A Practical Site-specific Method for the Detection of Bulky DNA Damages.

Helix-distorting DNA damages block RNA and DNA polymerase, compromising cell function and fate. In human cells, these damages are removed primarily by nucleotide excision repair (NER). Here, we describe damage-sensing PCR (dsPCR), a PCR-based method for the detection of these DNA damages. Exposure to DNA damaging agents results in lower PCR signal in comparison to non-damaged DNA, and repair is measured as the restoration of PCR signal over time. We show that the method successfully detects damages induced by ultraviolet (UV) radiation, by the carcinogenic component of cigarette smoke benzo[a]pyrene diol epoxide (BPDE) and by the chemotherapeutic drug cisplatin. Damage removal measured by dsPCR in a heterochromatic region is less efficient than in a transcribed and accessible region. Furthermore, lower repair is measured in repair-deficient knock-out cells. This straight-forward method could be applied by non-DNA repair experts to study the involvement of their gene-of-interest in repair. Furthermore, this method is fully amenable for high-throughput screening of DNA repair activity.

N-acetyl cysteine turns EPAC activators into potent killers of acute lymphoblastic leukemia cells.

Today, the majority of patients with pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL) survive their disease, but many of the survivors suffer from life-limiting late effects of the treatment. ALL develops in the bone marrow, where the cells are exposed to cAMP-generating prostaglandin E2. We have previously identified the cAMP signaling pathway as a putative target for improved efficacy of ALL treatment, based on the ability of cAMP signaling to reduce apoptosis induced by DNA damaging agents. In the present study, we have identified the antioxidant N-acetyl cysteine (NAC) as a powerful modifier of critical events downstream of the cell-permeable cAMP analog 8-(4-chlorophenylthio) adenosine-3', 5'- cyclic monophosphate (8-CPT). Accordingly, we found NAC to turn 8-CPT into a potent killer of ALL cells in vitro both in the presence and absence of DNA damaging treatment. Furthermore, we revealed that NAC in combination with 8-CPT is able to delay the progression of ALL in a xenograft model in NOD-scid IL2Rγnull mice. NAC was shown to rely on the ability of 8-CPT to activate the guanine-nucleotide exchange factor EPAC, and we demonstrated that the ALL cells are killed by apoptosis involving sustained elevated levels of calcium imposed by the combination of the two drugs. Taken together, we propose that 8-CPT in the presence of NAC might be utilized as a novel strategy for treating pediatric ALL patients, and that this powerful combination might be exploited to enhance the therapeutic index of current ALL targeting therapies.

Role of condensates in modulating DNA repair pathways and its implication for chemoresistance.

For cells, it is important to repair DNA damage, such as double-strand and single-strand DNA breaks, because unrepaired DNA can compromise genetic integrity, potentially leading to cell death or cancer. Cells have multiple DNA damage repair pathways that have been the subject of detailed genetic, biochemical, and structural studies. Recently, the scientific community has started to gain evidence that the repair of DNA double-strand breaks may occur within biomolecular condensates and that condensates may also contribute to DNA damage through concentrating genotoxic agents used to treat various cancers. Here, we summarize key features of biomolecular condensates and note where they have been implicated in the repair of DNA double-strand breaks. We also describe evidence suggesting that condensates may be involved in the repair of other types of DNA damage, including single-strand DNA breaks, nucleotide modifications (e.g., mismatch and oxidized bases), and bulky lesions, among others. Finally, we discuss old and new mysteries that could now be addressed considering the properties of condensates, including chemoresistance mechanisms.

Unwinding mechanism of SARS-CoV helicase (nsp13) in the presence of Ca2+, elucidated by biochemical and single-molecular studies.

