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At least 19 recordsLinked to original sources
PubMed1993

Substrate recognition and selectivity in the type IC DNA modification methylase M.EcoR124I.

The type I DNA modification methylase M.EcoR124I binds sequence specifically to DNA and protects a 25bp fragment containing its cognate recognition sequence from digestion by exonuclease III. Using modified synthetic oligonucleotide duplexes we have investigated the catalytic properties of the methylase, and have established that a specific adenine on each strand of DNA is the site of methylation. We show that the rate of methylation of each adenine is increased at least 100 fold by prior methylation at the other site. However, this is accompanied by a significant decrease in the affinity of the methylase for these substrates according to competitive gel retardation assays. In contrast, methylation of an adenine in the recognition site which is not a target for the enzyme results in only a small decrease in both DNA binding affinity and rate of methylation by the enzyme.

Base Sequence↗
PubMed1972

DNA modification methylase activity of Escherichia coli restriction endonucleases K and P.

The highly purified restriction endonucleases of E. coli K and coliphage P1 transfer methyl groups from S-adenosylmethionine to adenine residues of unmodified DNA. Incubation of unmodified DNA with endonucleases K or P and S-adenosylmethionine renders the DNA resistant to restriction. The enzymes, therefore, have both restriction endonuclease and modification methylase activities.

Chromatography, DEAE-Cellulose↗
PubMed1988

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'.

DNA↗
PubMed1985

Analysis of products of DNA modification by methylases: a procedure for the determination of 5- and N4-methylcytosines in DNA.

Although many different methods are used for the identification of methylated heterocyclic bases in DNA not all of them possess the ability to discriminate N4-methylcytosine (m4C) and 5-methylcytosine (m5C). Therefore, some of the methods need additional reexamination. This paper reinvestigates some chromatographic systems (thin-layer chromatography, paper chromatography, electrophoresis) most widely used in the analysis of minor bases occurring in nucleic acids according to their ability to separate m4C and m5C. A simple procedure for the preparation of the sample and a chromatographic system for its analysis was developed. The recommended chromatographic systems may be used for the simultaneous separation of not only of m4C and m5C but also both methylated cytosines together with N6-methyladenine and 7-methylguanine.

5-Methylcytosine↗
PubMed1994

Interaction of EcoP1 modification methylase with S-adenosyl-L-methionine: a UV-crosslinking study.

EcoP1 modification methylase was radioactively labeled when incubated with S-adenosyl-L-[methyl-3H]methionine in the presence of ultraviolet light. Crosslinking of the enzyme as detected by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel followed by fluorography and autoradiography, was shown to be specific by a number of criteria. More importantly, EcoP1 modification methylase was also radioactively labeled with S-adenosyl-L-[carboxyl-14C]methionine demonstrating that labeling involved binding of the entire AdoMet molecule rather than methylation of the protein. Further, c2 EcoP1 mutant DNA modification methylases which show negligible or very little methylation activity, correspondingly formed a weak or no adduct upon crosslinking. These results suggest that photolabeling of EcoP1 DNA modification methylase occurs at the AdoMet binding site.

Carbon Radioisotopes↗
PubMed2006

Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase.

We used engineered zinc finger peptides (ZFPs) to bind selectively to predetermined sequences in human mtDNA. Surprisingly, we found that engineered ZFPs cannot be reliably routed to mitochondria by using only conventional mitochondrial targeting sequences. We here show that addition of a nuclear export signal allows zinc finger chimeric enzymes to be imported into human mitochondria. The selective binding of mitochondria-specific ZFPs to mtDNA was exemplified by targeting the T8993G mutation, which causes two mitochondrial diseases, neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and also maternally inherited Leigh's syndrome. To develop a system that allows the monitoring of site-specific alteration of mtDNA we combined a ZFP with the easily assayed DNA-modifying activity of hDNMT3a methylase. Expression of the mutation-specific chimeric methylase resulted in the selective methylation of cytosines adjacent to the mutation site. This is a proof of principle that it is possible to target and alter mtDNA in a sequence-specific manner by using zinc finger technology.

