The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.     

Sequence specificity of the wild-type dam+) and mutant (damh) forms of bacteriophage T2 DNA adenine methylase

Non-glucosylated, non-methylated phage T2 DNA was methylated in vitro with partially purified wild-type (dam⁺) or mutant (dam^h) T2 DNA adenine methylase. The radioactively labeled methyladenine-containing DNA was enzymatically degraded and the resulting oligonucleotides were separated according to chain length by DEAE-cellulose chromatography. Following “fingerprinting” by two-dimensional electrophoresis, we determined the sequence for various di-, tri- and tetranucleotides containing radioactive N⁶-methyldeoxyadenosine. From this analysis we conclude that both T2 dam⁺ and T2 dam^h contain the sequence 5′…G-mA -Py…3′.     

Analysis of the genetic requirements for viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants.

RecBCD protein, necessary for Escherichia coli dam mutant viability, is directly required for DNA repair. Recombination genes recF+, recN+, recO+, and recQ+ are not essential for dam mutant viability; they are required for recBC sbcBC dam mutant survival. mutH, mutL, or mutS mutations do not suppress subinduction of SOS genes in dam mutants.     

Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli

The gene coding for the sequence-specific modification methylase methM . BspI of Bacillus sphaericus R has been cloned in Escherichia coli by means of plasmid pBR322. The selection was based on the expression of the cloned gene which rendered the recombinant plasmid resistant to BspI restriction endonuclease cleavage. The gene is carried by a 9 kb BamHI fragment and by a smaller 2.5 kb EcoRI fragment derived from the BamHI fragment. The Bsp-specific methylase level was found to be higher in the recombinant clones than in the parental strain. The methylase gene is probably located on the Bacillus sphaericus chromosome, and not on a plasmid known to be carried by this strain. The recombinant clones do not exhibit an BspI restriction endonuclease activity.     

Studies on the substrate specificity of the DNA methylase activity from Escherichia coli K-12.

A partially purified extract of DNA methylases from E. coli K-12 containing DNA-adenine as well as DNA-cytosine methylase activities has been examined with respect to different DNA species as substrates. The results show that the natural content of 6-MAP) in the applied DNA represses the DNA-adenine methylase activity. On the other hand, 5-MC, already present in the substrate does not influence the activity of the DNA-cytosine methylase. DNA from Micrococcus radiodurans, which is completely free of methylated bases served as comparison. Since netropsin preferentially binds to AT-rich regions of DNA, the influence of this oligopeptide antibiotic on the methylation of DNA was investigated. As expected the antibiotic predominantly inhibits adenine methylation of DNA. The degree of inhibition depends on the molar ratio of netropsin to DNA phosphate.     

Sequence specificity of DNA adenine methylase in the protozoan Tetrahymena thermophila.

The sequence specificity of the Tetrahymena DNA-adenine methylase was determined by nearest-neighbor analyses of in vivo and in vitro methylated DNA. In vivo all four common bases were found to the 5' side of N6-methyladenine, but only thymidine was 3'. Homologous DNA already methylated in vivo and heterologous Micrococcus luteus DNA were methylated in vitro by a partially purified DNA-adenine methylase activity isolated from Tetrahymena macronuclei. The in vitro-methylated sequence differed from the in vivo sequence in that both thymidine and cytosine were 3' nearest neighbors of N6-methyladenine.     

Sequence and functional-group specificity for cleavage of DNA junctions by RuvC of Escherichia coli.

RuvC is the DNA junction-resolving enzyme of Escherichia coli. While the enzyme binds to DNA junctions independently of base sequence, it exhibits considerable sequence selectivity for the phosphodiester cleavage reaction. We have analyzed the sequence specificity using a panel of DNA junctions, measuring the rate of cleavage of each under single-turnover conditions. We have found that the optimal sequence for cleavage can be described by (A approximately T)TT downward arrow(C>G approximately A), where downward arrow denotes the position of backbone scission. Cleavage is fastest when the cleaved phosphodiester linkage is located at the point of strand exchange. However, cleavage is possible one nucleotide 3' of this position when directed by the sequence, with a rate that is 1 order of magnitude slower than the optimal. The maximum sequence discrimination occurs at the central TT in the tetranucleotide site, where any alteration of sequence results in a rate reduction of at least 100-fold and cleavage is undetectable for some changes. However, certain sequences in the outer nucleotides are strongly inhibitory to cleavage. Introduction of base analogues around the cleavage site reveals a number of important functional groups and suggests that major-groove contacts in the center of the tetranucleotide are important for the cleavage process. Since RuvC binds to all the variant junctions with very similar affinity, any contacts affecting the rate of cleavage must be primarily important in the transition state. Introduction of the optimal cleavage sequence into a three-way DNA junction led to relatively efficient cleavage by RuvC, at a rate only 3-fold slower than the optimal four-way junction. This is consistent with a protein-induced alteration in the conformation of the DNA.     

