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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↗
PubMed1986

Escherichia coli K-12 restricts DNA containing 5-methylcytosine.

We have observed that plasmids containing certain cloned modification methylase genes of type II restriction-modification systems cannot be transformed into many laboratory strains of Escherichia coli K-12. The investigation of this phenomenon, reported here, has revealed (i) DNA containing 5-methylcytosine is biologically restricted by these strains, while DNA containing 6-methyladenine is not; (ii) restriction is due to two genetically distinct systems that differ in their sequence specificities, which we have named mcrA and mcrB (for modified cytosine restriction). Since 5-methylcytosine containing DNA is widespread in nature, the Mcr systems probably have a broad biological role. Mcr restriction may seriously interfere with molecular cloning of 5-methylcytosine-containing foreign DNAs. The Mcr phenotypes of some commonly used strains of E. coli K-12 are reported.

5-Methylcytosine↗
PubMed2000

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway.

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.

Catalysis↗
PubMed1982

DNA methyltransferase-dependent transcription of the phage Mu mom gene.

The phage Mu mom gene controls an unusual DNA modification. Expression of the mom function requires an active host (dam+) DNA adenine methylase [S-adenosyl-L-methionine:DNA (6-aminopurine)-methyltransferase]; in dam- hosts, Mu development is normal except that the viral DNA does not undergo the mom modification. The present communication compares transcription of the mom gene in dam+ versus dam- cells. 32P-labeled probes were prepared by nick-translation of a purified mom gene-containing restriction fragment and of virion DNA, respectively. These probes were hybridized with various RNAs blotted onto nitrocellulose filters (after fractionation by agarose gel electrophoresis). The salient findings are: (i) mom-specific RNA was readily detected in dam+ lysogenic cells, but only after induction of the Mu prophage; (ii) the level of mom RNA was decreased at least to 1/20th in induced dam- Mu lysogens; and (iii) little difference, if any, was observed between dam+ and dam- cells with respect to total Mu transcripts produced after prophage induction. These results are in accord with the known pattern of mom gene expression and Mu development. They show that the host (dam+) DNA adenine methylase activity is required for transcription of the mom gene. This represents a unique example where a DNA methylase exerts a positive regulatory role in mRNA transcription; alternative mechanisms for this process will be discussed.

Coliphages↗
PubMed1999

Evidence of horizontal transfer of the EcoO109I restriction-modification gene to Escherichia coli chromosomal DNA.

A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5'-(A/G)GGNCC(C/T)-3', was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.

Amino Acid Sequence↗
PubMed2002

Mobility of a restriction-modification system revealed by its genetic contexts in three hosts.

The flow of genes among prokaryotes plays a fundamental role in shaping bacterial evolution, and restriction-modification systems can modulate this flow. However, relatively little is known about the distribution and movement of restriction-modification systems themselves. We have isolated and characterized the genes for restriction-modification systems from two species of Salmonella, S. enterica serovar Paratyphi A and S. enterica serovar Bareilly. Both systems are closely related to the PvuII restriction-modification system and share its target specificity. In the case of S. enterica serovar Paratyphi A, the restriction endonuclease is inactive, apparently due to a mutation in the subunit interface region. Unlike the chromosomally located Salmonella systems, the PvuII system is plasmid borne. We have completed the sequence characterization of the PvuII plasmid pPvu1, originally from Proteus vulgaris, making this the first completely sequenced plasmid from the genus Proteus. Despite the pronounced similarity of the three restriction-modification systems, the flanking sequences in Proteus and Salmonella are completely different. The SptAI and SbaI genes lie between an equivalent pair of bacteriophage P4-related open reading frames, one of which is a putative integrase gene, while the PvuII genes are adjacent to a mob operon and a XerCD recombination (cer) site.

Amino Acid Sequence↗
PubMed2006

DNA-methyltransferase SsoII as a bifunctional protein: features of the interaction with the promoter region of SsoII restriction-modification genes.

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.

Catalytic Domain↗
PubMed1980

Methylation of foreign DNA sequences in eukaryotic cells.

The herpesvirus thymidine kinase gene has been used to introduce foreign DNA sequences into mouse L cells by DNA-mediated gene transfer. These inserted genes were then assayed for methylation at the specific sequence C-C-G-G by using the restriction enzyme isoschizomers Hpa II and Msp I. Despite the fact that 70% of the cellular C-C-G-G sites are methylated, herpesvirus sequences, plasmid DNA, and growth hormone gene DNA were found to remain unmethylated in 90% of the clones that contain these genes. DNA that had been methylated in vitro with Hpa II methylase was also inserted into L cells. The presence of this modification in the vector DNA did not, however, guarantee that these sequences remained methylated in the recipient clones. Only 10% of all transformed clones were found to contain methylated C-C-G-G sequences in the vector DNA, and these modifications were stable for 25-50 generations. Hha I and Mbo I were used to probe for methyl groups at these restriction sites, but none of the inserted sequences acquired these modifications. These results are discussed in relation to various models put forth to explain the process of methylation in eukaryotic cells.

DNA↗
PubMed1999

Shaping the genome--restriction-modification systems as mobile genetic elements.

