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PubMed1989

Cloning and structure of the BepI modification methylase.

The gene coding for a CGCG specific DNA methylase has been cloned in E. coli from Brevibacterium epidermidis. The enzyme, named BepI methylase, is probably the cognate methylase of the FnuDII isoschizomer BepI endonuclease isolated from this strain. The expression of BepI methylase in E. coli is dependent on the orientation of the cloned fragment suggesting that the gene is transcribed from a promoter on the plasmid vector. No BepI endonuclease could be detected in the clones producing BepI methylase. The nucleotide sequence of the BepI methylase gene has been determined, it predicts a protein of 403 amino acids (MR: 45,447). Analysis of the amino acid sequence deduced from the nucleotide sequence revealed similarities between the BepI methylase and other cytosine methylases. M. BepI methylates the external cytosine in its recognition sequence.

Amino Acid Sequence↗

PubMed1984

HhaI methylase and restriction endonuclease as probes for B to Z DNA conformational changes in d(GCGC) sequences.

The capacity of the modification methylase (MHhaI) and restriction endonuclease (HhaI) form Haemophilus haemolyticus to methylate and cleave, respectively, recognition sites which are in right-handed B or left-handed Z structures was determined in vitro. Plasmids containing tracts of (dC-dG) as well as numerous individual d(GCGC) sites distributed around the vector were studied. Negative supercoiling was used to convert the (dC-dG) tracts (approximately 30 bp in length) from a right-handed to a left-handed conformation. (Methyl-3H)-SAM was used to localize and quantitate modified d(GCGC) recognition sites, whereas cleavage by HhaI was used to detect unmethylated sites. In the left-handed Z-form, the (dC-dG) blocks were not methylated by MHhaI and not cleaved by HhaI. A two-dimensional gel analysis of a family of 33 topoisomers treated with MHhaI revealed that the lack of methylation in the (dC-dG) blocks was directly correlated to the supercoil-induced B to Z transition in these segments. These results are significant with respect to enzyme-DNA interactions in general and provide the basis for using HhaI and MHhaI as probes for different DNA structures and conformational transitions under physiological conditions.

Base Sequence↗

PubMed1988

Multiple DNA secondary structures in perfect inverted repeat inserts in plasmids. Right-handed B-DNA, cruciforms, and left-handed Z-DNA.

The capabilities of five recombinant plasmids, containing relatively long (approximately 60-100 base pairs) perfect inverted repeat (IR) inserts, to support supercoil stabilized non-B-DNA structures were studied in vitro. The IRs were also alternating purine-pyrimidine sequences, thus, each could form either left-handed Z-DNA or cruciforms. Single-strand specific endonucleases, restriction endonucleases and methylases, and OsO4 modifications were used to characterize the DNA structures. Two-dimensional gel electrophoretic studies indicated that three of the five IRs formed both cruciforms and Z-DNA. (C-G) containing inserts preferred to form Z-DNA, whereas (T-G) sequences favored cruciforms. In vivo supercoil relaxation experiments demonstrated the existence of cruciforms in Escherichia coli. The physiological significance of these structures is discussed.

Base Sequence↗

PubMed1984

Resistance of DNA from filamentous and unicellular cyanobacteria to restriction endonuclease cleavage.

Chromosomal DNA from nine species of filamentous cyanobacteria as diverse as Nostoc, Gloeotrichia and Plectonema is suggested to be extensively modified (methylated) by its resistance to cleavage by a number of restriction endonucleases. A remarkably similar pattern of DNA modification in these species contrasts with the known heterogeneity of their type II restriction endonuclease content. In particular, Nostoc PCC 73102, which lacks detectable sequence-specific endonucleases, is shown to possess extensive DNA modification. The use of isoschizomers demonstrates the presence of a methylase in the filamentous strains analogous to the dam enzyme of Escherichia coli. As a preliminary to assessing the significance of the DNA modification, a study of susceptibility to restriction endonuclease cleavage of the genomes of five unicellular cyanobacteria revealed considerable variation between the different strains. The significance of the DNA modification patterns elucidated is discussed in terms of the restriction endonuclease content and cellular differentiation of the relevant cyanobacterial strains.