Alleviation of type I restriction in adenine methylase (dam) mutants of Escherichia coli

Summary The host-controlled EcoK-restriction of unmodified phage .O is alleviated in dam mutants of Escherichia coli by 100- to 300-fold. In addition, the EcoK modification activity is substantially decreased in dam ^- strains. We show that type I restriction (EcoB, EcoD and EcoK) is detectably alleviated in dam mutants. However, no relief of EcoRI restriction (Type II) occurs in dam ^- strains and only a slight effect of dam mutation on EcoP1 restriction (Type III) is observed. We interpret the alleviation of the type I restriction in dam ^- strains to be a consequence of induction of the function which interferes with type I restriction systems.     

Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M.SssI).

We describe here the cloning, characterization and expression in E. coli of the gene coding for a DNA methylase from Spiroplasma sp. strain MQ1 (M.SssI). This enzyme methylates completely and exclusively CpG sequences. The Spiroplasma gene was transcribed in E. coli using its own promoter. Translation of the entire message required the use of an opal suppressor, suggesting that UGA triplets code for tryptophan in Spiroplasma. Sequence analysis of the gene revealed several UGA triplets, in a 1158 bp long open reading frame. The deduced amino acid sequence revealed in M.SssI all common domains characteristic of bacterial cytosine DNA methylases. The putative sequence recognition domain of M.SssI showed no obvious similarities with that of the mouse DNA methylase, in spite of their common sequence specificity. The cloned enzyme methylated exclusively CpG sequences both in vivo and in vitro. In contrast to the mammalian enzyme which is primarily a maintenance methylase, M.SssI displayed de novo methylase activity, characteristic of prokaryotic cytosine DNA methylases.     

The cloning, purification and characterization of the Eco RV modification methylase.

The gene for the Eco RV methylase has been cloned into a plasmid under control of the strong lambda PL promoter and overexpressed in E. coli. This plasmid, pVIC1, gives reliable overexpression of the methylase at levels of about 20% of total protein. Maximum yields of soluble protein are achieved after about 6 hours of induction. If the cells are harvested later than this much of the enzyme is found in the pellet fraction following centrifugation. A two column purification scheme using phosphocellulose and Blue-Sepharose chromatography has been developed. This yielded pure methylase in amounts of 5mg per gram E. coli cell paste. The enzyme is monomeric and methylates the first deoxyadenosine residue in its recognition sequence GATATC.     

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.     

Viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants requires increased expression of specific genes in the SOS regulon

Summary We have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam ^-). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD and dinF) were found to express higher levels of -galactosidase in dam ^- strains than in isogenic dam ⁺ strains. The attempted construction of dam ^- strains that were also mutant in one of several SOS genes indicated that the viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for dam ^- strain viability.     

Sequence and expression of the Escherichia coli K1 neuC gene product.

The nucleotide sequence of the neuC gene of the Escherichia coli K1 capsule gene cluster encodes a protein with a predicted molecular weight of 44,210 containing 391 amino acids. A chimeric protein with beta-galactosidase fused to the carboxy terminus of the neuC gene product (P7) was constructed and purified. Its amino-terminal sequence confirmed the prediction from the nucleotide sequence that the neuC gene overlaps the distal end of the neuA gene by a single base pair. Both the neuA and neuC genes are coexpressed under the control of a single upstream T7 or tac promoter, suggesting that neuA and neuC are part of an operon.     

Twelve species of the nucleoid-associated protein from Escherichia coli. Sequence recognition specificity and DNA binding affinity.

The genome of Escherichia coli is composed of a single molecule of circular DNA with the length of about 47,000 kilobase pairs, which is associated with about 10 major DNA-binding proteins, altogether forming the nucleoid. We expressed and purified 12 species of the DNA-binding protein, i.e. CbpA (curved DNA-binding protein A), CbpB or Rob (curved DNA-binding protein B or right arm of the replication origin binding protein), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q(beta)), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of td(-) phenotype A). The sequence specificity of DNA binding was determined for all the purified nucleoid proteins using gel-mobility shift assays. Five proteins (CbpB, DnaA, Fis, IHF, and Lrp) were found to bind to specific DNA sequences, while the remaining seven proteins (CbpA, Dps, Hfq, H-NS, HU, IciA, and StpA) showed apparently sequence-nonspecific DNA binding activities. Four proteins, CbpA, Hfq, H-NS, and IciA, showed the binding preference for the curved DNA. From the apparent dissociation constant (K(d)) determined using the sequence-specific or nonspecific DNA probes, the order of DNA binding affinity were determined to be: HU > IHF > Lrp > CbpB(Rob) > Fis > H-NS > StpA > CbpA > IciA > Hfq/Dps, ranging from 25 nM (HU binding to the non-curved DNA) to 250 nM (Hfq binding to the non-curved DNA), under the assay conditions employed.