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Environmental, endogenous and therapeutic alkylating agents can react with internucleotide phosphate groups in DNA to yield alkyl phosphotriester (PTE) adducts. Alkyl-PTEs are induced at relatively high frequencies and are persistent in mammalian tissues; however, their biological consequences in mammalian cells have not been examined. Herein, we assessed how alkyl-PTEs with different alkyl group sizes and stereochemical configurations (S P and R P diastereomers of Me and nPr) affect the efficiency and fidelity of transcription in mammalian cells. We found that, while the R P diastereomer of Me- and nPr-PTEs constituted moderate and strong blockages to transcription, respectively, the S P diastereomer of the two lesions did not appreciably perturb transcription efficiency. In addition, none of the four alkyl-PTEs induced mutant transcripts. Furthermore, polymerase η assumed an important role in promoting transcription across the S P-Me-PTE, but not any of other three lesions. Loss of other translesion synthesis (TLS) polymerases tested, including Pol κ, Pol ι, Pol ξ and REV1, did not alter the transcription bypass efficiency or mutation frequency for any of the alkyl-PTE lesions. Together, our study provided important new knowledge about the impact of alkyl-PTE lesions on transcription and expanded the substrate pool of Pol η in transcriptional bypass.

To cope with unrepaired DNA lesions, cells are equipped with DNA damage tolerance mechanisms, including translesion synthesis (TLS). While TLS polymerases are well documented in facilitating replication across damaged DNA templates, it remains unknown whether TLS polymerases participate in transcriptional bypass of DNA lesions in cells. Herein, we employed the competitive transcription and adduct bypass assay to examine the efficiencies and fidelities of transcription across N2-alkyl-2-deoxyguanosine (N2-alkyl-dG, alkyl = methyl, ethyl, n-propyl, or n-butyl) lesions in HEK293T cells. We found that N2-alkyl-dG lesions strongly blocked transcription and elicited CC → AA tandem mutations in nascent transcripts, where adenosines were misincorporated opposite the lesions and their adjacent 5 nucleoside. Additionally, genetic ablation of Pol η, but not Pol κ, Pol ι, or Pol ζ, conferred marked diminutions in the transcriptional bypass efficiencies of the N2-alkyl-dG lesions, which is exacerbated by codepletion of Rev1 in Pol η-deficient background. We also observed that the repair of N2-nBu-dG was not pronouncedly affected by genetic depletion of Pol η or Rev1. Hence, our results provided insights into transcriptional perturbations induced by N2-alkyl-dG lesions and expanded the biological functions of TLS DNA polymerases.

Chemically induced DNA adducts can lead to mutations and cancer. Unfortunately, because common analytical methods (e.g., liquid chromatography-mass spectrometry) require adducts to be digested or liberated from DNA before quantification, information about their positions within the DNA sequence is lost. Advances in nanopore sequencing technologies allow individual DNA molecules to be analyzed at single-nucleobase resolution, enabling us to study the dynamic of epigenetic modifications and exposure-induced DNA adducts in their native forms on the DNA strand. We applied and evaluated the commercially available Oxford Nanopore Technology (ONT) sequencing platform for site-specific detection of DNA adducts and for distinguishing individual alkylated DNA adducts. Using ONT and the publicly available ELIGOS software, we analyzed a library of 15 plasmids containing site-specifically inserted O6- or N2-alkyl-2-deoxyguanosine lesions differing in sizes and regiochemistries. Positions of DNA adducts were correctly located, and individual DNA adducts were clearly distinguished from each other.

Exogenous and endogenous chemicals can react with DNA to produce DNA lesions that may block DNA replication. Not much is known about the roles of polymerase (Pol) ν and Pol θ in translesion synthesis (TLS) in cells. Here we examined the functions of these two polymerases in bypassing major-groove O6-alkyl-2-deoxyguanosine (O6-alkyl-dG) and minor-groove N2-alkyl-dG lesions in human cells, where the alkyl groups are ethyl, n-butyl (nBu), and, for O6-alkyl-dG, pyridyloxobutyl. We found that Pol ν and Pol θ promote TLS across major-groove O6-alkyl-dG lesions. O6-alkyl-dG lesions mainly induced G→A mutations that were modulated by the two TLS polymerases and the structures of the alkyl groups. Simultaneous ablation of Pol ν and Pol θ resulted in diminished mutation frequencies for all three O6-alkyl-dG lesions. Depletion of Pol ν alone reduced mutations only for O6-nBu-dG, and sole loss of Pol θ attenuated the mutation rates for O6-nBu-dG and O6-pyridyloxobutyl-dG. Replication across the two N2-alkyl-dG lesions was error-free, and Pol ν and Pol θ were dispensable for their replicative bypass. Together, our results provide critical knowledge about the involvement of Pol ν and Pol θ in bypassing alkylated guanine lesions in human cells.

Drug resistance is a major challenge in the treatment of breast cancer. Many causes and mechanisms lead to the occurrence of drug resistance in breast cancer. Exosomes and their contents (DNA, mRNA, protein, and non-coding RNA) are important mediators of intercellular communication and play a role in tumor progression, metastasis, and recurrence. Among them, non-coding RNA carried by exosomes plays a crucial role in limiting drug efficacy. This article reviews the latest research progress on the relationship between exosomal non-coding RNA and drug resistance of breast cancer at home and abroad.

Endogenous metabolites and exogenous chemicals can induce covalent modifications on DNA, producing DNA lesions. The N2 of guanine was shown to be a common alkylation site in DNA; however, not much is known about the influence of the size of the alkyl group in N2-alkyldG lesions on cellular DNA replication or how translesion synthesis (TLS) polymerases modulate DNA replication past these lesions in human cells. To answer these questions, we employ a robust shuttle vector method to investigate the impact of four N2-alkyldG lesions (i.e., with the alkyl group being a methyl, ethyl, n-propyl, or n-butyl group) on DNA replication in human cells. We find that replication through the N2-alkyldG lesions was highly efficient and accurate in HEK293T cells or isogenic CRISPR-engineered cells with deficiency in polymerase (Pol) ζ or Pol η. Genetic ablation of Pol ι, Pol κ, or Rev1, however, results in decreased bypass efficiencies and elicits substantial frequencies of G → A transition and G → T transversion mutations for these lesions. Moreover, further depletion of Pol ζ in Pol κ- or Pol ι-deficient cells gives rise to elevated rates of G → A and G → T mutations and substantially decreased bypass efficiencies. Cumulatively, we demonstrate that the error-free replication past the N2-alkyldG lesions is facilitated by a specific subset of TLS polymerases, and we find that longer alkyl chains in these lesions induce diminished bypass efficiency and fidelity in DNA replication.

Genomic integrity is constantly challenged by exposure to environmental and endogenous genotoxic agents. Reactive oxygen species (ROS) represent one of the most common types of DNA damaging agents. While ROS mainly induce single-nucleobase lesions, epimeric 2-deoxyribose lesions can also be induced upon hydrogen atom abstraction from the C1, C3, or C4 carbon and the subsequent incorrect chemical repair of the resulting carbon-centered radicals. Herein, we investigated the replicative bypass of the C1- and C3-epimeric lesions of the four 2-deoxynucleosides in HEK293T human embryonic kidney epithelial cells. Our results revealed distinct bypass efficiencies and mutagenic properties of these two types of epimeric lesions. Replicative bypasses of all C1-epimeric lesions except α-dA are mutagenic in HEK293T cells, and their mutagenic properties are further modulated by translesion synthesis (TLS) DNA polymerases. By contrast, none of the four C3-epimeric lesions are mutagenic, and the replicative bypass of these lesions is not compromised upon depletion of polymerase η, ι, κ, or ζ. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of C1 and C3 epimeric lesions, and reveal the roles of TLS DNA polymerases in bypassing these lesions in human cells.

As one of CFETR’s candidate blankets, water cooled ceramic breeder (WCCB) blanket undertakes important nuclear-related functions such as tritium breeding, nuclear heat extraction, and neutron shielding. The reliability of neutronics design directly affects the realization of CFETR tritium self-sustaining goal. In order to verify the reliability of the neutronics design tool, i.e. MCNP and FNEDL databases, in the WCCB blanket neutronics design, the WCCB scaling-down mock up was developed to carry out the neutronics experiment in the DT neutron environment, including two layers of lithium titanate, two layers of beryllium, three layers of cooling plate, and one layer of tungsten armor. The neutronics performance of the mock up are similar with the real blanket module. The mock up neutronics experiments were carried out using DT neutron generator, the simulated value (C) and experimental value (E) of neutronics parameters represented by tritium production rate (TPR) were analyzed. For TPR validation, two independent detection technologies were used, including the Li2TiO3 pellets as off-line TPR detectors, and the developed lithium glass detectors as on-line TPR detectors. The TPR validation was complemented by evaluating the predicted neutron-induced reaction rates of the Au, Zr foils. The experiments on the mock up were analyzed by MCNP-4C code and FENDL-3.0 nuclear data libraries, and the source term for Monte Carlo simulation was built using the custom-developed model based on depth profiling of tritium in the tritide target. The analysis results show that the C/E of TPR in the axis of two lithium titanate layers is 0.97 and 1.08 respectively, while there is a large deviation between the calculated and experimental values of TPR in the edge region of the mock up. According to the experimental results from Li2TiO3 pellets, the C/E of TPR at the edge of two lithium titanate layers is 0.65 and 0.82, respectively. The TPR is obviously underestimated by simulation at the edge of lithium titanate layers, confirmed by the C/E results of 197Au(n, γ)198Au reaction rate at the edge of lithium titanate layers, which are 0.72 and 0.90, respectively. It is indicated that there are unexpected scattered neutrons in the edge region of the mock up, resulting in the higher TPR than expected. The further experiments are needed to verify the influence of the scattered neutron on the TPR at the edge region of the mock up. To investigate the TPR gap between simulation and the experimental results from lithium glass detectors, the contribution of the fast neutrons on the 6Li(n, t)4He should be further studied. Also the other suggestions are provided for further experimental research, refering to the radiation protections and nuclear measurements.