Detection Of DNA Double-strand Breaks Research Articles

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3′-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

P-406 Late paternal effect rooted in sperm DNA double-strand breaks (DSBs) correlates with the likelihood of miscarriage in patients with poor ovarian response

Abstract Study question Can paternal risk factors be minimized and prevented in ICSI procedure? Summary answer The influence of DSBs can be prevented in ICSI cases with normal ovarian response, whereas DSB is exclusively associated with miscarriage in poor ovarian reserve. What is known already Sperm DNA fragmentation (SDF) is intricately linked to male fertility capacity, with a high prevalence affecting 20% of men with unexplained infertility and up to 50% of those with idiopathic infertility. Despite these statistics, the association between paternal DNA damages and clinical outcomes is controversial. Variations in SDF effects may be influenced by female factors associated with maternal DNA repair activity. To elucidate this interrelationship, we assessed SDF effects on diverse ovarian responses using POSEIDON stratification. The goal was to identify potential paternal risk factors for ART as a preventative solution and devising preventative strategies. Study design, size, duration This retrospective study included 323 couples who underwent ICSI cycles at Lee Women’s Hospital. Normal responders (n = 89) were identified based on female age (<35 years) and AMH levels (≥1.2 ng/mL). Patients (n = 234) fulfilling POSEIDON group 1–4 criteria were categorized as poor ovarian response (POR). Clinical outcomes assessed included clinical pregnancy, miscarriage, and live birth rates. Participants/materials, setting, methods Paternal risk factors include male age, semen parameters (CEROS II CASA, Hamilton-Thorne), and two types of SDF evaluation. LensHooke® R10 kit (Bonraybio, Taiwan) was used to detect total SDF (tSDF), which includes single-strand breaks and double-strand breaks (DSBs). LensHooke® R11 kit was specifically used for DSB detection. Kruskal-Wallis test was conducted to compare controls and cases of ART failure. Univariate and multivariate logistic regression analyses were employed to identify potential factors. Main results and the role of chance We sought to evaluate paternal effects during the early gestation stage by comparing parental characteristics in groups with clinical pregnancy and those without pregnancy. No significant differences in paternal indications were observed between the groups. Notably, significantly lower AMH levels and advanced maternal age were prevalent in pregnancy failures across both normal responders and POR cases, highlighting maternal factors’ dominance in these outcomes. When dissecting paternal effects during the later gestation stage, male factors did not show a significant difference between the group with live birth and the group with miscarriage among normal responders. Nevertheless, several paternal pathogenic indications were identified between the groups in POR. Miscarriages were associated with compromised sperm total motility (68% vs. 54.5%, p = 0.01), reduced normal morphology rate (5.5% vs. 2.5%, p = 0.03), and elevated DSB levels (7.5% vs. 18.8%, p = 0.01). Furthermore, multivariate regression analysis identified high DSBs as an exclusive paternal factor in miscarriage risk (Odds ratio: 1.19, 95% confidence interval 1.04–1.36, p = 0.01). In the ROC curve, a DSBs threshold of 19%, based on the R11 technique, offers significant value in predicting miscarriage with an accuracy of 81%. Limitations, reasons for caution The limitations of the current study are single-center trial, and relatively small sample size. Further clinical trials are needed to robustly establish the relationship between DSB and miscarriage. Wider implications of the findings The impact of DSBs is overlooked in normal responders but not in POR due to different levels of maternal DNA repair activity. These results advocates for (1) the necessity of DSBs (R11) assessment for POR, and (2) egg donation as a potential strategy for couples facing high DSBs and POR. Trial registration number CS2-20012

Identification of ATM-dependent long non-coding RNAs induced in response to DNA damage

