Base Damage Research Articles

Structural and functional coupling in cross-linking uracil-DNA glycosylase UDGX.

Enzymes in uracil-DNA glycosylase (UDG) superfamily are involved in removal of deaminated nucleobases such as uracil, methylcytosine derivatives such as formylcytosine and carboxylcytosine, and other base damage in DNA repair. UDGX is the latest addition of a new class to the UDG superfamily with a sporadic distribution in bacteria. UDGX type enzymes have a distinct biochemical property of cross-linking itself to the resulting AP site after uracil removal. Built on previous biochemical and structural analyses, this work comprehensively investigated the kinetic and enzymatic properties of Mycobacterium smegmatis UDGX. Kinetics and mutational analyses, coupled with structural information, defined the roles of E52, D56, D59, F65 of motif 1, H178 of motif 2 and N91, K94, R107 and H109 of motif 3 play in uracil excision and cross-linking. More importantly, a series of quantitative analyses underscored the structural coupling through inter-motif and intra-motif interactions and subsequent functional coupling of the uracil excision and cross-linking reactions. A catalytic model is proposed, which underlies this catalytic feature unique to UDGX type enzymes. This study offers new insight on the catalytic mechanism of UDGX and provides a unique example of enzyme evolution.

Oxidative guanine base damage plays a dual role in regulating productive ALT-associated homology-directed repair

Cancer cells maintain telomeres by upregulating telomerase or alternative lengthening of telomeres (ALT) via homology-directed repair at telomeric DNA breaks. 8-Oxoguanine (8oxoG) is a highly prevalent endogenous DNA lesion in telomeric sequences, altering telomere structure and telomerase activity, but its impact on ALT is unclear. Here, we demonstrate that targeted 8oxoG formation at telomeres stimulates ALT activity and homologous recombination specifically in ALT cancer cells. Mechanistically, an acute 8oxoG induction increases replication stress, as evidenced by increased telomere fragility and ATR kinase activation at ALT telomeres. Furthermore, ALT cells are more sensitive to chronic telomeric 8oxoG damage than telomerase-positive cancer cells, consistent with increased 8oxoG-induced replication stress. However, telomeric 8oxoG production in G2 phase, when ALT telomere elongation occurs, impairs telomeric DNA synthesis. Our study demonstrates that a common oxidative base lesion has a dual role in regulating ALT depending on when the damage arises in the cell cycle.

Artesunate in glioblastoma therapy: Case reports and review of clinical studies

BackgroundArtesunate, a derivative of the active ingredient artemisinin from Artemisia annua L. used for centuries in the traditional Chinese medicine, is being applied as front-line drug in malaria treatment. As it is cytotoxic for cancer cells, trials are ongoing to include this drug as supplement in cancer therapy. In glioblastoma cells, artesunate was shown to induce oxidative stress, DNA base damage and double-strand breaks (DSBs), apoptosis, and necroptosis. It also inhibits DNA repair functions and bears senolytic activity. Compared to ionizing radiation, DNA damages accumulate over the whole exposure period, which makes the agent unique in its genotoxic profile. Artesunate has been used in adjuvant therapy of various cancers. PurposeAs artesunate has been used in adjuvant therapy of different types of cancer and clinical trials are lacking in brain cancer, we investigated its activity in glioma patients with focus on possible side effects. Study designBetween 2014 and 2020, twelve patients were treated with artesunate for relapsing glioma and analyzed retrospectively: 8 males and 4 females, median age 45 years. Histology: 4 glioblastomas WHO grade 4, 5 astrocytomas WHO grade 3, 3 oligodendrogliomas grade 2 or 3. All patients were pretreated with radiation and temozolomide-based chemotherapy. Artesunate 100 mg was applied twice daily p.o. combined with dose-dense temozolomide alone (100 mg/m2 day 1–5/7, 10 patients) or with temozolomide (50 mg/m2 day 1–5/7) plus lomustine (CCNU, 40 mg day 6/7). Blood count, C-reactive protein (CRP), liver enzymes, and renal parameters were monitored weekly. ResultsApart from one transient grade 3 hematological toxicity, artesunate was well tolerated. No liver toxicity was observed. While 8 patients with late stage of the disease had a median survival of 5 months after initiation of artesunate treatment, 4 patients with treatment for remission maintenance showed a median survival of 46 months. We also review clinical trials that have been performed in other cancers where artesunate was included in the treatment regimen. ConclusionsArtesunate administered at a dose of 2 × 100 mg/day was without harmful side effects, even if combined with alkylating agents used in glioma therapy. Thus, the phytochemical, which is also utilized as food supplement, is an interesting, well tolerated supportive agent useful for long-term maintenance treatment. Being itself cytotoxic on glioblastoma cells and enhancing the cytotoxicity of temozolomide as well as in view of its senolytic activity, artesunate has clearly a potential to enhance the efficacy of malignant brain cancer therapy.

