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The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase-AMP complex and the ligase-AMP-DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase-AMP complex that persist in the ligase-AMP-DNA complex. Notably, these complexes have DNA bending angles of ≈0°, 20°, 60°, or 100°. The mechanism of parallel reactions challenges the conventional notion of simple sequential reaction steps occurring among multiple conformations. The results provide insights into the dynamic conformational changes and the versatile attributes of T4 DNA ligase and suggest that the parallel multiple reaction pathways may correspond to diverse T4 DNA ligase functions. This mechanism may potentially have evolved as an adaptive strategy across evolutionary history to navigate complex environments.

Despite the improvements in outcomes for patients with multiple myeloma (MM) over the past decade, the disease remains incurable, and even those patients who initially respond favorably to induction therapy eventually suffer from relapse. Consequently, there is an urgent need for the development of novel therapeutic agents and strategies to enhance the treatment outcomes for patients with MM. The proteasome inhibitor bortezomib (BTZ) elicits endoplasmic reticulum (ER) stress and oxidative stress in MM cells, subsequent DNA damage, ultimately inducing cell apoptosis. Poly (ADP-ribose) polymerase 1 (PARP1) acts as a pivotal enzyme for DNA repair and thus deficient PARP1 renders cells more susceptible to DNA-damaging agents. Conceivably, targeting PARP1 may enhance BTZ-induced DNA damage and cell death in MM cells. In this study, Colony formation, CCK-8, and EdU-labeling assays were conducted to evaluate the effects on MM cell proliferation. The ZIP score was used to assess synergy. Apoptosis and intercellular ROS levels were analyzed using flow cytometry and fluorescence microscopy, respectively. Immunofluorescence and Western blot analyses were used to assess protein expression. The correlation between PARP1 expression levels and the clinical prognosis was examined by tumor-related databases and bioinformatics. The results show that PARP1 is overexpressed in patient MM cells and is associated with a poor prognosis. PARP1 inhibitor niraparib decreases MM cell growth and arrests cell cycle progression at the G2/M phase. When combined with BTZ, it synergistically increases DNA damage, inhibits proliferation, and induces apoptosis. Mechanistically, Niraparib facilitates BTZ-induced ROS elevation, causing DNA double-strand breaks (DSBs), and simultaneously inhibits lesion repair by impeding the expression of repair proteins XRCC1 (X-ray repair cross-complementing protein 1) and POLβ (DNA polymerase beta). Overall, Niraparib plus bortezomib represent a promising approach for treatment of MM. • Proteasome inhibitor bortezomib induces oxidative stress and DNA damage in MM cells. • PARP1 inhibitor niraparib exerts synergistic antimyeloma effect with bortezomib partially via ROS overproduction. • Niraparib downregulates XRCC1 and POLβ and inhibits DNA damage repair. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) tumors exhibit extensive genomic, epigenomic, and transcriptional diversity, with significant intratumoral heterogeneity, complicating standard treatment approaches involving radiation (RT) and the DNA-alkylating agent temozolomide (TMZ). In this study, we employed an integrative multi-omics approach, including targeted proteomics, transcriptomics, genomics, and DNA methylation profiling, to investigate the response of a representative panel of GBM patient-derived cancer stem cells (CSCs) to astrocytic differentiation and RT and TMZ treatments. Differentiated CSC progenies retained the expression of key stemness genes and survival pathways, while activating the BMP-Smad signaling pathway and upregulating extracellular matrix components. This was associated with increased resistance to TMZ, though not to RT, across all models. We identified TP53 status as a critical determinant of transcriptional response to both RT and TMZ, which was also modulated by the differentiation state and treatment modality in wildtype (wt) p53 GBM cells. Both mutant and wt p53 models exhibited significant activation of the DNA-damage associated interferon (IFN) response in CSCs and differentiated cells, implicating this pathway in the GBM response to therapy. We observed that activation of NF-κB was positively correlated with the levels of O-6-methylguanine-DNA methyltransferase (MGMT) protein, a direct DNA repair enzyme leading to TMZ resistance, regardless of MGMT promoter methylation status, further supporting the clinical potential for inhibition of NF-kB signaling in GBM treatment. Our integrative analysis of the impact of GBM cell developmental states, in the context of genomic and molecular diversity of patient-derived models, provides valuable insights for pre-clinical studies aimed at optimizing treatment strategies. [ABSTRACT FROM AUTHOR]

Glioblastoma (GB) is a highly malignant brain tumor with a poor prognosis, with a median survival of only 14.6 months despite aggressive treatments. Resistance to chemotherapy, particularly temozolomide (TMZ), is a significant challenge. The DNA repair enzyme MGMT and glioblastoma stem cells (GSCs) often mediate this resistance. Recent studies highlight the therapeutic potential of natural compounds, particularly delphinidins, found in deep purple berries. Delphinidins are known for their ability to inhibit NF-κB signaling, a critical pathway for GB progression, chemoresistance, and MGMT expression. Our research demonstrates that glycosylated delphinidins have potential adjuvant use in the treatment of GB, offering a promising natural strategy to combat TMZ resistance. Specifically, we observed that delphinidin 3,5 di-glucoside has potent anticancer effects when used alone. Meanwhile, delphinidin 3 glucoside acted in synergy with temozolomide to decrease cell viability, highlighting its potential as an adjuvant. It also exerted a faster and more sustained inhibition of NF-κB, highlighting its potential for long-lasting therapeutic effects. These findings open new avenues for targeted therapies against glioblastoma, particularly to overcome treatment resistance. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM), the most prevalent primary malignant brain tumor, remains challenging to treat due to extensive inter- and intra-tumor heterogeneity. This variability demands combination treatments to improve therapeutic outcomes. A significant obstacle in treating GBM is the expression of O6-methylguanine-DNA methyltransferase, a DNA repair enzyme that reduces the efficacy of the standard alkylating agent, temozolomide, in about 50% of patients. This underscores the need for novel, more targeted therapies. Our study investigates the metabolic–epigenetic impact of combining SN-38, a novel topoisomerase inhibitor inducing DNA double-strand breaks, with rabusertib, a checkpoint kinase 1 inhibitor. We identified this synergistic combination through high-throughput drug screening across a panel of GBM cell lines using a cancer drug library combined with SN-38. A secondary metabolic screening with the PEDS algorithm demonstrated a synergistic modulation of purine, one-carbon, and redox metabolism. Furthermore, the combined treatment led to the significant depletion of epigenetically relevant metabolites such as 5-methyl-cytosine, acetyl-lysine, and trimethyl-lysine. Reduced intermediates of the glutathione cycle indicated increased cellular stress following combinatorial treatment. Overall, the combination of SN-38 and rabusertib synergistically disrupts metabolites associated with epigenetic adaptations, leading to cytotoxicity independent of O6-methylguanine-DNA methyltransferase status, thereby underpinning this combination as a promising candidate for combinatorial therapy in GBM. [ABSTRACT FROM AUTHOR]

DNA-joining by ligase and polymerase enzymes has provided the foundational tools for generating recombinant DNA and enabled the assembly of gene and genome-sized synthetic products. Xenobiotic nucleic acid (XNA) analogues of DNA and RNA with alternatives to the canonical bases, so-called 'unnatural' nucleobase pairs (UBP-XNAs), represent the next frontier of nucleic acid technologies, with applications as novel therapeutics and in engineering semi-synthetic biological organisms. To realise the full potential of UBP-XNAs, researchers require a suite of compatible enzymes for processing nucleic acids on a par with those already available for manipulating canonical DNA. In particular, enzymes able to join UBP-XNA will be essential for generating large assemblies and also hold promise in the synthesis of single-stranded oligonucleotides. Here, we review recent and emerging advances in the DNA-joining enzymes, DNA polymerases and DNA ligases, and describe their applications to UBP-XNA manipulation. We also discuss the future directions of this field which we consider will involve two-pronged approaches of enzyme biodiscovery for natural UBP-XNA compatible enzymes, coupled with improvement by structure-guided engineering. [ABSTRACT FROM AUTHOR]

