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.     

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.     

Determination of methylation specificity of DsaV methyltransferase by a simple biochemical method.

We have developed a simple new method that can identify the base methylated by a sequence-specific DNA methyltransferase and have used it to identify the cytosine that is methylated by DsaV methyltransferase (M. DsaV) within its recognition sequence 5'-CCNGG. The method utilizes the fact that exonuclease III of E. coli does not degrade DNA ends with 3' overhangs and cannot hydrolyze a phosphorothioate linkage. DNA duplexes containing phosphorothioate linkages at specific positions were methylated with M. DsaV in the presence of [methyl-3H] S-adenosylmethionine and were subjected to exonuclease III digestion. The pattern of [methyl-3H] dCMP release from the duplexes was consistent with the methylation of the internal cytosine in CCNGG, but not of the outer cytosine. To establish the accuracy of this method, we confirmed the known specificity of EcoRII methyltransferase by the method. We also confirmed the specificity of M. DsaV using an established biochemical method that involves the use of a type IIS restriction enzyme. Methylation of CCWGG (W = A or T) sequences at the internal cytosines is native to E. coli and is not restricted by the modified cytosine restriction (Mcr) systems. Surprisingly, the gene for M. DsaV was significantly restricted by the McrBC system. We interpret this to mean that M. DsaV may occasionally methylate at sequences other than CCNGG or may occasionally methylate the outer cytosine in its recognition sequence.     

Determination of the adenine-specific methylase recognition site of Shigella sonnei 47, using hydrazinolysis with subsequent separation of purine oligonucleotides by thin layer chromatography on DEAE- cellulose

A procedure for separation of oligopurine blocks of different length and composition by two-dimensional thin layer chromatography on DEAE-cellulose plates has been developed. This method allows a comparative analysis of the purine isostich content in the DNAs of various origin. In case of methylated DNA, the method permits to compare the substrate specificity of different enzymes responsible for the adenine residue methylation in the DNA. In combination with enzymatic treatment of labeled methylated isostichs, the method described can be used for the deciphering of the methylated sequences as well as for constructing, in a number of cases, the recognition site of adenine-specific methylases. Thus, it was demonstrated that methylase SsoI recognizes the 5...G-A-A-T-T-C ... 3' sequence and methylates its adenine residue nearest to the 5'-end.     

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.     

Sequence specificity of isolated DNA-adenine methylases from Mycobacterium smegmatis (butyricum) and Shigella sonnei 47 cells

A set of four individual DNA-adenine methylases differing in pI (isoelectric point) values (MMbu4.2, MMbu6.4, MMbu7.3, and MMbu8.7), and a sole methylating enzyme with the same base specificity (MSso9.5) are present in M. smegmatis (butyricum) and Sh. sonnei 47 cells, respectively. The sequence specificity of each of those was studied 'in vitro' by a combined approach that comprised isostich (purine tract) analysis and identification of the immediate neighbourhood of the methylated base within the sequence methylated. The MSso9.5 recognition site has been established as the hexanucleotide 'palindromic' 5'-G-A-A-T-T-C-3' sequence which is structurally similar to the analogous MEco RI recognition site. However, in contrast to MEco RI, MSso9.5 methylates the 5'-end adenine residue in the sequence and thus it appears to be an isometimer of MEco RI. By means of the same approach, the partial nucleotide sequences methylated by each of the four individual M. butyricum enzymes were determined. MMbu7.3 and MMbu8.7 exhibit the identical sequence specificity upon methylation of the degenerative trinucleotide 5'-Py-A-Py-3' sequence and thus these enzymes are assumed to represent the different molecular forms of the methylase. MMbu4.2 methylates the 5'-G-G-A-3' sequence and thus it is of a great value as the tool for negating effects of the RBam HI and RAva II-type restriction. MMbu6.4 is of a particular interest on account of its unique DNA methylation pattern which is distinguished in the pronounced clustering of purine bases in the 5'-Pu-Pu-Pu-Pu-Pu-3' sequence methylated.     

Detection of 5-methylcytosine in DNA sequences.

