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.     

Determination of DNA methylase activity in animal tissue extracts

An express method for measuring the level of in vitro DNA methylation in homogenates and nuclei from animal tissues as well as during initial steps of DNA methylase isolation and purification when methylase activity is low and hardly testable by other methods has been suggested. The method is based on the measuring the radioactivity incorporated in filter adsorbed DNA (acid-insoluble material) 3H-label from S-adenosile-L-methionine as a result of in vitro DNA methylation. The advantage of the method consists in the replacement of a long-duration repeated deproteinization procedure traditionally used by a relatively simple procedure (15 min incubation of the mixture at 80 degrees C with 10 volumes of the 8M urea, 5 mM EDTA, 5% n-butanol, 2% sodium dodecilsulfate, 1 M sodium chloride solution) and the absence of any loss of DNA. The method is fit for the fast serial assay of DNA methylase activity taking into consideration that about one third of the total acid-insoluble radioactivity is due to the radioactivity in 5-methylcytosine residues in 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.     

High-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter

Three different expression systems were constructed for the high-level production of TaqI restriction endonuclease in recombinant Escherichia colicells. In system [R], the TaqI endonuclease gene was cloned and expressed under the control of the strong T7 RNA polymerase promoter. To protect cellular DNA, methylase protection was provided by constitutive co-expression of TaqI methylase activity either by cloning the TaqI methylase gene on a second plasmid (system [R,M]) or by constructing a recombinant plasmid harboring both the endonuclease and methylase genes (system [R+M]). In batch shake flasks containing complex media, co-expression of the methylase gene in systems [R,M] and [R+M] resulted in a 2- and 3-fold increase in volumetric productivity over system [R], yielding activities of 250x10(6) U l(-1) and 350x10(6) U l(-1), which were 28 and 39 times higher than the data in the literature, respectively. Under controlled bioreactor conditions in chemically defined medium, co-expression of methylase activity greatly improved the yield and specific TaqI endonuclease productivity of the recombinant cells, and reduced acetic acid excretion levels. System [R,M] is preferable for high expression levels at longer operation periods, while system [R+M] is well-suited for high expression levels in short-term bioreactor operation.     

Molecular cloning and expression in Escherichia coli of two modification methylase genes of Bacillus subtilis

Two modification methylase genes of Bacillus subtilis R were cloned in Escherichia coli by using a selection procedure which is based on the expression of these genes. Both genes code for DNA-methyltransferases which render the DNA of the cloning host E. coli HB101 insensitive to the BspRI (5'-GGCC) endonuclease of Bacillus sphaericus R. One of the cloned genes is part of the restriction-modification (RM) system BsuRI of B. subtilis R with specificity for 5'-GGCC. The other one is associated with the lysogenizing phage SP beta B and produces the methylase M.BsuP beta BI with specificity for 5'-GGCC. The fragment carrying the SP beta B-derived gene also directs the synthesis in E. coli of a third methylase activity (M.BsuP beta BII), which protects the host DNA against HpaII and MspI cleavage within the sequence 5'-CCGG. Indirect evidence suggests that the two SP beta B modification activities are encoded by the same gene. No cross-hybridization was detected either between the M.BsuRI and M.BsuP beta B genes or between these and the modification methylase gene of B. sphaericus R, which codes for the enzyme M.BspRI with 5'-GGCC specificity.     

Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox.

Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.     

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'.     

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.     

DNA-dependent protein methylase activity in bull seminal plasma.

The existence of a DNA-dependent protein methylase activity without any concomitant DNA methylase activity was demonstrated in bull seminal plasma. The enzyme utilized S-adenosyl-L-methionine as a methyl donor, and endogenous seminal plasma protein as the substrate. There was no demonstrable enzyme activity when the seminal plasma was preheated at 100 degrees for 10 min, or when the enzyme reaction mixture was incubated at 4 degrees. The protein methylase required a heterologous DNA source, had optimal activity at pH 8.1, and was enhanced in the presence of Mg2+, NH4+, and reduced glutathione. After the methylated protein product was separated from DNA by extraction with 0.2 M HCl, the incorporated radioactivity was shown to be totally solubilized by incubating the protein either with Pronase or 1 M NaOH, while RNase and DNase had no effect. Approximately 70% of the enzymatically synthesized amino acids in the protein product were tentatively identified as O-methylated amino acid ethers by virtue of their elution from a Dowex 50 H+ column with 0.2 M pyridine, and their stability to acid and base hydrolysis. The partially purified methylated product was shown by Sephadex G-50 chromatography to consist of three distinct radioactive proteins with molecular weights of approximately 21,000, 15,000, and 10,000.     

Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.

In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transformation frequencies of oriC plasmids into E. coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation. Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase. oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells. This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase. Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E. coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity. Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin.     

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.     

ScrFI restriction-modification system of Lactococcus lactis subsp. cremoris UC503: cloning and characterization of two ScrFI methylase genes.