Middle-aged obesity, characterized by excessive fat accumulation and systemic energy imbalance, often precedes various health complications. Recent research has unveiled a surprising link between DNA damage response and energy metabolism. Here, we explore the role of Eepd1, a DNA repair enzyme, in regulating adipose tissue function and obesity onset. Eepd1 is primarily expressed in adipose tissue, where its downregulation or deletion accelerates obesity development. We show that Eepd1 ablation hinders PKA activation, thereby inhibiting lipolysis and thermogenesis in adipose tissue. Notably, cold exposure enhances Eepd1's myristoylation, facilitating its anchorage to adipocyte membranes and subsequent activation of PKA, while a mutation at the myristoylation site of Eepd1 disrupts this process. Moreover, individuals with obesity exhibit reduced Eepd1 expression. Pharmacological restoration of Eepd1 with Retigabine dihydrochloride effectively mitigates obesity. This study reveals Eepd1's unexpected role in promoting adipose lipolysis and thermogenesis, underscoring its potential as a promising therapeutic target to combat obesity. Middle-aged obesity, marked by excessive fat accumulation, is closely linked to DNA damage. Here, the authors show that the DNA repair enzyme Eepd1, primarily expressed in adipose tissue, in its myristoylated form, promotes lipolysis and thermogenesis by activating PKA to combat obesity. [ABSTRACT FROM AUTHOR]

Previous data show that the knockdown of the MYBL1 gene in the MDA-MB-231 cell line leads to the downregulation of VCPIP1 gene expression. In addition, MYBL1 and VCPIP1 genes are co-expressed and dysregulated in some of the same triple negative breast cancer patient samples. We propose that the co-expression of the two genes is attributed to the MYBL1 transcription factor regulation of the VCPIP1 gene. We identify the MYBL1 transcription factor binding site upstream of the VCPIP1 start site and show that the MYBL1 protein can bind to the sequence identified in the VCPIP1 promoter region. Combined with the results from the knockdown study, these data support the ability of MYBL1 to regulate the VCPIP1 gene. The VCPIP1 gene functions as a deubiquitinating enzyme involved in DNA repair, protein positioning, and the assembly of the Golgi apparatus during mitotic signaling. The transcriptional regulation of VCPIP1 by the MYBL1 gene could implicate MYBL1 in these processes, which might contribute to tumor processes in TNBC. Although both genes are involved in cell cycle regulatory mechanisms, converging signaling mechanisms have not been identified. In a separate study, we performed sequence alignment of the MYBL1 transcript variants and identified an exon unique to the canonical variant. Probes that specifically target the unique MYBL1 exon show that the exon is overexpressed in tumor cell lines compared to non-tumor breast cells. We are classifying this unique MYBL1 exon as a tumor-associated exon. [ABSTRACT FROM AUTHOR]

Exposure to reactive oxygen species (ROS) can induce DNA-protein crosslinks (DPCs), unusually bulky DNA lesions that block replication and transcription and play a role in aging, cancer, cardiovascular disease, and neurodegenerative disorders. Repair of DPCs depends on the coordinated efforts of proteases and DNA repair enzymes to cleave the protein component of the lesion to smaller DNA-peptide crosslinks which can be processed by tyrosyl-DNA phosphodiesterases 1 and 2, nucleotide excision and homologous recombination repair pathways. DNA-dependent metalloprotease SPRTN plays a role in DPC repair, and SPRTN-deficient mice exhibit an accelerated aging phenotype and develop liver cancer early in life. We investigated the role of the SPRTN enzyme in the repair of DPCs produced by a free radical mechanism. Sprtn-deficient MEF cells treated with ionizing radiation had higher levels of total DPCs and exhibited greater sensitivity upon exposure to hydrogen peroxide and other crosslinking agents including cisplatin, phosphoramide mustard, and 1,2,3,4-diepoxybutane. Using a sensitive and accurate nanoLC-ESI+-MS/MS assay, we specifically measured the radical-induced crosslinking of thymidine in DNA crosslinking of thymidine in DNA to tyrosine in proteins (dT-Tyr) in the tissues of SPRTN hypomorphic (SprtnH/H) and wild type mice. Genomic DNA isolated from the tissues of SPRTN hypomorphic (SprtnH/H) mice exhibited higher levels of dT-Tyr in the liver, brain, heart, and kidney than wild-type animals. Overall, our results are consistent with the understanding that SPRTN has a role in maintaining genomic integrity upon exposure to ionizing radiation and endogenous reactive oxygen species. [ABSTRACT FROM AUTHOR]

The ability to control gene expression is vital for elucidating gene functions and developing next‐generation therapeutics. Current techniques are challenged by the lack of cell‐specific control designs or immunogenicity risk from foreign proteins. We develop a DNA repair inducible ribozyme switch that enables cell‐specific control of gene expression in cells and in vivo. This strategy designs plasmids with a DNA lesion (8‐oxoG and O6‐MeG) site‐specifically installed within the ribozyme encoding region, generating active hammerhead ribozyme for mRNA degradation due to transcriptional mutagenesis, whereas DNA repair yields a single‐base mismatch in the ribozyme to abrogate its activity. This strategy is demonstrated to allow specific control of gene expression in cancer cells with overexpressed DNA repair enzymes such as MutY DNA glycosylase and O6‐methylguanine‐DNA‐methyltransferases. It also shows the capability of conditionally regulating the expression of different proteins for signal reporting and gene editing, enabling DNA repair monitoring and targeted gene therapy in cancer cells. This strategy is demonstrated using the inducible CRISPR/Cas9 system for in vivo editing of oncogenic Polo‐like kinase 1 in a mouse model, resulting in significant tumor growth suppression. The DNA repair inducible ribozyme switch may provide a compact system for cell‐specific gene expression control toward precise gene therapy. [ABSTRACT FROM AUTHOR]

Tyrosyl-DNA phosphodiesterases 1 and 2 (TDP1 and TDP2) are important DNA repair enzymes that remove various adducts from the 3′- and 5′-ends of DNA, respectively. The suppression of the activity of these enzymes is considered as a promising adjuvant therapy for oncological diseases in combination with topoisomerase inhibitors. The simultaneous inhibition of TDP1 and TDP2 may result in greater antitumor effects, as these enzymes can mimic each other's functions. We have previously shown that usnic acid-based sulfides can act as dual inhibitors, with TDP1 activity in the low micromolar range and their TDP2 at 1 mM. The oxidation of their sulfide moieties to sulfoxides led to an order of magnitude decrease in their cytotoxicity potential, while their TDP1 and TDP2 activity was preserved. In this work, we synthesized new series of usnic acid-based sulfides and their oxidized analogues, i.e., sulfoxides and sulfones, to systematically study these irregularities. The new compounds inhibit TDP1 with IC50 values (the concentration of inhibitor required to reduce enzyme activity by half) in the 0.33–25 μM range. Most sulfides and some sulfoxides and sulfones inhibit TDP2 with an IC50 = 138−421 μM. In addition, the most active compounds synergized (×4) with topotecan on the HeLa cell line as well as causing dose-dependent DNA damage, as confirmed by Comet assay. Sulfides with the 6-methylbenzoimidazol-2-yl substituent (8f, IC50 = 0.33/138 μM, TDP1/2) and sulfones containing a pyridine-2-yl fragment (12k, IC50 = 2/228 μM, TDP1/2) are the most potent derivatives and, therefore, are promising for further development. [ABSTRACT FROM AUTHOR]

