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PubMed1987

Cloning of two type II methylase genes that recognise asymmetric nucleotide sequences: FokI and HgaI.

The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells. FokI methylase gene is contained on a 3.80 kb piece of F. okeanokoites DNA. Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI. Unmodified lambda DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI. The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H. galinarum DNA. This plasmid is resistant to digestion by HgaI. Neither the FokI nor the HgaI restriction endonuclease was detected in either clone. This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.

Cloning, Molecular↗
PubMed1980

Molecular cloning of EcoRII endonuclease and methylase genes.

The genes for restriction-modification system EcoRII have been cloned from plasmid N3 DNA using RSF2124 as a vector plasmid. The hybrid plasmids designated pFK321 and pFK322 contained a 5.8 megadaltons EcoRI--fragment derived from N3 DNA including the genes for restriction-modification system EcoRII and a gene for resistance to sulfanilamide.

Bacteriophage lambda↗
PubMedDate not supplied

[Specificity of new restrictases and methylases. Unusual modification of cytosine at position 4].

Fourteen restriction endonucleases and 4 methylases were isolated and purified from 14 strains of Citrobacter freundii and Escherichia coli, which were isolated from natural sources. To determine the nucleotide sequence recognized by the endonucleases a comparison of DNA cleavage patterns, the evaluation of the cleavage frequency of some DNA with known recognition sequences and mapping was used. It was determined that Cfr101 is a new enzyme recognizing 5'PuCCGGPy. Other restriction enzymes isolated were isoschizomers of: Cfr5I, Cfr11I, Eco60I, Eco61I--EcoRII; Cfr4I, Cfr8I, Cfr13I--Sau96I; Cfr6I--PvuII, Cfr9I--SmaI, Eco26I--HgiJII; Eco32I--EcoRV; Eco52I--XmaIII; Eco56I--NaeI. Some of the enzymes in C. freundii and E. coli were found for the first time. The methylases MCfrI; MCfr6I, MCfr9I and MCfr10I recognize the same nucleotide sequence as specific endonucleases isolated from the same strain. DNA modification in vitro by MCfrI and MCfr10I yields 5-methylcytosine and 4-methylcytosine by MCfr6I and MCfr9I.

Adenoviridae↗
PubMed1985

[Separation of modification methylation and restriction enzymes from Shigella sonnei 47].

Two systems for DNA host specificity have been demonstrated for Shigella sonnei cells, SsoI and SsoII. The aim of the present work was to separate the modificating methylases and restriction endonucleases from Shigella sonnei and to study the modificating functions of methylases MSsoI and MSsoII. The possibilities to separate the methylation and restriction enzymes by column chromatography on affine, ionoexchange and hydrophobic sorbents were analyzed. The scheme for separation of methylases and restriction endonucleases of Shigella sonnei was elaborated, consisting of the fractioning of total preparation on phenyl-sepharose and subsequent isoelectrofocusing on ampholines. The modification functions of MSsoI and MSsoII methylases obtained by this technique and devoid of concomitant restriction endonucleases were studied. The in vitro experiments have shown the acceptor DNA methylated by MSsoI or MSsoII to be RSsoI or RSsoII restriction proof.

DNA Restriction Enzymes↗
PubMed1983

DNA restriction--modification enzymes of phage P1 and plasmid p15B. Subunit functions and structural homologies.

We have purified the type III restriction enzymes EcoP1 and EcoP15 to homogeneity from bacteria that contain the structural genes for the enzymes cloned on small, multicopy plasmids and which overproduce the enzymes. Both of the enzymes contain two different subunits. The molecular weights of the subunits are the same for both enzymes and antibodies prepared against one enzyme cross-react with both subunits of the other. Bacteria containing a plasmid derivative in which a large part of one of the structural genes has been deleted have a restriction- modification+ phenotype and contain only the smaller of the two subunits. This subunit therefore must be the one that both recognizes the specific DNA sequence and methylates it in the modification reaction (the restriction enzyme itself also acts as a modification methylase). We have purified the P1 and P15 modification subunits from these deletion derivatives and have shown that in vitro they have the expected properties: they are sequence-specific modification methylases. In addition, we have demonstrated that strains carrying the full restriction/modification system also contain a pool of free modification subunits that might be responsible for in vivo modification.