Two genes from the total genomic DNA of dairy starter culture Lactococcus lactis subsp. cremoris UC503, encoding ScrFI modification enzymes, have been cloned and expressed in Escherichia coli. No homology between the two methylase genes was detected, and inverse polymerase chain reaction of flanking chromosomal DNA indicated that both were linked on the Lactococcus genome. Neither clone encoded the cognate endonuclease. The DNA sequence of one of the methylase genes (encoded by pCI931M) was determined and consisted of an open reading frame 1,170 bp long, which could encode a protein of 389 amino acids (M(r), 44.5). The amino acid sequence contained the highly characteristic motifs of an m5C methylase. Extensive regions of homology were observed with the methylases of NlaX, EcoRII, and Dcm.     

Nucleotide sequence of the DdeI restriction-modification system and characterization of the methylase protein.

The DdeI restriction-modification system was previously cloned and has been maintained in E. coli on two separate and compatible plasmids (1). The nucleotide sequence of the endonuclease and methylase genes has now been determined; it predicts proteins of 240 amino acids, Mr = 27,808, and 415 amino acids, Mr = 47,081, respectively. Inspection of the DNA sequence shows that the 3' end of the methylase gene had been deleted during cloning. The clone containing the complete methylase gene was made and compared to that containing the truncated gene; only clones containing the truncated form support the endonuclease gene in E. coli. Bal-31 deletion studies show that methylase expression in the Dde clones is also dependent upon orientation of the gene with respect to pBR322. The truncated and complete forms of the methylase protein were purified and compared; the truncated form appears to be more stable and active in vitro. Finally, comparison of the deduced amino acid sequence of M. DdeI with that of other known cytosine methylases shows significant regions of homology.     

鼠肝细胞癌变中DNA甲基化作用的研究

Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments.     

Isolation and characterization of two proteins possessing Hpa II methylase activity

Two proteins exhibiting Hpa II methylase activity have been purified to homogeneity from Haemophilus parainfluenzae and their physical and catalytic properties have been studied. Separation of the two Hpa II methylase activities was achieved by DEAE-Sephadex A-50 chromatography. In subsequent steps, each methylase was purified separately by chromatography on Sephacryl S-200, phosphocellulose, and hydroxylapatite. The proteins have molecular weights of 38,500 +/- 1,000 (Hpa II) and 41,500 +/- 1,000 (Hpa II') as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Sedimentation equilibrium analyses of the native proteins yield molecular weights of 38,800 +/- 3,000 and 42,200 +/- 3,000 for Hpa II and Hpa II', respectively, indicating that both enzymes are composed of a single subunit. Furthermore, both methylases exhibit identical specificity in the methylation of the nucleotide sequence dC-C-G-G in simian virus 40 (SV40) DNA and in a short synthetic oligonucleotide duplex. Although pH, temperature, and salt optima are the same for both enzymes, homogeneous Hpa II' methylase is more stable than Hpa II methylase. Preliminary peptide mapping indicates that the two enzymes are structurally related, suggesting the possibility that Hpa II' methylase may represent a precursor form of Hpa II methylase.     

Partial purification and characterisation of S-adenosylmethionine:protein-histidine N-methyltransferase from rabbit skeletal muscle

A new class of protein methylase (S-adenosylmethionine:protein-histidine N-methyltransferase) which methylates histidine residues of protein substrates using S-adenosylmethionine as the methyl donor has been partially purified from rabbit skeletal muscle, 22-fold with a yield of 56%. The enzyme activity was monitored using denatured myofibrils from young rabbit skeletal muscle as the methyl acceptor protein substrate. The enzyme was localised in the myofibrillar fraction and myofibrils isolated in pure form represented the enzyme-substrate complex. The enzyme was solubilised in 0.275 M KCl. The methylase showed no requirement for any metal ion and has a pH optimum of 8.0. It was shown to require a reducing agent like mercaptoethanol for its activity. It was also shown that cardiac and skeletal muscle forms of actins obtained from different species serve as the efficient methyl acceptor protein substrates. Since the enzyme was found to methylate specifically the histidine residues of actin we propose to designate this new methylase as protein methylase IV, to distinguish it from the already known protein methylases I, II and III.     

Mouse ascites DNA methylase: characterisation of size, proteolytic breakdown and nucleotide recognition

We have purified the DNA methylase from mouse ascites tumour cells to a specific activity of 11,500 units per mg protein using denatured Micrococcus luteus DNA as methyl acceptor. Methyl groups are transferred to cytosines almost exclusively in CpG dinucleotides. The purified enzyme contains two polypeptides of molecular mass 185 and 160 kDa, and an antiserum raised in a rabbit to the purified enzyme specifically reacts with these two proteins in crude extracts. The two proteins can be partially separated by affinity chromatography when activity is associated with the 185 kDa protein which can be proteolytically degraded to give polypeptides of 170 and later 100 and 50 kDa. Only the 185 kDa methylase is lost when cells are treated with azadeoxycytidine and this is the predominant form firmly bound in the nucleus of dividing cells. Antibody bound to the 185 kDa band in protein blots will itself bind native DNA methylase, which can be detected by its binding 14C-labelled, azacytosine-containing DNA.