Genotoxic Lesions Research Articles

The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks.

DNA-protein crosslinks (DPCs) are endogenous and chemotherapy-induced genotoxic DNA lesions and, if not repaired, lead to embryonic lethality, neurodegeneration, premature ageing, and cancer. DPCs are heavily polyubiquitinated, and the SPRTN protease and 26S proteasome emerged as two central enzymes for DPC proteolysis. The proteasome recognises its substrates by their ubiquitination status. How SPRTN protease, an essential enzyme for DPC proteolysis, achieves specificity for DPCs still needs to be discovered. We found that the N-terminal SPRTN catalytic region (SprT) possesses a ubiquitin-binding domain named the U biquitin interface of S prT D omain (USD). Using multiple biochemical, biophysical, and structural approaches, we reveal that USD binds ubiquitin chains. SPRTN binding to ubiquitin chains via USD leads to ∼ 67-fold higher activation of SPRTN proteolysis towards polyubiquitinated DPCs than the unmodified DPCs. This study reveals the ubiquitination of DPCs is the key signal for SPRTN's substrate specificity and rapid proteolysis.

The epistatic relationship of Drosophila melanogaster CtIP and Rif1 in homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR-white) assay, tracking across indels by decomposition (TIDE) analysis, and P{wIw_2 kb 3'} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1Δ/Δ single mutants, and DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1Δ/Δ single mutants. However, experiments in DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection.

Chemoprotective Effect of Eugenol on Repeated dose Doxorubicin, Induced Genotoxic Lesions in Mouse Bone Marrow

In present study, an attempt has been made to evaluate the possible in vivo chemoprotective potential of eugenol against doxorubicin induced genotoxic lesion in bone marrow. Swiss albino female mice were administered Eugenol (10 mg/kg/day, orally) 15 days before doxorubicin administration as well as concomitantly with doxorubicin. Genotoxic lesions were induced by a repeated dose intraperitoneal injection of doxorubicin (5mg/kg b.w. i.p. upto 9 days (alternate day). Micronuclei assay and COMET assay were carried out to determine genetic damage in bone marrow. Histopathological evaluation of bone marrow was also done to study lesions at cellular level. The study results demonstrated that eugenol significantly mitigated Doxorubicin induced micronuclei formation and DNA damage in the bone marrow niche. Histopathological observations revealed that Doxorubicn-intoxication resulted in massive structural impairment of bone marrow which was reduced by eugenol administration. Amelioration by eugenol is more significant in mice those received eugenol 15 days prior to Doxorubicin administration suggesting its chemoprotection. The present study suggests eugenol has the promising chemoprotective effect against Doxorubin-induced genetic lesions in mice bone marrow.

No benefit from TMZ treatment in glioblastoma with truly unmethylated MGMT promoter: Reanalysis of the CE.6 and the pooled Nordic/NOA-08 trials in elderly glioblastoma patients.

The treatment of elderly/ frail patients with glioblastoma is a balance between avoiding undue toxicity, while not withholding effective treatment. It remains debated, whether these patients should receive combined chemo-radiotherapy with temozolomide (RT/TMZ→TMZ) regardless of the O6-methylguanine DNA methyltransferase gene promoter (MGMTp) methylation status. MGMT is a well-known resistance factor blunting the treatment effect of TMZ, by repairing the most genotoxic lesion. Epigenetic silencing of the MGMTp sensitizes glioblastoma to TMZ. For risk-adapted treatment, it is of utmost importance to accurately identify patients, who will not benefit from TMZ treatment. Here, we present a reanalysis of the clinical trials CE.6 and the pooled NOA-08 and Nordic trials in elderly glioblastoma patients that compared RT to RT/TMZ→TMZ, or RT to TMZ, respectively. For 687 patients with available MGMTp methylation data, we applied a cutoff discerning truly unmethylated glioblastoma, established in a pooled analysis of 4 clinical trials for glioblastoma, with RT/TMZ→TMZ treatment, using the same quantitative methylation-specific MGMTp PCR assay. When applying this restricted cutoff to the elderly patient population, we confirmed that glioblastoma with truly unmethylated MGMTp derived no benefit from TMZ treatment. In the Nordic/NOA-08 trials, RT was better than TMZ, suggesting little or no benefit from TMZ. For evidence-based treatment of glioblastoma patients validated MGMTp methylation assays should be used that accurately identify truly unmethylated patients. Respective stratified management of patients will reduce toxicity without compromising outcomes and allow testing of more promising treatment options.

