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.     

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.     

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.     

Methylated Bases of Bacillus megaterium KM Nucleic Acids: Comparison with Escherichia coli

Parallel studies were performed with methionineless derivatives of Escherichia coli 15 T(-) and Bacillus megaterium KM: T(-). Methylated bases are present in the total cell ribonucleic acid (RNA) of B. megaterium. The level of RNA methylation in E. coli is about 60% greater than that in B. megaterium. Although E. coli deoxyribonucleic acid (DNA) was found to contain 0.12% 5-methylcytosine (5-MC) and 0.24% 6-methylaminopurine (6-MA), methylated bases were not detected in the DNA of B. megaterium. Assuming a molecular weight of 7 x 10(9) daltons for B. megaterium DNA, it was calculated that this organism could not contain more than one molecule of 5-MC or 6-MA per genome, and that possibly no methylated bases were present. Methylated bases were also not detected in the DNA of thymine-starved B. megaterium. Crude extracts of this organism possess RNA methylase activity but no detectable DNA methylase activity.     

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.     

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.     

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.     

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.     

Bacterial DNA containing methylated CpG motifs retains immunostimulatory activity in synergy with modified lipopolysaccharides

We previously described the immunostimulatory activity of CIA07, a combination of bacterial DNA fragments and modified LPS, and demonstrated that CIA07 has antitumor activity in a mouse bladder cancer model. In this study, we investigated whether methylation of the CpG motifs on the bacterial DNA fragments affects the immunostimulatory potential of CIA07. E. coli DNA fragments were methylated with CpG methylase, and then combined with modified LPS for experiments. Our results revealed that methylated CIA07 (mCIA07) and unmethylated CIA07 were equally active in inducing cytokine secretion from human whole blood cells. In addition, both methylated DNA fragments and mCIA07 retained the ability to activate expression and nuclear translocation of NF-kappaB in RAW 264.7 cells. Finally, methylated DNA fragments and mCIA07 exhibited an antitumor activity comparable to those of their unmethylated counterparts in our mouse bladder cancer model. These data demonstrate that CpG methylation of E. coli DNA does not abrogate the immunostimulatory activity of DNA fragments or CIA07, suggesting that the synergistic activity by bacterial DNA in combination with LPS may be independent of the methylation status of CpG motifs.     

Genetic transformation of Geobacillus kaustophilus HTA426 by conjugative transfer of host-mimicking plasmids

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, 10(-5)-10(-3) recipient(-1)). pSTE33T showed lower efficiency (10(-7)-10(-6) recipient(-1)) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.     

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.     

Identification and sequence analysis of a methylase gene in Porphyromonas gingivalis.

A gene from the periodontal organism Porphyromonas gingivalis has been identified as encoding a DNA methylase. The gene, referred to as pgiIM, has been sequenced and found to contain a reading frame of 864 basepairs. The putative amino acid sequence of the encoded methylase was 288 amino acids, and shared 47% and 31% homology with the Streptococcus pneumoniae DpnII and E. coli Dam methylases, respectively. The activity and specificity of the pgi methylase (M.PgiI) was confirmed by cloning the gene into a dam- strain of E. coli (JM110) and performing a restriction analysis on the isolated DNA with enzymes whose activities depended upon the methylation state of the DNA. The data indicated that M.PgiI, like DpnII and Dam, methylated the adenine residue within the sequence 5'-GATC-3'.     

Site-specific methylases induce the SOS DNA repair response in Escherichia coli.

Expression of the site-specific adenine methylase HhaII (GmeANTC, where me is methyl) or PstI (CTGCmeAG) induced the SOS DNA repair response in Escherichia coli. In contrast, expression of methylases indigenous to E. coli either did not induce SOS (EcoRI [GAmeATTC] or induced SOS to a lesser extent (dam [GmeATC]). Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response. Cytosine methylases foreign to E. coli (MspI [meCCGG], HaeIII [GGmeCC], BamHI [GGATmeCC], HhaI [GmeCGC], BsuRI [GGmeCC], and M.Spr) also induced SOS, whereas one indigenous to E. coli (EcoRII [CmeCA/TGG]) did not. SOS induction by cytosine methylation required the rglB locus, which encodes an endonuclease that cleaves DNA containing 5-hydroxymethyl- or 5-methylcytosine (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986).     