Targeted nuclear irradiation with a proton microbeam induces oxidative DNA base damage and triggers the recruitment of DNA glycosylases OGG1 and NTH1

DNA is the major target of radiation therapy of malignant tumors. Ionizing radiation (IR) induces a variety of DNA lesions, including chemically modified bases and strand breaks. The use of proton beam therapy for cancer treatment is ramping up, as it is expected to reduce normal tissue damage. Thus, it is important to understand the molecular mechanisms of recognition, signaling, and repair of DNA damage induced by protons in the perspective of assessing not only the risk associated with human exposure to IR but also the possibility to improve the efficacy of therapy. Here, we used targeted irradiation of nuclear regions of living cells with controlled number of protons at a high spatio-temporal resolution to detect the induced base lesions and characterize the recruitment kinetics of the specific DNA glycosylases to DNA damage sites. We show that localized irradiation with 4 MeV protons induces, in addition to DNA double strand breaks (DSBs), the oxidized bases 7,8-dihydro-8-oxoguanine (8-oxoG) and thymine glycol (TG) at the site of irradiation. Consistently, the DNA glycosylases OGG1 and NTH1, capable of excising 8-oxoG and TG, respectively, and initiating the base excision repair (BER) pathway, are recruited to the site of damage. To our knowledge, this is the first direct evidence indicating that proton microbeams induce oxidative base damage, and thus implicating BER in the repair of DNA lesions induced by protons.

Protective effect of Opuntia ficus indica cladode extract against gamma radiation-induced DNA strand breaks and base damage

Radiation therapy-related toxicity is becoming increasingly important as cancer treatment outcomes improve. It is common for radiation to damage cancer cells and normal cells by hydrolyzing H2O which leading to free radicals production. Response transcription factors and signal transduction pathways are activated, resulting in DNA damage. Natural antioxidants have attracted a lot of attention. My study aimed to assess the effectiveness of cactus cladode extract (CCE) in protecting DNA plasmids against γ-radiation. The samples were irradiated with 137Cs γ-rays at different doses in the absence or presence of CCE. Agarose gel electrophoresis analysis shows that the aqueous CCE prevents radiation-induced DNA damage. Indeed, the yields of SSB and DSBs are (78 ± 3) x10−4/Gy/plasmid and (0.75 ± 0.09) x 10−4/Gy/plasmid, respectively. However, the greatest fits for the yields for SSB and DSB forming, in the presence of CCE1 (0.5 μg), are (44 ± 2) x10−4/Gy/plasmid and (0.55 ± 0.05) x 10−4/Gy/plasmid, respectively. Despite, the yields for SSB and DSB formation in the presence of CCE2 (1 μg), are (31 ± 2) x10−4/Gy/plasmid and (0.48 ± 0.09) x 10−4/Gy/plasmid, respectively. While, the yields for SSB and DSB formation, in the presence of CCE3 (1.5 μg), are (22 ± 2) x10−4/Gy/plasmid and (0.4 ± 0.02) x 10−4/Gy/plasmid, respectively. Moreover, the CCE efficiently inhibit DNA base damage. A decrease of 70%, 83%, and 87% of the Nth-sensitive sites was found when the sample was irradiated with CCE1, CCE2, and CCE3, respectively. Moreover, we calculated a decrease of 66%, 79%, and 82% of the Fpg-sensitive sites when the sample was irradiated with CCE1, CCE2, and CCE3, respectively. In conclusion, my results indicate that the CCE is a potent radioprotector and hydroxyl radical scavenger; it efficiently inhibits DNA SSBs, DSBs and base damage. It suggests that this extract may be able to protect against genotoxicity, apoptosis, and nephrotoxicity.

