Kinetic and catalytic properties of dimeric KpnI DNA methyltransferase

KpnI DNA-(N(6)-adenine)-methyltransferase (KpnI MTase) is a member of a restriction-modification (R-M) system in Klebsiella pneumoniae and recognizes the sequence 5'-GGTACC-3'. It modifies the recognition sequence by transferring the methyl group from S-adenosyl-l-methionine (AdoMet) to the N(6) position of adenine residue. KpnI MTase occurs as a dimer in solution as shown by gel filtration and chemical cross-linking analysis. The nonlinear dependence of methylation activity on enzyme concentration indicates that the functionally active form of the enzyme is also a dimer. Product inhibition studies with KpnI MTase showed that S-adenosyl-l-homocysteine is a competitive inhibitor with respect to AdoMet and noncompetitive inhibitor with respect to DNA. The methylated DNA showed noncompetitive inhibition with respect to both DNA and AdoMet. A reduction in the rate of methylation was observed at high concentrations of duplex DNA. The kinetic analysis where AdoMet binds first followed by DNA, supports an ordered bi bi mechanism. After methyl transfer, methylated DNA dissociates followed by S-adenosyl-l-homocysteine. Isotope-partitioning analysis showed that KpnI MTase-AdoMet complex is catalytically active.     

DNA methyltransferases of the cyanobacterium Anabaena PCC 7120

From the characterization of enzyme activities and the analysis of genomic sequences, the complement of DNA methyltransferases (MTases) possessed by the cyanobacterium ANABAENA PCC 7120 has been deduced. ANABAENA has nine DNA MTases. Four are associated with Type II restriction enzymes (AVAI, AVAII, AVAIII and the newly recognized inactive AVAIV), and five are not. Of the latter, four may be classified as solitary MTases, those whose function lies outside of a restriction/modification system. The group is defined here based on biochemical and genetic characteristics. The four solitary MTases, DmtA/M.AVAVI, DmtB/M.AVAVII, DmtC/M. AVAVIII and DmtD/M.AVAIX, methylate at GATC, GGCC, CGATCG and rCCGGy, respectively. DmtB methylates cytosines at the N4 position, but its sequence is more similar to N6-adenine MTases than to cytosine-specific enzymes, indicating that it may have evolved from the former. The solitary MTases, appear to be of ancient origin within cyanobacteria, while the restriction MTases appear to have arrived by recent horizontal transfer as did five now inactive Type I restriction systems. One Mtase, M.AVAV, cannot reliably be classified as either a solitary or restriction MTase. It is structurally unusual and along with a few proteins of prokaryotic and eukaryotic origin defines a structural class of MTases distinct from all previously described.     

Crystal structure of MboIIA methyltransferase

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.     

Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis

Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5'-CAGCAG-3'. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.     

Identification and preliminary characterization of an O6-methylguanine DNA repair methyltransferase in the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae contains a DNA repair methyltransferase (MTase) that repairs O6-methylguanine. Methyl groups are irreversibly transferred from O6-methylguanine in DNA to a 25-kilodalton protein in S. cerevisiae cell extracts, and methyl transfer is accompanied by the formation of S-methylcysteine. The yeast MTase is expressed at approximately 150 molecules/cell in exponentially growing yeast cultures but is not detectable in stationary phase cells. Unlike mammalian and bacterial MTases, the yeast MTase is very temperature-sensitive, having a half-life of about 4 min at 37 degrees C, which may explain why others have failed to detect it. Like other DNA repair MTases, the S. cerevisiae MTase repairs O6-methylguanine more efficiently in double-stranded DNA than in single-stranded DNA. Synthesis of the yeast DNA MTase is apparently not inducible by sublethal exposures to alkylating agent, but rather MTase activity is depleted in cells exposed to low doses of alkylating agent. Judging from its molecular weight and substrate specificity, the yeast DNA MTase is more closely related to mammalian MTases than to Escherichia coli MTases.     

DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [N6-adenine] DNA methyltransferases

A genetic selection method, the P22 challenge-phage assay, was used to characterize DNA binding in vivo by the prokaryotic beta class [N:6-adenine] DNA methyltransferase M.RSR:I. M.RSR:I mutants with altered binding affinities in vivo were isolated. Unlike the wild-type enzyme, a catalytically compromised mutant, M.RSR:I (L72P), demonstrated site-specific DNA binding in vivo. The L72P mutation is located near the highly conserved catalytic motif IV, DPPY (residues 65-68). A double mutant, M.RSR:I (L72P/D173A), showed less binding in vivo than did M.RSR:I (L72P). Thus, introduction of the D173A mutation deleteriously affected DNA binding. D173 is located in the putative target recognition domain (TRD) of the enzyme. Sequence alignment analyses of several beta class MTases revealed a TRD sequence element that contains the D173 residue. Phylogenetic analysis suggested that divergence in the amino acid sequences of these methyltransferases correlated with differences in their DNA target recognition sequences. Furthermore, MTases of other classes (alpha and gamma) having the same DNA recognition sequence as the beta class MTases share related regions of amino acid sequences in their TRDs.     

Characterization of the major DNA repair methyltransferase activity in unadapted Escherichia coli and identification of a similar activity in Salmonella typhimurium.

Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did not appear to be induced in response to chronic DNA alkylation at least in this ada deletion strain. DNA MTase II was much more heat labile than Ada. Heat lability studies indicated that more than 95% of the MTase in unadapted E. coli was DNA MTase II. We discuss the possible implications of these results for the mechanism of induction of the adaptive response. A similarly active 19-kDa O6MeG-O4-methylthymine DNA MTase was identified in Salmonella typhimurium.     

Structural bases for substrate recognition and activity in Meaban virus nucleoside-2'-O-methyltransferase

Viral methyltransferases are involved in the mRNA capping process, resulting in the transfer of a methyl group from S-adenosyl-L-methionine to capped RNA. Two groups of methyltransferases (MTases) are known: (guanine-N7)-methyltransferases (N7MTases), adding a methyl group onto the N7 atom of guanine, and (nucleoside-2'-O-)-methyltransferases (2'OMTases), adding a methyl group to a ribose hydroxyl. We have expressed and purified two constructs of Meaban virus (MV; genus Flavivirus) NS5 protein MTase domain (residues 1-265 and 1-293, respectively). We report here the three-dimensional structure of the shorter MTase construct in complex with the cofactor S-adenosyl-L-methionine, at 2.9 angstroms resolution. Inspection of the refined crystal structure, which highlights structural conservation of specific active site residues, together with sequence analysis and structural comparison with Dengue virus 2'OMTase, suggests that the crystallized enzyme belongs to the 2'OMTase subgroup. Enzymatic assays show that the short MV MTase construct is inactive, but the longer construct expressed can transfer a methyl group to the ribose 2'O atom of a short GpppAC(5) substrate. West Nile virus MTase domain has been recently shown to display both N7 and 2'O MTase activity on a capped RNA substrate comprising the 5'-terminal 190 nt of the West Nile virus genome. The lack of N7 MTase activity here reported for MV MTase may be related either to the small size of the capped RNA substrate, to its sequence, or to different structural properties of the C-terminal regions of West Nile virus and MV MTase-domains.     

Structure, function and mechanism of exocyclic DNA methyltransferases

DNA MTases (methyltransferases) catalyse the transfer of methyl groups to DNA from AdoMet (S-adenosyl-L-methionine) producing AdoHcy (S-adenosyl-L-homocysteine) and methylated DNA. The C5 and N4 positions of cytosine and N6 position of adenine are the target sites for methylation. All three methylation patterns are found in prokaryotes, whereas cytosine at the C5 position is the only methylation reaction that is known to occur in eukaryotes. In general, MTases are two-domain proteins comprising one large and one small domain with the DNA-binding cleft located at the domain interface. The striking feature of all the structurally characterized DNA MTases is that they share a common core structure referred to as an 'AdoMet-dependent MTase fold'. DNA methylation has been reported to be essential for bacterial virulence, and it has been suggested that DNA adenine MTases (Dams) could be potential targets for both vaccines and antimicrobials. Drugs that block Dam could slow down bacterial growth and therefore drug-design initiatives could result in a whole new generation of antibiotics. The transfer of larger chemical entities in a MTase-catalysed reaction has been reported and this represents an interesting challenge for bio-organic chemists. In general, amino MTases could therefore be used as delivery systems for fluorescent or other reporter groups on to DNA. This is one of the potential applications of DNA MTases towards developing non-radioactive DNA probes and these could have interesting applications in molecular biology. Being nucleotide-sequence-specific, DNA MTases provide excellent model systems for studies on protein-DNA interactions. The focus of this review is on the chemistry, enzymology and structural aspects of exocyclic amino MTases.     