A restriction enzyme gene is often linked to a modification methylase gene the role of which is to protect a recognition site on DNA from breakage by the former. Loss of some restriction-modification gene complexes leads to cell death through restriction breakage in the genome. Their behavior as genomic parasites/symbionts may explain the distribution of restriction sites and clarify certain aspects of bacterial recombination repair and mutagenesis. A comparison of bacterial genomes supports the hypothesis that restriction-modification gene complexes are mobile elements involved in various genome rearrangements and evolution.

Bacteria↗
PubMed2004

Initiation of translocation by Type I restriction-modification enzymes is associated with a short DNA extrusion.

Recognition of 'foreign' DNA by Type I restriction-modification (R-M) enzymes elicits an ATP-dependent switch from methylase to endonuclease activity, which involves DNA translocation by the restriction subunit HsdR. Type I R-M enzymes are composed of three (Hsd) subunits with a stoichiometry of HsdR2:HsdM2:HsdS1 (R2-complex). However, the EcoR124I R-M enzyme can also exist as a cleavage deficient, sub-assembly of HsdR1:HsdM2:HsdS1 (R1-complex). ATPS was used to trap initial translocation complexes, which were visualized by Atomic Force Microscopy (AFM). In the R1-complex, a small bulge, associated with a shortening in the contour-length of the DNA of 8 nm, was observed. This bulge was found to be sensitive to single-strand DNA nucleases, indicative of non-duplexed DNA. R2-complexes appeared larger in the AFM images and the DNA contour length showed a shortening of approximately 11 nm, suggesting that two bulges were formed. Disclosure of the structure of the first stage after the recognition-translocation switch of Type I restriction enzymes forms an important first step in resolving a detailed mechanistic picture of DNA translocation by SF-II DNA translocation motors.

Biological Transport↗
PubMed1999

[Expression detection and location analysis of BstNI isoschizomer restriction-modification system gene].

Some genetic markers of E. coli HB101 and JM110 were identified, two bacterial strains were used as recipients respectively to detect the expression of a restriction endonuclease(R) gene and a methylase(M) gene of BstNI isoschizomer restriction-modification system. DNA fragment containing the R-M genes was deleted unilaterally with exoIII and 23 deletion subclones were obtained. According to the Enzyme activity of each subclone, R and M gene were located respectively at the regions of 0.2-->1.4 kb and 1.5-->3.3 kb from cloning site PstI. Analysis showed that the R. M system belongs to type II, two genes are controlled by the different promoters; the recognition sequence of this system is the same as that of DNA-cytosine methyltransferase(Dcm), the latter's methylation function can resist the R enzyme. It was interesting that the recombinant plasmid with an R+ M- genotype appeared to be lethal to dcm+ hosts yet. This indicated that the M gene closely linking to R gene is of critical importance for the existence of the R-M system in process of evolution.

Chromosome Mapping↗
PubMed1986

Isolation of a deoxycytidylate methyl transferase capable of protecting DNA uniquely against cleavage by endonuclease R.Aqu I (isoschizomer of Ava I).

A sequence-specific modification methylase (M.AquI) was isolated and purified from Agmenellum quadruplicatum (Synechococcus PCC 7002). This enzyme uniquely methylates the deoxycytidylate residue in the sequence *CYCGRG indicated by the asterisk. It was shown to protect DNA against cleavage by restriction endonucleases AvaI, SmaI and XhoI, which recognize the sequences CYCGRG, CCCGGG, and CTCGAG, respectively.

Base Sequence↗
PubMed2002

Circular permutation of DNA cytosine-N4 methyltransferases: in vivo coexistence in the BcnI system and in vitro probing by hybrid formation.

Sequence analysis of the BcnI restriction-modification system from Bacillus centrosporus revealed four open reading frames (bcnIC, bcnIR, bcnIB and bcnIA) that are arranged as two converging collinear pairs. One pair encodes a putative small regulatory protein, C.BcnI, and the restriction endonuclease R.BcnI. The other two gene products are the DNA cytosine-N4 methyltransferases M.BcnIA and M.BcnIB, which differ by circular permutation of conserved sequence motifs. The BcnI methyltransferases are isospecific on double-stranded DNA [methylation specificity CC(C/G)GG], but M.BcnIA can also methylate the target sites in single-stranded DNA. Functional analysis shows that bcnIA is dispensable (bcnIB is capable of protecting the DNA against the in vivo activity of bcnIR); in contrast, no stable clones were obtained if bcnIB alone was deleted from the system. By analogy with the DpnII system, the second methylase M.BcnIA may play a role in the transformation proficiency of its gram-positive host. The interchangeability of homologous elements in the beta class of cytosine-N4 methylases was probed by hybrid formation between M.BcnIB and its closest homolog M.Cfr9I (CCCGGG) employing a novel semi-random strategy combined with selection for catalytic activity. The fusion points in the active hybrids mapped in a narrow region located between sequence motifs X and I. Our data illustrate that recombination of two related sequences by circular permutation may serve as an evolutionary mechanism for creating new specificities of amino MTases.