Adenosine Triphosphate↗
PubMed2003

M.BstF5I-2 and M.BstF5I-4 DNA methyltransferases from BstF5I restriction-modification system of Bacillus stearothermophilus F5.

The BstF5I restriction-modification system from Bacillus stearothermophilus F5 includes four site-specific DNA methyltransferases, thus differing from all known restriction-modification systems. Here we demonstrated for the first time that one bacterial cell can possess two pairs of methylases with identical substrate specificities (methylases BstF5I-1 and BstF5I-3 recognize GGATG, whereas methylases BstF5I-2 and BstF5I-4 recognize CATCC) that modify adenine residues on both DNA strands. Different chromatographic methods provide homogenous preparations of methylases BstF5I-2 and BstF5I-4. We estimated the principal kinetic parameters of the reaction of transfer of methyl group from the donor S-adenosyl-L-methionine to the recognition site 5;-CATCC-3; catalyzed by BstF5I-2 and BstF5I-4 DNA [N6-adenine]-methyltransferases from the BstF5I restriction-modification system.

Bacteriophage lambda↗
PubMed1978

Phenotypically cryptic EcoRI endonuclease activity specified by the ColE1 plasmid.

An endonuclease having EcoRI specificity is produced by bacteria containing the ColE1 plasmid. Such bacterial cells fail to express restriction or modification functions in vivo, and phage or plasmid DNA obtained from ColE1-containing cells has unmodified EcoRI sites that are cleaved in vitro by purified EcoRI endonuclease or by enzyme extracted from bacteria that carry ColE1. No EcoRI DNA methylase activity associated with ColE1 has been detected. The finding of phenotypically cryptic ColE1-dependent EcoRI endonuclease activity and the absence of any detectable EcoRI modification system in ColE1-containing cells suggest a control mechanism that appears to prevent functional expression of the ColE1-determined enzyme in vivo.

Bacteriocin Plasmids↗
PubMed1988

Effect of in vitro DNA methylation on beta-globin gene expression.

When the human beta-globin gene was methylated at every cytosine residue and was inserted into mouse fibroblasts by DNA-mediated gene transfer, the transcription of the gene was strongly inhibited. This methylation also prevented expression and induction of the gene in mouse erythroleukemia cells. By using partially methylated hybrid molecules, it was shown that methylation-sensitive negative regulatory elements are located in both the 5' and 3' ends of the beta-globin gene but not in the 90-base-pair region usually associated with promoter activity. To further investigate the role of DNA methylation in the regulation of the beta-globin gene, 50-base-pair poly(dG-dC) tracts were introduced into various sites in a mouse-human hybrid gene, and these inserts were methylated by means of the Hha I methylase. Heavy methylation of these artificially added sites had no effect on either transcription initiation or elongation, suggesting that DNA modification operates through fixed endogenous sites in the gene domain.

Animals↗
PubMed1976

[DNA-methylase activities from animal mitochondria and nuclei: different specificity of DNA methylation].

DNA-methylase activities which methylate cytosine residues in homo- and heterologous DNA were detected in mitochondria and nuclei from rat liver and beef heart. Adenine modifying DNA-methylases in mitochondria and nuclei were not found. DNA from mitochondria and nuclei differ significantly in the methylation degree and in the pattern of the 5-methyl-cytosine distribution by pyrimidine isostichs as DNA in vivo and in vitro being methylated. Mitochondrial DNA methylase has the maximum activity at 30 degrees and pH 7.8 this enzyme(s) differ(s) from the nuclear one(s) in the pH dependence of its activity. After exhaustive in vitro methylation of various DNA by the nuclear enzyme DNA-methylase from mitochondria additionally introduces CH3 groups from S-adenosylmethionine into these DNA (about 3 times more CH3 groups than nuclear enzyme). Nuclear DNA-methylase also methylates DNA which is previously fully-methylated by the mitochondrial enzyme, but to a lesser degree. In conditions of exhaustive DNA methylation mitochondrial enzyme introduces into E. coli B DNA about four times more methyl groups as compared to the nuclear one. After the methylation of E. coli B DNA by mitochondrial enzyme the label (3H-methyl) was detected predominantly in mono-, and in case of nuclear enzyme--in di- and tripyrimidine fragments. Mitochondrial DNA-methylase differs from the nuclear one in the nature of recognized DNA sequences; these enzymes seems to be represented by different proteins. The mitochondrial enzyme methylates shorter nucleotide sequences in DNA as compared to the nuclear DNA-methylase. All these data suggest there exist organoid specificity of genome methylation in animal cell and the modification-restriction systems in animal nucleus and mitochondria are different in character.

