Partial purification of a ribosomal ribonucleic acid methylase from rat liver nuclei and methylation of undermethylated nuclear ribonucleic acid from regenerating liver of ethionine-treated rat

S-Adenosylmethionine-dependent ribosomal RNA (rRNA) methylase has been purified approx. 90-fold from rat liver nuclei. The partially purified methylase catalyzes the methylation of base and ribose in hypomethylated nuclear rRNA prepared from the regenerating rat liver after treatment with ethionine and adenine. The enzyme has an apparent molecular weight of about 3 x 10(4) and a sedimentation coefficient of 3.0 S. The enzyme is optimally active at pH 9.5 and sensitive to p-chloromercuribenzoate. Thiol-protecting reagents, such as dithiothreitol, are necessary for its activity, and the enzyme requires no divalent cations for its full activity. This enzyme did not efficiently transfer the methyl group to nuclear rRNA from normal rat liver, compared with hypomethylated nuclear rRNA. Methyl groups were mainly incorporated into pre-rRNA larger than 28 S, and the extent of 2'-O-methylation of ribose by this enzyme was greater than that of base methylation in the hypomethylated rRNA. No other nucleic acids, including transfer RNA (tRNA) and microsomal RNA from normal as well as ethionine-treated rat livers, tRNA from Escherichia coli, yeast RNA, and DNA from rat liver and calf thymus, were significantly methylated by this methylase. These results suggest that partially purified rRNA methylase from rat liver nuclei incorporates methyl groups into hypomethylated pre-rRNA from S-adenosylmethionine.     

Cloning of the BamHI methyl transferase gene from Bacillus amyloliquefaciens

We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E. coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E. coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.     

The CpG-specific methylase SssI has topoisomerase activity in the presence of Mg2+.

A prokaryotic CpG-specific methylase from Spiroplasma, SssI methylase, is now widely used to study the effect of CpG methylation in mammalian cells, and can processively modify cytosines in CpG dinucleotides in the absence of Mg2+. In the presence of Mg2+, we found (i) that the methylation reaction is distributive rather than processive as a result of the decreased affinity of SssI methylase for DNA, and (ii) that a type I-like topoisomerase activity is present in SssI methylase preparations. This topoisomerase activity was still present in SssI methylase further purified by either SDS-polyacrylamide or isoelectric focusing gel electrophoresis. We show that methylase and topoisomerase activities are not functionally interdependent, since conditions exist where only one or the other enzymatic activity is detectable. The catalytic domains of SssI methylase and prokaryotic topoisomerases show similarity at the amino acid level, further supporting the idea that the topoisomerase activity is a genuine activity of SssI methylase. Mycoplasmas, including Spiroplasma, have the smallest genomes of all living organisms; thus, this condensation of two enzymatic activities into the same protein may be a result of genome economy, and may also have functional implications for the mechanism of methylation.     

Reduced methyl group acceptance of 1-beta-D-arabinofuranosylcytosine-containing DNA polymers

Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.     

A DNA-modification methylase from Bacillus stearothermophilus V.

A type II modification methylase (M BstVI) was partially purified from the thermophilic bacterium Bacillus stearothermophilus V. The methylase catalyses the transfer of methyl groups from S-adenosyl-L-methionine to unmodified double-stranded DNA. The product of methylation was identified by paper chromatography as N6-methyladenine. Since M BstVI protects DNA against cleavage by BstVI and XhoI restriction endonucleases, it follows that it methylates the adenine residue in the sequence 5'-C-T-C-G-A-G-3'.     

Phycobiliprotein methylation. Effect of the gamma-N-methylasparagine residue on energy transfer in phycocyanin and the phycobilisome