Amino Acid Sequence↗
PubMed1992

Structure and evolution of the XcyI restriction-modification system.

The XcyI restriction-modification system from Xanthomonas cyanopsidis recognizes the sequence, CCCGGG. The XcyI endonuclease and methylase genes have been cloned and sequenced and were found to be aligned in a head to tail orientation with the methylase preceding and overlapping the endonuclease by one base pair. The nucleotide sequence codes for an N4 cytosine methyltransferase with a predicted molecular weight of 33,500 and an endonuclease comprised of 333 codons and a molecular weight of 36,600. Sequence comparisons revealed significant similarity between the XcyI, CfrI and SmaI methylisomers. In contrast, no similarity was detected between the primary structures of the XcyI and SmaI endonucleases. The XcyI restriction-modification system is highly homologous to the XmaI genes, although the DNA sequences flanking the genes rapidly diverge. The sequence of the XcyI endonuclease contains two motifs which have recently been identified as essential to the activity of the EcoRV endonuclease.

Amino Acid Sequence↗
PubMedDate not supplied

S1 nuclease mapping of the phage Mu mom gene promoter: a model for the regulation of mom expression.

The mom gene of bacteriophage Mu encodes a DNA modification function. Expression of this modification requires the host Escherichia coli Dam (DNA-adenine methylase) function and the transacting phage Mu Dad function. The mom gene was subcloned into a variety of sites on plasmid pBR322. Insertions were made into the HincII and PvuI sites within the amp gene and into the ClaI site of the tet gene promoter. The only clones found were those in which the orientation of the mom gene prevents its transcription from the vector promoter(s), suggesting that constitutive expression of mom from a foreign promoter can occur independently of Dad function but is lethal for the cell. Employing S1 nuclease mapping, we have identified two Mu mRNA transcripts: (1) the gin transcript extends into the gin-mon intercistronic divide and terminates downstream from the BclI site; and (2) the mom transcript appears to initiate about 74 bp upstream from the BclI site, 12 bp downstream from a promoter-like sequence. Production of the mom transcript is dependent on the host Dam activity and on Dad transactivation. In contrast, the gin transcript is produced independently of Dam and Dad functions; the gin transcript may extend into the mom gene, but it appears to be either degraded at the 3' end or differentially terminated. We propose that regulation of mom gene transcription involves both positive and negative regulatory proteins, and that binding of the Dad protein (a "late" Mu protein) is required for transcription initiation by the host RNA polymerase. However, Dad protein action may be inhibited by prior binding of a repressor to the mom operator, located farther upstream. We propose that this repressor (encoded by a phage or host gene) binds to the operator only when there is no active Dam enzyme present, i.e., when there is no methylation of (or methylase binding to) the G-A-T-C sites within the mom operator.

Base Sequence↗
PubMed1979

[Isolation and properties of DNA-cytosine-methylase I from Escherichia coli MRE 600].

DNA-cytosine-methylase I was isolated and purified to homogeneity. The yield made up to about 30% of total activity. The enzyme molecular weight as determined by centrifugation in a sucrose gradient, by gel filtration and by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate was found to be 45,000. The Michaelis constant was 1,8 . 10(-6) M for SAM and 2 . 10(-4) M for DNA. DNA-cytosine-methylase I modifies phage lambda DNA in 60 sites. This modification does not protect DNA from the effects of restriction endonucleases HpaII and BsuRI. The enzyme methylates DNA in the nucleotide sequence: 5'...Pur-MC-C-G-G-Pyr...3'.

DNA (Cytosine-5-)-Methyltransferases↗
PubMed1984

Site-specific cleavage of DNA at 8- and 10-base-pair sequences.

A method is described for cutting DNA at specific sites that are 8 and 10 base pairs long. The DNA is first treated with a specific methylase, either the restriction-modification enzyme M. Taq I, which converts the 4-base sequence T-C-G-A to T-C-G-mA, or the similar enzyme M. Cla I, which converts the 6-base sequence A-T-C-G-A-T to A-T-C-G-mA-T. The DNA is then cleaved with Dpn I, a restriction endonuclease that recognizes the sequence G-mA-T-C. Dpn I is unique in that it cuts only DNA that is methylated at adenine in both strands of its recognition sequence. In DNAs that are not otherwise methylated at adenine in both strands of the sequence G-A-T-C, cleavage by Dpn I occurs only at the following sequences: in the case of M. Taq I methylation, 5' T-C-G-mA - T-C-G-mA 3' 3' mA-G-C - T-mA-G-C - T 5'; in the case of M. Cla I methylation, 5' A - T-C-G-mA - T-C-G-mA-T 3' 3' T-mA-G-C - T-mA-G-C - T-A 5'. Specific cutting and cloning at these methylase/Dpn I-generated sites is shown experimentally. Further, we describe how the above technique can be extended to generate Dpn I cleavage sites of up to 12 base pairs. In DNA that contains equal amounts of each base distributed at random, 8- and 10-base-pair recognition sequences occur, on the average, approximately once every 65,000 and 1,000,000 base pairs, respectively. Potential applications, including the development of cloning vectors and a rapid method for chromosome walking, are discussed.

Base Composition↗