The recent outbreak of COVID-19 has created a serious health crisis with fatFal infectious viral diseases, such as Severe Acute Respiratory Syndrome (SARS). The nsp13, a helicase of coronaviruses is an essential element for viral replication that unwinds secondary structures of DNA and RNA, and is thus considered a major therapeutic target for treatment. The replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, in association with viral replication organelles, called virus-induced cytosolic double-membrane vesicles (DMVs). In addition, an increase in cytosolic Ca2+ concentration accelerates viral replication. However, the molecular mechanism of nsp13 in the presence of Ca2+ is not well understood. In this study, we applied biochemical methods and single-molecule techniques to demonstrate how nsp13 achieves its unwinding activity while performing ATP hydrolysis in the presence of Ca2+. Our study found that nsp13 could efficiently unwind double stranded (ds) DNA under physiological concentration of Ca2+ of cytosolic DMVs. These findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs.

A red light-triggered chemical tool for sequence-specific alkylation of G-quadruplex and I-motif DNA.

The importance of non-canonical DNA structures such as G-quadruplexes (G4) and intercalating-motifs (iMs) in the fine regulation of a variety of cellular processes has been recently demonstrated. As the crucial roles of these structures are being unravelled, it is becoming more and more important to develop tools that allow targeting these structures with the highest possible specificity. While targeting methodologies have been reported for G4s, this is not the case for iMs, as evidenced by the limited number of specific ligands able to bind the latter and the total absence of selective alkylating agents for their covalent targeting. Furthermore, strategies for the sequence-specific covalent targeting of G4s and iMs have not been reported thus far. Herein, we describe a simple methodology to achieve sequence-specific covalent targeting of G4 and iM DNA structures based on the combination of (i) a peptide nucleic acid (PNA) recognizing a specific sequence of interest, (ii) a pro-reactive moiety enabling a controlled alkylation reaction, and (iii) a G4 or iM ligand orienting the alkylating warhead to the reactive residues. This multi-component system allows for the targeting of specific G4 or iM sequences of interest in the presence of competing DNA sequences and under biologically relevant conditions.

Products Generated by Amine-Catalyzed Strand Cleavage at Apurinic/Apyrimidinic Sites in DNA: New Insights from a Biomimetic Nucleoside Model System.

Abasic sites are common in cellular and synthetic DNA. As a result, it is important to characterize the chemical fate of these lesions. Amine-catalyzed strand cleavage at abasic sites in DNA is an important process in which conversion of small amounts of the ring-opened abasic aldehyde residue to an iminium ion facilitates ß-elimination of the 3'-phosphoryl group. This reaction generates a trans-α,ß-unsaturated iminium ion on the 3'-terminus of the strand break as an obligate intermediate. The canonical product expected from amine-catalyzed cleavage at an AP site is the corresponding trans-α,ß-unsaturated aldehyde sugar remnant resulting from hydrolysis of this iminium ion. Interestingly, a handful of studies have reported noncanonical 3'-sugar remnants generated by amine-catalyzed strand cleavage, but the formation and properties of these products are not well-understood. To address this knowledge gap, a nucleoside system was developed that enabled chemical characterization of the sugar remnants generated by amine-catalyzed ß-elimination in the 2-deoxyribose system. The results predict that amine-catalyzed strand cleavage at an AP site under physiological conditions has the potential to reversibly generate noncanonical cleavage products including cis-alkenal, 3-thio-2,3-dideoxyribose, and 2-deoxyribose groups alongside the canonical trans-alkenal residue on the 3'-terminus of the strand break. Thus, the model reactions provide evidence that the products generated by amine-catalyzed strand cleavage at abasic sites in cellular DNA may be more complex that commonly thought, with trans-α,ß-unsaturated iminium ion intermediates residing at the hub of interconverting product mixtures. The results expand the list of possible 3'-sugar remnants arising from amine-catalyzed cleavage of abasic sites in DNA that must be chemically or enzymatically removed for the completion of base excision repair and single-strand break repair in cells.

Unexpected Complexity in the Products Arising from NaOH-, Heat-, Amine-, and Glycosylase-Induced Strand Cleavage at an Abasic Site in DNA.

