DNA methylases of Hemophilus influenzae Rd. II. Partial recognition site base sequences

A small percentage of the adenine bases in Hemophilus influenzae strain Rd DNA are methylated in the 6-amino position. The methyl groups are introduced specifically by at least four different DNA methylases (I, II, III and IV). A method is described for determining the 3′ and 5′ nearest-neighbor bases to methylated adenine so as to reveal the specificity of each methylase. Tritium-labeled methyl groups are introduced into the DNA. The DNA is then digested to dinucleotides using the Bacillus subtilis phage SP3 DNase, followed by removal of the terminal 5′-phosphoryl group with phosphatase to produce dinucleoside monophosphates. These are analyzed by Aminex A25 (Bio-Rad) chromatography. Dinucleoside monophosphate species containing the 3′ neighbor or the 5′ neighbor are resolved so that a trinucleotide is determined that contains the centrally placed methylated adenine. H. influenzae Rd DNA contains seven dinucleoside monophosphate species, about 80% representing GpmA and mApT in approximately equal amount. DNA methylases I, II, III and IV introduce methyl groups into sequences containing the trinucleotides CpmApC, PupmApC, NpmApA and GpmApT, respectively. The sequence methylated by NDA methylase II is consistent with the recognition site determined by Kelly and Smith (1970) for the H. influenzae restriction enzyme, endonuclease R.     

Sequence specificity of the P1 modification methylase (M.Eco P1) and the DNA methylase (M.Eco dam) controlled by the Escherichia coli dam gene.

Labeled oligonucleotides have been fractionated from pancreatic DNase digests of DNA that had been methylated in vitro with the P1 modification enzyme (M·Eco P1) or with the DNA-adenine methylase (M·Eco dam) controlled by the Escherichia coli dam gene. The sequences of methylated oligonucleotides were established for M·Eco dam modification of calf thymus DNA. The results show that M·Eco dam inethylates adenine residues contained in the twofold symmetrical sequence, 5′ … G-A-T-C … 3′. The sequence for the site methylated by M·Eco P1 has also been deduced; we propose that M·Eco P1 modification produces the following methylated pentameric sequence: 5′ … A-G-A¹-C-Py … 3′ (where $\text{A}{}^1\text{= N}{}^6$ methyladenine and Py is C or T).     

Methylation of parental and progeny DNA strands in Physarum polycephalum

Although 5-methylcytosine comprises 4 to 8% of the cytosine residues in the major nuclear DNA of Physarum polycephalum (Evans &; Evans, 1970), only 1 % of the cytosine residues of progeny DNA become methylated during replication. Further methylation occurs during the same and subsequent mitotic cycles, so that 6 to 7 cycles after its synthesis, 5-methylcytosine comprises 5 to 7% of the DNA-cytosine residues of a single generation of DNA. The extent of methylation occurring during the S period has been measured by the determination of the specific activity of the precursor (S-adenosylmethionine) and the product (DNA-5-methylcytosine) and by comparison of the radioactivity in DNA-cytosine and DNA-5-methylcytosine after incorporation of [¹⁴C]deoxycytidine. Continuing methylation of parental DNA has been shown, by density shift experiments and by the conversion of prelabeled DNA-cytosine to DNA-5-methylcytosine. The DNA-5-methylcytosine once formed was found to be stable.     

A genetic and functional analysis of the unusually large variable region in the M·Alu I DNA-(cytosine C5 )-methyltransferase

The M·AluI DNA-(cytosine C5)-methyltransferase (5mC methylase) acts on the sequence 5′-AGCT-3′. The amino acid sequences of known 5mC methylases contain ten conserved motifs, with a variable region between Motifs VIII and IX that contains one or more “target-recognizing domains” (TRDs) responsible for DNA sequence specificity. Monospecific 5mC methylases are believed to have only one TRD, while multispecific 5mC methylases have as many as five. M·AluI has the second-largest variable region of all known 5mC methylases, and sequence analysis reveals five candidate TRDs. In testing whether M·AluI is in fact monospecific it was found that AGCT methylation represents only 80–90% of the methylating activity of this enzyme, while control experiments with the enzyme M·HhaI gave no unexplained activity. Because individual TRDs can be deleted from multispecific methylases without general loss of activity, a series of insertion and deletion mutants of the M·AluI variable region were prepared. All deletions that removed more than single amino acids from the variable region caused significant loss of activity; a sensitive in vivo assay for methylase activity based on McrBC restriction suggested that the central portion of the variable region is particularly important. In some cases, multispecific methylases can accommodate a TRD from another multispecific methylase, thereby acquiring an additional specificity. When TRDs were moved from a multispecific methylase into two different locations in the variable region of M·AluI, all hybrid enzymes had greatly reduced activity and no new specificities. M·AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.     