E-cigarettes (E.cigs) cause inflammation and damage to human organs, including the lungs and heart. In the gut, E.cig vaping promotes inflammation and gut leakiness. Further, E.cig vaping increases tumorigenesis in oral and lung epithelial cells by inducing mutations and suppressing host DNA repair enzymes. It is well known that cigarette (cig) smoking increases the risk of colorectal cancer (CRC). To date, it is unknown whether E.cig vaping impacts CRC development. A mouse model of human familial adenomatous polyposis (CPC-APC) was utilized wherein a mutation in the adenomatous polyposis coli (APC) gene, CDX2-Cre-APCMin/+, leads to the development of colon adenomas within 11–16 weeks. Mice were exposed to air (controls), E.cig vaping, cig, or both (dual exposure). After 4 weeks of 2 h exposures per day (1 h of each for dual exposures), the colon was collected and assessed for polyp number and pathology scores by microscopy. Expression of inflammatory cytokines and cancer stem cell markers were quantified. DNA damage such as double-strand DNA breaks was evaluated by immunofluorescence, western blot, and gene-specific long amplicon qPCR. DNA repair enzyme levels (NEIL-2, NEIL-1, NTH1, and OGG1) were quantified by western blot. Proliferation markers were assessed by RT-qPCR and ELISA. CPC-APC mice exposed to E.cig, cig, and dual exposure developed a higher number of polyps compared to controls. Inflammatory proteins, DNA damage, and cancer stemness markers were higher in E-cig, cig, and dual-exposed mice as well. DNA damage was found to be associated with the suppression of DNA glycosylases, particularly with NEIL-2 and NTH1. E.cig and dual exposure both stimulated cancer cell stem markers (CD44, Lgr-5, DCLK1, and Ki67). The effect of E.cigs on polyp formation and CRC development was less than that of cigs, while dual exposure was more tumorigenic than either of the inhalants alone. E.cig vaping promotes CRC by stimulating inflammatory pathways, mediating DNA damage, and upregulating transcription of cancer stem cell markers. Critically, combining E.cig vaping with cig smoking leads to higher levels of tumorigenesis. Thus, while the chemical composition of these two inhalants, E.cigs and cigs, is highly disparate, they both drive the development of cancer and when combined, a highly common pattern of use, they can have additive or synergistic effects. [ABSTRACT FROM AUTHOR]

DNA repair dysregulation is a key driver of cancer development. Understanding the molecular mechanisms underlying DNA repair dysregulation in cancer cells is crucial for cancer development and therapies. Here, we report that enhancer of zeste homolog 2 (EZH2) directly methylates poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), an essential enzyme involved in DNA repair, and regulates its activity. Functionally, EZH2-catalyzed methylation represses PARP1 catalytic activity, down-regulates the recruitment of x-ray repair cross-complementing group-1 to DNA lesions and its associated DNA damage repair; on the other hand, it protects the cells from nicotinamide adenine dinucleotide overconsumption upon DNA damage formation. Meanwhile, EZH2-mediated methylation regulates PARP1 transcriptional and oncogenic activity, at least in part, through impairing PARP1-E2F1 interaction and E2F1 transcription factor activity. EZH2 and PARP1 inhibitors synergistically suppress prostate cancer growth. Collectively, our findings uncover an insight of EZH2 functions in fine-tuning PARP1 activity during DNA damage repair and cancer progression, which provides a rationale for combinational targeting EZH2 and PARP1 in cancer. [ABSTRACT FROM AUTHOR]

The Fukushima Daiichi Nuclear Power Plant accident caused the release of large amounts of radioactive material into the environment. Radiation from radionuclides cause DNA lesions, mainly via oxidation, which adversely affect wild organisms by damaging their germ cells. Here, we investigated the effects of radiation on the reproductive organs of Japanese field mice (Apodemus speciosus) by estimating the dose rate of radiation exposure, the accumulation of DNA lesions, and the expression of DNA repair enzymes. In highly contaminated areas, mouse testes received a radiation dose rate > 0.1 mGy/d. According to the International Commission on Radiological Protection, there is a very low probability of effects in the reference rat species at this exposure level. The results of the current study do not definitively conclude that the expression of 8-oxoguanine DNA glycosylase 1 and superoxide dismutase in mouse testes increase with dose rate and lifetime dose. However, 8-hydroxy-2′-deoxyguanosine accumulation increases in a dose rate- and lifetime dose-dependent manner in mouse testes, but is not observed in the sperm of the cauda epididymis. These results suggest that, although DNA lesions occurred in male germ cells of Fukushima mice, most were successfully repaired by DNA repair enzymes at the observed gene expression level. [ABSTRACT FROM AUTHOR]

Age‐related macular degeneration (AMD) is a major cause of vision loss in older adults. AMD is caused by degeneration in the macula of the retina. The retina is the highest oxygen consuming tissue in our body and is prone to oxidative damage. DNA damage is one hallmark of aging implicated in loss of organ function. Genome instability has been associated with several disorders that result in premature vision loss. We hypothesized that endogenous DNA damage plays a causal role in age‐related retinal changes. To address this, we used a genetic model of systemic depletion of expression of the DNA repair enzyme ERCC1‐XPF. The neural retina and retinal pigment epithelium (RPE) from Ercc1−/Δ mice, which models a human progeroid syndrome, were compared to age‐matched wild‐type (WT) and old WT mice. By 3‐months‐of age, Ercc1−/Δ mice presented abnormal optokinetic and electroretinogram responses consistent with photoreceptor dysfunction and visual impairment. Ercc1−/Δ mice shared many ocular characteristics with old WT mice including morphological changes, elevated DNA damage markers (γ‐H2AX and 53BP1), and increased cellular senescence in the neural retinal and RPE, as well as pathological angiogenesis. The RPE is essential for the metabolic health of photoreceptors. The RPE from Ercc1−/Δ mice displayed mitochondrial dysfunction causing a compensatory glycolytic shift, a characteristic feature of aging RPE. Hence, our study suggests spontaneous endogenous DNA damage promotes the hallmarks of age‐related retinal degeneration. [ABSTRACT FROM AUTHOR]

Cationic antimicrobial peptides (AMPs), also called host defence peptides, have established antimicrobial and anticancer activities. Conjugation of an AMP to a bioactive molecule with complementary activity can address some of the clinical limitations of the peptide candidate. This approach has been particularly applied in antimicrobial applications of AMPs, but it remains relatively less explored in the generation of anticancer candidates. In this study, two usnic acid derivatives, based on hydrazinothiazole and benzylidenefuranone pharmacophore moieties, respectively, were conjugated to L-K6, a lysine/leucine-rich AMP, through a new pyrazole ligation intrinsically driven by the cargo molecule. Both components, the usnic acid derivative and the peptide, are selectively active against cancer cells, by targeting the human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) and through DNA damage, respectively. The two conjugates, based on a hydrazone linkage, exhibited pleiotropic effects, ranging from reduction in the activity of the parent drugs to their conservation or even enhancement. Notably, the conjugates retained some anti-TDP1 activity and displayed intermediate, or even higher, cytotoxicities against glioblastoma cells, compared to their individual components. [ABSTRACT FROM AUTHOR]