DNA damage response (DDR) is a complex process, essential for cell survival. Especially deleterious type of DNA damage are DNA double-strand breaks (DSB), which can lead to genomic instability and malignant transformation if not repaired correctly. The central player in DSB detection and repair is the ATM kinase which orchestrates the action of several downstream factors. Recent studies have suggested that long non-coding RNAs (lncRNAs) are involved in DDR. Here, we aimed to identify lncRNAs induced upon DNA damage in an ATM-dependent manner. DNA damage was induced by ionizing radiation (IR) in immortalized lymphoblastoid cell lines derived from 4 patients with ataxia-telangiectasia (AT) and 4 healthy donors. RNA-seq revealed 10 lncRNAs significantly induced 1 h after IR in healthy donors, whereas none in AT patients. 149 lncRNAs were induced 8 h after IR in the control group, while only three in AT patients. Among IR-induced mRNAs, we found several genes with well-known functions in DDR. Gene Set Enrichment Analysis and Gene Ontology revealed delayed induction of key DDR pathways in AT patients compared to controls. The induction and dynamics of selected 9 lncRNAs were confirmed by RT-qPCR. Moreover, using a specific ATM inhibitor we proved that the induction of those lncRNAs is dependent on ATM. Some of the detected lncRNA genes are localized next to protein-coding genes involved in DDR. We observed that induction of lncRNAs after IR preceded changes in expression of adjacent genes. This indicates that IR-induced lncRNAs may regulate the transcription of nearby genes. Subcellular fractionation into chromatin, nuclear, and cytoplasmic fractions revealed that the majority of studied lncRNAs are localized in chromatin. In summary, our study revealed several lncRNAs induced by IR in an ATM-dependent manner. Their genomic co-localization and co-expression with genes involved in DDR suggest that those lncRNAs may be important players in cellular response to DNA damage.

DNA as the main target in radiotherapy-ahistorical overview from first isolation to anti-tumour immune response.

DNA damage is one of the foremost mechanisms of irradiation at the biological level. After the first isolation of DNA by Friedrich Miescher in the 19th century, the structure of DNA was described by Watson and Crick. Several Nobel Prizes have been awarded for DNA-related discoveries. This review aims to describe the historical perspective of DNA in radiation biology. Over the decades, DNA damage has been identified and quantified after irradiation. Depending on the type of sensing, different proteins are involved in sensing DNA damage and repairing the damage, if possible. For double-strand breaks, the main repair mechanisms are non-homologous end joining and homologous recombination. Additional mechanisms are the Fanconi anaemia pathway and base excision repair. Different methods have been developed for the detection of DNA double-strand breaks. Several drugs have been developed that interfere with different DNA repair mechanisms, e.g., PARP inhibitors. These drugs have been established in the standard treatment of different tumour entities and are being applied in several clinical trials in combination with radiotherapy. Over the past decades, it has become apparent that DNA damage mechanisms are also directly linked to the immune response in tumours. For example, cytosolic DNA fragments activate the innate immune system via the cGAS STING pathway.

The potential use of DNA double-strand breaks detection in various fields of medicine

The article discusses the relevance of determining DNA double-strand breaks (DSBs) using the analysis of γ-H2AX foci as a marker of DNA instability in various conditions. The issues of the formation of DSBs and the peculiarities of their detection in various tissues are highlighted. Changes in the intensity of DSBs formation during the use of radiological diagnostic methods, stress, increased physical exertion, some oncological and rheumatic diseases, as well as the dynamics of DNA repair on the background of various methods of therapy were analyzed.

QUANTIFICATION OF DNA DOUBLE-STRAND BREAKS IN BENIGN AND MALIGNANT BREAST DISEASES

Relevance: Double-strand DNA breaks are the most dangerous DNA damage. Analysis of foci of phosphorylated histone protein H2AX (γH2AX) is currently the most sensitive method for detecting DNA double-strand breaks. This protein modification can become a biomarker of cellular stress, especially in diagnosing and monitoring neoplastic diseases. In this study, we utilized novel pattern recognition algorithms on the AKLIDES® platform to automatically analyze immunofluorescent images of γH2AX foci and compare the results with visual scores. The γH2AX foci formation on peripheral blood mononuclear cells of women with breast cancer or benign breast tumors was studied.
 The article aimed to quantify DNA double-strand breaks in peripheral blood lymphocytes in women with breast cancer and benign breast masses to identify a possible biomarker.
 Methods: γ-H2AX foci in lymphocytes were analyzed using the automated AKLIDES system in patients with breast cancer (n=29) and benign breast tumors (n=24).
 Results: When comparing the indicators of the main and control groups in the channel of ruptures “FITC,” a statistically significant difference was found in the indicators “Foci dia” (p=0.0382), “Focilnt mean” (p=0.0166), “Colocalisation” (p=0.0486). In the repair channel “AРС,” significant differences were found in the indicators “Nuclei BGInt” (p=0.0166) and the indicator “Focilnt mean” (p=0.0118).
 Conclusion: The revealed changes of DNA double-strand breaks along the FITC break channels and APC repair between the main and control groups can possibly serve as a breast cancer diagnostic marker.