The phycobiliproteins contain a conserved unique modified residue, gamma-N-methylasparagine at beta-72. This study examines the consequences of this methylation for the structure and function of phycocyanin and of phycobilisomes. An assay for the protein asparagine methylase activity was developed using [methyl-3H]S-adenosylmethionine and apophycocyanin purified from Escherichia coli containing the genes for the alpha and beta subunits of phycocyanin from Synechococcus sp. PCC 7002 as substrates. This assay permitted the partial purification, from Synechococcus sp. PCC 6301, of the activity that methylates phycocyanin and allophycocyanin completely at residue beta-72. Using the methylase assay, two independent nitrosoguanidine-induced mutants of Synechococcus sp. PCC 7942 were isolated that do not exhibit detectable phycobiliprotein methylase activity. These mutants, designated pcm 1 and pcm 2, produce phycocyanin and allophycocyanin unmethylated at beta-72. The phycobiliproteins in these mutants are assembled into phycobilisomes and can be methylated in vitro by the partially purified methylase from Synechococcus sp. PCC 6301. The mutants produce phycobiliproteins in amounts comparable to those of wild-type and the mutant and wild-type phycocyanins are equivalent with respect to thermal stability profiles. Monomeric phycocyanins purified from these strains show small spectral shifts that correlate with the level of methylation. Phycobilisomes from the mutant strains exhibit defects in energy transfer, both in vivo and in vitro, that are also correlated with deficiencies in methylation. Unmethylated or undermethylated phycobilisomes show greater emission from phycocyanin and allophycocyanin and lower fluorescence emission quantum yields than do fully methylated particles. The results support the conclusion that the site-specific methylation of phycobiliproteins contributes significantly to the efficiency of directional energy transfer in the phycobilisome.     

A possible role of chromatin and tightly-bound chromatin proteins on enzyme-catalyzed methylation of DNA

Upon extensive digestion with DNAaseI of placenta chromatin matrix, previously "stripped" from its loosely-bound components by high-salt extraction, a fraction is obtained that contains almost no endogenous DNA methylase activity but whose DNA, if still included in this whole fraction--not if it has been purified to a protein-free condition--is a good substrate for externally added enzyme. This chromatin matrix can even cause a significant stimulation of methylation of single-stranded Micrococcus luteus DNA by placental methylase. In vivo, this phenomenon may have possible counterparts in the existence of highly-methylated regions of chromatin loops that appear to be protected by tightly-bound protein components from digestion of the "stripped loops" with DNAaseI.     

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.     

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.     

Reduced methyl group acceptance of 1-β-d-arabinofuranosylcytosine-containing DNA polymers

Previous studies have shown that 1-β-d-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.     

鼠肝DNA甲基化酶的纯化及物理化学性质的研究

以Ｗｉｓｔａｒ大鼠肝为材料,确立了一个简便的纯化鼠肝ＤＮＡ甲基化酶的程序,包括：细胞的超声破碎、去内源核酸、硫酸铵盐析、磷酸纤维素亲和层析、ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０柱层析及ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤。用不同浓度聚丙烯酰胺凝胶电泳和孔梯度凝胶电泳检测,纯化后的酶已达电泳均一,且酶的比活力提高１１２倍。以聚丙烯酰胺孔梯度凝胶电泳测得其天然酶的分子量为３６５ｋＤ,以ＳＤＳ－聚丙烯酰胺凝胶电泳测得该酶有两种亚基,大亚基为９５ｋＤ,小亚基为８５ｋＤ,推测该酶由两个大亚基和两个小亚基组成。     

Development and validation of an approach to produce large‐scale quantities of CpG‐methylated plasmid DNA

The prokaryotic CpG‐specific DNA methylase from Spiroplasma, SssI methylase, has been extensively used to methylate plasmid DNA in vitro to investigate the effects of methylation in vertebrate systems. Currently available methods to produce CpG‐methylated plasmid DNA have certain limitations and cannot generate large quantities of methylated DNA without cost or problems of purity. Here we describe an approach in which the SssI methylase gene has been introduced into the Escherichia coli bacterial genome under the control of an inducible promoter. Plasmid DNA propagated in this bacterium under conditions which induce the methylase gene result in significant (> 90%) CpG methylation. Methylated DNA produced by this approach behaves similarly to methylated DNA produced in vitro using the purified methylase. The approach is scalable allowing for the production of milligram quantities of methylated plasmid DNA.     

Characterization of DNA binding activities of over-expressedKpnI restriction endonuclease and modification methylase

The genes encoding theKpnI restriction endonuclease and methyltransferase fromKlebsiella pneumoniae have been cloned and expressed inEscherchia coli using a two plasmid strategy. The gene forKpnI methylase with its promoter was cloned and expressed in pACYC184. Even though the methylase clone is in a low copy number plasmid pACMK, high level expression of methylase is achieved. A hyper-expressing clone ofKpnI endonuclease, pETRK was engineered by cloning the R gene into the T7 expression system. This strategy resulted in over-expression ofKpnI endonuclease to about 15–30% of cellular protein. Both the enzymes were purified using a single Chromatographic step to apparent homogeneity. The yield of purified endonuclease and methylase from one liter of culture was approximately 30 and 6 mg respectively. Electrophoretic mobility shift assays show that both the enzymes are capable of binding to specific recognition sequence in the absence of any cofactors. The complexes ofKpnI methyl transferase and endonuclease with their cognate site exhibit distinctive behaviour with respect to ionic requirement.     