• Astrocytes-derived EVs reduce cytotoxicity in HBECs treated with Na 2 Cr 2 O 7. • Exposure to Na 2 Cr 2 O 7 suppressed immune response genes. • Astrocyte-derived EVs affect lncRNA expression that regulates brain development. • Exposure to Na 2 Cr 2 O 7 is associated with an increase in 8OHdG genomic levels. This study investigates the therapeutic effect of astrocyte-derived extracellular vesicles (EVs) in mitigating neurotoxicity-induced transcriptome changes, mitochondrial function, and base excision repair mechanisms in human brain endothelial cells (HBECs). Neurodegenerative disorders are marked by inflammatory processes impacting the blood–brain barrier (BBB) that involve its main components- HBECs and astrocytes. Astrocytes maintain homeostasis through various mechanisms, including EV release. The effect of these EVs on mitigating neurotoxicity in HBECs has not been investigated. This study assesses the impact of astrocyte-derived EVs on global transcriptome changes, cell proliferation, cytotoxicity, oxidative DNA damage, and mitochondrial morphology in HBECs exposed to the neurotoxic reagent Na 2 Cr 2 O 7. Exposure to Na 2 Cr 2 O 7 for 5 and 16 h induced oxidative DNA damage, measured by an increase in genomic 8OHdG, while the EVs reduced the accumulation of the adduct. A neurotoxic environment caused a non-statistically significant upregulation of the DNA repair enzyme OGG1 while the addition of astrocyte-derived EVs was associated with the same level of expression. EVs caused increased cell proliferation and reduced cytotoxicity in Na 2 Cr 2 O 7 -treated cells. Mitochondrial dysfunction associated with a reduced copy number and circular morphology induced by neurotoxic exposure was not reversed by astrocyte-derived EVs. High-throughput RNA sequencing revealed that exposure to Na 2 Cr 2 O 7 suppressed immune response genes. The addition of astrocyte-derived EVs resulted in the dysregulation of long noncoding RNAs impacting genes associated with brain development and angiogenesis. These findings reveal the positive impact of astrocytes-derived EVs in mitigating neurotoxicity and as potential therapeutic avenues for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

In the last decade, several novel functions of the mammalian Apurinic/Apyrimidinic Endodeoxyribonuclease 1 (APE1) have been discovered, going far beyond its canonical function as DNA repair enzyme and unveiling its potential roles in cancer development. Indeed, it was shown to be involved in DNA G-quadruplex biology and RNA metabolism, most importantly in the miRNA maturation pathway and the decay of oxidized or abasic miRNAs during oxidative stress conditions. In recent years, several noncanonical pathways of miRNA biogenesis have emerged, with a specific focus on guanosine-rich precursors that can form RNA G-quadruplex (rG4) structures. Here, we show that several miRNA precursors, dysregulated upon APE1 depletion, contain an rG4 motif and that their corresponding target genes are up-regulated after APE1 depletion. We also demonstrate, both by in vitro assays and by using different cancer cell lines, that APE1 can modulate the folding of an rG4 structure contained in pre-miR- 92b, with a mechanism strictly dependent on lysine residues present in its N-terminal disordered region. Furthermore, APE1 cellular depletion alters the maturation process of miR-92b, mainly affecting the shuttling between the nucleus and cytosol. Bioinformatic analysis of APE1-regulated rG4-containing miRNAs supports the relevance of our findings in cancer biology. Specifically, these miRNAs exhibit high prognostic significance in lung, cervical, and liver tumors, as suggested by their involvement in several cancer-related pathways. [ABSTRACT FROM AUTHOR]

The base excision repair enzyme thymine DNA glycosylase (TDG) protects against mutations by removing thymine or uracil from guanine mispairs and functions in active DNA demethylation by excising 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Post-translational modification of TDG by SUMO (small ubiquitin-like modifier) reduces its glycosylase activity but the mechanism remains unclear. We investigated this problem using biochemical and biophysical approaches and a TDG construct comprising residues 82 to 340 (of 410) that includes the SUMOylation site and the motif for non-covalent SUMO binding. Single turnover kinetics experiments were collected at multiple enzyme concentrations ([E]) and the hyperbolic dependence of activity (kobs) on [E] yielded the maximal glycosylase activity (kmax), the enzyme concentration giving half-maximal activity (K0.5), and the catalytic efficiency (kmax/K0.5). Sumoylation of TDG (or TDG82-340) causes large reductions in catalytic efficiency for G·T, G·U, G·fC, and G·caC DNA substrates, due largely to weakened substrate af- finity (increased K0.5). 19F NMR experiments show that sumoylation of TDG82-340 reduces productive binding to G·U mispairs and dramatically impairs binding to G·T mispairs. A mutation in the TDG SUMO-interacting motif (SIM), E310Q, shown previously to perturb the noncovalent binding of SUMO to unmodified TDG, rescues the glycosylase activity of sumoylated TDG82-340. Similarly, NMR studies show the mutation restores the productive binding of sumoylated TDG82-340 to G·U and G·T pairs. Together, the results indicate that intramolecular SUMO-SIM interactions mediate the adverse effect of sumoylation on TDG activity and suggest a model whereby the disruption of SUMO-SIM interactions enables productive binding of sumoylated TDG to substrate sites in DNA. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) presents significant medical challenges, largely due to the genetic diversity of Mycobacterium tuberculosis, which enhances the resilience and resistance of the pathogen to first-line treatments. In response to the global rise of drug-resistant TB, second-line antitubercular drugs like bedaquiline (BDQ), linezolid (LZD), and clofazimine (CFZ) have become critical treatment options. Understanding the molecular changes these drugs induce is essential for optimizing TB therapy. To contribute to this effort, we investigated their impact on genome maintenance and stability using Mycobacterium smegmatis as a model organism. Using mutation accumulation assays and whole-genome sequencing, we found that the second-line antibiotics did not significantly increase mutation rates, unlike the positive control UV treatment. However, upon BDQ treatment, we detected mutations in transporter proteins and transcription factors without any increase in the minimal inhibitory concentration. Additionally, BDQ and CFZ were found to alter DNA repair pathways and reduce cellular dNTP levels, particularly CFZ, which depleted dGTP, impacting DNA synthesis. CFZ also upregulated DNA repair enzymes, enhancing error-free repairs. Despite minimal mutagenic effects, both drugs displayed distinct impacts on cellular mechanisms, suggesting additional modes of action. [ABSTRACT FROM AUTHOR]

Among cancer-related deaths worldwide, colorectal cancer ranks second, accounting for 1.2% of deaths in those under 50 years and 0.6% of deaths in those between 50 and 54 years. The anticancer drug 5-fluorouracil is widely used to treat colorectal cancer. Due to a better understanding of the drug's mechanism of action, its anticancer activity has been increased through a variety of therapeutic alternatives. Clinical use of 5-FU has been severely restricted due to drug resistance. The chemoresistance mechanism of 5-FU is challenging to overcome because of the existence of several drug efflux transporters, DNA repair enzymes, signaling cascades, classical cellular processes, cancer stem cells, metastasis, and angiogenesis. Curcumin, a potent phytocompound derived from Curcuma longa, functions as a nuclear factor (NF)-κB inhibitor and sensitizer to numerous chemotherapeutic drugs. Piperine, an alkaloid found in Piper longum, inhibits cancer cell growth, causing cell cycle arrest and apoptosis. This review explores the mechanism of 5-FU-induced chemoresistance in colon cancer cells and the role of curcumin and piperine in enhancing the sensitivity of 5-FU-based chemotherapy. Clinical trial registration: Not applicable [ABSTRACT FROM AUTHOR]