QUANTIFICATION OF DNA DOUBLE-STRAND BREAKS IN BENIGN AND MALIGNANT BREAST DISEASES

Relevance: Double-strand DNA breaks are the most dangerous DNA damage. Analysis of foci of phosphorylated histone protein H2AX (γH2AX) is currently the most sensitive method for detecting DNA double-strand breaks. This protein modification can become a biomarker of cellular stress, especially in diagnosing and monitoring neoplastic diseases. In this study, we utilized novel pattern recognition algorithms on the AKLIDES® platform to automatically analyze immunofluorescent images of γH2AX foci and compare the results with visual scores. The γH2AX foci formation on peripheral blood mononuclear cells of women with breast cancer or benign breast tumors was studied.
 The article aimed to quantify DNA double-strand breaks in peripheral blood lymphocytes in women with breast cancer and benign breast masses to identify a possible biomarker.
 Methods: γ-H2AX foci in lymphocytes were analyzed using the automated AKLIDES system in patients with breast cancer (n=29) and benign breast tumors (n=24).
 Results: When comparing the indicators of the main and control groups in the channel of ruptures “FITC,” a statistically significant difference was found in the indicators “Foci dia” (p=0.0382), “Focilnt mean” (p=0.0166), “Colocalisation” (p=0.0486). In the repair channel “AРС,” significant differences were found in the indicators “Nuclei BGInt” (p=0.0166) and the indicator “Focilnt mean” (p=0.0118).
 Conclusion: The revealed changes of DNA double-strand breaks along the FITC break channels and APC repair between the main and control groups can possibly serve as a breast cancer diagnostic marker.

Ultrasound-induced cavitation renders prostate cancer cells susceptible to hyperthermia: Analysis of potential cellular and molecular mechanisms.

Background: Focused ultrasound (FUS) has become an important non-invasive therapy for prostate tumor ablation via thermal effects in the clinic. The cavitation effect induced by FUS is applied for histotripsy, support drug delivery, and the induction of blood vessel destruction for cancer therapy. Numerous studies report that cavitation-induced sonoporation could provoke multiple anti-proliferative effects on cancer cells. Therefore, cavitation alone or in combination with thermal treatment is of great interest but research in this field is inadequate. Methods: Human prostate cancer cells (LNCap and PC-3) were exposed to 40s cavitation using a FUS system, followed by water bath hyperthermia (HT). The clonogenic assay, WST-1 assay, and Transwell® invasion assay, respectively, were used to assess cancer cell clonogenic survival, metabolic activity, and invasion potential. Fluorescence microscopy using propidium iodide (PI) as a probe of cell membrane integrity was used to identify sonoporation. The H2A.X assay and Nicoletti test were conducted in the mechanism investigation to detect DNA double-strand breaks (DSBs) and cell cycle arrest. Immunofluorescence microscopy and flow cytometry were performed to determine the distribution and expression of 5α-reductase (SRD5A). Results: Short FUS shots with cavitation (FUS-Cav) in combination with HT resulted in, respectively, a 2.2, 2.3, and 2.8-fold decrease (LNCap) and a 2.0, 1.5, and 1.6-fold decrease (PC-3) in the clonogenic survival, cell invasiveness and metabolic activity of prostate cancer cells when compared to HT alone. FUS-Cav immediately induced sonoporation in 61.7% of LNCap cells, and the combination treatment led to a 1.4 (LNCap) and 1.6-fold (PC-3) increase in the number of DSBs compared to HT alone. Meanwhile, the combination therapy resulted in 26.68% of LNCap and 31.70% of PC-3 with cell cycle arrest in the Sub-G1 phase and 35.37% of PC-3 with cell cycle arrest in the G2/M phase. Additionally, the treatment of FUS-Cav combined with HT block the androgen receptor (AR) signal pathway by reducing the relative Type I 5α-reductase (SRD5A1) level to 38.28 ± 3.76% in LNCap cells, and decreasing the relative Type III 5α-reductase 3 (SRD5A3) level to 22.87 ± 4.88% in PC-3 cells, in contrast, the relative SRD5A level in untreated groups was set to 100%. Conclusion: FUS-induced cavitation increases the effects of HT by interrupting cancer cell membranes, inducing the DSBs and cell cycle arrest, and blocking the AR signal pathway of the prostate cancer cells, with the potential to be a promising adjuvant therapy in prostate cancer treatment.

In Vitro High-Throughput Genotoxicity Testing Using γH2AX Biomarker, Microscopy and Reproducible Automatic Image Analysis in ImageJ-A Pilot Study with Valinomycin.

(1) Background: The detection of DNA double-strand breaks in vitro using the phosphorylated histone biomarker (γH2AX) is an increasingly popular method of measuring in vitro genotoxicity, as it is sensitive, specific and suitable for high-throughput analysis. The γH2AX response is either detected by flow cytometry or microscopy, the latter being more accessible. However, authors sparsely publish details, data, and workflows from overall fluorescence intensity quantification, which hinders the reproducibility. (2) Methods: We used valinomycin as a model genotoxin, two cell lines (HeLa and CHO-K1) and a commercial kit for γH2AX immunofluorescence detection. Bioimage analysis was performed using the open-source software ImageJ. Mean fluorescent values were measured using segmented nuclei from the DAPI channel and the results were expressed as the area-scaled relative fold change in γH2AX fluorescence over the control. Cytotoxicity is expressed as the relative area of the nuclei. We present the workflows, data, and scripts on GitHub. (3) Results: The outputs obtained by an introduced method are in accordance with expected results, i.e., valinomycin was genotoxic and cytotoxic to both cell lines used after 24 h of incubation. (4) Conclusions: The overall fluorescence intensity of γH2AX obtained from bioimage analysis appears to be a promising alternative to flow cytometry. Workflow, data, and script sharing are crucial for further improvement of the bioimage analysis methods.

Chromatin landscape, DSB levels, and cKU-70/80 contribute to patterning of meiotic DSB processing along chromosomes in C. elegans.

Programmed DNA double-strand break (DSB) formation is essential for achieving accurate chromosome segregation during meiosis. DSB repair timing and template choice are tightly regulated. However, little is known about how DSB distribution and the choice of repair pathway are regulated along the length of chromosomes, which has direct effects on the recombination landscape and chromosome remodeling at late prophase I. Here, we use the spatiotemporal resolution of meiosis in the Caenorhabditis elegans germline along with genetic approaches to study distribution of DSB processing and its regulation. High-resolution imaging of computationally straightened chromosomes immunostained for the RAD-51 recombinase marking DSB repair sites reveals that the pattern of RAD-51 foci throughout pachytene resembles crossover distribution in wild type. Specifically, RAD-51 foci occur primarily along the gene-poor distal thirds of the chromosomes in both early and late pachytene, and on both the X and the autosomes. However, this biased off-center distribution can be abrogated by the formation of excess DSBs. Reduced condensin function, but not an increase in total physical axial length, results in a homogeneous distribution of RAD-51 foci, whereas regulation of H3K9 methylation is required for the enrichment of RAD-51 at off-center positions. Finally, the DSB recognition heterodimer cKU-70/80, but not the non-homologous end-joining canonical ligase LIG-4, contributes to the enriched off-center distribution of RAD-51 foci. Taken together, our data supports a model by which regulation of the chromatin landscape, DSB levels, and DSB detection by cKU-70/80 collaborate to promote DSB processing by homologous recombination at off-center regions of the chromosomes in C. elegans.

Discovery of novel DNA-damaging agents through phenotypic screening for DNA double-strand break.

DNA double-strand breaks (DSBs) seriously damage DNA and promote genomic instability that can lead to cell death. They are the source of conditions such as carcinogenesis and aging, but also have important applications in cancer therapy. Therefore, rapid detection and quantification of DSBs in cells are necessary for identifying carcinogenic and anticancer factors. In this study, we detected DSBs using a flow cytometry-based high-throughput method to analyze γH2AX intensity. We screened a chemical library containing 9600 compounds and detected multiple DNA-damaging compounds, although we could not identify mechanisms of action through this procedure. Thus, we also profiled a representative compound with the highest DSB potential, DNA-damaging agent-1 (DDA-1), using a bioinformatics-based method we termed "molecular profiling." Prediction and verification analysis revealed DDA-1 as a potential inhibitor of topoisomerase IIα, different from known inhibitors such as etoposide and doxorubicin. Additional investigation of DDA-1 analogs and xenograft models suggested that DDA-1 is a potential anticancer drug. In conclusion, our findings established that combining high-throughput DSB detection and molecular profiling to undertake phenotypic analysis is a viable method for efficient identification of novel DNA-damaging compounds for clinical applications.

Butyrate Enhances γ-H2AX Induced by Benzo[a]pyrene.

Benzo[a]pyrene (BaP) is known to form DNA adduct following metabolic activation, which causes phosphorylation of histone H2AX (γ-H2AX). Recent studies have shown that histone deacetylase (HDAC) inhibitors enhanced BaP-induced CYP1A1 gene expression. In this study, we examined the relationship between the HDAC inhibitor-augmented metabolic activation and BaP-induced γ-H2AX. Sodium butyrate (SB), a typical HDAC inhibitor, enhanced BaP-induced γ-H2AX. The enhanced DNA damage was further confirmed by biased sinusoidal field gel electrophoresis, which detects DNA double-strand breaks. SB remarkably augmented BaP-induced CYP1A1 gene expression, and CYP1A1-overexpressing cells showed elevated generation of γ-H2AX. Furthermore, SB enhanced intracellular oxidation after treatment with BaP. These results suggested that SB-induced CYP1A1 upregulation facilitated BaP metabolism, which might result in excess DNA adducts or oxidative DNA damages, leading to augmentation of γ-H2AX.

Cellular susceptibility and oxidative stress response to menadione of logarithmic, quiescent, and nonquiescent Saccharomyces cerevisiae cell populations

The aim of the present study was to compare cellular susceptibility and oxidative stress response of S. cerevisiae logarithmic (log), quiescent (Q), and non-quiescent (NQ) cell populations to menadione – a well-known inducer of oxidative stress. Three main approaches were used: microbiological – cell survival, molecular – constant field gel electrophoresis for detection of DNA double-strand breaks (DSB), and biochemical – measurement of reactive oxygen species (ROS) levels, oxidized proteins, lipid peroxidation, glutathione, superoxide dismutase (SOD) and catalase on S. cerevisiae haploid strain BY4741. The doses causing 20% (LD20) and 50% (LD50) lethality were calculated. The effect of menadione as a well-known oxidative stress inducer is compared in the log, Q, and NQ cells. Survival data reveal that Q cells are the most susceptible to menadione with LD50 corresponding to 9 µM menadione. On the other hand, dose-dependent DSB induction is found only in Q cells confirming the results shown above. No effect on DSBs levels is observed in log and NQ cells. Further, the oxidative stress response of the cell populations is clarified. Results show significantly higher levels of SOD and ROS in Q cells than in log cells after the treatment with 100 µM menadione. On the other side, higher induction of oxidized proteins, malondialdehyde, and glutathione is observed after menadione treatment of log cells. Our study provides evidence that Saccharomyces cerevisiae quiescent cells are the most susceptible to the menadione action. It might be suggested that the DNA damaging and genotoxic action of menadione in Saccharomyces cerevisiae quiescent cells could be related to ROS production.

