Damage In Human Cells Research Articles

Mitotic chromatin marking governs asymmetric segregation of DNA damage.

The faithful segregation of intact genetic material and the perpetuation of chromatin states through mitotic cell divisions are pivotal for maintaining cell function and identity across cell generations. However, most exogenous mutagens generate long-lasting DNA lesions that are segregated during mitosis. How this segregation is controlled is unknown. Here, we uncover a mitotic chromatin-marking pathway that governs the segregation of UV-induced damage in human cells. Our mechanistic analyses reveal two layers of control: histone ADP-ribosylation, and the incorporation of newly synthesized histones at UV damage sites, that both prevent local mitotic phosphorylations on histone H3 serine residues. Functionally, this chromatin-marking pathway drives the asymmetric segregation of UV damage in the cell progeny with consequences on daughter cell fate. We propose that this mechanism may help preserve the integrity of stem cell compartments during asymmetric cell divisions.

Quercetin and kaempferol from saffron petals alleviated hydrogen peroxide-induced oxidative damage in B16 cells.

Saffron petals are usually considered as waste after saffron harvest. However, saffron petals contain many important phytochemical components (e.g. quercetin and kaempferol), which may alleviate oxidative damage in human cells. The contents of flavonoids and crocin in different parts of saffron were analyzed. The protective effects of flavonoids from saffron on oxidative damage of B16 cells were investigated. Saffron stigma contained high contents of crocin and picrocrocin, whereas flavonoid content (quercetin, 4.03 ± 0.33 mg g-1 DW; kaempferol, 47.80 ± 0.60 mg g-1 DW) was higher in saffron petals than in other parts. Incubation of B16 cells with quercetin (10-30 μmol L-1) and kaempferol (20-30 μmol L-1) obtained from saffron extracts could significantly increase the total antioxidant capacity (T-AOC) and the activity of NADPH:dehydrogenase quinone-1 (NQO1) to alleviate H2O2-induced oxidative damage. Quercetin was better than kaempferol in increasing NQO1 activity and T-AOC. Quercetin extracted from saffron petals could induce NQO1 expression through regulating kelch-like ECH-associated protein-1/nuclear factor erythroid 2-related factor-2 signaling pathway to protect B16 cells from oxidative damage. The content of kaempferol-3-O-sophoroside and quercetin-3-O-sophoroside was higher in saffron petals than in other parts of saffron. The kaempferol and quercetin obtained from saffron petals could enhance the activity of antioxidant enzyme NQO1 and T-AOC in B16 cells. This indicated that saffron petals, as a potential functional food, may prevent diseases caused by oxidative stress. © 2024 Society of Chemical Industry.

Labilization of the DNA structure in peripheral blood lymphocytes of COVID-19 patients

Introduction. Available data indicate the SARS-CoV-2 coronavirus to be potent of impairing DNA repair processes and cause oxidative stress, which can lead to the accumulation of DNA damage in human cells. However, the DNA-damaging effect of the virus has not yet been sufficiently studied. The purpose of the research was to study the ability of SARS-CoV-2 to cause DNA damage in human peripheral blood lymphocytes. Materials and methods. One hundred forty COVID-19 patients and 24 donors of the control group are included in the study. The level of DNA fragmentation in lymphocytes was determined by alkaline DNA-comet assay. Statistical differences between the mean medians of the «%DNA in the comet tail» (tail DNA%) were assessed using Student’s t-test. The Jeffers test was used to compare the proportions of cells with different levels of DNA-damage. Statistical differences between groups were assessed using the Mann-Whitney test. Results. In the COVID-19 patients, an increase in the level of breaks and alkali-labile sites in DNA was revealed when compared to controls (p = 0.025). In the group of patients infected with SARS-CoV-2, the proportion of comets with DNA damage of up to 5% decreased (p = 0.009), while the proportion of comets containing more than 10% DNA tail increased (p = 0.000). The number of atypical comets compared to the control increased by 3.7 and 5.9 times with mild and moderate severity of the disease, respectively (r = 0.993; p = 0.001). In the association with diseases – coronary heart disease (CHD) and diabetes mellitus type II (DM type 2), the level of DNA fragmentation in lymphocytes statistically significantly increased compared to the group of patients without these diseases. Limitations. A limitation is the lack of data on DNA-structure damage in severe COVID-19 disease. Conclusion. SARS-CoV-2 infection leads to labilization of the DNA structure in human peripheral blood lymphocytes. The level of DNA damage depends on the severity of COVID-19 and the presence of comorbid diseases: CHD and DM type 2. The results of the study are important for understanding the mechanisms of action of the virus on human immunocompetent cells.