Base Sequence↗

PubMed1981

Purification and characterization of two new modification methylases: MClaI from Caryophanon latum L and MTaqI from Thermus aquaticus YTI.

A method for detecting Type II modification methylases and determining their methylation site by assaying the ability of methylated DNA to be cleaved by heterologous restriction enzymes is described and applied to the isolation of the restriction modification methylases from Thermus thermophilus HB8, Thermus aquaticus YTI and Caryophanon latum L. M.TaqI is shown to have a methylation specificity identical to M.ThI (TCGmeA). M.ClaI methylates at adenine and protects a subset of TthI sites indicating that it methylates the sequence ATCGmeAT. Methylation by M.ThI also protects against cleavage by SalI, XhoI and at some HindII, AccI and MboI sites.

Adenine↗

PubMed1985

DNA methylation in mammalian nuclei.

A novel system to study the methylation of newly synthesized DNA in isolated nuclei was developed. Approximately 2.5% of cytosine residues incorporated into nascent DNA became methylated by endogenous methylase(s), and the level of DNA modification was reduced by methylation inhibitors. DNA synthesis and methylation were dependent on separate cytosol factors. The cytosol factor or factors required for DNA methylation were sensitive to trypsin digestion and were precipitable by (NH4)2SO4, suggesting that they were proteinaceous. Time-course experiments revealed a short lag of approximately 20 s between synthesis and methylation in nuclei. The DNAs produced in these nuclei were a mixed population of low molecular weight fragments and higher molecular weight fragments shown to be short extension of existing replicons. The methylation level found in low molecular weight DNA was lower than that found in bulk L1210 DNA, indicating that further methylation events might take place after ligation of small fragments. These data suggest that newly synthesized DNA is a good substrate for methylase enzymes and that nuclear cytoplasmic interactions may be important in controlling inheritance of methylation patterns.

Animals↗

PubMed1990

Identification of peptides involved in S-adenosylmethionine binding in the EcoRI DNA methylase. Photoaffinity laveling with 8-azido-S-adenosylmethionine.

The Mr 38,050 monomeric EcoRI DNA methylase is part of a bacterial restriction-modification system. The methylase transfers the methyl group from S-adenosylmethionine (AdoMet) to the second adenine in the double-stranded DNA sequence 5'-GAATTC-3'. We have used the radiolabeled photoaffinity analog 8-azido-S-adenosylmethionine (8-N3-AdoMet) to identify peptides at the AdoMet binding site in the binary methylase-cofactor analog complex. The dissociation constants in the absence of DNA for the analog and AdoMet are 12.9 and 4.8 microM, respectively. The apparent kcat and Km values, obtained with the double-stranded DNA substrate 5'-CGCGAATTCGCG-3', are 5.0 s-1 and 0.710 microM (8-N3-AdoMet) and 4.3 s-1 and 0.335 microM (AdoMet). Photolabeling by 8-N3-AdoMet occurs upon irradiation with ultraviolet light and is inhibited by AdoMet. Digestion of the adducted methylase with subtilisin generated several radiolabeled peptides. Peptide sequencing from independent photolabeling experiments revealed two radiolabeled peptides containing amino acids 206-212 and 213-221. Instability of the adducted peptides precluded assignment of modified amino acids.

Affinity Labels↗

PubMed1989

Cloning and characterization of the genes encoding the MspI restriction modification system.

The genes encoding the MspI restriction modification system, which recognizes the sequence 5' CCGG, have been cloned into pUC9. Selection was based on expression of the cloned methylase gene which renders plasmid DNA insensitive to MspI cleavage in vitro. Initially, an insert of 15 kb was obtained which, upon subcloning, yielded a 3 kb EcoRI to HindIII insert, carrying the genes for both the methylase and the restriction enzyme. This insert has been sequenced. Based upon the sequence, together with appropriate subclones, it is shown that the two genes are transcribed divergently with the methylase gene encoding a polypeptide of 418 amino acids, while the restriction enzyme is composed of 262 amino acids. Comparison of the sequence of the MspI methylase with other cytosine methylases shows a striking degree of similarity. Especially noteworthy is the high degree of similarity with the HhaI and EcoRII methylases.

