An essential role for sodium in the bicarbonate transporting system of the cyanobacterium Anabaena variabilis

The Eco dam methylase is active on denatured DNA and single-stranded synthetic oligonucleotides containing GATC sites. The results suggest that on interaction with single-stranded oligonucleotides the Eco dam methylase is able to form a duplex structure within the GATC site, and that this duplex site is a substrate for enzyme.     

Effect of the structure of oligonucleotide substrates on interaction with methylase Eco dam

The interaction of Eco dam methylase with various synthetic oligonucleotide substrates was investigated. The "imperfect" duplexes contained a normal GATC recognition sequence in one chain of the enzyme recognition site and had some defects in the complementary chain, i.e., the absence of one or several nucleotide residues or the presence of S-methyl thiophosphate groups at the 3'-termini. The 3'-S-methyl thiophosphate residue has the same effect on the methylation of oligonucleotide complexes as does the absence of internucleotide phosphate in the analogous complexes. The presence of both GA dinucleotides in the recognition site is necessary for a productive enzyme-substrate interaction. The experimental data suggest that Eco dam methylase does form a symmetrical enzyme-substrate complex which is similar to that formed by type II restriction enzymes.     

Study of the substrate-induced changes in the state of Eco dam methylase using a method of small-angle x-ray scattering

Interaction of Ecodam methylase (E.C. 2.1.1) with synthetic oligonucleotide substrates of various primary structure was studied by the small angle X-ray scattering method. Complex formation between the enzyme and substrates occurs after addition of double-stranded oligonucleotides to the methylase. In the presence of 1 M NaC1 (when the enzyme is inactive) addition of the synthetic substrates does not result in complex formation. Comparison of the experimental scattering parameters with the calculated ones has been made. The best coincidence of these data is obtained for the model which proposed Ecodam methylase dimer formation in the course of its interaction with the substrates.     

Chemical synthesis and properties of oligonucleotide substrates for restriction endonuclease BamHI and methyltransferase Eco dam

Oligodeoxyribonucleotides which form a number of duplexes, containing the recognition sequences for endonuclease BamHI and DNA methylase Eco dam, were synthesised by the phosphotriester approach. Furthermore, synthesis of 3'-phosphorylated oligodeoxyribonucleotides from corresponding S-methyl phosphorothioate triester oligomers is described. The synthetic duplexes are characterized by some defects in the recognition sequences for endonuclease BamHI and methylase Eco dam, viz. nick, absence of an internucleotide phosphate, modifications (including partial single-strandedness) of the recognition site. Interaction of the enzymes with these synthetic substrates was investigated.     

DNA-methylase Sau 3A: isolation and various properties

DNA-methylase Sau 3A has been isolated for the first time from Staphylococcus aureus 3A cells and purified by column chromatography on phosphocellulose PII, heparin-Sepharose and blue Sepharose. The purified enzyme methylates the GATC sequence with the formation of GATm5C as can be evidenced from the protection of DNA from digestion with restrictases Sau 3A and Bam HI, the lack of the C3H3-group incorporation into Sau 3A DNA-restricts and the formation of a single methylated base m5C. Sau 3A methylase modifies only a two-filament (but not one-filament) DNA. Thus, methylase Sau 3A modifies the both DNA chains in the recognition site during a single binding act. The 5-azacytidine-containing DNA inhibits by 95% the activity of methylase Sau 3A. Ado-met is the single methyl group donor for methylase Sau 3A. The presence of m6A in the recognition site does not affect the activity of methylase Sau 3A. The practical recommendations for the use of M. Sau 3A, alongside with M. Eco dam, for the study of dam methylation by additional methylation of the DNA in vitro in the presence of [methyl-3H]-S-adenosyl-methionine are given.     

The double role of methyl donor and allosteric effector of S-adenosyl-methionine for Dam methylase of E. coli.

The turnover of DNA-adenine-methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC.     

Relaxed specificity of the EcoRV restriction endonuclease

The EcoRV restriction endonuclease normally shows a high specificity for its recognition site on DNA, GATATC. In standard reactions, it cleaves DNA at this site several orders of magnitude more readily than at any alternative sequence. But in the presence of dimethyl sulphoxide and at high pH, the EcoRV enzyme cleaves DNA at several sites that differ from its recognition site by one nucleotide. Of the 18 (3 X 6) possible sequences that differ from GATATC by one base, all were cleaved readily except for the following 4 sites: TATATC, CATATC, GATATA and GATATG. However, two of the sites that could be cleaved by EcoRV in the presence of dimethyl sulphoxide, GAGATC and GATCTC, were only cleaved on DNA that lacked dam methylation: both contain the sequence GATC, the recognition site for the dam methylase of Escherichia coli.     

Mycoplasma restriction: identification of a new type of restriction specificity for DNA containing 5-methylcytosine.

Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.     

Sequence and substrate specificity of isolated DNA methylases from Escherichia coli C

Two DNA methylase activities of Escherichia coli C, the mec (designates DNA-cytosine-methylase gene, which is also designated dcm) and dam gene products, were physically separated by DEAE-cellulose column chromatography. The sequence and substrate specificity of the two enzymes were studied in vitro. The experiments revealed that both enzymes show their expected sequence specificity under in vitro conditions, methylating symmetrically on both DNA strands. The mec enzyme methylates exclusively the internal cytosine residue of CCATGG sequences, and the dam enzyme methylates adenine residues at GATC sites. Substrate specificity experiments revealed that both enzymes methylate in vitro unmethylated duplex DNA as efficiently as hemimethylated DNA. The results of these experiments suggest that the methylation at a specific site takes place by two independent events. A methyl group in a site on one strand of the DNA does not facilitate the methylation of the same site on the opposite strand. With the dam methylase it was found that the enzyme is incapable of methylating GATC sites located at the ends of DNA molecules.     

