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The aim of the present study is to provide computational insight using dispersion-corrected density-functional calculations into the reactivity properties of modified cytosine in the gas phase and in aqueous solution, whereby special emphasis is put on systems that are obtained through demethylation and methylation. Since this field is relatively incipient, our goal is to identify relationships between reactivity and stability for the modified compounds to understand their biological functionalities. Our results show that addition of a methyl, hydroxylmethyl, formyl, or carboxyl group reduces the length of the nearest hydrogen bond between the cytosine-guanine (CG) base pair and increases the length of the longest hydrogen bond of the DNA base pair. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems. [Display omitted] • DarTG1 is a toxin-antitoxin system for reversible ADP-ribosylation of guanosine bases • The toxin DarT1 links ADP-ribose to the amino group of guanines in ssDNA • NADARs function as antitoxins by reversing DarT1-catalyzed guanine ADP-ribosylation • Guanine de-ADP-ribosylation activity is conserved in eukaryotic NADARs The toxin-antitoxin system DarTG provides control of bacterial growth and protection against bacteriophages. Schuller et al. characterize the DarTG1 subfamily as a module for reversible ADP-ribosylation of guanosine bases. Its antitoxin belongs to the NADAR enzyme family, in which hydrolytic activity on guanine-ADP-ribosylated DNA is conserved among eukaryotic and non-DarT1 toxin-associated members. [ABSTRACT FROM AUTHOR]

We report herein a set of calculations designed to examine the effects of epigenetic modifications on the structure of DNA. The incorporation of methyl, hydroxymethyl, formyl and carboxy substituents at the 5-position of cytosine is shown to hardly affect the geometry of CG base pairs, but to result in rather larger changes to hydrogen-bond and stacking binding energies, as predicted by dispersion-corrected density functional theory (DFT) methods. The same modifications within double-stranded GCG and ACA trimers exhibit rather larger structural effects, when including the sugar-phosphate backbone as well as sodium counterions and implicit aqueous solvation. In particular, changes are observed in the buckle and propeller angles within base pairs and the slide and roll values of base pair steps, but these leave the overall helical shape of DNA essentially intact. The structures so obtained are useful as a benchmark of faster methods, including molecular mechanics (MM) and hybrid quantum mechanics/molecular mechanics (QM/MM) methods. We show that previously developed MM parameters satisfactorily reproduce the trimer structures, as do QM/MM calculations which treat bases with dispersion-corrected DFT and the sugar-phosphate backbone with AMBER. The latter are improved by inclusion of all six bases in the QM region, since a truncated model including only the central CG base pair in the QM region is considerably further from the DFT structure. This QM/MM method is then applied to a set of double-stranded DNA heptamers derived from a recent X-ray crystallographic study, whose size puts a DFT study beyond our current computational resources. These data show that still larger structural changes are observed than in base pairs or trimers, leading us to conclude that it is important to model epigenetic modifications within realistic molecular contexts.

Essential oils have recognized antimicrobial and antifungal properties which allow their utilization in agriculture like an alternative to pesticides, but their utilization requires the knowledge of all the potential structural changes and damages produced by the interaction with the vegetal organisms. In this paper, we investigated the effects of two essential oils, the tea tree oil (TTO) and the mixture of clove and rosemary oils (C + R), on the molecular structure of Vicia faba roots by Fourier transform infrared (FTIR) and Fourier near infrared transform (FTNIR) spectroscopy. FTIR spectroscopy showed structural modifications of the absorption bands related to DNA (1100 and 1050 cm, carbohydrate backbones, and nucleotide bands within 900 and 850 cm), proteins (1700 and 1600 cm amide I band, 1580 and 1520 cm amide II band), and lipids (methylene group of aliphatic chains between 2950 and 2800 cm). The changes in the secondary structures of proteins consisted of a denaturation depending on increased presence of random coil structures. In addition, in the samples treated with TTO oils, we observed the presence of protein oxidation, an effect negligible instead for the C + R-treated samples. The modified shapes of the infrared methyl bands of aliphatic chains suggested an increased lipid disorder which could also determine lipid peroxidation. The changes observed for the DNA structures at the highest concentration of the above essential oils can be related to the genotoxic effect of eugenol, an important constituent of both TTO and C + R mixture oils. FTNIR spectroscopy showed the modified shape of the second overtone bands belonging to methyl and methylene groups, between 8500 and 8000 cm. This confirmed the increased lipid disorders already observed by FTIR spectroscopy. The results obtained on the probe organism V. faba show that FTIR and FTNIR spectroscopy can become a useful support to the conventional cytogenetic tests used in the evaluation of the allelopathic uses of essential oils in agriculture. [ABSTRACT FROM AUTHOR]