Effects of Intra-Base Pair Proton Transfer on Dissociation and Singlet Oxygenation of9-Methyl-8-Oxoguanine-1-Methyl-Cytosine Base-Pair Radical Cations.

8-Oxoguanosine is the most common oxidatively generated base damage and pairs with cytidine within duplex DNA. The 8-oxoguanosine-cytidine lesion, if not recognized and removed, not only leads to G-to-T transversion mutations but renders the base pair being more vulnerable to the ionizing radiation and singlet oxygen (1O2) damage. Herein, reaction dynamics of a prototype Watson-Crick base pair [9MOG·1MC]·+, consisting of 9-methyl-8-oxoguanine radical cation (9MOG·+) and 1-methylcystosine (1MC), was examined using mass spectrometry coupled with electrospray ionization. We first detected base-pair dissociation in collisions with the Xe gas, which provided insight into intra-base pair proton transfer of 9MOG·+·1MC[[EQUATION]][9MOG - HN1]··[1MC + HN3']+ and subsequent non-statistical base-pair separation. We then measured the reaction of [9MOG·1MC]·+ with 1O2, revealing the two most probable pathways, C5-O2 addition and HN7-abstraction at 9MOG. Reactions were entangled with the two forms of 9MOG radicals and base-pair structures as well as multi-configurations between open-shell radicals and 1O2 (that has a mixed singlet/triplet character). These were disentangled by utilizing approximately spin-projected density functional theory, coupled-cluster theory and multi-referential electronic structure modeling. The work delineated base-pair structural context effects and determined relative reactivity toward 1O2 as [9MOG - H]· > 9MOG·+ > [9MOG - HN1]··[1MC + HN3']+ ≥ 9MOG·+·1MC.

DNA mismatch repair protects the genome from oxygen-induced replicative mutagenesis.

DNA mismatch repair (MMR) corrects mismatched DNA bases arising from multiple sources including polymerase errors and base damage. By detecting spontaneous mutagenesis using whole genome sequencing of cultured MMR deficient human cell lines, we show that a primary role of MMR is the repair of oxygen-induced mismatches. We found an approximately twofold higher mutation rate in MSH6 deficient DLD-1 cells or MHL1 deficient HCT116 cells exposed to atmospheric conditions as opposed to mild hypoxia, which correlated with oxidant levels measured using electron paramagnetic resonance spectroscopy. The oxygen-induced mutations were dominated by T to C base substitutions and single T deletions found primarily on the lagging strand. A broad sequence context preference, dependence on replication timing and a lack of transcriptional strand bias further suggested that oxygen-induced mutations arise from polymerase errors rather than oxidative base damage. We defined separate low and high oxygen-specific MMR deficiency mutation signatures common to the two cell lines and showed that the effect of oxygen is observable in MMR deficient cancer genomes, where it best correlates with the contribution of mutation signature SBS21. Our results imply that MMR corrects oxygen-induced genomic mismatches introduced by a replicative process in proliferating cells.

Chemical characterization of Callingcard Vine (Entada polystachya (L.) DC. var. polystachya) aqueous seed extract and evaluation of its cytotoxic, genotoxic and mutagenic properties.