Col E1 DNA has methylated cytosine in the sequence 5'-CC*(A/T)GG-3' and methylated adenine in the sequence 5'-GA*TC-3' at the positions indicated by asterisks(*). When the Maxam-Gilbert DNA sequencing method is applied to this DNA, the methylated cytosine (5-methylcytosine) is found to be less reactive to hydrazine than are cytosine and thymine, so that a band corresponding to that base does not appear in the pyrimidine cleavage patterns. The existence of the methylated cytosine can be confirmed by analyzing the complementary strand or unmethylated DNA. In contrast, the methylated adenine (probably N6-methyladenine) cannot be distinguished from adenine with standard conditions for cleavage at adenine.     

Sse8387I, a useful eight base cutter for mammalian genome analysis (influence of methylation on the activity of Sse8387I).

To develop restriction enzymes that are useful for genome analysis, we previously performed screening and isolated Sse8387I from Streptomyces sp. strain 8387. Sse8387I is a restriction enzyme that recognizes 5'-CCTGCA/GG-3' and cleaves DNA at the site shown by the diagonal (Nucleic Acid Res., 18, 5637-5640). The present study evaluated the effects of methylation that is important when Sse8387I is used for genome analysis. Sse8387I lost cleavage activity after methylation of adenine or methylation of cytosine at any site in the recognition sequence. However, the recognition sequence of Sse8387I contains no CG sequence, which is the mammalian methylation sequence. In addition, we evaluated the effects of methylation of CG at sites other than the recognition sequence. The cleavage activity of Sse8387I was maintained even when CG sequences were present immediately before or after, or near the recognition sequence, and cytosine was methylated. These results suggest that CG methylation does not affect the cleavage activity of Sse8387I. Therefore, Sse8387I seems to be very useful for mammalian genome analysis.     

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.     

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.     

Fractionation and purification of DNA methylases and specific endonucleases from cells of Escherichia coli SK]

Fractionation and purification of DNA methylases and specific endonucleases from E. coli SK responsible for DNA specificity to host prokaryotic cells were studied. The most efficient purification was achieved by precipitation of proteins by 0.6 saturated ammonium sulfate with subsequent chromatography on KM-cellulose and concentration of fractions by dialysis against glycerol. Under these conditions the methylase activity produced 4 discrete fractions. Due to purification the specific activity of methylases increased 11--20-fold in various fractions. Methylase from the first (A) and fourth (BII) peaks catalyzed the methylation of cytosine to produce 5-methylcytosine; methylase from the third peak (BI) methylated adenine to produce 6-methylaminopurine. The chemical specificity of the second peak (B) methylase could not be established due to very high lability of the enzyme in this fraction. Specific endonuclease was found in the gradient zones eluted by 0.1--0.2 M and 0.65--0.75 M NaCl. It is assumed that those enzymes providing for DNA hydrolysis up to the formation of high--molecular discrete fragments, are restricting endonucleases of the SK system. The results obtained strongly suggest the existence of several types of methylases and restricting endonucleases in E. coli SK cells.     

Identification and sequence analysis of a methylase gene in Porphyromonas gingivalis.

A gene from the periodontal organism Porphyromonas gingivalis has been identified as encoding a DNA methylase. The gene, referred to as pgiIM, has been sequenced and found to contain a reading frame of 864 basepairs. The putative amino acid sequence of the encoded methylase was 288 amino acids, and shared 47% and 31% homology with the Streptococcus pneumoniae DpnII and E. coli Dam methylases, respectively. The activity and specificity of the pgi methylase (M.PgiI) was confirmed by cloning the gene into a dam- strain of E. coli (JM110) and performing a restriction analysis on the isolated DNA with enzymes whose activities depended upon the methylation state of the DNA. The data indicated that M.PgiI, like DpnII and Dam, methylated the adenine residue within the sequence 5'-GATC-3'.     

Non-C-G recognition sequences of DNA cytosine-5-methyltransferase from rat liver

The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.     

