Single-site DNA cleavage by Type III restriction endonuclease requires a site-bound enzyme and a trans-acting enzyme that are ATPase-activated.

Endonucleolytic cleavage of DNA by Type III restriction-modification (RM) enzymes requires long-range communication between at least two recognition sites in inverted orientation. This results in convergence of two nuclease domains, one each from the enzymes loaded at the recognition sites with one still bound to the site. The nucleases catalyze scission of the single-strands leading to double-strand DNA break. An obscure feature of the Type III RM enzymes EcoP1I and EcoP15I is their ability to cleave DNA having a single recognition site under certain conditions. Here we demonstrate that single-site cleavage is the result of cooperation between an enzyme bound to the recognition site in cis and one in trans. DNA cleavage is catalyzed by converging nucleases that are activated by hydrolysis-competent ATPase in presence of their respective DNA substrates. Furthermore, a single activated nuclease cannot nick a strand on its own, and requires the partner. Based on the commonalities in the features of single-site and two-site cleavage derived from this study, we propose that their mechanism is similar. Furthermore, the products of two-site cleavage can act as substrates and activators of single-site cleavage. The difference in the two modes lies in how the two cooperating enzymes converge, which in case of single-site cleavage appears to be via 3D diffusion.

A Novel Tool for Microbial Genome Editing Using the Restriction-Modification System.

Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.

Molecular characterisation of a type III restriction-modification system in Campylobacter upsaliensis.

Two examples of Campylobacter upsaliensis RM3195 and JV21 strains are shown to carry putative type III restriction (res)-modification (mod) enzyme gene clusters, following genome sequence analyses. It is suggested that the cluster is composed of at least three structural genes, res, internal methylase gene and mod, in the strains, based on the nucleotide sequence information. A ribosome binding site, a putative promoter consisting of a consensus sequence at the -10-like structure and a semiconserved T-rich region and a putative intrinsic p-independent transcriptional terminator were identified for the gene cluster in the two strains. Using two primer pairs, f-/r-res and f-/r-mod, 34 of 41 C. upsaliensis isolates generated two expected amplicons of the res and mod gene segments, and using another primer pair, the same number of isolates also generated an amplicon of the res and mod gene segments cluster, including the third internal methylase gene. Thus, C. upsaliensis isolates frequently carried putative type III R-M gene clusters, encoding the three enzymes. Interestingly, two possible overlaps were identified within the three tandem structural genes. In addition, the type III R-M gene cluster loci appear to be very similar among the C. upsaliensis isolates and very different from other thermophilic campylobacters.

Phytopathogenic bacteria phenotype conversion as a result of their lysogenisation by coliphage P1.

A set of lysogenic strains of phytopathogenic bacteria Erwinia "horticola" and Erwinia amylovora associated with woody plants was obtained using bacteriophage P1 Cmc1ts100. The phenotype conversion from Cm(S) to Cm(R) was shown to be connected with introducing of authentic prophage DNA of 94.8 kb as a single-copy plasmid into the cells. Prophage state is unstable: P1 plasmid is spontaneously lost with high frequency by the cells. In lysogenic cells the prophage genes of type III restriction-modification complex EcoP1I are actively expressed. The system formed by E. "horticola" 450 and 60 as well as their lysogenic derivatives and specific bacteriophages provides an opportunity to divide the latter into three groups according to the level of restriction in the course of their interaction with the enzyme EcoP1I. The difference in phage responses to the endonuclease presence in a lysogenized host presumably correlates with the number of enzyme recognition sequences and the adsorption sites availability. After the prophage plasmid DNA curing the characteristic value of phage sensitivity of cells is changed. The lysogenic strains obtained in this work allow for the exploration of EcoP1I restriction-modification gene complex interaction with polyvalent phages able to grow not only on E. coli, but also on such phytopathogens as E. "horticola" and E. amylovora.

Type III restriction-modification enzymes: a historical perspective.

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

Functional consequences of mutating conserved SF2 helicase motifs in the Type III restriction endonuclease EcoP15I translocase domain.

For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II-VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I-VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2-7 fold) in all enzyme variants tested.

Characterization of type II and III restriction-modification systems from Bacillus cereus strains ATCC 10987 and ATCC 14579.