The mechanism by which DNA adenine methylase and PapI activate the pap epigenetic switch

The expression of pyelonephritis-associated pili (Pap) in uropathogenic Escherichia coli is epigenetically controlled by a reversible OFF to ON switch. In phase OFF cells, the global regulator Lrp is bound to pap sites proximal to the pilin promoter, whereas in phase ON cells, Lrp is bound to promoter distal sites. We have found that the local regulator PapI increases the affinity of Lrp for the sequence "ACGATC," which contains the target "GATC" site for DNA adenine methylase (Dam) and is present in both promoter proximal and distal sites. Mutational analyses show that methylation of the promoter proximal GATC(prox) site by Dam is required for transition to the phase ON state by specifically blocking PapI-dependent binding of Lrp to promoter proximal sites. Furthermore, our data support the hypothesis that PapI-dependent binding of Lrp to a hemimethylated GATC(dist) site generated by DNA replication is a critical component of the switch mechanism.     

Interaction of SeqA and Dam methylase on the hemimethylated origin of Escherichia coli chromosomal DNA replication

Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam methylase. Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end of oriC were examined. We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers. Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R. On the other hand, Dam methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences. The binding specificity and higher affinity of SeqA protein over Dam methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam methylase to the newly replicated oriC for the sequestration of chromosomal replication. Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam methylase, and most SeqA protein spontaneously dissociated 10 min after binding. Also, SeqA protein delayed the in vitro methylation of hemimethylated 13-mers by Dam methylase. These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.     

Presence of N6-methyladenine in GATC sequences of Bacillus popilliae and Bacillus lentimorbus KLN2.

Nine strains of Bacillus popilliae and Bacillus lentimorbus KLN2 contain N6-methyladenine in GATC sequences, as determined by using the restriction enzymes MboI and DpnI. Among eight other Bacillus species examined, all, except one strain of Bacillus brevis (ATCC 9999), lacked adenine methylation in GATC. A methylase with Escherichia coli dcm site specificity was not present in any of the Bacillus species studied.     

DNA methylation: sequences flanking C-G pairs modulate the specificity of the human DNA methylase.

Synthetic single-stranded oligodeoxynucleotides of known sequence have been used as in vitro substrates for a partially purified HeLa cell DNA methylase. Although most oligonucleotides tested cannot be used by the HeLa DNA methylase in vitro, we have found a unique 27mer, containing 2 C-G pairs, that is an excellent substrate for the enzyme. Analysis of the methylation of the 27mer, its derivatives and other oligomer substrates reveal that the HeLa DNA methylase does not significantly methylate an oligomer which contains just one C-G pair. In addition, only one of the two C-G pairs in the 27mer is methylated and this methylation is abolished if the other C-G pair is converted to a C-A pair. Furthermore, the HeLa enzyme apparently cannot methylate C-G pairs located in compounds containing a high A + T content. The most efficient methylation occurs with multiple separated C-G pairs in a compound with a high G + C content (greater than 65%). The results suggest that clustering of C-G pairs in regions of the DNA high in G + C content may be the preferred site for DNA methylation in vivo.     

Proteins encoded by the DpnII restriction gene cassette. Two methylases and an endonuclease

Proteins encoded by three genes in the DpnII restriction enzyme cassette of Streptococcus pneumoniae were purified and characterized. Large amounts of the proteins were produced by subcloning the cassette in an Escherichia coli expression system. All three proteins appear to be dimers composed of identical polypeptide subunits. One is the DpnII endonuclease, and the other two are DNA adenine methylase active at 5' GATC 3' sites. Inactivation of enzyme activity by insertions into the genes and comparison of the DNA sequence with the amino-terminal sequence of amino acid residues in the proteins demonstrated the following correspondence between genes and enzymes. The promoter-proximal gene in the operon, dpnM, encodes a 33 X 10(3) Mr polypeptide that gives rise to a potent DNA methylase. The next gene, dpnA, encodes the 31 x 10(3) Mr polypeptide of a weaker and less-specific methylase. The third gene, dpnB, encodes the 34 x 10(3) Mr polypeptide of the endonuclease. Although the endonuclease polypeptide is initiated from an ordinary ribosome-binding site, each of the methylase polypeptide begins at an atypical site with a consensus sequence entirely different from that of Shine & Dalgarno. This presumptive novel ribosome-binding site is well recognized in both S. pneumoniae and E. coli.     

Expression of the Escherichia coli dam methylase in Saccharomyces cerevisiae: effect of in vivo adenine methylation on genetic recombination and mutation.

The Escherichia coli DNA adenine methylase (dam) gene has been introduced into Saccharomyces cerevisiae on a yeast-E. coli shuttle vector. Sau3AI, MboI, and DpnI restriction enzyme digests and Southern hybridization analysis indicated that the dam gene is expressed in yeast cells and methylates GATC sequences. Analysis of digests of total genomic DNA indicated that some GATC sites are not sensitive to methylation. The failure to methylate may reflect an inaccessibility to the methylase due to chromosome structure. The effects of this in vivo methylation on the processes of recombination and mutation in mitotic cells were determined. A small but definite general increase was found in the frequency of mitotic recombination. A similar increase was observed for reversion of some auxotrophic markers; other markers demonstrated a small decrease in mutation frequency. The effects on mutation appear to be locus (or allele) specific. Recombination in meiotic cells was measured and was not detectably altered by the presence of 6-methyladenine in GATC sequences.