Quantification of Intracellular DNA-Protein Cross-Links with N7-Methyl-2'-Deoxyguanosine and Their Contribution to Cytotoxicity.

The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo, but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPCMdG), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPCMdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.

Hybrid CdSe/ZnS Quantum Dot-Gold Nanoparticle Composites Assembled by Click Chemistry: Toward Affordable and Efficient Redox Photocatalysts Working with Visible Light.

A new modular, easy-to-synthesize photocatalyst was prepared by assembling colloidal CdSe/ZnS quantum dots (QD) and gold nanoparticles (AuNP) via their ligands thanks to copper-catalyzed azide to alkyne cycloaddition (CuAAC) click chemistry. The resulting composite (QD-AuNP) photocatalyst was tested with a benchmark photoredox system previously reported by our group, for which QD alone acted as a photocatalyst but with a modest quantum yield (QY = 0.06%) and turnover number (TON = 350 in 3 h) due to poor charge separation. After optimization, the QD-AuNP composites exhibited much improved photocatalytic performances: up to five times higher TON (2600 in 3 h) and up to 24 times faster reaction in the first 10 min of visible irradiation. Such an improvement is attributed to an efficient electron transfer from QD to AuNP in the photoexcited QD-AuNP composites, which ensures a much better charge separation than that in QD alone. This was confirmed by studying both (i) the quenching of the QD photoluminescence during the synthesis of the QD-AuNP composites and (ii) the blue shift of the AuNP plasmon absorption band due to the accumulation of up to 7400 electrons per AuNP in QD-AuNP composites under visible light irradiation in the presence of electron donors.

Perinatal foodborne titanium dioxide exposure-mediated dysbiosis predisposes mice to develop colitis through life.

BACKGROUND: Perinatal exposure to titanium dioxide (TiO2), as a foodborne particle, may influence the intestinal barrier function and the susceptibility to develop inflammatory bowel disease (IBD) later in life. Here, we investigate the impact of perinatal foodborne TiO2 exposure on the intestinal mucosal function and the susceptibility to develop IBD-associated colitis. Pregnant and lactating mother mice were exposed to TiO2 until pups weaning and the gut microbiota and intestinal barrier function of their offspring was assessed at day 30 post-birth (weaning) and at adult age (50 days). Epigenetic marks was studied by DNA methylation profile measuring the level of 5-methyl-2'-deoxycytosine (5-Me-dC) in DNA from colic epithelial cells. The susceptibility to develop IBD has been monitored using dextran-sulfate sodium (DSS)-induced colitis model. Germ-free mice were used to define whether microbial transfer influence the mucosal homeostasis and subsequent exacerbation of DSS-induced colitis. RESULTS: In pregnant and lactating mice, foodborne TiO2 was able to translocate across the host barriers including gut, placenta and mammary gland to reach embryos and pups, respectively. This passage modified the chemical element composition of foetus, and spleen and liver of mothers and their offspring. We showed that perinatal exposure to TiO2 early in life alters the gut microbiota composition, increases the intestinal epithelial permeability and enhances the colonic cytokines and myosin light chain kinase expression. Moreover, perinatal exposure to TiO2 also modifies the abilities of intestinal stem cells to survive, grow and generate a functional epithelium. Maternal TiO2 exposure increases the susceptibility of offspring mice to develop severe DSS-induced colitis later in life. Finally, transfer of TiO2-induced microbiota dysbiosis to pregnant germ-free mice affects the homeostasis of the intestinal mucosal barrier early in life and confers an increased susceptibility to develop colitis in adult offspring. CONCLUSIONS: Our findings indicate that foodborne TiO2 consumption during the perinatal period has negative long-lasting consequences on the development of the intestinal mucosal barrier toward higher colitis susceptibility. This demonstrates to which extent environmental factors influence the microbial-host interplay and impact the long-term mucosal homeostasis.

The thiolation of uridine 34 in tRNA, which controls protein translation, depends on a [4Fe-4S] cluster in the archaeum Methanococcus maripaludis.