Genomic Features of Homologous Recombination Deficiency in Breast Cancer: Impact on Testing and Immunotherapy.

Genomic instability is one of the well-established hallmarks of cancer. The homologous recombination repair (HRR) pathway plays a critical role in correcting the double-stranded breaks (DSB) due to DNA damage in human cells. Traditionally, the BRCA1/2 genes in the HRR pathway have been tested for their association with breast cancer. However, defects in the HRR pathway (HRD, also termed 'BRCAness'), which has up to 50 genes, have been shown to be involved in tumorigenesis and treatment susceptibility to poly-ADP ribose polymerase inhibitors (PARPis), platinum-based chemotherapy, and immune checkpoint inhibitors (ICIs). A reliable consensus on HRD scores is yet to be established. Emerging evidence suggests that only a subset of breast cancer patients benefit from ICI-based immunotherapy. Currently, albeit with limitations, the expression of programmed death-ligand 1 (PDL1) and tumor mutational burden (TMB) are utilized as biomarkers to predict the favorable outcomes of ICI therapy in breast cancer patients. Preclinical studies demonstrate an interplay between the HRR pathway and PDL1 expression. In this review, we outline the current understanding of the role of HRD in genomic instability leading to breast tumorigenesis and delineate outcomes from various clinical trials. Furthermore, we discuss potential strategies for combining HRD-targeted therapy with immunotherapy to achieve the best healthcare outcomes in breast cancer patients.

Phaeodactylum tricornutum as a stable platform for pilot scale production and investigation of the viability of spirulina fucoxanthin as a commercial lipolysis active novel compound

The nutraceutical market has experienced a significant growth over the last years, due to changing consumer preferences towards products of natural and organic origin. One of the main areas of interest in this field is microalgae-derived carotenoids, whose antioxidant activity protects against oxidative damage in human cells. The diatom Phaeodactylum tricornutum has been widely studied due to its high fucoxanthin content (antioxidant carotenoid). However, pilot and industrial scale experiments to realize continuous and stable production of P. tricornutum and extracted fucoxanthin are limited. For this reason, this research focuses on the study of the fucoxanthin production, going through culture, harvesting, scaling procedure and encapsulation stage. Finally, the optimal culture conditions for a fucoxanthin production of 2.5 mg/day/L were verified. Furthermore, the antioxidant capacity showed a decrease during storage, but still presented a capacity of 18 months, with improved performance when encapsulated with spirulina. The study carried out on encapsulated fucoxanthin-spirulina shows an inhibitory effect on lipogenesis at all concentrations tested in the 3T3-L1 cell line and a lipolytic efficacy at concentrations of 0.2 mg/mL and 0.1 mg/mL in the 3T3-L1 cell line.

Light Quality Potentiates the Antioxidant Properties of Brassica rapa Microgreen Extracts against Oxidative Stress and DNA Damage in Human Cells.