Sequence specificity of the wild-type dam+) and mutant (damh) forms of bacteriophage T2 DNA adenine methylase

Non-glucosylated, non-methylated phage T2 DNA was methylated in vitro with partially purified wild-type (dam⁺) or mutant (dam^h) T2 DNA adenine methylase. The radioactively labeled methyladenine-containing DNA was enzymatically degraded and the resulting oligonucleotides were separated according to chain length by DEAE-cellulose chromatography. Following “fingerprinting” by two-dimensional electrophoresis, we determined the sequence for various di-, tri- and tetranucleotides containing radioactive N⁶-methyldeoxyadenosine. From this analysis we conclude that both T2 dam⁺ and T2 dam^h contain the sequence 5′…G-mA -Py…3′.     

Cloning and sequence comparison ofAvaI andBsoBI restriction-modification systems

AvaI andBsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5′CYCGRG3′ and cleave between the first C and second Y to generate a four-base 5′ extension. TheAvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned intoEscherichia coli by the methylase selection method. TheBsoBI restriction endonuclease gene (bsoBIR) and part of theBsoBI methylase gene (bsoBIM) were cloned by the “endo-blue” method (SOS induction assay), and the remainder ofbsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated thatAvaI andBsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. TheavaIM gene precedesavaIR in theAvaI RM system, while thebsoBIR gene is located upstream ofbsoBIM in theBsoBI RM system. BothAvaI andBsoBI methylases contain motifs conserved among the N⁴ cytosine methylases.     

Procaryotic and eucaryotic traits of DNA methylation in spiroplasmas (mycoplasmas).

Differences in the type of base methylated (cytosine or adenine) and in the extent of methylation were detected by high-pressure liquid chromatography in the DNAs of five spiroplasmas. Nearest neighbor analysis and digestion by restriction enzyme isoschizomers also revealed differences in methylation sequence specificity. Whereas in Spiroplasma floricola and Spiroplasma sp. strain PPS-1 5-methylcytosine was found on the 5' side of each of the four major bases, the cytosine in Spiroplasma apis DNA was methylated only when its 3' neighboring base was adenine or thymine. In Spiroplasma sp. strain MQ-1 over 95% of the methylated cytosine was in C-G sequences. Essentially all of the C-G sequences in the MQ-1 DNA were methylated. Partially purified extracts of S. apis and Spiroplasma sp. strain MQ-1 were used to study substrate and sequence specificity of the methylase activity. Methylation by the MQ-1 enzyme was exclusively at C-G sequences, resembling in this respect eucaryotic DNA methylases. However, the MQ-1 methylase differed from eucaryotic methylases by showing high activity on nonmethylated DNA duplexes, low activity with hemimethylated DNA duplexes, and no activity on single-stranded DNA.     

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.     

In vitro methylation of bacteriophage lambda DNA by wild type (dam+) and mutant (damh) forms of the phage T2 DNA adenine methylase.

The wild-type (dam⁺) and mutant (dam^h) forms of the bacteriophage T2 DNA adenine methylase have been partially purified; these enzymes methylate the sequence, 5/t' … G-A-Py … 3′ (Hattman et al., 1978a). However, in vitro methylation studies using phage λ DNA revealed the following: (1) T2 dam⁺ and dam^h enzymes differ in their ability to methylate λ DNA; under identical reaction conditions the T2 dam^h enzyme methylated λ DNA to a higher level than did the dam⁺ enzyme. However, the respective methylation sites are equally distributed on the l and r strands. (2) Methylation with T2 dam^h, but not T2 dam⁺ protected λ against P1 restriction. This was demonstrated by transfection of Escherichia coli (P1) spheroplasts and by cleavage with R·EcoP1. (3) T2 dam⁺ and dam^h were similarly capable of methylating G-A-T-C sequences on λ DNA; e.g. λ·dam₃ DNA (contains no N⁶-methyladonine) methylated with either enzyme was made resistant to cleavage by R·DpnII. In contrast, only the T2 dam^h modified DNA was resistant to further methylation by M·EcoP1 (which methylates the sequence 5′ … A-G-A-C-Py … 3′; Hattman et al., 1978b). (4) λ·dam₃ DNA was partially methylated to the same level with T2 dam⁺ or T2 dam^h; the two enzymes produced different patterns of G-A-C versus G-A-T methylation. We propose that the T2 dam⁺ enzyme methylates G-A-C sequences less efficiently than the T2 dam^h methylase; this property does not entirely account for the large difference in methylation levels produced by the two enzymes.     