The genomes of two Bacillus cereus strains (ATCC 10987 and ATCC 14579) have been sequenced. Here, we report the specificities of type II/III restriction (R) and modification (M) enzymes. Found in the ATCC 10987 strain, BceSI is a restriction endonuclease (REase) with the recognition and cut site CGAAG 24-25/27-28. BceSII is an isoschizomer of AvaII (G/GWCC). BceSIII cleaves at ACGGC 12/14. The BceSIII C terminus resembles the catalytic domains of AlwI, MlyI, and Nt.BstNBI. BceSIV is composed of two subunits and cleaves on both sides of GCWGC. BceSIV activity is strongly stimulated by the addition of cofactor ATP or GTP. The large subunit (R1) of BceSIV contains conserved motifs of NTPases and DNA helicases. The R1 subunit has no endonuclease activity by itself; it strongly stimulates REase activity when in complex with the R2 subunit. BceSIV was demonstrated to hydrolyze GTP and ATP in vitro. BceSIV is similar to CglI (GCSGC), and homologs of R1 are found in 11 sequenced bacterial genomes, where they are paired with specificity subunits. In addition, homologs of the BceSIV R1-R2 fusion are found in many sequenced microbial genomes. An orphan methylase, M.BceSV, was found to modify GCNGC, GGCC, CCGG, GGNNCC, and GCGC sites. A ParB-methylase fusion protein appears to nick DNA nonspecifically. The ATCC 14579 genome encodes an active enzyme Bce14579I (GCWGC). BceSIV and Bce14579I belong to the phospholipase D (PLD) family of endonucleases that are widely distributed among Bacteria and Archaea. A survey of type II and III restriction-modification (R-M) system genes is presented from sequenced B. cereus, Bacillus anthracis, and Bacillus thuringiensis strains.

Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity.

For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.

Methyleugenol genotoxicity in the Fischer 344 rat using the comet assay and pathway-focused gene expression profiling.

Methyleugenol (MEG), a constituent of human food, induces malignant tumors in multiple tissues of rats and mice. Although MEG forms DNA adducts and induces unscheduled DNA synthesis in rat liver, it is negative in many in vitro genetic toxicity assays. In the present study, we evaluated MEG-induced DNA damage in the rat using (1) the alkaline Comet assay, (2) the oxidative Comet assay, and (3) expression profiling of genes associated with DNA damage pathways. Male F344 rats received single oral doses of 400 or 1000 mg/kg body weight (bw) MEG and DNA damage was assessed by the Comet assay in liver, bladder, bone marrow, kidney, and lung 3 h and 24 h later. MEG failed to produce any increase in DNA damage. In addition, rats were given a single oral dose of 2000 mg/kg bw MEG, and Comet assays were performed with liver, bone marrow, and bladder 1, 3, 6, and 8 h later. With one exception (bone marrow at 8 h), no DNA damage was detected. Enzyme-modified Comet assays were conducted in parallel with standard Comet assays in liver. Whereas no MEG-induced DNA damage was detected following formamidopyrimidine DNA glycosylase digestion, digestion with endonuclease III resulted in increases in DNA damage at the 6- and 8-h sampling times. Gene expression analysis on the livers from MEG-exposed rats showed significant reduction in genes associated with DNA repair. The results indicate that MEG induces DNA damage in rat liver and that oxidative DNA damages may be partly responsible for the genotoxicity of MEG in rodents.

Evidence for an evolutionary antagonism between Mrr and Type III modification systems.

The Mrr protein of Escherichia coli is a laterally acquired Type IV restriction endonuclease with specificity for methylated DNA. While Mrr nuclease activity can be elicited by high-pressure stress in E. coli MG1655, its (over)expression per se does not confer any obvious toxicity. In this study, however, we discovered that Mrr of E. coli MG1655 causes distinct genotoxicity when expressed in Salmonella typhimurium LT2. Genetic screening enabled us to contribute this toxicity entirely to the presence of the endogenous Type III restriction modification system (StyLTI) of S. typhimurium LT2. The StyLTI system consists of the Mod DNA methyltransferase and the Res restriction endonuclease, and we revealed that expression of the LT2 mod gene was sufficient to trigger Mrr activity in E. coli MG1655. Moreover, we could demonstrate that horizontal acquisition of the MG1655 mrr locus can drive the loss of endogenous Mod functionality present in S. typhimurium LT2 and E. coli ED1a, and observed a strong anti-correlation between close homologues of MG1655 mrr and LT2 mod in the genome database. This apparent evolutionary antagonism is further discussed in the light of a possible role for Mrr as defense mechanism against the establishment of epigenetic regulation by foreign DNA methyltransferases.

A type III-like restriction endonuclease functions as a major barrier to horizontal gene transfer in clinical Staphylococcus aureus strains.

