N6-methyladenine: the other methylated base of DNA

Contrary to mammalian DNA, which is thought to contain only 5-methylcytosine (m5C), bacterial DNA contains two additional methylated bases, namely N6-methyladenine (m6A), and N4-methylcytosine (m4C). However, if the main function of m5C and m4C in bacteria is protection against restriction enzymes, the roles of m6A are multiple and include, for example, the regulation of virulence and the control of many bacterial DNA functions such as the replication, repair, expression and transposition of DNA. Interestingly, even if adenine methylation is usually considered a bacterial DNA feature, the presence of m6A has been found in protist and plant DNAs. Furthermore, indirect evidence suggests the presence of m6A in mammal DNA, raising the possibility that this base has remained undetected due to the low sensitivity of the analytical methods used. This highlights the importance of considering m6A as the sixth element of DNA.     

Molecular characterization of a putative plant homolog of MBD4 DNA glycosylase

Methyl-CpG-binding domain 4 (MBD4) DNA glycosylase is involved in excision of spontaneous deamination products of cytosine and 5-methylcytosine in animals, but it is unknown whether related proteins perform similar functions in plants. We report here the isolation and biochemical characterization of a putative MBD4 homolog from Arabidopsis thaliana, designated as MBD4L (MBD4-like). The plant enzyme lacks the MBD domain present in mammalian MBD4 proteins, but conserves a DNA glycosylase domain with critical residues for substrate recognition and catalysis, and it is more closely related to MBD4 homologs than to other members of the HhH-GPD superfamily. Arabidopsis MBD4L excises uracil and thymine opposite G, and the presence of halogen substituents at C5 of the target base greatly increases its excision efficiency. No significant activity is detected on cytosine derivatives such as 5-methylcytosine or 5-hydroxymethylcytosine. The enzyme binds to the abasic site product generated after excision, which decreases its catalytic turnover in vitro. Both the full-length protein and a N-terminal truncated version retaining the catalytic domain exhibit a preference for a CpG sequence context, where most plant DNA methylation is found. Our results suggest that an important function of Arabidopsis MBD4L is to protect the plant genome from the mutagenic consequences of cytosine and 5-methylcytosine deamination.     

DNA from Aspergillus flavus contains 5-methylcytosine

DNA from Aspergillus sp. has been reported not to contain 5-methylcytosine. However, it has been found that Aspergillus nidulans responds to 5-azacytidine, a drug that is a strong inhibitor of DNA methyltransferases. Therefore, we have re-examined the occurrence of 5-methylcytosine in DNA from Aspergillus flavus by using a highly sensitive and specific method for detection of modified bases in genomic DNA comprising high-performance liquid chromatography separation of nucleosides, labeling of the nucleoside with deoxynucleoside kinase and two-dimensional thin-layer chromatography. Our results show that 5-methylcytosine is present in DNA from A. flavus. We estimate the relative amounts of 5-methylcytosine to cytosine to be approximately 1/400.     

Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2-(13)C]cytosine and [2-(13)C]5-methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 microg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.     

Analysis of products of DNA modification by methylases: a procedure for the determination of 5- and N4-methylcytosines in DNA

Although many different methods are used for the identification of methylated heterocyclic bases in DNA not all of them possess the ability to discriminate N4-methylcytosine (m4C) and 5-methylcytosine (m5C). Therefore, some of the methods need additional reexamination. This paper reinvestigates some chromatographic systems (thin-layer chromatography, paper chromatography, electrophoresis) most widely used in the analysis of minor bases occurring in nucleic acids according to their ability to separate m4C and m5C. A simple procedure for the preparation of the sample and a chromatographic system for its analysis was developed. The recommended chromatographic systems may be used for the simultaneous separation of not only of m4C and m5C but also both methylated cytosines together with N6-methyladenine and 7-methylguanine.     

Methylation Modifications in Eukaryotic Messenger RNA

RNA methylation modifications have been found for decades of years, which occur at different RNA types of numerous species, and their distribution is species-specific. However, people rarely know their biological functions. There are several identified methylation modifications in eukaryotic messenger RNA （mRNA）, such as NT-methylguanosine （mVG） at the cap, Nr-methyl-2＇-O-methyladenosine （m6Am）, 2＇-O-methylation （Nm） within the cap and the internal positions, and internal N6-methyladenosine （m6A） and 5-methylcytosine （mSC）. Among them, mTG cap was studied more clearly and found to have vital roles in several important mRNA processes like mRNA translation, stability and nuclear export, m6A as the most abundant modification in mRNA was found in the 1970s and has been proposed to function in mRNA splicing, translation, stability, transport and so on. mrA has been discovered as the first RNA reversible modification which is demethylated directly by human fat mass and obesity associated protein （FRO） and its homolog protein, alkylation repair ho- molog 5 （ALKBH5）. b-TO has a special demethylation mechanism that demethylases m6A to A through two over-oxidative intermediate states： N6-hydroxymethyladenosine （hm6A） and Nr-formyladenosine （frA）. The two newly discovered m6A demethylases, bTO and ALKBH5, significantly control energy homeostasis and spermatogenesis, respectively, indicating that the dynamic and reversible mrA, analogous to DNA and histone modifications, plays broad roles in biological kingdoms and brings us an emerging field ＂RNA Epige- netics＂. 5-methylcytosine （5mC） as an epigenetic mark in DNA has been studied widely, but mSC in mRNA is seldom explored. The bisulfide sequencing showed mSC is another abundant modification in mRNA, suggesting that it might be another RNA epigenetic mark. This review focuses on the main methylation modifications in mRNA to describe their formation, distribution, function and demethylation from the current knowledge and to provide future 19erspectives on functional studies.     