Callingcard Vine (Entada polystachya (L.) DC. var. polystachya - Fabaceae) is a common plant in coastal thickets from western Mexico through Central America to Colombia and Brazil, especially in Amazon biome. It has been popularly used as a urinary burning reliever and diuretic. However, the plant chemical constituents are poorly understood and Entada spp. genotoxic potential have not been previously investigated. In the present study we determined the chemical composition of the aqueous E. polystachya crude seed extract (EPCSE) and evaluated the cytotoxic, genotoxic and mutagenic properties of EPCSE in Salmonella typhimurium and Chinese hamster fibroblast (V79) cells. Cytotoxic activity was also evaluated in tumor cell lines (HT29, MCF7 and U87) and non-malignant cells (MRC5). The chemical analysis by High Resolution Mass Spectrometry (HRMS) of EPCSE indicated the presence of saponin and chalcone. The results of the MTT and clonal survival assays suggest that EPCSE is cytotoxic to V79 cells. Survival analysis showed higher IC50 in non-tumor compared with tumor cell lines. EPCSE showed induction of DNA strand breaks as revealed by the alkaline comet assay and micronucleus test. Using the modified comet assay, it was possible to detect the induction of oxidative DNA base damage by EPCSE in V79 cells. Consistently, the extract induced increase lipid peroxidation (TBARS), superoxide dismutase (SOD) and catalase (CAT) activities in V79 cells. In addition, EPCSE induced mutations in S. typhimurium TA98 and TA100 strains, confirming a mutagenic potential. Taken together, our results suggest that EPCSE is cytotoxic and genotoxic to V79 cells and mutagenic to S. typhimurium. These properties can be related to the pro-oxidant ability of the extract and induction of DNA lesions. Additionally, EPCSE could inhibit the growth of tumor cells, especially human colorectal adenocarcinoma (HT29) cell line, and can constitute a possible source of antitumor natural agents.

Quantifying telomere transcripts as tool to improve risk assessment for genetic instability and genotoxicity.

Telomere repeat-containing RNAs (TERRA) are transcribed from telomeres as long non-coding RNAs and are part of the telomere structure with protective function. The genetic stability of cells requires telomeric repeats at the ends of chromosomes. Maintenance of telomere length (TL) is essential for proliferative capacity and chromosomal integrity. In contrast, telomere shortening is a recognized risk factor for carcinogenesis and a biomarker of aging due to the cumulative effects of environmental exposures and life experiences such as trauma or stress. In this context, telomere repeats are lost due to cell proliferation, but are also susceptible to stress factors including reactive oxygen species (ROS) inducing oxidative base damage. Quantitative PCR (qPCR) of genomic DNA is an established method to analyze TL as a tool to detect genotoxic events. That same qPCR method can be applied to RNA converted into cDNA to quantify TERRA as a useful tool to perform high-throughput screenings. This short review summarizes relevant qPCR studies using both TL and TERRA quantification, provides an overall view of the molecular mechanisms of telomere protection against ROS by TERRA, and summarizes the presented studies comparing the results at DNA and RNA levels, which indicate that fluctuations at transcript level might reflect a short-term response. Therefore, we conclude that performing both of these measurements together will improve genotoxicity studies.

Chlorine oxide radical (ClO[rad]) enables the enhanced degradation of antibiotic resistance genes during UV/chlorine treatment by selectively inducing base damage