Plants are an inexhaustible source of bioactive compounds beneficial for contrasting oxidative stress, leading to many degenerative pathologies. Brassica rapa L. subsp. rapa is well known for its nutraceutical properties among edible vegetable species. In our work, we aimed to explore an eco-friendly way to enhance the beneficial dietary phytochemicals in this vast world of crop-growing plants at selected light quality conditions. White broad-spectrum (W) and red-blue (RB) light regimes were used for growing brassica microgreens. The organic extracts were tested on keratinocytes upon oxidative stress to explore their capability to act as natural antioxidant cell protectors. Our results show that both W and RB extracts caused a notable reduction in reactive oxygen species (ROS) levels induced by H2O2. Interestingly, according to its higher contents of polyphenols and flavonoids, the RB was more efficient in reducing ROS amount and DNA damage than the W extract, particularly at the lowest concentration tested. However, at higher concentrations (up to 100 μg/mL), the antioxidant effect reached a plateau, and there was little added benefit. These findings confirm that RB light effectively increases the antioxidant compounds in Brassica rapa L. microgreens, thus contributing to their enhanced activity against oxidative-induced genotoxicity compared to microgreens grown under W light.

Application of the Cytokinesis-Block Micronucleus Assay for High-Dose Exposures Using Imaging Flow Cytometry

The cytokinesis-block micronucleus assay is a well-established method to assess radiation-induced genetic damage in human cells. This assay has been adapted to imaging flow cytometry (IFC), allowing automated analysis of many cells, and eliminating the need to create microscope slides. Furthermore, to improve the efficiency of assay performance, a small-volume method previously developed was employed. Irradiated human blood samples were cultured, stained, and analyzed by IFC to produce images of the cells. Samples were run using both manual and 96-well plate automated acquisition. Multiple parameter-based image features were collected for each sample, and the results were compared to confirm that these acquisition methods are functionally identical. This paper details the multi-parametric analysis developed and the resulting calibration curves up to 10 Gy. The calibration curves were created using a quadratic random coefficient model with Poisson errors, as well as a logistic discriminant function. The curves were then validated with blinded, irradiated samples, using relative bias and relative mean square error. Overall, the accuracy of the dose estimates was adequate for triage dosimetry (within 1 Gy of the true dose) over 90% of the time for lower doses and about half the time for higher doses, with the lowest success rate between 5 and 6 Gy where the calibration curve reached its peak and there was the smallest change in MN/BNC with dose. This work describes the application of a novel multi-parametric analysis that fits the calibration curves and allows dose estimates up to 10 Gy, which were previously limited to 4 Gy. Furthermore, it demonstrates that the results from samples acquired manually and with the autosampler are functionally similar.

Genome-scale mapping of DNA damage suppressors through phenotypic CRISPR-Cas9 screens

To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.

Dynamics and Molecular Interactions of GPI-Anchored CD59.

CD59 is a GPI-anchored cell surface receptor that serves as a gatekeeper to controlling pore formation. It is the only membrane-bound inhibitor of the complement membrane attack complex (MAC), an immune pore that can damage human cells. While CD59 blocks MAC pores, the receptor is co-opted by bacterial pore-forming proteins to target human cells. Recent structures of CD59 in complexes with binding partners showed dramatic differences in the orientation of its ectodomain relative to the membrane. Here, we show how GPI-anchored CD59 can satisfy this diversity in binding modes. We present a PyLipID analysis of coarse-grain molecular dynamics simulations of a CD59-inhibited MAC to reveal residues of complement proteins (C6:Y285, C6:R407 C6:K412, C7:F224, C8β:F202, C8β:K326) that likely interact with lipids. Using modules of the MDAnalysis package to investigate atomistic simulations of GPI-anchored CD59, we discover properties of CD59 that encode the flexibility necessary to bind both complement proteins and bacterial virulence factors.