Mammalian life depends on two distinct pathways of DNA damage tolerance

DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.

Hepatocellular Carcinoma: Aetiology and Mechanisms of Development. A Literature Review

Liver cancer remains a global challenge of healthcare, with the incidence growing worldwide. According to various authors, over 1 million patients will be diagnosed with liver cancer each year by 2025. The molecular pathogenesis of HCC varies with respect to genotoxic lesions and aetiologies. Although our understanding of the HCC pathophysiology and drivers tends to improve, it is still distant from translation into clinical practice. About 25 % of HCC cases are associated with variant mutations. HCC pathophysiology is a complex multi-step process. The interaction of various factors underlies the early stages of malignant hepatocyte transformation towards the development of HCC. Overall, about 20–25 % of HCC patients have at least one potential driver mutation. Obesity should also be noted as being associated with a higher risk of HCC and various other cancers. Despite many issues in the HCC pathogenesis being already known, the unresolved questions remain. Modern molecular genetic diagnostics and animal modelling of malignant tumours are expanding our horizons of knowledge in this field.

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine.

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.

Revisiting the Function of p21CDKN1A in DNA Repair: The Influence of Protein Interactions and Stability.

The p21CDKN1A protein is an important player in the maintenance of genome stability through its function as a cyclin-dependent kinase inhibitor, leading to cell-cycle arrest after genotoxic damage. In the DNA damage response, p21 interacts with specific proteins to integrate cell-cycle arrest with processes such as transcription, apoptosis, DNA repair, and cell motility. By associating with Proliferating Cell Nuclear Antigen (PCNA), the master of DNA replication, p21 is able to inhibit DNA synthesis. However, to avoid conflicts with this process, p21 protein levels are finely regulated by pathways of proteasomal degradation during the S phase, and in all the phases of the cell cycle, after DNA damage. Several lines of evidence have indicated that p21 is required for the efficient repair of different types of genotoxic lesions and, more recently, that p21 regulates DNA replication fork speed. Therefore, whether p21 is an inhibitor, or rather a regulator, of DNA replication and repair needs to be re-evaluated in light of these findings. In this review, we will discuss the lines of evidence describing how p21 is involved in DNA repair and will focus on the influence of protein interactions and p21 stability on the efficiency of DNA repair mechanisms.

Effects of N7-Alkylguanine Conformation and Metal Cofactors on the Translesion Synthesis by Human DNA Polymerase η.

Non-enzymatic alkylation on DNA often generates N7-alkyl-2'-deoxyguanosine (N7alkylG) adducts as major lesions. N7alkylG adducts significantly block replicative DNA polymerases and can be bypassed by translesion synthesis (TLS) polymerases such as polymerase η (polη). To gain insights into the bypass of N7alkylG by TLS polymerases, we conducted kinetic and structural studies of polη catalyzing across N7BnG, a genotoxic lesion generated by the carcinogenic N-nitrosobenzylmethylamine. The presence of templating N7BnG in the polη catalytic site decreased the replication fidelity by ∼9-fold, highlighting the promutagenicity of N7BnG. The catalytic efficiency for dCTP incorporation opposite N7BnG decreased ∼22-fold and ∼7-fold compared to the incorporation opposite undamaged guanine in the presence of Mg2+ and Mn2+, respectively. A crystal structure of the complexes grown with polη, templating N7BnG, incoming dCTP, and Mg2+ ions showed the lack of the incoming nucleotide and metal cofactors in the polη catalytic site. Interestingly, the templating N7BnG adopted a syn conformation, which has not been observed in the published N7alkylG structures. The preferential formation of syn-N7BnG conformation at the templating site may deter the binding of an incoming dCTP, causing the inefficient bypass by polη. In contrast, the use of Mn2+ in place of Mg2+ in co-crystallization yielded a ternary complex displaying an anti-N7BnG:dCTP base pair and catalytic metal ions, which would be a close mimic of a catalytically competent state. We conclude that certain bulky N7-alkylG lesions can slow TLS polymerase-mediated bypass by adopting a catalytically unfavorable syn conformation in the replicating base pair site.