Selenite: a good inhibitor of rat-liver DNA methylase

DNA methylase from rat liver was partially purified through a DEAE sephacel column and characterized in an in vitro assay with respect to time, protein, DNA and S-adenosylmethionine curves. The Km for S-adenosylmethionine was 2.5 microM. Sodium selenium inhibited the methylation of DNA in a dose dependent fashion when added to the assay. It was also demonstrated that selenite non-competitively inhibits rat-liver DNA methylase with a Ki of 6.7 microM. Dithiothreitol had no effect on selenite inhibition and increasing amounts of DNA did not alter the inhibition. However, increasing amounts of protein overcame the inhibition, suggesting that selenite is reacting with the DNA methylase protein. DNA methylase isolated from selenite treated animals had only 43% of the activity as enzyme from control rats. It appears that selenite is a good inhibitor of DNA methylase.     

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.     

Myelin basic protein inhibits histone-specific protein methylase I

Bovine brain myelin basic protein, free of associated proteolytic activity, was found to be a specific inhibitor of histone-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23) purified from bovine brain. 50% of the methyl group incorporation into the histone substrate catalyzed by the methylase I was inhibited by myelin basic protein at a concentration of 0.326 mM. However, neither of the peptide fragments (residues 1-116 and residues 117-170) generated by the chemical cleavage of myelin basic protein at the tryptophan residue retained the inhibitory activity for histone-specific protein methylase I. Proteins such as gamma-globulin, bovine serum albumin, bovine pancreatic ribonuclease and polyarginine did not exhibit significant inhibitory activity toward the enzyme. The Ki value for myelin basic protein was estimated to be 3.42 X 10(-5) M for histone-specific protein methylase I and the nature of the inhibition was uncompetitive toward histone substrate.     

DNA methylases of Hemophilus influenzae Rd. I. Purification and properties

Hemophilus influenzae strain Rd DNA contains small amounts of 5-methylcytosine (0.012%) and significantly greater amounts of N-6-methyladenine (0.34%). Four DNA adenine methylases have been identified and purified from crude extracts of H. influenzae Rd by means of phosphocellulose chromatography. Each of the four enzymes requires (S-adenosyl-l-methionine as a methyl group donor and each differs in its ability to methylate various DNAs in vitro. DNA methylase I is related to the genetically described modification-restriction system in H. influenzae Rd, and is presumably the modification enzyme for that system. DNA methylase II introduces approximately 130 methyl groups into a phage T7 DNA molecule and protects T7 DNA from the H. influenzae Rd restriction enzyme, endonuclease R, described by Smith and Wilcox (1970). These findings indicate that DNA methylase II is the modification enzyme corresponding to endonuclease R. A third modification-restriction system, which does not affect T7 DNA, has been detected in H. influenzae Rd. DNA methylase III is apparently the modification enzyme for this system. The biological function of DNA methylase IV remains unknown.     

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor.

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.     

Isolation and characterization of DNA cytosine 5-methyltransferase from human placenta

DNA cytosine 5-methyltransferase has been extensively purified (about 2600-fold) from the soft tissue of human placenta by chromatography on DEAE-cellulose and hydroxyapatite, and by an affinity step on agarose-immobilized S-adenosylhomocysteine. The isolated enzyme has a molecular weight of 135,000 and methylates DNA from various sources in native and heat-denatured forms. The synthetic copolymer poly(dG-dC) . poly(dG-dC) is methylated in B- and Z-conformation to about the same extent. DNA containing hemimethylated sites was isolated from P815 cells grown in the presence of 5-azacytidine. This P815 DNA was used to measure the "maintenance' DNA methylase activity, whereas 5-methylcytosine-free procaryotic DNA served as a substrate for the "de novo' DNA methylase activity in our enzyme preparation. The crude extract as well as the highly purified DNA methylase are capable of transferring methyl groups to these two types of substrate. The fact that both types of activity co-chromatograph during the isolation procedure suggests that one enzyme molecule may exercise both the "maintenance' and "de novo' activity.