Chemoprofiling and In vitro antioxidant potentials of methanol water extracts from Nigerian ginger (Zingiber officinale)

Ginger (Zingiber officinale) is one of the popular spices in Nigeria. It is highly utilised over the world as spice as well as for the treatment of inflammation, arthritis and other conditions involving reactive oxygen species (ROS), which are known to damage human cells. The proximate, phytochemical composition and antioxidant properties of the plant are reported in this work. Extraction by maceration was used for the pulverized rhizomes with methanol-water (50:50). Further partitioning of the extract was carried out using n-hexane, ethyl acetate and butanol into different fractions according to their polarity. The antioxidant activity was evaluated with 1,1-diphenyl-2-icrylhydrazyl (DPPH) radical scavenging assay, ferric reducing antioxidant power (FRAP) assay, nitric oxide radical scavenging activity (NOSA) and lipid peroxidation scavenging activity (LPSA). The proximate content was determined using conventional chemical methods. The FTIR analyses of n-hexane fraction of Z. officinale revealed some functional groups that may be present in two likely classes of secondary metabolites: terpenes and alkaloids. The ethyl acetate fraction produced flavonoid (121.31 mg/100 g; quercetin) and alkaloids because of the functional groups associated with them, while the butanol fraction with functional groups present revealed cardiac glycosides (4.88 mg/100 g; digoxin) and alkaloids (45.19 mg/100 g). The plant also contained reducing sugar (64.62 mg/100 g glucose), steroids (8.65 mg/100 g; cholesterol), tannins (56.82 mg/100 g; tannic acid) and phenolic compounds (110.64 mg/100 g; gallic acid) from the phytochemical analyses. The proximate analysis revealed that it has a high carbohydrate content (54.54%), but a relatively modest crude fibre (10.21%), moisture content (10.02%), total ash (9.30%), crude fat (8.80%) and crude protein (7.12%). The presence of these phyto-compounds could explain its customary use as oxidative stress inhibitor.

NEIL3-mediated proteasomal degradation facilitates the repair of cisplatin-induced DNA damage in human cells

Anti-neoplastic effect of DNA cross-linking agents such as cisplatin, mitomycin C, and psoralen is attributed to their ability to induce DNA interstrand cross-links (ICLs), which block replication, transcription, and linear repair pathways by preventing DNA strand separation and trigger apoptosis. It is generally agreed that the Fanconi anemia (FA) pathway orchestrates the removal of ICLs by the combined actions of various DNA repair pathways. Recently, attention has been focused on the ability of the NEIL3-initiated base excision repair pathway to resolve psoralen- and abasic site-induced ICLs in an FA-independent manner. Intriguingly, overexpression of NEIL3 is associated with chemo-resistance and poor prognosis in many solid tumors. Here, using loss- and gain-of-function approaches, we demonstrate that NEIL3 confers resistance to cisplatin and participates in the removal of cisplatin–DNA adducts. Proteomic studies reveal that the NEIL3 protein interacts with the 26S proteasome in a cisplatin-dependent manner. NEIL3 mediates proteasomal degradation of WRNIP1, a protein involved in the early step of ICL repair. We propose that NEIL3 participates in the repair of ICL-stalled replication fork by recruitment of the proteasome to ensure a timely transition from lesion recognition to repair via the degradation of early-step vanguard proteins.

Determination of (+)-Catechin and Antioxidant Activity in Faloak (Sterculia quadrifida R. Br) Stem Bark Infusion

Oxidative stress is a condition that can damage human cells and tissues and has been linked to a number of illnesses, including cancer, cardiovascular disease, autoimmune disorders, and neurological diseases. Oxidative stress conditions can be brought on by pollution, radiation exposure, and an unhealthy lifestyle. Antioxidants are substances that can be used to both prevent and treat oxidative stress. This study aimed to identify and quantify (+)-catechin levels and antioxidant activity of the stem bark of Sterculia quadrifida R. Br extracted by the infusion method, a method similar to traditional medicine processing generally in the community. Determination of (+)-catechin and antioxidant activity of S. quadrifida were evaluated by HPLC and DPPH assay, respectively. Quantification of (+) catechin content by HPLC system with wavelength 280 nm and antioxidant activity by spectrophotometry method with wavelength 517 nm. The results show that the mean value of (+)-catechin level was 7.786% and the IC50 value of the antioxidant activity was 51.5 ug/mL having a moderate antioxidant activity category. S. quadrifida stem bark infusion can be utilized as a medication candidate for the prevention or treatment of a variety of disorders caused by oxidative stress.