Hydrolytic loss of nucleobases from the deoxyribose backbone of DNA is one of the most common unavoidable types of damage in synthetic and cellular DNA. The reaction generates abasic sites in DNA, and it is important to understand the properties of these lesions. The acidic nature of the α-protons of the ring-opened abasic aldehyde residue facilitates the ß-elimination of the 3'-phosphoryl group. This reaction is expected to generate a DNA strand break with a phosphoryl group on the 5'-terminus and a trans-α,ß-unsaturated aldehyde residue on the 3'-terminus; however, a handful of studies have identified noncanonical sugar remnants on the 3'-terminus, suggesting that the products arising from strand cleavage at apurinic/apyrimidinic sites in DNA may be more complex than commonly thought. We characterized the strand cleavage induced by the treatment of an abasic site-containing DNA oligonucleotide with heat, NaOH, piperidine, spermine, and the base excision repair glycosylases Fpg and Endo III. The results showed that under multiple conditions, cleavage at an abasic site in a DNA oligomer generated noncanonical sugar remnants including cis-α,ß-unsaturated aldehyde, 2-deoxyribose, and 3-thio-2,3-dideoxyribose products on the 3'-terminus of the strand break.

Discovery of DNA-Targeting HDAC Inhibitors with Potent Antitumor Efficacy In Vivo That Trigger Antitumor Immunity.

We observed a synergistic antiproliferation effect with combined use of a DNA minor groove binder and a histone deacetylase (HDAC) inhibitor. Inspired by this result, a new series of benzimidazole-hydroxamate hybrids were designed and synthesized to target both DNA minor groove and HDAC. The most active compounds 9k and 9l not only exhibited improved HDAC inhibitory activities compared to SAHA but also possessed potent antiproliferation activities against tumor cells. Importantly, compounds 9k and 9l showed good in vivo antitumor efficacies in both HEL xenograft model and murine melanoma model. We also found that 9k and 9l promote the antigen presentation and activate T cells, thereby triggering antitumor immunity. Moreover, these inhibitors reshaped the tumor immune microenvironment by inhibiting the recruitment of Treg cells and promoting the polarization of tumor-infiltrating macrophages to M2 type with antitumor activity. Our study validated the effectiveness of incorporating a DNA-binding fragment in HDAC inhibitors as novel multitargeting antitumor agents.

CSB-independent, XPC-dependent transcription-coupled repair in Drosophila.

Drosophila melanogaster has been extensively used as a model system to study ionizing radiation and chemical-induced mutagenesis, double-strand break repair, and recombination. However, there are only limited studies on nucleotide excision repair in this important model organism. An early study reported that Drosophila lacks the transcription-coupled repair (TCR) form of nucleotide excision repair. This conclusion was seemingly supported by the Drosophila genome sequencing project, which revealed that Drosophila lacks a homolog to CSB, which is known to be required for TCR in mammals and yeasts. However, by using excision repair sequencing (XR-seq) genome-wide repair mapping technology, we recently found that the Drosophila S2 cell line performs TCR comparable to human cells. Here, we have extended this work to Drosophila at all its developmental stages. We find TCR takes place throughout the life cycle of the organism. Moreover, we find that in contrast to humans and other multicellular organisms previously studied, the XPC repair factor is required for both global and transcription-coupled repair in Drosophila.

Oxidative stress, DNA, and membranes targets as modes of antibacterial and antibiofilm activity of facile synthesized biocompatible keratin-copper nanoparticles against multidrug resistant uro-pathogens.