Animals↗
PubMed2006

Involvement of the LlaKR2I methylase in expression of the AbiR bacteriophage defense system in Lactococcus lactis subsp. lactis biovar diacetylactis KR2.

The native lactococcal plasmid, pKR223, from Lactococcus lactis subsp. lactis biovar diacetylactis KR2 encodes two distinct bacteriophage-resistant mechanisms, the LlaKR2I restriction and modification (R/M) system and the abortive infection (Abi) mechanism, AbiR, that impedes bacteriophage DNA replication. This study completed the characterization of AbiR, revealing that it is the first Abi system to be encoded by three genes, abiRa, abiRb, and abiRc, arranged in an operon and that it requires the methylase gene from the LlaKR2I R/M system. An analysis of deletion and insertion clones demonstrated that the AbiR operon was toxic in L. lactis without the presence of the LlaKR2I methylase, which is required to protect L. lactis from AbiR toxicity. The novelty of the AbiR system resides in its original gene organization and the unusual protective role of the LlaKR2I methylase. Interestingly, the AbiR genetic determinants are flanked by two IS982 elements generating a likely transposable AbiR composite. This observation not only substantiated the novel function of the LlaKR2I methylase in the AbiR system but also illustrated the evolution of the LlaKR2I methylase toward a new and separate cellular function. This unique structure of both the LlaKR2I R/M system and the AbiR system may have contributed to the evolution of the LlaKR2I methylase toward a novel role comparable to that of the cell cycle-regulated methylases that include Dam and CcrM methylases. This new role for the LlaKR2I methylase offers a unique snapshot into the evolution of the cell cycle-regulated methylases from an existing R/M system.

Bacteriophages↗
PubMed2000

SINE retroposons can be used in vivo as nucleation centers for de novo methylation.

SINEs (short interspersed elements) are an abundant class of transposable elements found in a wide variety of eukaryotes. Using the genomic sequencing technique, we observed that plant S1 SINE retroposons mainly integrate in hypomethylated DNA regions and are targeted by methylases. Methylation can then spread from the SINE into flanking genomic sequences, creating distal epigenetic modifications. This methylation spreading is vectorially directed upstream or downstream of the S1 element, suggesting that it could be facilitated when a potentially good methylatable sequence is single stranded during DNA replication, particularly when located on the lagging strand. Replication of a short methylated DNA region could thus lead to the de novo methylation of upstream or downstream adjacent sequences.

5-Methylcytosine↗
PubMed1978

Overmethylation of DNAs by the EcoRI methylase.

EcoRI methylase is able to catalyze methy incorporation into DNA at sequences other than the canonical EcoRI site. At high enzyme concentrations and over a wide range of pH and ionic strengths, EcoRI methylase modifies polyoma DNA (which contains one EcoRI site) at a number of sites. This modification prevents EcoRI endonuclease activity, and thus is presumably at or near the EcoRI sequences (5') NAATTN.

Adenine↗
PubMed1997

DNA distortion and base flipping by the EcoRV DNA methyltransferase. A study using interference at dA and T bases and modified deoxynucleosides.