The tumor suppressor breast cancer 1, early onset (BRCA1) plays an important role in maintaining the genome integrity, actively participating in DNA repair via homologous recombination. The level of functional final BRCA1 protein is precisely regulated in cells. Epigenetic regulation of BRCA1 gene expression is one of the most important mechanisms for BRCA1 level fine-tuning. In this review the main epigenetic mechanisms of BRCA1 expression regulation are presented, including DNA methylation, histone covalent modifications, and regulation with transcription factors. Another important function of BRCA1 emerging in recent years is its role in the regulation of breast cancer stem cell development. Moreover, based on its multiple functions, BRCA1 is also arising as a powerful biomarker. In particular, it could be used as a prognostic marker of the course of the disease and as a predictive marker of the efficiency of platinum-based chemotherapy. [ABSTRACT FROM AUTHOR]

During transcription elongation, RNA polymerase II (pol II) travels along the DNA template across thousands to millions of nucleotides and accurately synthesizes the complementary RNA transcripts. Apart from its canonical function as a key enzyme for DNA-dependent RNA synthesis, pol II also functions as a selective sensor to recognize DNA lesions or epigenetic modifications. [ABSTRACT FROM AUTHOR]

We report herein a set of calculations designed to examine the effects of epigenetic modifications on the structure of DNA. The incorporation of methyl, hydroxymethyl, formyl and carboxy substituents at the 5-position of cytosine is shown to hardly affect the geometry of CG base pairs, but to result in rather larger changes to hydrogen-bond and stacking binding energies, as predicted by dispersion-corrected density functional theory (DFT) methods. The same modifications within double-stranded GCG and ACA trimers exhibit rather larger structural effects, when including the sugar-phosphate backbone as well as sodium counterions and implicit aqueous solvation. In particular, changes are observed in the buckle and propeller angles within base pairs and the slide and roll values of base pair steps, but these leave the overall helical shape of DNA essentially intact. The structures so obtained are useful as a benchmark of faster methods, including molecular mechanics (MM) and hybrid quantum mechanics/molecular mechanics (QM/MM) methods. We show that previously developed MM parameters satisfactorily reproduce the trimer structures, as do QM/MM calculations which treat bases with dispersion-corrected DFT and the sugar-phosphate backbone with AMBER. The latter are improved by inclusion of all six bases in the QM region, since a truncated model including only the central CG base pair in the QM region is considerably further from the DFT structure. This QM/MM method is then applied to a set of double-stranded DNA heptamers derived from a recent X-ray crystallographic study, whose size puts a DFT study beyond our current computational resources. These data show that still larger structural changes are observed than in base pairs or trimers, leading us to conclude that it is important to model epigenetic modifications within realistic molecular contexts.

Over 50 years of efforts, cellular reprogramming opens a new door for disease modeling and regenerative medicine. Although induction of pluripotency by transcription factors has become common, only a small portion of basic mechanisms of epigenetic modifications during this process have been revealed. To clearly understand reprogramming and devise ways to promote full transition towards pluripotency, we must gain insight from comprehensive characterizations of cells at distinct reprogramming stages, which involves gene expression profiling, chromatin state maps of key activating and repressive marks, and DNA modifications. Here, we review recent advances in epigenetic reprogramming to pluripotency with a focus on the principal molecular regulators and attach importance to the combination of high-throughput sequencing and systematic biology approaches in uncovering underlying molecular mechanisms of this unique platform in future researches. [ABSTRACT FROM AUTHOR]