Escherichia coli and Enterococcus faecalis are two of the most prevalent uro-pathogens and are difficult to treat as they acquire multidrug-resistant traits. In this study, the main objective was to develop biocompatible copper nanoparticles using chicken feather keratin protein (CuNPs-K) and to investigate their impact on multidrug-resistant (MDR) uro-pathogens, E. coli and E. faecalis, under both single and mixed culture conditions. CuNPs-K were characterised by UV-Vis spectroscopy, dynamic light scattering, X-ray diffraction, Fourier transform infrared spectroscopy, and docking experiments. The MIC values of CuNPs-K against single and mixed planktonic cultures were 50 µg/ml and 75 µg/ml, respectively. CuNPs-K efficiently disrupted the biofilm of single and mixed uro-pathogen cultures by eliminating sessile cells. This biofilm disruption may be attributed to a decline in the production of extracellular polymeric substances in both single and mixed bacterial cultures treated with CuNPs-K. Moreover, selective antimicrobial activity was determined by selectivity assays using T24 cells. CuNPs-K targets both the bacterial membrane and DNA with elevated reactive oxygen species (ROS) as their bactericidal mode of action. This comprehensive antimicrobial activity of CuNPs-K was further confirmed in vivo by using the zebra fish model. In this study, CuNPs-K effectively reduced bacterial load with increased survivability of infected zebrafish. All these results suggest that CuNPs-K can be explored as an exceptional antibacterial agent against MDR uro-pathogenic E. coli and E. faecalis.

Cytotoxic Ruthenium(II) Complexes Containing a Dangling Pyridine: Selectivity for Diseased Cells Mediated by pH-Dependent DNA Binding.

Ruthenium(II) complexes of the type [Ru(bpy)2(L1/L2/L3)]PF6 [where bpy = 2,2'-bipyridine, H(L1) = N-(pyrid-2-yl)salicylaldimine (1), H(L2) = N-(6-methylpyrid-2-yl)salicylaldimine (2), and H(L3) = N-(4,6-dimethylpyrid-2-yl)salicylaldimine (3)] have been isolated. The X-ray structures of 1-3 reveal distorted octahedral coordination geometry with a planar ruthenium phenolate moiety. They exhibit interpair dimeric association in their solid state such as (a) π-π-stacking interactions (1-3) and (b) C-H···π interactions (2). The 1H NMR spectral data shed light on the characteristics of metal-ligand bonding and chelate ring conformations. The complexes exhibit strong metal-to-ligand charge-transfer transitions in the visible region. The complexes also undergo two successive metal-based oxidative processes corresponding to the RuII/RuIII and RuIII/RuIV couples. Resonance Raman studies strongly suggest that the lowest unoccupied molecular orbital of 1-3 is localized at the bpy ligand. Absorption, emission, and circular dichroic spectral measurements for 1-3 with calf-thymus DNA reveal a groove binding mode of interaction. Interestingly, all of the complexes exhibit pH-dependent DNA damage, and the pH at which the damage is highest corresponds to the pH conditions of the cancer cells. The DNA damage is in the order of 3 > 2 > 1, in which a hydrolytic mechanism dominates. The protein binding properties of the complexes examined by the tryptophan quenching measurements suggest a static mechanism. The positive ΔH and ΔS values indicate that the force acting between the complexes and bovine serum albumin (BSA) is mainly a hydrophobic interaction, and thus BSA may act as a targeted drug-delivery vehicle for ruthenium(II) complexes (K ∼ 105). It is noteworthy that 3 exhibits selectivity with high cytotoxicity against breast cancer cells (EVSA-T and MCF-7), and its potency is comparable to that of cisplatin.

Quantification of single-strand DNA lesions caused by the topoisomerase II poison etoposide using single DNA molecule imaging.

DNA-damaging agents, such as radiation and chemotherapy, are common in cancer treatment, but the dosing has proven to be challenging, leading to severe side effects in some patients. Hence, to be able to personalize DNA-damaging chemotherapy, it is important to develop fast and reliable methods to measure the resulting DNA damage in patient cells. Here, we demonstrate how single DNA molecule imaging using fluorescence microscopy can quantify DNA-damage caused by the topoisomerase II (TopoII) poison etoposide. The assay uses an enzyme cocktail consisting of base excision repair (BER) enzymes to repair the DNA damage caused by etoposide and label the sites using a DNA polymerase and fluorescently labeled nucleotides. Using this DNA-damage detection assay we find a large variation in etoposide induced DNA-damage after in vitro treatment of blood cells from healthy individuals. We furthermore used the TopoII inhibitor ICRF-193 to show that the etoposide-induced damage in DNA was TopoII dependent. We discuss how our results support a potential future use of the assay for personalized dosing of chemotherapy.

