Methodologies for the detection and sequencing of the epigenetic-like oxidative DNA modification, 8-oxo-7,8-dihydroguanine.

The human genome is constantly threatened by endogenous and environmental DNA damaging agents that can induce a variety of chemically modified DNA lesions including 8-oxo-7,8-dihydroguanine (OG). Increasing evidence has indicated that OG is not only a biomarker for oxidative DNA damage but also a novel epigenetic-like modification involved in regulation of gene expression in mammalian cells. Here we summarize the recent progress in OG research focusing on the following points: (i) the mechanism of OG production in organisms and its biological consequences in cells, (ii) the accurate identification of OG in low-abundance genomes and complex biological backgrounds, (iii) the development of OG sequencing methods.

The Comparison Between Traditional Versus 3D Printing Combined With Computer Navigation Technique in the Management of Orbital Blowout Fractures.

Background: Three-dimensional printing (3D printing) technology and computer navigation technology have been gradually applied in surgeries for orbital blowout fractures. This study compared the efficacy of traditional techniques (group I) and 3D printing combined with computer navigation technology-assisted techniques (group II) in the management of orbital blowout fractures.

Methods: All patients treated for orbital blowout fractures in the Affiliated Hospital of Yangzhou University from March 2018 to February 2021 were reviewed.

Photothermal Synergistic Effect Induces Bimetallic Cooperation to Modulate Product Selectivity of CO Reduction on Different CeO Crystal Facets.

Product selectivity of solar-driven CO reduction and HO oxidation reactions has been successfully controlled by tuning the spatial distance between Pt/Au bimetallic active sites on different crystal facets of CeO catalysts. The replacement depth of Ce atoms by monatomic Pt determines the distance between bimetallic sites, while Au clusters are deposited on the surface. This space configuration creates a favourable microenvironment for the migration of active hydrogen species (*H).

Concentrated solar CO reduction in HO vapour with >1% energy conversion efficiency.

HO dissociation plays a crucial role in solar-driven catalytic CO methanation, demanding high temperature even for solar-to-chemical conversion efficiencies <1% with modest product selectivity. Herein, we report an oxygen-vacancy (V) rich CeO catalyst with single-atom Ni anchored around its surface V sites by replacing Ce atoms to promote HO dissociation and achieve effective photothermal CO reduction under concentrated light irradiation. The high photon flux reduces the apparent activation energy for CH production and prevents V from depletion.

Development of an Endonuclease V-Assisted Analytical Method for Sequencing Analysis of Deoxyinosine in DNA.

Deoxyinosine (dI) is a highly mutagenic lesion that preferentially pairs with deoxycytidine during replication, which may induce A to G transition and ultimately contribute to carcinogenesis. Therefore, finding the site of dI modification in DNA is of great value for both basic research and clinical applications. Herein, we developed a novel method to sequence the dI modification site in DNA, which utilizes endonuclease V (EndoV)-dependent deamination repair to specifically label the modification site with biotin-14-dATP that allows the affinity enrichment of dI-bearing DNA for sequencing.

Next-Generation Sequencing-Based Analysis of the Effects of - and -Methyldeoxyadenosine Adducts on DNA Transcription.

DNA methylation can occur naturally or be induced by various environmental and chemotherapeutic agents. The regioisomeric - and -methyldeoxyadenosine (1mdA and 6mdA, respectively) represent an important class of methylated DNA adducts. In this study, we developed a shuttle vector- and next-generation sequencing-based assay to quantitatively assess the effects of 1mdA and 6mdA on the accuracy and efficiency of DNA transcription.

Next-Generation Sequencing-Based Analysis of the Roles of DNA Polymerases ν and θ in the Replicative Bypass of 8-Oxo-7,8-dihydroguanine in Human Cells.