Functional analysis of an acid adaptive DNA adenine methyltransferase from Helicobacter pylori 26695

HP0593 DNA-(N(6)-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5'-GCAG-3'and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co(2+), Mn(2+), and also Mg(2+) stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.     

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework.     

DNA methylation in Neisseria gonorrhoeae and other Neisseriae

It has been reported in the literature that Neisseria gonorrhoeae DNA is modified by the methyltransferases (MTases) M.NgoI, M.NgoII, and M.NgoIII, as well as three other cytosine MTases and one adenine MTase, even if the corresponding restriction endonucleases are not present. We envisioned the possibility of cloning one of the N. gonorrhoeae MTase-encoding genes for use as a species-specific DNA probe. We therefore undertook a survey of methylation patterns of several clinical isolates of N. gonorrhoeae and N. meningitidis as well as ATCC strains of other Neisseriae. We found, from digestion patterns with isoschizomers, one N. gonorrhoeae strain that lacked M.NgoII and two that lacked M.NgoIII. All N. meningitidis strains (save one) were resistant to digestion with NlaIV thus possessing an MTase like NgoV, and one was resistant to SstII, thus having an NgoIII-like MTase. None were resistant to isoschizomers of NgoI, NgoIII and NgoIV. Some other Neisseriae had an MTase with NlaIV (NgoV) specificity, but none had NgoI, II, III or IV specificity, except for the Branhamella-like N. caviae-ovis group and N. lactamica where these specificities were present in at least one strain of this group. Therefore, among the Neisseriae other than N. caviae only M.NgoI is N. gonorrhoeae-specific.     

Diversity of DNA methyltransferases that recognize asymmetric target sequences

DNA methyltransferases (MTases) are a group of enzymes that catalyze the methyl group transfer from S-adenosyl-L-methionine in a sequence-specific manner. Orthodox Type II DNA MTases usually recognize palindromic DNA sequences and add a methyl group to the target base (either adenine or cytosine) on both strands. However, there are a number of MTases that recognize asymmetric target sequences and differ in their subunit organization. In a bacterial cell, after each round of replication, the substrate for any MTase is hemimethylated DNA, and it therefore needs only a single methylation event to restore the fully methylated state. This is in consistent with the fact that most of the DNA MTases studied exist as monomers in solution. Multiple lines of evidence suggest that some DNA MTases function as dimers. Further, functional analysis of many restriction-modification systems showed the presence of more than one or fused MTase genes. It was proposed that presence of two MTases responsible for the recognition and methylation of asymmetric sequences would protect the nascent strands generated during DNA replication from cognate restriction endonuclease. In this review, MTases recognizing asymmetric sequences have been grouped into different subgroups based on their unique properties. Detailed characterization of these unusual MTases would help in better understanding of their specific biological roles and mechanisms of action. The rapid progress made by the genome sequencing of bacteria and archaea may accelerate the identification and study of species- and strain-specific MTases of host-adapted bacteria and their roles in pathogenic mechanisms.     

Purification, cloning and sequence analysis of RsrI DNA methyltransferase: lack of homology between two enzymes, RsrI and EcoRI, that methylate the same nucleotide in identical recognition sequences.

RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced. This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI. The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da. A fragment of R. sphaeroides chromosomal DNA exhibited M.RsrI activity in E. coli and was used to sequence the rsrIM gene. The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15. In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology. Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes. The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl. Acids Res. (1989) 17, this issue).     

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGAT-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.     

