The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.
A mutant (designated mec−) has been isolated from Escherichia coli C which has lost DNA-cytosine methylase activity and the ability to protect phage λ against in vivo restriction by the RII endonuclease. This situation is analogous to that observed with an E. coli K-12 mec− mutant; thus, the E. coli C methylase appears to have overlapping sequence specificity with the K-12 and RII enzymes; (the latter methylases have been shown previously to recognize the same sequence). Covalently closed, supertwisted double-standed DNA (RFI) was isolated from C mec+ and C mec− cells infected with bacteriophage φX174. φX· mec− RFI is sensitive to in vitro cleavage by R·EcoRII and is cut twice to produce two fragments of almost equal size. In contrast, φX·mec+ RFI is relatively resistant to in vitro cleavage by R·EcoRII. R·BstI, which cleaves mec+/RII sites independent of the presence or absence of 5-methylcytosine, cleaves both forms of the RFI and produces two fragments similar in size to those observed with R· EcoRII. These results demonstrate that φX·mec+ RFI is methylated in vivo by the host mec+ enzyme and that this methylation protects the DNA against cleavage by R·EcoRII. This is consistent with the known location of two mec+/ RII sequences (viz., [Formula: see text]) on the φX174 map. Mature singlestranded virion DNA was isolated from φX174 propagated in C mec+ or C mec− in the presence of l-[methyl-3H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that φX·mec+ DNA had a 10-fold-higher ratio of [3H]5-methylcytosine to [3H]cytosine compared to φX·mec−. Since φX·mec+ contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of φX174 is mediated by the host mec+ enzyme only. These results are not consistent with the conclusions of previous reports that φX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.
The HpaII restriction-modification system from Haemophilus parainfluenzae recognizes the DNA sequence CCGG. The gene for the HpaII methylase has been cloned into E. coli and its nucleotide sequence has been determined. The DNA of the clones is fully protected against cleavage by the HpaII restriction enzyme in vitro, indicating that the methylase gene is active in E. coli. The clones were isolated in an McrA-strain of E. coli; attempts to isolate them in an McrA+ strain were unsuccessful. The clones do not express detectable HpaII restriction endonuclease activity, suggesting that either the endonuclease gene is not expressed well in E. coli, or that it is not present in its entirety in any of the clones that we have isolated. The derived amino acid sequence of the HpaII methylase shows overall similarity to other cytosine methylases. It bears a particularly close resemblance to the sequences of the HhaI, BsuFI and MspI methylases. When compared with three other methylases that recognize CCGG, the variable region of the HpaII methylase, which is believed to be responsible for sequence specific recognition, shows some similarity to the corresponding regions of the BsuFI and MspI methylases, but is rather dissimilar to that of the SPR methylase.
The dcm locus of Escherichia coli K-12 has been shown to code for a methylase that methylates the second cytosine within the sequence 5'-CC(A/T)GG-3'. This sequence is also recognized by the EcoRII restriction-modification system coded by the E. coli plasmid N3. The methylase within the EcoRII system methylates the same cytosine as the dcm protein. We have isolated, from a library of E. coli K-12 DNA, two overlapping clones that carry the dcm locus. We show that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain. We also show that the cloned gene codes for a methylase, that it complements mutations in the EcoRII methylase, and that it protects EcoRII recognition sites from cleavage by the EcoRII endonuclease. We found no phage restriction activity associated with the dcm clones.
Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.
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.
A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco57I was cloned and expressed in E.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco57I endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7I, TaqI, CviBIII, PstI, BamHI and HincII methylases. Homologs of this sequence are also found within the sequences of the PaeR7I, PstI and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.
We have determined the nature of the deoxyribonucleic acid (DNA) modification governed by the SA host specificity system of Salmonella typhimurium. Two lines of evidence indicate that SA modification is based on methylation of DNA-adenine residues. (i) The SA+ locus of Salmonella was transferred into Escherichia coli B, a strain that does not contain 5-methylcytosine in its DNA; although the hybrid strain was able to confer SA modification, its DNA still did not contain 5-methylcytosine. (ii) the N6-methyladenine content of phage L DNA was measured after growth in various host strains; phage lacking SA modification contained fewer N6-methyladenine residues per DNA. We also investigated the possibility, suggested by others (32), that SA modification protects phage DNA against restriction by the RII host specificity system. Phages lambda, P3, and L were grown in various SA+ and SA- hosts and tested for their relative plating ability on strains containing or lacking RII restriction; the presence or absence of SA modification had no effect on RII restriation. In vitro studies revealed, however, that Salmonella DNA is protected against cleavage by purified RII restriction endonuclease (R-EcoRII). This protection is not dependent on SA modification; rather, it appears to be due to methylation by a DNA-cytosine methylase which has overlapping specificity with the RII modification enzyme, but which is not involved in any other known host specificity system.