Amino Acid Sequence↗

PubMedDate not supplied

[Isolation and properties of DNA-cytosine methyltransferase from Escherichia coli C].

The method of isolation and partial purification of DNA-cytosine-methyltransferase (DC-methylase) from E. coli C is described. The enzyme underwent approximately 100-fold purification. The obtained preparation of DC-methylase can be additionally considerably purified by sedimentation in sucrose gradient. Native molecular weight of DC-methylase from E. coli C. is 70,000. The activity of enzyme does not depend on the Mg2+ ions. DC-methylase E. coli C provides DNA of lambda phage in vitro with full resistance against restriction endonuclease EcoRII. In DNA methylated by DC-methylase the modified cytosine, mainly in C-MC and C-MC-T sequences, corresponds to the pyrimidine sequences of specific site EcoRII. DNA of lambda.B phage contains approximately 80 sites for modification by DC-methylase E. coli C. The results obtained point to the same specificity in vitro of DNA-cytosine-methylase E. coli C and DNA-methylase EcoRII.

Binding Sites↗

PubMed1980

A DNA methylase from Thermus thermophilus HB8.

A DNA methylase was purified in a homogeneous state from a extremely thermophilic bacterium, Thermus thermophilus HB8, by chromatography on, successively, phosphocellulose, CM-cellulose, and heparin-Sepharose. The molecular weight of the enzyme was determined to be about 44,000 by gel filtration on a Sephadex G-100 column and 41,000 by SDS-poly-acrylamide gel electrophoresis, and these findings suggest a single polypeptide enzyme. The enzyme develops maximum activity around pH 7.4 and at 70 degrees C. Enzymatic activity is completely inhibited by 0.2 M NaCl or 2 mM HgCl2. The enzyme transfers methyl groups from S-adenosyl-L-methionine to a double stranded DNA. The sole product of the reaction was identified as N-6-methyl adenine after hydrolysis of the DNA with formic acid. The enzyme kinetics obey the Michaelis-Menten equation and Km values for S-adenosylmethionine and lambda phage DNA were determined to be 0.8 muM and 10 microgram/ml, respectively. The enzyme does not transfer methyl groups to TthHB8I endonuclease digested DNA as well as the host (T. thermophilus HB8) DNA. The number of methyl groups of the fully methylated phiX174 RF DNA was about twice as many as TthHB8I endonuclease sites on the DNA. The distribution of the methyl groups of phiX174 RF DNA among the HaeIII fragments was the same as that of TthHB8I endonuclease sites, suggesting that this DNA methylase is the other component of the modification-restriction system including TthHB8I endonuclease. The enzyme probably recognizes the sequence, 5'-TCGA-3', in a double stranded DNA and probably methylates adenine in the above sequence.

Bacteriophage phi X 174↗

PubMedDate not supplied

[Specificity of action of DNA methylases BcnI, CfrI and Cfr10I].

The site specificity of three DNA methylases BcnI, CfrI and Cfr10I was determined to be 5'Cm4C(C/G)GG, 5'PyGGm5CCPu and 5'Pum5CCGGPy, respectively. Using the modification methylases under investigation with known restriction endonucleases, fourteen new DNA cleavage specificities can be created. Some aspects of the use of restriction endonucleases in DNA methylation analysis are discussed.

Bacillus↗

PubMed1981

The effect of sequence specific DNA methylation on restriction endonuclease cleavage.

Sequence specific DNA methylation sometimes results in the protection of some or all of a restriction endonucleases' cleavage sites. This is usually, but not always, the result of methylation of one or both strands of DNA at the site characteristic of the corresponding "cognate" modification methylase. The known effects of sequence specific methylation on restriction endonucleases are compiled.