The lack of precise spatiotemporal gene modulation and therapy impedes progress in medical applications. Herein, a 980 nm near‐infrared (NIR) light‐controlled nanoplatform, namely URMT, is developed, which can allow spatiotemporally controlled photodynamic therapy and trigger the enzyme‐activated gene expression regulation in tumors. URMT is constructed by engineering an enzyme‐activatable antisense oligonucleotide, which combined with an upconversion nanoparticle (UCNP)‐based photodynamic nanosystem, followed by the surface functionalization of triphenylphosphine (TPP), a mitochondria‐targeting ligand. URMT allows for the 980 nm NIR light‐activated generation of reactive oxygen species, which can induce the translocation of a DNA repair enzyme (namely apurinic/apyrimidinic endonuclease 1, APE1) from the nucleus to mitochondria. APE1 can recognize the basic apurinic/apyrimidinic (AP) sites in DNA double‐strands and perform cleavage, thereby releasing the functional single‐strands for gene regulation. Overall, an augmented antitumor effect is observed due to NIR light‐controlled mitochondrial damage and enzyme‐activated gene regulation. Altogether, the approach reported in this study offers high spatiotemporal precision and shows the potential to achieve precise and specific gene regulation for targeted tumor treatment. [ABSTRACT FROM AUTHOR]

Base excision repair is the main pathway involved in active DNA demethylation. 5-formylcytosine and 5-carboxylcytosine, two oxidized moieties of methylated cytosine, are recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. Using single molecule fluorescence experiments, we study TDG in the presence and absence of 5-formylcytosine. TDG exhibits multiple modes of linear diffusion, including hopping and sliding, in search of base modifications. TDG active site variants and truncated N-terminus, reveals these variants alter base modification search and recognition mechanism of TDG. On DNA containing an undamaged nucleosome, TDG is found to either bypass, colocalize with, or encounter but not bypass the nucleosome. Truncating the N-terminus reduces the number of interactions with the nucleosome. Our findings provide mechanistic insights into how TDG searches for modified DNA bases in chromatin. Thymine DNA glycosylase is an essential DNA repair enzyme involved in oxidative demethylation of 5- methylC. In this study the authors have tracked specific and non-specific DNA binding of TDG at the single molecule level. TDG is found to hop and slide on DNA and found to interact with nucleosomes. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV), along with other prominent respiratory RNA viruses such as influenza and SARS-CoV-2, significantly contributes to the global incidence of respiratory tract infections. These pathogens induce the production of reactive oxygen species (ROS), which play a crucial role in the onset and progression of respiratory diseases. However, the mechanisms by which viral RNA manages ROS-induced base oxidation remain poorly understood. Here, we reveal that 8-oxo-7,8-dihydroguanine (8-oxoGua) is not merely an incidental byproduct of ROS activity but serves as a strategic adaptation of RSV RNA to maintain genetic fidelity by hijacking the 8-oxoguanine DNA glycosylase 1 (OGG1). Through RNA immunoprecipitation and next-generation sequencing, we discovered that OGG1 binding sites are predominantly found in the RSV antigenome, especially within guanine-rich sequences. Further investigation revealed that viral ribonucleoprotein complexes specifically exploit OGG1. Importantly, inhibiting OGG1's ability to recognize 8-oxoGua significantly decreases RSV progeny production. Our results underscore the viral replication machinery's adaptation to oxidative challenges, suggesting that inhibiting OGG1's reading function could be a novel strategy for antiviral intervention. Author summary: Respiratory tract infections caused by RSV present a significant public health concern, especially affecting infants and the elderly. Effective antiviral strategies are urgently needed against this non-segmented, negative-sense RNA virus of the Pneumoviridae family. Our research reveals how RSV maintains replication fitness despite oxidative damage, a common exacerbator of respiratory conditions. We have identified a mechanism where RSV preserves its genome integrity under oxidative stress using the DNA repair enzyme OGG1. This enzyme ensures that the viral RNA accurately pairs 8-oxoGua with cytosine, crucial for maintaining the fidelity of the viral genome. Targeting the interplay between OGG1 and RSV RNA offers a promising approach for developing novel antiviral treatments, potentially countering the virus's evolutionary adaptations. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3) are the two most prevalent polyglutamine (polyQ) neurodegenerative diseases, caused by CAG (encoding glutamine) repeat expansion in the coding region of the huntingtin (HTT) and ataxin-3 (ATXN3) proteins, respectively. We have earlier reported that the activity, but not the protein level, of an essential DNA repair enzyme, polynucleotide kinase 3'-phosphatase (PNKP), is severely abrogated in both HD and SCA3 resulting in accumulation of double-strand breaks in patients' brain genome. While investigating the mechanistic basis for the loss of PNKP activity and accumulation of DNA double-strand breaks leading to neuronal death, we observed that PNKP interacts with the nuclear isoform of 6-phosphofructo-2-kinase fructose-2,6-bisphosphatase 3 (PFKFB3). Depletion of PFKFB3 markedly abrogates PNKP activity without changing its protein level. Notably, the levels of both PFKFB3 and its product fructose-2,6 bisphosphate (F2,6BP), an allosteric modulator of glycolysis, are significantly lower in the nuclear extracts of postmortem brain tissues of HD and SCA3 patients. Supplementation of F2,6BP restored PNKP activity in the nuclear extracts of patients' brain. Moreover, intracellular delivery of F2,6BP restored both the activity of PNKP and the integrity of transcribed genome in neuronal cells derived from the striatum of the HD mouse. Importantly, supplementing F2,6BP rescued the HD phenotype in Drosophila, suggesting F2,6BP to serve in vivo as a cofactor for the proper functionality of PNKP and thereby, of brain health. Our results thus provide a compelling rationale for exploring the therapeutic use of F2,6BP and structurally related compounds for treating polyQ diseases. [ABSTRACT FROM AUTHOR]

Developing effective inhibitors of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) has been challenging because of the enzyme shallow catalytic pocket and non-specific substrate binding interactions. Recently, we discovered a quinolone-binding hot spot in TDP1's active site proximal to the evolutionary conserved Y204 and F259 residues that position DNA. Sulfur (VI) fluoride exchange (SuFEx) is a biocompatible click chemistry reaction that enables acylation of protein residues, including tyrosine. Selective protein modifications can provide insights into the biological roles of proteins and inform ligand design. As we report herein, we used SuFEx chemistries to prepare covalent TDP1-bound binders showing site-specific covalent bonds with Y204. Our work presents the first application of SuFEx chemistries to TDP1 ligands. It validates the ability to covalently modify specific TDP1 residues by designed targeting and adds to the chemical biology resource toolbox for studying TDP1. Developing effective inhibitors of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) is challenging because of the enzyme's shallow catalytic pocket and non-specific substrate binding interactions. Here, the authors use Sulfur (VI) fluoride exchange chemistries to prepare covalent TDP1-bound binders showing site-specific covalent bonds with the Y204 residue that position DNA. [ABSTRACT FROM AUTHOR]

Obstructive sleep apnea syndrome (OSAS), characterized by chronic intermittent hypoxia (CIH), is an independent risk factor for aggravating non‐alcoholic steatohepatitis (NASH). The prevailing mouse model employed in CIH research is inadequate for the comprehensive exploration of the impact of CIH on NASH development due to reduced food intake observed in CIH‐exposed mice, which deviates from human responses. To address this issue, a pair‐feeding investigation with CIH‐exposed and normoxia‐exposed mice is conducted. It is revealed that CIH exposure aggravates DNA damage, leading to hepatic fibrosis and inflammation. The analysis of genome‐wide association study (GWAS) data also discloses the association between Eepd1, a DNA repair enzyme, and OSAS. Furthermore, it is revealed that CIH triggered selective autophagy, leading to the autophagic degradation of Eepd1, thereby exacerbating DNA damage in hepatocytes. Notably, Eepd1 liver‐specific knockout mice exhibit aggravated hepatic DNA damage and further progression of NASH. To identify a therapeutic approach for CIH‐induced NASH, a drug screening is conducted and it is found that Retigabine dihydrochloride suppresses CIH‐mediated Eepd1 degradation, leading to alleviated DNA damage in hepatocytes. These findings imply that targeting CIH‐mediated Eepd1 degradation can be an adjunctive approach in the treatment of NASH exacerbated by OSAS. [ABSTRACT FROM AUTHOR]