Thiolation of uridine 34 in the anticodon loop of several tRNAs is conserved in the three domains of life and guarantees fidelity of protein translation. U34-tRNA thiolation is catalyzed by a complex of two proteins in the eukaryotic cytosol (named Ctu1/Ctu2 in humans), but by a single NcsA enzyme in archaea. We report here spectroscopic and biochemical experiments showing that NcsA from Methanococcus maripaludis (MmNcsA) is a dimer that binds a [4Fe-4S] cluster, which is required for catalysis. Moreover, the crystal structure of MmNcsA at 2.8 Å resolution shows that the [4Fe-4S] cluster is coordinated by three conserved cysteines only, in each monomer. Extra electron density on the fourth nonprotein-bonded iron most likely locates the binding site for a hydrogenosulfide ligand, in agreement with the [4Fe-4S] cluster being used to bind and activate the sulfur atom of the sulfur donor. Comparison of the crystal structure of MmNcsA with the AlphaFold model of the human Ctu1/Ctu2 complex shows a very close superposition of the catalytic site residues, including the cysteines that coordinate the [4Fe-4S] cluster in MmNcsA. We thus propose that the same mechanism for U34-tRNA thiolation, mediated by a [4Fe-4S]-dependent enzyme, operates in archaea and eukaryotes.

Structural and functional insights into the activation of the dual incision activity of UvrC, a key player in bacterial NER.

Bacterial nucleotide excision repair (NER), mediated by the UvrA, UvrB and UvrC proteins is a multistep, ATP-dependent process, that is responsible for the removal of a very wide range of chemically and structurally diverse DNA lesions. DNA damage removal is performed by UvrC, an enzyme possessing a dual endonuclease activity, capable of incising the DNA on either side of the damaged site to release a short single-stranded DNA fragment containing the lesion. Using biochemical and biophysical approaches, we have probed the oligomeric state, UvrB- and DNA-binding abilities and incision activities of wild-type and mutant constructs of UvrC from the radiation resistant bacterium, Deinococcus radiodurans. Moreover, by combining the power of new structure prediction algorithms and experimental crystallographic data, we have assembled the first model of a complete UvrC, revealing several unexpected structural motifs and in particular, a central inactive RNase H domain acting as a platform for the surrounding domains. In this configuration, UvrC is maintained in a 'closed' inactive state that needs to undergo a major rearrangement to adopt an 'open' active state capable of performing the dual incision reaction. Taken together, this study provides important insight into the mechanism of recruitment and activation of UvrC during NER.

Fine Tuning of Quantum Dots Photocatalysts for the Synthesis of Tropane Alkaloid Skeletons.

Several types of Quantum Dots (QDs) (CdS, CdSe and InP, as well as core-shell QDs such as type I InP-ZnS, quasi type-II CdSe-CdS and inverted type-I CdS-CdSe) were considered for generating α-aminoalkyl free radicals. The feasibility of the oxidation of the N-aryl amines and the generation of the desired radical was evidenced experimentally by quenching of the photoluminescence of the QDs and by testing a vinylation reaction using an alkenylsulfone radical trap. The QDs were tested in a radical [3+3]-annulation reaction giving access to tropane skeletons and that requires the completion of two consecutive catalytic cycles. Several QDs such as CdS core, CdSe core and inverted type I CdS-CdSe core-shell proved to be efficient photocatalysts for this reaction. Interestingly, the addition of a second shorter chain ligand to the QDs appeared to be essential to complete the second catalytic cycle and to obtain the desired bicyclic tropane derivatives. Finally, the scope of the [3+3]-annulation reaction was explored for the best performing QDs and isolated yields that compare well with classical iridium photocatalysis were obtained.

A noncanonical response to replication stress protects genome stability through ROS production, in an adaptive manner.

Cells are inevitably challenged by low-level/endogenous stresses that do not arrest DNA replication. Here, in human primary cells, we discovered and characterized a noncanonical cellular response that is specific to nonblocking replication stress. Although this response generates reactive oxygen species (ROS), it induces a program that prevents the accumulation of premutagenic 8-oxoguanine in an adaptive way. Indeed, replication stress-induced ROS (RIR) activate FOXO1-controlled detoxification genes such as SEPP1, catalase, GPX1, and SOD2. Primary cells tightly control the production of RIR: They are excluded from the nucleus and are produced by the cellular NADPH oxidases DUOX1/DUOX2, whose expression is controlled by NF-κB, which is activated by PARP1 upon replication stress. In parallel, inflammatory cytokine gene expression is induced through the NF-κB-PARP1 axis upon nonblocking replication stress. Increasing replication stress intensity accumulates DNA double-strand breaks and triggers the suppression of RIR by p53 and ATM. These data underline the fine-tuning of the cellular response to stress that protects genome stability maintenance, showing that primary cells adapt their responses to replication stress severity.