Stage-specific DNA methylation in a fungal plant pathogen.

Significantly more 5-methylcytosine residues were found in the DNA from the dormant sclerotia of Phymatotrichum omnivorum than in the DNA from the metabolically active mycelia of the fungus, as shown by high-pressure liquid chromatography of acid-hydrolyzed DNA digests and by restriction of the DNA with the isoschizomers MspI and HpaII. N6-Methyladenine was not detected in GATC sequences in the DNA isolated from either stage.     

Enzymic removal of 5-methylcytosine from DNA by a human DNA-glycosylase.

DNA 5-methylcytosine is a major factor in the silencing of mammalian genes; it is involved in gene expression, differentiation, embryogenesis and neoplastic transformation. A decrease in DNA 5-methylcytosine content is associated with activation of specific genes. There is much evidence indicating this to be an enzymic process, with replacement of 5-methylcytosine by cytosine. We demonstrate here enzymic release of 5-methylcytosines from DNA by a human 5-methylcytosine-DNA glycosylase activity, which affords a possible mechanism for such replacement. This activity generates promutagenic apyrimidinic sites, which can be related to the high frequency of mutations found at DNA 5-methylcytosine loci. The recovery of most released pyrimidines as thymines indicates subsequent deamination of free 5-methylcytosines by a 5-methylcytosine deaminase activity. This prevents possible recycling of 5-methylcytosine into replicative DNA synthesis via a possible 5-methyl-dCTP intermediate synthesized through the pyrimidine salvage pathway. Taken together, these findings indicate mechanisms for removal of 5-methylcytosines from DNA, hypermutability of DNA 5-methylcytosine sites, and exclusion of 5-methylcytosines from DNA during replication.     

DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine.

While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues.     

Tissue-specific differences in DNA methylation in various mammals

The only naturally occurring modified base in vertebrate DNA is 5-methylcytosine. Using a precise high-performance liquid chromatographic analysis of DNA enzymatically digested to deoxynucleosides, we have shown that rats, mice and four types of monkey display tissue-specific as well as species-specific differences in the extent of methylation of their cytosine residues. Several similarities in the patterns of tissue-specific DNA methylation in these mammals and in the previously studied human samples were observed. Compared to most other types of DNA examined, brain and thymus DNAs were hypermethylated, which suggests that this hypermethylation is a determinant or a necessary byproduct of mammalian differentiation. In all of the studied rodents and primates, the highly repeated DNA sequence fraction was more methylated than the moderately repetitive or single copy fractions. The tissue-specific differences in overall DNA methylation showed no correlation with what is known about average cell turnover rates nor with the percentage of the genome that is transcribed. Liver regeneration in the rat following partial hepatectomy did not detectably alter 5-methylcytosine levels in liver DNA. A considerable increase in the extent of methylation of total liver DNA was observed during normal development of the rat. The latter phenomenon may be due to a major change in the cellular composition of the liver.     

Bacterial DNA methylation and gene transfer efficiency

The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences. These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam(-), dcm(-)) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam(-), dcm(-)) counterpart. Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene transfer efficiency. However, the persistence of the use of (dam(+), dcm(+)) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in eukaryotic cells or organisms.     

DNA methylation in mycobacteria: Absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences

Abstract The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.     

DNA damage and mutations produced by chloroacetaldehyde in a CpG-methylated target gene

Chloroacetaldehyde (CAA) is a metabolite of the human carcinogen vinyl chloride. CAA produces several types of DNA adducts including the exocyclic base adducts 3,N(4)-ethenocytosine, 1,N(6)-ethenoadenine, N(2),3-ethenoguanine, and 1,N(2)-ethenoguanine. Adducts of CAA with 5-methylcytosine have not yet been characterized. Here we have analyzed the mutational spectra produced by CAA in the supF gene of the pSP189 shuttle vector when present in either an unmethylated or CpG-methylated state. The vectors were replicated in human nucleotide excision repair-deficient XP-A fibroblasts. The mutational spectra obtained with the unmethylated and methylated supF target genes were generally similar with a preponderance of C/G to T/A transitions and C/G to A/T transversions. CAA-induced DNA adducts were mapped along the supF gene by using thermostable thymine DNA glycosylase (TDG) in conjunction with ligation-mediated PCR or by a Taq polymerase stop assay. Prominent CAA-induced TDG-sensitive sites were seen at several CpG positions but were independent of methylation. Methylated CpG sites were sites of CAA-induced mutations but were not the major mutational hotspots. Taq polymerase arrest sites were observed at numerous sequence positions in the supF gene and reflected the rather broad distributions of mutations along the sequence. We conclude that methylated CpG sites are not preferential targets for chloroacetaldehyde-induced mutagenesis.     