Chemical modification of DNA is a common strategy to improve the properties of oligonucleotides, particularly for therapeutics and nanotechnology. Existing synthetic methods essentially rely on phosphoramidite chemistry or the polymerization of nucleoside triphosphates but are limited in terms of size, scalability, and sustainability. Herein, we report a robust alternative method for the de novo synthesis of modified oligonucleotides using template-dependent DNA ligation of shortmer fragments. Our approach is based on the fast and scaled accessibility of chemically modified shortmer monophosphates as substrates for the T3 DNA ligase. This method has shown high tolerance to chemical modifications, flexibility, and overall efficiency, thereby granting access to a broad range of modified oligonucleotides of different lengths (20 → 120 nucleotides). We have applied this method to the synthesis of clinically relevant antisense drugs and ultramers containing diverse modifications. Furthermore, the designed chemoenzymatic approach has great potential for diverse applications in therapeutics and biotechnology. Chemical modification of DNA is a common strategy to improve the properties of oligonucleotides. Herein, the authors report a robust method based on the template-dependent DNA ligation of shortmer fragments by DNA ligases, which is compatible with numerous chemical modifications and applicable to short (~20 nt) and longer (<100 nt) sequences. [ABSTRACT FROM AUTHOR]

Background: Bladder cancer, a highly fatal disease, poses a significant threat to patients. Positioned at 19q13.2-13.3, LIG1, one of the four DNA ligases in mammalian cells, is frequently deleted in tumour cells of diverse origins. Despite this, the precise involvement of LIG1 in BLCA remains elusive. This pioneering investigation delves into the uncharted territory of LIG1's impact on BLCA. Our primary objective is to elucidate the intricate interplay between LIG1 and BLCA, alongside exploring its correlation with various clinicopathological factors. Methods: We retrieved gene expression data of para-carcinoma tissues and bladder cancer (BLCA) from the GEO repository. Single-cell sequencing data were processed using the "Seurat" package. Differential expression analysis was then performed with the "Limma" package. The construction of scale-free gene co-expression networks was achieved using the "WGCNA" package. Subsequently, a Venn diagram was utilized to extract genes from the positively correlated modules identified by WGCNA and intersect them with differentially expressed genes (DEGs), isolating the overlapping genes. The "STRINGdb" package was employed to establish the protein-protein interaction (PPI) network.Hub genes were identified through the PPI network using the Betweenness Centrality (BC) algorithm. We conducted KEGG and GO enrichment analyses to uncover the regulatory mechanisms and biological functions associated with the hub genes. A machine-learning diagnostic model was established using the R package "mlr3verse." Mutation profiles between the LIG1^high and LIG1^low groups were visualized using the BEST website. Survival analyses within the LIG1^high and LIG1^low groups were performed using the BEST website and the GENT2 website. Finally, a series of functional experiments were executed to validate the functional role of LIG1 in BLCA. Results: Our investigation revealed an upregulation of LIG1 in BLCA specimens, with heightened LIG1 levels correlating with unfavorable overall survival outcomes. Functional enrichment analysis of hub genes, as evidenced by GO and KEGG enrichment analyses, highlighted LIG1's involvement in critical function such as the DNA replication, cellular senescence, cell cycle and the p53 signalling pathway. Notably, the mutational landscape of BLCA varied significantly between LIG1high and LIG1low groups. Immune infiltrating analyses suggested a pivotal role for LIG1 in immune cell recruitment and immune regulation within the BLCA microenvironment, thereby impacting prognosis. Subsequent experimental validations further underscored the significance of LIG1 in BLCA pathogenesis, consolidating its functional relevance in BLCA samples. Conclusions: Our research demonstrates that LIG1 plays a crucial role in promoting bladder cancer malignant progression by heightening proliferation, invasion, EMT, and other key functions, thereby serving as a potential risk biomarker. [ABSTRACT FROM AUTHOR]

Introduction: Glioblastoma, which affects a large number of patients every year and has an average overall lifespan of around 14.6 months following diagnosis stands out as the most lethal primary invasive brain tumor. Currently, surgery, radiation, and chemotherapy with temozolomide (TMZ) are the three major clinical treatment approaches. However, the ability to treat patients effectively is usually limited by TMZ resistance. Naringin, a bioflavonoid with anti-cancer, antioxidant, metal-chelating, and lipid-lowering effects, has emerged as a promising therapeutic option. Methods: To explore the targets and pathways of naringin and TMZ in glioblastoma network pharmacology, cell line-based ELISA, flow cytometry, immunocytochemistry, western blotting, and LC-HRMS based metabolomics study were used. Results: The findings through the network pharmacology suggested that the key targets of naringin in the chemosensitization of glioblastoma would be Poly [ADP-ribose] polymerase 1 (PARP-1), O-6-Methylguanine-DNA Methyltransferase (MGMT), and caspases. The functional enrichment analysis revealed that these targets were significantly enriched in important pathways such as p53 signaling, apoptosis, and DNA sensing. Further, the results of the in-vitro study in U87-MG and T98-G glioblastoma cells demonstrated that TMZ and naringin together significantly reduced the percentage of viability and inhibited the DNA repair enzymes PARP-1 and MGMT, and PI3K/AKT which led to chemosensitization and, in turn, induced apoptosis, which was indicated by increased p53, caspase-3 expression and decreased Bcl2 expression. Additionally, a metabolomics study in T98-G glioblastoma cells using liquid chromatography high-resolution mass spectrometry (LC-HRMS) revealed downregulation of C8-Carnitine (-2.79), L-Hexanoylcarnitine (-4.46), DLCarnitine (-2.46), Acetyl-L-carnitine (-3.12), Adenine (-1.3), Choline (-2.07), Propionylcarnitine (-1.69), Creatine (-1.33), Adenosine (-0.84), Spermine (-1.42), and upregulation of Palmitic Acid (+1.03) and Sphingosine (+0.89) in the naringin and TMZ treatment groups. Discussion: In conclusion, it can be said that naringin in combination with TMZ chemosensitized TMZ antiglioma response and induced apoptosis in tumor cells. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction and genomic instability are key hallmarks of aging. The aim of this study was to evaluate whether maintenance of physical capacities at very old age is associated with key hallmarks of aging. To investigate this, we measured mitochondrial bioenergetics, mitochondrial DNA (mtDNA) copy number and DNA repair capacity in peripheral blood mononuclear cells from centenarians. In addition, circulating levels of NAD+/NADH, brain-derived neurotrophic factor (BDNF) and carbonylated proteins were measured in plasma and these parameters were correlated to physical capacities. Centenarians without physical disabilities had lower mitochondrial respiration values including ATP production, reserve capacity, maximal respiration and non-mitochondrial oxygen-consumption rate and had higher mtDNA copy number than centenarians with moderate and severe disabilities (p < 0.05). In centenarian females, grip strength had a positive association with mtDNA copy number (p < 0.05), and a borderline positive trend for activity of the central DNA repair enzyme, APE 1 (p = 0.075), while a negative trend was found with circulating protein carbonylation (p = 0.07) in the entire cohort. Lastly, a trend was observed for a negative association between BDNF and activity of daily living disability score (p = 0.06). Our results suggest that mechanisms involved in maintaining mitochondrial function and genomic stability may be associated with maintenance of physical function in centenarians. [ABSTRACT FROM AUTHOR]