Staphylococcus aureus is an versatile pathogen that can cause life-threatening infections. Depending on the clinical setting, up to 50% of S. aureus infections are caused by methicillin-resistant strains (MRSA) that in most cases are resistant to many other antibiotics, making treatment difficult. The emergence of community-acquired MRSA drastically changed the picture by increasing the risk of MRSA infections. Horizontal transfer of genes encoding for antibiotic resistance or virulence factors is a major concern of multidrug-resistant S. aureus infections and epidemiology. We identified and characterized a type III-like restriction system present in clinical S. aureus strains that prevents transformation with DNA from other bacterial species. Interestingly, our analysis revealed that some clinical MRSA strains are deficient in this restriction system, and thus are hypersusceptible to the horizontal transfer of DNA from other species, such as Escherichia coli, and could easily acquire a vancomycin-resistance gene from enterococci. Inactivation of this restriction system dramatically increases the transformation efficiency of clinical S. aureus strains, opening the field of molecular genetic manipulation of these strains using DNA of exogenous origin.

Creating bacterial strains from genomes that have been cloned and engineered in yeast.

We recently reported the chemical synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium.

Haemophilus influenzae phasevarions have evolved from type III DNA restriction systems into epigenetic regulators of gene expression.

Phase variably expressed (randomly switching) methyltransferases associated with type III restriction-modification (R-M) systems have been identified in a variety of pathogenic bacteria. We have previously shown that a phase variable methyltransferase (Mod) associated with a type III R-M system in Haemophilus influenzae strain Rd coordinates the random switching of expression of multiple genes, and constitutes a phase variable regulon--'phasevarion'. We have now identified the recognition site for the Mod methyltransferase in H. influenzae strain Rd as 5'-CGAAT-3'. This is the same recognition site as the previously described HinfIII system. A survey of 59 H. influenzae strains indicated significant sequence heterogeneity in the central, variable region of the mod gene associated with target site recognition. Intra- and inter-strain transformation experiments using Mod methylated or non-methylated plasmids, and a methylation site assay demonstrated that the sequence heterogeneity seen in the region encoding target site specificity does correlate to distinct target sites. Mutations were identified within the res gene in several strains surveyed indicating that Res is not functional. These data suggest that evolution of this type III R-M system into an epigenetic mechanism for controlling gene expression has, in some strains, resulted in loss of the DNA restriction function.

DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes.

EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.

Structural domains in the type III restriction endonuclease EcoP15I: characterization by limited proteolysis, mass spectrometry and insertional mutagenesis.

The Type III restriction endonuclease EcoP15I forms a hetero-oligomeric enzyme complex that consists of two modification (Mod) subunits and two restriction (Res) subunits. Structural data on Type III restriction enzymes in general are lacking because of their remarkable size of more than 400 kDa and the laborious and low-yield protein purification procedures. We took advantage of the EcoP15I-overexpressing vector pQEP15 and affinity chromatography to generate a quantity of EcoP15I high enough for comprehensive proteolytic digestion studies and analyses of the proteolytic fragments by mass spectrometry. We show here that in the presence of specific DNA the entire Mod subunit is protected from trypsin digestion, whereas in the absence of DNA stable protein domains of the Mod subunit were not detected. In contrast, the Res subunit is comprised of two trypsin-resistant domains of approximately 77-79 kDa and 27-29 kDa, respectively. The cofactor ATP and the presence of DNA, either specific or unspecific, are important stabilizers of the Res subunit. The large N-terminal domain of Res contains numerous functional motifs that are predicted to be involved in ATP-binding and hydrolysis and/or DNA translocation. The C-terminal small domain harbours the catalytic center. Based on our data, we conclude that both structural Res domains are connected by a flexible linker region that spans 23 amino acid residues. To confirm this conclusion, we have investigated several EcoP15I enzyme mutants obtained by insertion mutagenesis in and around the predicted linker region within the Res subunit. All mutants tolerated the genetic manipulation and did not display loss of function or alteration of the DNA cleavage position.

A mutation in the Mod subunit of EcoP15I restriction enzyme converts the DNA methyltransferase to a site-specific endonuclease.

A closer inspection of the amino acid sequence of EcoP15I DNA methyltransferase revealed a region of similarity to the PDXn(D/E)XK catalytic site of type II restriction endonucleases, except for methionine in EcoP15I DNA methyltransferase instead of proline. Substitution of methionine at position 357 by proline converts EcoP15I DNA methyltransferase to a site-specific endonuclease. EcoP15I-M357P DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically EcoP151-M357P.DNA methyltransferase specifically binds to the recognition sequence 5'-CAGCAG-3' and cleaves DNA asymmetrically, 5'-CAGCAG(N)(10)-3', as indicated by the arrows, in presence of magnesium ions.