Investigating Alkylated Prodigiosenes and Their Cu(II)-Dependent Biological Activity: Interactions with DNA, Antimicrobial and Photoinduced Anticancer Activity.

Prodigiosenes are a family of red pigments with versatile biological activity. Their tripyrrolic core structure has been modified many times in order to manipulate the spectrum of activity. We have been looking systematically at prodigiosenes substituted at the C ring with alkyl chains of different lengths, in order to assess the relevance of this substituent in a context that has not been investigated before for these derivatives: Cu(II) complexation, DNA binding, self-activated DNA cleavage, photoinduced cytotoxicity and antimicrobial activity. Our results indicate that the hydrophobic substituent has a clear influence on the different aspects of their biological activity. The cytotoxicity study of the Cu(II) complexes of these prodigiosenes shows that they exhibit a strong cytotoxic effect towards the tested tumor cell lines. The Cu(II) complex of a prodigiosene lacking any alkyl chain excelled in its photoinduced anticancer activity, thus demonstrating the potential of prodigiosenes and their metal complexes for an application in photodynamic therapy (PDT). Two derivatives along with their Cu(II) complexes showed also antimicrobial activity against Staphylococcus aureus strains.

Mouse Embryonic Fibroblasts Isolated From Nthl1 D227Y Knockin Mice Exhibit Defective DNA Repair and Increased Genome Instability.

Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

Detection and structural analysis of pyrimidine-derived radicals generated on DNA using a profluorescent nitroxide probe.

The oxidative damage of DNA is associated with aging and the development of various diseases. Although nucleoside-derived radicals play an important role in DNA oxidation, their analysis methods are limited. Herein, we propose a fluorometric detection and structural analysis of radicals on the surface of oxidatively damaged DNA using a profluorescent nitroxide probe combined with liquid chromatography-fluorometry and high-resolution tandem mass spectrometry.

Protective effect of hawthorn vitexin on the ethanol-injured DNA of BRL-3A hepatocytes.

ABSTRACT: Vitexin is a natural active ingredient in hawthorn leaves, which has a wide range of anti-tumor effects. This study was conducted to assess the protective effect of hawthorn vitexin on the ethanol-injured DNA of hepatocytes in vitro and to explore its mechanism. The effect of different concentrations of hawthorn vitexin on ethanol-injured hepatocytes was detected via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method to study the protective effect of hawthorn vitexin on ethanol-injured DNA damage in hepatocytes. Single-cell gel electrophoresis was used to observe the effect of hawthorn vitexin on ethanol-induced DNA damage in hepatocytes, and the Olive tail moment was measured. Cell physiological and biochemical indexes, such as superoxide dismutase activity, malonaldehyde content, and glutathione peroxidase activity, were detected with kits. The mRNA expression of the superoxide dismutase gene was measured via real-time quantitative polymerase chain reaction. It was showed that 0.2, 0.4, and 0.8 mg mL-1 hawthorn vitexin could significantly repair hepatocyte growth and ethanol-induced DNA damage. This effect was closely related to the improvement in superoxide dismutase, malonaldehyde, and glutathione peroxidase. Hawthorn vitexin could be used to repair ethanol-injured hepatocytes through antioxidation effects, and showed potential for the treatment of liver injury.

An assessment of the mutational load caused by various reactions used in DNA encoded libraries.

