Sequence specificity of DNA methylases from Bacillus amyloliquefaciens and Bacillus brevis

DNA methylation in Bacillus amyloliquefaciens strain H (Bam)² and Bacillus brevis (Bbv) has been examined by a variety of techniques. In vivo labelling studies revealed that Bam DNA contains no N⁶-methyladenine (MeAde), but contains 5-methylcytosine (MeCyt); approximately 0·7% of the cytosine residues are methylated.DNA methylase activity was partially purified from both Bam and Bbv; the Bam enzyme preparation transferred methyl groups from S-adenosyl-l-[methyl-³H]methionine ([³H]AdoMet) to specific DNA cytosine residues only; in agreement with Vanyushin & Dobritsa (1975), the Bbv enzyme preparation methylated both DNA adenine and cytosine residues. The (partial) sequence specificity of the methylases was determined by analyzing [³H]methyl-labelled dinucleotides obtained from enzymatic digests of DNA methylated in vitro. Bam and Bbv each contain a DNA-cytosine methylase with overlapping sequence specificity; e.g. both enzymes produce G-C1, C1-A and C1-T. This is consistent with a single, twofold symmetrical methylation sequence of 5′ … G-C1-(A or T)-G-C … 3′; this was observed by Vanyushin & Dobritsa (1975) for a different Bbv strain. Bam contains a second DNA-cytosine methylase (not present in Bbv), which produces T-C1 and C1-T. We propose that this methylase is the BamI modification enzyme, and that the modified sequence is 5′ … G-G-A-T-C1-C … 3′.Bbv appears to contain two DNA-adenine methylases which produce the (partial) methylated sequences, 5′ … G-A1-T … 3′ and 5′ … A-A1-G … 3′, respectively; in the former case, all the G-A-T-C sites on Bbv DNA appear to be methylated.     

Differential methylation of mitochondrial and nuclear DNA in cultured mouse, hamster and virus-transformed hamster cells. In vivo and in vitro methylation

Information has been lacking as to whether mitochondrial DNA of animal cells is methylated. The methylation patterns of mitochondrial and nuclear DNAs of several mammalian cell lines have therefore been compared by four methods: (1) in vivo transfer of the methyl group from [methyl-³H]methionine; (2) in vivo incorporation of [³²P]orthophosphate and a combination of (1) and (2); (3) in vivo incorporation of [³H]deoxycytidine; (4) in vitro methylation of DNAs with ³H-labeled S-adenosylmethionine as methyl donor and DNA methylase preparations from L cell nuclei. The cell lines were mouse L cells, $\text{BHK21}\text{C13}$, $\text{C13}\text{B4}$ (baby hamster kidney cells transformed by the Bryan strain of Rouse sarcoma virus), and PyY (BHK cells transformed by polyoma virus). DNA bases were separated chromatographically, using 5-methylcytosine, 6-methylaminopurine and, in some cases, 7-methylguanine as markers.Mitochondrial DNA was found to be significantly less methylated than nuclear DNA with respect to 5-methylcytosine in all cell types studied and by all methods used. The relative advantages and disadvantages of each method have been discussed. The level of 5-methylcytosine in mitochondrial DNA as compared with that in nuclear DNA was estimated as one-fourth to one-fourteenth in various cell lines. The estimated 5-methylcytosine content per circular mitochondrial DNA molecule (mol. wt 10 × 10⁶) was about 12 methylcytosine residues for L cells and 24, 30 and 36 methylcytosine residues for BHK, B4 and PyY cells, respectively. Relative to cytosine residues, the estimate was one 5-methylcytosine per 500 cytosine residues of mitochondrial DNA and one 5-methylcytosine per 36 cytosine residues of nuclear DNA from L-cells. The values for methylcytosine of mitochondrial DNA are presumed to be maximal. PyY cells as compared with other cells had the highest methylcytosine content of both mitochondrial and nuclear DNA as estimated by method (3). No methylation of nuclear DNA was observed in confluent L cells.Evidence for the presence of DNA methylase activity associated with mitochondrial fractions was obtained. This activity could be distinguished from other cellular DNA methylase activity by differential response to mercaptoethanol. Radioactivity from ³H-labeled S-adenosylmethionine was found only in 5-methyl-cytosine of DNA.     

The kinetics of DNA methylation in cultures of a mouse adrenal cell line

Direct measurements of the methylation of newly-synthesized DNA were made in cultures of a clonal mouse adrenal cortex cell line, Y129OS3, by (1) following the incorporation of radioactivity from methionine-(methyl)-C¹⁴ into a segment of DNA which had been density-labeled with bromouracil and (2) labeling DNA cytosine with C¹⁴-deoxycytidine and then following the appearance of radioactivity in DNA 5-methylcytosine. The results establish that during exponential growth the DNA of this cell line is methylated entirely within a few minutes of its synthesis. Using the second technique described above accurate, sensitive measurements of DNA methylation levels can be made by comparing radioactivity in 5-methylcytosine to radioactivity in cytosine plus 5-methylcytosine. In this cell line 5-methylcytosine accounts for 4.3 ± 0.2% of the DNA cytosine. Some apparent contradictions between these results and those of other workers are discussed.     

Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging

The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as ¹⁵N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HpaII (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HpaII methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HpaII methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.     

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m⁵C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m⁵C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.     

Mutants of the N-3 R-Factor Conditionally Defective in hspII Modification and Deoxyribonucleic Acid-Cytosine Methylase Activity

The N-3 drug resistance (R) factor specifies a deoxyribonucleic acid (DNA)-cytosine methylase and a DNA restriction-modification (hspII) system. We have isolated three independent mutants that are conditionally defective in their ability to modify bacteriophage lambda and to methylate DNA-cytosine residues. The ratio of 5-methylcytosine to N(6)-methyladenine in bacterial DNA and in the DNA of phages lambda and fd was determined after labeling with [methyl-(3)H]methionine at various growth temperatures. Although the ability of the wild-type N-3 factor to modify phage lambda and to methylate DNA-cytosine residues was unaffected with increasing temperature, two of the mutants exhibited a parallel loss in modification and cytosine methylation ability. The ability of the third mutant to carry out these functions was dependent on the presence or absence of an amber suppressor mutation in the host genome. These results offer further support for the notion that hspII modification is mediated by a DNA-cytosine methylase. Evidence is also presented that the modification methylase is responsible for the in vivo methylation of phage fd DNA (which is not subject to hspII restriction in vivo).     

Distribution of N<Superscript>6</Superscript>-Methyladenine in DNA of T2 Phage and its Host <Emphasis Type="Italic">Escherichia coli</Emphasis> B

N⁶-METHYLADENINE (6-MeAde) and 5-methylcytosine occur as minor bases in bacterial and phage DNA^1–7 and seem to result from the selective methylation of adenine and cytosine residues by specific DNA methylases⁸. Methylation is the final stage in DNA synthesis and is essential for the phenomenon of host modification of phages^9–11; it is one of the mechanisms controlling DNA replication in the cell^{12, 13}. A study of the distribution of minor bases in DNA is therefore important not only for the elucidation of the specificity and mechanism of action of DNA methylases but also for an understanding of the purpose of this methylation. We believe that in Escherichia coli, DNA methylase exerts its action on adenine residues in chain terminating triplets: 6-MeAde may serve as a signal for gene termination in this system.     

Mechanistic Insights into Cytosine-N3 Methylation by DNA Methyltransferase DNMT3A

Recently, it has been discovered that different DNA-(cytosine C5)-methyltransferases including DNMT3A generate low levels of 3mC [Rosic et al. (2018), Nat. Genet., 50, 452–459]. This reaction resulted in the co-evolution of DNMTs and ALKB2 DNA repair enzymes, but its mechanism remained elusive. Here, we investigated the catalytic mechanism of DNMT3A for cytosine N3 methylation. We generated several DNMT3A variants with mutated catalytic residues and measured their activities in 5mC and 3mC generation by liquid chromatography linked to tandem mass spectrometry. Our data suggest that the methylation of N3 instead of C5 is caused by an inverted binding of the flipped cytosine target base into the active-site pocket of the DNA methyltransferase, which is partially compatible with the arrangement of catalytic amino acid residues. Given that all DNA-(cytosine C5)-methyltransferases have a common catalytic mechanism, it is likely that other enzymes of this class generate 3mC following the same mechanism.     

5-Methylcytosine content and methylation status in six millet DNAs

High performance liquid chromatographic analysis of the total nuclear DNAs of 6 millets plant species indicates that the 5-methylcytosine content ranges from 3% in barn yard millet to 9.6% in great millet while the fraction of cytosines methylated varies between 14% in little millet to 31 % in pearl millet. Digestion of millet DNAs with MspI/HpaII suggests that CpG methylation is more in great millet DNA while CpC methylation is more in the other 5 millet DNAs. Digestion of millet DNAs with MboI, Sau3AI andDpnI indicates that some of the^5’ GATC^3’ sequences are methylated at adenine and/or cytosine residues except in little millet where adenine methylation of the^5’GATC^3’ sequences is insignificant and there is a predominance of cytosine methylation in these sequences.     

Heat- and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA

5-Methylcytosine residues in DNA underwent deamination at high temperatures. Furthemore, their rate of deamination at neutral or alkaline pH was greater than that of cytosine residues in DNA. As sources of [¹⁴C]5-methylcytosine-containing DNA, we used bacteriophage XP-12 DNA, in which 5-methylcytosine residues completely replace C residues, and calf thymus DNA experimentally substituted with [¹⁴C]5-methylcytosine residues. Upon incubation at 95°C in a physiological buffer or at 60°C in 1 M NaOH, the respective rates of deamination of 5-methylcytosine residues were about 3- and 1.5-times those of cytosine residues. Under the same conditions, the free 5-methyldeoxycytidine was converted to thymidine more rapidly than deoxycytidine was converted to deoxyuridine. The reactions at physiological pH and elevated temperature suggest that deamination of 5-methylcytosine residues may yield a significant portion of spontaneous mutations in vivo, especially in view of the lack of thymine-specific mismatch repair systems with specificity and efficiency comparable to that of uracil excision repair systems.     