Prokaryotic DNA[cytosine-C5]methyltransferases (m5C-methylases) share a common architectural arrangement of ten conserved sequence motifs. A series of eleven hybrids have been constructed between the HpaII (recognition sequence: Cm5CGG) and HhaI (recognition sequence: Gm5CGC) DNA-methylases. The hybrids were over-expressed in E.coli and their in vivo methylation phenotypes investigated. Six were inactive by our assay while five of them retained partial methylation activity and full specificity. In all five cases the specificity matched that of the parent methylase which contributed the so-called variable region, located between conserved motifs VIII and IX. This was the only sequence held in common between the active hybrids and for the first time provides unequivocal evidence that the specificity determinants of the mono-specific m5C-methylases are located within the variable region. Correlation of the hybrid methylase structure with the efficiency of methylation suggests that conserved motif IX may interact with the variable region whereas motif X most probably interacts with the N-terminal half of the molecule.
Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions.
NruI and Sbo13I are restriction enzyme isoschizomers with the same recognition sequence 5' TCG↓CGA 3' (cleavage as indicated↓). Here we report the cloning of NruI and Sbo13I restriction-modification (R-M) systems in E. coli. The NruI restriction endonuclease gene (nruIR) was cloned by PCR and inverse PCR using primers designed from the N-terminal amino acid sequence. The NruI methylase gene (nruIM) was derived by inverse PCR walking.
The amino acid sequences of NruI endonuclease and methylase are very similar to the Sbo13I R-M system which has been cloned and expressed in E. coli by phage selection of a plasmid DNA library. Dot blot analysis using rabbit polyclonal antibodies to N6mA- or N4mC-modified DNA indicated that M.NruI is possibly a N6mA-type amino-methyltransferase that most likely modifies the external A in the 5' TCGCGA 3' sequence. M.Sbo13I, however, is implicated as a probable N4mC-type methylase since plasmid carrying sbo13IM gene is not restricted by Mrr endonuclease and Sbo13I digestion is not blocked by Dam methylation of the overlapping site. The amino acid sequence of M.NruI and M.Sbo13I did not show significant sequence similarity to many known amino-methyltransferases in the α, β, and γ groups, except to a few putative methylases in sequenced microbial genomes.
The order of the conserved amino acid motifs (blocks) in M.NruI/M.Sbo13I is similar to the γ. group amino-methyltranferases, but with two distinct features: In motif IV, the sequence is DPPY instead of NPPY; there are two additional conserved motifs, IVa and Xa as extension of motifs IV and X, in this family of enzymes. We propose that M.NruI and M.Sbo13I form a subgroup in the γ group of amino-methyltransferases.
Macrolide-lincosamide-streptogramin B resistance specified by Streptococcus sanguis plasmid pAM77 involves an adenine methylase, whose synthesis, demonstrable both phenotypically and by analysis of methionine-labeled proteins made in Bacillus subtilis minicells, is inducible by erythromycin, lincomycin, and streptogramin type B antibiotics. Localization of the methylase structural gene, including its control region in DNA fragments obtained with restriction endonucleases, has been deduced from DNA blot experiments with characterized target and probe DNAs from other streptococci, combined with DNA sequence analysis and comparison of the putative streptococcal methylase sequence with that of a cognate methylase in staphylococcal plasmid pE194. The streptococcal methylase migrates electrophoretically in polyacrylamide gels with the mobility of a 29,000-dalton protein. The sequence organization of the putative streptococcal methylase mRNA leader sequence partially resembles its staphylococcal counterpart and can support a similar mechanism of secondary structure rearrangement leading to methylase synthesis. The deduced 5' leader sequence preceding the pAM77 methylase structural gene sequence comprises approximately 155 nucleotides within which one can identify a putative control peptide 36 amino acid residues in length (in contrast to 19 in the pE194 peptide) and at least 14 possible classes of overlapping inverted complementary repeat sequences (in contrast to 3 in the pE194 control region), one of which can sequester the sequence AGGAG 7 nucleotides upstream from the putative (methionine) start codon of the streptococcal methylase. Comparison of the pAM77 and pE194 methylase amino acid sequences and their respective nucleotide sequences shows 51% conservation of amino acid residues (124 of 244) and 59% conservation of nucleotide residues (433 of 738), which suggests a common origin for the two methylase structural gene sequences. Differences in mRNA base composition associated with conserved amino acid residues occur mostly in the third nucleotide ("wobble") position of codons and may reflect adaptation of methylase genes to optimal expression in host cells with differing codon use patterns.