The EcoRV DNA methyltransferase introduces a CH3 group at the 6-amino position of the first dA in the duplex sequence d(GATATC). It has previously been reported that the methylase contacts the four phosphates (pNpNpGpA) at, and preceding, the 5'-end of the recognition sequence as well as the single dG in this sequence (Szczelkun, M. D., Jones, H., and Connolly, B. A. (1995) Biochemistry 34, 10734-10743). To study the possible role of the dA and T bases within the ATAT sequence, interference studies have been carried out using diethylpyrocarbonate and osmium tetroxide. The methylase bound very strongly to hemimethylated oligonucleotides modified at the second AT, of the ATAT sequence, in the unmethylated strand of the duplex. This probably arises because these modifications facilitate DNA distortion that follows the binding of the nucleic acid to the protein. Oligonucleotides containing modified bases at both the target dA base and its complementary T were used to determine whether this dA methylase flips out its target base in a similar manner to that observed for dC DNA methylases. In binary EcoRV methylase-DNA complexes, analogues that weakened the base pair caused an increase in affinity between the protein and the nucleic acid. In contrast, in ternary EcoRV methylase-DNA-sinefungin (an analogue of the natural co-factor, S-adenosyl-L-methionine (AdoMet)) complexes, only small differences in affinity were observed between the normal dA-T base pair and the analogues. These results are almost identical to those seen with DNA dC methylases (Klimasauskas, S., and Roberts R. J. (1995) Nucleic Acid Res. 23, 1388-1395; Yang, S. A., Jiang-Cheng, S., Zingg, J. M., Mi, S., and Jones, P. A. (1995) Nucleic Acids Res. 23, 1380-1387) and support a base-flipping mechanism for DNA dA methylases.

DNA-Binding Proteins↗
PubMed1989

Involvement of E. coli dcm methylase in Tn3 transposition.

The effects of DNA methyltransferases on Tn3 transposition were investigated. The E. coli dam (deoxyadenosine methylase) gene was found to have no effect on Tn3 transposition. In contrast, Tn3 was found to transpose more frequently in dcm+ (deoxycytosine methylase) cells than in dcm- mutants. When the EcoRII methylase gene was introduced into dcm- cells (E. coli strain GM208), the frequency of Tn3 transposition in GM208 was dramatically increased. The EcoRII methylase recognizes and methylates the same sequence as does the dcm methylase. These results suggest that deoxycytosine methylase modified DNA may be a preferred target for Tn3 transposition. Experiments were also performed to determine whether the Tn3 transposase was involved in DNA modification. Plasmid DNA isolated from dcm- E. coli containing the Tn3 transposase gene was susceptible to ApyI digestion but resistant to EcoRI digestion, suggesting that Tn3 transposase modified the dcm recognition sequence. In addition, restriction enzymes TaqI, AvaII, BglI and HpaII did not digest this DNA completely, suggesting that the recognition sequences of TaqI, AvaII, BglI and HpaII were modified by Tn3 transposase to a certain degree. The type(s), the extent and mechanism(s) of this modification remain to be investigated.

Base Sequence↗
PubMedDate not supplied

[The effect of the phage lambda ral gene on the level of synthesis of the EcoK restriction endonuclease beta-subunit].

E. coli hsd genes were subcloned from lambda 642 (ral+) into lambda L47.1 vector (ral-after replacement). The influence of bacteriophage lambda ral gene on the expression efficiency of hsdS kappa, hsdM kappa genes was investigated. It was shown, that its presence in vitro enhanced the synthesis of beta-subunit, hsdM gene product, and the increase of modification in vivo was observed. It is proposed that the increase of modification rate of lambda phage fully unmodified DNA is connected with the appearance of E. coli DNA methylase consisting of beta- and gamma-subunits but lacking alpha-subunit.

Bacteriophage lambda↗
PubMed1989

Sequence motifs characteristic of DNA[cytosine-N4]methyltransferases: similarity to adenine and cytosine-C5 DNA-methylases.

The sequences coding for DNA[cytosine-N4]methyltransferases MvaI (from Micrococcus varians RFL19) and Cfr9I (from Citrobacter freundii RFL9) have been determined. The predicted methylases are proteins of 454 and 300 amino acids, respectively. Primary structure comparison of M.Cfr9I and another m4C-forming methylase, M.Pvu II, revealed extended regions of homology. The sequence comparison of the three DNA[cytosine-N4]-methylases using originally developed software revealed two conserved patterns, DPF-GSGT and TSPPY, which were found similar also to those of adenine and DNA[cytosine-C5]-methylases. These data provided a basis for global alignment and classification of DNA-methylase sequences. Structural considerations led us to suggest that the first region could be the binding site of AdoMet, while the second is thought to be directly involved in the modification of the exocyclic amino group.

Amino Acid Sequence↗