 7 nM), and Talazoparib (IC₅₀ = 1 nM), which were optimized with docking scores between −9.0 to −9.3 kcal/mol and validated by in vitro and in vivo assays, achieving 60–80% inhibition of tumor growth in BRCA-mutated models and achieving up to 21-month improvement in progression-free survival in clinical trials of BRCA-mutated breast and ovarian cancer patients. These strategies enable site-specific hopping into the PARP1 nicotinamide-binding pocket to enhance inhibitor affinity and specificity and reduce off-target activity. Employing computation and experimental verification in a hybrid strategy have brought next-generation inhibitors to the clinic with accelerated development, higher efficacy, and personalized treatment for breast cancer patients. Future approaches, including AI-aided generative models and multi-omics integration, have the promise to further refine inhibitor design, paving the way for precision oncology. 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