Arsenic is a naturally occurring metal carcinogen found in the Earth's crust. Millions of people worldwide are chronically exposed to arsenic through drinking water and food. Exposure to inorganic arsenic has been implicated in many diseases ranging from acute toxicities to malignant transformations. Despite the well-known deleterious health effects of arsenic exposure, the molecular mechanisms in arsenic-mediated carcinogenesis are not fully understood. Since arsenic is non-mutagenic, the mechanism by which arsenic causes carcinogenesis is via alterations in epigenetic-regulated gene expression. There are two possible ways by which arsenic may modify the epigenome-indirectly through an arsenic-induced generation of reactive oxygen species which then impacts chromatin remodelers, or directly through interaction and modulation of chromatin remodelers. Whether directly or indirectly, arsenic modulates epigenetic gene regulation and our understanding of the direct effect of this modulation on chromatin structure is limited. In this chapter we will discuss the various ways by which inorganic arsenic affects the epigenome with consequences in health and disease., Competing Interests: Conflict of interest statement The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Mapping DNA modifications at the base resolution is now possible at the genome level thanks to advances in sequencing technologies. Long-read sequencing data can be used to identify modified base patterns. However, the downstream analysis of Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) data requires the integration of genomic annotation and comprehensive filtering to prevent the accumulation of artifact signals. We present in this chapter, a linear workflow to fully analyze modified base patterns using the DNA Modification Annotation (DNAModAnnot) package. This workflow includes a thorough filtering based on sequencing quality and false discovery rate estimation and provides tools for a global analysis of DNA modifications. Here, we provide an application example of this workflow with PacBio data and guide the user by explaining expected outputs via a fully integrated Rmarkdown script. This protocol is presented with tips showing how to adapt the provided code for annotating epigenomes of any organism according to the user needs., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Methylation of the fifth carbon of cytosine was the first epigenetic modification to be discovered in DNA. Recently, three new DNA modifications have come to light: hydroxymethylcytosine, formylcytosine, and carboxylcytosine, all generated by oxidation of methylcytosine by Ten Eleven Translocation ( TET) enzymes. These modifications can initiate full DNA demethylation, but they are also likely to participate, like methylcytosine, in epigenetic signalling per se. A scenario is emerging in which coordinated regulation at multiple levels governs the participation of TETs in a wide range of physiological functions, sometimes via a mechanism unrelated to their enzymatic activity. Although still under construction, a sophisticated picture is rapidly forming where, according to the function to be performed, TETs ensure epigenetic marking to create specific landscapes, and whose improper build-up can lead to diseases such as cancer and neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Abstract: The reactions of glyoxal with 2′-deoxyguanosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions resulted in the formation of two previously undetected adducts in addition to the known reaction product 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx-dG). The adducts were isolated and purified by reversed-phase liquid chromatography and structurally characterised by UV absorbance, mass spectrometry, 1H and 13C NMR spectroscopy. The hitherto unknown adducts were identified as: 5-carboxymethyl-3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx2-dG) and N 2-(carboxymethyl)-9-(2′-deoxy-β-d-erythro-pentofuranosyl)-purin-6(9H)-one (Gx1-dG). Both adducts were shown to arise from Gx-dG. Gx-dG and Gx2-dG were found to be unstable and partly transformed to Gx1-dG, which is a stable adduct and seems to be the end-product of the glyoxal reaction with 2′-deoxyguanosine. All adducts formed in the reaction of glyoxal with 2′-deoxyguanosine were observed in calf thymus DNA. Also in DNA, Gx1-dG was the only stable adduct. The transformation of Gx-dG to Gx1-dG seemed to take place in single-stranded DNA and therefore, Gx1-dG may be a potentially reliable biomarker for glyoxal exposure and may be involved in the genotoxic properties of the compound. [Copyright &y& Elsevier]

Abstract: The Escherichia coli isolate CT596 excludes infection by the Myoviridae T4 ip1 − phage that lacks the encapsidated IPI* protein normally injected into the host with the phage DNA. Screening of a CT596 genomic library identified adjacent genes responsible for this exclusion, gmrS (942 bp) and gmrD (708 bp) that are encoded by a cryptic prophage DNA. The two genes are necessary and sufficient to confer upon a host the ability to exclude infection by T4 ip1 − phage and other glucosyl-hydroxymethylcytosine (glc-HMC) Tevens lacking the ip1 gene, yet allow infection by phages with non-glucoslyated cytosine (C) DNA that lack the ip1 gene. A plasmid expressing the ip1 gene product, IPI*, allows growth of Tevens lacking ip1 on E. coli strains carrying the cloned gmrS/gmrD genes. Members of the Teven family carry a diverse and, in some cases, expanded set of ip1 locus genes. In vivo analysis suggests a family of gmr genes that specifically target sugar-HMC modified DNA have evolved to exclude Teven phages, and these exclusion genes have in turn been countered by a family of injected exclusion inhibitors that likely help determine the host range of different glc-HMC phages. [Copyright &y& Elsevier]