Compared to individual UV or chlorine disinfection, the combined UV and chlorine (i.e., UV/chlorine) can substantially promote the degradation of antibiotic resistance genes (ARGs) in the effluent by generating radicals. However, the mechanisms of ARG degradation induced by radicals during UV/chlorine treatment remain largely unknown, limiting further enhancement of ARG degradation by process optimization. Herein, we aimed to uncover the role of different radicals in ARG degradation and the molecular mechanisms of ARG degradation by radicals in UV/chlorine process. The ClO was proven to be responsible for the enhanced ARG degradation during UV/chlorine treatment, while the other radicals (OH, Cl, and Cl2−) played a minor role. This is because ClO possessed both high steady-state concentration and high reactivity toward ARGs (rate constant: 4.29 × 1010 M−1 s−1). The ClO might collaborate with free chlorine to degrade ARG. The ClO degraded ARGs by selectively attacking guanine and thymine but failed to induce strand breakage, while chlorine could break the strand of ARGs. Ultimately, ClO cooperated with chlorine to degrade ARGs quickly by hydroxylation and chlorination of bases and produce many chlorine- and nitrogen-containing products as revealed by high-resolution mass spectrometry. The uncovered degradation mechanisms of ARGs by UV/chlorine provide useful guidelines for process optimization to achieve deep removal of effluent ARGs.

Inactive PARP1 causes embryonic lethality and genome instability in a dominant-negative manner.

PARP1 (poly-ADP ribose polymerase 1) is recruited and activated by DNA strand breaks, catalyzing the generation of poly-ADP-ribose (PAR) chains from NAD+. PAR relaxes chromatin and recruits other DNA repair factors, including XRCC1 and DNA Ligase 3, to maintain genomic stability. Here we show that, in contrast to the normal development of Parp1-null mice, heterozygous expression of catalytically inactive Parp1 (E988A, Parp1+/A) acts in a dominant-negative manner to disrupt murine embryogenesis. As such, all the surviving F1 Parp1+/A mice are chimeras with mixed Parp1+/AN (neoR retention) cells that act similarly to Parp1+/-. Pure F2 Parp1+/A embryos were found at Mendelian ratios at the E3.5 blastocyst stage but died before E9.5. Compared to Parp1-/- cells, genotype and expression-validated pure Parp1+/A cells retain significant ADP-ribosylation and PARylation activities but accumulate markedly higher levels of sister chromatid exchange and mitotic bridges. Despite proficiency for homologous recombination and nonhomologous end-joining measured by reporter assays and supported by normal lymphocyte and germ cell development, Parp1+/A cells are hypersensitive to base damages, radiation, and Topoisomerase I and II inhibition. The sensitivity of Parp1+/A cells to base damages and Topo inhibitors exceed Parp1-/- controls. The findings show that the enzymatically inactive PARP1 dominant negatively blocks DNA repair in selective pathways beyond wild-type PARP1 and establishes a crucial physiological difference between PARP1 inactivation vs. deletion. As a result, the expression of enzymatically inactive PARP1 from one allele is sufficient to abrogate murine embryonic development, providing a mechanism for the on-target side effect of PARP inhibitors used for cancer therapy.

A Study of Skull Base According to Gera Classification in a Korean Population

Background and Objectives The leakage of cerebrospinal fluid due to damage to the ethmoid roof and cribriform plate (CP) is one of the most serious complications of endoscopic sinus surgery (ESS). For the risk of skull base injury during ESS, the Keros classification has been widely used, but recently the Gera classification has been introduced as a tool for risk assessment. This study evaluated the risk of skull base damage using the Gera classification in Koreans.Subjects and Method We analyzed 592 left and right sides of 296 paranasal sinus CTs, and measure the angle between the lateral lamella of CP and the lateral extension of the horizontal plane passing through CP, namely the Gera angle. The angles were classified into three categories: the type I (>80°, low risk), type II (45° to 80°, medium risk), and type III (<45°, high risk).Results The type III, which is vulnerable to skull base damage, accounted for 11.7% of the study subjects. The Gera angle in females (64.0°±12.3°) was greater than that in males (61.3°± 14.0°), showing a significant gender difference (p=0.015). Although there was no difference between the left and the right side of the Gera angle (p=0.155), there was a significant difference between the left and the right side in the Gera classification (p<0.001). Age and the Gera angle showed a negative correlation (r=-0.116, p=0.005).Conclusion The type III of the Gera classification was observed in 11.7% of the total study group. For the type III, extra care is required during ESS.

Mitochondrial OGG1 expression reduces age-associated neuroinflammation by regulating cytosolic mitochondrial DNA.