Fractionation and specificity of DNA methylases from the Escherichia coli SK cells

Summary Specificity of DNA methylation enzymes from the E. coli SK cells and conditions for their separation have been investigated. Column chromatography on carboxymethylcellulose permits fractionation of methylase activity into six discrete peaks whose specificity to the methylated base has been determined in vitro with H³-SAM as precursor. All methylases specific for adenine produced 6-methylaminopurine, all methylases specific for cytosine yielded 5-methylcytosine.The first enzymatic activity peak containing cytosine methylase free of traces of adenine-methyiating activity (E₁), and the second peak containing both the enzymes (E₂) were not adsorbed on the ion exchanger and went off the column with the effluent (column buffer). Adenine specific methylase E₂ is retarded to a small extent during the passage through the column. The second adenine methylases (W) was characterized by weak bonds with the ion exchanger and was removed when washing the column with column buffer. The elution with NaCl gradient produced successively three enzymatic activity peaks: adenine methylase (G_I), cytosine methylase (G_II), and one more adenine methylase (G_III) removed from the column by 0.16 m, 0.24 m and 0.43 m NaCl respectively.Using a new modification of the complementary methylation test, the specificity with regard to recognition site was examined for all enzymes, except for W and G_III, which were extremely unstable. The adenine methylases E₂ and G_I and the cytosine methylases E₁ and G_II were shown to recognize different sites and to be different enzymes. In view of the drastic differences in their chromatographic behaviour and physical stability, the adenine methylases W and G_III are probably also different enzymes.     

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.     

Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M.SssI).

We describe here the cloning, characterization and expression in E. coli of the gene coding for a DNA methylase from Spiroplasma sp. strain MQ1 (M.SssI). This enzyme methylates completely and exclusively CpG sequences. The Spiroplasma gene was transcribed in E. coli using its own promoter. Translation of the entire message required the use of an opal suppressor, suggesting that UGA triplets code for tryptophan in Spiroplasma. Sequence analysis of the gene revealed several UGA triplets, in a 1158 bp long open reading frame. The deduced amino acid sequence revealed in M.SssI all common domains characteristic of bacterial cytosine DNA methylases. The putative sequence recognition domain of M.SssI showed no obvious similarities with that of the mouse DNA methylase, in spite of their common sequence specificity. The cloned enzyme methylated exclusively CpG sequences both in vivo and in vitro. In contrast to the mammalian enzyme which is primarily a maintenance methylase, M.SssI displayed de novo methylase activity, characteristic of prokaryotic cytosine DNA methylases.     

Isolation and Characterization of Site-Specific DNA-methyltransferases from Bacillus coagulans K

Two site-specific DNA methyltransferases, M.BcoKIA and M.BcoKIB, were isolated from the thermophilic strain Bacillus coagulans K. Each of the methylases protects the recognition site 5'-CTCTTC-3'/5'-GAAGAG-3' from cleavage with the cognate restriction endonuclease BcoKI. It is shown that M.BcoKIB is an N6-adenine specific methylase and M.BcoKIA is an N4-cytosine specific methylase. According to bisulfite mapping, M.BcoKIA methylates the first cytosine in the sequence 5'-CTCTTC-3'.     

Sequence specificity of isolated DNA-cytosine methylases from Shigella sonnei 47 cells

Five individual DNA-cytosine methylases differing in pI (isoelectric point) values are present in Shigella sonnei 47-cells. The sequence specificity of each of those was determined 'in vitro' by a highly efficient combined approach that included pyrimidine tract (isostic) analysis, identification of the immediate neighbourhood of the methylated base within the recognition sequence and the calculation method. The enzyme with pI 5.3 (MSso5.3) is the counterpart of the RSso 47 II in the Sso 47 II restriction-modification system and methylates the internal cytosine residue of the 'palindromic' 5'-C-C-N-G-G-3' sequence. The enzymes with pI 6.2 (MSso6.2) and 7.4 (MSso7.4) exhibit identical specificity upon methylation of the 'palindromic' 5'-Py-C-N-G-Pu-3' sequence, but differ in the pI values of the proteins. The enzyme with pI 4.2 (MSso4.2) recognizes the unique tetranucleotide 5'-C-C-C-C-3' sequence and methylates the second cytosine residue at the 5'-end of the sequence. The enzyme with pI 8.4 (MSso8.4) methylates the central cytosine residue within the degenerative trinucleotide 5'-(PuC)-C-C-3' sequence. MSso5.3, MSso6.2, and MSso7.4 are presumed to belong to the 'family' of sequence-specific (Eco RII-like) enzymes. These DNA-cytosine methylases are likely to be evolutionary related to Eco RII and to have undergone a sufficient genetic drift so as to recognize similar (but more degenerative) nucleotide sequences.     