DNA encoded libraries have become an essential hit-finding tool in early drug discovery. Recent advances in synthetic methods for DNA encoded libraries have expanded the available chemical space, but precisely how each type of chemistry affects the DNA is unstudied. Available assays to quantify the damage are limited to write efficiency, where the ability to ligate DNA onto a working encoded library strand is measured, or qPCR is performed to measure the amplifiability of the DNA. These measures read signal quantity and overall integrity, but do not report on specific damages in the encoded information. Herein, we use next generation sequencing (NGS) to measure the quality of the read signal in order to quantify the truthfulness of the retrieved information. We identify CuAAC to be the worst offender in terms of DNA damage amongst commonly used reactions in DELs, causing an increase of G â†’ T transversions. Furthermore, we show that the analysis provides useful information even in fully elaborated DELs; indeed we see that vestiges of the synthetic history, both chemical and biochemical, are written into the mutational spectra of NGS datasets.

Precious metal complexes of bis(pyridyl)allenes: synthesis and catalytic and medicinal applications.

The incorporation of donor-type substituents on the allene core opens up the possibility of coordination complexes in which the metal is bonded to the donor groups, with or without interaction with the double bond system. Despite the challenges in the synthesis of such allene-containing metal complexes, their unique 3D environments and dual functionality (allene and metal) could facilitate catalysis and interaction with chemical and biological systems. Bis(pyridyl)allenes are presented here as robust ligands for novel Pd(II), Pt(IV) and Au(III) complexes. Their synthesis, characterisation and first application as catalysts of benchmark reactions for Pd, Pt and Au are presented with interesting reactivity and selectivities. The complexes have also been probed as antimicrobial and anticancer agents with promising activities, and the first studies on their unusual interaction with several DNA structures will open new avenues for research in the area of metallodrugs with new mechanisms of action.

The Inhibition Effect of Photo-Cross-Linking between Probes in Photo-Induced Double Duplex Invasion DNA.

Double duplex invasion (DDI) DNA is a useful antigene method that inhibits expression of genomic DNA. We succeeded in performing photoinduced-DDI (pDDI) using ultrafast photo-cross-linking. 5-Cyanouracil (CN U) has been used in pDDI to inhibit photo-cross-linking between probes, but its importance has not been clarified. Therefore, in this study, we evaluated the effect of spacer (S) and d-spacer (dS) that exhibit photo-cross-linking ability similar to that of CN U. CN U exhibited the highest pDDI efficiency, and S, dS, and T were not very different. The photo-cross-linking inhibitory effect was better with S and dS than with thymidine (T). Conversely, the thermal stability was significantly lower with S and dS than with T. The results suggest that the pDDI efficiency is determined by the balance between the photo-cross-linking inhibitory effect and the thermal stability, which is the introduction efficiency for double-stranded DNA. Therefore, CN U, which has a photo-cross-linking inhibitory effect and a high Tm value, showed the highest inhibitory efficiency.

Biomarkers of DNA Oxidation Products: Links to Exposure and Disease in Public Health Studies.

Environmental exposure can increase the production of reactive oxygen species and deplete cellular antioxidants in humans, resulting in oxidatively generated damage to DNA that is both a useful biomarker of oxidative stress and indicator of carcinogenic hazard. Methods of oxidatively damaged DNA analysis have been developed and used in public health research since the 1990s. Advanced techniques detect specific lesions, but they might not be applicable to complex matrixes (e.g., tissues), small sample volume, and large-scale studies. The most reliable methods are characterized by (1) detecting relevant DNA oxidation products (e.g., premutagenic lesions), (2) not harboring technical problems, (3) being applicable to complex biological mixtures, and (4) having the ability to process a large number of samples in a reasonable period of time. Most effort has been devoted to the measurements of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG), which can be analyzed by chromatographic, enzymic, and antibody-based methods. Results from validation trials have shown that certain chromatographic and enzymic assays (namely the comet assay) are superior techniques. The enzyme-modified comet assay has been popular because it is technically simpler than chromatographic assays. It is widely used in public health studies on environmental exposures such as outdoor air pollution. Validated biomarker assays on oxidatively damaged DNA have been used to fill knowledge gaps between findings in prospective cohort studies and hazards from contemporary sources of air pollution exposures. Results from each of these research fields feed into public health research as approaches to conduct primary prevention of diseases caused by environmental or occupational agents.