The KpnI restriction-modification (KpnI RM) system was previously cloned and expressed in E. coli. The nucleotide sequences of the KpnI endonuclease (R.KpnI) and methylase (M. KpnI) genes have now been determined. The sequence of the amino acid residues predicted from the endonuclease gene DNA sequence and the sequence of the first 12 NH2-terminal amino acids determined from the purified endonuclease protein were identical. The kpnIR gene specifies a protein of 218 amino acids (MW: 25,115), while the kpnIM gene codes for a protein of 417 amino acids (MW: 47,582). The two genes transcribe divergently with a intergeneic region of 167 nucleotides containing the putative promoter regions for both genes. No protein sequence similarity was detected between R.KpnI and M.KpnI. Comparison of the amino acid sequence of M.KpnI with sequences of various methylases revealed a significant homology to N6-adenine methylases, a partial homology to N4-cytosine methylases, and no homology to C5-methylases.
Two DNA methylase activities of Escherichia coli C, the mec (designates DNA-cytosine-methylase gene, which is also designated dcm) and dam gene products, were physically separated by DEAE-cellulose column chromatography. The sequence and substrate specificity of the two enzymes were studied in vitro. The experiments revealed that both enzymes show their expected sequence specificity under in vitro conditions, methylating symmetrically on both DNA strands. The mec enzyme methylates exclusively the internal cytosine residue of CCATGG sequences, and the dam enzyme methylates adenine residues at GATC sites. Substrate specificity experiments revealed that both enzymes methylate in vitro unmethylated duplex DNA as efficiently as hemimethylated DNA. The results of these experiments suggest that the methylation at a specific site takes place by two independent events. A methyl group in a site on one strand of the DNA does not facilitate the methylation of the same site on the opposite strand. With the dam methylase it was found that the enzyme is incapable of methylating GATC sites located at the ends of DNA molecules.
BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The BslI restriction-modification system from Bacillus species was cloned and expressed in Escherichia coli. The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), the bslIRα gene, and the bslIRβ gene. The α and β subunits of BslI can be expressed independently in E. coli in the absence of BslI methylase (M.BslI) protection. BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (αβ), heterotetramers (α2β2), and possibly oligomers in solution. Two β subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer BslI complex (α2β2). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is α2β2. M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the β group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 in BslI sites in the first or second cytosine are resistant to BslI digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion. Two putative zinc fingers are found in the α subunit of BslI endonuclease.
Two different cytosine DNA-methylases, NI and GII, are present in Escherichia coli SK. The GII methylase recognizes the five-member symmetric sequence: 5'...NpCpCpApGpGpN...3'. This sequence is identical with the recognition site of the hsp II type determined by RII plasmid but, in contrast to RII methylase, the GII enzyme methylates cytosine located on the 5' side of the site. By analogy with the isoshizomery of the restricting endonucleases, RII and GII DNA methylaeses may be called isomethymers which recognize the same site but methylate different bases. Since the phage of the SK and hsp II phenotypes is effectively restricted in respective cells it may be assumed that the isomethymeric modification does not provide any protection against the corresponding restrictases. NI methylase recognizes the five-member symmetric site which represents an inverted sequence of the GII site: 5'...NpGpGpApCpCpN...3'. In this case cytosine at the 3'-end of the recognition site is methylated.
The turnover of DNA-adenine-methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC.
A gene encoding a putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in Escherichia coli. The protein was purified to over 90% purity by chromatography. The purified enzyme, SauUSI, predominantly cleaves modified DNA containing 5mC and 5-hydroxymethylcytosine. Cleavage of 5mC-modified plasmids indicated that the sites S5mCNGS (S = C or G) are preferentially digested. The endonuclease activity requires the presence of adenosine triphosphate (ATP) or dATP whereas the non-hydrolyzable γ-S-ATP does not support activity. SauUSI activity was inhibited by ethylenediaminetetraacetic acid. It is most active in Mg++ buffers. No companion methylase gene was found near the SauUSI restriction gene. The absence of a cognate methylase and cleavage of modified DNA indicate that SauUSI belongs to type IV restriction endonucleases, a group that includes EcoK McrBC and Mrr. SauUSI belongs to a family of highly similar homologs found in other sequenced S. aureus, S. epidermidis and S. carnosus genomes. More distant SauUSI orthologs can be found in over 150 sequenced bacterial/archaea genomes. Finally, we demonstrated the biological function of the type IV REase in restricting 5mC-modified plasmid DNA by transformation into clinical S. aureus strain SA564, and in restricting phage λ infection when the endonuclease is expressed in E. coli.