Amino Acid Sequence↗
PubMed2005

A nonradioactive DNA methyltransferase assay adaptable to high-throughput screening.

We have developed a nonradioactive assay method for DNA methyltransferases based on the ability to protect substrate DNA from restriction. DNA immobilized to a microplate well was treated sequentially with methyltransferase and an appropriate endonuclease. The amount of methylated DNA product is reflected by a proportional decrease in endonuclease cleavage, which is in turn reflected by increased retention of the end-labeled affinity probe. A single universal substrate was designed to assay multiple methyltransferases including those that do not have a cognate endonuclease. The methodology developed is suited to screen a large number of compounds for inhibitors of various methyltransferases.

Base Sequence↗
PubMed2004

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions.

We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially independent on the outer surface of the dimer. The solution behavior suggests the formation of HhaI dimers as well. The same HhaI dimer interface is also observed in the previously characterized binary (M.HhaI-AdoMet) and ternary (M.HhaI-DNA-AdoHcy) complex structures, crystallized in different space groups. The dimer is characterized either by a non-crystallographic two-fold symmetry or a crystallographic symmetry. The dimer interface involves three segments: the amino-terminal residues 2-8, the carboxy-terminal residues 313-327, and the linker (amino acids 179-184) between the two functional domains--the catalytic methylation domain and the DNA target recognition domain. Both the amino- and carboxy-terminal segments are part of the methylation domain. We also examined protein-protein interactions of other structurally characterized DNA MTases, which are often found as a 2-fold related 'dimer' with the largest dimer interface area for the group-beta MTases. A possible evolutionary link between the Type I and Type II restriction-modification systems is discussed.

Crystallization↗
PubMed2000

Differential distribution of novel restriction-modification systems in clonal lineages of Neisseria meningitidis.

Using representational difference analysis, we isolated novel meningococcal restriction-modification (R-M) systems. NmeBI, which is a homologue of the R-M system HgaI of Pasteurella volantium, was present in meningococci of the ET-5 complex and of lineage III. NmeAI was found in serogroup A, ET-37 complex, and cluster A4 meningococci. NmeDI was harbored by meningococci of the ET-37 complex and of cluster A4, but not by serogroup A meningococci. Two of the R-M systems, NmeBI and NmeDI, were located at homologous positions between the phenylalanyl-tRNA synthetase genes pheS and pheT, which appeared to be a preferential target for the insertion of foreign DNA in meningococci. The distribution of the three R-M systems was tested with 103 meningococcal strains comprising 49 sequence types. The vast majority of the strains had either NmeBI, NmeAI, or both NmeAI and NmeDI. Using cocultivation experiments, we could demonstrate that NmeBI, which was present in ET-5 complex meningococci, was responsible for a partial restriction of DNA transfer from meningococci of the ET-37 complex to meningococci of the ET-5 complex.

Clone Cells↗
PubMed1990

Cloning and characterization of the HpaII methylase gene.

The HpaII restriction-modification system from Haemophilus parainfluenzae recognizes the DNA sequence CCGG. The gene for the HpaII methylase has been cloned into E. coli and its nucleotide sequence has been determined. The DNA of the clones is fully protected against cleavage by the HpaII restriction enzyme in vitro, indicating that the methylase gene is active in E. coli. The clones were isolated in an McrA-strain of E. coli; attempts to isolate them in an McrA+ strain were unsuccessful. The clones do not express detectable HpaII restriction endonuclease activity, suggesting that either the endonuclease gene is not expressed well in E. coli, or that it is not present in its entirety in any of the clones that we have isolated. The derived amino acid sequence of the HpaII methylase shows overall similarity to other cytosine methylases. It bears a particularly close resemblance to the sequences of the HhaI, BsuFI and MspI methylases. When compared with three other methylases that recognize CCGG, the variable region of the HpaII methylase, which is believed to be responsible for sequence specific recognition, shows some similarity to the corresponding regions of the BsuFI and MspI methylases, but is rather dissimilar to that of the SPR methylase.