High allelic diversity in the methyltransferase gene of a phase variable type III restriction-modification system has implications for the fitness of Haemophilus influenzae.

Phase variable restriction-modification (R-M) systems are widespread in Eubacteria. Haemophilus influenzae encodes a phase variable homolog of Type III R-M systems. Sequence analysis of this system in 22 non-typeable H.influenzae isolates revealed a hypervariable region in the central portion of the mod gene whereas the res gene was conserved. Maximum likelihood (ML) analysis indicated that most sites outside this hypervariable region experienced strong negative selection but evidence of positive selection for a few sites in adjacent regions. A phylogenetic analysis of 61 Type III mod genes revealed clustering of these H.influenzae mod alleles with mod genes from pathogenic Neisseriae and, based on sequence analysis, horizontal transfer of the mod-res complex between these species. Neisserial mod alleles also contained a hypervariable region and all mod alleles exhibited variability in the repeat tract. We propose that this hypervariable region encodes the target recognition domain (TRD) of the Mod protein and that variability results in alterations to the recognition sequence of this R-M system. We argue that the high allelic diversity and phase variable nature of this R-M system have arisen due to selective pressures exerted by diversity in bacteriophage populations but also have implications for other fitness attributes of these bacterial species.

Characterization of the Type III restriction endonuclease PstII from Providencia stuartii.

A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5'-CTGATG(N)(25-26/27-28)-3'. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis ( approximately 40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25-26 nt downstream of the top strand sequence to generate a two base 5'-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5'-CATCAG-3'. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein-protein contacts to activate endonuclease activity.

Restriction endonucleases: classification, properties, and applications.

Restriction endonucleases have become a fundamental tool of molecular biology with many commercial vendors and extensive product lines. While a significant amount has been learned about restriction enzyme diversity, genomic organization, and mechanism, these continue to be active areas of research and assist in classification efforts. More recently, one focus has been their exquisite specificity for the proper recognition sequence and the lack of homology among enzymes recognizing the same DNA sequence. Some questions also remain regarding in vivo function. Site-directed mutagenesis and fusion proteins based on known endonucleases show promise for custom-designed cleavage. An understanding of the enzymes and their properties can improve their productive application by maintaining critical digest parameters and enhancing or avoiding alternative activities.

Evolutionary relationship of Alw26I, Eco31I and Esp3I, restriction endonucleases that recognise overlapping sequences.

Type II restriction endonucleases (ENases) have served as models for understanding the enzyme-based site-specific cleavage of DNA. Using the knowledge gained from the available crystal structures, a number of attempts have been made to alter the specificity of ENases by mutagenesis. The negative results of these experiments argue that the three-dimensional structure of DNA-ENase complexes does not provide enough information to enable us to understand the interactions between DNA and ENases in detail. This conclusion calls for alternative approaches to the study of structure-function relationships related to the specificity of ENases. Comparative analysis of ENases that manifest divergent substrate specificities, but at the same time are evolutionarily related to each other, may be helpful in this respect. The success of such studies depends to a great extent on the availability of related ENases that recognise partially overlapping nucleotide sequences (e.g. sets of enzymes that bind to recognition sites of increasing length). In this study we report the cloning and sequence analysis of genes for three Type IIS restriction-modification (RM) systems. The genes encoding the ENases Alw26I, Eco31I and Esp3I (whose recognition sequences are 5'-GTCTC-3', 5'-GGTCTC-3' and 5'-CGTCTC-3', respectively) and their accompanying methyltransferases (MTases) have been cloned and the deduced amino acid sequences of their products have been compared. In pairwise comparisons, the degree of sequence identity between Alw26I, Eco31I and Esp3I ENases is higher than that observed hitherto among ENases that recognise partially overlapping nucleotide sequences. The sequences of Alw26I, Eco31I and Esp3I also reveal identical mosaic patterns of sequence conservation, which supports the idea that they are evolutionarily related and suggests that they should show a high level of structural similarity. Thus these ENases represent very attractive models for the study of the molecular basis of variation in the specific recognition of DNA targets. The corresponding MTases are represented by proteins of unusual structural and functional organisation. Both M. Alw26I and M. Esp3I are represented by a single bifunctional protein, which is composed of an m(6)A-MTase domain fused to a m(5)C-MTase domain. In contrast, two separate genes encode the m(6)A-MTase and m(5)C-MTase in the Eco31I RM system. Among the known bacterial m(5)C-MTases, the m(5)C-MTases of M. Alw26I, M. Eco31I and M. Esp3I represent unique examples of the circular permutation of their putative target recognition domains together with the conserved motifs IX and X.