Presence and possible functions of DNA methylation in plastid genomes of higher plants have been highly controversial. While a number of studies presented evidence for the occurrence of both cytosine and adenine methylation in plastid genomes and proposed a role of cytosine methylation in the transcriptional regulation of plastid genes, several recent studies suggested that at least cytosine methylation may be absent from higher plant plastid genomes. To test if either adenine or cytosine methylation can play a regulatory role in plastid gene expression, we have introduced cyanobacterial genes for adenine and cytosine DNA methyltransferases (methylases) into the tobacco plastid genome by chloroplast transformation. Using DNA cleavage with methylation-sensitive and methylation-dependent restriction endonucleases, we show that the plastid genomes in the transplastomic plants are efficiently methylated. All transplastomic lines are phenotypically indistinguishable from wild-type plants and, moreover, show no alterations in plastid gene expression. Our data indicate that the expression of plastid genes is not sensitive to DNA methylation and, hence, suggest that DNA methylation is unlikely to be involved in the transcriptional regulation of plastid gene expression.
Chloroplast; Adenine methylation; Cytosine methylation; Dam methylation; Plastid transformation; Nicotiana tabacum
DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.
A new E. coli strain has been constructed that contains the dinD1::LacZ+ fusion and is deficient in methylation-dependent restriction systems (McrA-, McrBC-, Mrr-). This strain has been used to clone restriction endonuclease genes directly into E. coli. When E. coli cells are not fully protected by the cognate methylase, the restriction enzyme damages the DNA in vivo and induces the SOS response. The SOS-induced cells form blue colonies on indicator plates containing X-gal. Using this method the genes coding for the thermostable restriction enzymes Taql (5'TCGA3') and Tth111l (5'GACNNNGTC3') have been successfully cloned in E. coli. The new strain will be useful to clone other genes involved in DNA metabolism.
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.
A procedure is described for the partial purification of the deoxyribonucleic acid (DNA)-cytosine methylases controlled by the RII plasmid and by the Escherichia coli mec+ gene. The two enzymes exhibit similar but distinct chromatographic behavior on diethylaminoethyl-cellulose and phosphocellulose. Preliminary studies on the two methylases indicate that they are indistinguishable with respect to their Km for S-adenosylmethionine and their pH (in tris (hydroxymethyl)aminomethane buffer) and NaCl concentration optima. In vitro methylation of various phage lambda DNA substrates by the mec'r RII enzyme modifies the DNA to a form that is completely resistant to double-stranded cleavage by the RII restriction endonuclease (R-EcoRII). These results are consistent with our earlier proposal that the mec8ethylase recognizes RII host specificity sites.
Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mismatches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previously we have shown that a plasmid containing an 11-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa. The genes responsible for these phenotypes have been identified by DNA sequence analysis. The gene essential for cytosine methylation, dcm, is predicted to code for a 473-amino-acid protein and is not required for VSP repair. It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRII methylase. The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein. This gene, named vsr, is not essential for DNA methylation. Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm. The two genes appear to be transcribed from a common promoter but are in different translational registers. This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation.
Differences in the type of base methylated (cytosine or adenine) and in the extent of methylation were detected by high-pressure liquid chromatography in the DNAs of five spiroplasmas. Nearest neighbor analysis and digestion by restriction enzyme isoschizomers also revealed differences in methylation sequence specificity. Whereas in Spiroplasma floricola and Spiroplasma sp. strain PPS-1 5-methylcytosine was found on the 5' side of each of the four major bases, the cytosine in Spiroplasma apis DNA was methylated only when its 3' neighboring base was adenine or thymine. In Spiroplasma sp. strain MQ-1 over 95% of the methylated cytosine was in C-G sequences. Essentially all of the C-G sequences in the MQ-1 DNA were methylated. Partially purified extracts of S. apis and Spiroplasma sp. strain MQ-1 were used to study substrate and sequence specificity of the methylase activity. Methylation by the MQ-1 enzyme was exclusively at C-G sequences, resembling in this respect eucaryotic DNA methylases. However, the MQ-1 methylase differed from eucaryotic methylases by showing high activity on nonmethylated DNA duplexes, low activity with hemimethylated DNA duplexes, and no activity on single-stranded DNA.