DNA polymerase (Pol) ν and Pol θ are two specialized A-family DNA polymerases that function in the translesion synthesis of certain DNA lesions. However, the biological functions of human Pols ν and θ in cellular replicative bypass of 8-oxo-7,8-dihydroguanine (8-oxoG), an important carcinogenesis-related biomarker of oxidative DNA damage, remain unclear. Herein, we showed that depletion of Pols ν and θ in human cells could cause an elevated hypersensitivity to oxidative stress induced by hydrogen peroxide.

Transcriptional Perturbations of 2,6-Diaminopurine and 2-Aminopurine.

2,6-Diaminopurine (Z) is a naturally occurring adenine (A) analog that bacteriophages employ in place of A in their genetic alphabet. Recent discoveries of biogenesis pathways of Z in bacteriophages have stimulated substantial research interest in this DNA modification. Here, we systematically examined the effects of Z on the efficiency and fidelity of DNA transcription.

DNA Polymerase η Promotes the Transcriptional Bypass of -Alkyl-2'-deoxyguanosine Adducts in Human Cells.

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 -alkyl-2'-deoxyguanosine (-alkyl-dG, alkyl = methyl, ethyl, -propyl, or -butyl) lesions in HEK293T cells.

YTHDF2 Binds to 5-Methylcytosine in RNA and Modulates the Maturation of Ribosomal RNA.

5-Methylcytosine is found in both DNA and RNA; although its functions in DNA are well established, the exact role of 5-methylcytidine (mC) in RNA remains poorly defined. Here we identified, by employing a quantitative proteomics method, multiple candidate recognition proteins of mC in RNA, including several YTH domain-containing family (YTHDF) proteins. We showed that YTHDF2 could bind directly to mC in RNA, albeit at a lower affinity than that toward -methyladenosine (mA) in RNA, and this binding involves Trp, a conserved residue located in the hydrophobic pocket of YTHDF2 that is also required for mA recognition.

Engineering a 3D DNA-Logic Gate Nanomachine for Bispecific Recognition and Computing on Target Cell Surfaces.

Among the vast number of recognition molecules, DNA aptamers generated from cell-SELEX exhibit unique properties for identifying cell membrane biomarkers, in particular protein receptors on cancer cells. To integrate all recognition and computing modules within a single structure, a three-dimensional (3D) DNA-based logic gate nanomachine was constructed to target overexpressed cancer cell biomarkers with bispecific recognition. Thus, when the Boolean operator "AND" returns a true value, it is followed by an "ON" signal when the specific cell type is presented.

Cytotoxic and mutagenic properties of minor-groove -alkylthymidine lesions in human cells.

Endogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove -alkylthymidine (-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven -alkyldT lesions, with the alkyl group being a Me, Et, Pr, Pr, Bu, Bu, or Bu, are recognized by the DNA replication machinery in human cells.

Position-dependent effects of regioisomeric methylated adenine and guanine ribonucleosides on translation.

Reversible methylation of the N6 or N1 position of adenine in RNA has recently been shown to play significant roles in regulating the functions of RNA. RNA can also be alkylated upon exposure to endogenous and exogenous alkylating agents. Here we examined how regio-specific methylation at the hydrogen bonding edge of adenine and guanine in mRNA affects translation.

Replication studies of carboxymethylated DNA lesions in human cells.

Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.

Translesion synthesis of O4-alkylthymidine lesions in human cells.

Environmental exposure, endogenous metabolism and cancer chemotherapy can give rise to alkylation of DNA, and the resulting alkylated thymidine (alkyldT) lesions were found to be poorly repaired and persistent in mammalian tissues. Unrepaired DNA lesions may compromise genomic integrity by inhibiting DNA replication and inducing mutations in these processes. In this study, we explored how eight O-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, (R)-sBu and (S)-sBu, are recognized by DNA replication machinery in HEK293T human embryonic kidney cells.

The Functions of Serine 687 Phosphorylation of Human DNA Polymerase η in UV Damage Tolerance.

