Evidence that Escherichia coli virus T1 induces a DNA methyltransferase

DNA of Escherichia coli virus T1 is resistant to MboI cleavage and appears to be heavily methylated. Analysis of methylation by the isoschizomeric restriction enzymes Sau3AI and DpnI revealed that recognition sites for E. coli DNA adenine methylase (dam methylase) are methylated. The same methylation pattern was found for virus T1 DNA grown on an E. coli dam host, indicating a T1-specific DNA methyltransferase.     

Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.

In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transformation frequencies of oriC plasmids into E. coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation. Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase. oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells. This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase. Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E. coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity. Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin.     

Effect of dam methylation on the activity of the E. coli replication origin, oriC.

Methylation of GATC sites by the dam methylase is required for efficient initiation of DNA replication at the replication origin, oriC, of Escherichia coli. This is demonstrated by the inability of minichromosomes to be maintained in dam mutant strains. The requirement for methylated GATC sites is less stringent in vitro than in vivo. The time required for complete methylation of the origin region apparently determines the minimal spacing of replication forks on the chromosome.     

P1 plasmid replication requires methylated DNA.

Plasmids driven by the plasmid replication origin of bacteriophage P1 cannot be established in Escherichia coli strains that are defective for the DNA adenine methylase (dam). Using a composite plasmid that has two origins, we show that the P1 origin cannot function even in a plasmid that is already established in a dam strain. An in vitro replication system for the P1 origin was developed that uses as a substrate M13 replicative-form DNA containing the minimal P1 origin. The reaction mixture contains a crude extract of E. coli and purified P1 RepA protein. In addition to being RepA dependent, synthesis was shown to be dependent on methylation of the dam methylase-sensitive sites of the substrate DNA. As the P1 origin contains five such sites in a small region known to be critical for origin function, it can be concluded that methylation of these sites is a requirement for initiation. This suggests that the postreplicational methylation of the origin may control reinitiation and contribute to the accuracy of the highly stringent copy-number control of the origin in vivo.     

Sequence and substrate specificity of isolated DNA methylases from Escherichia coli C

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.     

Hemimethylation prevents DNA replication in E. coli

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication. We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites. These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E. coli.     

The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis.

The nucleotide sequence recognized and cleaved by the restriction endonuclease MboI is 5' GATC and is identical to the central tetranucleotide of the restriction sites of BamHI and BglII. Experiments on the restriction of DNA from Escherichia coli dam and dam+ confirm the notion that GATC sequences are adenosyl-methylated by the dam function of E. coli and thereby are made refractory to cleavage by MboI. On the basis of this observation the degree of dam methylation of various DNAs was examined by cleavage with MboI and other restriction endonucleases. In plasmid DNA essentially all of the GATC sequences are methylated by the dam function. The DNA of phage lambda is only partially methylated, extended methylation is observed in the DNA of a substitution mutant of lambda, lambda gal8bio256, and in the lambda derived plasmid, lambdadv93, which is completely methylated. In contrast, phage T7 DNA is not methylated by dam. A suppression of dam methylation of T7 DNA appears to act only in cis dam. A suppression of dam methylation of T7 DNA appears to act only in cis since plasmid DNA replicated in a T7-infected cell is completely methylated. The results are discussed with respect to the participation of the dam methylase in different replication systems.     

DNA Adenine Methylase Mutants of Salmonella Typhimurium and a Novel Dam-Regulated Locus

Mutants of Salmonella typhimurium lacking DNA adenine methylase were isolated; they include insertion and deletion alleles. The dam locus maps at 75 min between cysG and aroB, similar to the Escherichia coli dam gene. Dam(-) mutants of S. typhimurium resemble those of E. coli in the following phenotypes: (1) increased spontaneous mutations, (2) moderate SOS induction, (3) enhancement of duplication segregation, (4) inviability of dam recA and dam recB mutants, and (5) suppression of the inviability of the dam recA and dam recB combinations by mutations that eliminate mismatch repair. However, differences between S. typhimurium and E. coli dam mutants are also found: (1) S. typhimurium dam mutants do not show increased UV sensitivity, suggesting that methyl-directed mismatch repair does not participate in the repair of UV-induced DNA damage in Salmonella. (2) S. typhimurium dam recJ mutants are viable, suggesting that the Salmonella RecJ function does not participate in the repair of DNA strand breaks formed in the absence of Dam methylation. We also describe a genetic screen for detecting novel genes regulated by Dam methylation and a locus repressed by Dam methylation in the S. typhimurium virulence (or ``cryptic') plasmid.     