Abstract: The Escherichia coli CT596 prophage exclusion genes gmrS and gmrD were found to encode a novel type IV modification-dependent restriction nuclease that targets and digests glucosylated (glc)-hydroxymethylcytosine (HMC) DNAs. The protein products GmrS (36 kDa) and GmrD (27 kDa) were purified and found to be inactive separately, but together degraded several different glc-HMC modified DNAs (T4, T2 and T6). The GMR enzyme is able to degrade both α–glucosy-HMC T4 DNA and β–glucosyl-HMC T4 DNA, whereas no activity was observed against non-modified DNAs including unmodified T4 cytosine (C) DNA or non-glucosylated T4 HMC DNA. Enzyme activity requires NTP, favors UTP, is stimulated by calcium, and initially produces 4 kb DNA fragments that are further degraded to low molecular mass products. The enzyme is inhibited by the T4 phage internal protein I* (IPI*) to which it was found to bind. Overall activities of the purified GmrSD enzyme are in good agreement with the properties of the cloned gmr genes in vivo and suggest a restriction enzyme specific for sugar modified HMC DNAs. IPI* thus represents a third generation bacteriophage defense against restriction nucleases of the Gmr type. [Copyright &y& Elsevier]

Abstract: In search for new platinum-based anticancer drugs, four cisplatin analogues, which contain pyrazole rings as non-leaving ligands, have been synthesized: cis-PtCl2(3,5-DM HMPz)2, cis-PtCl2(Pz)2, cis-PtCl2(ClMPz)2, and cis-PtCl2(HMPz)2, where Pz=pyrazole, H=hydroxyl, M=methyl. We tested their cytotoxicity, apoptosis induction ability, DNA damaging and modification properties comparing them in respect to the parent compound. The cytotoxic activity of these platinum pyrazole complexes toward the murine leukemia cell line was 2.9–3.8 times lower than actvity of cisplatin. The tested compounds varied in their mechanism of action by producing different DNA lesions. The most interesting compound seems to be the complex with chloromethyl groups at N1 of pyrazole rings, which exhibited the highest ability to form bifunctional adducts with DNA in vitro. [Copyright &y& Elsevier]

A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq , NOMeseq, and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m6A-SMAC-seq and m6A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A novel siphovirus, vB_PagS_MED16 (MED16) was isolated in Lithuania using Pantoea agglomerans strain BSL for the phage propagation. The double-stranded DNA genome of MED16 (46,103 bp) contains 73 predicted open reading frames (ORFs) encoding proteins, but no tRNA. Our comparative sequence analysis revealed that 26 of these ORFs code for unique proteins that have no reliable identity when compared to database entries. Based on phylogenetic analysis, MED16 represents a new genus with siphovirus morphology. In total, 35 MED16 ORFs were given a putative functional annotation, including those coding for the proteins responsible for virion morphogenesis, phage–host interactions, and DNA metabolism. In addition, a gene encoding a preQ0 DNA deoxyribosyltransferase (DpdA) is present in the genome of MED16 and the LC–MS/MS analysis indicates 2′-deoxy-7-amido-7-deazaguanosine (dADG)-modified phage DNA, which, to our knowledge, has never been experimentally validated in genomes of Pantoea phages. Thus, the data presented in this study provide new information on Pantoea-infecting viruses and offer novel insights into the diversity of DNA modifications in bacteriophages. [ABSTRACT FROM AUTHOR]

DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification. [ABSTRACT FROM AUTHOR]