Cancer, a widespread challenge to global health, remains a puzzle of intricate molecular dynamics. This review article delves into the mystery of cancer, with a keen focus on understanding the contributory role of thymidylate synthase (TS) in cancer. TS, a vital enzyme in DNA synthesis and repair, emerges as a significant player in the narrative of cancer development. The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) is a major step in producing DNA. Numerous malignancies, including those of the breast, colon, lung, and ovary, have been linked to dysregulation of TS activity. Overexpression or mutations of TS lead to uncontrolled cell proliferation and tumorigenesis molecular interactions and signalling pathways involving TS come under scrutiny, revealing the nuanced connections that propel its involvement in cancer progression. Beyond overexpression and mutations, there emerges a subtle layer of regulation that involves microRNAs (miRNAs). These tiny particles attach to the TS messenger RNA, causing translational repression or its degradation, which in turn affects TS activity. Moving towards the therapeutic realm, thymidylate synthase inhibition acts as a promising anti-cancer strategy. Targeting TS with small-molecule inhibitors could provide a novel approach to treat various cancers. By reducing the number of available nucleotides, TS inhibition would slow down or halt cancer cell division, thus depriving the tumor of the building blocks required for its proliferation and growth. The aim is to assess the viability and effectiveness of targeting TS to halt or slow down cancer progression. There is growing evidence that, in comparison to traditional TS inhibitors, few novel antifolate TS inhibitors are effective against a wider variety of neoplasms, such as lung carcinomas. It has been discovered that TS inhibitors increase cancer tissues' sensitivity to chemotherapy and radiation, increasing their vulnerability to these treatments. This article aims to provide a comprehensive insight into TS, examining its cellular details, detailing the heterocyclic moieties and molecular foundations, and providing a promising future outlook. Thymidylate synthase enzyme in cancer progression [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae is a genetically tractable, affordable, and extensively documented eukaryotic single-cell model organism. This budding yeast is amenable for the development of genetic and biochemical experiments and is frequently used to investigate the function, activity, and mechanism of mammalian proteins. However, yeast contains a cell wall that hinders select assays including organelle isolation. Lytic enzymes, with Zymolyase as the most effective and frequently used tool, are utilized to weaken the yeast cell wall resulting in yeast spheroplasts. Spheroplasts are easily lysed by, for example, osmotic-shock conditions to isolate yeast nuclei or mitochondria. However, during our studies of the DNA repair enzyme tyrosyl-DNA phosphodiesterase I (Tdp1), we encountered a negative effect of Zymolyase. We observed that Zymolyase treatment affected the steady-state protein levels of Tdp1. This was revealed by inconsistencies in technical and biological replicate lysates of plasmid-born galactose-induced expression of Tdp1. This off-target effect of Zymolyase is rarely discussed in articles and affects a select number of intracellular proteins, including transcription factors and assays such as chromatin immunoprecipitations. Following extensive troubleshooting, we concluded that the culprit is the Ser-protease, Zymolyase B, component of the Zymolyase enzyme mixture that causes the degradation of Tdp1. In this study, we report the protocols we have used, and our final protocol with an easy, affordable adaptation to any assay/protocol involving Zymolyase. [ABSTRACT FROM AUTHOR]

Aim of the study: Polymorphisms of DNA repair enzyme gene may alter the ability of damage repair, ischemic stroke susceptibility and outcome. This study aimed to explore the association of polymorphisms in PARP1 and the effects of interactions between genes in Chinese.Materials and methods: A total of 500 patients and 500 healthy controls were enrolled for genotyping. Results: Clinical information analysis revealed higher levels of alcohol and smoking exposure in patients with ischemic stroke, as well as chronic conditions such as diabetes, hypertension, and higher serum triglycerides concentration. In addition, Polymorphism in PARP1 rs8679 was significantly associated with the decreased ischemic stroke risk. Patients harboring the PARP1 rs8679 AG/GG genotype had a better initial stroke, and as for the mRNA level of PARP1, it was suppressed with mutant genotype in comparison with the wild genotype. Finally, the suppressed of PARP1 was induced by gain-binding ability of miR-124-5p through 3'UTR directly binding.Conclusions: In conclusion, our study demonstrates that the SNP rs8679 in PARP1 3′-UTR might act as a protective factor for the outcome of patients with ischemic stroke. [ABSTRACT FROM AUTHOR]

Background: By complexing poly (ADP-ribose) (PAR) in reaction to broke strand, PAR polymerase1 (PARP1) acts as the key enzyme participated in DNA repair. However, recent studies suggest that unrepaired DNA breaks results in persistent PARP1 activation, which leads to a progressively reduce in hexokinase1 (HK1) activity and cell death. PARP-1 is TCF-4/β-A novel co activator of gene transactivation induced by catenin may play a role in the development of colorectal cancer. The molecular mechanism of PARP1 remains elusive. Methods: 212 colorectal cancer (CRC) patients who had the operation at our hospital were recruited. PARP1 expression was evaluated by immunohistochemistry. Stable CRC cell lines with low or high PARP1 expression were constructed. Survival analysis was computed based on PARP1 expression. The cell proliferation was tested by CCK-8 and Colony formation assay. The interaction of PARP1 and XRCC2 was detected by immunoprecipitation (IP) analysis. Results: Compared with matching adjacent noncancerous tissue, PARP1 was upregulated in CRC tissue which was correlated with the degree of differentiation, TNM stage, depth of invasion, metastasis, and survival. In addition, after constructing CRC stable cell lines with abnormal expression of PARP1, we found that overexpression of PARP1 promoted proliferation, and demonstrated the interaction between PARP1 and XRCC2 in CRC cells through immunoprecipitation (IP) analysis. Moreover, the inhibitor of XRCC2 can suppress the in vitro proliferation arousing by upregulation of PARP1. Conclusions: PARP1 was upregulated in CRC cells and promoted cell proliferation. Furthermore, the expression status of PARP1 was significantly correlated with some clinicopathological features and 5-year survival. [ABSTRACT FROM AUTHOR]

A number of new conjugates of dehydroabiethylamine and adamantane was obtained using a three-step synthetic scheme in order to study the effect of linker type and length on biological activity. The inhibitory activity of the synthesized compounds toward the DNA repair enzyme Tyrosyl-DNA phosphodiesterase 1 was studied. The compounds are potent inhibitors, exhibiting activity in micromolar concentrations. [ABSTRACT FROM AUTHOR]

The importance of DNA repair enzymes in maintaining genomic integrity is highlighted by the hypothesis that DNA damage by reactive oxygen/nitrogen species produced inside the host cell is essential for the mutagenesis process. Endonuclease III (Nth), formamidopyrimide (Fpg) and endonuclease VIII (Nei) DNA glycosylases are essential components of the bacterial base excision repair process. Mycobacterium leprae lost both fpg/nei genes during the reductive evolution event and only has the nth (ML2301) gene. This study aims to characterize the mutations in the nth gene of M. leprae strains and explore its correlation with drug-resistance. A total of 91 M. leprae positive DNA samples extracted from skin biopsy samples of newly diagnosed leprosy patients from NSCB Hospital Jabalpur were assessed for the nth gene as well as drug resistance-associated loci of the rpoB, gyrA and folP1 genes through PCR followed by Sanger sequencing. Of these 91 patients, a total of two insertion frameshift mutations, two synonymous and seven nonsynonymous mutations were found in nth in seven samples. Sixteen samples were found to be resistant to ofloxacin and one was found to be dapsone resistant as per the known DRDR mutations. No mutations were found in the rpoB region. Interestingly, none of the nth mutations were identified in the drug-resistant associated samples. The in-silico structural analysis of the non-synonymous mutations in the Nth predicted five of them were to be deleterious. Our results suggest that the mutations in the nth gene may be potential markers for phylogenetic and epidemiological studies. [ABSTRACT FROM AUTHOR]

Background and purpose: DNA damage can lead to carcinogenesis if replication proceeds without proper repair. This study focused on the purification of a novel quercetin derivative present in Terminalia chebula fruit and studied its protective role in hepatoma cells due to H2O2-DNA damage. Experimental approach: The pure compound obtained from the silica gel column was subjected to structural characterization using spectroscopic techniques. MTT assay was employed to select a non-toxic concentration of the isolated compounds on HepG2 and Chang liver cells. The antigenotoxic property of the compound on HepG2 and Chang liver cells was carried out by alkaline comet assay. Analyses of expression levels of mRNA for two DNA repair enzymes, OGG1 and NEIL1, in HepG2 and Chang liver cells, were carried out using the RT-PCR method. Findings/Results: The pure compound obtained from the fraction-5 of diethyl ether extract was identified as a novel quercetin derivative and named 7-(but-2-en-1-yloxy)-2-(4(but-2-en-1-yloxy)-3-hydroxyphenyl)-3- (hexa-2,4-dien-1-yloxy)-6-hydroxy-4H-chromen-4-one. This compound recorded modest toxicity at the highest concentration tested (percentage cell viability at 100 µg/mL was 64.71 ± 0.38 for HepG2 and 45.32 ± 0.07 for Chang liver cells). The compound has demonstrated noteworthy protection against H2O2-induced DNA damage in both cell lines. Analyses of mRNA expression levels for enzymes OGG1 and NEIL1 enzymes in HepG2 and Chang liver cells asserted the protective role of the isolated compound against H2O2-induced DNA damage. Conclusion and implication: The protective effect of a novel quercetin derivative isolated from T. chebula in the hepatoma cells is reported here for the first time. [ABSTRACT FROM AUTHOR]

Background: 8-Oxoguanine DNA glycosylase (OGG1), a well-known DNA repair enzyme, has been demonstrated to promote lung fibrosis, while the specific regulatory mechanism of OGG1 during pulmonary fibrosis remains unclarified. Methods: A bleomycin (BLM)-induced mouse pulmonary fibrosis model was established, and TH5487 (the small molecule OGG1 inhibitor) and Mitochondrial division inhibitor 1 (Mdivi-1) were used for administration. Histopathological injury of the lung tissues was assessed. The profibrotic factors and oxidative stress-related factors were examined using the commercial kits. Western blot was used to examine protein expression and immunofluorescence analysis was conducted to assess macrophages polarization and autophagy. The conditional medium from M2 macrophages was harvested and added to HFL-1 cells for culture to simulate the immune microenvironment around fibroblasts during pulmonary fibrosis. Subsequently, the loss- and gain-of function experiments were conducted to further confirm the molecular mechanism of OGG1/PINK1. Results: In BLM-induced pulmonary fibrosis, OGG1 was upregulated while PINK1/Parkin was downregulated. Macrophages were activated and polarized to M2 phenotype. TH5487 administration effectively mitigated pulmonary fibrosis, M2 macrophage polarization, oxidative stress and mitochondrial dysfunction while promoted PINK1/Parkin-mediated mitophagy in lung tissues of BLM-induced mice, which was partly hindered by Mdivi-1. PINK1 overexpression restricted M2 macrophages-induced oxidative stress, mitochondrial dysfunction and mitophagy inactivation in lung fibroblast cells, and OGG1 knockdown could promote PINK1/Parkin expression and alleviate M2 macrophages-induced mitochondrial dysfunction in HFL-1 cells. Conclusion: OGG1 inhibition protects against pulmonary fibrosis, which is partly via activating PINK1/Parkin-mediated mitophagy and retarding M2 macrophage polarization, providing a therapeutic target for pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

The year 2022 was marked by the mpox outbreak caused by the human monkeypox virus (MPXV), which is approximately 98% identical to the vaccinia virus (VACV) at the sequence level with regard to the proteins involved in DNA replication. We present the production in the baculovirus-insect cell system of the VACV DNA polymerase holoenzyme, which consists of the E9 polymerase in combination with its co-factor, the A20-D4 heterodimer. This led to the 3.8 Å cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. The model of the holoenzyme was constructed from high-resolution structures of the components of the complex and the A20 structure predicted by AlphaFold 2. The structures do not change in the context of the holoenzyme compared to the previously determined crystal and NMR structures, but the E9 thumb domain became disordered. The E9-A20-D4 structure shows the same compact arrangement with D4 folded back on E9 as observed for the recently solved MPXV holoenzyme structures in the presence and the absence of bound DNA. A conserved interface between E9 and D4 is formed by a cluster of hydrophobic residues. Small-angle X-ray scattering data show that other, more open conformations of E9-A20-D4 without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Biolayer interferometry (BLI) showed that the E9-D4 interaction is indeed weak and transient in the absence of DNA although it is very important, as it has not been possible to obtain viable viruses carrying mutations of key residues within the E9-D4 interface. Author summary: The 2022 outbreak of mpox is caused by monkeypox virus closely related to the best-studied model, vaccinia virus. Genome replication, which takes place largely autonomously in the cytosol of the infected cell, is still not really understood. Viral DNA synthesis involves a DNA repair enzyme, the uracil-DNA glycosylase D4 linked to the structural protein A20 forming the processivity factor, which in turn binds to the DNA polymerase E9 forming the complex required for processive DNA synthesis. Here we present the 3.8 Å structure of the apo form of vaccinia virus polymerase holoenzyme E9-A20-D4 obtained by cryo-electron microscopy. Several recent structures from monkeypox virus, with or without bound DNA, and our structure provide static views of the complex revealing a previously undescribed contact between E9 and D4. Our small-angle X-ray scattering data show that other, more open conformations without the E9-D4 contact exist in solution using the flexibility of two hinge regions in A20. Based on site-directed mutagenesis and binding studies, we show that the contact between E9 and D4, which serves to encircle the template strand for processive DNA synthesis, is essential, although the interaction is weak and transient in the absence of DNA. [ABSTRACT FROM AUTHOR]

In the cell, DNA polymerase β (Polβ) is involved in many processes aimed at maintaining genome stability and is considered the main repair DNA polymerase participating in base excision repair (BER). Polβ can fill DNA gaps formed by other DNA repair enzymes. Single-nucleotide polymorphisms (SNPs) in the POLB gene can affect the enzymatic properties of the resulting protein, owing to possible amino acid substitutions. For many SNP-associated Polβ variants, an association with cancer, owing to changes in polymerase activity and fidelity, has been shown. In this work, kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring polymorphic variants G274R, G290C, and R333W. Previously, the amino acid substitutions at these positions have been found in various types of tumors, implying a specific role of Gly-274, Gly-290, and Arg-333 in Polβ functioning. All three polymorphic variants had reduced polymerase activity. Two substitutions—G274R and R333W—led to the almost complete disappearance of gap-filling and primer elongation activities, a decrease in the deoxynucleotide triphosphate–binding ability, and a lower polymerization constant, due to alterations of local contacts near the replaced amino acid residues. Thus, variants G274R, G290C, and R333W may be implicated in an elevated level of unrepaired DNA damage. [ABSTRACT FROM AUTHOR]