Optimization of ultrasonic-assisted polysaccharide extraction from Hyperici Perforati Herba using response surface methodology and assessment of its antioxidant activity

This study performed a comprehensive investigation of Hyperici Perforati Herba polysaccharide (HPHP) regarding the development and optimization of extraction methods, elucidation of structure and characteristics, and determination of antioxidant activities. An ultrasonic-assisted extraction method, which offered advantages in terms of the extraction yield and energy efficiency, was developed by response surface analysis. The following optimum conditions were determined: a crushing degree at 65 mesh, ultrasonic time at 50 min and temperature of 43 °C. Through enzyme-mediated deproteination via the Sevag method, activated carbon depigmentation, and DEAE-52 and Sephadex G-100 column elution, three HPHPs were obtained, and their monosaccharides mainly included mannose, galactose, glucose and arabinose. The molar weights were 8.347, 1.199 and 22.426 kDa, respectively. The HPHP structures were an amorphous aggregate of spherical-like shapes with a rough surface of pores and crevices, which presented characteristic Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectra of polysaccharides. Their main glucosidic linkage is the α-type configuration. Moreover, HPHPs exhibited strong scavenging activity for DPPH·, ABTS·+, OH· and O2·− radicals; good ferric reducing power; and effective protection against oxidative damage in human cells. Overall, the results of this work underpinned a fundamental understanding of HPHPs, thus providing a potential antioxidant for further research and development.

Teixobactin kills bacteria by a two-pronged attack on the cell envelope

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1–3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a β-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

EFFECTS OF RADIATION FROM MOBILE PHONES ON FERTILITY AND THE QUALITY OF SEMEN IN REGULAR MOBILE PHONE USERS

Objective: The use of mobile phones over extended periods of time is associated with genotoxicity. The Specific Absorption Rate (SAR) values of mobile phones indicate that they release less radiofrequency radiation, which is discovered to be within a safer limit. This is the case because SAR values can be measured. The use of mobile phones for extended periods of time may also cause DNA damage in human cells. Methods: The purpose of this study is to analyse the impact that radiation from mobile phones have on fertility by analysing the quality of the sperm of people who use their phones often. Results: The research was carried out on a total of 150 people who were between the ages of 20 and 40 and divided into three groups (frequent mobile users, moderate mobile users and less mobile phone users). Prior to collecting samples from the subjects, permission was obtained from them. The researcher conducted individual interviews with each participant in the study in order to collect information for filling out a structured questionnaire. Analyses and comparisons were made between the three groups on the properties of the sperm, including their motility and shape. Conclusion: The findings of a number of research indicate that radiations released by mobile phones have an effect on male fertility by causing permanent alterations in the morphology of the semen. The current research demonstrates that there is a problem with the fertilising ability of sperm due to defects and changes in sperm morphology. According to the findings of this research, radiations released by mobile phones will have an effect on male fertility. The genotoxic impact may be brought to the attention of regular users of mobile phones and the required measures can be taken via the use of biosensors, which offer a warning signal or alarm when the radiations level exceeds the usual limit.

Interplay between H3K36me3, methyltransferase SETD2, and mismatch recognition protein MutSα facilitates processing of oxidative DNA damage in human cells

