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.     

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.     

The oriC unwinding by dam methylation in Escherichia coli.

It has been shown that dam methylation is important in the regulation of initiation of DNA replication in E.coli. The question then arises as to whether dam methylation in the oriC region mediates any structural changes in DNA involved in the regulation of initiation of DNA replication. We demonstrate that the thermal melting temperature of the oriC region is lowered by adenine methylation at GATC sites. The regulation of initiation of DNA replication by dam methylation may be attributed to the ease of unwinding at GATC sites in oriC.     

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.     

Influence of DNA adenine methylase on the sensitivity of Escherichia coli to near-ultraviolet radiation and hydrogen peroxide

Near-ultraviolet (NUV) radiation and hydrogen peroxide (H2O2) inactivation studies were performed on Escherichia coli K-12 DNA adenine methylation (dam) mutants and on cells that carry plasmids which overexpress Dam methylase. Lack of methylation resulted in increased sensitivity to NUV and H2O2 (a photoproduct of NUV). In a dam mutant carrying a dam plasmid, the levels of Dam enzyme and resistance to NUV and H2O2 were restored. However, using a multicopy dam+ plasmid strain, increasing the methylase above wildtype levels resulted in an increase in sensitivity of the cells rather than resistance.     

Detection and analysis of enzymatic DNA methylation of oligonucleotide substrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry was employed to analyze DNA methylation carried out by the Escherichia coli dam DNA methyltransferase using oligonucleotide substrates with molecular masses of 5000-10,000 Da per strand. The mass spectrometry assay offers several advantages: (i) it directly shows the methylation as the increase in the mass of the substrate DNA, (ii) it is nonradioactive, (iii) it is quantitative, and (iv) it can be automated for high-throughput applications. Since unmethylated and methylated DNA are detected, the ratio of methylation can be determined directly and accurately. Furthermore, the assay allows detection individually of the methylation of several substrates in competition, offering an ideal setup to analyze the specificity of DNA interacting with enzymes. We could not identify methylation at any noncanonical site, indicating that the dam MTase is a very specific enzyme. Finally, MALDI-TOF mass spectrometry permitted assessment of the number of methyl groups incorporated into each DNA strand, thereby, allowing study of mechanistic details such as the processivity of the methylation reaction. We provide evidence that the dam MTase modifies DNA in a processive reaction, confirming earlier findings.     

Roles of DNA adenine methylation in controlling replication of the REPI replicon of plasmid pColV-K30

DNA adenine methylation controls DNA replication of plasmids containing the prototypic REPI replicon by affecting protein recognition and by altering the helical stability of the origin. Denaturing gradient gel electrophoresis shows that adenine methylated origin DNA is more easily melted than unmethylated. However, because an added DNA adenine methylation (dam) site at the origin, whether in or out of phase with other helically aligned dam sites, actually prevents replication, we conclude that destabilization of the helix is not the exclusive function of adenine methylation in REPI replication. We find that the conformation and degree of methylation at the origin, features which are important for protein recognition, are essential for replication. In fact, RepI, a protein required for replication initiation at REPI replicons, contains a region homologous with a domain in proteins which specifically recognize and bind 5'-GATC-3'. We propose that the dam sites in the origin play a dual role: one is destabilization of the helix, and the other is protein recognition.     

Biological role of DNA methylation: sequence-specific single-strand breaks associated with hypomethylation of GATC sites in Escherichia coli DNA.

The effect of methylation of GATC sites in Escherichia coli DNA on the formation of single-strand breaks was studied with dam+, dam mutant, and Dam-overproducer strains. Single-strand breaks have been observed in dam mutant cells predominantly at TpT and, to a lesser extent, at CpC. In dam mutant cells harboring pTP166 (a plasmid containing the dam gene), no such nicks were observed.     

Developmental abnormalities associated with deoxyadenosine methylation in transgenic tobacco

As in other higher eukaryotes, DNA methylation in plants is predominantly found at deoxycytosine residues, while deoxyadenosine residues are not methylated at significant levels. ^6mdA methylation has been successfully introduced into yeast and Drosophila via expression of a heterologous methyltransferase, but similar attempts in tobacco had, up until now, proved unsuccessful despite the correct expression of a methyltransferase construct. It was unclear whether this result reflected the failure of heterologous methyltransferases to enter the nucleus, or whether ^6mdA methylation, which has been shown to interfere with promoter activity, was toxic for plants. Here we show that ^6mdA methylation can be successfully introduced into transgenic tobacco plants via expression of the bacterial dam enzyme. The efficiency of ^6mdA methylation was directly proportional to expression levels of the dam construct, and methylation of all GATC sites was observed in a highly expressing line. Increasing expression levels of the enzyme in different plants correlated with increasingly abnormal phenotypes affecting leaf pigmentation, apical dominance, and leaf and floral structure. Whilst introduction of dam -specific methylation does not cause any developmental abnormalities in yeast or Drosophila , our data suggest that methylation of deoxyadenine residues in plants interferes with the expression of genes involved in leaf and floral development.     

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.     

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.     

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.     

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.     

Inactivation of deoxyadenosine methyltransferase (dam) attenuates Haemophilus influenzae virulence

Mutants in deoxyadenosine methyltransferase (dam) from many Gram-negative pathogens suggest multiple roles for Dam methylase: directing post-replicative DNA mismatch repair to the correct strand, guiding the temporal control of DNA replication and regulating the expression of multiple genes (including virulence factors) by differential promoter methylation. Dam methylase (HI0209) in strain Rd KW20 was inactivated in Haemophilus influenzae strains Rd KW20, Strain 12 and INT-1; restriction with Dam methylation-sensitive enzymes DpnI and DpnII confirmed the absence of Dam methylation, which was restored by complementation with a single copy of dam ectopically expressed in cis. Despite the lack of increased mutation frequency, the dam mutants had a 2-aminopurine-susceptible phenotype that could be suppressed by secondary mutations in mutS, suggesting a role for Dam in H. influenzae DNA mismatch repair. Invasion of human brain microvascular endothelial cells (HBMECs) and human respiratory epithelial cells (NCI-H292) by the dam mutants was significantly attenuated in all strains, suggesting the absence of a Dam-regulated event necessary for uptake or invasion of host cells. Intracellular replication was inhibited only in the Strain 12 dam mutant, whereas in the infant rat model of infection, the INT-1 dam mutant was less virulent. Dam activity appears to be necessary for both in vitro and in vivo virulence in a strain-dependent fashion and may function as a regulator of gene expression including virulence factors.     

Bacteriophage Mu-induced modification of DNA is dependent upon a host function.

The DNA of bacteriophage Mu, extracted from induced lysates, is partially resistant to digestion by the endonuclease BalI. This modification of DNA is controlled by the Mu modification function (mom), which acts in conjunction with the dam (DNA-adenine methylation) function of Escherichia coli. Since the BalI recognition site is apparently different from the dam recognition site, these results imply that either the specificity of the dam function is changed by the mom function or the mom function requires the dam function for its activity.     

The great GATC: DNA methylation in E. coli

In Escherichia coli the methylation of the adenine in the sequence 5'-GATC-3' is catalysed by the dam gene product, a DNA adenine methylase. We review the proposed roles for this methylation, and the sequence it modifies, in mismatch repair, DNA-protein interaction, gene expression, the initiation of chromosome replication, chromosome segregation, chromosome structure and the occurrence of mutational hotspots.