DNA polymerase η (polη) is a Y-family translesion synthesis polymerase that plays a key role in the cellular tolerance toward UV irradiation-induced DNA damage. Here, we identified, for the first time, the phosphorylation of serine 687 (Ser(687)), which is located in the highly conserved nuclear localization signal (NLS) region of human polη and is mediated by cyclin-dependent kinase 2 (CDK2). We also showed that this phosphorylation is stimulated in human cells upon UV light exposure and results in diminished interaction of polη with proliferating cell nuclear antigen (PCNA).

Roles of Aag, Alkbh2, and Alkbh3 in the Repair of Carboxymethylated and Ethylated Thymidine Lesions.

Environmental and endogenous genotoxic agents can result in a variety of alkylated and carboxymethylated DNA lesions, including N3-ethylthymidine (N3-EtdT), O(2)-EtdT, and O(4)-EtdT as well as N3-carboxymethylthymidine (N3-CMdT) and O(4)-CMdT. By using nonreplicative double-stranded vectors harboring a site-specifically incorporated DNA lesion, we assessed the potential roles of alkyladenine DNA glycosylase (Aag); alkylation repair protein B homologue 2 (Alkbh2); or Alkbh3 in modulating the effects of N3-EtdT, O(2)-EtdT, O(4)-EtdT, N3-CMdT, or O(4)-CMdT on DNA transcription in mammalian cells. We found that the depletion of Aag did not significantly change the transcriptional inhibitory or mutagenic properties of all five examined lesions, suggesting a negligible role of Aag in the repair of these DNA adducts in mammalian cells.

Mass Spectrometry-Based Quantitative Strategies for Assessing the Biological Consequences and Repair of DNA Adducts.

The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of chemically modified DNA lesions. Unrepaired DNA lesions may elicit cytotoxic and mutagenic effects and contribute to the development of human diseases including cancer and neurodegeneration. Understanding the deleterious outcomes of DNA damage necessitates the investigation about the effects of DNA adducts on the efficiency and fidelity of DNA replication and transcription.

Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo.

Aberrant transcription induced by DNA damage may confer risk for the development of cancer and other human diseases. Traditional methods for measuring lesion-induced transcriptional alterations often involve extensive colony screening and DNA sequencing procedures. Here we describe a protocol for the quantitative assessment of the effects of DNA lesions on the efficiency and fidelity of transcription in vitro and in mammalian cells.

Identification and Functional Characterizations of N-Terminal α-N-Methylation and Phosphorylation of Serine 461 in Human Poly(ADP-ribose) Polymerase 3.

Poly(ADP-ribose) polymerase 3 (PARP3) is a member of the PARP family enzymes which catalyze the ADP-ribosylation of proteins. PARP3 plays an important role in DNA damage repair and mitotic progression. In this study, we identified, using mass spectrometric techniques, two novel post-translational modification sites in PARP3, α-N-methylation and phosphorylation of serine 461 (S461).

Transcriptional inhibition and mutagenesis induced by N-nitroso compound-derived carboxymethylated thymidine adducts in DNA.

N-nitroso compounds represent a common type of environmental and endogenous DNA-damaging agents. After metabolic activation, many N-nitroso compounds are converted into a diazoacetate intermediate that can react with nucleobases to give carboxymethylated DNA adducts such as N3-carboxymethylthymidine (N3-CMdT) and O(4)-carboxymethylthymidine (O(4)-CMdT). In this study, we constructed non-replicative plasmids carrying a single N3-CMdT or O(4)-CMdT, site-specifically positioned in the transcribed strand, to investigate how these lesions compromise the flow of genetic information during transcription.

Transcriptional bypass of regioisomeric ethylated thymidine lesions by T7 RNA polymerase and human RNA polymerase II.

Alkylative damage to DNA can be induced by environmental chemicals, endogenous metabolites and some commonly prescribed chemotherapeutic agents. The regioisomeric N3-, O(2)- and O(4)-ethylthymidine (N3-, O(2)- and O(4)-EtdT, respectively) represent an important class of ethylated DNA lesions. Using nonreplicative double-stranded vectors containing an N3-EtdT, O(2)-EtdT or O(4)-EtdT at a defined site in the template strand, herein we examined the effects of these lesions on DNA transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells.