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PubMed1992

Regulation of the BamHI restriction-modification system by a small intergenic open reading frame, bamHIC, in both Escherichia coli and Bacillus subtilis.

BamHI, from Bacillus amyloliquefaciens H, is a type II restriction-modification system recognizing and cleaving the sequence G--GATCC. The BamHI restriction-modification system contains divergently transcribed endonuclease and methylase genes along with a small open reading frame oriented in the direction of the endonuclease gene. The small open reading frame has been designated bamHIC (for BamHI controlling element). It acts as both a positive activator of endonuclease expression and a negative repressor of methylase expression of BamHI clones in Escherichia coli. Methylase activity increased 15-fold and endonuclease activity decreased 100-fold when bamHIC was inactivated. The normal levels of activity for both methylase and endonuclease were restored by supplying bamHIC in trans. The BamHI restriction-modification system was transferred into Bacillus subtilis, where bamHIC also regulated endonuclease expression when present on multicopy plasmid vectors or integrated into the chromosome. In B. subtilis, disruption of bamHIC caused at least a 1,000-fold decrease in endonuclease activity; activity was partially restored by supplying bamHIC in trans.

Bacillus subtilis↗

PubMed1986

Isolation and characterization of the M.EaeI modification methylase.

The modification enzyme (M.EaeI) corresponding to the restriction endonuclease EaeI was partially purified from Enterobacter aerogenes PW201. The M.EaeI enzyme methylates the innermost cytosine residue in each strand of the family of related sequences that constitute the EaeI recognition site to give: 5'-Y-G-G-5mC-C-R-3' where 5mC is 5-methylcytosine. M.EaeI protects these sites against cleavage by HaeIII, and protects overlapping 5'-C-C-G-G-3' sites against cleavage by both HpaII and MspI.

Binding Sites↗

PubMed1992

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.

Adenine↗

PubMed1996

"Small is beautiful": major modifications in DNA structure or dynamics by small substituents or ligands.

This short review assembles the contributions of the author's laboratory to the structural aspects of DNA. DNA was modified by small ligands and/or substituents. There are three aspects to this work: a) Protonation of guanosine and DNA and the formation of triple- and quadruple-strands of guanosine, its nucleotides, their polymers and DNA. b) Substitution of the 2'-position of deoxyribose by the most polar atom, fluorine: studies on 2'-deoxy-2'-fluro-nucleosides, -nucleotides and their polymers, studied both by structural and biological methods. c) The effect of introducing the methyl group in the large groove of DNA: NMR studies of oligonucleotides containing N6-methylated adenine residues, and enzymatic and molecular biology work on Dam methylase are reported.

Adenine↗

PubMed1987

Interaction of AluI, Cfr6I and PvuII restriction-modification enzymes with substrates containing either N4-methylcytosine or 5-methylcytosine.

The cleavage specificity of R.Cfr6I, an isoschizomer of PvuII restriction endonuclease was determined to be 5'CAG decreases CTG and the methylation specificity of Cfr6I and PvuII methylases, 5'CAG4mCTG. Thus, M.Cfr6I and M.PvuII are new additions to the list of methylases with N4-methylcytosine specificity. Neither of the above RM enzymes acts on the substrates containing either N4-methylcytosine or 5-methylcytosine in a cognate methylation position.

5-Methylcytosine↗

PubMed1987

Organization and sequence of the hsd genes of Escherichia coli K-12.

The nucleotide sequence of the hsdR and M genes, together with that for hsdS comprises an 8400 base segment spanning the entire hsd region of Escherichia coli K-12. The three hsd genes are transcribed in the same direction, but from two promoters. hsdR and hsdM are separated by 492 base-pairs, whereas the termination codon of hsdM overlaps the initiation codon of hsdS. pres precedes hsdR, and our data indicate a transcription termination signal in the interval between hsdR and pmod, as expected if transcription of hsdM and S is dependent on pmod. Transcription from pres is not influenced by the products of the hsdM and S genes, and the mechanism whereby restriction is prevented when the hsd region is transferred to a modification-deficient cell remains to be elucidated. A segment of the predicted amino acid sequence of the M polypeptide shares homology with a variety of adenine methylases and may identify part of the active site for methylation of specific adenine residues. The R polypeptide shows homology with a variety of ATPases, and pronounced regions of alpha-helical structure are predicted, one of which is amphipathic.

Amino Acid Sequence↗

PubMed1988

Cloning and sequencing the HinfI restriction and modification genes.

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).

Amino Acid Sequence↗

PubMedDate not supplied

[Covalent binding of Cys142 from SsoII methyltransferase with DNA duplexes, containing a phosphoryldisulfide internucleotide group].

DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (M.SsoII). The possibility of duplex-M.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of M.SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA-enzyme complex.

Amino Acid Sequence↗

PubMed1992

Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV).

The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.

Base Sequence↗

PubMed1983

Enzymatic methylation of chicken erythrocyte DNA modified by two carcinogens, 2-(N-acetoxyacetylamino) fluorene and methylnitrosourea.

Both DNA-AAF and MNU-alkylated DNA are methylated less than nonmodified DNA by rat brain nuclei cytosine 5-methyltransferase purified either by chromatography on DEAE cellulose or by Dyematrex. The inhibition of methylation is proportional to the modification of the DNA, and DNA having a given percentage of bases modified with MNU is less methylated than DNA modified to the same extent with AAF. Moreover, DNA-AAF irreversibly inhibits the methylation of native DNA, whereas MNU-alkylated DNA does not inhibit the methylation of native DNA. The AAF-substituted DNA has a higher affinity for the enzyme than native DNA. However, this is probably not due to the AAF-induced local destabilization of the DNA helix, since heat-denatured DNA shows a lower affinity for the enzyme than double-stranded DNA. Addition of DNA-AAF to the enzyme preincubated with native DNA inhibits methylation, but only after a lag period. This agrees with the model in which the methylase walks along the strand to methylate cytosine residues before being detached from the DNA. AAF bound to guanine residues may block the movement of the enzyme along the helix. The in vitro hypomethylation of DNA, caused by carcinogens, could explain the in vivo observations made by several authors and could have significance in gene activity, cellular differentiation, and oncogenesis.

2-Acetylaminofluorene↗

PubMed1996

Use of a non-selective transformation technique to construct a multiply restriction/modification-deficient mutant of Neisseria gonorrhoeae.

A technique that allows for easy identification of transformants of Neisseria gonorrhoeae in the absence of selective pressure has been developed. A suicide vector that contains a gonococcal DNA uptake sequence was constructed to aid in DNA uptake. In this transformation procedure, a limiting number of cells is incubated with an excess amount of DNA, and the mixture is plated onto a non-selective medium. At least 20% of the resulting colonies contained cells that had been transformed. This strategy was utilized to construct specific deletions of the S.N goI, II, IV, V and VII restriction-modification (R/M) genes. All five deletions were successfully incorporated into the chromosome of FA19, producing strain JUG029. Strain JUG029 could be transformed with non-methylated plasmid DNA while strain FA19 could not be transformed with such DNA. The development of a simple, non-selective transformation technique, coupled with the construction of a strain that is more permissive for DNA-mediated transformation, will aid in genetic manipulations of the gonococcus.

Base Sequence↗

PubMed1987

Inhibition of methylation at two internal N6-methyladenosine sites caused by GAC to GAU mutations.

We previously have mapped N6-methyladenosine (m6A) sites within the genomic RNA of Rous sarcoma virus (RSV). The results of that study and of experiments using inhibitors of methylation suggest that m6A might be involved in mRNA processing events. We describe an approach for directly analyzing the function of m6A in RNA and for studying the sequence specificity of the m6A methylase. Two sites of methylation in RSV (nucleotides 7414 and 7424) were altered by oligonucleotide-directed mutagenesis. The highly conserved GAC consensus sequence at those sites was changed to GAU. The new sequences were no longer methylated in the RSV genomic RNA; the GAC sequence was required for efficient base modification at those two adenosines. The altered m6A pattern did not affect viral RNA processing or the viral life cycle within infected cells.

Adenosine↗

PubMed1993

The M.AluI DNA-(cytosine C5)-methyltransferase has an unusually large, partially dispensable, variable region.

The DNA methyltransferase of the AluI restriction-modification system, from Arthrobacter luteus, converts cytosine to 5-methylcytosine in the sequence AGCT. The gene for this methyltransferase, aluIM, was cloned into Escherichia coli and sequenced. A 525-codon open reading frame was found, consistent with deletion evidence, and the deduced amino acid sequence revealed all ten conserved regions common to 5-methylcytosine methyltransferases. The aluIM sequence predicts a protein of M(r) 59.0k, in agreement with the observed M(r), making M.AluI the largest known methyltransferase from a type II restriction-modification system. M.AluI also contains the largest known variable region of any monospecific DNA methyltransferase, larger than that of most multispecific methyltransferases. In other DNA methyltransferases the variable region has been implicated as the sequence-specific target recognition domain. An in-frame deletion that removes a third of this putative target-recognition region leaves the Alu I methyltransferase still fully active.

Amino Acid Sequence↗

PubMed2000

Cloning, expression, and purification of a thermostable nonhomodimeric restriction enzyme, BslI.

BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The BslI restriction-modification system from Bacillus species was cloned and expressed in Escherichia coli. The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), the bslIRalpha gene, and the bslIRbeta gene. The alpha and beta subunits of BslI can be expressed independently in E. coli in the absence of BslI methylase (M.BslI) protection. BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (alphabeta), heterotetramers (alpha(2)beta(2)), and possibly oligomers in solution. Two beta subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer BslI complex (alpha(2)beta(2)). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is alpha(2)beta(2). M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the beta group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 in BslI sites in the first or second cytosine are resistant to BslI digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion. Two putative zinc fingers are found in the alpha subunit of BslI endonuclease.

Amino Acid Sequence↗

PubMed1996

Cloning and nucleotide sequence of the AgeI methylase gene from Agrobacterium gelatinovorum IAM 12617, a marine bacterium.

The AgeI restriction-modification system from a marine bacterium, Agrobacterium gelatinovorum IAM 12617, recognizes the nucleotide sequence ACCGGT. The gene coding for the AgeI methylase (M.AgeI) was cloned into Escherichia coli DH5 alpha MCR, and the nucleotides of the gene were sequenced. The M.AgeI gene coded for a protein of 429 amino acid residues (molecular mass, 47,358 daltons). The deduced amino acid sequence of M.AgeI was compared with those of other methylases and showed that there are high degrees of similarity in some cytosine-5 methylases.

Amino Acid Sequence↗

PubMed1995

Interaction of the MvaI and SsoII methyltransferases with DNAs altered at the central base pair of the recognition sequence.

The interaction of the MvaI and SsoII DNA methyltransferases (MTases; M.MVaI and M.SsoII, respectively) with a set of synthetic DNA duplexes, containing a M.MvaI and M.SsoII recognition site (CCWGG), was investigated. In these DNA duplexes dA or dT of the recognition site was replaced by nucleoside analogs with modified sugar moieties and heterocyclic bases (2'-deoxy-2'-fluorouridine (flU), 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine (xT), 1-(beta-D-3'-deoxy-threo-pentofuranosyl)uracil (tU)), or by 1,3-propanediol (Prd). A new approach for monitoring methylation of each strand of DNA duplexes by MTases was developed. It allowed the determination of the influence of the modification in one DNA strand on the methylation of the other. In most cases, for both M.MvaI and M.SsoII, sugar analog-containing duplexes showed inhibition of methylation of only the modified strand. Prd-containing DNA duplexes were not substrates for M.MvaI. M.SsoII did not methylate DNA duplexes in which the dT residue was replaced by Prd.

Amino Acid Sequence↗

PubMed1983

DNA modification, differentiation, and transformation.

Substantial evidence has accumulated over the last 5 years that the methylation of cytosine residues in vertebrate DNA is implicated in the control of gene expression. We have used analogs of cytidine, modified in the 5 position, as specific inhibitors of DNA methylation to probe the relationship between this process and cellular differentiation. 5-Azacytidine effected marked changes in the differentiated state of cultured cells and induced the formation of biochemically differentiated muscle, fat, and chondrocytes from mouse fibroblast cell lines. Since the analog is a powerful inhibitor of DNA methylation, we suggest that this inhibition is causally related to the mechanism of phenotypic conversion. DNA extracted from cells treated with 5-azacytidine was hemimethylated and was used as an efficient acceptor of methyl groups in an in vitro reaction in the presence of eukaryotic methylases. In vitro methylation was inhibited if the substrate DNA was preincubated with a diverse range of chemical carcinogens including benzo(a)pyrene diolepoxide. Thus, chemical carcinogens may induce changes in gene expression by alteration of cellular methylation patterns. Recent experiments have also demonstrated that freshly explanted diploid fibroblasts from mice, hamsters, and humans lose substantial quantities of 5-methylcytosine during cell division and aging in culture. Taken together, these experiments suggest that the genomic distribution of 5-methylcytosine might have importance in normal differentiation and also in the aberrant gene expression found in cancer and senescence in culture.

5-Methylcytosine↗

PubMed1982

DNA methylation in eukaryotes.

The DNA of higher eukaryotes contains one minor base, namely 5-methylcytosine. The distribution of this minor base between different species and different DNA fractions will be considered together with the actual sequences methylated. The properties of the enzyme responsible for DNA modification will be reviewed, particular note being paid to the efficiency of methylation of different DNA substrates. Various possible functions of the 5-methylcytosine in DNA will be considered and particular attention will be paid to the finding that specific modified bases present in DNA not undergoing transcription are absent in the same genes when these are being actively transcribed.

5-Methylcytosine↗