Aging is accompanied by a decline in DNA repair efficiency, which leads to the accumulation of different types of DNA damage. Age-associated chronic inflammation and generation of reactive oxygen species exacerbate the aging process and age-related chronic disorders. These inflammatory processes establish conditions that favor accumulation of DNA base damage, especially 8-oxo-7,8 di-hydroguanine (8-oxoG), which in turn contributes to various age associated diseases. 8-oxoG is repaired by 8-oxoG glycosylase1 (OGG1) through the base excision repair (BER) pathway. OGG1 is present in both the cell nucleus and in mitochondria. Mitochondrial OGG1 has been implicated in mitochondrial DNA repair and increased mitochondrial function. Using transgenic mouse models and cell lines that have been engineered to have enhanced expression of mitochondria-targeted OGG1 (mtOGG1), we show that elevated levels of mtOGG1 in mitochondria can reverse aging-associated inflammation and improve functions. Old male mtOGG1Tg mice show decreased inflammation response, decreased TNFα levels and multiple pro-inflammatory cytokines. Moreover, we observe that male mtOGG1Tg mice show resistance to STING activation. Interestingly, female mtOGG1Tg mice did not respond to mtOGG1 overexpression. Further, HMC3 cells expressing mtOGG1 display decreased release of mtDNA into the cytoplasm after lipopolysacchride induction and regulate inflammation through the pSTING pathway. Also, increased mtOGG1 expression reduced LPS-induced loss of mitochondrial functions. These results suggest that mtOGG1 regulates age-associated inflammation by controlling release of mtDNA into the cytoplasm.

Effect of 8-Hydroxyguanine DNA Glycosylase 1 on the Function of Immune Cells.

Excess reactive oxygen species (ROS) can cause an imbalance between oxidation and anti-oxidation, leading to the occurrence of oxidative stress in the body. The most common product of ROS-induced base damage is 8-hydroxyguanine (8-oxoG). Failure to promptly remove 8-oxoG often causes mutations during DNA replication. 8-oxoG is cleared from cells by the 8-oxoG DNA glycosylase 1 (OGG1)-mediated oxidative damage base excision repair pathway so as to prevent cells from suffering dysfunction due to oxidative stress. Physiological immune homeostasis and, in particular, immune cell function are vulnerable to oxidative stress. Evidence suggests that inflammation, aging, cancer, and other diseases are related to an imbalance in immune homeostasis caused by oxidative stress. However, the role of the OGG1-mediated oxidative damage repair pathway in the activation and maintenance of immune cell function is unknown. This review summarizes the current understanding of the effect of OGG1 on immune cell function.

Spontaneous allelic variant in deafness-blindness gene Ush1g resulting in an expanded phenotype.

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1gbw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1gbw/bw mice had no substantial retinal degeneration compared with Ush1gbw /+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.

DNA Protection against Damages Induced by Low-Energy Electrons: Absolute Cross Sections for Arginine-DNA Complexes.

Histone proteins protect cellular DNA from radiation damage. We find that arginine, a major component of histone proteins, protects DNA from lesions induced by low-energy secondary electrons generated by radiation. Thin films of 7 ± 2, 12 ± 4, and 17 ± 4 nm thicknesses containing arginine-plasmid-DNA complexes in molar ratio of [Arg2+]/[PO4-] = 16 are irradiated in vacuum with 5 and 10 eV electrons. Damage yields are measured for base damages, cross-links, single-strand breaks (SSBs), double-strand breaks, and other clustered lesions. Most damage results from dissociative electron attachment. Absolute cross sections (ACSs) for all damage types are extracted from yields at different film thicknesses. Compared with bare DNA, these ACSs are reduced by factors of up to 4.4 in Arg-DNA complexes. SSB protection is the highest. Potentially lethal cluster lesions decrease by factors of up to 2.2. ACSs are critical input parameters in modeling radiation-induced damage and assessing protection factors under simulated cellular conditions.

