In dinoflagellates, a unique and extremely divergent genomic and nuclear organization has evolved. The highly unusual features of dinoflagellate nuclei and genomes include permanently condensed liquid crystalline chromosomes, primarily packaged by proteins other than histones, genes organized in very long unidirectional gene arrays, a general absence of transcriptional regulation, high abundance of the otherwise very rare DNA modification 5-hydroxymethyluracil (5-hmU), and many others. While most of these fascinating properties are originally identified in the 1970s and 1980s, they have not yet been investigated using modern genomic tools.
In this work, we address some of the outstanding questions regarding dinoflagellate genome organization by mapping the genome-wide distribution of 5-hmU (using both immunoprecipitation-based and basepair-resolution chemical mapping approaches) and of chromatin accessibility in the genome of the Symbiodiniaceae dinoflagellate Breviolum minutum. We find that the 5-hmU modification is preferentially enriched over certain classes of repetitive elements, often coincides with the boundaries between gene arrays, and is generally correlated with decreased chromatin accessibility, the latter otherwise being largely uniform along the genome. We discuss the potential roles of 5-hmU in the functional organization of dinoflagellate genomes and its relationship to the transcriptional landscape of gene arrays.
Our results provide the first window into the 5-hmU and chromatin accessibility landscapes in dinoflagellates.

5-Hydroxymethyluracil (5hmU) is an oxidation derivative of thymine in the genomes of various organisms and may serve as both an epigenetic mark and a cancer biomarker. However, the current 5hmU assays usually have drawbacks of laborious procedures, low specificity, and unsatisfactory sensitivity. Herein, we demonstrate the click chemistry-mediated hyperbranched amplification-driven dendritic nanoassembly for genome-wide analysis of 5hmU in breast cell lines and human breast tissues. The proposed strategy possesses good selectivity, ultralow background, and high sensitivity with a detection limit of 83.28 aM. This method can accurately detect even a 0.001% 5hmU level in the mixture. Moreover, it can determine 5hmU at single-cell level and distinguish the expressions of 5hmU in tissues of normal persons and breast cancer patients, holding great promise in 5hmU-related biological research and clinical diagnosis.

For many decades, uracil has been an antineoplastic agent used in combination with tegafur to treat various human cancers, including breast, prostate, and liver cancer. Therefore, it is necessary to explore the molecular features of uracil and its derivatives. Herein, the molecule\'s 5-hydroxymethyluracil has been thoroughly characterized by NMR, UV-Vis, and FT-IR spectroscopy by means of experimental and theoretical analysis. Density functional theory (DFT) using the B3LYP method at 6-311++G(d,p) was computed to achieve the optimized geometric parameters of the molecule in the ground state. For further investigation and computation of the NLO, NBO, NHO analysis, and FMO, the improved geometrical parameters were utilized. The potential energy distribution was used to allocate the vibrational frequencies using the VEDA 4 program. The NBO study determined the relationship between the donor and acceptor. The molecule\'s charge distribution and reactive regions were highlighted using the MEP and Fukui functions. Maps of the hole and electron density distribution in the excited state were generated using the TD-DFT method and PCM solvent model in order to reveal electronic characteristics. The energies and diagrams for the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) were also provided. The HOMO-LUMO band gap estimated the charge transport within the molecule. When examining the intermolecular interactions in 5-HMU, Hirshfeld surface analysis was used, and fingerprint plots were also produced. The molecular docking investigation involved docking 5-HMU with six different protein receptors. Molecular dynamic simulation has given a better idea of the binding of the ligand with protein.

5-hydroxymethyluracil was originally identified as an oxidatively modified DNA base derivative. Recent evidence suggests that its formation may result from the oxidation of thymine in a reaction that is catalyzed by TET proteins. Alternatively, it could be generated through the deamination of 5-hydroxymethylcytosine by activation-induced cytidine deaminase. The standard method for evaluating 5-hydroxymethyluracil content is the highly sensitive and highly specific isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). Despite many advantages, this method has one great limitation. It is not able to measure compounds at a single-cell level. Our goal was to develop and optimize a method based on flow cytometry that allows the evaluation of 5-hydroxymethyluracil levels at a single cell level in peripheral leukocytes.