A subclass of archaeal U8-tRNA sulfurases requires a [4Fe-4S] cluster for catalysis.

Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC + C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe-4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe-4S] cluster is coordinated by the three cysteines of the CXXC + C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.

In vitro reconstitution of an efficient nucleotide excision repair system using mesophilic enzymes from Deinococcus radiodurans.

Nucleotide excision repair (NER) is a universal and versatile DNA repair pathway, capable of removing a very wide range of lesions, including UV-induced pyrimidine dimers and bulky adducts. In bacteria, NER involves the sequential action of the UvrA, UvrB and UvrC proteins to release a short 12- or 13-nucleotide DNA fragment containing the damaged site. Although bacterial NER has been the focus of numerous studies over the past 40 years, a number of key questions remain unanswered regarding the mechanisms underlying DNA damage recognition by UvrA, the handoff to UvrB and the site-specific incision by UvrC. In the present study, we have successfully reconstituted in vitro a robust NER system using the UvrABC proteins from the radiation resistant bacterium, Deinococcus radiodurans. We have investigated the influence of various parameters, including temperature, salt, protein and ATP concentrations, protein purity and metal cations, on the dual incision by UvrABC, so as to find the optimal conditions for the efficient release of the short lesion-containing oligonucleotide. This newly developed assay relying on the use of an original, doubly-labelled DNA substrate has allowed us to probe the kinetics of repair on different DNA substrates and to determine the order and precise sites of incisions on the 5' and 3' sides of the lesion. This new assay thus constitutes a valuable tool to further decipher the NER pathway in bacteria.

Reactivity of Singlet Oxygen with DNA, an Update.

The reactivity of singlet oxygen with DNA constituents and in particular with the guanine base has been studied during more than four decades but the exact mechanisms by which such a reactive oxygen species reacts with DNA is still a matter of debate. In this review article, a summary of the data that were obtained from several laboratories and using complementary experimental and theoretical approaches are presented. Reaction mechanisms of 1 O2 with guanine and its oxidation product 8-oxo7,8-dihydroguanine are presented both at the nucleoside level and when the base is inserted into DNA since significant differences have been observed. Efforts have been made to propose tentative mechanisms to explain the conflicting results that were sometimes reported and hypotheses have been put forward to tentatively explain still contradictory observations.

Iron-sulfur biology invades tRNA modification: the case of U34 sulfuration.

Sulfuration of uridine 34 in the anticodon of tRNAs is conserved in the three domains of life, guaranteeing fidelity of protein translation. In eubacteria, it is catalyzed by MnmA-type enzymes, which were previously concluded not to depend on an iron-sulfur [Fe-S] cluster. However, we report here spectroscopic and iron/sulfur analysis, as well as in vitro catalytic assays and site-directed mutagenesis studies unambiguously showing that MnmA from Escherichia coli can bind a [4Fe-4S] cluster, which is essential for sulfuration of U34-tRNA. We propose that the cluster serves to bind and activate hydrosulfide for nucleophilic attack on the adenylated nucleoside. Intriguingly, we found that E. coli cells retain s2U34 biosynthesis in the ΔiscUA ΔsufABCDSE strain, lacking functional ISC and SUF [Fe-S] cluster assembly machineries, thus suggesting an original and yet undescribed way of maturation of MnmA. Moreover, we report genetic analysis showing the importance of MnmA for sustaining oxidative stress.

Radiation Dose-Enhancement Is a Potent Radiotherapeutic Effect of Rare-Earth Composite Nanoscintillators in Preclinical Models of Glioblastoma.