Despite widespread interest for understanding how modified bases have evolved their contemporary functions, limited experimental evidence exists for measuring how close an organism is to accidentally creating a new, modified base within the framework of its existing genome. Here, we describe the biochemical and structural basis for how a single-point mutation in E. coli 's naturally occurring cytosine methyltransferase can surprisingly endow a neomorphic ability to create the unnatural DNA base, 5-carboxymethylcytosine (5cxmC), in vivo. Mass spectrometry, bacterial genetics, and structure-guided biochemistry reveal this base to be exclusively derived from the natural but sparse secondary metabolite carboxy- S -adenosyl-L-methionine (CxSAM). Our discovery of a new, unnatural DNA modification reveals insights into the substrate selectivity of DNA methyltransferase enzymes, offers a promising new biotechnological tool for the characterization of the mammalian epigenome, and provides an unexpected model for how neomorphic bases could arise in nature from repurposed host metabolites. • Mutagenesis unexpectedly identifies a neomorphic activity in a DNA methyltransferase • Bacterial genetics uncovers the modification 5-carboxymethylcytosine (5cxmC) • Structure-guided biochemistry reveals enzymatic basis for DNA carboxymethylation • 5cxmC provides a model for how gain-of-function genomic bases may arise in nature In Wang and Kohli, 5-carboxymethylcytosine is revealed as an unnatural genomic DNA modification that can be generated in vivo from a unique combination of a sparse natural host metabolite and a neomorphic cytosine DNA methyltransferase. [ABSTRACT FROM AUTHOR]

Archaea often thrive in environmental extremes, enduring levels of heat, pressure, salinity, pH, and radiation that prove intolerable to most life. Many environmental extremes raise the propensity for DNA damaging events and thus, impact DNA stability, placing greater reliance on molecular mechanisms that recognize DNA damage and initiate accurate repair. Archaea can presumably prosper in harsh and DNA-damaging environments in part due to robust DNA repair pathways but surprisingly, no DNA repair pathways unique to Archaea have been described. Here, we review the most recent advances in our understanding of archaeal DNA repair. We summarize DNA damage types and their consequences, their recognition by host enzymes, and how the collective activities of many DNA repair pathways maintain archaeal genomic integrity. [ABSTRACT FROM AUTHOR]

Methyl-CpG binding protein 2 (MeCP2) is a multifunctional epigenetic reader playing a role in transcriptional regulation and chromatin structure, which was linked to Rett syndrome in humans. Here, we focus on its isoforms and functional domains, interactions, modifications and mutations found in Rett patients. Finally, we address how these properties regulate and mediate the ability of MeCP2 to orchestrate chromatin compartmentalization and higher order genome architecture. [ABSTRACT FROM AUTHOR]

Analytical techniques based on mass spectrometry allow to analyze DNA modifications in body fluids. Here we describe two chromatographic methods that can be used for the simultaneous determination of the modified DNA bases and nucleosides in the same urine sample: isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) and high-performance liquid chromatography coupled with gas chromatography and mass spectrometry (HPLC/GC/MS).

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.

Computational analysis of digital images provides a robust and unbiased way to compare and investigate the amount (pixel intensity) and spatial distribution of DNA modifications. The DNA modifications in the cells are visualized by fluorescence labeling and the images are captured by confocal microscopy. The key advantage of the confocal over conventional microscope is that it images only a thin optical section around the focal plane of the microscope therefore it can precisely record signals only from the focal plane inside the nucleus. In this chapter, we will describe in detail several analysis methods to visualize and quantify the DNA modification signals including how to investigate codistribution of such signals when using dual labeling.

Collectively, the dsDNA tailed bacteriophages (Caudovirales) contain the largest chemical diversity of naturally occurring deoxynucleotides in DNA observed to date. The continuing discovery of new modifications in phages suggest many more are waiting to be found. Thus, methods for the observation and characterization of noncanonical nucleosides are timely. We present here protocols for extraction of genomic DNA from bacteriophage particles, enzymatic hydrolysis of DNA to free nucleosides, and examination of nucleoside composition by HPLC and mass spectrometry.

Background: Bee venom is a promising agent for cancer treatment due to its selective cytotoxic potential for cancer cells through apoptotic pathways. However, there is no evidence for changes in the epigenome and mitochondrial DNA copy numbers after bee venom application. The purpose of this study was to determine the impact of bee venom on cytosine modifications and mitochondrial DNA copy number variation., Methods: A broad range of methods was applied to elucidate the impact of bee venom on neoplastic cells. These included MTT assay for detection of cytotoxicity, immunostaining of cytosine modifications and mitochondria, assessment of cellular morphology by flow cytometry, and quantification of mitochondrial DNA copy numbers using QPCR., Results: Bee venom-induced cell death was selective for cancer cells, where it triggered a response characterized by alteration of cytosine modification. In contrast, normal cells were more resistant to DNA modifications. Furthermore, application of the venom resulted in variation of mitochondrial membrane permeability and mitochondrial DNA copy numbers, together with alterations in cell morphology, manifesting as reduced affected cell size., Conclusion: The study findings suggest that bee venom can be used as a selective DNA (de)methylating agent in cancer. Various agents (such as decitabine and 5-azacytidine) have been synthesized and developed for cancer treatment, and a range of syntheses and preparation and application methods have been described for these patented drugs. However, to the best of our knowledge, no previous research has investigated the use of bee venom or any component thereof for epigenetic therapy in cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