Immunocytochemistry can be instrumental in assessing the spatial distribution and relative levels of epigenetic modifications. Although conventional immunostaining has been utilized for the detection of 5-methylcytosine (5mC) in animal cells and tissues for several decades, the sensitivity of techniques based on the use of fluorophore-conjugated secondary antibodies is not always sufficient for studying DNA modifications that are less abundant in DNA compared with 5mC. Here we describe a protocol for sensitive immunocytochemistry that utilizes peroxidase-conjugated secondary antibodies coupled with catalyzed reporter deposition and allows for detection of low-abundance noncanonical bases (e.g., 5-carboxylcytosine, 5caC, 5-formylcytosine, 5fC, 5-hydroxymethyluracil, 5hmU) in mammalian DNA. This method can be employed for evaluation of the levels and nuclear distribution of DNA modifications and permits their colocalization with protein markers in animal cells.

5-Hydroxymethyluracil ( 5hmU ) is found in the genomes of a diverse range of organisms as another kind of 5-hydroxymethylpyrimidine, with the exception of 5-hydroxymethylcytosine ( 5hmC ). The biological function of 5hmU has not been well explored due to lacking both specific 5hmU recognition and single-cell analysis methods. Here we report differentiated visualization of single-cell 5hmU and 5hmC with microfluidic hydrogel encoding (sc 5hmU / 5hmC -microgel). Single cells and their genomic DNA after cell lysis can be encapsulated in individual agarose microgels. The 5hmU sites are then specifically labeled with thiophosphate for the first time, followed by labeling 5hmC with azide glucose. These labeled bases are each encoded into respective DNA barcode primers by chemical cross-linking. In situ amplification is triggered for single-molecule fluorescence visualization of single-cell 5hmU and 5hmC . On the basis of the sc 5hmU / 5hmC -microgel, we reveal cell type-specific molecular signatures of these two bases with remarkable single-cell heterogeneity. Utilizing machine learning algorithms to decode four-dimensional signatures of 5hmU / 5hmC , we visualize the discrimination of nontumorigenic, carcinoma and highly invasive breast cell lines. This strategy provides a new route to analyze and decode single-cell DNA epigenetic modifications.

Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2\'-deoxyribo-5-hydroxyuridin (HdU) and 2\'-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF-), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.

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In Caenorhabditis elegans, two DNA glycosylases, UNG-1 and NTH-1, and two AP endonucleases, APN-1 and EXO-3, have been characterized from the base-excision repair (BER) pathway that repairs oxidatively modified DNA bases. UNG-1 removes uracil, while NTH-1 can remove 5-hydroxymethyluracil (5-hmU), an oxidation product of thymine, as well as other lesions. Both APN-1 and EXO-3 can incise AP sites and remove 3\'-blocking lesions at DNA single strand breaks, and only APN-1 possesses 3\'- to 5\'-exonulease and nucleotide incision repair activities. We used C. elegans mutants to study the role of the BER pathway in processing 5-hmU. We observe that ung-1 mutants exhibited a decrease in brood size and lifespan, and an elevated level of germ cell apoptosis when challenged with 5-hmU. These phenotypes were exacerbated by RNAi downregulation of apn-1 in the ung-1 mutant. The nth-1 or exo-3 mutants displayed wild type phenotypes towards 5-hmU. We show that partially purified UNG-1 can act on 5-hmU lesion in vitro. We propose that UNG-1 removes 5-hmU incorporated into the genome and the resulting AP site is cleaved by APN-1 or EXO-3. In the absence of UNG-1, the 5-hmU is removed by NTH-1 creating a genotoxic 3\'-blocking lesion that requires the action of APN-1.

Nucleosides, nucleotides and 2\'-deoxyribonucleoside triphosphates (dNTPs) containing 5-(hydroxymethyl)uracil protected with photocleavable groups (2-nitrobenzyl-, 6-nitropiperonyl or 9-anthrylmethyl) were prepared and tested as building blocks for the polymerase synthesis of photocaged oligonucleotides and DNA. Photodeprotection (photorelease) reactions were studied in detail on model nucleoside monophosphates and their photoreaction quantum yields were determined. Photocaged dNTPs were then tested and used as substrates for DNA polymerases in primer extension or PCR. DNA probes containing photocaged or free 5-hydroxymethylU in the recognition sequence of restriction endonucleases were prepared and used for the study of photorelease of caged DNA by UV or visible light at different wavelengths. The nitropiperonyl-protected nucleotide was found to be a superior building block because the corresponding dNTP is a good substrate for DNA polymerases, and the protecting group is efficiently cleavable by irradiation by UV or visible light (up to 425 nm).