Relevant Aspects of Drug Genetic Toxicology

Genotoxic lesions are not only a cause of genetic pathologies and cancer, but also the most common and significant factor of the etiopathology of cardiovascular and neurodegenerative disorders, reproductive losses, infertility, and aging. This requires careful monitoring of the use of potential genotoxicants including medicinal products (MPs), which are a group of compounds intentionally and routinely used by humans. Genotoxicity assesment is highly essential in preclinical drug safety studies. The aim of the study was to analyse the current situation and reveal possible ways of addressing methodological and regulatory issues in genotoxicology to enable proper conduction of drug safety preclinical studies. The article summarises basic concepts of the modern genetic toxicology development and highlights the history of research aimed at identifying mutagenic, clastogenic, and aneugenic effects. The authors analyse regulatory aspects of genotoxicological studies of MPs and consider issues of improving the strategy for genotoxicity testing. The paper gives information about the genotoxicity tests approved for MPs, recommendations for interchangeability of tests in relation to particular study characteristics. The authors carried out a comparative analysis of the tests’ pros and cons with an emphasis that the study of each MP is a separate scientific task. They discuss interpretation of results and prediction of MP carcinogenic potential in genotoxicological studies. Recommendations are given for the optimisation of the MP genotoxicity assessment strategy, considering its partial integration into general toxicity studies. The article stresses the urgent need to develop registration methods for genotoxic events in germ cells, assesses the prospects of new tests, and reviews new trends in drug genotoxicology fundamental research.

Reproductive health of parents

The reproductive health of parents is the onset and further physiological course of pregnancy, childbirth and health of the unborn child. Saving of reproductive health can be considered as protecting the family from factors with “reproductive toxicity”, that is lead to morphological and functional changes in the germ cells, organs and systems of embryo. The risk of infertility and miscarriage is significantly higher in couples where a man and a woman have balanced chromosomal rearrangements or gene polymorphisms. In the future, we will be able to block genotoxic lesions of the genome at the gene level.Research and development of experience on this issue continues. Now there are already some promising methods, which include nutritional support at the stage of pregnancy planning using folic acid and myo-inositol. New guidelines have been developed for folic acid, metafolin, MTHFR gene variants, and others. Neural tube defects are more common in people with MTHFR 677 CT and TT genotypes in countries where food folate fortification is not used, so they did not get enough of it. Women who are planning a pregnancy and have previously children with malformations are recommended to intake folic acid 4000 µg daily, starting 1 month before pregnancy and during the first 3 months of gestation.However, getting ready for pregnancy is much more than 3 or 12 months before conception. Preparing for pregnancy is a healthy lifestyle for parents from the moment they are conceived to their reproductive years. Saving of reproductive health is protection against genotoxic damage. Genotoxic substances act in different periods of life with different intensity of strength and time, therefore, the program for maintaining reproductive health should begin from the moment of conception of future parents. At the same time, the reproductive health saving program applies to both women and men.

Blue light induces DNA damage in normal human skin keratinocytes.

The generation of DNA damage by ultra-violet radiations (UV) is well established, and both the nature of the DNA lesions and their respective DNA repair pathways have largely been described. Besides UV rays, visible light constitutes a very important part of the sun spectrum where blue light is considered a significant contributor to premature aging. However, blue light-induced DNA damage has not been deeply explored yet. In the present study, we assessed in human skin keratinocytes the DNA and chromosome damaging activities of blue light rays (415nm) as well as their associated DNA repair mechanisms. Our results demonstrated that blue light induced dose-dependent DNA damage in human keratinocytes. Both oxidative and cyclobutane-pyrimidine-dimer (CPD) DNA lesions were generated. They were repaired through base excision repair (BER) and nucleotide excision repair (NER) pathways, respectively. Moreover, by using the micronucleus assay we demonstrated, for the first time, that a blue wavelength exerted a clastogenic/aneugenic effect in human keratinocytes, leading to chromosome aberration. We concluded that, in normal human keratinocytes, blue light creates genotoxic lesions which might accelerate or at least contribute to premature skin aging.

Abstract SP125: Rescue DNA repair pathway and tumor immunity

Abstract Defects in DNA damage response (DDR) is a major factor that predispose normal cells to acquire oncogenic mutations. However, after a tumor develops, cancer cells manage their survival by repairing DNA damage resulting from unchecked DNA replication. Cancer cells can use the alternative or backup (rescue) DNA repair programs to overcome their DDR defects and cope with genotoxic lesions. For example, breast cancer cells with deficiency in homologous recombination (HR) can repair their DNA by either relying on other highly expressed HR-related proteins (such as RAD51 or PARP1), or by using other DNA repair mechanisms such as break-induced replication, alternative nonhomologous end joining (ALT-NHEJ) and basal excision repair (BER). Since DNA repair pathways operate in related networks in cancers in general and breast cancers in particular, it is likely that factor/s that play important roles in multiple DNA repair pathways may serve as a critical component of backup DNA repair pathways that breast cancer cells get addicted to survive and to become therapy resistant. One way these rescue DNA repair pathway factor/s may promote breast cancer progression is by shaping tumor immune microenvironment. Consistent with that, DNA damage and DNA repair pathways in general have been shown to regulate multiple arms of the immune response including antigenicity or tumor mutational burden (TMB) and regulation of immune checkpoint protein levels. Our studies suggest that rescue DNA repair pathways may support tumor progression and therapy response by promoting tumor cell intrinsic and extrinsic mechanisms of immunosuppression. Our results reveal that factors associated with backup DNA repair pathwaysmay promote immunosuppression by altering cGAS-STING and Type I Interferon (IFN) signaling pathways. Furthermore, using a specific inhibitor that targets rescue DNA repair pathway signaling, we show altered levels of several proteins involved in antigen processing and presentation, including proteasome/Immunoproteasome subunits and MHC I protein subunits, which are essential for the detection of cancer cells by cytotoxic T cells. Importantly, our pre-clinical and clinical data suggest that approaches aimed at successfully targeting rescue DNA repair factor(s) may promote anti-tumor immunity and will have promising clinical outcomes for treating breast cancer patients. Citation Format: M Rao. Rescue DNA repair pathway and tumor immunity [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP125.

Physical and Genetic Assays for the Study of DNA Joint Molecules Metabolism and Multi-invasion-Induced Rearrangements in S. cerevisiae.

DNA double-strand breaks (DSBs) are genotoxic lesions that can be repaired in a templated fashion by homologous recombination (HR). HR is a complex pathway that involves the formation of DNA joint molecules (JMs) containing heteroduplex DNA. Various types of JMs are formed throughout the pathway, including displacement loops (D-loops), multi-invasions (MI), and double Holliday junction intermediates. Dysregulation of JM metabolism in various mutant contexts revealed the propensity of HR to generate repeat-mediated chromosomal rearrangements. Specifically, we recently identified MI-induced rearrangements (MIR), a tripartite recombination mechanism initiated by one end of a DSB that exploits repeated regions to generate rearrangements between intact chromosomal regions. MIR occurs upon MI-JM processing by endonucleases and is suppressed by JM disruption activities. Here, we detail two assays: a physical assay for JM detection in Saccharomyces cerevisiae cells and genetic assays to determine the frequency of MIRin various chromosomal contexts. These assays enable studying the regulation of the HR pathway and the consequences of their defectsfor genomic instability by MIR.

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like γ-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that γ-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Molecular Mechanisms of PARP-1 Inhibitor 7-Methylguanine

7-Methylguanine (7-MG), a natural compound that inhibits DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1), can be considered as a potential anticancer drug candidate. Here we describe a study of 7-MG inhibition mechanism using molecular dynamics, fluorescence anisotropy and single-particle Förster resonance energy transfer (spFRET) microscopy approaches to elucidate intermolecular interactions between 7-MG, PARP-1 and nucleosomal DNA. It is shown that 7-MG competes with substrate NAD+ and its binding in the PARP-1 active site is mediated by hydrogen bonds and nonpolar interactions with the Gly863, Ala898, Ser904, and Tyr907 residues. 7-MG promotes formation of the PARP-1–nucleosome complexes and suppresses DNA-dependent PARP-1 automodification. This results in nonproductive trapping of PARP-1 on nucleosomes and likely prevents the removal of genotoxic DNA lesions.

Dipeptide repeat proteins inhibit homology-directed DNA double strand break repair in C9ORF72 ALS/FTD

BackgroundThe C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated.Methods and resultsUsing DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls.ConclusionsCollectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.

Detection of Bleomycin-Induced DNA Double-Strand Breaks in Escherichia coli by Pulsed-Field Gel Electrophoresis Using a Rotating Gel Electrophoresis System.

DNA double-strand break (DSB) is one of the most genotoxic lesions, and unrepaired DSBs can lead to chromosomal instability and eventually cause cell death. Quantitative markers, such as phosphorylated histone H2AX (γ-H2AX) and p53-binding protein 1 (53BP1) foci in mammalian cells, are not available for the detection of DSBs in prokaryotes. Therefore, as an alternative method, pulsed-field gel electrophoresis (PFGE) is widely used to analyze broken DNA molecules by separating them from intact DNA. Here, we examined the accumulation of bleomycin (BLM)-induced DSBs by PFGE, using a rotating gel electrophoresis (RGE) system. We defined two sets of parameters with distinct advantages; the first one focuses on the analysis of the size of the broken DNA fragments, whereas the second allows for the direct comparison of the accumulation of DSBs among strains and treatments. This method represents a powerful tool for the study of genomic integrity and the characterization of genotoxic substances.

Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates.

Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the ‘HepaCometChip’, enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.

Formic Acid of ppm Enhances LC-MS/MS Detection of UV Irradiation-Induced DNA Dimeric Photoproducts.

Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are genotoxic DNA lesions and mainly generated on thymine-thymine (T-T) dinucleotides upon UV irradiation. Regarding the sensitivity, specificity, and accuracy of analytical methods, it is of first choice to develop a reliable assay for simultaneous detection of these DNA lesions using liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, the dilemma is the low detection sensitivity of the phosphate-containing dimeric photoproducts even using most favorable negative-ion mode for LC-MS/MS analysis. Unexpectedly, we observed that the detection sensitivity of T-T CPD and 6-4PP could be significantly improved using formic acid/acetic acid (∼ppm) as an additive of the mobile phase for reversed-phase LC-MS/MS analysis. This is the first demonstration of the enhancement of LC-MS/MS signals by formic acid/acetic acid in negative-ion mode. Of note, these acidic agents are often used for positive-ion mode in LC-MS assays. Benefited from the developed method, we could quantify both T-T CPD and 6-4PP in mouse embryonic stem cells upon UVC irradiation at low dosage. This sensitive method is applicable to the screening and identification of genes involved in formation, signaling, and repair of UV lesion.