Impact of Tumor Treating Fields (TTFields) on DNA Damage Repair in Mesothelioma

Tumor Treating Fields (TTFields) are low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields delivered to solid tumors noninvasively and locoregionally. TTFields have demonstrated a promising median overall survival in patients with malignant pleural mesothelioma (MPM), an aggressive thoracic cancer with poor prognosis, without increases in systemic toxicity (STELLAR clinical trial). Consequently, TTFields therapy combined with pemetrexed and a platinum-based chemotherapy agent are approved as first line treatment for unresectable MPM in the US and Europe. While efficacy of TTFields for MPM treatment is well-established, the underlying mechanism of action needs further elucidation. This study investigated the TTFields effect on DNA damage and repair in MPM and tested the combination of TTFields with DNA damaging agents such as cisplatin and pemetrexed, in vitro and in vivo.TTFields frequencies ranging from 100-400 kHz were applied to human MPM cell lines (NCI-H2052 and MSTO-211H) to identify the most effective frequency. To detect DNA double strand breaks (DSB), the effect of optimal TTFields frequency on the formation of ɣH2AX foci was examined by fluorescent microscopy. Levels of DNA damage repair related proteins were evaluated via immunoblotting of cell lysates. The combined cytotoxic effect of TTFields with cisplatin or pemetrexed was tested in vitro. Efficacy of TTFields concomitant with both cisplatin and pemetrexed was examined in C57BL/6 mice inoculated subcutaneously with RN-5 cells by measuring tumor volume and DNA damage within the tumor.The optimal TTFields frequency was identified as 150 kHz in both MPM cell lines, displaying significant cytotoxicity and formation of DNA DSB. These effects were accompanied by increased expression of p21 and p27, proteins involved in DNA damage-induced cell cycle arrest, and reduced expression of proteins from the Fanconi Anemia (FA) DNA repair pathway - FANCA, FANCD2, FANCJ, and BRCA1. Co-treatment of TTFields with cisplatin or pemetrexed augmented efficacy versus each treatment alone, with the TTFields-pemetrexed combination displaying an additive effect, and the co-administration of TTFields with cisplatin demonstrating synergistic interaction. In vivo, tumor volume fold change was significantly decreased for the combination of TTFields with chemotherapy (cisplatin + pemetrexed) versus control, while levels of DNA damage within the tumor were increased.Efficacy of TTFields for the treatment of MPM is associated with increased DNA damage, elevated levels of DNA-damage related cell cycle arrest proteins, and reduced expression of FA pathway proteins. This latter effect may account for the synergistic interaction displayed for the TTFields-cisplatin combination, as cisplatin is known to cause DNA damage that requires the FA pathway for repair.

Involvement of HIF-1α in the Detection, Signaling, and Repair of DNA Double-Strand Breaks after Photon and Carbon-Ion Irradiation.

Simple SummaryHypoxia-Inducible Factor 1α (HIF-1α), the main regulator of the oxygen homeostasis, promotes cancer cell survival through proliferation, angiogenesis, metastasis and radioresistance. Previously, our group demonstrated that silencing HIF-1α under hypoxia leads to a substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and carbon-ions, probably resulting from an accumulation of deleterious complex DNA damage. In this study, we aimed at determining the potential role of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, under hypoxia, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). Silencing HIF-1α under hypoxia led us to demonstrate the involvement of this transcriptional regulator in DSB repair in non-CSCS and CSC, thus highlighting its targeting together with radiation as a promising therapeutic strategy against radioresistant tumor cells in hypoxic niches.Hypoxia-Inducible Factor 1α (HIF-1α), which promotes cancer cell survival, is the main regulator of oxygen homeostasis. Hypoxia combined with photon and carbon ion irradiation (C-ions) stabilizes HIF-1α. Silencing HIF-1α under hypoxia leads to substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and C-ions. Thus, this study aimed to clarify a potential involvement of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). After confirming the nucleoshuttling of HIF-1α in response to both exposure under hypoxia, we showed that silencing HIF-1α in non-CSCs and CSCs decreased the initiation of the DSB detection (P-ATM), and increased the residual phosphorylated H2AX (γH2AX) foci. While HIF-1α silencing did not modulate 53BP1 expression, P-DNA-PKcs (NHEJ-c) and RAD51 (HR) signals decreased. Altogether, our experiments demonstrate the involvement of HIF-1α in the detection and signaling of DSBs, but also in the main repair pathways (NHEJ-c and HR), without favoring one of them. Combining HIF-1α silencing with both types of radiation could therefore present a potential therapeutic benefit of targeting CSCs mostly present in tumor hypoxic niches.

Emerging Technologies for Genome-Wide Profiling of DNA Breakage.