Effects of High Levels of DNA Adenine Methylation on Methyl-Directed Mismatch Repair in ESCHERICHIA COLI

Two methods were used in an attempt to increase the efficiency and strand selectivity of methyl-directed mismatch repair of bacteriophage lambda heteroduplexes in E. coli. Previous studies of such repair used lambda DNA that was only partially methylated as the source of methylated chains. Also, transfection was carried out in methylating strains. Either of these factors might have been responsible for the incompleteness of the strand selectivity observed previously. In the first approach to increasing strand selectivity, heteroduplexes were transfected into a host deficient in methylation, but no changes in repair frequencies were observed. In the second approach, heteroduplexes were prepared using DNA that had been highly methylated in vitro with purified DNA adenine methylase as the source of methylated chains. In heteroduplexes having a repairable cI/+ mismatch, strand selectivity was indeed enhanced. In heteroduplexes with one chain highly methylated and the complementary chain unmethylated, the frequency of repair on the unmethylated chain increased to nearly 100%. Heteroduplexes with both chains highly methylated were not repaired at a detectable frequency. Thus, chains highly methylated by DNA adenine methylase were refractory to mismatch repair by this system, regardless of the methylation of the complementary chain. These results support the hypothesis that methyl-directed mismatch repair acts to correct errors of replication, thus lowering the mutation rate.     

Two restriction endonucleases from Bacillus globiggi.

The sites of action of the restriction enzyme Bgl II on lambda DNA are mapped. This enzyme recognises the sequence 5' ...AGATCT...3' and makes staggered cuts producing sticky ends. In lambda DNA, the second A in this sequence is methylated about 50% of the time by a bacterial methylase absent in E. coli dam. In contrast to Bgl II, Bgl I makes many cuts in lambda DNA and produces 5' terminals which are not substrates for polynucleotide kinase.     

DNA-methylase Sau 3A: isolation and various properties

DNA-methylase Sau 3A has been isolated for the first time from Staphylococcus aureus 3A cells and purified by column chromatography on phosphocellulose PII, heparin-Sepharose and blue Sepharose. The purified enzyme methylates the GATC sequence with the formation of GATm5C as can be evidenced from the protection of DNA from digestion with restrictases Sau 3A and Bam HI, the lack of the C3H3-group incorporation into Sau 3A DNA-restricts and the formation of a single methylated base m5C. Sau 3A methylase modifies only a two-filament (but not one-filament) DNA. Thus, methylase Sau 3A modifies the both DNA chains in the recognition site during a single binding act. The 5-azacytidine-containing DNA inhibits by 95% the activity of methylase Sau 3A. Ado-met is the single methyl group donor for methylase Sau 3A. The presence of m6A in the recognition site does not affect the activity of methylase Sau 3A. The practical recommendations for the use of M. Sau 3A, alongside with M. Eco dam, for the study of dam methylation by additional methylation of the DNA in vitro in the presence of [methyl-3H]-S-adenosyl-methionine are given.     

A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA.

A deoxyribonuclease specific for methylated DNA was isolated from Diplococcus pneumoniae. The enzyme, an endonuclease, degrades DNA for Escherichia coli to fragments of average molecular weight about half a million; it forms discrete fragments from phage lambda DNA. Methyl-deficient E. coli DNA is not attacked, neither is DNA from Micrococcus radiodurans, which contains no methylated adenine or cytosine. Nor is DNA from D. pneumoniae or phage T7 attacked. However, DNA from M. radiodurans, D. pneumoniae, and T7 is attacked after methylation with and E. coli extract. Methylated T7 DNA is degraded to discrete fragments. Although the genetic transforming activity of normal DNA from D. pneumoniae is not affected by the enzyme, transforming activity of methylated DNA is destroyed. The enzyme is designated endonuclease R Dpn I. Under certain conditions another enzyme of complementary specificity can be isolated. This enzyme, designated endonuclease R Dpn II, produces a similar pattern of fragments from the DNA of T7 without prior methylation of the DNA. It also degrades normal DNA for D. pneumoniae. It is suggested that this pair of enzymes plays a role in some unknown control process, which would involve a large fraction of the specific base sequences that are methylated in E. coli DNA and are present but not methylated in DNA from other sources.