qDNAmod: a statistical model-based tool to reveal intercellular heterogeneity of DNA modification from SMRT sequencing data

In an isogenic cell population, phenotypic heterogeneity among individual cells is common and critical for survival of the population under different environment conditions. DNA modification is an important epigenetic factor that can regulate phenotypic heterogeneity. The single molecule real-time (SMRT) sequencing technology provides a unique platform for detecting a wide range of DNA modifications, including N6-methyladenine (6-mA), N4-methylcytosine (4-mC) and 5-methylcytosine (5-mC). Here we present qDNAmod, a novel bioinformatic tool for genome-wide quantitative profiling of intercellular heterogeneity of DNA modification from SMRT sequencing data. It is capable of estimating proportion of isogenic haploid cells, in which the same loci of the genome are differentially modified. We tested the reliability of qDNAmod with the SMRT sequencing data of Streptococcus pneumoniae strain ST556. qDNAmod detected extensive intercellular heterogeneity of DNA methylation (6-mA) in a clonal population of ST556. Subsequent biochemical analyses revealed that the recognition sequences of two type I restriction–modification (R-M) systems are responsible for the intercellular heterogeneity of DNA methylation initially identified by qDNAmod. qDNAmod thus represents a valuable tool for studying intercellular phenotypic heterogeneity from genome-wide DNA modification.     

Gene silencing and endogenous DNA methylation in mammalian cells

It is known that transformed mammalian cells can spontaneously inactivate genes at low frequency by the de novo methylation of promoter sequences. It is usually assumed that this is due to DNA methyl transferase activity, but an alternative possibiity is that 5-methyldCTP is present in these cells and can be directly incorporated into DNA. The ongoing repair of DNA containing 5-methylcytosine will produce 5-methyldeoxycytidine monophosphate (5-methyldCMP), so the question arises whether this can be phosphorylated to 5-methyldCTP. We have tested this using three strains of CHO cells with different levels of 5-methyldCMP deaminase activity. That with the lowest enzyme activity, designated HAM⁻, has previously been shown to incorporate tritium labelled 5-methyldeoxycytidine into 5-methylcytosine in DNA, with a greater amount of label in thymine. This strain is phenotypically unstable producing cells resistant to bromodeoxyuridine (BrdU) and 6-thioguanine (6-TG) at high frequency. In contrast, the strain with the highest 5-methyldCMP deaminease, designated HAM⁺, is extremely stable, and the starting strain K1 HAM^s1 is intermediate between the HAM⁻ and HAM⁺ phenotypes. We have also shown that human diploid fibroblast strain MRC-5 has a phenotype like HAM⁺, whereas its SV40 transformed derivative, MRC-5V2 resembles HAM⁻ in having low 5-methyl dCMP deaminase activity, and is phenotypically unstable with regard to 6-TG resistance. It seems that 5-methyldCMP deaminase can be down-regulated in transformed cells, and this can promote de novo methylation by incorporation of 5-methyldCTP derived from 5-methyldCMP.     

Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA

Human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in genomic DNA, in a recently discovered cytosine demethylation pathway. We present here the crystal structures of the hTDG catalytic domain in complex with duplex DNA containing either 5caC or a fluorinated analog. These structures, together with biochemical and computational analyses, reveal that 5caC is specifically recognized in the active site of hTDG, supporting the role of TDG in mammalian 5-methylcytosine demethylation.     

Determination of trace amounts of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography

A high-performance liquid chromatographic method to separate five major bases (cytosine, thymine, guanine, adenine, and uracil) and three minor methylated bases (5-methylcytosine, N6-methyladenine, and 7-methylguanine) has been developed using a volatile mobile phase under isocratic conditions. It is extended to quantitate 5-methylcytosine in trace amounts (1 in 20,000 cytosine residues). The suitability of the method has been verified by estimating 5-methylcytosine in DNAs of phi X174 and pBR322. The method has been applied to quantitate the extent of cytosine methylation in DNA of larval silk glands of Bombyx mori. Our results confirm that the pupal DNA of Drosophila melanogaster does not contain detectable amounts of 5-methylcytosine.     

On the impossibility of the incorporation of 5-methylcytosine and its nucleosides into higher plant DNA

No radioactivity was detected in 5-methylcytosine isolated from wheat DNA after incubation of wheat seedlings with 3H-labelled 5-methylcytosine, 5-methylcytidine and 5-methyldeoxycytidine. No label from 3H-5-methylcytosine was found in DNA of seedlings. After incubation of seedlings with 3H-labelled nucleosides of 5-methylcytosine, radioactivity was discovered only in thymine of DNA. Thus 5-methylcytosine and its nucleosides can not be used in plants as direct precursors of 5-methyl cytosine residues in DNA, but nucleosides of 5-methylcytosine may be deaminated to thymidine (or deoxythymidine) and subsequently incorporated into DNA.     

N4-methylcytosine as a minor base in bacterial DNA.

The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.