Amino Acid Sequence↗
PubMed2005

Isolation and characterization of biochemical properties of DNA methyltransferase FauIA modifying the second cytosine in the nonpalindromic sequence 5'-CCCGC-3'.

A gene encoding DNA methyltransferase (methylase) FauIA of the restriction-modification system FauI from Flavobacterium aquatile (recognizing sequence 5'-CCCGC-3') was cloned in pJW vector. The latter was used for transformation of E. coli RRI cells followed by subsequent thermoinduction and biomass elaboration. Highly purified DNA methyltransferase FauIA preparation was obtained using chromatography on different sorbents. The molecular mass of the isolated enzyme of about 39 kD corresponds to its theoretical value. The enzyme was characterized by temperature and pH optima of 33 degrees C and pH 7.5, respectively. Methylation of a synthetic oligonucleotide by FauIA methylase followed by its cleavage with various restrictases and analysis of the resultant restriction fragments revealed that FauIA methylase modified the second cytosine residue in the sequence 5'-CCCGC-3'. Kinetic analysis revealed Km and catalytic constant values of 0.16 microM and 0.05 min(-1), respectively.

Amino Acid Sequence↗
PubMedDate not supplied

Repair-modification and evolution of the eukaryotic genome organization.

For a complete reconstruction of the damaged unmethylated islands, in theory, the conventional excision-repair is sufficient. For a complete reconstruction of the damaged methylated domains, a coupling has to take place involving the excision-repair (able to reestablish their ATGC-language) plus the DNA-methylase (able to reestablish their modified ATGC5mC-language). This coupling, defined as "repair-modification," is essentially functioning during the S-phase, because the DNA-polymerase beta (pol beta) is active during the whole cell cycle, whereas the DNA-methylase (met) is active in S and appears to be repressed or inactive during the major part of G1 and during the phases G2 and M. Consequently, after damage, some silent genes might become expressed during these phases, if it is true that DNA methylation is inversely proportional to transcription. Repair-modification should, therefore, exert a continuous differential pressure on evolution of given parts of the genome, when they are methylated to a different extent. According to Darwinian concepts, repair-modification would lead to a high variability, especially of uncoding DNA sequences (if hypermethylated), whereas on the basis of this variability, selection might favor transposition of specific regulatory elements into given transcriptional units. In these, the conservative nature of the coding elements (if unmethylated) would obviously be ensured by the conventional excision-repair.

Animals↗
PubMed2006

Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland.

AIMS: Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. METHODS AND RESULTS: Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. CONCLUSIONS: Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.

Base Sequence↗
PubMed1994

Identification of the XorII methyltransferase gene and a vsr homolog from Xanthomonas oryzae pv. oryzae.

The gene encoding the XorII methyltransferase (M.XorII) was cloned from Xanthomonas oryzae pv. oryzae and characterized in Escherichia coli. The M.XorII activity was localized to a 3.1 kb BamHI-BstXI fragment, which contained two open reading frames (ORFs) of 1272 nucleotides (424 amino acids) and 408 nucleotides (136 amino acids). Ten polypeptide domains conserved in other M5 cytosine methyltransferases (MTases) were identified in the deduced amino acid sequence of the 1272 ORF. E. coli Mrr+ strains were transformed poorly by plasmids containing the XorII MTase gene, indicating the presence of at least one MCG in the recognition sequence for M.XorII (CGATCG). The 408 nucleotide ORF was 36% identical at the amino acid level to sequences of the E. coli dem-vsr gene, which is required for very short patch repair. X. oryzae pv. oryzae genomic DNA that is resistant to digestion by PvuI and XorII hybridizes with a 7.0 kb fragment containing the XorII MTase gene and vsr homolog, whereas DNA from strains that lack M.XorII activity do not hybridize with the fragment.

Amino Acid Sequence↗
PubMed2000

Diversity of restriction-modification gene homologues in Helicobacter pylori.

The complete genome sequences of two Helicobacter pylori strains have recently become available. We have searched them for homologues of restriction-modification genes. One strain (26695) carried 52 such homologues, and the other (J99) carried 53. Their sequence alignments were arranged in the form of a phylogenetic tree and compared with the tree based on rRNA. The trees showed that the homologues are scattered among diverse groups of bacteria. They also revealed high polymorphism within the species--there are 42 pairs with high homology, 10 specific to 26695, and 11 specific to J99. Many of the restriction-modification homologues were characterized by a GC content lower than that of the average gene in the genome. Some of the restriction-modification homologues showed a different codon use bias from the average genes. These observations are interpreted in terms of horizontal transfer of the restriction-modification genes.

Base Composition↗
PubMed2000

Comparison between Pyrococcus horikoshii and Pyrococcus abyssi genome sequences reveals linkage of restriction-modification genes with large genome polymorphisms.

Recent work suggests that restriction-modification gene complexes are mobile genetic elements that insert themselves into the genome and cause various genome rearrangements. In the present work, the complete genome sequences of Pyrococcus horikoshii and Pyrococcus abyssi, two species in a genus of hyperthermophilic archaeon (archaebacterium), were compared to detect large genome polymorphisms linked with restriction-modification gene homologs. Sequence alignments, GC content analysis, and codon usage analysis demonstrated the diversity of these homologs and revealed a possible case of relatively recent acquisition (horizontal transfer). In two cases out of the six large polymorphisms identified, there was insertion of a DNA segment with a modification gene homolog, accompanied by target deletion (simple substitution). In two other cases, homologous DNA segments carrying a modification gene homolog were present at different locations in the two genomes (transposition). In both cases, substitution (insertion/deletion) in one of the two loci was accompanied by inversion of adjacent chromosomal segment. In the fifth case, substitution by a DNA segment carrying type I restriction, modification, and specificity gene homologs was likewise accompanied by adjacent inversion. In the last case, two homologous DNA segments, were found at different loci in the two genomes (transposition), but only one of them had insertion of a modification homolog and an unknown ORF. The possible relationship of these polymorphisms to attack by restriction enzymes on the chromosome will be discussed.

Chromosome Inversion↗
PubMed1999

Characterization of a CACAG pentanucleotide repeat in Pasteurella haemolytica and its possible role in modulation of a novel type III restriction-modification system.

In a previous study, a recombinant plasmid that contains a CACAG pentanucleotide repeat was isolated from a Pasteurella haemolytica A1 library. Southern hybridization analysis using a (CACAG)5probe indicated the presence of two loci that contain the pentanucleotide repeats on the genome of P.haemolytica A1. Additional hybridization analyses against genomic DNA from related microorganisms indicated that the repeats are only present in P.haemolytica and Pasteurella trehalosi T3. The various serotypes of P.haemolytica werefound to have either one or two of the CACAG repeat-containing loci. Examination of the locus designated Rpt2 by PCR and sequence analysis indicated that the number of CACAG repeats could change upon serial subculture which most likely occurs as a result of DNA slipped-strand mispairing. A plasmid carrying the Rpt2 locus was isolated and characterized. Sequenceanalysis indicated that the CACAG repeats are contained within the 5'-end of a gene that showed homology to mod genes of type III restriction-modification systems. A second open reading frame downstream was identified which showed homology to res genes of type III restriction-modification systems. Both the modification and restriction proteins could be expressed and polypeptides of the expected sizes were detected by SDS-PAGE. Restriction activity could also be detected in crude cytoplasmic extracts of Escherichia coli strains carrying the mod and res genes on recombinant plasmids.

Amino Acid Sequence↗