Oxidative DNA damage contributes to aging and the pathogenesis of numerous human diseases including cancer. 8-hydroxyguanine (8-oxoG) is the major product of oxidative DNA lesions. Although OGG1-mediated base excision repair is the primary mechanism for 8-oxoG removal, DNA mismatch repair has also been implicated in processing oxidative DNA damage. However, the mechanism of the latter is not fully understood. Here, we treated human cells defective in various 8-oxoG repair factors with H2O2 and performed biochemical, live cell imaging, and chromatin immunoprecipitation sequencing analyses to determine their response to the treatment. We show that the mismatch repair processing of oxidative DNA damage involves cohesive interactions between mismatch recognition protein MutSα, histone mark H3K36me3, and H3K36 trimethyltransferase SETD2, which activates the ATM DNA damage signaling pathway. We found that cells depleted of MutSα or SETD2 accumulate 8-oxoG adducts and fail to trigger H2O2-induced ATM activation. Furthermore, we show that SETD2 physically interacts with both MutSα and ATM, which suggests a role for SETD2 in transducing DNA damage signals from lesion-bound MutSα to ATM. Consistently, MutSα and SETD2 are highly coenriched at oxidative damage sites. The data presented here support a model wherein MutSα, SETD2, ATM, and H3K36me3 constitute a positive feedback loop to help cells cope with oxidative DNA damage.

Evaluation of Spontaneous DNA Damage Using the Alkaline Comet Assay in Lymphocyte Cells of Humans Living in the High Level Natural Radiation Area of Mamuju, Indonesia

To evaluate the biological impacts of high background radiation exposures that are represented by spontaneous deoxyribonucleic acid (DNA) damage, an evaluation on lymphocyte cells from residents of Mamuju, West Sulawesi, Indonesia was tested. The mean annual dose received by individuals in this area is about 10.40 mSv. Of the 177 adult subjects studied, 102 were from high-level natural radiation areas of Mamuju and 75 subjects were from a nearby normal-level natural radiation area. Both areas are similar in living situations. DNA strand breaks and other parameters of study and control group were determined using a standardized comet assay. Our results showed that chronic low-level natural radiation had resulted in significantly higher (p<0.001) DNA damage based on the three parameters of the assay (tail length, tail DNA, and tail moment) compared to those of control. There was a positive correlation between the level of DNA damage and age, where people aged 40 years and older had a higher level of DNA damage than those under 40 year. The level of DNA damage was also found to be higher in females compared to that of males. It was concluded that chronic exposure to natural radiation in Mamuju had induced spontaneous DNA damage in human cells after long-term exposure which was dependent on age and sex.

Crosstalk between CRISPR-Cas9 and the human transcriptome

CRISPR-Cas9 expression independent of its cognate synthetic guide RNA (gRNA) causes widespread genomic DNA damage in human cells. To investigate whether Cas9 can interact with endogenous human RNA transcripts independent of its guide, we perform eCLIP (enhanced CLIP) of Cas9 in human cells and find that Cas9 reproducibly interacts with hundreds of endogenous human RNA transcripts. This association can be partially explained by a model built on gRNA secondary structure and sequence. Critically, transcriptome-wide Cas9 binding sites do not appear to correlate with published genome-wide Cas9 DNA binding or cut-site loci under gRNA co-expression. However, even under gRNA co-expression low-affinity Cas9-human RNA interactions (which we term CRISPR crosstalk) do correlate with published elevated transcriptome-wide RNA editing. Our findings do not support the hypothesis that human RNAs can broadly guide Cas9 to bind and cleave human genomic DNA, but they illustrate a cellular and RNA impact likely inherent to CRISPR-Cas systems.

The Candida albicans virulence factor candidalysin must self-assemble in solution to form membrane pores

Pathogenic Candida albicans is responsible for systemic infections resulting in high morbidity and mortality to vulnerable populations. This is in large part due to the diverse virulence factors C. albicans employs to invade and damage human cells. One of these, candidalysin (CL), was recently found to be required for infection. It was proposed that CL promotes infection by cell membrane disruption. However, the specific mechanism by which this occurs is unknown. Atomic force microscopy (AFM) imaging of supported lipid bilayers treated with CL revealed static and dynamic pore populations, suggesting that membrane disruption occurs through pore formation.

Aberrant RNA methylation triggers recruitment of an alkylation repair complex

Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase invitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.