5-Methylcytosine is not detectable in Saccharomyces cerevisiae DNA.

We examined the DNA of Saccharomyces cerevisiae by both HpaII-MspI restriction enzyme digestion and high-performance liquid chromatography analysis for the possible presence of 5-methylcytosine. Both of these methods failed to detect cytosine methylation within this yeast DNA; i.e., there is less than 1 5-methylcytosine per 3,100 to 6,000 cytosine residues.     

Analysis of TET Expression/Activity and 5mC Oxidation during Normal and Malignant Germ Cell Development

During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of IDH1 (IDH1 ^R132) and IDH2 (IDH2 ^R172) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected.     

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.     

Repair methylation of parental DNA in synchronized cultures of Novikoff hepatoma cells.

Parental and filial DNA strands were isolated from a Novikoff rat hepatoma cell line, synchronized by S-phase arrest with excess thymidine, that had completed up to one round of DNA replication in the presence of (14-C-methyl)methionine and (6-3-H) bromodeoxyuridine. Both strands were methylated, the proportion of total methyl label in parental DNA increasing slightly with time in S-phase. The studies were repeated with (14-C-methyl)methionine and (3-H)deoxycytidine to determine if parental methylation occurred on extant or repair-inserted cytosine residues. Both (14-C) and (3-H) were found in parental DNA. The (14-C)/(3-H) ration of parental DNA-5-methylcytosine was about twice that in filial DNA while the (3-H) data showed twice the concentration of 5-methylcytosine in parental compared to filial DNA. Thus parental methylation occurred on repair-inserted cytosine residues and resulted in overmethylation. That the DNA damage and repair was due to 5-phase arrest was shown by repeating the studies using a sequential mitotic-G1 arrest method. With this method little (14-C) or (3-H) was found in parental DNA. We conclude that S-phase arrest leads to DNA damage and repair with subsequent overmethylation of repair-inserted cytosines; that sequential mitotic-G1 arrest minimizes DNA damage; and, that the latter technique, suitable for synchronization of large quantities of cells, may prove useful in relatively artifact-free studies of eukaryotic DNA replication.     

Effect of trypsin on DNA methylation in isolated nuclei from developing sea urchin embryos

Nuclei isolated from the developing sea urchin embryo $\text{Paracentrotus lividus}$ and incubated in the presence of [³H-methyl] S-adenosylmethionine methylate their own DNA. Addition of small amounts of trypsin produces a 20-fold increase in DNA methylation. The time kinetics and the specificity of the trypsin activation of DNA methylation are described. The only products of the reaction are 5-methylcytosine and thymine. DNA adenine, guanine and cytosine are not labeled. The distribution of the counts between 5-methyl-cytosine and thymine is variable. While 5-methylcytosine originates by enzymatic methylation of DNA cytosines, the origin of the labeled thymine cannot be inferred with certainty.     

Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli

In Escherichia coli, cytosine DNA methylation is catalyzed by the DNA cytosine methyltransferase (Dcm) protein and occurs at the second cytosine in the sequence 5'CCWGG3'. Although the presence of cytosine DNA methylation was reported over 35?years ago, the biological role of 5-methylcytosine in E.?coli remains unclear. To gain insight into the role of cytosine DNA methylation in E.?coli, we (1) screened the 72 strains of the ECOR collection and 90 recently isolated environmental samples for the presence of the full-length dcm gene using the polymerase chain reaction; (2) examined the same strains for the presence of 5-methylcytosine at 5'CCWGG3' sites using a restriction enzyme isoschizomer digestion assay; and (3) quantified the levels of 5-methyl-2'-deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm-mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5-methylcytosine ranges from 0.86% to 1.30% of the cytosines. We also demonstrate that Dcm reduces the expression of ribosomal protein genes during stationary phase, and this may explain the highly conserved nature of this DNA modification pathway.     

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.     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

DNA methylation in the fungi

A systematic study on the incidence and patterns of cytosine methylation in the fungi has been carried out by restriction and nearest-neighbor analysis of DNAs isolated from undifferentiated cells of several fungal species. With respect to DNA modification, the fungi appear to be a heterogeneous group, with a 5-methylcytosine content ranging from undetectable levels (less than or equal to 0.1% of cytosine residues methylated in 18 out of 20 species tested) to low but detectable levels (e.g. congruent to 0.2 and congruent to 0.5% of the total cytosines methylated in Sporotrichum dimorphosporum and Phycomyces blakesleeanus, respectively). In the species where it has been detected, 5-methylcytosine is located mostly at CpG doublets, and the methylated sites are clustered in long tracts (10-30 kilobase pairs) separated from essentially unmethylated regions. This methylated compartment, which comprises a small fraction (1-11%) of the total DNA, contains at least a specific set of repetitive sequences. These results contrast with the higher 5-methylcytosine content found in the fungus Physarum polycephalum and in vertebrates and higher plants.