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

High-resolution, ultrasensitive and quantitative DNA double-strand break labeling in eukaryotic cells using i-BLESS.

DNA double-strand breaks (DSBs) are implicated in various physiological processes, such as class-switch recombination or crossing-over during meiosis, but also present a threat to genome stability. Extensive evidence shows that DSBs are a primary source of chromosome translocations or deletions, making them a major cause of genomic instability, a driving force of many diseases of civilization, such as cancer. Therefore, there is a great need for a precise, sensitive, and universal method for DSB detection, to enable both the study of their mechanisms of formation and repair as well as to explore their therapeutic potential. We provide a detailed protocol for our recently developed ultrasensitive and genome-wide DSB detection method: immobilized direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (i-BLESS), which relies on the encapsulation of cells in agarose beads and labeling breaks directly and specifically with biotinylated linkers. i-BLESS labels DSBs with single-nucleotide resolution, allows detection of ultrarare breaks, takes 5 d to complete, and can be applied to samples from any organism, as long as a sufficient amount of starting material can be obtained. We also describe how to combine i-BLESS with our qDSB-Seq approach to enable the measurement of absolute DSB frequencies per cell and their precise genomic coordinates at the same time. Such normalization using qDSB-Seq is especially useful for the evaluation of spontaneous DSB levels and the estimation of DNA damage induced rather uniformly in the genome (e.g., by irradiation or radiomimetic chemotherapeutics).

Impact of hypoxia on the double-strand break repair after photon and carbon ion irradiation of radioresistant HNSCC cells

DNA double-strand breaks (DSBs) induced by photon irradiation are the most deleterious damage for cancer cells and their efficient repair may contribute to radioresistance, particularly in hypoxic conditions. Carbon ions (C-ions) act independently of the oxygen concentration and trigger complex- and clustered-DSBs difficult to repair. Understanding the interrelation between hypoxia, radiation-type, and DNA-repair is therefore essential for overcoming radioresistance. The DSBs signaling and the contribution of the canonical non-homologous end-joining (NHEJ-c) and homologous-recombination (HR) repair pathways were assessed by immunostaining in two cancer-stem-cell (CSCs) and non-CSCs HNSCC cell lines. Detection and signaling of DSBs were lower in response to C-ions than photons. Hypoxia increased the decay-rate of the detected DSBs (γH2AX) in CSCs after photons and the initiation of DSB repair signaling (P-ATM) in CSCs and non-CSCs after both radiations, but not the choice of DSB repair pathway (53BP1). Additionally, hypoxia increased the NHEJ-c (DNA-PK) and the HR pathway (RAD51) activation only after photons. Furthermore, the involvement of the HR seemed to be higher in CSCs after photons and in non-CSCs after C-ions. Taken together, our results show that C-ions may overcome the radioresistance of HNSCC associated with DNA repair, particularly in CSCs, and independently of a hypoxic microenvironment.

Considering Cell Proliferation to Optimize Detection of Radiation-Induced 53BP1 Positive Foci in 15 Mouse Strains Ex Vivo.

Due to high metabolic activity, proliferating cells continuously generate free radicals, which induce DNA double-strand breaks (DSB). Fluorescently tagged nuclear foci of DNA repair protein 53 binding protein-1 (53BP1) are used as a standard metric for measuring DSB formation at baseline and in response to environmental insults such as radiation. Here we demonstrate that the background level of spontaneous 53BP1+ foci formation can be modeled mathematically as a function of cell confluence, which is a metric of their proliferation rate. This model was validated using spontaneous 53BP1+ foci data from 72 cultures of primary skin fibroblasts derived from 15 different strains of mice, showing a ∼10-fold decrease from low to full confluence that is independent of mouse strain. On the other hand, the baseline level of spontaneous 53BP1+ foci in a fully confluent cell population was strain-dependent, suggesting genomic associations, and correlated with radiation sensitivity based on previous measurements in the same cell lines. Finally, we have developed an online open-access tool to correct for the effect of cell confluence on 53BP1+ foci-based quantification of DSB. This tool provides guidelines for the number of cells required to reach statistical significance for the detection of DSB induced by low doses of ionizing radiation as a function of confluence and time postirradiation.

Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1.

Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.