5-Hydroxymethylcytosine (5hmC) is an abundant DNA modification in human and mouse brain, as well as in embryonic stem cells, while severely depleted in multiple types of cancer. Assays for 5hmC detection and quantification, both on a locus-specific and global level, are limited in number and often resource-intensive. Immunodetection of 5hmC through antibodies remains a cost-effective and widely accessible approach. This chapter describes an ELISA-based protocol for 5hmC detection and quantification in genomic or in vitro modified DNA. It is based on the passive adsorption of DNA onto a solid polystyrene surface and the specific detection of 5hmC, which generates a measurable chemiluminescent signal, proportional to the amount of immobilized 5hmC. The assay utilizes a standard curve for interpolation of 5hmC percentage and a loading standard for monitoring loading precision.

Cytosine DNA methylation (5-methylcytsone, 5mC) is the major DNA modification found in the genomes of animals and plants. Although the roles of 5mC and its oxidized derivatives in the regulation of gene expression are relatively well attested and extensively explored, a number of recent studies imply that noncytosine DNA modifications may also convey specific biological functions and act as "epigenetic" marks in multicellular organisms. Here we review experimental evidence for the presence of noncytosine epigenetic modifications in metazoans and plants focusing on two "unusual" DNA bases, 5-hydroxymethyluracil (5hmU) and N6-methyladenine (6mA), and suggest potential explanations for inconsistencies in the currently available data on abundance and potential biological roles of these DNA modifications in mammals.

5-Methylcytosine (5mC) is an epigenetic mark known to contribute to the regulation of gene expression in a wide range of biological systems. Ten Eleven Translocation (TET) dioxygenases oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in metazoans and fungi. Moreover, two recent reports imply the existence of other species of modified cytosine in unicellular alga Chlamydomonas reinhardtii and malaria parasite Plasmodium falciparum. Here we provide an overview of the spectrum of cytosine modifications and their roles in demethylation of DNA and regulation of gene expression in different eukaryotic organisms.

Epigenetic modifications on DNA include canonical 5‐methylcytosine (5mC) and its iteratively oxidized derivatives mediated by TET dioxygenase: 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine (5fC), and 5‐carboxycytosine (5caC). All of them have been recognized to play important roles in gene expression regulation and to be indispensable for normal mammalian reproduction and development. In order to understand the mechanisms underlying these roles, over the past few decades, technical advances have been made to comprehensively profile the distribution pattern of bulk epigenetic modifications in genomic DNA. Recently, single‐cell methods have identified the epigenetic patterns even within an individual cell. This review focuses on single‐cell DNA epigenetic modifications sequencing technologies and their applications, and gives prospects on the development of single‐cell methods based on bisulfite‐free strategy. [ABSTRACT FROM AUTHOR]

Covalent DNA modifications, such as 5-methylcytosine (5mC), are increasingly the focus of numerous research programs. In eukaryotes, both 5mC and 5-hydroxymethylcytosine (5hmC) are now recognized as stable epigenetic marks, with diverse functions. Bacteria, archaea, and viruses contain various other modified DNA nucleobases. Numerous databases describe RNA and histone modifications, but no database specifically catalogues DNA modifications, despite their broad importance in epigenetic regulation. To address this need, we have developed DNAmod: the DNA modification database. DNAmod is an open-source database (https://dnamod.hoffmanlab.org) that catalogues DNA modifications and provides a single source to learn about their properties. DNAmod provides a web interface to easily browse and search through these modifications. The database annotates the chemical properties and structures of all curated modified DNA bases, and a much larger list of candidate chemical entities. DNAmod includes manual annotations of available sequencing methods, descriptions of their occurrence in nature, and provides existing and suggested nomenclature. DNAmod enables researchers to rapidly review previous work, select mapping techniques, and track recent developments concerning modified bases of interest. [ABSTRACT FROM AUTHOR]