AdoMet-dependent methylation, DNA methyltransferases and base flipping

Twenty AdoMet-dependent methyltransferases (MTases) have been characterized structurally by X-ray crystallography and NMR. These include seven DNA MTases, five RNA MTases, four protein MTases and four small molecule MTases acting on the carbon, oxygen or nitrogen atoms of their substrates. The MTases share a common core structure of a mixed seven-stranded beta-sheet (6 downward arrow 7 upward arrow 5 downward arrow 4 downward arrow 1 downward arrow 2 downward arrow 3 downward arrow) referred to as an 'AdoMet-dependent MTase fold', with the exception of a protein arginine MTase which contains a compact consensus fold lacking the antiparallel hairpin strands (6 downward arrow 7 upward arrow). The consensus fold is useful to identify hypothetical MTases during structural proteomics efforts on unannotated proteins. The same core structure works for very different classes of MTase including those that act on substrates differing in size from small molecules (catechol or glycine) to macromolecules (DNA, RNA and protein). DNA MTases use a 'base flipping' mechanism to deliver a specific base within a DNA molecule into a typically concave catalytic pocket. Base flipping involves rotation of backbone bonds in double-stranded DNA to expose an out-of-stack nucleotide, which can then be a substrate for an enzyme-catalyzed chemical reaction. The phenomenon is fully established for DNA MTases and for DNA base excision repair enzymes, and is likely to prove general for enzymes that require access to unpaired, mismatched or damaged nucleotides within base-paired regions in DNA and RNA. Several newly discovered MTase families in eukaryotes (DNA 5mC MTases and protein arginine and lysine MTases) offer new challenges in the MTase field.     

M.(phi)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme.

In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and eukaryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order. In addition, one variable region, which includes the target-recognizing domain(s) (TRDs) characteristic for each enzyme, has been localized in one region between the same blocks of these conserved elements. This conservation in the order of conserved and variable sequences suggests stringent structural constraints in the primary structure to obtain the correct folding of the enzymes. Here we report the characterization of a new type of a multispecific MTase, M.(phiphi)BssHII, which is expressed as two isoforms. Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conventional location, one TRD located at a non-canonical position at its N-terminus. Isoform II is represented by the same MTase, but without the N-terminal TRD. The N-terminal TRD provides HaeII methylation specificity to isoform I. The TRD is fully functional when engineered into either the conventional variable region of M.(phiphi)BssHII or the related monospecific M.phi3TII MTase. The implications of this structural plasticity with respect to the evolution of MTases are discussed.     

Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases

Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m5U in RNA, m5C in RNA and m5C in DNA. The DNA:m5C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods. On the other hand, the sequence-structure-function relationships of RNA:m5C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes. Sequence analyses and homology modeling of the yeast tRNA:m5C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m5C MTase active site. The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical. The newly identified residues include a Lys residue in motif I and an Asp in motif IV. A conserved Gln found in motif X was found to be dispensable for MTase activity. Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m5C MTase homolog PH1374. Theoretical and experimental analyses revealed that RNA:m5C MTases share a number of features with either RNA:m5U MTases or DNA:m5C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases. We infer that RNA:m5C MTases evolved from RNA:m5U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m5C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.     

Cloning the KpnI restriction-modification system in Escherichia coli

The genes encoding the KpnI restriction and modification (R-M) system from Klebsiella pneumoniae, recognizing the sequence, 5'-GGTAC decreases C-3', were cloned and expressed in Escherichia coli. Although the restriction endonuclease (ENase)- and methyltransferase (MTase)-encoding genes were closely linked, initial attempts to clone both genes as a single DNA fragment in a plasmid vector resulted in deletions spanning all or part of the gene coding for the ENase. Initial protection of the E. coli host with MTase expressed on a plasmid was required to stabilize a compatible plasmid carrying both the ENase- and the MTase-encoding genes on a single DNA fragment. However, once established, the MTase activity can be supplied in cis to the kpnIR gene, without an extra copy of kpnIM. A chromosomal map was generated localizing the kpnIR and kpnIM genes on 1.7-kb and 3.5-kb fragments, respectively. A final E. coli strain was constructed, AH29, which contained two compatible plasmids: an inducible plasmid carrying the kpnIR gene which amplifies copy number at elevated temperatures and a pBR322 derivative expressing M.KpnI. This strain produces approx. 10 million units of R.KpnI/g of wet-weight cells, which is several 1000-fold higher than the level of R.KpnI produced by K. pneumoniae. In addition, DNA methylated with M.KpnI in vivo does not appear to be restricted by the mcrA, mcrB or mrr systems of E. coli.