To improve the prognosis of glioblastoma, innovative radiotherapy regimens are required to augment the effect of tolerable radiation doses while sparing surrounding tissues. In this context, nanoscintillators are emerging radiotherapeutics that down-convert X-rays into photons with energies ranging from UV to near-infrared. During radiotherapy, these scintillating properties amplify radiation-induced damage by UV-C emission or photodynamic effects. Additionally, nanoscintillators that contain high-Z elements are likely to induce another, currently unexplored effect: radiation dose-enhancement. This phenomenon stems from a higher photoelectric absorption of orthovoltage X-rays by high-Z elements compared to tissues, resulting in increased production of tissue-damaging photo- and Auger electrons. In this study, Geant4 simulations reveal that rare-earth composite LaF3:Ce nanoscintillators effectively generate photo- and Auger-electrons upon orthovoltage X-rays. 3D spatially resolved X-ray fluorescence microtomography shows that LaF3:Ce highly concentrates in microtumors and enhances radiotherapy in an X-ray energy-dependent manner. In an aggressive syngeneic model of orthotopic glioblastoma, intracerebral injection of LaF3:Ce is well tolerated and achieves complete tumor remission in 15% of the subjects receiving monochromatic synchrotron radiotherapy. This study provides unequivocal evidence for radiation dose-enhancement by nanoscintillators, eliciting a prominent radiotherapeutic effect. Altogether, nanoscintillators have invaluable properties for enhancing the focal damage of radiotherapy in glioblastoma and other radioresistant cancers.

Dose-dependent genomic DNA hypermethylation and mitochondrial DNA damage in Japanese tree frogs sampled in the Fukushima Daiichi area.

The long-term consequences of the nuclear disaster at the Fukushima Daiichi Nuclear Power Plant (FDNPP) that occurred on March 2011, have been scarcely studied on wildlife. We sampled Japanese tree frogs (Dryophytes japonicus), in a 50 -km area around the FDNPP to test for an increase of DNA damages and variation of DNA methylation level. The ambient dose rate ranged between 0.4 and 2.8 µGy h-1 and the total estimated dose rate absorbed by frogs ranged between 0.3 and 7.7 µGy h-1. Frogs from contaminated sites exhibited a dose-dependent increase of global genomic DNA methylation level (5-mdC and 5-hmdC) and of mitochondrial DNA damages. Such DNA damages may indicate a genomic instability, which may induce physiological adaptations governed by DNA methylation changes. This study stresses the need for biological data combining targeted molecular methods and classic ecotoxicology, in order to better understand the impacts on wildlife of long term exposure to low ionizing radiation levels.

Impairment of Base Excision Repair in Dermal Fibroblasts Isolated From Nevoid Basal Cell Carcinoma Patients.

The nevoid basal cell carcinoma syndrome (NBCCS), also called Gorlin syndrome is an autosomal dominant disorder whose incidence is estimated at about 1 per 55,600-256,000 individuals. It is characterized by several developmental abnormalities and an increased predisposition to the development of basal cell carcinomas (BCCs). Cutaneous fibroblasts from Gorlin patients have been shown to exhibit an increased sensitivity to ionizing radiations. Mutations in the tumor suppressor gene PTCH1, which is part of the Sonic Hedgehog (SHH) signaling pathway, are responsible for these clinical manifestations. As several genetic mutations in the DNA repair genes are responsible of photo or radiosensitivity and high predisposition to cancers, we hypothesized that these effects in Gorlin syndrome might be due to a defect in the DNA damage response (DDR) and/or the DNA repair capacities. Therefore, the objective of this work was to investigate the sensitivity of skin fibroblasts from NBCCS patients to different DNA damaging agents and to determine the ability of these agents to modulate the DNA repair capacities. Gorlin fibroblasts showed high radiosensitivity and also less resistance to oxidative stress-inducing agents when compared to control fibroblasts obtained from healthy individuals. Gorlin fibroblasts harboring PTCH1 mutations were more sensitive to the exposure to ionizing radiation and to UVA. However, no difference in cell viability was shown after exposure to UVB or bleomycin. As BER is responsible for the repair of oxidative DNA damage, we decided to assess the BER pathway efficacy in Gorlin fibroblasts. Interestingly, a concomitant decrease of both BER gene expression and BER protein activity was observed in Gorlin fibroblasts when compared to control. Our results suggest that low levels of DNA repair within Gorlin cells may lead to an accumulation of oxidative DNA damage that could participate and partly explain the radiosensitivity and the BCC-prone phenotype in Gorlin syndrome.

Chromatin recruitment of OGG1 requires cohesin and mediator and is essential for efficient 8-oxoG removal.

One of the most abundant DNA lesions induced by oxidative stress is the highly mutagenic 8-oxoguanine (8-oxoG), which is specifically recognized by 8-oxoguanine DNA glycosylase 1 (OGG1) to initiate its repair. How DNA glycosylases find small non-helix-distorting DNA lesions amongst millions of bases packaged in the chromatin-based architecture of the genome remains an open question. Here, we used a high-throughput siRNA screening to identify factors involved in the recognition of 8-oxoG by OGG1. We show that cohesin and mediator subunits are required for re-localization of OGG1 and other base excision repair factors to chromatin upon oxidative stress. The association of OGG1 with euchromatin is necessary for the removal of 8-oxoG. Mediator subunits CDK8 and MED12 bind to chromatin and interact with OGG1 in response to oxidative stress, suggesting they participate in the recruitment of the DNA glycosylase. The oxidative stress-induced association between the cohesin and mediator complexes and OGG1 reveals an unsuspected function of those complexes in the maintenance of genomic stability.

A "Population Dynamics" Perspective on the Delayed Life-History Effects of Environmental Contaminations: An Illustration with a Preliminary Study of Cadmium Transgenerational Effects over Three Generations in the Crustacean Gammarus.

We explore the delayed consequences of parental exposure to environmentally relevant cadmium concentrations on the life-history traits throughout generations of the freshwater crustacean Gammarus fossarum. We report the preliminary results obtained during a challenging one-year laboratory experiment in this environmental species and propose the use of population modeling to interpret the changes in offspring life-history traits regarding their potential demographic impacts. The main outcome of this first long-term transgenerational assay is that the exposure of spawners during a single gametogenesis cycle (3 weeks) could result in severe cascading effects on the life-history traits along three unexposed offspring generations (one year). Indeed, we observed a decrease in F1 reproductive success, an early onset of F2 offspring puberty with reduced investment in egg yolk reserves, and finally a decrease in the growth rate of F3 juveniles. However, the analysis of these major transgenerational effects by means of a Lefkovitch matrix population model revealed only weak demographic impacts. Population compensatory processes mitigating the demographic consequences of parental exposure seem to drive the modification of life-history traits in offspring generations. This exploratory study sheds light on the role of population mechanisms involved in the demographic regulation of the delayed effects of environmental toxicity in wild populations.

Solid-phase synthesis of branched oligonucleotides containing a biologically relevant dCyd341 interstrand crosslink DNA lesion.

Branched oligonucleotides containing a biologically relevant DNA lesion, dCyd341, which involves an interstrand crosslink between a cytosine base on one strand and a ribose moiety on the opposite strand, were prepared in a single automated solid-phase synthesis. For this, we first prepared the phosphoramidite analogue of dCyd341 bearing an orthogonal levulinyl protecting group. Then, following the synthesis of the first DNA strand containing dCyd341, the levulinic group was removed and the synthesis was then continued from the free base hydroxyl group at the branching point, using traditional phosphoramidites. The synthesized oligonucleotides were fully characterized by MALDI-TOF/MS and were enzymatically digested, and the presence of the lesion was confirmed by HPLC-MS/MS and the sequence was finally controlled upon exonuclease digestion followed by MALDI-TOF/MS analysis. The developed strategy was successfully employed for the preparation of several short linear and branched oligonucleotides containing the aforementioned lesion.

Probing interaction of a trilysine peptide with DNA underlying formation of guanine-lysine cross-links: insights from molecular dynamics.

DNA-protein cross-links constitute bulky DNA lesions that interfere with the cellular machinery. Amongst these stable covalently tethered adducts, the efficient nucleophilic addition of the free amino group of lysines onto the guanine radical cation has been evidenced. In vitro addition of a trilysine peptide onto a guanine radical cation generated in a TGT oligonucleotide is so efficient that competitive addition of a water molecule, giving rise to 8-oxo-7,8-dihydroguanine, is not observed. This suggests a spatial proximity between guanine and lysine for the stabilization of the prereactive complex. We report all-atom microsecond scale molecular dynamics simulations that probe the structure and interactions of the trilysine peptide (KKK) with two oligonucleotides. Our simulations reveal a strong, electrostatically driven yet dynamic interaction, spanning several association modes. Furthermore, the presence of neighbouring cytosines has been identified as a factor favoring KKK binding. Relying on ab initio molecular dynamics on a model system constituted of guanine and methylammonium, we also corroborate a mechanistic pathway involving fast deprotonation of the guanine radical cation followed by hydrogen transfer from ammonium leaving as a result a nitrogen reactive species that can subsequently cross-link with guanine. Our study sheds new light on a ubiquitous mechanism for DNA-protein cross-links also stressing out possible sequence dependences.