Base Sequence↗

PubMed1990

Incorporation of a complete set of deoxyadenosine and thymidine analogues suitable for the study of protein nucleic acid interactions into oligodeoxynucleotides. Application to the EcoRV restriction endonuclease and modification methylase.

A complete set of dA and T analogues designed for the study of protein DNA interactions has been prepared. These modified bases have been designed by considering the groups on the dA and T bases that are accessible to proteins when these bases are incorporated into double-helical B-DNA [Seeman, N. C., Rosenberg, J. M., & Rich, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 804-808]. Each of the positions on the two bases, having the potential to interact with proteins, have been subject to nondisruptive, conservative change. Typically a particular group (e.g., the 6-NH2 of dA or the 5-CH3 of T) has been replaced with a hydrogen atom. Occasionally keto groups (the 2- and 4-keto oxygen atoms of T) have been replaced with sulfur. The base set has been incorporated into the self-complementary dodecamer d(GACGATATCGTC) at the central d(ATAT) sequence. Melting temperature determination shows that the modified bases do not destabilize the double helix. Additionally, circular dichroism spectroscopy shows that almost all the altered bases have very little effect on overall oligodeoxynucleotide conformation and that most of the modified oligomers have a B-DNA type structure. d(GATATC) is the recognition sequence for the EcoRV restriction modification system. Initial rate measurements (at a single oligodeoxynucleotide concentration of 20 microM) have been carried out with both the EcoRV restriction endonuclease and modification methylase. This has enabled a preliminary identification of the groups of the dA and T bases within the d(GATATC) sequence that make important contacts to both proteins.

Adenosine↗

PubMedDate not supplied

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.

Bacillus subtilis↗

PubMed2002

An in vitro screening method for DNA cytosine-C5-methylase inhibitor.

A specific inhibitor of DNA cytosine C5-methylases would be useful for studying genomic imprinting, X-chromosome inactivation, carcinogenesis, and regulation of tissue-specific gene expression, for these physiological phenomena appears to be regulated through DNA methylation in promoter sequences. This paper reports a novel convenient in vitro assay method for screening DNA cytosine C5-methylase inhibitor. Our method uses a commercially available Hae III methylase (cytosine C5 methylase), its corresponding Hae III endonuclease, and lambda DNA as their substrate.

Adenosine↗

PubMed1993

Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121.

We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extracts. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5'-G/GNCC-3') in this site to generate a three base 5' overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10(3) viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.

Base Sequence↗

PubMed1978

[Sensitivity of chromosomal and plasmid E. coli DNA to restriction endonuclease Eco RII].

It was shown that E. coli C, E. coli MRE 600 DNA, and also plasmid DNA of Col E1, RSF 2124 from E. coli K-12, and plasmid DNA from E. coli MRE 600 were completely resistant against restriction endonuclease R. Eco RII. Plasmid DNAs of Col E1, RSF 2124 amplificated for 4 hours in the presence of chloramphenicol are sensitive to R. Eco RII but after 16-hour amplification in the presence of chloramphenicol these DNAs acquire complete resistance against R. Eco RII. These data point to the slower rate of modification of DNA in vivo by DC-methylases of Eco RII type in comparison with DNA methylase Eco RII.

Chloramphenicol↗

PubMed1984

The EcoA restriction and modification system of Escherichia coli 15T-: enzyme structure and DNA recognition sequence.

The EcoA restriction enzyme from Escherichia coli 15T- has been isolated. It proves to be an unusual enzyme, clearly related functionally to the classical type I restriction enzymes. The basic enzyme is a two subunit modification methylase. Another protein species can be purified which by itself has no enzymatic activities but which converts the modification methylase to an ATP and S-adenosylmethionine-dependent restriction endonuclease. The DNA recognition sequence of EcoA has an overall structure that is very similar to previously determined type I sequences. It is: 5'-GAGNNNNNNNGTCA-3' 3'-CTCNNNNNNNCAGT-5' where N can be any nucleotide. Modification methylates the adenosyl residue in the specific trinucleotide and the adenosyl residue in the lower strand of the specific tetranucleotide.

Base Sequence↗