Relaxed specificity of the EcoRV restriction endonuclease

The EcoRV restriction endonuclease normally shows a high specificity for its recognition site on DNA, GATATC. In standard reactions, it cleaves DNA at this site several orders of magnitude more readily than at any alternative sequence. But in the presence of dimethyl sulphoxide and at high pH, the EcoRV enzyme cleaves DNA at several sites that differ from its recognition site by one nucleotide. Of the 18 (3 X 6) possible sequences that differ from GATATC by one base, all were cleaved readily except for the following 4 sites: TATATC, CATATC, GATATA and GATATG. However, two of the sites that could be cleaved by EcoRV in the presence of dimethyl sulphoxide, GAGATC and GATCTC, were only cleaved on DNA that lacked dam methylation: both contain the sequence GATC, the recognition site for the dam methylase of Escherichia coli.     

Expression of the Escherichia coli dam methylase in Saccharomyces cerevisiae: effect of in vivo adenine methylation on genetic recombination and mutation.

The Escherichia coli DNA adenine methylase (dam) gene has been introduced into Saccharomyces cerevisiae on a yeast-E. coli shuttle vector. Sau3AI, MboI, and DpnI restriction enzyme digests and Southern hybridization analysis indicated that the dam gene is expressed in yeast cells and methylates GATC sequences. Analysis of digests of total genomic DNA indicated that some GATC sites are not sensitive to methylation. The failure to methylate may reflect an inaccessibility to the methylase due to chromosome structure. The effects of this in vivo methylation on the processes of recombination and mutation in mitotic cells were determined. A small but definite general increase was found in the frequency of mitotic recombination. A similar increase was observed for reversion of some auxotrophic markers; other markers demonstrated a small decrease in mutation frequency. The effects on mutation appear to be locus (or allele) specific. Recombination in meiotic cells was measured and was not detectably altered by the presence of 6-methyladenine in GATC sequences.     

The Escherichia coli dam DNA methyltransferase modifies DNA in a highly processive reaction

The Escherichia coli dam adenine-N6 methyltransferase modifies DNA at GATC sequences. It is involved in post-replicative mismatch repair, control of DNA replication and gene regulation. We show that E. coli dam acts as a functional monomer and methylates only one strand of the DNA in each binding event. The preferred way of ternary complex assembly is that the enzyme first binds to DNA and then to S-adenosylmethionine. The enzyme methylates an oligonucleotide containing two dam sites and a 879 bp PCR product with four sites in a fully processive reaction. On lambda-DNA comprising 48,502 bp and 116 dam sites, E. coli dam scans 3000 dam sites per binding event in a random walk, that on average leads to a processive methylation of 55 sites. Processive methylation of DNA considerably accelerates DNA methylation. The highly processive mechanism of E. coli dam could explain why small amounts of E. coli dam are able to maintain the methylation state of dam sites during DNA replication. Furthermore, our data support the general rule that solitary DNA methyltransferase modify DNA processively whereas methyltransferases belonging to a restriction-modification system show a distributive mechanism, because processive methylation of DNA would interfere with the biological function of restriction-modification systems.     

Unusual Modification of Bacteriophage Mu DNA

Bacteriophage Mu DNA was labeled after induction in the presence of [2-(3)H]adenine or [8-(3)H]adenine. Both Mu mom(+).dam(+) DNA and Mu mom(-).dam(+) DNA have similar N(6)-methyladenine (MeAde) contents, as well as similar frequencies of MeAde nearest neighbors. Both DNAs are sensitive to in vitro cleavage by R.DpnI but resistant to cleavage by R.DpnII. These results indicate that the mom(+) protein does not alter the sequence specificity of the host dam(+) methylase to produce MeAde at new sites. However, we have discovered a new modified base, denoted A(x), in Mu mom(+).dam(+) DNA; approximately 15% of the adenine residues are modified to A(x). Although the precise nature of the modification is not yet defined, analysis by electrophoresis and chromatography indicates that the N(6)-amino group is not the site of modification, and that the added moiety contains a free carboxyl group. A(x) is not present in Mu mom(+).dam(+) or Mu mom(-).dam(+) phage DNA or in cellular DNA from uninduced Mu mom(+).dam(+) lysogens. These results suggest that expression of the dam(+) and mom(+) genes are required for the A(x) modification and that this modification is responsible for protecting Mu DNA against certain restriction nucleases. Mu mom(+).dam(-) DNA and Mu mom(-).dam(-) DNA contain a very low level of MeAde (ca. 1 MeAde per 5,000 adenine residues). Since the only nearest neighbor to MeAde appears to be cytosine, we suggest that the methylated sequence is 5'... C-A(*)-C... 3' and that this methylation is mediated by the EcoK modification enzyme.     

Direct role of the Escherichia coli Dam DNA methyltransferase in methylation-directed mismatch repair.