Site-specificity of DNA methylases from Shigella sonnei 47 cells

A complex approach involving isoplith analysis, enzymatic treatment of methylated isopliths and a computer analysis of experimental data has been used for determining site specificity of six methylases from Shigella sonnei 47 cells termed according to their specificity for a nitrous base and pI as MC4.2, MC5.3, MC6.2, MC7.4, MC8.4 and MA9.5. It has been found that the recognition site of MA9.5 is a palyndrome six-member structure of the 5'...GAATTC...3' type and that this enzyme is an isometimer with respect to MEcoRI. It has been demonstrated for the first time for methylases that the recognition site of MC4.2 is represented by a non-symmetrical four-member sequence, 5'...NCCCCN...3' characterized by unique blocking of cytosines. MC8.4 possesses a broad specificity of substrate recognition and methylates the cytosine residue within the composition of the non-symmetrical unique sequence 5'...N (C/Pu) CCN...3', whose 5'-terminal base is depleted in three nucleotides. MC5.3 methylates the 3'-terminal cytosine residue within the composition of the pentanucleotide palindrome recognition site, 5'...CCNGG...3'. MC6.2 and MC7.4 possess identical pentanucleotide recognition sites of 5'...(Py)CNG(Pu)...3', but are distinguished in pI. The latter finding has been shown for the first time for different methylases within one strain.     

Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections.

Methylations of highly specific sites in the promoter and 5' regions of eucaryotic genes have been shown to shut off gene activity and thus play a role in the long-term regulation of gene expression. It was therefore of interest to investigate whether site-specific DNA methylations could also play a role in adenovirus DNA in productive infections. It has been reported earlier that adenovirion DNA is not detectably methylated (U. Günthert, M. Schweiger, M. Stupp, and W. Doerfler, Proc. Natl. Acad. Sci. USA 73:3923-3927, 1976). In the present study, evidence for the methylation of cytidine residues in 5'-CCGG-3' and 5'-GCGC-3' sequences or the methylation of adenine residues in 5'-GATC-3' and 5'-TCGA-3' sequences in intranuclear adenovirus type 2(Ad2) DNA isolated and analyzed early (5 h) or late (24 h) after infection could not be obtained. In Ad2 DNA, 22.5% of all 5'-CG-3' dinucleotides reside in 5'-CCGG-3' and 5'-GCGC-3' sequences. Intranuclear viral DNA was examined by restriction endonuclease cleavage by using HpaII, MspI, HhaI, DpnI, or TaqI and Southern blot hybridizations. The HindIII fragments of Ad2 DNA served as hybridization probes. The data rendered it very unlikely that free intracellular adenovirus DNA in productively infected cells was extensively methylated. Thus, DNA methylation was not a likely element in the regulation of free adenovirus DNA expression in productively infected cells.     

Arthrobacter viscosus DNA is modified at GATC sequence

Arthrobacter viscosus DNA was resistance to digestion by restriction enzymes that are sensitive to methylation of the cytosine residue (but not of adenine) within the GATC recognition sequence. Restriction enzymes sensitive to methylation of cytosine in other recognition sequences were not affected. A. viscosus DNA thus appeared to contain methylated cytosine specifically at the GATC sequence.     

Molecular analysis of N6-methyladenine patterns in Tetrahymena thermophila nuclear DNA.

We have cloned two DNA fragments containing 5'-GATC-3' sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila. Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites. Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena. Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA. In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth. This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation. We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns.     

How M.MspI and M.HpaII decide which base to methylate.

The HpaII methylase (M.HpaII) recognizes the sequence CCGG and methylates the inner cytosine residue. The MspI methylase (MspI) recognizes the same sequence but methylates the outer cytosine residue. Both methylases have the usual architecture of 10 well-conserved motifs surrounding a variable region, responsible for sequence specific recognition, that is quite different in the two methylases. We have constructed hybrids between these two methylases and studied their methylation properties. A hybrid containing the variable region and C-terminal sequences from M.MspI methylates the outer cytosine residue. A second hybrid identical to the first except that the variable region derives from the M.HpaII methylates the inner cytosine residue. Thus the choice of base to be methylated within the recognition sequence is determined by the variable region.     

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.