α1-Adrenoceptor agonist methoxamine inhibits base excision repair via inhibition of apurinic/apyrimidinic endonuclease 1 (APE1).

Methoxamine (Mox) is a well-known α1-adrenoceptor agonist, clinically used as a longer-acting analogue of epinephrine. 1R,2S-Mox (NRL001) has been also undergoing clinical testing to increase the canal resting pressure in patients with bowel incontinence. Here we show, that Mox hydrochloride acts as an inhibitor of base excision repair (BER). The effect is mediated by the inhibition of apurinic/apyrimidinic endonuclease APE1. We link this observation to our previous report showing the biologically relevant effect of Mox on BER - prevention of converting oxidative DNA base damage to double-stranded breaks. We demonstrate that its effect is weaker, but still significant when compared to a known BER inhibitor methoxyamine (MX). We further determined Mox's relative IC 50 at 19 mmol L-1, demonstrating a significant effect of Mox on APE1 activity in clinically relevant concentrations.

A new G-quadruplex-specific photosensitizer inducing genome instability in cancer cells by triggering oxidative DNA damage and impeding replication fork progression.

Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.

Development of a Repair Enzyme Fluorescent Probe to Reveal the Intracellular DNA Damage Induced by Benzo[a]pyrene in Living Cells

Pollutant exposure causes a series of DNA damage in cells, resulting in the initiation and progression of diseases and even cancers. An investigation of the DNA damage induced by pollutants in living cells is significant to evaluate the cytotoxicity, genotoxicity, and carcinogenicity of environmental exposure, providing critical insight in the exploration of the etiologies of diseases. In this study, we develop a repair enzyme fluorescent probe to reveal the DNA damage caused by an environmental pollutant in living cells by single-cell fluorescent imaging of the most common base damage repair enzyme named human apurinic/apyrimidinic endonuclease 1 (APE1). The repair enzyme fluorescent probe is fabricated by conjugation of an APE1 high affinity DNA substrate on a ZnO2 nanoparticle surface to form a ZnO2@DNA nanoprobe. The ZnO2 nanoparticle serves as both a probe carrier and a cofactor supplier, releasing Zn2+ to activate APE1 generated by pollutant exposure. The AP-site in the DNA substrate of the fluorescent probe is cleaved by the activated APE1, releasing fluorophore and generating fluorescent signals to indicate the position and degree of APE1-related DNA base damage in living cells. Subsequently, the developed ZnO2@DNA fluorescent probe is applied to investigate the APE1-related DNA base damage induced by benzo[a]pyrene (BaP) in living human hepatocytes. Significant DNA base damage by BaP exposure is revealed, with a positive correlation of the damage degree with exposure time in 2-24 h and the concentration in 5-150 μM, respectively. The experimental results demonstrate that BaP has a significant effect on the AP-site damage, and the degree of DNA base damage is time-dependent and concentration-dependent.

DNA mutilation: A telltale sign of cancer inception.

DNA damage is a discrepancy in its chemical structure precipitated by a multitude of factors. Most DNA damages can be repaired efficiently through diverse restorative mechanisms subjective to the type of damage. DNA-damaging agents elicit a medley of cellular retorts like cell cycle arrest, followed by DNA repair mechanisms or apoptosis. An unrepaired DNA damage in a nonreplicating cell does not generally engender mutations but a similar scenario in replicating cell routes to permanent modification of genetic material shrugging to carcinogenesis. DNA mutilation can be allied to disarray in bases, debasement of backbone, or crosslinks. Base damages or backbone damages like single-strand and double-strand DNA breaks are usually produced by reactive oxygen species and ionizing radiation. This substantial DNA damage has broadly been considered to be caused by various exogenous and endogenous agents with variable rates of causality and decrees of risk, sourcing toward cancer or other diseases, necessitating furtherance in diagnostics at sequential points. The purpose of this article is to review in detail the various types of DNA damages, their contributory factors, and recent developments in their identification.