The T4 dam+ gene has been cloned (S. L. Schlagman and S. Hattman, Gene 22:139-156, 1983) and transferred into an Escherichia coli dam-host. In this host, the T4 Dam DNA methyltransferase methylates mainly, if not exclusively, the sequence 5'-GATC-3'; this sequence specificity is the same as that of the E. coli Dam enzyme. Expression of the cloned T4 dam+ gene suppresses almost all the phenotypic traits associated with E. coli dam mutants, with the exception of hypermutability. In wild-type hosts, 20- to 500-fold overproduction of the E. coli Dam methylase by plasmids containing the cloned E. coli dam+ gene results in a hypermutability phenotype (G.E. Herman and P. Modrich, J. Bacteriol. 145:644-646, 1981; M.G. Marinus, A. Poteete, and J.A. Arraj, Gene 28:123-125, 1984). In contrast, the same high level of T4 Dam methylase activity, produced by plasmids containing the cloned T4 dam+ gene, does not result in hypermutability. To account for these results we propose that the E. coli Dam methylase may be directly involved in the process of methylation-instructed mismatch repair and that the T4 Dam methylase is unable to substitute for the E. coli enzyme.     

A mutation in the DNA adenine methylase gene (dam) of Salmonella typhimurium decreases susceptibility to 9-aminoacridine-induced frameshift mutagenesis

A mutant of Salmonella typhimurium with a reduced response to mutation induction by 9-aminoacridine (9AA) has been isolated. The mutation (dam-2) is located in the DNA adenine methylase gene. The dam-2 mutant strain exhibits a level of sensitivity to 2-aminopurine (2AP) intermediate between that of the dam+ and the DNA adenine methylation-deficit dam-1 strain, and 2AP sensitivity was reversed by introduction of a mutH mutation or of the plasmid pMQ148 (which carries a functional Escherichia coli dam+ gene). However, the dam-2 strain is not grossly defective in DNA adenine methylase activity. Whole cell DNA appears full methylated at -GATC- sites. The levels of 9AA required to induce equivalent levels of frameshift mutagenesis in the dam-2 strain were approximately 2-fold higher than for the dam+ strain. Introduction of pMQ148 dam+ reduced the level of 9AA required for induction of frameshift mutations 4-fold in the dam-2 strain and 2-fold in the dam+ strain. The dam-2 mutation had no effect on the levels of ICR191 required for induction of frameshift mutations, but introduction of pMQ148 reduced the ICR191-induced mutagenesis 2-fold. The dam+/pMQ148, dam-2/pMQ148 and dam-1/pMQ148 strains showed identical dose-response curves for both 9AA and ICR191. These results are consistent with a slightly reduced (dam-2) or increased (pMQ148) rate of methylation at the replication fork. The 2AP sensitivity of the dam-2 strain cannot be simply explained. Furthermore, addition of methionine to the assay medium reverses the 2AP sensitivity of the dam-2 strain, but has no effect on 9AA mutagenesis.     

Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease.

The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme. The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine. This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease. Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification.     

Methylation of GATC sites is required for precise timing between rounds of DNA replication in Escherichia coli.

We have used the Koppes and Nordstr?m (Cell 44:117-124, 1986) CsCl density transfer approach for analysis of DNA from exponentially growing, isogenic Escherichia coli dam+ and dam mutant cells to show that timing between DNA replication initiation events is precise in the dam+ cells but is essentially random in the dam cells. Thus, methylation of one or more GATC sites, such as those found in unusual abundance within the origin, oriC, is required for precise timing between rounds of DNA replication, and precise timing between initiation events is not required for cell viability. Both the dam-3 point mutant and the delta(dam)100 complete deletion mutant were examined. The results were independent of the mismatch repair system; E. coli mutH cells showed precise timing, whereas timing in the isogenic E. coli mutH delta(dam)100 double mutant was random. The mechanism is thus different from the role of Dam methylation in mismatch repair and probably involves conversion of hemimethylated GATC sites present in daughter origins just after initiation to a fully methylated state.     

Restriction of two-replicon shuttle Escherichia coli-Streptomyces plasmids in Streptomyces lividans strain 66

The shuttle Escherichia coli - Streptomyces plasmids were used to transform S. lividans 66. Plasmid DNAs isolated from this strain transform it 10-1000-fold more efficiently than DNAs from E. coli. Rare transformant cured from most restricted plasmid is more efficient recipient of plasmid DNA from E. coli and has the property of R +/- M+ mutant. Restriction in S. lividans 66 correlates with the appearance in DNA from E. coli of the sites susceptible to Scg2I restriction endonuclease. The latter was isolated earlier from recombinant strain Rcg2, a hybrid between S. griseus Kr. 15 and S. coelicolor A3(2). Scg2I possesses the recognition sequence CCTAGG, like EcoRII, MvaI and Eco dcm methylase. The DNA resistant to Scg2I cleavage retained this ability after in vitro modification by EcoRII methylase. So, the resistance of DNA to Scg2I cleavage is not connected with methylation at 4th and 5th position of second cytosine in the recognition sequence. Neither restriction of plasmid DNA in S. lividans 66 is dependent on dcm modification in E. coli, though its dependence on dam modification is not excluded. It is assumed that the restriction in S. lividans 66 is specified by endonuclease analogous to Scg2I.