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.     

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.     

Mode of action of the Spiroplasma CpG methylase M.SssI.

The cytosine DNA methylase from the wall-less prokaryote, Spiroplasma strain MQ1 (M.SssI) methylates completely and exclusively CpG-containing sequences, thus showing sequence specificity which is similar to that of mammalian DNA methylases. M.SssI is shown here to methylate duplex DNA processively as judged by kinetic analysis of methylated intermediates. The cytosine DNA methylases, M.HpaII and M.HhaI, from other prokaryotic organisms, appear to methylate in a non-processive manner or with a very low degree of processivity. The Spiroplasma enzyme interacts with duplex DNA irrespective to the presence of CpG sequences in the substrate DNA. The enzyme proceeds along a CpG-containing DNA substrate molecule methylating one strand of DNA at a time.     

DNA protection with the DNA methylase M . BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII

BamHI fragments of the Bacillus brevis var. GB plasmid pAD1 have been cloned in Escherichia coli HB101 using pBR322 plasmid as a vector. The analysis of the recombinant plasmids showed that additional PstI sites had appeared in cloned fragments of pAD1. Methylation of the recombinant plasmids in vitro by enzymes from B. brevis GB cells blocks cleavage at these additional PstI sites of cloned pAD1 fragments and at the PstI site of pBR322. Among DNA methylases of B. brevis GB, the cytosine DNA methylase M . BbvI is the most likely agent modifying the recognition sequences of PstI. The methylase can modify cytosine residues in PstI or PvuII sites if these recognition sequences are linked to G at 5'- or to C at 3'-termini. In particular, in vitro methylation of the SV40 DNA by B. brevis GB methylases protects one of the two PstI sites and two of the three PvuII sites. The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.     

Complementary action of restriction enzymes endo R-DpnI and Endo R-DpnII on bacteriophage f1 DNA

Bacteriophage f1 duplex DNA was isolated from Escherichia coli strains containing different DNA methylases and assayed for its sensitivity to endonucleolytic cleavage by the enzymes endo R · DpnI and endo R · DpnII. The former enzyme is specific for methylated DNA, the latter for unmethylated DNA (Lacks &; Greenberg, 1975). The E. coli dam methylase was found to be responsible for making f1 resistant to endo R · DpnII and sensitive to endo R · DpnI. Endo R · DpnI cleaved f 1 DNA from dam⁺ cells at four sites. Additional methylation by enzymes other than the dam methylase gave no further cleavage. Endo R · DpnII cleaved f1 DNA from dam^? cells also at four sites to give restriction fragments identical to those obtained with endo R · DpnI cleavage. Thus, the two enzymes are complementary in that they recognize and cleave within the same DNA sequence, one if the DNA is methylated, the other if it is unmethylated. DNA duplexes containing one methylated strand (dam ⁺) and one unmethylated strand (dam^?) were prepared in vitro. These methylated hybrids were refractory to endonucleolytic cleavage by both endo R · DpnI and endo R · DpnII. Neither enzyme, therefore, appears to make even a single strand break at a methylated/unmethylated hybrid site.     

Effects of 5-azacytidine induced DNA demethylation on methyltransferase gene expression and resveratrol production in cell cultures of Vitis amurensis

DNA becomes methylated in vivo through the action of a specific group of enzymes known as methyltransferases or methylases. Plants are known to possess the methyltransferases (Met), chromo methyltransferases (CMT), and domainrearranged methyltransferases (DRM) methylase families, which affect cytosine methylation within different contexts. DNA methylation has been proposed to play a role in secondary plant metabolism, but there is a lack of valid data connecting these two processes. In this study, we treated control and transformed with rolB gene from Agrobacterium rhizogenes cell cultures of Vitis amurensis with the demethylation agent 5-azacytidine (azaC). The purpose of the current investigation was to study effects of induced DNA demethylation on methyltransferase gene expression in connection to resveratrol production, a naturally occurring polyphenol that has a wide range of intriguing biological properties. Using semi-quantitative and real-time PCR, we showed that rolB gene transformation of V. amurensis cells decreased Met and CMT expression, but significantly increased DRM expression. AzaC treatment of the control and the rolB-transgenic calli significantly increased expression of all methylases (excluding Met). Following 3 months of azaC treatment, we detected significantly elevated levels of rolB gene expression in the transgenic calli. In current paper, we discuss how methylase expression may influence resveratrol biosynthesis and rolB transgene expression. Effects of azaC application are discussed.